tag:blogger.com,1999:blog-56974334807023975222024-02-21T07:03:50.819+05:30MicroboidsSharing Knowledge improves Knowledge...
Knowledge should come at as less cost as possible.Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.comBlogger291125tag:blogger.com,1999:blog-5697433480702397522.post-2190368540232676862020-08-17T18:51:00.000+05:302020-08-17T18:51:10.111+05:30Laboratory Series#19: Viral Transport medium<div class="separator"><div style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em; text-align: justify;">In the current scenario of coping with large diagnostic load, a lot of people have asked me on use of VTM in Viral diagnostics. Here are the absolute basics.</div><div style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><div style="text-align: justify;">Viral transport medium is a liquid media that can sustain the virus numbers, infectivity and reduce or inhibit any possible contaminants (such as bacteria or fungus). As already noted, VTM is not a universal requirement. In some cases, the sample may be submitted for analysis as it is. Samples including CSF, blood, BAL and urine samples can be simply sent in a cold chain. For other samples such as tissue biopsies, nasal swabs, skin swabs etc. VTM is preferred. There a large number of commercial and inhouse formulations of VTM available. Each of these supports good recovery of a few range of viruses. The basic components of all these viral transport media is a buffer solution with protective components such as proteins or charcoal and a PH indicator. A widely accepted principle is that VTMs are isotonic in nature. However, this is not true since there are several transport media which have high tonicity due to high sucrose content and have been successfully used. Note that VTM in itself dilutes the concentration of the virus from the specimen since the VTM is subsequently used for the testing procedure. Further, additional steps such as vigorous vortexing and centrifugation may be required to suspend the virus into medium especially for swabs. This allows for the removal of viruses that are otherwise strongly adsorbed onto the swab surface.</div><div style="text-align: justify;"><br /></div><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjkEL_sHcFQNRTE7bO3qsv_kIAwEyvhmnQxUXiEKIc6aLy4d6iOACXotbgii35Yt154sVibvkWWH78hvrYXNHYbA-209qtUp9oQu6NMXrm2hoDroQNxjU7NF0slXD9FNG4srbKZmYEY2JK6/s748/VTM.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="400" data-original-width="748" height="342" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjkEL_sHcFQNRTE7bO3qsv_kIAwEyvhmnQxUXiEKIc6aLy4d6iOACXotbgii35Yt154sVibvkWWH78hvrYXNHYbA-209qtUp9oQu6NMXrm2hoDroQNxjU7NF0slXD9FNG4srbKZmYEY2JK6/w640-h342/VTM.png" width="640" /></a></td></tr><tr><td class="tr-caption" style="text-align: center;"><b>Figure 1:</b> Universal Viral Transport Medium. <a href="https://www.bd.com/en-us/offerings/capabilities/specimen-collection/swab-based-specimen-collection/bd-universal-viral-transport-system" target="_blank">Source</a></td></tr></tbody></table><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><b>Cell culture medium:</b></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Most cell culture medium (CCM) contain buffer solutions, a minimal set of nutrients, and a PH indicator. They are also used in the laboratory for attaining cell growth and supporting virus culture in-vitro. In this view, several CCMs have been studied and standardized for alternative use as VTM. The most common example include Eagle’s minimum essential medium with 10% fetal bovine albumin for recovery of HSV from genital swabs. However, they were found to be less efficient especially if longer time is involved. Currently, modified CCMs are used for transport of specimens only if it involves a very short time (< 30 min).</div></div></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><b>Balanced Salt Solutions:</b></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Balanced Salt solutions (BSS) are extremely useful in controlling the PH which is a major determinant of stability. The most commonly used formulation is phosphate-buffered saline or Hanks BSS. The solutions were combined with other proteins (ex: Albumin, gelatin), buffers (Ex: HEPES buffer) or others (Ex: Agar, Mucopolysaccharides, gelatin, Charcoal) to enhance the virus viability.</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><b>Buffered Sugar solutions:</b></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Combining of buffers with sugars such as Sorbitol or Sucrose in concentrations served as a strong cryoprotectant and have been previously used as transport mediums. However, they cause a significant reduction in the number of enveloped viruses and hence their use has been limited.</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><b>Bentonite based Solution:</b></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Bentonite is an absorbent clay that acts as an excellent cation- exchange resin. They have been used as a plain or with a combination of proteins that enhance stability. The combination has been found to provide extreme stability for several viruses for up to 21 days.</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><div align="center"><table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none;"><tbody><tr><td style="border: 1pt solid; padding: 0cm 5.4pt;" valign="top"><p align="center" class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: center;"><b><span lang="" style="font-family: Optima; font-size: 11pt;">Virus Transport medium<o:p></o:p></span></b></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p align="center" class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: center;"><b><span lang="" style="font-family: Optima; font-size: 11pt;">Main Contents<o:p></o:p></span></b></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p align="center" class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: center;"><b><span lang="" style="font-family: Optima; font-size: 11pt;">Examples of Best Supported Viruses<o:p></o:p></span></b></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Stuart’s Medium<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Agar<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Thioglycolic acid<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Sodium glycerophosphate<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Calcium chloride<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Methylene blue<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Herpes Simplex Virus- 1 & 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Virocult combination<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">M</span><span lang="" style="font-family: Optima; font-size: 11pt;">odified Stuart medium<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Phosphate buffer<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">D-glucose<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Lactalbumin hydrolysate<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Chloramphenicol & Cycloheximide<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="font-family: Optima; font-size: 11pt;">Herpes Simplex Virus<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="font-family: Optima; font-size: 11pt;">Varicella-Zoster Virus<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="font-family: Optima; font-size: 11pt;">Influenza Type A& B.<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Hanks </span><span lang="" style="font-family: Optima; font-size: 11pt;">Buffered BSA<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Hanks BSS<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">1% Bovine Serum Albumin (BSA)<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Sodium bicarbonate<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Phenol Red<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">*With or without antibiotics (Amphotericin B, Penicillin G and streptomycin).<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNoSpacing" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="font-family: Optima; font-size: 11pt;">Parainfluenza virus<o:p></o:p></span></p><p class="MsoNoSpacing" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="font-family: Optima; font-size: 11pt;">Enterovirus<o:p></o:p></span></p><p class="MsoNoSpacing" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="font-family: Optima; font-size: 11pt;">Adenovirus<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Bentonite Transport medium<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Tris buffer<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">EDTA<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Bentonite coated with rabbit serum<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">HSV<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Influenza viruses<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Rubella viruses<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Carr-Scarborough<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Phosphate-buffered sucrose<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">L-15 medium<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Glutamic acid<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Bovine albumin<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Gentamicin<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Most commonly used for Chlamydia recovery.<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Varicella-Zoster Virus<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Leibovitz-SPG<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Leibovitz Buffer<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">S</span><span lang="" style="font-family: Optima; font-size: 11pt;">ucrose<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Phosphate buffer<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Glutamate<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">BSA<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">HSV<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Rhinovirus<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Charcoal VTM (CVTM)<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Phosphate-buffered saline<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Potassium chloride<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Charcoal<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Agar<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Adenovirus<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Modified Leibovitz-Emory<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Same as CVTM. Agarose use instead of Agar<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Adenovirus<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt;" valign="top"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Richards media<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 187pt;" valign="top" width="249"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span face="" lang="" style="font-family: "courier new"; font-size: 11pt;"></span><span lang="" style="font-family: Optima; font-size: 11pt;">Phosphate buffer<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Sucrose<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Amino acids<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Bovine serum<o:p></o:p></span></p><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">phenol red<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 160.15pt;" valign="top" width="214"><p class="MsoNormal" style="font-family: "times new roman", serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt; text-align: justify;"><span lang="" style="font-family: Optima; font-size: 11pt;">Most respiratory viruses<o:p></o:p></span></p></td></tr></tbody></table></div></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><b>Table 1:</b> <span style="font-family: Optima;">Various types of VTMs available for use and their contents.</span></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Since many different VTMs do not support the recovery of a plethora of viruses more recently formulations have been made that support almost every virus. These are commercially available and known as UVTM (Universal VTMs) and are available from most of the standard suppliers. Alcohol-based VTM (Ex: CyMol VTM) are also commercially available. They rapidly inactivate the virus but maintain the integrity and structure of the virus. They are compatible with downstream applications such as PCR and immunofluorescence, but not culture.</div>Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-85734134523151922122020-07-15T17:36:00.005+05:302020-07-15T17:58:48.034+05:30COVID 19 Vaccine Pipeline<div style="text-align: justify;">In follow up with the earlier posts on the current situation of COVID19 there is an ever increased focussed on the development of a vaccine for the current COVID19 pandemic. As of today, there are 23 candidate vaccines currently in various phase of a clinical trial and another 137 candidates in the preclinical evaluation phase. Vaccine design and development is a long process, though some aspects of it can be fast-tracked in an emergency situation such as the current pandemic. Figure 1 provides with an approximate timeline of events for vaccine development. As you can see depending on the success of each phase a complete market-ready vaccine can still take years of R&D into it.</div><div style="text-align: justify;"><br /></div><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEijT9q7qHfFr_ODFpkKfoh70ERQ5cH013BdC2ZICGrrdnisq_9JznDYiUaqYhtcb3JmaCk6-7GXg8Vycqwt_NUrZVGGMaGjQdM6ew3M1al6vvUUfhLel8BQI1D9UZ-NBMxp580ACxilIk_K/s1118/Figure+1.jpg" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="630" data-original-width="1118" height="360" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEijT9q7qHfFr_ODFpkKfoh70ERQ5cH013BdC2ZICGrrdnisq_9JznDYiUaqYhtcb3JmaCk6-7GXg8Vycqwt_NUrZVGGMaGjQdM6ew3M1al6vvUUfhLel8BQI1D9UZ-NBMxp580ACxilIk_K/w640-h360/Figure+1.jpg" width="640" /></a></td></tr><tr><td class="tr-caption" style="text-align: center;"><b>Figure 1:</b> Vaccine development timeline. <a href="https://www.gsk.com/en-gb/research-and-development/development/how-we-develop-new-vaccines/">Source</a></td></tr></tbody></table><div><br /></div><br /><div style="text-align: justify;">There is currently a lack of clarity on various factors of the immune response against SARS CoV2. Though as expected for several viruses, T cell response seems to be important. Studies are currently implying that healthy people have a larger percentage of T cells (Especially helper T cells and Killer T cells) that are reactive to SARS CoV 2 compared to those who are really sick. Interestingly, people who have not been apparently exposed to the virus also have some sort of T cell memory against SARS CoV 2. These are mostly cross-reactive cells, indicating exposure to other coronaviruses. Studies that have looked at the humoral response indicate that though people produce specific antibodies, neutralising antibodies are low in quantity. Several varieties of lymphocytes including CD4+ T cells, CD8+ T cells, B cells, and NK cells have shown a significant association with inflammatory status in COVID-19 CD8+ T cell appears to be the key. Concerns in the rapid deployment of a COVID19 vaccine include the idea that there is a lack of long-term immunity and the possibility of ADE (Antibody-dependent enhancement). The best available picture at present indicates that sterilising immunity and its memory is probably available for about 3-5 months at best, in a natural infection course. In essence, an ideal vaccine has to achieve a longterm neutralising immune response and no ADE.</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Here is a summary list of vaccines and some of their features that are currently in one of the clinical testing phases.</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><div align="center"><table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none; width: 565px;"><tbody><tr><td style="border: 1pt solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><b><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Candidate Vaccine<o:p></o:p></span></b></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><b><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Developer<o:p></o:p></span></b></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><b><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Design<o:p></o:p></span></b></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><b><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Evaluation Phase<o:p></o:p></span></b></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="background-color: white; background-position: initial initial; background-repeat: initial initial; color: #002060; font-family: optima; font-size: 10.5pt;">PiCoVacc</span><span style="color: #002060; font-family: optima; font-size: 10.5pt;"><o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Sinovac<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inactivated SARS CoV 2 CN2 strain with alum<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 3<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">ChAdOx1-S<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">University of Oxford & AstraZeneca<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 3<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">AD5 Vectored COVID19 Vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">CanSino Biological Inc. & Beijing Institute of Biotechnology<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">LNP encapsulated vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Moderna & National Institute of Allergy and Infectious Diseases<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">mRNA-1273 lipid nanoparticle encapsulated mRNA-based vaccine that encodes for a full-length, prefusion stabilized spike (S) protein of SARS-CoV-2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">INO-4800<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inovio Pharmaceuticals & International Vaccine Institute<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="background-color: white; background-position: initial initial; background-repeat: initial initial; color: #002060; font-family: optima; font-size: 10.5pt;">Plasmid pGX9501, encoding for the full length of the Spike glycoprotein of SARS-CoV-2 is administered through electroporation (Celectra device)</span><span style="color: #002060; font-family: optima; font-size: 10.5pt;"><o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">AG0301-COVID19<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Osaka University<o:p></o:p></span></p><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">AnGes<o:p></o:p></span></p><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Takara Bio<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">DNA vaccine encoding antigens from SARS-CoV-2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">ZyCov-D Vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Cadila Health Care Ltd<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">DNA vaccine encoding antigens from SARS-CoV-2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1 / 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inactivated Vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Wuhan Institute of Biological Products & Sinopharm<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inactivated SARS CoV 2 strain<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1 / 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Covaxin<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Bharat Biotech<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inactivated Whole-Virion<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1 / 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">NVX CoV2373<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Novavax<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Full length recombinant SARS CoV-2 glycoprotein nanoparticle vaccine adjuvanted with Matrix M<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1 / 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">BNT162b1 and BNT162b2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">BioNTech/Fosun Pharma/Pfizer<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Nucleoside modified RNAs are formulated in lipid nanoparticles. BNT162b1 encodes a SARS-CoV-2 receptor-binding domain (RBD) antigen, while BNT162b2 encodes the virus’ full-length spike protein antigen.<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1 / 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">GX-19<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Genexine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">DNA vaccine expressing SARS-CoV-2 S-protein antigen.<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1 / 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inactivated Vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Chinese Academy of Medical Sciences (IMBCAMS) & Yunnan Center for Disease Control and Prevention.<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Inactivated SARS CoV 2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Gam-COVID-Vac Lyo.<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Gamaleya Institute<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Adenoviral-based vaccine against SARS-CoV-2<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 2<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Protein subunit vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Clover Biopharmaceuticals, GSK and Dynavax Technologies<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">S-Trimer protein that resembles the coronavirus spike protein</span><span style="font-family: optima; font-size: 10.5pt;"><o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Protein Subunit vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Institute of Microbiology, Chinese Academy of Sciences & Zhifei Longcom<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Chinese Hamster Ovary cells producing a modified form of the coronavirus spike protein Adjuvanted recombinant protein (RBD-Dimer).<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Covax19<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Vaxine Pty & MedyTox<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Spike protein along with an adjuvant called Advax</span><span style="color: #002060; font-family: optima; font-size: 10.5pt;"><o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Protein Subunit<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">University of Queensland, GSK and Dynavax Technologies<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Molecular clamp” technology containing<o:p></o:p></span></p><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">stabilized Spike protein<o:p></o:p></span></p><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;"> </span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">COVAC1<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Imperial College London<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">LNP containig a self-amplifying ribonucleic acid (saRNA) vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">CVnCoV<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Curevac<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">mRNA encoding the spike protein of SARS-CoV-2 packaged into lipid nanoparticles.<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">ARCoV<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">People's Liberation Army (PLA) Academy of Military Sciences, Suzhou Abogen Biosciences and Walvax Biotechnology<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">SARS CoV 2 mRNA Vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr><tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 99.65pt;" valign="top" width="133"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">VLP vaccine<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 141.1pt;" valign="top" width="188"><p class="Default" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Medicago Inc, GSK and Dynavax<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 127.55pt;" valign="top" width="170"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span lang="" style="color: #002060; font-family: optima; font-size: 10.5pt;">Plant derived virus like particles<o:p></o:p></span></p></td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 55.35pt;" valign="top" width="74"><p class="MsoNormal" style="font-family: calibri, sans-serif; font-size: 12pt; margin: 0cm 0cm 0.0001pt;"><span style="color: #002060; font-family: optima; font-size: 10.5pt;">Phase 1<o:p></o:p></span></p></td></tr></tbody></table></div></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;"></div><div style="text-align: justify;"><b>References:</b></div><div style="text-align: justify;"><br /></div>1. Weiskopf et al. Phenotype and kinetics of SARS-CoV-2–specific T cells in COVID-19 patients with acute respiratory distress syndrome. <a href="https://www.blogger.com/#">Link</a><br /><br />2. Yarmarkovich et al. Identification of SARS-CoV-2 Vaccine Epitopes Predicted to Induce Long-Term Population-Scale Immunity. <a href="https://www.blogger.com/#">Link</a><div><br /></div><div>3. Chen et al. The SARS-CoV-2 Vaccine Pipeline: an Overview. Curr Trop Med Rep. 2020 Mar 3;1-4. <a href="https://pubmed.ncbi.nlm.nih.gov/32219057/">Link</a></div>Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-89860403250183915882020-06-22T20:25:00.001+05:302020-06-22T20:39:27.830+05:30 COVID19: Why breaking the chain has been so difficult<div style="text-align: justify;">The latest of the pandemic (COVID19) seems to have elicited an extraordinary infodemic response. There are all sorts of stuff written all over the digital media that it is almost impossible to keep up. Each day thousands of new cases are reported on a global scale, with current numbers being more than 9 million. Though the actual case fatality ratio is low, the total number of deaths as per official records has crossed 4.5 lakhs. That is a serious issue to be considered. Also, on average, 40000 fresh cases are recorded every day. Figure 1, shows you the current scenario of COVID19 in countries where high numbers of diagnosis are recorded. </div><div style="text-align: justify;"><br /></div><table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: left;"><tbody><tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEigpBVIu-r4i-I3qv6oBwxic0YAF0jA-PFIO-laHVaWMYWfuCBvwH49HIBT0ip_e9esDMrlNP5XxUIRv-hbnIX_IBZWv-bkZCzTIye54HW6IUW_6OF5LC7KrhenBGmp-jaAjn4RHITHfYrF/s993/Figure+1.jpg" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="393" data-original-width="993" height="254" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEigpBVIu-r4i-I3qv6oBwxic0YAF0jA-PFIO-laHVaWMYWfuCBvwH49HIBT0ip_e9esDMrlNP5XxUIRv-hbnIX_IBZWv-bkZCzTIye54HW6IUW_6OF5LC7KrhenBGmp-jaAjn4RHITHfYrF/w640-h254/Figure+1.jpg" title="COVID 19 Cases" width="640" /></a><br /><br /></td></tr><tr><td class="tr-caption" style="text-align: center;"><b>Figure 1:</b> COVID 19- Number of cases and deaths. Based on data available as on 22nd June 2020. <a href="https://www.worldometers.info/coronavirus/" target="_blank">Source</a></td></tr></tbody></table><div style="text-align: justify;"><br /></div><div style="text-align: justify;">So the question stands, "Why have we terribly failed to contain SARS CoV 2?"</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Based on serological surveys and molecular testing results available at this time indicates that a large number of cases are totally asymptomatic. Interestingly, there is some evidence to assume that the asymptomatic cases are most likely to be the source of spread. They have a higher viral load, longer shedding time but no symptoms (<a href="https://www.virology.ws/2020/06/18/virus-and-antibodies-during-asymptomatic-sars-cov-2-infections-2/" target="_blank">Link</a>). Though the exact numbers vary between studies and opinions, it appears that approximately 20% of the population is asymptomatic. So that is the first challenge. We need to find all the asymptomatic cases which are spreading.</div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Second, is even more interesting. The virus once established, leads to symptoms by the 5th or 6th day (Varies from 2- 14 days). But, the person is infectious (able to spread), <a href="https://virologydownunder.com/politically-infectious-period/">approximately 48 hrs before he has had any symptoms </a>(in most people). So even in the symptomatic cases, by the time a diagnosis is made they have most likely transmitted to a few persons. By the time a potential primary contact is traced, they are most likely to have established the infection. The window period is extremely small. Assuming the average incubation time to appearance of symptoms is 5 days, and thus infectious period begins on 3rd day (See Figure 2), by the time the laboratory diagnosis is made, the primary contact has already began its infectious period. This presymptomatic infectious period is extremely hard to identify. Thus, the window period available to identify the primary contact and isolate them such that they don't transmit (and thereby break the transmission chain) is extremely small. It is probably not more than 24 hrs which is available to the public health authority.</div><div style="text-align: justify;"><br /></div><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjqLp7uF-GM319cLbgrvIU6LYAvhLJJ-CgQt8ul0FDZlyn-2IAK7R2Jo3lijYVZvQ_RGnsEvn4v2YxkAr0H3LKrS2ovl9pqEYP6VTDXoAPdRcFabD1fj-9x8mabMkvcakIxjvf4DPjfy1bS/s1128/Figure+2.jpg" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="685" data-original-width="1128" height="388" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjqLp7uF-GM319cLbgrvIU6LYAvhLJJ-CgQt8ul0FDZlyn-2IAK7R2Jo3lijYVZvQ_RGnsEvn4v2YxkAr0H3LKrS2ovl9pqEYP6VTDXoAPdRcFabD1fj-9x8mabMkvcakIxjvf4DPjfy1bS/w640-h388/Figure+2.jpg" width="640" /></a></td></tr><tr><td class="tr-caption" style="text-align: center;"><b>Figure 2:</b> Timeline of events indicating the rapidity of the spread of SARS CoV 2 and the window period available to identify that the primary contact can start spreading.<br /></td></tr></tbody></table><div style="text-align: justify;"><br /></div><div style="text-align: justify;"><br /></div><div style="text-align: justify;">Now combine the above two and you begin to see why COVID19 spread so fast and controlling it was really demanding. Asymptomatics are spreading which we don't know unless tested and the primary contact of the symptomatic case has to be identified and isolated at an extraordinary speed. This is exactly the reason why mass-scale testing, identification and subsequent isolation is extremely useful.</div><div style="text-align: justify;"><br /></div>Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-25771237800414057152020-05-28T12:18:00.000+05:302020-06-22T20:39:27.829+05:30SARS CoV 2: Is it evolving & how many Subtypes?<div dir="ltr" style="text-align: left;" trbidi="on">
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For almost 5 months now, the centre stage of infectious diseases has been taken by SARS CoV 2. The pandemic has hit every region of the globe. At the time of writing this post, a total of more than 3.52 lakh deaths have been confirmed (See Fig 1. For up to date case statistics check this <a href="https://www.worldometers.info/coronavirus/">link</a>). There is an unprecedented level of research being thrown into the development of vaccines and drugs. But there is one question that's being asked which needs to be answered. Is the SARS CoV 2 actually evolving and how many types are really known?</div>
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgiVhx9Z9dohFUDpcXmYpgnDkGYwLqrIFqfrwTi7hGp9zewgnaeAchsHKaDuoUMsxzIBM_FGAFmv0vxyS0-x-Ks7uyDdocwSQQvGtTmgn6e0VstOV2E50DybfjYJkiOR_lFZg3dOCGd-MYp/s1600/Figure+1.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="513" data-original-width="1161" height="282" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgiVhx9Z9dohFUDpcXmYpgnDkGYwLqrIFqfrwTi7hGp9zewgnaeAchsHKaDuoUMsxzIBM_FGAFmv0vxyS0-x-Ks7uyDdocwSQQvGtTmgn6e0VstOV2E50DybfjYJkiOR_lFZg3dOCGd-MYp/s640/Figure+1.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 1:</b> SARS CoV 2 Statistics as available on 26th May 2020.</td></tr>
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To date, more than 32,139 viral genomic sequences of SARS CoV 2 are available for researchers to mine (<a href="https://www.gisaid.org/epiflu-applications/next-hcov-19-app/">Link</a>). There are also several other databases including These include <a href="https://www.viprbrc.org/brc/home.spg?decorator=corona">ViPR</a>, <a href="https://covid-19.uniprot.org/">COVID-19 UniProtKB</a>, <a href="https://db.cngb.org/datamart/disease/DATAdis19/">hCov</a>-19 and <a href="http://clingen.igib.res.in/covid19genomes/">COVID-19 GenomePedia</a> that are currently tracking mutations.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: justify;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgsPGq21h36LeciHdPYx5LOpOrHytbrvzlo9u_yHeDQQKjXCfgYwevJzQl9hevFJHjBsomk4iD6C9OjELnysHWN7-K-CQbK5Rs0YUi8-2d5lbsYEf6qprLmQTvokSmj7SLfCsmkczeX-U31/s1600/Figure+2.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="474" data-original-width="971" height="195" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgsPGq21h36LeciHdPYx5LOpOrHytbrvzlo9u_yHeDQQKjXCfgYwevJzQl9hevFJHjBsomk4iD6C9OjELnysHWN7-K-CQbK5Rs0YUi8-2d5lbsYEf6qprLmQTvokSmj7SLfCsmkczeX-U31/s400/Figure+2.png" width="400" /></a></td></tr>
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<b>Figure 2: </b>Map showing 3176 of 3207 genomes sampled between</div>
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Jan 2020 and Apr 2020. <a href="https://nextstrain.org/">Source</a></div>
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An analysis of data earlier (Figure 2), shows the spread of various virus genomes. Of course, with more virus strains being sequenced this data looks much different every day. But the graphic definitely gives an idea on the distribution of strains. As per the most recent paper by <a href="https://europepmc.org/article/ppr/ppr151293">Junior et al</a>, there appear to be at least 16 genomic subtypes of SARS CoV 2 of which 6 subtypes are prominent.</div>
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So does that mean the virus is evolving and branching? The answer, unfortunately, is not fairly simple.<br />
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One group of scientists would argue that genomic level variation is seen consistently and is groupable suggesting that there is a branching of lineages. Indeed, as seen from the map in Fig 2 a cluster can be clearly made out indicating that certain subtypes are predominant in various geographical regions. This means two things. First, there is a subtype distinctive enough by their genomic signature. Second, the predominance of a subtype in a geographical area indicates that the virus has evolved to its local population.</div>
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Not everyone is convinced on the other side of the debate. Viruses being <a href="https://varuncnmicro.blogspot.com/2015/03/quasi-species-discussion.html">quasispecies</a> and mutations being quite common, many scientists opine that just mutations don't qualify for being called as a subtype. In theory, a virus becomes a subgroup if there is a different phenotypic character associated with that genetic change. For example, they bind to a different receptor, has altered cell specificity, there is a drastic change in virulence property etc.</div>
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There is only one strain of SARS-CoV-2. The first virus isolate, taken from a Wuhan patient in December 2019, is the same strain as the most recent isolate taken anywhere else in the world in May 2020. So far no one has shown that any of these virus isolates differ in any fundamental property.</div>
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- Vincent Racaniello (<a href="https://www.virology.ws/2020/05/07/there-is-one-and-only-one-strain-of-sars-cov-2/">Link</a>)</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: justify;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiHJgECQQ2ID3XBreNx7RXVftKAhpBL0uvxBVUl0EHnleH8-m5t-9z7XyhXlFz8xIeg_40MFaS9_f7JIkThQFFeZ_2wg5A6li6Kk-YW3NKz4RHJokCrQgnNZLhCSg_350fQx13D-AS7Bgxw/s1600/Picture1.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="775" data-original-width="1037" height="298" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiHJgECQQ2ID3XBreNx7RXVftKAhpBL0uvxBVUl0EHnleH8-m5t-9z7XyhXlFz8xIeg_40MFaS9_f7JIkThQFFeZ_2wg5A6li6Kk-YW3NKz4RHJokCrQgnNZLhCSg_350fQx13D-AS7Bgxw/s400/Picture1.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 3:</b> Founder effect vs Bottleneck effect. </td></tr>
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The blog post quoted above relies on the principle of "Founder effect". Let's take a small detour to understand what is "Founder effect". To explain in simplest possible terms, when a very small number of starting genetic material is propagated and established, you get a founder effect. Such a "founded" population will actually have a lower genetic variation and appear distinctive, from the original population. In reality, the founded population has dominated and thus the genetic sequencing will show predominance with very low numbers from the original population (Referred to as "noise"). Do not confuse this with the concept of "Bottleneck evolution" though <a href="https://www.differencebetween.com/difference-between-founder-effect-and-bottleneck-effect/">both are related concepts</a>. Fig 3 gives you a comparison of both for reference.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjLRYmOYl10_HrdElCErZDmP_5BfJb5LdQJ72CPwkW98Xfk7ptYnt3S_53kcfU1EWOzqYbtx1p64U8SnS2AjicN6M7DagaPaYET_iOWKMfuXuwiFLdGpcKmWx4Y5KHDzQhPMboc5YjZ08qa/s1600/Screenshot+2020-05-28+at+12.36.28+PM.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="500" data-original-width="991" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjLRYmOYl10_HrdElCErZDmP_5BfJb5LdQJ72CPwkW98Xfk7ptYnt3S_53kcfU1EWOzqYbtx1p64U8SnS2AjicN6M7DagaPaYET_iOWKMfuXuwiFLdGpcKmWx4Y5KHDzQhPMboc5YjZ08qa/s400/Screenshot+2020-05-28+at+12.36.28+PM.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Table:</b> Mutations observed in Spike Protein of SARS CoV 2. <a href="https://www.biorxiv.org/content/10.1101/2020.04.29.069054v2.full.pdf">Source</a></td></tr>
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In the context of SARS CoV 2, the argument is that SARS CoV 2 from the Wuhan strain has been seeded through small introductions (Most likely through international travel) in various parts of the globe which has lead to colonisation of a founding quasispecies. Hence the observation is due to founder effect and not evolution. Further supporting this line of argument is that there is no study that has shown evidence that there is a significant phenotypic level change due to sequence including the recent paper showing a D614G change in spike protein. At this point, we have no idea if this change has any phenotypic significant. Indeed the paper observes "<i>There was, however, no significant correlation found between D614G status and hospitalization status; although the G614 mutation was slightly enriched among the ICU subjects, this was not statistically significant". </i>The summary of mutations observed in spike protein is shown in the table. The location of D614G mutation is shown in Figure 4.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgpl65sJww_VXvqogNQlvDKmtCiVTA8N9eZObcVPf309Mcm1kIuGVFFnZ7XJNO08TMYw-tGmkvp_Of2YNTOX-r_ioZDWqC6VgQTYzkrGeDbteneavNkXP1yo70wueZ5TGAnGEgwnCqo4_gS/s1600/Figure+4.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="505" data-original-width="849" height="380" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgpl65sJww_VXvqogNQlvDKmtCiVTA8N9eZObcVPf309Mcm1kIuGVFFnZ7XJNO08TMYw-tGmkvp_Of2YNTOX-r_ioZDWqC6VgQTYzkrGeDbteneavNkXP1yo70wueZ5TGAnGEgwnCqo4_gS/s640/Figure+4.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 4:</b> Location of D614G mutation in Spike protein of SARS CoV 2. <a href="https://www.thinkpragati.com/podcast/the-pragati-podcast/8678/sars-cov-2-mutation-evolution-a2a/">Source</a></td></tr>
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Coming back to the core question, "Is the SARS CoV 2 actually evolving and how many types are really known?". At this point in time, based on the above arguments my understanding is that the virus is not really evolving (No evidence of evolution), and there is only one real subtype. However, there are several genotypes (Genomic subtypes) that do not differ in their phenotypic properties. The genotyping is useful only to understand the transmission dynamics. In practice, all the isolates to date are the same with reference to their phenotypic characters of virulence and pathogenesis.</div>
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<b>References:</b></div>
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1. Tung Phan.Genetic diversity and evolution of SARS-CoV-2. Infection, Genetics and Evolution. <a href="https://www.sciencedirect.com/science/article/pii/S1567134820300915">Link</a></div>
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2. Korber et al. Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. <a href="https://www.biorxiv.org/content/10.1101/2020.04.29.069054v2.full">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-58905083828777670882020-05-08T11:47:00.001+05:302020-06-22T20:39:27.829+05:30Should we blame Gain of Function Research: SARS CoV 2 Context<div dir="ltr" style="text-align: left;" trbidi="on">
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In the last few weeks, I have been receiving a lot of questions asking and challenging if the SARS CoV 2 is Gain of function (GoF) research that has gone wrong. The questions have been coming after seeing my comment on an article in nature news (<a href="https://www.nature.com/news/engineered-bat-virus-stirs-debate-over-riskyresearch-1.18787">Link</a>). Considering there is a lot of media hype on the story, and a lot of public actually believe that SARS CoV 2 is a DURC (Dual-use Research of Concern) and GoF Product some clarity is needed.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfUxrmSV42ZITzDq364b4Hqkv1k69QpTDwOTlaYpHJDK-hYeTZVVOyLR8f8j9QSAZKqtGuzOufHsLeOR7AEuXtRZIiwRvb1WMPF5bfqyRIFPIiJXaYJNlHp0xSux_CiO1DfiqR7NfYVcNa/s1600/Shi+Zhengli.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="506" data-original-width="918" height="110" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfUxrmSV42ZITzDq364b4Hqkv1k69QpTDwOTlaYpHJDK-hYeTZVVOyLR8f8j9QSAZKqtGuzOufHsLeOR7AEuXtRZIiwRvb1WMPF5bfqyRIFPIiJXaYJNlHp0xSux_CiO1DfiqR7NfYVcNa/s200/Shi+Zhengli.jpg" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo:</b> Shi Zhengli. <a href="https://www.wionews.com/world/chinas-bat-woman-shi-zhengli-goes-missing-297076">Source</a></td></tr>
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So why is this even a debate? The question comes based on the earlier research by Shi Zhengli’s group where there was GoF research conducted on coronavirus to understand its transmission and pathogenic capabilities. Further, her recent disappearance has substantially pushed the debate forward (<a href="https://www.blogger.com/blogger.g?blogID=5697433480702397522#editor/target=post;newUi=1;postID=5890508382877767088">Link</a>). Even worse, the genetic sequence of SARS CoV 2 is 96.2% similar to the virus isolated in the Wuhan lab in 2013. There was also a story published on the internet with a Hollywood movie like plot claiming that this was a biological weapon that had accidentally leaked. There are many versions of this story circulating on the internet. These claims have had so much visibility, there is an actual investigation federal level investigation into the whole matter (<a href="https://www.dailymail.co.uk/news/article-8253669/Pentagon-steps-investigation-coronavirus-linked-Chinese-biological-warfare.html">Link</a>).</div>
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Genetic homology and relatedness is a great way to tell relatedness. The more is the genetic similarity, the closer are the isolates. So automatically the question comes if the virus is more than 96% similar to whats isolated at Wuhan lab in 2013, that has to be the source, right? The answer (Need not be) is slightly technical. In simplification, another SARS CoV strain from Civet has shown 99.8% identity which is perfectly in agreement with natural transmission possibilities. The SHC14-MA15 which is the chimeric virus (Lab engineered strain for GoF research) is extremely different from the SARS CoV 2 sequences (only 93% similar). In genetic terms, a change of more than 1% is considered as highly divergent and lower relatedness. I especially like the very detailed analysis published by Yuri Deigin. There is a very detailed analysis of the possibility of engineering SARS CoV 2 (<a href="https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748">Link</a>) which considers both sides of the argument. At this point, it is not possible for any research to establish or refute the "Laboratory origin" theory of SARS CoV 2. However, the proofs available tend to indicate that it is a naturally occurring outbreak.</div>
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So let me put the question hanging in there. Assuming that the SARS CoV 2 leaked from the BSL-4 Wuhan Institute (Please note I don't imply it did), does it mean GoF research conducted at the Wuhan lab is responsible for the outbreak?</div>
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I urge you to go back and read my blog on GoF research (<a href="https://varuncnmicro.blogspot.com/2014/07/ferrets-are-not-humans.html">Link</a> to my previous post). There is a certain way you do scientific experiments which are accepted by the international community based on years of backing research. For example, when the avian H1N1 was modified to see if it can infect human cell lines in a BSL-4 Lab, they did it to understand what mutations are required and to what extent which will make it infective to humans. This arms the scientific community with prior knowledge on what's likely to happen and how (if at all it happens). So if the strain hits, we don't start fresh from scratch in trying to understand what has happened. The reason we are ahead of the curve in this pandemic is a lot of molecular details are understood for Coronavirus is published GoF research. Indeed a bat originated coronavirus outbreak was predicted a year earlier as a clear possibility. An excerpt from the paper reads</div>
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<span style="background-color: white; font-family: "merriweather"; font-size: 16px;"><span style="color: blue;">"Fifteen years after the first highly pathogenic human coronavirus caused the severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak, another severe acute diarrhoea syndrome coronavirus (SADS-CoV) devastated livestock production by causing fatal diseases in pigs. Both outbreaks began in China and were caused by coronaviruses of bat origin. This increased the urgency to study bat coronaviruses in China to understand their potential of causing another virus outbreak".</span></span><br />
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<span style="background-color: white; font-family: Merriweather; font-size: 16px; text-align: justify;"><span style="color: blue;">“It is highly likely that future SARS or MERS-like coronavirus</span></span><br />
<span style="background-color: white; font-family: Merriweather; font-size: 16px; text-align: justify;"><span style="color: blue;">outbreaks will originate from bats, and there is an increased probability that this will occur in China”.</span></span></div>
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<span style="background-color: white; font-family: "merriweather"; font-size: 16px;">-<a href="https://www.mdpi.com/1999-4915/11/3/210">Source paper</a></span></div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-sbU1saG4eAY8eups8S1gZiixfqEDOMXyJp2QVP2GYNn-apBqMWSklNTH8a6CXhTRnJIkHLKaWUHKpSSTFYmO8GLVQrZ_8xaxxy0jmxlI9mWuCjDqXZtgxBzj6ftYelqWW-QKCIzlpiu_/s1600/GoF.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="225" data-original-width="225" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-sbU1saG4eAY8eups8S1gZiixfqEDOMXyJp2QVP2GYNn-apBqMWSklNTH8a6CXhTRnJIkHLKaWUHKpSSTFYmO8GLVQrZ_8xaxxy0jmxlI9mWuCjDqXZtgxBzj6ftYelqWW-QKCIzlpiu_/s1600/GoF.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Polling Results from Science<br />
Advisory Board. Data from 2015.</td></tr>
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Coming back to the question on the light of apropos, a BSL-4 lab is highly regulated and there is a strict code of conduct on how strains are handled in it. If at any point in time, it is proved that SARS COV 2 is indeed a lab generated virus, by accident it means that they have lapsed from scientific integrity and action will be taken accordingly. But claiming that regulated GoF research is to blame and the whole world has to stop such research is "uncontrolled stupidity". </div>
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At this point, I should answer a question. Is the GoF research a biological weapon program in context to SARS CoV 2. If SARS CoV 2 is a biological weapon detail of this virus would not have been published by the Chinese when the research was in progress. Experiments conducted for research into understanding the biology by academic and industrial scientists are very carefully planned and done at well contained biological safety labs. The results of such research are published and used to build vaccines and therapeutics. Biological weapons research is not done by academic or industrial scientists. Contrarily, they are done in military facilities only by some countries (it is assumed so), the results of which are never published. Those research are not called as GoF research (Since it doesn't follow the GoF protocol) and do not come under the control of IRBs. The logic of not communicating such research openly is obvious.</div>
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I have given you the background. In summary, the GoF research conducted on coronaviruses has helped us understand and even predict the outbreaks. There is no evidence as of yet (out of huge numbers of papers that have actually analysed the question) that it is a laboratory strain or deliberately released virus, but rather appears as naturally occurring. So consider the risk-benefit ratio and I will let the reader decide if we should blame Gain of function research.</div>
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<b>References:</b></div>
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1. Michael J. Selgelid. Gain-of-Function Research: Ethical Analysis. Sci Eng Ethics. 2016; 22(4): 923–964. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996883/">Link</a></div>
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2. Duprex, W., Fouchier, R., Imperiale, M. et al. Gain-of-function experiments: time for a real debate. Nat Rev Microbiol. 2015;13, 58–64. <a href="https://www.nature.com/articles/nrmicro3405">Link</a><br />
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3. Declan Butler. Engineered bat virus stirs debate over risky research. <a href="https://www.nature.com/news/engineered-bat-virus-stirs-debate-over-riskyresearch-1.18787">Link</a><br />
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4. Liu et al. No credible evidence supporting claims of the laboratory engineering of SARS-CoV-2. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054935/#">Emerg Microbes Infect</a>. 2020; 9(1): 505–507. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054935/">Link</a><br />
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5. Fan et al. Bat Coronaviruses in China. <a href="https://www.mdpi.com/1999-4915/11/3/210">Link</a></div>
Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-50852366569911253592020-02-23T11:28:00.004+05:302020-02-23T11:39:36.957+05:30Lab Series#18: The Levey-Jennings Chart<div dir="ltr" style="text-align: left;" trbidi="on">
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhGD38e5NCfSWMSse9Gu4xQl4DrqDSLJhu7W8kWzUW9xSF3TpztmFmoxSqLdbytd-vbLLWu21lsfxtEupyZokzeUDf1OvwXbxQ-XhiboBzB475okonGhwVI8-4h8ASuM9_AKhbfFvxFQa3R/s1600/LJ.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="173" data-original-width="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhGD38e5NCfSWMSse9Gu4xQl4DrqDSLJhu7W8kWzUW9xSF3TpztmFmoxSqLdbytd-vbLLWu21lsfxtEupyZokzeUDf1OvwXbxQ-XhiboBzB475okonGhwVI8-4h8ASuM9_AKhbfFvxFQa3R/s1600/LJ.png" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">LJ chart. <a href="https://en.wikipedia.org/wiki/Laboratory_quality_control">Source</a></td></tr>
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A huge reader base of this blog is somehow connected to health care and a majority are connected to a laboratory that deals with medical samples. A strong requirement of the work nature is to be producing consistent results. But how do you actually measure that the results produced are consistent and reliable? Well, one of the methods it is to measure the variations and see if it is acceptable. There is a huge number of tests that can be potentially done to estimate the variations and check if the results that are produced are indeed acceptable. The point of this post is to talk about one such test- "LJ Chart", simply because the method is universally done to ascertain the authenticity of results. But before I jump into the calculations, it is important to understand some background concepts.</div>
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<span style="color: #741b47;"><b>A Scenario to Consider...</b></span></div>
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Let us start with a scenario. Assume that you have received a blood sample for measuring HIV Viral Load. The method of estimation that you are using is a Real-time PCR based kit. Any method of estimation uses some sort of chemistry, involving some set of steps, each of which uses reagents and some delivery devices. In the above example, we aliquot several reagents of predetermined volume sequentially, using a pipette and then look for the sequence amplification using a <a href="https://varuncnmicro.blogspot.com/2015/05/lab-series-5-dna-amplification-in-lab.html">Real time PCR method</a>. Any analytical measurement has an inbuilt error into it. Consider this. The International System of Units (<a href="https://www.bipm.org/en/measurement-units/">SI units</a>) identifies a total of 7 base units and 22 derived units. Based on these standards, a first copy is made which is used to calibrate further downstream. For example, a copy of the international standard of 1 Kg is issued against which the national level standard is calibrated. Because of the process of calibration, a small error is inevitably introduced. This, in turn, is used for calibrating other regional level systems and other industries which offer calibration and so on with a small level of error introduced at each level. So, in the end, a well-calibrated pipette which was supposed to deliver you a 100 uL of water, will actually deliver you anywhere between 99.9999 uL and 100.0001 uL (The actual numbers will vary. But you get the illustrative idea). Now consider repeated process and addition of multiple reagents (Each having its own set of undefinable errors). Thus, even in the best hands, there is an uncontrollable level of error. Add to it a level of manual handling and the levels of variations shoot up significantly. At some point, if not well controlled, the error rate is too much and can be called as "Failure to achieve reproducibility and quality".</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<b><span style="color: #741b47;">True value, Accuracy and Precision</span></b></div>
<div style="text-align: justify;">
<b><span style="color: #073763;"><br /></span></b></div>
<div style="text-align: justify;">
<b><span style="color: #073763;">True Value</span></b></div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
"True value" is a hypothetical concept. This represents the actual concentration of the analyte. Let's say in this above example, the true value is 300 copies/ml. However, because of the error inbuilt to the technique, let's say the final result was 302 copies/ml. I would say that the testing of the sample was exceptionally good. But there's a problem. I don't know the true value (If I had known then why will I run the test). Since in reality, we cannot ever find the true value and thus the error cannot be actually quantified, we use "conventional true value". It is also known as "Assigned" or "Reference" value. As per the International vocabulary of basic and general terms in
metrology (VIM) document,<br />
<br />
<div style="text-align: center;">
"The true value would be obtainable by a perfect measurement, that is a
measurement without measurement error. The true value is by nature unobtainable."</div>
</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<b><span style="color: #073763;">Accuracy</span></b></div>
<div style="text-align: right;">
</div>
<div style="text-align: justify;">
<b><span style="color: #073763;"><br /></span></b></div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi4Sr6NMgQqwtYDE0SCY3r_PaYUkQaaj8vwxc8oENiIaB3QXiBMQU_6YDZnyrs87aTLWwS2kBMkNpEZnHwe-HS_yGElVBWEK8qer6GdSkQ1nNdfbGKr8y1BBAmmx7ua3dL0P45bzJ7Ihpls/s1600/Figure+1.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="349" data-original-width="498" height="224" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi4Sr6NMgQqwtYDE0SCY3r_PaYUkQaaj8vwxc8oENiIaB3QXiBMQU_6YDZnyrs87aTLWwS2kBMkNpEZnHwe-HS_yGElVBWEK8qer6GdSkQ1nNdfbGKr8y1BBAmmx7ua3dL0P45bzJ7Ihpls/s320/Figure+1.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 1: </b>Accuracy and Precision. <a href="https://www.webpages.uidaho.edu/veg_measure/Modules/Lessons/Module%202(Sampling)/2_3_Accuracy_and_bias.htm">Source</a></td></tr>
</tbody></table>
<div style="text-align: justify;">
The classical definition states that accuracy is the closeness of the agreement between the result of a measurement and a true value. Mathematically, accuracy can be calculated if the true value is known. In the case scenario, accuracy= 99.33%. But since the true value is not known, we calculate this based on the reference value. We will talk more about how to find the reference value later. The nearer the result is to the true value, the better is the accuracy. See Figure 1.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<b><span style="color: #073763;">Precision</span></b></div>
<div style="text-align: justify;">
<b><span style="color: #073763;"><br /></span></b></div>
<div style="text-align: justify;">
Precision, in comparison to true value and accuracy, is a real quantifiable measurement. Precision refers to the agreement of a set of results among themselves. In the above example, let's say I repeated the test 20 times, and each time I got the value as 302 copies/ml then my precision is 100 %. Now let's say, I performed the test 20 times and each time I got 350 copies/ml each time, my precision remains 100% but my accuracy is far from reality. On the contrary, if testing is accurate every time, the precision is bound to be very tight.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
In reality, we don't express the accuracy and precision as a percentage but rather as a statistical function called a <a href="https://statisticsbyjim.com/basics/measures-central-tendency-mean-median-mode/">central tendency</a> (Mean ± Standard deviation). In Figure 1, the central point (Bull's eye) is the True value. Measurements near to the true value are accurate and clustering of measurement indicates precision. The Levey-Jennings Chart is basically a mathematical model that helps you say if the accuracy and precision of the measurements are good enough to call your result as reproducible, thereby aiding quality control.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<span style="color: #741b47;"><b>Constructing an LJ (</b><b>Levey-Jennings) </b><b>Chart</b></span></div>
<div style="text-align: justify;">
<b><br /></b></div>
<div style="text-align: justify;">
There is a lot of history associated with quality control charts (Read more about it <a href="http://www.ijbls.org/upfile/Issues/201591894942.pdf">here</a>). What I want to emphasize is that Stanley Levey and Εlmer Jennings adapted this method from Shewhart charts.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
For constructing an LJ chart you need the following.</div>
<div style="text-align: justify;">
<ol>
<li>Quality Control Material </li>
<li>An estimate of the True Value</li>
<li>An estimate of the variations that are inherent to the testing.</li>
</ol>
</div>
<div>
<div style="text-align: justify;">
A quality control material is a material that is in all aspects similar to what is otherwise tested (In this case patient material), however, the value of analyte is already known. The quality control materials are preferentially a commercially available material. For example, ready-made quality control materials are available with known HIV RNA copies in it. In a few cases, such materials are not available. In these cases, an in house material can be prepared by pooling a few samples. If using a commercial material the values provided by the manufacturer are taken as conventional true value (As I already stated above a real true value is always unknown). For the in house materials, it is assumed that the average (Mean value) of multiple different runs of the same sample is the conventional true value. This is based on the assumption that all the errors are random in nature and hence the average value will be extremely close to the true value.</div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
To generate an estimate of allowed variations the IQC material is tested multiple times. It is recommended that the test is run 20 times, but has to be done at least 10 times to generate a reasonably accurate standard deviation (SD). For example, let us say I have a QC material which is known to have 300 copies/ml of HIV. I will run the same QC 20 different times and all the values that I have obtained will be used to compute a mean and standard deviation. I have created an example here for you to follow.</div>
<br />
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none; color: black;"><tbody>
<tr><td style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 71.25pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<a href="https://www.blogger.com/null" name="_GoBack"></a><b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Run Number<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 70.9pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Copies/ml<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 51.35pt;" valign="top" width="68"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Run Number<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Copies/ml<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Run Number<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Copies/ml<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Run Number<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: solid solid solid none; border-top-color: windowtext; border-top-width: 1pt; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">Copies/ml<o:p></o:p></span></b></div>
</td></tr>
<tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 71.25pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">1<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 70.9pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">301<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.35pt;" valign="top" width="68"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">6<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">298<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">11<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">302<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">16<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">297<o:p></o:p></span></div>
</td></tr>
<tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 71.25pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">2<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 70.9pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">302<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.35pt;" valign="top" width="68"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">7<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">299<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">12<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">301<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">17<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">298<o:p></o:p></span></div>
</td></tr>
<tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 71.25pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">3<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 70.9pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">300<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.35pt;" valign="top" width="68"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">8<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">301<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">13<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">299<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">18<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">301<o:p></o:p></span></div>
</td></tr>
<tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 71.25pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">4<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 70.9pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">298<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.35pt;" valign="top" width="68"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">9<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">300<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">14<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">298<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">19<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">299<o:p></o:p></span></div>
</td></tr>
<tr><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-left-color: windowtext; border-left-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid; padding: 0cm 5.4pt; width: 71.25pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">5<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 70.9pt;" valign="top" width="95"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">299<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.35pt;" valign="top" width="68"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">10<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">302<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">15<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">303<o:p></o:p></span></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<b><span style="font-size: 11pt; line-height: 16.866666793823242px;">20<o:p></o:p></span></b></div>
</td><td style="border-bottom-color: windowtext; border-bottom-width: 1pt; border-right-color: windowtext; border-right-width: 1pt; border-style: none solid solid none; padding: 0cm 5.4pt; width: 51.4pt;" valign="top" width="69"><div align="center" class="MsoNormal" style="font-family: Calibri, sans-serif; font-size: 12pt; line-height: 18.399999618530273px; margin: 0cm 0cm 0.0001pt; text-align: center;">
<span style="font-size: 11pt; line-height: 16.866666793823242px;">302<o:p></o:p></span></div>
</td></tr>
</tbody></table>
<br />
<div style="text-align: center;">
<b>Table 1: </b>Example data for creating baseline data. Mean= 300; SD= 1.747</div>
<br />
<div style="text-align: justify;">
Now, all you need is some statistical calculations. In simplest terms, what you need is to calculate mean and SD. If you don't know how to calculate mean and SD, please see the <a href="https://www.mathsisfun.com/data/standard-deviation-formulas.html">link</a>. You must note that the logic of using mean here is that the generated data is based on a <a href="https://statisticsbyjim.com/basics/normal-distribution/">normal distribution</a>. If you are unsure, check if the data is normally distributed or not by performing a <a href="https://www.statisticshowto.datasciencecentral.com/shapiro-wilk-test/">Shapiro-Wilk Test</a>. In Table 1, the data is normally distributed. You can perform this check directly online by using this <a href="http://sdittami.altervista.org/shapirotest/ShapiroTest.html">Link</a>.</div>
<br />
<div style="text-align: justify;">
A common problem that is encountered in building the baseline data is an outlier. In Table 1, assume that Run 19 produced a result of 315 copies. Just one variable changes your entire data (Mean= 300.8; SD= 3.76). Such a variable is called an outlier. So how do you actually decide if its an outlier? There are a lot of ways you can detect an outlier in the data (See this <a href="https://towardsdatascience.com/ways-to-detect-and-remove-the-outliers-404d16608dba">link</a>). Personally, I use a statistical analysis method called a Grubb's test to identify an outlier. You can perform this test using an <a href="https://www.graphpad.com/quickcalcs/Grubbs1.cfm">online tool</a>. If there is a significant outlier (p >0.05), the baseline data can be constructed by omitting that outlier. Another method is to just check if there is any variable that itself is more than the 3SD mark and omit that. Since, by default a value outside ± 3SD indicates out of quality, the value in itself doesn't qualify to be included for baseline data construction. As I will show later smaller is the SD, tighter is your LJ analysis. So just by removing one outlier, your baseline data quality, in this case, will be good. For plotting the baseline graph, just take a graph-paper and mark the middle line as Mean. Then mark the upper line as Mean +1SD and the lower line as Mean-1SD. Next mark ± 2SD and ± 3SD. For your comparison, I have shown a comparison of the LJ baseline data for Table 1 and how it changes when the 19th variable is changed to 315 copies.</div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjDGjCFQg6ut2gYyS4YbqI9v97dFbPgmEtN0v-Sagubw2lWIEzk-wtBjsMRvCS7IKnygY9719SOE9h1KzkvpXOmPVmtb75xYYHvX0GQhQXtktX6Ef0MQd-l2iwRIntOXu4PJ7SJP0ByaNdA/s1600/Fig+2.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="837" data-original-width="1600" height="334" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjDGjCFQg6ut2gYyS4YbqI9v97dFbPgmEtN0v-Sagubw2lWIEzk-wtBjsMRvCS7IKnygY9719SOE9h1KzkvpXOmPVmtb75xYYHvX0GQhQXtktX6Ef0MQd-l2iwRIntOXu4PJ7SJP0ByaNdA/s640/Fig+2.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 2:</b> LJ Chart baseline. middle line indicates Mean. The red lines indicates ± 2SD. Note the difference in the 2 charts.</td></tr>
</tbody></table>
<div style="text-align: justify;">
<br />
Now all you need to do is run your QC sample whenever I am running a test along with the the unknowns. For example, in the example scenario, along with the patient samples (unknown) I will also run a QC just like a sample. Whatever value I get, I will mark it on the graph. So here's the rules of how to interpret the QC values.</div>
<div style="text-align: justify;">
</div>
<ol>
<li>If the QC value is in between ± 2SD the QC is considered as "<b>Passed"</b>. You can be sure that the quality of testing is good.</li>
<li>If the QC value is more than ± 2SD but less than ± 3SD the QC is considered as <b>"Passed with warning"</b>. The testing is still good enough to be reported. However, there is some problem that has begun to creep in. This is usually because the pipette or the equipment requires calibration and the reagents needs to be checked.</li>
<li>If the QC value is more than the ± 3SD the QC is considered as <b>"Failed"</b>. This indicates that the test results are not acceptable and some component of testing has failed to function to the mark. The results obtained needs to be disqualified and the cause of problem has to be identified through a root cause analysis. Once the problem is rectified, the test has to be repeated along with QC.</li>
</ol>
<div>
<div style="text-align: justify;">
Let's now assume that I check for QC everyday. Connecting all the dots on the graph provides me with my LJ chart for the month. Table 2 shows 15 QC runs. Try building the LJ graph on your own. The graph should look like Figure 2.</div>
</div>
<div>
<br /></div>
<div>
</div>
<br />
<table border="0" cellpadding="0" cellspacing="0" class="MsoNormalTable" style="border-collapse: collapse; text-align: justify;">
<tbody>
<tr>
<td style="border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 71.25pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>Run Number</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 70.9pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>Copies/ml</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 51.35pt;" valign="top" width="68"><div class="MsoNoSpacing" style="text-align: center;">
<b>Run Number</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="top" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<b>Copies/ml</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 51.4pt;" valign="top" width="69"><div class="MsoNoSpacing" style="text-align: center;">
<b>Run Number</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="top" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<b>Copies/ml</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
</tr>
<tr>
<td style="border-top: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 71.25pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>1</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 70.9pt;" valign="bottom" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">299.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.35pt;" valign="top" width="68"><div class="MsoNoSpacing" style="text-align: center;">
<b>6</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">306.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.4pt;" valign="top" width="69"><div class="MsoNoSpacing" style="text-align: center;">
<b>11</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">302.00<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="border-top: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 71.25pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>2</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 70.9pt;" valign="bottom" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">302.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.35pt;" valign="top" width="68"><div class="MsoNoSpacing" style="text-align: center;">
<b>7</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">302.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.4pt;" valign="top" width="69"><div class="MsoNoSpacing" style="text-align: center;">
<b>12</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">297.00<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="border-top: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 71.25pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>3</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 70.9pt;" valign="bottom" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">303.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.35pt;" valign="top" width="68"><div class="MsoNoSpacing" style="text-align: center;">
<b>8</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">301.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.4pt;" valign="top" width="69"><div class="MsoNoSpacing" style="text-align: center;">
<b>13</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">301.00<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="border-top: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 71.25pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>4</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 70.9pt;" valign="bottom" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">304.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.35pt;" valign="top" width="68"><div class="MsoNoSpacing" style="text-align: center;">
<b>9</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">299.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.4pt;" valign="top" width="69"><div class="MsoNoSpacing" style="text-align: center;">
<b>14</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">302.00<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="border-top: none; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; width: 71.25pt;" valign="top" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<b>5</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 70.9pt;" valign="bottom" width="95"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">305.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.35pt;" valign="top" width="68"><div class="MsoNoSpacing" style="text-align: center;">
<b>10</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">298.00<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 51.4pt;" valign="top" width="69"><div class="MsoNoSpacing" style="text-align: center;">
<b>15</b><b><span style="font-size: 12.0pt;"><o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; width: 57.45pt;" valign="bottom" width="77"><div class="MsoNoSpacing" style="text-align: center;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt;">297.00<o:p></o:p></span></div>
</td>
</tr>
</tbody></table>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<b>Table 2:</b> Hypothetical QC runs.</div>
</div>
<div>
<div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhSrdGGEauRIKTVQJHbgejkdP4o9pG4nPzBmMLFttmAk0poTJNNhsJxYkrnINbMTq7lGw0pK4ZQ1NM78-put1pbVSkkfnoRT_Z9nqHCAsSHiBXor91X4Xw_RO69vadniwF_gvbYsowu5Rdv/s1600/Fig+3.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="1082" data-original-width="1600" height="432" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhSrdGGEauRIKTVQJHbgejkdP4o9pG4nPzBmMLFttmAk0poTJNNhsJxYkrnINbMTq7lGw0pK4ZQ1NM78-put1pbVSkkfnoRT_Z9nqHCAsSHiBXor91X4Xw_RO69vadniwF_gvbYsowu5Rdv/s640/Fig+3.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 3:</b> LJ chart </td></tr>
</tbody></table>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
Now consider this same data in context with what would have happened if your baseline did include the outlier. The QC value of 306 copies/ml would be then considered as QC passed (Not even a warning; In this case the value would have been between ± 2SD; See Figure 2). Remember, lower is your SD for baseline data, the better is your LJ chart and the control.</div>
<div style="text-align: justify;">
<br /></div>
<br />
<div style="text-align: justify;">
That brings into question, when do you consider that your LJ chart is good? There is no perfect answer for this. In general SD is a measure of how much deviation is actually there in your data. In general, we say the SD should not be more than 10% of your mean. Again consider the above scenario. If the SD was 10% (=30), then your ± 3SD will be in the range of 210 to 390 copies/ml. A variation in the run amounting to more than this, is not reliable. In this case, the LJ chart will be tolerant to a very high variation. Imagine, the true value is 300 copies, but analysis shows its 215 copies and still, the run is considered as a pass. That had be far from accurate (about 71.6% of true value). To simplify this we calculate <a href="https://en.wikipedia.org/wiki/Coefficient_of_variation">Coefficient of variation</a> (CV). A CV of < 0.1 or more indicates that the SD is within 10%. For table 1, CV= 0.0058.</div>
<br />
<div style="text-align: justify;">
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiuIK6GGxOur4TQUkU7ncG7e5INZf6A5Fir5Qb_ccGDJQh7Z0vNN3q6lXTISKenARPkiFIwq_54Od-IMM919FIeeGcpcpoCDjzTZ3PhRkDu00w2xyIKJ68x8yE9JpZrurNXMsGv2deRoUgx/s1600/Formula.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em; text-align: justify;"><img border="0" data-original-height="207" data-original-width="979" height="82" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiuIK6GGxOur4TQUkU7ncG7e5INZf6A5Fir5Qb_ccGDJQh7Z0vNN3q6lXTISKenARPkiFIwq_54Od-IMM919FIeeGcpcpoCDjzTZ3PhRkDu00w2xyIKJ68x8yE9JpZrurNXMsGv2deRoUgx/s400/Formula.png" width="400" /></a></div>
<b><span style="color: #741b47;"><br /></span></b>
<b><span style="color: #741b47;">Errors identifiable in LJ Chart</span></b></div>
<div style="text-align: justify;">
<b><span style="color: #741b47;"><br /></span></b></div>
<div style="text-align: right;">
</div>
<div style="text-align: justify;">
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhTtJIBlyal6EOxBd9cgGCyKapvyCwWoyrTZ1rDGKpNFKqJKsUYMYZ4s8PcH7HaqH18unQDVlVjGYk7o8n7zpNdsNl0Usp7kmNhXRtvyldj0HddGV71lHjxYOpFS8evselvd6rC26My8aVp/s1600/1.gif" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="272" data-original-width="471" height="184" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhTtJIBlyal6EOxBd9cgGCyKapvyCwWoyrTZ1rDGKpNFKqJKsUYMYZ4s8PcH7HaqH18unQDVlVjGYk7o8n7zpNdsNl0Usp7kmNhXRtvyldj0HddGV71lHjxYOpFS8evselvd6rC26My8aVp/s320/1.gif" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 4:</b> Random and Systematic error. <a href="https://www.e-education.psu.edu/natureofgeoinfo/c5_p5.html">Source</a></td></tr>
</tbody></table>
There are two types of errors that can be identified when looking into the LJ chart- Random and Systematic. A random error is used to indicate that the deviation from mean as seen in the graph, is due to an uncontrollable influence. The LJ graph in this case will show readings on both the sides of mean randomly. This error cannot be eliminated entirely, though can be reduced with good calibration of equipment's used and good laboratory practices. In contrast, systematic error is usually due to a error in the procedure which skews the results into one side. In this case, the error will follow a trend. Figure 4 and Table 3 shows a comparison of the error types. </div>
<div style="text-align: justify;">
<br /></div>
<div style="text-align: justify;">
<div align="center">
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableLightShadingAccent1" style="border-collapse: collapse; border: none; mso-border-bottom-alt: solid #4F81BD 1.0pt; mso-border-bottom-themecolor: accent1; mso-border-top-alt: solid #4F81BD 1.0pt; mso-border-top-themecolor: accent1; mso-padding-alt: 0in 5.4pt 0in 5.4pt; mso-yfti-tbllook: 1184;">
<tbody>
<tr>
<td style="border-bottom: solid #4F81BD 1.0pt; border-left: none; border-right: none; border-top: solid #4F81BD 1.0pt; mso-border-bottom-themecolor: accent1; mso-border-top-themecolor: accent1; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 5;">
<b><span style="color: #365f91; font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Feature<o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid #4F81BD 1.0pt; border-left: none; border-right: none; border-top: solid #4F81BD 1.0pt; mso-border-bottom-themecolor: accent1; mso-border-top-themecolor: accent1; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 1;">
<b><span style="color: #365f91; font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Random Error<o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid #4F81BD 1.0pt; border-left: none; border-right: none; border-top: solid #4F81BD 1.0pt; mso-border-bottom-themecolor: accent1; mso-border-top-themecolor: accent1; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 1;">
<b><span style="color: #365f91; font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Systematic Error<o:p></o:p></span></b></div>
</td>
</tr>
<tr>
<td style="background: #D3DFEE; border: none; mso-background-themecolor: accent1; mso-background-themetint: 63; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 68;">
<b><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Common Causes<o:p></o:p></span></b></div>
</td>
<td style="background: #D3DFEE; border: none; mso-background-themecolor: accent1; mso-background-themetint: 63; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoListParagraphCxSpFirst" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; margin-left: 21.9pt; margin-right: 0in; margin-top: 0in; mso-add-space: auto; mso-list: l0 level1 lfo1; mso-yfti-cnfc: 64; text-indent: -.25in;">
<!--[if !supportLists]--><span style="font-family: "symbol"; font-size: 10.0pt; line-height: 150%;">·<span style="font-family: "times new roman"; font-size: 7pt; font-stretch: normal; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Environmental variation<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpMiddle" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; margin-left: 21.9pt; margin-right: 0in; margin-top: 0in; mso-add-space: auto; mso-list: l0 level1 lfo1; mso-yfti-cnfc: 64; text-indent: -.25in;">
<!--[if !supportLists]--><span style="font-family: "symbol"; font-size: 10.0pt; line-height: 150%;">·<span style="font-family: "times new roman"; font-size: 7pt; font-stretch: normal; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Instrumental variation<o:p></o:p></span></div>
<div class="MsoListParagraphCxSpLast" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; margin-left: 21.9pt; margin-right: 0in; margin-top: 0in; mso-add-space: auto; mso-list: l0 level1 lfo1; mso-yfti-cnfc: 64; text-indent: -.25in;">
<!--[if !supportLists]--><span style="font-family: "symbol"; font-size: 10.0pt; line-height: 150%;">·<span style="font-family: "times new roman"; font-size: 7pt; font-stretch: normal; line-height: normal;">
</span></span><!--[endif]--><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Variation in reagents<span style="color: #365f91; mso-themecolor: accent1; mso-themeshade: 191;"><o:p></o:p></span></span></div>
</td>
<td style="background: #D3DFEE; border: none; mso-background-themecolor: accent1; mso-background-themetint: 63; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 64;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Incorrect usage or protocol<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="border: none; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 4;">
<b><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Variation and directionality<o:p></o:p></span></b></div>
</td>
<td style="border: none; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Varies
and nonpredictable.<o:p></o:p></span></div>
</td>
<td style="border: none; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Roughly
constant and unidirectional<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="background: #D3DFEE; border: none; mso-background-themecolor: accent1; mso-background-themetint: 63; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 68;">
<b><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Reproducibility<o:p></o:p></span></b></div>
</td>
<td style="background: #D3DFEE; border: none; mso-background-themecolor: accent1; mso-background-themetint: 63; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 64;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">No<o:p></o:p></span></div>
</td>
<td style="background: #D3DFEE; border: none; mso-background-themecolor: accent1; mso-background-themetint: 63; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 64;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Yes<o:p></o:p></span></div>
</td>
</tr>
<tr>
<td style="border-bottom: solid #4F81BD 1.0pt; border: none; mso-border-bottom-themecolor: accent1; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in; mso-yfti-cnfc: 4;">
<b><span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Error can be eliminated with good practices<o:p></o:p></span></b></div>
</td>
<td style="border-bottom: solid #4F81BD 1.0pt; border: none; mso-border-bottom-themecolor: accent1; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">No<o:p></o:p></span></div>
</td>
<td style="border-bottom: solid #4F81BD 1.0pt; border: none; mso-border-bottom-themecolor: accent1; padding: 0in 5.4pt 0in 5.4pt;" valign="top"><div class="MsoNormal" style="line-height: 150%; margin-bottom: .0001pt; margin-bottom: 0in;">
<span style="font-family: "arial" , "sans-serif"; font-size: 10.0pt; line-height: 150%;">Yes<o:p></o:p></span></div>
</td>
</tr>
</tbody></table>
</div>
</div>
<div style="text-align: justify;">
<br />
<div style="text-align: center;">
<b>Table 3:</b> Comparison of Random and Systematic error.</div>
<br />
Regularly building a LJ graph for every variable that is quantitatively tested in the laboratory takes time and is sometimes biased by human intervention. So I have created an excel template which can be directly used for constructing an LJ graph. Just follow the steps shown in Figure 5 to generate your data. You can download the excel template from this <a href="https://drive.google.com/file/d/1gx-L8Yv2tH8OHSDS_LHQiQ_FLSKqJrwm/view?usp=sharing">Link</a>.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGkDvGLbuHcONQQOsUSQWEjPPy3jP1XmWoBQbRxYLC7pQcAQ4DOOkG877Ulw0kDF5NlTVb-Hhy72FqKnLmHgvaZG2MKooi_qy4BX3GXnAG5kOBv2og-v3hJMYY5hjzzCAANYMAWy2fRmit/s1600/Figure+5.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="1444" data-original-width="1600" height="576" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGkDvGLbuHcONQQOsUSQWEjPPy3jP1XmWoBQbRxYLC7pQcAQ4DOOkG877Ulw0kDF5NlTVb-Hhy72FqKnLmHgvaZG2MKooi_qy4BX3GXnAG5kOBv2og-v3hJMYY5hjzzCAANYMAWy2fRmit/s640/Figure+5.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 5:</b> Using the Excel to generate LJ charts.</td></tr>
</tbody></table>
<br />
Now that you are able to construct the LJ charts lets consider a few more arguments. The LJ chart is a useful tool to see how good is the variation. In other words you are measuring the precision, with reference to a conventional true value. Just like any statistics tool, LJ chart quality depends on the baseline data that has been used. if the measurement was not precise enough (High SD) the chart will become tolerant to high error rate. The closer the values are to the mean, more accurate is your measurement capabilities. We call it as "GiGo" (Garbage in- Grabage out). If the quality of input data is bad, the output will also be bad.<br />
<br />
LJ chart can also be used to determine other important aspects. For example, in declaring outbreaks. Lets say for example you have seen DEN-2 serotype diagnosis at the rate of 15 cases / week (with SD of 2 cases) for the past 6 months at a particular geographical location. And now, you diagnose 22 cases (> +3SD), in the same context. You can confidently declare it as an outbreak scenario for that week in the locality. Any situation where you are looking into significant change the concept of <span style="text-align: left;">±3SD can be evoked. The only disadvantage being that LJ can be used only when quantification can be achieved in numbers.</span><br />
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<b>References</b></div>
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1. International vocabulary of basic and general terms in
metrology (VIM). 3rd Edition. <a href="http://www.geste.mecanica.ufrgs.br/medterm/ISO_VEM.pdf">Link</a></div>
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2. Petros Karkalousos, Angelos Evangelopoulos. The History of Statistical Quality Control in Clinical Chemistry
and Haematology (1950 – 2010). International Journal of Biomedical Laboratory Science (IJBLS). 2015. <a href="http://www.ijbls.org/upfile/Issues/201591894942.pdf">Link</a><br />
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3. Coskun A. Modified Levey-Jennings charts for calculated laboratory tests. Clin Chem Lab Med. 2006;44(4):387-90. <a href="https://www.ncbi.nlm.nih.gov/pubmed/16599829">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-71384776826698322562020-02-05T15:40:00.000+05:302020-06-22T20:39:27.830+05:30Follow up Post: Wuhan Coronavirus<div dir="ltr" style="text-align: left;" trbidi="on">
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I must admit that my previous post on <a href="https://varuncnmicro.blogspot.com/2020/02/what-we-know-about-wuhan-coronavirus-to.html">Wuhan Coronavirus</a> (nCoV 2019) is not in any way complete information. Some of the details I skipped deliberately to provide an overview and avoid long boring read. Apparently, I should have given more information, considering that there is so much information floating around.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: justify;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg8sa6xremmFdVgpFYB_jxXLaRpspBrlJnx0aW3kq476eOo9TS4p6YKmmZDlha4qlT2iI2bvykxxkbQ_Hm_sEW-SlMy-gIgrrOy29E7AKBohAe-wwUYGvOlv3r5NiOYFJJjixxX_5ZvWW52/s1600/R+affinis.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="529" data-original-width="550" height="191" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg8sa6xremmFdVgpFYB_jxXLaRpspBrlJnx0aW3kq476eOo9TS4p6YKmmZDlha4qlT2iI2bvykxxkbQ_Hm_sEW-SlMy-gIgrrOy29E7AKBohAe-wwUYGvOlv3r5NiOYFJJjixxX_5ZvWW52/s200/R+affinis.jpg" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 1:</b> <i>R affinis</i> bat. <a href="http://www.bio.bris.ac.uk/research/bats/China%20bats/rhinolophusaffinis.htm">Source</a></td></tr>
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To the best of my understanding, we have not really understood what was the source from which the infection was acquired. Based on the sequencing results, the best match that has been found to be a coronavirus sequence isolated from <i>Rhinolophus affinis</i> (More commonly known as the Intermediate Horseshoe Bat) which was isolated in Yunnan Province in 2013 (More than 96% identity of sequence). The sequence identity in itself cannot ascertain that the virus made the jump to humans through a bat, but the circumstantial evidence is indicative. There are some important details from the paper by Zhou et al. The nCoV whole-genome sequences show similarity to the SARS-CoV. Further, the spike gene which encodes for receptor binding protein- S was found to be highly different from other CoVs, though it still uses the same ACE2 receptor for entry. Figure 1, from the paper, shows that there are some important changes in the S protein which is involved in cell entry.</div>
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiQlGtBT8vjNfh3H-v4LBZ9dAAYga1F-KWf78g_LJt9G3SJI894fW9mdJYLT5dRaOULmCNYxd2M_0B58acKdiJ7-fOXhrOBLzRVOrlEs_opM3yYhEv-u7W4yCzE6O3nhr1cYAgltnIwXeBl/s1600/Figure+1.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="682" data-original-width="793" height="550" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiQlGtBT8vjNfh3H-v4LBZ9dAAYga1F-KWf78g_LJt9G3SJI894fW9mdJYLT5dRaOULmCNYxd2M_0B58acKdiJ7-fOXhrOBLzRVOrlEs_opM3yYhEv-u7W4yCzE6O3nhr1cYAgltnIwXeBl/s640/Figure+1.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 1: </b>Amino acid changes in the S protein indicating significant changes. <a href="https://www.nature.com/articles/s41586-020-2012-7">Source: Zhou et al.</a></td></tr>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEji331IjtxTbbDxiqQvBe1Q20vQDzQ8zeEwUVNrFNDz5giIPFbl4qJkcnbSDDCusgg7KixBmODH-RKGVxGKGIC9V4hbjFMEyHU3B2V8wJFLgu0LYEMKxuFfiGIm0zrNI-bSGcWu1Pq9Haev/s1600/Batsoup.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="362" data-original-width="623" height="185" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEji331IjtxTbbDxiqQvBe1Q20vQDzQ8zeEwUVNrFNDz5giIPFbl4qJkcnbSDDCusgg7KixBmODH-RKGVxGKGIC9V4hbjFMEyHU3B2V8wJFLgu0LYEMKxuFfiGIm0zrNI-bSGcWu1Pq9Haev/s320/Batsoup.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 2:</b> Bat Soup. <a href="https://www.thesun.co.uk/news/10801901/china-coronavirus-outbreak-wuhan/">Source</a></td></tr>
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Though the exact mechanism of how it jumped or when it made the jump is not clear at this point of time, the current running hypothesis is that the "jump" happened at the Huanan Fish Market which sells exotic wild meat some which are also illegal. Interestingly, about 45% of the initial cluster of cases were not connected to the Wuhan market. This brings up two possibilities. First, the Wuhan market is not the primary place where the W happened. Alternatively, the percentage of people who did not directly acquire it from Wuhan market probably got it through a human to human transmission (By contact with asymptomatic case). The most famous "bat soup theory" suggests that the virus was acquired and adapted by consuming bat soup. Personally, I have doubts over this since Coronaviruses, in general, are not resistant to heat. A more likely possibility is the consumption of raw exotic meat. The definitive evidence would be finding the virus sequence from bats in Wuhan market, where the ground zero is supposed to be. <a href="http://www.xinhuanet.com/english/2020-01/27/c_138735677.htm">According to a Chinese newspaper, environmental sampling was done</a> at the Wuhan seafood market for 585 samples. of these, 33 samples were found to be positive for nCoV. All of the positives were from the market’s western portion where the wildlife and exotic meat was sold.</div>
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That brings in the question if the first case was the one reported in December. This is something difficult to answer. The best answer that I can think is "we are not sure". The first case might have acquired it much earlier and been asymptomatic and simply not detected. There is no way to tell. We can just say, that first known case was the one reported. There is a fair chance that the virus was circulating much before in Wuhan. The very high number of reported cases spreading at a rapid rate can be indeed explained by the idea that the virus was in circulation much earlier before December. </div>
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In the context of the question, if bats are the source <a href="https://www.bloombergquint.com/china/where-did-coronavirus-come-from-why-bats-may-be-to-blame">Christian Walzer comments</a>, “These animals are live. You will see a bird on top of a domestic pig, and you might have snake and bats, all stacked together in wire-mesh cages. virus-laden fluids and secretions can mix, helping create new viruses, especially when the animals are slaughtered right in front of customers. If you planned it and thought, ‘I am going to make new viruses, that is exactly how you would do it.”</div>
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The second question that comes up is the epidemic severity of the nCoV in comparison to SARS-CoV and MERS CoV. I have compiled a comparison table for yourself to see. As can be noted, the nCoV has the lowest mortality rate as of yet, with the highest being MERS. But nCoV is leading in the total number of cases. However, the outbreak cannot be taken lightly. Remember, 1918 flu had a mortality rate of approximately 2.5 %. However, it still is considered as one of the grave pandemics. This was due to an extremely high speed of spreading at the time.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgfOK2V3ss4mQK-DUTkkUY8Bcw3ljgQ33gyKflbpgqA1bVWvd0hyphenhyphenFd4KxXsKH-dAIfdmrve7ToZnSsX8z9xjlnzmhyld3grZqsfXoOJQsmF1uwnLOJcuqZnlggLmWKrUSRThoFbVkm9Lwp7/s1600/Figure+1.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="347" data-original-width="1043" height="212" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgfOK2V3ss4mQK-DUTkkUY8Bcw3ljgQ33gyKflbpgqA1bVWvd0hyphenhyphenFd4KxXsKH-dAIfdmrve7ToZnSsX8z9xjlnzmhyld3grZqsfXoOJQsmF1uwnLOJcuqZnlggLmWKrUSRThoFbVkm9Lwp7/s640/Figure+1.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Table 1: </b>Comparison of SARS-CoV, MERS-CoV and nCoV-2019.</td></tr>
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The most common clinical symptoms of the infection include fever, cough and myalgia or fatigue. In almost all cases, the symptoms appear within 14 days. However, only a subset of cases actually requires hospitalisation and intensive treatment. Fig 2, shows the timelines of clinical symptoms after the onset of illness. The laboratory diagnosis is currently performed using RT-PCR from respiratory samples. The <a href="https://www.who.int/docs/default-source/coronaviruse/wuhan-virus-assay-v1991527e5122341d99287a1b17c111902.pdf">WHO protocol</a> uses 3 targets for identification- First-line screening detects the E gene; Confirmatory assay targets the RdRp gene and an additional confirmatory assay look for the N gene.</div>
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As discussed in the <a href="https://varuncnmicro.blogspot.com/2020/02/what-we-know-about-wuhan-coronavirus-to.html">previous post</a>, an experimental drug remdesivir is currently under investigation. The drug was first tried in the US for the first case detected. The patient improved in a day and within 4 days, the case was successfully treated by this drug. Another study by Wang et al tested the efficacy of ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir and favipiravir in an in-vitro model and found that remdesivir and chloroquine were highly effective. Remdesivir is an adenosine analogue, which incorporates into nascent viral RNA chains and results in premature termination, which has been previously demonstrated to be useful against SARS and MERS. Chloroquine has been previously shown to block SARS infection by increasing endosomal pH required for virus/cell fusion, as well as interfering with the glycosylation of cellular receptors. The recent research from <a href="https://www.ecowatch.com/andean-condor-2645030016.html">Thailand reports on successful treatment</a> with lopinavir and ritonavir with large doses of the flu drug oseltamivir. </div>
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The current scenario of the outbreak is not showing any signs of slowing down. A few experts based on the current data available suggest that the outbreak may escalate if sufficient measures are not taken. As <a href="https://www.ecowatch.com/coronavirus-treatment-thailand-doctors-2645011844.html?rebelltitem=3#rebelltitem3">Anthony S. FauciIt's comments</a>, "it is very, very transmissible, and it almost certainly is going to be a pandemic. But will it be catastrophic? I don't know". </div>
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<b>References:</b></div>
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1. Zhou et al. Pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. https://doi.org/10.1038/s41586-020-2012-7. <a href="https://www.nature.com/articles/s41586-020-2012-7">Link</a><br />
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2. Holshue et al. First Case of 2019 Novel Coronavirus in the United States. NEJM. 2020. <a href="https://www.nejm.org/doi/full/10.1056/NEJMoa2001191">Link</a><br /><br />
2. Wang et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Nature. 2020. https://doi.org/10.1038/s41422-020-0282-0. <a href="https://www.nature.com/articles/s41422-020-0282-0">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-33760063192733714302020-02-04T10:57:00.002+05:302020-06-22T20:39:27.829+05:30What we know about the Wuhan Coronavirus: To date<div dir="ltr" style="text-align: left;" trbidi="on">
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In the past 3 weeks, there has been massive media attention on a novel coronavirus strain which seems to be infecting and spreading at an accelerated rate. There is so much of internet content about it, with a majority of them being false news. Its time to write a piece on it and clear some air.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhCQWWHzRGezvJy_azF95EuRF2ip_bAiTe7Vvkp8-NohieawvPrBCHJdNJpSstKBoxqv5QuFLeIBaPlquW8CiAFCE7NJqhHWgOf2VEnVLK91_HwivONe32UYcdLWfSZQCbXrC_9bq5FIYHD/s1600/Figure+1.gif" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="435" data-original-width="500" height="173" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhCQWWHzRGezvJy_azF95EuRF2ip_bAiTe7Vvkp8-NohieawvPrBCHJdNJpSstKBoxqv5QuFLeIBaPlquW8CiAFCE7NJqhHWgOf2VEnVLK91_HwivONe32UYcdLWfSZQCbXrC_9bq5FIYHD/s200/Figure+1.gif" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 1:</b> Structure of Coronavirus.<br />
<a href="https://www.nature.com/articles/nm1143">Source</a></td></tr>
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Coronaviruses are enveloped non-segmented positive-sense RNA viruses belonging to the family Coronaviridae (Order Nidovirales). Historically, Coronavirus disease was first described in 1931. Only two human coronaviruses- HCoV-229E and HCoV-OC43 were known until the appearance of SARS-CoV and later MERS-CoV. Structurally, the coronavirus is spherically shaped with a few proteins (See Figure 1). Their RNA genome is one of the largest with about 30Kbp sequence packed into a helical capsid contributed by the nucleocapsid protein (N) and further surrounded by an envelope. The M (membrane), E (Envelope) and S (Spike) protein form the structural proteins. The S protein mediates cell entry through receptors most possibly by acting as a class I viral membrane fusion protein.<br />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTIns9BBjbJDnY0vNbQg9PgyJaBRf7Ysm0zQ-94fAatI6tlPl-j7xhv3QlgHIFd3tb5grt9P0GHY9ZLrVpwKOUhelRo-iAfPgZ6UEy_9TkJrlu3zTWzsnZhYvTARROA-wqQBMvfq81oRn0/s1600/Classification.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="428" data-original-width="924" height="185" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTIns9BBjbJDnY0vNbQg9PgyJaBRf7Ysm0zQ-94fAatI6tlPl-j7xhv3QlgHIFd3tb5grt9P0GHY9ZLrVpwKOUhelRo-iAfPgZ6UEy_9TkJrlu3zTWzsnZhYvTARROA-wqQBMvfq81oRn0/s400/Classification.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Table 1:</b> Classification of Coronaviruses.</td></tr>
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The complete set of known coronaviruses are currently classified into 4 genera. The details of these are summarised in Table 1. As can be seen, the beta coronaviruses have been the most problematic to humans. This is especially due to their adaptability to new environments through rapid mutation and recombination with relative ease.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhUzyvASFPweKl6LNT9ai0SCj1rB6ua2h1GTdh-vOQyQ4JJvyl90BFN6bN_karBzIIxfUIxHDi72B-kIgQC34jUIE5GkTEe-DQpCvdQILykdqAh5ozKGz8bxU-doz-tlzr075ibwQN4GXH8/s1600/Stats.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="581" data-original-width="741" height="312" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhUzyvASFPweKl6LNT9ai0SCj1rB6ua2h1GTdh-vOQyQ4JJvyl90BFN6bN_karBzIIxfUIxHDi72B-kIgQC34jUIE5GkTEe-DQpCvdQILykdqAh5ozKGz8bxU-doz-tlzr075ibwQN4GXH8/s400/Stats.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 2:</b> Current Coronavirus Statistics. <a href="https://www.worldometers.info/coronavirus/">Source</a></td></tr>
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The current growing epidemic of coronavirus designated as novel coronavirus 2019 was first reported in December 2019 from Wuhan in China viral pneumonia. Subsequently, a massive increase in the number of cases has been observed. At the time of writing this post, a <b>total of </b><span style="text-align: left;"><b>20,626 cases</b> have been confirmed </span><span style="text-align: left;">of which 2,790 (14%) are </span><span style="text-align: left;">in critical condition. There have been </span><span style="text-align: left;"><b>427 deaths</b> and </span><span style="text-align: left;">653 cases have been confirmed to have recovered completely (Live update is available through this <a href="https://www.worldometers.info/coronavirus/">link</a>). That makes the case fatality ratio to approximately 2.07%. The most recent available estimate of R<span style="font-size: x-small;">0 </span></span>for nCoV 2019 is approximately 4.08 (WHO estimates the value in the range of 1.4 - 2.5). That means the epidemic is currently in a growing stage. The epidemic was initially declared as a PHEIC (Public Health Emergency of International Concern) which has been now escalated to a level 4- Global emergency.</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiNVUCIZmzM0H-BnOmA7aSdBZRoquNo-ZRQgYlkC6A_POA7QNX48SBPGvYoEWNHN3e-Z76Rjxz1bDqeO4jWEL8i6HjO4E-8ifrCRhLU-W0kAbMpW_HRNnAjpry8t_8VS9abfk0wwFqgQxEn/s1600/World+Map.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="1156" data-original-width="1600" height="462" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiNVUCIZmzM0H-BnOmA7aSdBZRoquNo-ZRQgYlkC6A_POA7QNX48SBPGvYoEWNHN3e-Z76Rjxz1bDqeO4jWEL8i6HjO4E-8ifrCRhLU-W0kAbMpW_HRNnAjpry8t_8VS9abfk0wwFqgQxEn/s640/World+Map.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Figure 3:</b> Geographic distribution of nCoV-2019. <a href="http://Geographic distribution of 2019-nCov">Source: ECDC</a></td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEit8EARX3A5-5uLil14Y2Gob62l31ruxXLhe8RjwKNgwY9RxyIgtC2ZNeZE8QfpM9M99ODsaGdm67GXuDhBLbCbXa8Gmdlr26YLIt45aUKpPtThYVqzBYM-dFas73fVAZyeRQmLaAV0YvK6/s1600/Figure+4.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="350" data-original-width="584" height="238" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEit8EARX3A5-5uLil14Y2Gob62l31ruxXLhe8RjwKNgwY9RxyIgtC2ZNeZE8QfpM9M99ODsaGdm67GXuDhBLbCbXa8Gmdlr26YLIt45aUKpPtThYVqzBYM-dFas73fVAZyeRQmLaAV0YvK6/s400/Figure+4.png" width="400" /></a></td></tr>
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<b>Figure 4:</b> Phylogenetic tree showing Wuhan Coronavirus relationship.</div>
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<a href="https://ncbiinsights.ncbi.nlm.nih.gov/2020/01/13/novel-coronavirus/">Source</a>.</div>
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There has been a lot of media coverage in recent times suggesting that the Wuhan Coronavirus is a product of bioweapon research program that has leaked. I must say the news agencies have shown irresponsible behaviour here. This was further fuelled by a <a href="https://www.biorxiv.org/content/10.1101/2020.01.30.927871v1">paper posted in biorxiv</a> suggesting that the nCoV sequences had been engineered with HIV Gag sequence inserted into it. Most people even believed leading to the widespread circulation of the news. Thankfully, the paper has been retracted owing to lack of scientific robustness (<a href="https://www.statnews.com/2020/02/03/retraction-faulty-coronavirus-paper-good-moment-for-science/">Link</a>). There is currently no genomic level evidence that it is engineered. Indeed the phylogenetic tree clarifies that the sequence is very much similar to the Bat coronavirus sequence, thereby also indicating a possible origin source.</div>
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"Based on the virus genome and properties there is no indication whatsoever that it was an engineered virus. Most countries had largely abandoned their bioweapons research after years of work proved fruitless. The vast majority of new, nasty diseases come from nature".</div>
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-Richard Ebright & Tim Trevan. <a href="https://www.washingtonpost.com/world/2020/01/29/experts-debunk-fringe-theory-linking-chinas-coronavirus-weapons-research/">Source</a></div>
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Currently, the diagnosis of coronavirus has been done by RT-PCR from the respiratory samples (<a href="https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.3.2000045">Link</a>). Scientists from China have previously reported that it can be cultured in human cells and that it enters them through the same molecular receptor as the coronavirus that causes SARS. The virus is also cultured at the Peter Doherty Institute for Infection and Immunity (Doherty Institute) in Melbourne have successfully cultured the coronavirus. Quoting Dr Druce on this “Chinese officials released the genome sequence of this novel coronavirus, which is helpful for diagnosis, however, having the real virus means we now have the ability to actually validate and verify all test methods, and compare their sensitivities and specificities - it will be a game changer for diagnosis. The virus will be used as positive control material for the Australian network of public health laboratories, and also shipped to expert laboratories working closely with the World Health Organization (WHO) in Europe.” <a href="https://www.technologynetworks.com/cell-science/news/wuhan-coronavirus-successfully-grown-in-cell-culture-in-australia-330081">Source</a></div>
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In approximately a month the number of papers published on the Wuhan Coronavirus has already climbed to more than 60 papers (<a href="https://www.nature.com/articles/d41586-020-00253-8">Link</a>), evidence that a lot of labs are actually working on the virus at an extreme pace and there is a lot of data on clinical presentation and transmission. However, as of date, there are no approved specific coronavirus antiviral drugs. A clinical trial using a combination of lopinavir and ritonavir (Anti HIV drugs) based on its previously demonstrated use against SARS-CoV is currently in process. Another drug- <a href="https://www.genengnews.com/topics/drug-discovery/gilead-partnering-with-china-on-trial-of-remdesivir-as-coronavirus-treatment/">remdesivir</a> is also under testing. There was news circulating that the anti-HIV drug Nelfinavir, can be used for treatment <a href="https://www.motherjones.com/environment/2020/01/three-of-the-most-viral-claims-about-the-coronavirus-are-fake/">which is not true</a>. Rather, oseltamivir is currently used for treatment as per the publications.</div>
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Currently, the infection is spreading at a very high rate and with global attention, it is unlikely that many cases are missed. However, recent reports suggesting that many number of cases are asymptomatic and these asymptomatic cases can be good spreaders has challenged the perceptions we have for this infection. It must be noted that most coronavirus cases currently seen have not caused serious health damage and only a subset of the cases who have other predisposing factors are probably entering a critical stage. The WHO has urged the public to take <a href="https://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-public">simple hygiene precautions</a> such as frequent washing of hands to avoid catching an infection.</div>
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In summary, nCoV-2019 is currently an ascending epidemic slowly moving to pandemic proportions, with most cases still being reported from China. The case-fatality ratio is very low but early detection method is available. There are no specific antivirals but the research is currently in rapid progress.</div>
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<b>References:</b></div>
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1. R Lu et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020. pii: S0140-6736(20)30251-8. <a href="https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30251-8/fulltext">Link</a></div>
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2. Huang et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020. <a href="https://pubmed.ncbi.nlm.nih.gov/31986264-clinical-features-of-patients-infected-with-2019-novel-coronavirus-in-wuhan-china/">Link</a></div>
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3. Rothe et al. Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany. NEJM. 2020. DOI: 10.1056/NEJMc2001468 <a href="https://www.nejm.org/doi/10.1056/NEJMc2001468">Link</a></div>
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4. Li et al. Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia. NEJM. 2020. DOI: 10.1056/NEJMoa2001316 <a href="https://www.nejm.org/doi/full/10.1056/NEJMoa2001316">Link</a> </div>
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5. Wu et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020. https://doi.org/10.1038/s41586-020-2008-3. <a href="https://www.nature.com/articles/s41586-020-2008-3">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-12915330324886751042019-02-18T20:31:00.004+05:302019-02-18T20:32:10.871+05:30Dialister and Coprococcus species in the Gut Microbiome has a role in Mental Health<div dir="ltr" style="text-align: left;" trbidi="on">
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If I had asked you to identify what are the most important organs of the human body, you are very likely to miss out on "microbiome". Though in a traditional textbook sense we never think of Microbiome as a part of the human body there is growing evidence and debate that perhaps microbiome contributes to the human gene pool. Some scientists have begun calling it a "<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414803/">virtual endocrine organ</a>", thanks to many of the chemicals that they contribute. The concept that human is a <a href="https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0466-8">holobiont</a> and has implications on physiology is overwhelmingly taking over.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjhSbmDV0dIg-fOzVZXe6qsATlXR8_ax6axMMip6eG6rqSdS18_EIh1pacFuw0vcNCAU7wMiSdNRjrNc0_XS7_LTy0l6Z_wAGIgs9JMvcJi-kkkEI2Yr2quZcToUMBGI1s-0GhGzIynbD0C/s1600/F1.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="702" data-original-width="1280" height="348" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjhSbmDV0dIg-fOzVZXe6qsATlXR8_ax6axMMip6eG6rqSdS18_EIh1pacFuw0vcNCAU7wMiSdNRjrNc0_XS7_LTy0l6Z_wAGIgs9JMvcJi-kkkEI2Yr2quZcToUMBGI1s-0GhGzIynbD0C/s640/F1.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1: </b>The concept of humans being a holobiont. <a href="https://msystems.asm.org/content/1/2/e00028-16">Source</a></td></tr>
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The point of this post is not to discuss the concept of humans being a holobiont, but to talk about a recent paper on the importance of gut microbiome in mental health. If you haven't read my previous posts on this topic, I suggest you read my earlier posts <a href="http://varuncnmicro.blogspot.com/2015/08/microbes-for-thought.html">here</a> and <a href="http://varuncnmicro.blogspot.com/2016/05/the-link-between-brain-and-microbiome.html">here</a>.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjGfOTpgAt5mW6lp0yY7sB7Sadj65p6fErHLvVqiwpjK4A9hK35oGbdQZ2SR1DwjYbuY4fAdI0kxDUPQlJwj903afGxqNRGjfIs1yZXUTTHcYeJTcUKWUrXdVOM5R9ZlLvle8ZdnHUijxPI/s1600/F2.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="500" data-original-width="527" height="378" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjGfOTpgAt5mW6lp0yY7sB7Sadj65p6fErHLvVqiwpjK4A9hK35oGbdQZ2SR1DwjYbuY4fAdI0kxDUPQlJwj903afGxqNRGjfIs1yZXUTTHcYeJTcUKWUrXdVOM5R9ZlLvle8ZdnHUijxPI/s400/F2.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2: </b>Associations between QoL scores or depression and bacterial genera.<br /><a href="https://www.nature.com/articles/s41564-018-0337-x">Source</a></td></tr>
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Papers that have been published on the relationship between the microbiome and mental health have studied a few small numbers of cases which brings into question as to how valid the studies are in reality. So, a group of Dutch microbiologists from VIB-KU Leuven Center for Microbiology decided to look bioinformatically into the microbiome of 1,054 individuals enrolled in the Flemish Gut Flora Project (FGFP) and correlated their findings with the quality of life (QoL). They discovered that people presenting with depression had depleted levels of two bacteria- <i>Coprococcus species</i> and Dialister species irrespective of antidepressants. They also validated their results against a second independent cohort of 1,063 individuals from the Dutch LifeLinesDEEP (LLD) cohort and in a cohort of clinically depressed patients at the University Hospitals Leuven, Belgium. Fig 2 from the paper, shows a summary of microbial associations. With some additional bioinformatic analysis, they specifically looked for the presence of <i>Bacteroides enterotype 2, </i>which was based on their previous publication (<a href="https://www.nature.com/articles/nature24460">Link</a>) and found they indeed had a significant association with depression.</div>
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<a href="https://neurosciencenews.com/depression-gut-bacteria-10685/">Jeroen Raes summarises the findings in his comment</a>, "The relationship between gut microbial metabolism and mental health is a controversial topic in microbiome research. The notion that microbial metabolites can interact with our brain – and thus behaviour and feelings – is intriguing, but gut microbiome-brain communication has mostly been explored in animal models, with human research lagging behind. In our population-level study we identified several groups of bacteria that co-varied with human depression and QoL across populations. This finding adds more evidence pointing to the potentially dysbiotic nature of the Bacteroides 2 enterotype we identified earlier. Apparently, microbial communities that can be linked to intestinal inflammation and reduced wellbeing share a set of common features."</div>
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A small digression. <i>Dialister species</i> is known with reference to humans for some time as a <a href="https://aac.asm.org/content/51/12/4498">potential pathogen</a>, though very rare. They are small, anaerobic or microaerophilic gram-negative coccobacilli found to be involved with oral infections such as periodontitis, acute necrotizing ulcerative gingivitis, and endodontic infections especially <i>D. pneumosintes</i> and <i>D. invisus</i>. Other known human pathogens include <i>D micraerophilus and D propionicifaciens</i>. Coprococcus species are gram positive, anaerobic cocci knwon for its butyrate-producing capacity is a member of Clostridia group known to be a part of the human gut microbiome and found in faecal microbiome. Currently, three species are known- <i>Coprococcus catus, Coprococcus comes </i>and<i> Coprococcus eutactus. </i></div>
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Finding an association is one thing but how does it work? To understand possible mechanisms, the researchers developed a framework and based on literature curated and annotated 56 Gut–Brain modules (GBM). In a simplified sense, GBM is created by taking the genome of all the known microbiome and look for coding sequences which represent the ability to produce chemicals that can involve with neural function. For example, some of the <i>Fusobacterium </i>genomes that were analysed carried the potential to synthesize histamine. By comparing the GBM between microbiomes of people with a high or low QoL the researchers found 3 GBM's of interest based on data from the FGFP data. These include</div>
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1. Synthesis of 3,4-dihydroxyphenylacetic acid (DOPAC; positive correlation),</div>
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2. Isovaleric acid synthesis (via α -keto-acid decarboxylase pathway; positive correlation)</div>
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3. Histamine synthesis (Negative correlation).</div>
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However, when the findings were validated against the LLD metagenomic database, they found that only DOPAC synthesis could be replicated. Though Coprococcus did not have a genetic system to directly synthesise DOPAC, there were genes for converting 3,4-dihydroxy phenylacetaldehyde to DOPAC, in the genomes of <i>Coprococcus comes</i> and <i>Coprococcus catus</i>.</div>
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<a href="http://www.vib.be/en/research/departments/Pages/VIB-KU-Leuven-Center-for-Microbiolgy.aspx"></a></div>
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As the first author <a href="https://www.psychologytoday.com/ca/blog/the-athletes-way/201902/gut-microbiome-research-is-advancing-leaps-and-bounds">Mireia Valles-Colomer simplifies it</a>, “Many neuroactive compounds are produced in the human gut. We wanted to see which gut microbes could participate in producing, degrading, or modifying these molecules. Our toolbox not only allows to identify the different bacteria that could play a role in mental health conditions but also the mechanisms potentially involved in this interaction with the host. For example, we found that the ability of microorganisms to produce DOPAC, a metabolite of the human neurotransmitter dopamine, was associated with better mental quality of life."</div>
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There is a word of caution associated with these studies. The current paper is completely a computational-based study and they need to be validated. However, they do give a lead into the understanding that microbes are significantly connected with mental health and probably their gene pool contributes to human neuro-metabolism through the principle of holobiosis.</div>
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<b>References:</b><br />
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Clarke G, Stilling RM, Kennedy PJ, Stanton C, Cryan JF, Dinan TG. Gut Microbiota: The Neglected Endocrine Organ. Mol Endocrinol. 2014 Aug;28(8):1221-38 <a href="https://www.ncbi.nlm.nih.gov/pubmed/24892638">Link</a><div>
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<br />Maarten van de Guchte, Joël Doré. Humans as holobionts: implications for prevention and therapy. Microbiome 2018; 6:81. <a href="https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0466-8">Link</a><div>
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Mireia Valles-Colomer, Gwen Falony, Youssef Darzi, Ettje F. Tigchelaar, Jun Wang, Raul Y. Tito, Carmen Schiweck, Alexander Kurilshikov, Marie Joossens, Cisca Wijmenga, Stephan Claes, Lukas Van Oudenhove, Alexandra Zhernakova, Sara Vieira-Silva & Jeroen Raes. The neuroactive potential of the human gut microbiota in quality of life and depression. Nature Microbiology (2019). https://doi.org/10.1038/s41564-018-0337-x <a href="https://www.nature.com/articles/s41564-018-0337-x">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-72773386712646652182019-02-01T12:55:00.001+05:302019-02-01T12:55:39.397+05:30Blogger's Desk#12- Financial spending on R&D: India<div dir="ltr" style="text-align: left;" trbidi="on">
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Greetings</div>
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Though this is a blog space to discuss Microbiology, I have also used this platform to sometimes talk about science itself to a larger audience of interest. In April 2015, I wrote a post on the plight of Indian research scholars (<a href="http://varuncnmicro.blogspot.com/2015/04/bloggers-desk-5-alarming-difficulties.html">Link</a>). With time, nothing seems to have really improved in the country. The brightest of research scholars in the country are demonstrating on the streets to gain the attention of the government and seeking to solve the long-standing issues (<a href="https://www.nature.com/articles/d41586-019-00208-8">Link</a>). In this post, I am trying to provide a snapshot of the current financial status of research(ers) in India.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhOFiNo1TmENhazA8IgQomvxvPrvlA2XCC7mL9rLgV06jPN3NzP1I_Dj56EpSQRxZmJV3ieiwympkuxXoi2XFFBSeiSQZmBW6_Vk-u5L6D1tMciCukeSTg9gaC5noXLlQ4tWqWfPQeQDM19/s1600/F1.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="637" data-original-width="1219" height="334" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhOFiNo1TmENhazA8IgQomvxvPrvlA2XCC7mL9rLgV06jPN3NzP1I_Dj56EpSQRxZmJV3ieiwympkuxXoi2XFFBSeiSQZmBW6_Vk-u5L6D1tMciCukeSTg9gaC5noXLlQ4tWqWfPQeQDM19/s640/F1.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small; text-align: justify;"><b>Fig 1:</b> Investment on R&D by country. <a href="http://uis.unesco.org/apps/visualisations/research-and-development-spending/">Source</a></span></td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEilRIieFR1CdmFm5IcjNTw5jQg-BDjxU2_AInr-3wo28liYSJlGGvyYGC3lzMVNjUuj2S5PenwBTWJ0HNfUZ1biDeA9V1zP3oihnr0K3EL_cgpv1rdSUKNJIygelQnOIcP6XrTIl-3eup2t/s1600/F2.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="651" data-original-width="1600" height="162" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEilRIieFR1CdmFm5IcjNTw5jQg-BDjxU2_AInr-3wo28liYSJlGGvyYGC3lzMVNjUuj2S5PenwBTWJ0HNfUZ1biDeA9V1zP3oihnr0K3EL_cgpv1rdSUKNJIygelQnOIcP6XrTIl-3eup2t/s400/F2.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2:</b> %GDP expenditure on R&D</td></tr>
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The prime minister of India aims to place India at the t<a href="https://economictimes.indiatimes.com/news/politics-and-nation/pm-narendra-modi-adds-jai-anusandhan-to-jai-jawan-jai-kisan-and-jai-vigyan/articleshow/67363585.cms">op 3 on a global scale</a>. Evidently, though these are words of political interest, there is nothing much that has been done at the scale of reality for the long term. In a study published in 2013 by <a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066449">Meo et al</a>, it has been convincingly shown that there is a direct relationship between the spending on R&D and the quality of publications. In this light, India stands really down. Fig 2, shows a % GDP spending of India (in the year 2015) in comparison to some a few countries, as per the data available from the <a href="https://data.worldbank.org/indicator/GB.XPD.RSDV.GD.ZS">world bank</a>. Despite this situation, the Indian spend on R&D has <a href="https://economictimes.indiatimes.com/news/economy/finance/indias-rd-spend-stagnant-for-20-years-at-0-7-of-gdp/articleshow/62697271.cms">been the same at 0.6% GDP</a> from the year 1998 to 2017. This financial crunch in research funding is totally <a href="https://www.scimagojr.com/countryrank.php">reflected in our scientific publications.</a> India stands at the 9th position on a global scale in producing citable documents but ranks 184/239 when it comes to the average citations per document. A large chunk of the citations is contributed by self-citations. Clearly, the government is not funding enough research labs to do their research work.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjWeWmSSJYQC224QpybaDkblwvJg9fZMvlL4_eftVxuTR5y3HZ9jAXygZxK1uY6z4kmTMg8Pf6G-yBvp7NuLVN74xtJj-hO9yAvx-b097NNptfuEqT3Z0M69sjULGN-QcR2272r57ZQ_sDb/s1600/F3.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="464" data-original-width="757" height="196" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjWeWmSSJYQC224QpybaDkblwvJg9fZMvlL4_eftVxuTR5y3HZ9jAXygZxK1uY6z4kmTMg8Pf6G-yBvp7NuLVN74xtJj-hO9yAvx-b097NNptfuEqT3Z0M69sjULGN-QcR2272r57ZQ_sDb/s320/F3.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 3:</b> Stipend amounts received by PhD Scholars.</td></tr>
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There has been a recent demand from researchers like me, who are pursuing our PhD under government fellowship (Through agencies such as CSIR/UGC/DST/ICMR). The developments so far have <a href="https://www.ndtv.com/education/dst-issues-revised-fellowship-order-1985615">not been in favour of PhD scientists</a> as a whole. It is largely the opinion of lots of scientists that the brain drain is due to this unfavourable financial situation. In fact, India contributes as a <a href="http://www.pewresearch.org/fact-tank/2017/03/03/india-is-a-top-source-and-destination-for-worlds-migrants/">top source</a> for emigration globally. Figure 3 shows the trend of stipend received by PhD students for performing research, who have qualified through national level competitive examinations. It appears from the graph that there is an increase in the salaries of PhD's by year. However, in reality, this was very low from the beginning with reference to the cost of living and the value for the degree or how much the same talent is paid in other countries where research is a priority.<br />
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Apparently, if India wants to be one among the best for research output, there is a requirement for a substantial increase in financial allocation for research. The 20-year-old trend of 0.6% of GDP is absolutely insufficient for sustaining the increased quality of research(ers).</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-44107393993319709702019-01-19T11:35:00.001+05:302019-01-19T11:36:14.299+05:30Genetic Spying for diagnostics- SHERLOCK CRISPR System: An update<div dir="ltr" style="text-align: left;" trbidi="on">
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Greetings</div>
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There have been so many great microbiology stories that I should have written about in the year 2018, but I was so occupied I never got the time. Well, doesn't mean this blog has died. Of course, the "almost a year gap" in writing has slightly affected my style of blogging. But I will pick it up back in a few weeks. That being said, let us come back to talk some science.</div>
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In the field of diagnostics, speed and accuracy of diagnostics is an ever challenging factor. From classic microbiology techniques which takes at least 48 hours to identify and characterise a pathogen, we have come a long way to more modern molecular methods such as PCR which can report in a couple of hours. These days, bacterial diagnostics have become faster with the use of <a href="http://varuncnmicro.blogspot.com/2016/02/laboratory-series-10-maldi-tof-in.html">MALDI-TOF</a> instrumentation. For a quick and reliable diagnostics we still mostly rely on <a href="http://varuncnmicro.blogspot.com/2015/05/lab-series-5-dna-amplification-in-lab.html">PCR</a> and <a href="http://varuncnmicro.blogspot.com/2015/04/lab-series-2-dna-sequencing.html">NGS</a> methods. But they come with a high price tag and sophistication which are not directly "Field deployable". An alternative is immunologically based rapid diagnostic tests which take months to develop and validate and have lower capabilities than a genetic test.</div>
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On a side note, <a href="http://varuncnmicro.blogspot.com/2014/02/clustered-regularly-interspaced-short.html">the CRISPR-Cas syste</a>m has been widely harvested in genetic engineering. There is a great debate on who owns the intellectual property rights to CRISPR based gene editing technology which is an <a href="https://cen.acs.org/policy/litigation/Broad-prevails-over-Berkeley-CRISPR/96/web/2018/09">ongoing legal battle between the Broad and Berkley institute</a>. There is also news of a Chinese research team lead by <i>He Jiankui</i> claiming to have created the first CRISPR-edited twins girls named Lulu and Nana. However, there are strong doubts over the claim and the bioethical aspects of this work have been subject an international discussion (<a href="https://www.theatlantic.com/science/archive/2018/12/15-worrying-things-about-crispr-babies-scandal/577234/">Link</a>).</div>
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Cas 13 protein has some interesting properties. Cas13a is functionally comparable to Cas9. In addition to its programmable RNase activity, Cas13a shows a collateral activity leading to non-specific degradation of any nearby transcripts regardless of complementarity to the spacer. Upon recognition of a specific RNA sequence programmed Cas13a is activated which then cleaves the surrounding ssRNA molecules. By using a quenched fluorescent ssRNA which can be cleaved to release the reporter (The idea is similar to TaqMan chemistry in Real-time PCR), the signal can be easily read. This phenomenon was harnessed by a group of scientists from the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering & Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University to develop a technique called as "SHERLOCK" in April 2017. SHERLOCK is a fancy acronym for <b><u>S</u></b>pecific <b><u>H</u></b>igh-sensitivity <b><u>E</u></b>nzymatic <b><u>R</u></b>eporter un<b><u>LOCK</u></b>ing. Subsequently the same team in April 2018 reported an improved design further to develop a protocol known as SHERLOCK-HUDSON. HUDSON is an acronym for "<b><u>H</u></b>eating <b><u>U</u></b>nextracted <b><u>D</u></b>iagnostic <b><u>S</u></b>amples to <b><u>O</u></b>bliterate <b><u>N</u></b>ucleases). Originally SHERLOCK presented with a few limitations related to quantification. Subsequently, SHERLOCK V2 which uses a combination of Cas13, Cas12a, and Csm6, was reported which can achieve multiplex nucleic acid detection with enhanced sensitivity. Also, SHERLOCK used fluorescent detectors whereas, in the improved version the reporter was modified such that cleaved reporter could be detected on commercial lateral flow strips.</div>
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There are a couple of associated technologies here. HOLMES (one-<b><u>HO</u></b>ur <b><u>L</u></b>ow-cost <b><u>M</u></b>ultipurpose highly <b><u>E</u></b>fficient <b><u>S</u></b>ystem) platform for nucleic acid detection uses Cas 12 as the enzyme. DETECTR (<b><u>D</u></b>NA <b><u>E</u></b>ndonuclease <b><u>T</u></b>argeted <b><u>C</u></b>RISPR <b><u>T</u></b>rans <b><u>R</u></b>eporter which was independently developed at Doudna's Lab also uses Cas 12a with small changes in the details.</div>
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<tr><td class="tr-caption"><b>Fig 1:</b> Overview of the development of the CRISPR systems and their applications. <a href="https://www.sciencedirect.com/science/article/pii/S2405805X18300607">Source</a></td></tr>
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<b>Fig 2:</b> The principle of SHERLOCK and DETECTR detection methods.</div>
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<a href="https://blog.addgene.org/finding-nucleic-acids-with-sherlock-and-detectr">Source</a></div>
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SHERLOCK is mainly an RNA detection tool and has been demonstrated to be useful in diagnostics such as Zika Virus detection at attomolar concentrations. In contrast, DETECTR is a DNA detection method and has been demonstrated to be useful in identifying HPV. HUDSON is not a detection method, but rather a processing protocol where the virus particles are lysed and heat treated to work directly with SHERLOCK without the need for separate processing of samples. HOLMES works very much similar to other techniques except that it uses Cas12b in a Loop-Mediated Isothermal Amplification methodology. Figure 2 gives a schematic summary of the working of SHERLOCK and DETECTR.</div>
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Feng Zhang, whose lab is involved in developing the SHERLOCK technology <a href="https://www.broadinstitute.org/news/sherlock-team-advances-its-crispr-based-diagnostic-tool">commented</a>, "SHERLOCK provides an inexpensive, easy-to-use, and sensitive diagnostic method for detecting nucleic acid material — and that can mean a virus, tumour DNA, and many other targets. The SHERLOCK improvements now give us even more diagnostic information and put us closer to a tool that can be deployed in real-world applications. The technology demonstrates potential for many healthcare applications, including diagnosing infections in patients and detecting mutations that confer drug resistance or cause cancer, but it can also be used for industrial and agricultural applications where monitoring steps along the supply chain can reduce waste and improve safety".</div>
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<b>References:</b></div>
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Liu H, Wang L, Luo Y. Blossom of CRISPR technologies and applications in disease treatment. Synth Syst Biotechnol. 2018 Oct 22;3(4):217-228. <a href="https://www.ncbi.nlm.nih.gov/pubmed/30370342">Link</a></div>
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Myhrvold et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018 Apr 27;360(6387):444-448. <a href="https://www.ncbi.nlm.nih.gov/pubmed/29700266">Link</a></div>
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Gootenberg et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 28;356(6336):438-442. <a href="https://www.ncbi.nlm.nih.gov/pubmed/28408723">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-28899266289105280622019-01-18T19:15:00.001+05:302019-01-18T19:15:36.512+05:30Mitochondria participates actively in Phagocytosis.<div dir="ltr" style="text-align: left;" trbidi="on">
Greetings<br />
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Anyone who has studied biology knows about the process of phagocytosis. Phagocytosis, is a process by which a cell engulfs other cells or particles. In context with immune system, phagocyosis is an important mechanism in defense against many different microbes. In fact, the mechanism is so important that several pathogenic microbes have evolved and acquired specialised mechanism to evade killing by phagoctyosis.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjcx0NN9s-cdhZJom0OON4r6s1ct7w8Iko2Uwb3uHraJwArrqULDQK2AATI9VKwt08INTChhcgqwNrl4iU4KTtYsgKUrGf_fyxkibPWY6ZyjDtnFzskL2BqALqY4_8sJi21PhffMb_HITJf/s1600/F1.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="1052" data-original-width="1280" height="263" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjcx0NN9s-cdhZJom0OON4r6s1ct7w8Iko2Uwb3uHraJwArrqULDQK2AATI9VKwt08INTChhcgqwNrl4iU4KTtYsgKUrGf_fyxkibPWY6ZyjDtnFzskL2BqALqY4_8sJi21PhffMb_HITJf/s320/F1.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Mechanisms of escaping Phagocytic killing<br />
by Microbes. <a href="http://www.biochemsoctrans.org/content/41/2/475">Source</a></td></tr>
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There are basically two broad mechanisms of surviving phagocytosis. First, the microbe can completly evade being phagocytosed for example by using a capsule (Ex: <i>S pneumoniae</i>). Second, after being phagocytosed the microbe can survive being ripped off inside the phagosome either by inhibiting phagosome lysosome fusion (<i>Ex: M tuberculosis</i>), surviving inside phagolysosome (Ex: <i>Coxiella burnetii</i>) or escape from phagosome (Ex: <i>Shigella</i> <i>species</i>). Some microbes can even produce toxins that directly attack phagocytic cells (Ex: Leukocidins by <i>S aureus</i>). Figure 1 is a summary of microbial tools that allows escape from being killed by phagocytosis.<br />
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Though the exact details of phagocytic killing vary, the prinicple is pretty much the same globally. For example, the WBC first attaches the bacteria to its cell wall through a receptor and then internalises the bacteria inside a vacuole (Phagosome). The phagosome then attaches with a lysosome which consists of several enzymes to form a phagolysosome. This process is called as maturation. Simultaneously, the phagolysosome is acidifed by the multi-subunit vacuolar ATPase (V-ATPase), which pumps hydrogen ions into the lysosomal compartment that activates the degradative enzymes of the lysosome. The internal of phagolysosome contains enzymes such as nucleases, proteases, lipases, glucosidase etc which aids in digestion of its content. Figure 2, provides the major steps involved in phagocytosis and autophagy.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggCgn5yu-B5DIhbgr_z9wTGxXpvuRYHs3HTrS7Jm6pHOiwaz68PWtL8bFlTx5E3Z3Ac_glKE_rEs7wkLgU0Bo9YmfPII9xf1BX7qkWbXCe7yDqQ5kbiQ_tNRnZrUP8g6TdS5t_EFsXMk9P/s1600/F2.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="653" data-original-width="506" height="640" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggCgn5yu-B5DIhbgr_z9wTGxXpvuRYHs3HTrS7Jm6pHOiwaz68PWtL8bFlTx5E3Z3Ac_glKE_rEs7wkLgU0Bo9YmfPII9xf1BX7qkWbXCe7yDqQ5kbiQ_tNRnZrUP8g6TdS5t_EFsXMk9P/s640/F2.jpg" width="492" /></a></td></tr>
<tr><td class="tr-caption"><b>Fig 2:</b> Major steps in phagocytosis and autophagy. <a href="https://ajp.amjpathol.org/article/S0002-9440(13)00027-8/fulltext">Source</a></td></tr>
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In classic textbook case scenario, mitochondria doesnt appear in the scene. In 2011, researchers showed experimental evidence indicating that the activation of <a href="http://varuncnmicro.blogspot.com/2015/03/greetings-cells-are-amazing-bag-of-self.html">Toll like receptors</a>- TLR1, TLR2 and TLR4 lead to recruitment of mitochondria to macrophage phagosomes and enhanced mitochondrial Reactive oxygen species (ROS) production. Subsequently a detailed work in 2015 by another group (lead by <span style="text-align: left;"><a href="https://medicine.umich.edu/dept/microbiology-immunology/mary-oriordan-phd">Mary O'Riordan</a></span>) discovered that inositol-requiring enzyme 1α (IRE1α), a protein from endoplasmic reticulum was an important factor in phagocytosis killing. Further analysis using ROS tracker<span style="font-family: Minion; font-size: 10pt; text-align: left;"> </span>chloromethyl 2',7'-dichlorodihydrofluorescein diacetate showed that in a normally functioning macrophage ROS migrated from mitochondria to the phagosome to achieve an effective killing. Most probably the process of engulfment turns on the stress signal which leads to mitochondrial synthesis of mROS (mitochondrial ROS), packed and sent into the phagosome.</div>
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<tr><td class="tr-caption" style="text-align: center;"><b>Figure 3:</b> MDVs delivers mROS to the phagosome.<br /><a href="https://www.cell.com/cell-host-microbe/pdfExtended/S1931-3128(18)30543-2">Source</a></td></tr>
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Most recently, this understanding has been further explored by <a href="https://medicine.umich.edu/dept/microbiology-immunology/mary-oriordan-phd" style="text-align: left;">Mary O'Riordan</a> and her team. The study followed up the previous findings indicating that the TLR dependent IRE1α induced mitochondrial ROS (mostly mitochondrial hydrogen peroxide) that are delivered to phagosomes via mitochondria derived vesicles (MDVs). This study basically identified Sod2 as a key enzyme involved in mH<span style="font-size: x-small;">2</span>O<span style="font-size: x-small;">2</span> production in a TLR dependent mechanism. The pathway was also demonstrated to be operational via Parkin/Pink1-dependent mitochondrial stress pathway.</div>
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Abuaita (the first author of the paper) comments, "We discovered that macrophages sense invading MRSA and turn on the machinery to increase mitochondrial development of ROS". O'Riordan who has been working on the role of mitochondria in enhancing phagosome function <a href="https://www.sciencedaily.com/releases/2018/11/181112131554.htm">comments on the published study</a>, "ROS are also damaging to our own cells, so we hypothesized that there must be some delivery mechanism. Mitochondria have not traditionally been known to package and deliver substances to different parts of the cell. The immune system is full of redundancies. It has to, by definition; every bacteria, virus, or parasite that we know is a pathogen is one because it has evolved ways to avoid the immune system. The immune system also has a real diversity of purpose and mechanism. Being open to different ways of asking questions about the immune system and understanding the biology of these pathogens helped us to find the right experimental system to use."</div>
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<b>References:</b></div>
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Smith LM, May RC. Mechanisms of microbial escape from phagocyte killing. <i>Biochem Soc Trans</i>. 2013 Apr;41(2):475-90. <a href="https://www.ncbi.nlm.nih.gov/pubmed/23514140">Link</a></div>
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Richards DM, Endres RG. The mechanism of phagocytosis: two stages of engulfment. <i>Biophys J</i>. 2014 Oct 7;107(7):1542-53. <a href="https://www.ncbi.nlm.nih.gov/pubmed/25296306">Link</a></div>
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West AP, Brodsky IE, Rahner C, Woo DK, Erdjument-Bromage H, Tempst P, Walsh MC, Choi Y, Shadel GS, Ghosh S. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. <i>Nature</i>. 2011 Apr 28;472(7344):476-80. <a href="https://www.ncbi.nlm.nih.gov/pubmed/21525932">Link</a></div>
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Abuaita BH, Schultz TL, O'Riordan MX. Mitochondria-Derived Vesicles Deliver Antimicrobial Reactive Oxygen Species to Control Phagosome-Localized Staphylococcus aureus. Cell Host Microbe. 2018 Nov 14;24(5):625-636.e5. <a href="https://www.ncbi.nlm.nih.gov/pubmed/30449314">Link</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-69194662799921991722018-03-30T18:22:00.000+05:302018-03-30T18:22:35.462+05:30BTB# 13: Dye dilution method for understanding cell proliferation<div dir="ltr" style="text-align: left;" trbidi="on">
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Cell proliferation assays, are now very commmonly used in a variety of research experiments. With techniques becoming more and more accurate, gone are the days where we use a simple counting of cells to understand the cellular proliferation. On a very broad sense, cellular proliferation assays are of various types including- Cell counting, Cell cycle measurement, BrdU/EdU uptake method, Dye dilution method. Of all these, in this post I will discuss about Dye dilution method since they provide great and easy measurements.</div>
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In dye dilution method, cells uptake a fluorescent dye which is stably sitting inside the cell. In an ideal scenario, they are neither metabolised or removed from the cell. When the cell divides, the fluorescent dye is distributed equally between the two daughter cells. This can be captured as a fluorescent signal in flow cytometry. On a best day, this method can trace 5-7 generations of cell divisions. How do you apply this? Let us say, for example your experiment involves understanding what happens to a B cell after 4 rounds of replication, after you have stimulated them. For that you need to first know that the B cell has undergone 4 rounds of division.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTsqeTWrwNGgbuzF6qGhq5HGX8RIWZO4AVGBqgLuVeHfkr5o95JK2HxrP_qAXY_paur_X1SRwrv2RSNqh_OjFQclLy0jzKWrAxiwovl3UrOIaYEnBkMjgcmcHZJr6oKVYiZowoCJzarKlW/s1600/Fig+1-+Cell+trace.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="240" data-original-width="560" height="136" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTsqeTWrwNGgbuzF6qGhq5HGX8RIWZO4AVGBqgLuVeHfkr5o95JK2HxrP_qAXY_paur_X1SRwrv2RSNqh_OjFQclLy0jzKWrAxiwovl3UrOIaYEnBkMjgcmcHZJr6oKVYiZowoCJzarKlW/s320/Fig+1-+Cell+trace.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Theoretical proliferation analysis. <a href="https://www.thermofisher.com/in/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-70/celltrace-far-red-cell-proliferation-kit.html">Source</a></td></tr>
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In Figure 1, the parent cell is in generation 0 (G0), which has taken up the dye fully (In practice, the exact concentraion of dye depends on the total dye used and concentration of cell in the mix). When the cell divides, the daughter cell (generation 1) has half the concentration of dyes. The 3rd generation of cell has 1/4th of the G0 and so on. This is reflected in Flow cytometry data, reduced intensity indicates subsequent generation.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhMUTSOsemvix5DHuYkdlQfmqOGSI9dxd4aqk-syE_LtFFrDRlQO1EcBjGbutL2LFg7cqjSApIGt9l3bLGquqcdLPjJEFL7BgULS1wRx8-6Xn0E7bxtk5fEN4IhkzVu4DgJlFKpfn_2jAdc/s1600/Fig+2.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="301" data-original-width="289" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhMUTSOsemvix5DHuYkdlQfmqOGSI9dxd4aqk-syE_LtFFrDRlQO1EcBjGbutL2LFg7cqjSApIGt9l3bLGquqcdLPjJEFL7BgULS1wRx8-6Xn0E7bxtk5fEN4IhkzVu4DgJlFKpfn_2jAdc/s200/Fig+2.jpg" width="190" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2:</b> Typical plot using<br />CellTrace Violet. <a href="https://bitesizebio.com/20880/cell-proliferation-round-2-and-beyond-the-dye-dilution-method/">Source</a></td></tr>
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In an example (Fig 2) where cells have been stimulated and left for 48 hours before analysis, shows the histogram representation of data analysis. The plot shows that cells have made a maximum of 4 divisions (shown in red). The unfilled histogram shows the starting population of undivided cells. Note that the 1st generation has higher concentration of dye than the 2nd and hence is more brighter (hence the peak towards right). The height of the peak represents number of cells.</div>
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There are several such dyes available in the market. Some of the most common dyes that you might have heard about include CellVue Claret, Cell Trace Violet, eFlour etc. The choice of dye is based on several parameters such as if It is taken up by live cells, Intensity of staining, cellular retention, segregation between daughter cells.</div>
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There are two major classes of these dyes, based on where the dye is retained by the cells. </div>
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1. Intracellular dyes</div>
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2. Cell membrane dyes (Also known as lipophilic dyes)</div>
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<tr><td class="tr-caption" style="text-align: center;"><b>Table 1:</b> Common Cell Proliferation Dyes. <a href="https://expertcytometry.com/4-critical-components-in-cellular-proliferation-measurement/">Source</a></td></tr>
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Intracellular proliferation dyes as the name suggest binds to intracellular contents. One of the most commonly used dye under this banner is the CFDA-SE (Carboxyfluorescein diacetate succinimidyl ester). CFDA-SE enters the cell where it is acted upon by cellular esterases which cleave the compound into <a href="http://www.bdbiosciences.com/ds/pm/tds/565082.pdf">CFSE</a> (Fluoerescent compound) which stays intracellularly by binding to intracellular lysine residues and other amine sources. However, the CFSE has a toxic activity and hence these days, <a href="http://flowcytometry-embl.de/wp-content/uploads/2016/12/Cell-proliferation.pdf">Cell Trace Violet</a> is more commonly used. Cell membrane dyes are the ones, which bind to cell membrane contents and give a fluoerescent reading. <a href="https://www.ncbi.nlm.nih.gov/pubmed/18161520">CellVue Claret</a>, a red dye is one of the best example in this category. Table 1 gives the most Common Cell Proliferation Dyes that are in current use.</div>
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Most recently, a method called as HCS (High content screening) is begining to turn out to be more sensitive when compared with dye method. The reason being they are more sensitive and superb signal-to-noise ratios than other methods, owing to their automated measurement of synthesis of new DNA. In reality this system is an improved version of BrdU/EdU uptake method using an automated platform.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-41063537791402795222018-03-20T14:19:00.007+05:302018-03-20T14:20:04.943+05:30Disease X: The term is very misleading<div dir="ltr" style="text-align: left;" trbidi="on">
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Greetings</div>
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It has been nearly more than 5 months since I last wrote a blog piece here all thanks to a busy schedule. There have been too many things that I wish I would have written about. Anyways, thanks to all those readers who enquired if I would still be writing or I have quit. Nope I havent quit. I just took a Mega break.</div>
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The latest sensation in the field of infectious disease is something called as "Disease X". This fancy and scary term is talked about in virtually every leading news outlet. After witnessing some of the most discussed Global emergencies such as SARS, MERS and Ebola Disease X is being projected as the next deadly pandemic in the making. So what exactly is Disease X? In its most essential sense, Disease X is a hypothetical infectious agent that is expected to cause a devastating outbreak at a global scale.</div>
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According to the WHO, "Represents the knowledge that a serious international epidemic could be caused by a pathogen currently unknown to cause human disease". Dr. Anthony Fauci, further clarfies, "As experience has taught us more often than not the thing that is gonna hit us is something that we did not anticipate. Just the way we didn't anticipate Zika, we didn't think there would be an Ebola that would hit cities." <a href="https://edition.cnn.com/2018/03/12/health/disease-x-blueprint-who/index.html">Source</a></div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgFJE8DTfhrk8x85X5kGxv8fyzfa8sn9vrBQ_qC40wk3gQ_vxaOGQj5drjUeUufX0ArGGs7TBbqgAhpzwlSXfNaD8mQ62Fla8ervmhsCl7YlJrMbfOBFQFlglZnNQM_1RiDAdvTNwkgK7No/s1600/Disease+X+Headlines.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="378" data-original-width="679" height="221" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgFJE8DTfhrk8x85X5kGxv8fyzfa8sn9vrBQ_qC40wk3gQ_vxaOGQj5drjUeUufX0ArGGs7TBbqgAhpzwlSXfNaD8mQ62Fla8ervmhsCl7YlJrMbfOBFQFlglZnNQM_1RiDAdvTNwkgK7No/s400/Disease+X+Headlines.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 1:</b> Disease X.</td></tr>
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In May 2015, the WHO was requested to come up with a plan for identifing potential pathogens of pandemic capabilities and how it could be handled and tackled. The first list tentative list was published on 10th Decembe 2015 based on expert opinions and understanding. This document formed the basis for "List of Blueprint priority diseases". The <a href="http://www.who.int/medicines/ebola-treatment/WHO-list-of-top-emerging-diseases/en/">orignial list</a> of disease priorities included Crimean Congo haemorrhagic fever, Ebola virus disease and Marburg, Lassa fever, MERS and SARS coronavirus diseases, Nipah and Rift Valley fever.</div>
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In February 2015, WHO <a href="http://www.who.int/blueprint/priority-diseases/RDBlueprint-PrioritizationTool.pdf">published a objective methodology</a> called as "Methodology for Prioritizing Severe Emerging Diseases for Research and Development", which is aimed at determining what agents should be classified as priority. This is mainly based on factors such as research and understanding already available, pandemic potential, immunity etc. Based on the assessment, a <a href="http://www.who.int/blueprint/priority-diseases/en/">second revision</a> of List of Blueprint priority diseases was released. The agents include</div>
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<li>Crimean-Congo haemorrhagic fever (CCHF)</li>
<li>Ebola virus disease and Marburg virus disease</li>
<li>Lassa fever</li>
<li>Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS)</li>
<li>Nipah and henipaviral diseases</li>
<li>Rift Valley fever (RVF)</li>
<li>Zika</li>
<li><b>Disease X</b></li>
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It should be noted that this list is not absolute and is more of a guideline representing where research has to be prioritsed for epidemic/pandemic preparedness. Disease X is an unknown entity. This could be anything that we havent identified yet to cause devastating infection.</div>
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Disease X is thus a term given to known unknow that could potentially cause significant problem. In other words, "We have no idea whats on the next in the list". Take into consideration, WHO committee science adviser <a href="https://globalnews.ca/news/4079589/disease-x-world-health-organization-global-pandemic/">John-Arne Rottingen says</a>, “It may seem strange to be adding an ‘X,’ but the point is to make sure we prepare and plan flexibly in terms of vaccines and diagnostic tests. We want to see ‘plug and play’ platforms developed which will work for any, or a wide number of diseases; systems that will allow us to create countermeasures at speed.”</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-87299865778919917492017-10-18T11:43:00.004+05:302017-10-18T11:43:52.721+05:30Usutu virus<div dir="ltr" style="text-align: left;" trbidi="on">
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At least according to me flavivirus members are one of the most difficult to understand classification. Thats because, they have so many different types of viruses by their characters and too many different types. Almost every virus in this group has at least a part of its life cycle in an animal. Once in a while they pop up in a limited geographical region and are noticed by the global infection research community.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEie_cCWXHukqVoPzvWlLxUtR1ISH8I-s6ZAyvBnckBp7R7XBfN1yaKeVRktIW-XdPCo0pcd2I-4SOilL2RCY720y8GpkcyV53v_osZviRdtZduATWdFWmu5hME14utBB-UlCL0pmFMGsFdl/s1600/Turdus+merula.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="670" data-original-width="1000" height="133" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEie_cCWXHukqVoPzvWlLxUtR1ISH8I-s6ZAyvBnckBp7R7XBfN1yaKeVRktIW-XdPCo0pcd2I-4SOilL2RCY720y8GpkcyV53v_osZviRdtZduATWdFWmu5hME14utBB-UlCL0pmFMGsFdl/s200/Turdus+merula.jpg" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 1:</b> Eurasian blackbird (<i>Turdus merula</i>).<br /><a href="http://www.hbw.com/ibc/species/eurasian-blackbird-turdus-merula">Source</a></td></tr>
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One such virus which has recently grabbed some attention is Usutu virus. Usutu virus (USUV) is a member of flavivirus closely resembling Japanese encephalitis complex. It is a Group IV member, with + single stranded RNA genome. It was first identified in South Africa in 1959. USUV first came into significant notice in 2001 when there was a massive black bird mortality in eastern Vienna, Austria. Subsequently, this virus has been identified in several geographical locations. But until now, USUV was thought to be limited to bird population, except for 4 confirmed cases in humans ever to be reported. In a surveillance study, in Austria, 54 of 208 individuals tested positive for USUV-neutralizing antibodies, thus indicating exposure.</div>
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<b>Fig 1:</b> Phylogenetic analysis of several members of the </div>
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JEV group and selected other mosquito-borne flaviviruses.</div>
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<a href="https://wwwnc.cdc.gov/eid/article/8/7/02-0094-f2">Source</a></div>
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As a part of blood screening program in Austria, samples from July to August 2017, 12047 blood samples from donors in eastern Austria was screened for West Nile virus (WNV) infection. Of these 7 samples were found to be positive for WNV by nucleic acid testing. WNV positive samples were follow up tested by WNV- and Usutu virus (USUV)-specific RT- and RT-qPCR assays for confirmation, which showed that of the 7 positive, 6 were USUV positive only one was WNV positive. This was further confirmed using sequence analysis. Interestingly, none of these donors had or later developed any clinical symptoms.</div>
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Norbert Nowotny from Austria who is an author in the most recent paper on USUV <a href="https://www.sciencedaily.com/releases/2017/10/171013103319.htm">comments</a>, "Flavivirus-positive blood donations, both West Nile and Usutu virus positives are discarded and consequently do not pose any risk to recipients of blood donations. However, there are a number of European countries, in which West Nile virus infections did not yet occur but in which Usutu virus circulates. In these countries blood donations may not be screened for flaviviruses. On the other hand, blood recipients are frequently immunocompromised persons, in which an Usutu virus infection may result in severe disease. To increase awareness of this possibility was one of the main goals of our second study".</div>
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Considering that USUV seropositivity and genetic evidence of virus in healthy blood samples are found without any associated clinical symptoms it currently appears unlikely that this virus is significantly causing a human infection. But, given its close genetic relatedness to JEV and WNV, the virus can make a jump anytime, which is proposed to be monitored.</div>
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<b>Reference:</b></div>
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Bakonyi T, Jungbauer C, Aberle S, Kolodziejek J, Dimmel K, Stiasny K et al. Usutu virus infections among blood donors, Austria, July and August 2017-Raising awareness for diagnostic challenges. <i>Eurosurveillance</i>. 2017;22(41).</div>
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Ashraf U, Ye J, Ruan X, Wan S, Zhu B, Cao S. Usutu Virus: An Emerging Flavivirus in Europe. <i>Viruses</i>. 2015;7(1):219-238.</div>
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Bakonyi T, Erdélyi K, Brunthaler R, Dán Á, Weissenböck H, Nowotny N. Usutu virus, Austria and Hungary, 2010-2016. <i>Emerg Microbes Infect</i>. 2017 Oct 11;6(10):e85. doi: 10.1038/emi.2017.72.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-51015437790607554382017-10-05T12:44:00.002+05:302017-10-05T12:45:18.482+05:30Nobel Awards- 2017<div dir="ltr" style="text-align: left;" trbidi="on">
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-jB3sfLGbIMYHknrzpKIQSyhyphenhyphenZJlYyF54nGwCsCzkgVUsxG8acnkqIuNQ5yG0vWJSMyn8KFvdGdbPiWTFPx96pCD-fUFJSS9NMcxokXd4E3TBrDRKAX0NbI-Oi7wJkfrnFKe4B427-PfV/s1600/1.png" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="492" data-original-width="500" height="196" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-jB3sfLGbIMYHknrzpKIQSyhyphenhyphenZJlYyF54nGwCsCzkgVUsxG8acnkqIuNQ5yG0vWJSMyn8KFvdGdbPiWTFPx96pCD-fUFJSS9NMcxokXd4E3TBrDRKAX0NbI-Oi7wJkfrnFKe4B427-PfV/s200/1.png" width="200" /></a><a href="https://www.nobelprize.org/">Nobel Prize</a> is considered as the most prestigious award for research accademics. The recipients of the award are chosen by the Nobel foundation constituted by Nobel committee of Royal Swedish Academy of Sciences, Nobel committee of Karolinska Institutet and Norwegian Nobel Committee. The award consists of a citation, gold medal and money. More than the award amount, the fame is considered as far superior.</div>
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<b>1. Physiology / Medicine:</b></div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicQ6kRhSv4GPCTNAW29pz-tvRdc6xLAfX_jWjLxcELTxq56kik1cHSMvJuvRSozi1z2YzxE-oNi7b9_uuuPS85w05yK3fG-fMJG8AtVxzdRuj0zgoL7z6ZsabCGBYkvEoijoRGrN6Ujsf9/s1600/Photo+1.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="399" data-original-width="630" height="202" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicQ6kRhSv4GPCTNAW29pz-tvRdc6xLAfX_jWjLxcELTxq56kik1cHSMvJuvRSozi1z2YzxE-oNi7b9_uuuPS85w05yK3fG-fMJG8AtVxzdRuj0zgoL7z6ZsabCGBYkvEoijoRGrN6Ujsf9/s320/Photo+1.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 1: </b>Michael Rosbash (left), Jeffrey Hall (centre)<br />
and Michael Young (right). <a href="http://www.nature.com/news/medicine-nobel-awarded-for-work-on-circadian-clocks-1.22736">Source</a></td></tr>
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The award goes to three scientists- Jeffrey Hall and Michael Rosbash (from Brandeis University Waltham, Massachusetts) and Michael Young (Rockefeller University) for their studies on <a href="https://en.wikipedia.org/wiki/Circadian_rhythm">circadian clocks</a>. Circardian rhythm in simplest terms is a endogenous, entrainable biological system which oscillates over a period of cycling time aproximately 24hrs. It is widely seen across species from cyanobacteria to humans. The field of study called as "Chronobiology" has been shown to have important affects including human health. The functioning of circardain rhythm has been shown to be strongly affected in conditions such as sleep disorders and several mental health disorders such as Schizophrneia.</div>
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<b>2. Physics:</b></div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEinvjAnuepWwZ_KQQZFGVL6hOpPQHWGoPjNMVW2uNOkWkPy-f-vQrdbigJrINz-X6Uru_Fklk6YxgQ1N0Ap_KRXmJKjr-h-DI26Ha7oYIIK1LoVi90_e-5Xc-A3JgwjR6T0dtp_gX5xQufJ/s1600/Photo+2.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="355" data-original-width="630" height="179" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEinvjAnuepWwZ_KQQZFGVL6hOpPQHWGoPjNMVW2uNOkWkPy-f-vQrdbigJrINz-X6Uru_Fklk6YxgQ1N0Ap_KRXmJKjr-h-DI26Ha7oYIIK1LoVi90_e-5Xc-A3JgwjR6T0dtp_gX5xQufJ/s320/Photo+2.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 2:</b> Rainer Weiss (left), Barry Barish (centre), and<br />
Kip Thorne (right). <a href="http://www.nature.com/news/gravitational-wave-detection-wins-physics-nobel-1.22737">Source</a></td></tr>
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This award has been announced to three scientists- Rainer Weiss (Massachusetts Institute of Technology, Cambridge), Barry Barish and Kip Thorne (California Institute of Technology, Pasadena) for their work on detection of gravitational waves at Laser Interferometer Gravitational Wave Observatory (LIGO). <a href="https://en.wikipedia.org/wiki/Gravitational_wave">Gravitational waves</a> are ripples in the curvature of space-time that are generated in certain gravitational interactions and propagate as waves outward from their source at the speed of light. This phenomenon was predicted in 1916 by Albert Einstein on the basis of his theory of general relativity. Basically, when 2 massive objects such as black holes collide with each other they distort the space time around which travels through space. The pattern of this wave helps understand several astrophysical phenomenon.</div>
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<b>3. Chemistry:</b></div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhTtELIqWsraLjw8P1AgUg1GySitn9V5YNkYt46bc4gGYkSNAtbXF70qGk-HfZo2aveCY6rJl7SPcTYMM1FNapt86NKCk70j_llpeGYI86hYTMtdkfFCoOfgxJ5Mq7O1229tkC1mdiGgnzy/s1600/Photo+3.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="401" data-original-width="780" height="164" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhTtELIqWsraLjw8P1AgUg1GySitn9V5YNkYt46bc4gGYkSNAtbXF70qGk-HfZo2aveCY6rJl7SPcTYMM1FNapt86NKCk70j_llpeGYI86hYTMtdkfFCoOfgxJ5Mq7O1229tkC1mdiGgnzy/s320/Photo+3.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Photo 3: Jacques Dubochet (left), Joachim Frank (centre)<br />
and Richard Henderson (right). <a href="https://www.theguardian.com/science/live/2017/oct/04/the-2017-nobel-prize-in-chemistry-to-be-announced-live">Source</a></td></tr>
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The award has been given to Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge) Joachim Frank (Colombia university), and Jacques Dubochet (University of Lausanne, Switzerland) helped to develop cryo electron microscopy. <a href="https://en.wikipedia.org/wiki/Cryo-electron_microscopy">Cryo-electron microscopy</a> or famously referred to as cryo-EM is a type of electron microscopy where the sample is studied at highly frozen temperatures. Cryogenic processing gets around the problem of treating the cells or molecules with several reagents, instead it could be seen in its native form. Cryo EM is now commonly used to study structure of viruses and there are extended 3D versions of the technique. Cryo EM- method is also slowly replacing X ray crytsallography method to study biomolecule structure.</div>
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Apart from the above Nobel Prize in Literature, Nobel Peace Prize and Sveriges Riksbank Prize in Economic Sciences in Memory of Alfred Nobel are given, for which the winners are yet to be announced as of at the time of writing this post.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-19797448120407318882017-09-30T19:11:00.003+05:302017-09-30T19:11:26.366+05:30Which viruses maybe seen in human semen<div dir="ltr" style="text-align: left;" trbidi="on">
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One of the least understood questions in virology in a recent turn of outbreaks like Ebola and Zika are that there are reports of these viruses being transmitted sexually. Now that we know that semen by itself possibly harbours a microbiome (<a href="http://varuncnmicro.blogspot.com/2016/04/semen-microbiome.html">Link</a>) there definitely is a good chance that many different microbes can harbour in reproductive tissues such as testis. But there is no comprehensive literature on all possible viruses infecting the male reproductive tract. </div>
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A meta-analysis which searched for all the literature available on evidence of virus presence in semen turned up 3,818 PubMed search results. Screening all the literature, the authors concluded that 26 viruses can survive in human semen which can seed a viremia. A summary is shown in Table 1.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgoPbFgGewNUDORWwos5081kaKfZgXFov5cutxK9wDXldILBPTgxwHAhE2dKjv-Cl8_Ivh3gVh1CXbH07km7I3uDwe9gpjGY3nvUtglc0kcYEq6090g7GkwFh91SurjFbFsd2IRFeTE1E0n/s1600/Table+1.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="593" data-original-width="840" height="450" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgoPbFgGewNUDORWwos5081kaKfZgXFov5cutxK9wDXldILBPTgxwHAhE2dKjv-Cl8_Ivh3gVh1CXbH07km7I3uDwe9gpjGY3nvUtglc0kcYEq6090g7GkwFh91SurjFbFsd2IRFeTE1E0n/s640/Table+1.png" width="640" /></a></td></tr>
<tr><td class="tr-caption"><b>Table 1: </b>Viruses that are capable of causing viremia and found in human semen. <a href="https://wwwnc.cdc.gov/eid/article/23/11/17-1049-t1">Source</a></td></tr>
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It should be noted that not all viruses listed have a defined capability to be sexually transmitted. But their biological effects on variables such as sexual health is not known. It is also known what is the duration of residence and how far they can seed viremia, serve as latency and what is the likely concentration. As <a href="https://www.geek.com/science/human-semen-can-carry-at-least-27-different-viruses-1717058/">Alex Salam puts it</a>, "Clinicians need to consider the possibility that traditionally non-sexually transmitted viruses can persist in semen, and this, therefore, raises the possibility of sexual transmission. Detection means that evidence of viral genetic material or viral protein was found in semen. It’s important to note that this does not mean that the virus is viable, i.e., capable of replicating. To prove this, the virus needs to be isolated and grown in cells or animals. For many of the viruses, this test has not been done, so we don’t know whether virus is viable or not."</div>
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This paper needs to be followed up with laboratory tests to establish the results. Further, this list is definitely not complete. But at least we have a more likely and possible catalogue of which viruses may be found in semen.</div>
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<b>References:</b><div>
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Salam AP, Horby PW. The breadth of viruses in human semen. <i>Emerg Infect Dis</i>. 2017. https://doi.org/10.3201/eid2311.171049</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-17213130161592880482017-09-29T18:18:00.000+05:302017-09-29T18:18:29.644+05:30Zika Virus- Update III<div dir="ltr" style="text-align: left;" trbidi="on">
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjYOlm-aIaE9gCz1UdAfujiaS-z-2Z2DL_YKzilTz_qCjJjEFjjpSagqPISOdidNYg3Nra_AHdsdlovI0DdeMAqlVtNA76cXS7_SDLd66zOqtyeXB6K6d095mqGDYW9i8pFEZsHlTtMUqRe/s1600/Zika.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="800" data-original-width="800" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjYOlm-aIaE9gCz1UdAfujiaS-z-2Z2DL_YKzilTz_qCjJjEFjjpSagqPISOdidNYg3Nra_AHdsdlovI0DdeMAqlVtNA76cXS7_SDLd66zOqtyeXB6K6d095mqGDYW9i8pFEZsHlTtMUqRe/s200/Zika.png" width="200" /></a></td></tr>
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<b>Photo 1:</b> Digitally-colorized TEM of</div>
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Zika virus. <a href="https://en.wikipedia.org/wiki/Zika_virus">Source</a></div>
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The last I wrote about Zika was almost more than an year ago (<a href="http://varuncnmicro.blogspot.com/2016/08/zika-infection-updates-ii.html">Link</a>). Ever since, literature on Zika Virus (ZIKV) has grown tremendously. It is too much to ask for a summary of everything that is known about zika to date. For all my posts on Zika, refer this <a href="https://varuncnmicro.blogspot.in/search/label/Zika%20virus">link</a>. In this post I will give the most recent updates of interest.</div>
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Zika virus has roughly a global distribution and 79 countries are in the spotlight. The geographical regions affected are categorised into <a href="http://www.who.int/emergencies/zika-virus/situation-report/classification-table.pdf">4 categories</a> (Category 1-4). Category 1 represent countries with new introduction of Zika virus since 2015 with ongoing transmission. Category 2 are areas with either the evidence of virus circulation before 2015 or with ongoing transmission of the virus that is no longer in the new or re-introduction phase, but where there is no evidence of interruption. Category 3 are regions with interrupted transmission and with potential for future transmission. Category 4, where the <i>Aedes aegypti</i> mosquito that spreads the disease exists, but there has been no reported transmission of the virus.<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhC2wO34kWMLgYTMRbUvRHGXGDVt0OyqOgibW3IWlOfGaYO6IUjz9VvdCVdkIsDScxXj1iQqeafMO2vAqQCrQ22Pzoo7yfzBwX8sJiNP6YyyXGBMsHyYvuaeIoGvmJic1fr47KOfAPUwZLs/s1600/Fig+1.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="500" data-original-width="750" height="426" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhC2wO34kWMLgYTMRbUvRHGXGDVt0OyqOgibW3IWlOfGaYO6IUjz9VvdCVdkIsDScxXj1iQqeafMO2vAqQCrQ22Pzoo7yfzBwX8sJiNP6YyyXGBMsHyYvuaeIoGvmJic1fr47KOfAPUwZLs/s640/Fig+1.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Global map of ZIKV infection categorised by country. <a href="https://www.thesun.co.uk/travel/3279778/world-map-reveals-countries-where-tourists-are-still-at-risk-of-contracting-zika/">Source</a></td></tr>
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There are 2 hypothesis on why the ZIKV infection has emerged. The first is that the ZIKV infection was indeed causing infection earlier. But there was no targeted testing to detect them. The other is genetic hypothesis. As per this view, genetic change might have resulted in emergence of a virus strain with greater epidemic potential and virulence, causing epidemics with more severe disease. In a just published study a team of researchers have explored this hypothesis. The team strated with analysis of genetic differences between modern Zika strain and an ancestral strain isolated from a patient in Cambodia in 2010. Based on the initial analysis, a single serine to asparagine substitution (S139N) in the viral polyprotein was predicted as important. This was then proven with a mouse model which showed that virus with the S139N mutation caused the most damage to neuronal cells. As the author of the paper <a href="https://www.scientificamerican.com/article/a-single-mutation-helps-modern-zika-cause-birth-defects/">Chen-Feng Qin comments</a>, "Besides host factors, such as low immunity to the virus in affected communities, there are definitely some other unknown viral proteins or amino acids that may contribute to the complex pathogenesis of microcephaly—independently or synergistically. Our study identified a unique genetic determinant that links to severe microcephaly".</div>
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The exact mechanism of pathogen invasion is not known. There is some indication that the virus targets neuronal progenitors in the developing brain. Research has suggested that AXL receptor tyrosine kinase is the cellular receptor for the virus. But most probably this is not the only receptor. Given that the ZIKV infection targets glial cells, researchers have tried to use <a href="https://www.sciencedaily.com/releases/2017/05/170519084107.htm">Zika strain to attack glioblastoma</a> in a laboratory model.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg1gKW5Z3N03_sCAP9AjCAIuISC7F3iRcLGN09BJM6FXy1QOv0ghReXyKthjpeRMF4YMlXr294npOcHXxX-8uOX4Zo0dJE1Cs7XqP2seKHbxJjuTklDlBj11kftDhmDatBOOuc6vockfFcF/s1600/Photo+2.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="360" data-original-width="522" height="219" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg1gKW5Z3N03_sCAP9AjCAIuISC7F3iRcLGN09BJM6FXy1QOv0ghReXyKthjpeRMF4YMlXr294npOcHXxX-8uOX4Zo0dJE1Cs7XqP2seKHbxJjuTklDlBj11kftDhmDatBOOuc6vockfFcF/s320/Photo+2.jpg" width="320" /></a></td></tr>
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<b>Photo 2:</b> PET brain images post-infection</div>
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with Zika virus. <a href="https://www.sciencedaily.com/releases/2017/09/170919164125.htm">Source</a></div>
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In a recent research using PET scanning (using a probe <span style="background-color: #fcfcfc; color: #333333; font-family: Georgia, serif; font-size: 17px; letter-spacing: 0.10199999809265137px; text-align: start;">[</span><span style="box-sizing: border-box; color: #333333; font-family: Georgia, serif; font-size: 12.75px; letter-spacing: 0.10199999809265137px; line-height: 0; position: relative; text-align: start; top: -0.5em; vertical-align: baseline;">18</span><span style="background-color: #fcfcfc; color: #333333; font-family: Georgia, serif; font-size: 17px; letter-spacing: 0.10199999809265137px; text-align: start;">F]</span> DPA-714) scientists have imaged the brain inflammation following Zika virus infection in mice. They found that levels of Zika virus in the mouse brain increased from day 3 to day 10 post-infection. During the period, the mice showed a 2- to 6-fold increase in global brain neuroinflammation. The study highlited that despite Zika affecting local brain tissue parts, the inflammation extends to all parts of the brain.<br />
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The focus of research currently is the cross reactivity between Zika and Dengue. Zika and Dengue are closely related viruses. Both the viruses show antibody enhanced infection and hence there is the question as to how does dengue vaccine affect Zika infection. There is no clear answer to this question. There is some opinion that dengue infection enhances zika infection. More recently, it has been suggested that antibodies against DENV E-dimer epitope (EDE)<span style="background-color: white; color: #333333; font-family: "arial" , "helvetica" , "ms pゴシック" , "ms ゴシック" , "osaka" , "ms pgothic" , sans-serif; font-size: 14.494999885559082px;"> </span>not only neutralizes the dengue virus in mice, but also protects both adults and fetuses from Zika infection by neutralisation. The different findings in various papers is probably based on the various types of targetting antibody that is tested.<br />
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Zika has gained strong interntational attention and there is a great focus on studying the Zika transmission, pathogenesis, treatment and vaccine.<br />
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<b>References:</b></div>
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Yuan etal. A single mutation in the prM protein of Zika virus contributes to fetal microcephaly. <i>Science (2017)</i>. DOI: 10.1126/science.aam7120<br />
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David Baud, Duane J Gubler, Bruno Schaub, Marion C Lanteri, Didier Musso. An update on Zika virus infection. <i>Lancet (2017)</i>. http://dx.doi.org/10.1016/S0140-6736(17)31450-2</div>
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Estefania Fernandez, Wanwisa Dejnirattisai, Bin Cao, Suzanne M Scheaffer, Piyada Supasa, Wiyada Wongwiwat, Prabagaran Esakky, Andrea Drury, Juthathip Mongkolsapaya, Kelle H Moley, Indira U Mysorekar, Gavin R Screaton, Michael S Diamond. Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection. <i>Nature Immunology</i>, 2017; DOI: 10.1038/ni.3849.</div>
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Kyle Kuszpit, Bradley S. Hollidge, Xiankun Zeng, Robert G. Stafford, Sharon Daye, Xiang Zhang, Falguni Basuli, Joseph W. Golden, Rolf E. Swenson, Darci R. Smith, Thomas M. Bocan. [18F]DPA-714 PET Imaging Reveals Global Neuroinflammation in Zika Virus-Infected Mice. <i>Molecular Imaging and Biology</i>, 2017; DOI: 10.1007/s11307-017-1118-2</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-82441388539998202252017-09-17T13:18:00.004+05:302017-09-17T13:19:03.777+05:30Compound isolated from C difficile act against C difficile: Avidocin-CDs<div dir="ltr" style="text-align: left;" trbidi="on">
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<i>Clostridium difficile</i> represents a <a href="http://varuncnmicro.blogspot.in/2014/01/clostridium-difficile-on-note.html">unique problem</a> in health care system. <i>C difficile</i> is not affected by a wide range of antibiotics and they start showing up when gut microbiome is depleted due to antibiotics. C difficile is often treated with antibiotics such as vancomycin, metronidazole and fidaxomicin which cause further disruption of the resident microbiota leading thus increasing chances of relapse. <a href="http://varuncnmicro.blogspot.com/2012/02/c-diff-poop-rescue.html">A faecal microbiome transplant</a> (FTM) is an option but it comes with some challenges. For example, It is nearly impossible to rule out every possible infection from a healthy donor and we are technologically incompetent yet to find the right best combination of microbiome for the gut. The best answer would be an antibiotic that kills only and only C diff. That means we have to be extremely specific.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgxjVcURK732gnRxEaFInQhO1kz_ksc_-htFxlZjIZ-toO3UChhE7JVHflOWHDku06RcgEar-ST0-5hRGfx3AAOw5YA5F-Ey3RolEMa-D50gIeaQxh_OGDoBeVjpIBfy885PoqrBvzbAaWL/s1600/Photo+1.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="217" data-original-width="279" height="155" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgxjVcURK732gnRxEaFInQhO1kz_ksc_-htFxlZjIZ-toO3UChhE7JVHflOWHDku06RcgEar-ST0-5hRGfx3AAOw5YA5F-Ey3RolEMa-D50gIeaQxh_OGDoBeVjpIBfy885PoqrBvzbAaWL/s200/Photo+1.jpg" width="200" /></a></td></tr>
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<b>Fig 1:</b> Electron micrograph of negatively stained</div>
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purified diffocin particles isolated from CD4 strain.</div>
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<a href="http://jb.asm.org/content/194/22/6240.full"></a><a href="http://jb.asm.org/content/194/22/6240.full">Source</a></div>
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<i>C difficile</i> is a highly competitive bacteria. <i>C difficile</i> strains compete with each other and they produce an R-type bacteriocin (High-molecular-weight or phage tail-like) which is called as diffocins. In a generalised sense, R-type bacteriocins attack target cells by specific receptor-binding on the surface. This is followed by sheath contraction and insertion of the core through the envelope of the target bacterium. The diffocins are ultra specific in their activity and their receptor binding activity can be modified to attack <i>C difficile</i> in general by modifying its binding to specific receptors. </div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjZYE4XcrycpgzIvBaTbioQiryI76XJI3_tlADjS2_K2hPPxjZP9HQKlL4Et9KT4A8TYubYfuAr6BSTR6dGA4u1nYcoFKDLi9lXJjsDoLVXtMjHMQU3jZuY3iiZb_-Ahck8sZUfDRuERIAA/s1600/Table+1.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="517" data-original-width="615" height="269" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjZYE4XcrycpgzIvBaTbioQiryI76XJI3_tlADjS2_K2hPPxjZP9HQKlL4Et9KT4A8TYubYfuAr6BSTR6dGA4u1nYcoFKDLi9lXJjsDoLVXtMjHMQU3jZuY3iiZb_-Ahck8sZUfDRuERIAA/s320/Table+1.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Table 1:</b> Sensitivity of various bacteria to modified diffocins.<br /><a href="http://www.icds.si/icds-2012/docs/Posters/P1_Gebhart_et_al.pdf">Source</a></td></tr>
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Table 1 shows a summary of the sensitivity of various bacterial strains to heterologously expressed, recombinant diffocins. A company that is currently working on using these diffocins and their modified version- <a href="https://www.avidbiotics.com/technology/avidocin-proteins/">Avidbiotics</a> Corp (California based Biotech company) have named them as Avidocin-CDs. One of such constructs Av-CD291.2 had been found to have wide spectrum activity against a range of hypervirulent <i>C difficile</i> strains tested, without affecting the microbiome.</div>
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There is sufficient literature evidence to indicate that avidocins are highly specific for C difficile and able to survive transit through mouse GI tract.</div>
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In the latest paper, the story becomes better now. The team of scientists from the University of Sheffield, AvidBiotics Corp, and the University of Glasgow have published a study on Avidocin-CD291.2 on how it could be extended for clinical use.<span style="background-color: white; color: #414042; font-family: 'Open Sans', sans-serif; font-size: 13px;"> </span>Analysis of rare Av-CD291.2–resistant mutants enabled identification of S-layer protein A (SlpA) as the target. The paper showed that Av-CD291.2–resistant mutants lack an S-layer. The lack of S-layer also introduces a high sensitivity to innate immune molecules combined with sporulation defects. These S-layer mutant strains survived poorly in the standard charcoal medium which is used to transport <i>C difficile</i> strains. Interestingly, acquisition of Avidocin-CD resistance results in loss of toxin production and complete loss of virulence.</div>
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As Dr Robert P. Fagan, senior corresponding author <a href="https://www.sciencedaily.com/releases/2017/09/170906144949.htm">comments</a>, "We discovered that the weapons naturally produced by <i style="text-align: justify;">C difficile</i> and those engineered by our colleagues at AvidBiotics were using certain proteins in the S-layer to identify which strains to target. The <i>C difficile</i> S-layer is unique to these bacteria, which explains why Avidocin-CD killing is so specific. Scientists at AvidBiotics Corp were then able to engineer different versions of Avidocin-CD to target 12 of the 14 known types of S-layer."</div>
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<b>Reference:</b></div>
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Joseph A. Kirk, Dana Gebhart, Anthony M. Buckley, Stephen Lok, Dean Scholl, Gillian R. Douce, Gregory R. Govoni, Robert P. Fagan. New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. <i>Science Translational Medicine</i>, 2017; 9 (406): eaah6813 DOI: <a href="http://dx.doi.org/10.1126/scitranslmed.aah6813">10.1126/scitranslmed.aah6813</a></div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-66379134291874650002017-09-05T18:57:00.001+05:302017-09-05T18:57:52.063+05:30Hypervirulent Klebsiella pneumoniae<div dir="ltr" style="text-align: left;" trbidi="on">
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One of the most common ideas associated with clinical antibiotic resistance is the fitness factor. I have in multiple posts talking about this idea in my previous posts. The idea is that when a bacteria acquired genes for antibiotic resistance there is a fitness cost associated with it. Until and unless there is this constant antibiotic threat to the bacteria, it is not so useful for the bacteria to keep resistance genes, since there is a cost associated with maintaining that gene. There are several experiments that showed at least for some drugs that if you mix a sensitive and resistant phenotype of a given organism and allow it grow together on an antibiotic free condition, the sensitive strain tends to dominate easily.</div>
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The idea is all fine and good in laboratory conditions. In a series of 5 hospital acquired pneumoniae infection (All fatal) following surgery in a Chinese hospital, a <i>Klebsiella pneuomoniae</i> ST11 strain has been isolated which is not only an MDR (Multi drug resistant) but also a hypervirulent strain.</div>
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The study, in summary, basically went something like this. There were 5 back to back pneumoniae cases following surgery for traumatic cases who developed pneumoniae and died of it. The first one in the series was identified as the most probable patient zero (Index case). The other four patients were located in different wards with overlapping stays. In all the cases multiple samples were taken and all the <i>Klebsiella pneumoniae</i> strains isolated were fully characterised using phenotypic and genetic approaches. In total 21 non-repeated carbapenem-resistant <i>K pneumoniae</i> strains were recovered from various clinical specimens of the five patients</div>
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What was really interesting to me was that they did a string test, human neutrophil assay and a wax moth virulence test to establish its hypervirulence status. These tests are really valuable models and so I think I should explain these in a little bit of detail.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgU_i4OSwqbB-kOzs-dbstoNNu5nzEyR3qqHFVN89xsOBm5oeWsboQyABYZn7u1Q88LSAtequnCJZvIr2u0-msdHiES90IilcHv5L1zpSBZ53j5Nd5rD-IU-SmJ3qwQhK9QlhwFxgiDYN0I/s1600/Photo+1.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="341" data-original-width="698" height="156" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgU_i4OSwqbB-kOzs-dbstoNNu5nzEyR3qqHFVN89xsOBm5oeWsboQyABYZn7u1Q88LSAtequnCJZvIr2u0-msdHiES90IilcHv5L1zpSBZ53j5Nd5rD-IU-SmJ3qwQhK9QlhwFxgiDYN0I/s320/Photo+1.jpg" width="320" /></a></td></tr>
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<b>Photo 1:</b> Positive “String test” on a hypervirulent</div>
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strain of K. pneumoniae. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654609/">Source</a></div>
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String test (Don't confuse this with the <a href="https://microbeonline.com/string-test-laboratory-diagnosis-vibrio-cholerae/">string test done to identify Vibrio cholerae</a>), is a kind of qualitative marker used to test the hypermucoviscous phenotype seen in <i>Klebsiella pneumoniae</i> which are an indicator or hypervirulence. Basically, you grow the organism on a 5% sheep blood agar at 37°C overnight. <span style="background-color: white; font-family: "times new roman" , "stixgeneral" , serif; font-size: 15.999099731445313px;">The string test is positive when a bacteriology inoculation loop or needle is able to generate a viscous string > 5 mm in length by stretching bacterial colonies on an agar plate. See Photo 1. In this paper, authors reported that t</span>he string test was positive for all five strains, each producing strings longer than 20 mm.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3G2Otd_QV1J3CjlNok7Sv_VpheIXDf3isGY1vV01fAjhOItv19TmM4mv0lnuGaY8JvB1wgGgYv1fZ-2ZFeKAnUgmBM_bPPsfsJjQT-Mso-C3S7E_JN21-VZZpwLeotEo5ejbJAWc5wQZP/s1600/Fig+1.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="496" data-original-width="475" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3G2Otd_QV1J3CjlNok7Sv_VpheIXDf3isGY1vV01fAjhOItv19TmM4mv0lnuGaY8JvB1wgGgYv1fZ-2ZFeKAnUgmBM_bPPsfsJjQT-Mso-C3S7E_JN21-VZZpwLeotEo5ejbJAWc5wQZP/s320/Fig+1.png" width="306" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2:</b> Neutrophil Survival of K pneumoniae strains.<br />
<a href="http://www.sciencedirect.com/science/article/pii/S1473309917304899">Source</a></td></tr>
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<span style="font-family: "times new roman" , "stixgeneral" , serif;"><span style="background-color: white; font-size: 15.999099731445313px;">Neutrophil assay is a test to see how good the bacteria resist killing by the neutrophil in laboratory conditions. </span></span>In principle, neutrophils obtained from healthy volunteers. A well containing 10⁶ neutrophils and 10⁶ CFU of opsonised <i>K pneumoniae</i> in RPMI/H medium is prepared at 37°C. A small sample is taken at intervals and bacteria is plated on Luria broth agar. Survival is calculated as a percentage of CFUs with reference to controls. For the study, <i>K pneumoniae</i> 4 and 5 (representative strains), two classic ST11 strains FJ8 and FJ9 (known to be not hypervirulent strains) and two known K1 hypervirulent <i>K pneumoniae</i> strains 1088 and 91 along with PC <i>K pneumoniae </i>which was removed for its virulence plasmid were studied. Fig 1 shows the results. You can clearly see that the strains 4 and 5 were really evading the neutrophil killing.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi1zfsRFk2_tym2gshlpolFyLZqoqH2FCvA1ZxxH5LtoPC1bMbba8fYSt425gmZPH1GIcJBlnnCTHdvXHo4NyARHldtWYfH589Ys5Ylr5vd3a1bBVEYjQ5BMjU-YRfU4PrRZCTf-xEUCkcK/s1600/Fig+2.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="586" data-original-width="471" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi1zfsRFk2_tym2gshlpolFyLZqoqH2FCvA1ZxxH5LtoPC1bMbba8fYSt425gmZPH1GIcJBlnnCTHdvXHo4NyARHldtWYfH589Ys5Ylr5vd3a1bBVEYjQ5BMjU-YRfU4PrRZCTf-xEUCkcK/s320/Fig+2.png" width="257" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2:</b> Virulence potential of isolated strains.<br />
<a href="http://www.sciencedirect.com/science/article/pii/S1473309917304899">Source</a></td></tr>
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Another test for virulence was done using <a href="https://en.wikipedia.org/wiki/Galleria_mellonella"><i>Galleria mellonella</i></a> or honeycomb moth. Basically, the bacteria are incubated with the larvae and you look for the ability of the larva to survive. For this study, cultures of <i>K pneumoniae</i> strains were washed with PBS and larva was infected with the bacteria and survival rate of the larvae was studied. See Fig 2. </div>
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The study does provide a compelling evidence that the strains they isolated were hypervirulent. Yes, they were also MDR strains but most of the <i>K pneumoniae</i> strains isolated globally are MDRs. But mind you they are not super bugs. I wasn't sure if these strains were resistant to drugs like Colistin and from the data they appear tigecycline sensitive.</div>
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I take this study as a proof that ST11 type which is a common circulating type in Asia is capable of hypervirulence. They have a 170 kbp plasmid (pVir- CR-HvKP4) which makes it hypervirulent, multidrug resistant, and transmissible. Considering so many people are dying from <i>Klebsiella pneumoniae</i> infections it may be worthwhile to test how many percentages of it is actually hypervirulent.</div>
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<b>Reference</b></div>
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Danxia Gu, Ning Dong, Zhiwei Zheng, Di Lin, Man Huang, Lihua Wang, Edward Wai-Chi Chan, Lingbin Shu, Jiang Yu, Rong Zhang, Sheng Chen. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. <i>The Lancet Infectious Diseases,</i> 2017. https://doi.org/10.1016/S1473-3099(17)30489-9</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-62354133856599294182017-08-17T14:26:00.001+05:302017-08-17T21:09:30.792+05:30Gonococcus superbug: Current Status<div dir="ltr" style="text-align: left;" trbidi="on">
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2xKJMWWFQcFCRPedbO9qoWOL0unh1srjk9eodx1JsFYs-55lwl9VcCq8dAyGiqfYcrP9L47X7UY1x-3ogHeXrVIeoY2V47hLw9RsD-Hi1Djgo_W49jCeny8j4ubLny-h8HKsuETFiSbG5/s1600/Neisseria+gonorrhoeae.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="424" data-original-width="525" height="161" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2xKJMWWFQcFCRPedbO9qoWOL0unh1srjk9eodx1JsFYs-55lwl9VcCq8dAyGiqfYcrP9L47X7UY1x-3ogHeXrVIeoY2V47hLw9RsD-Hi1Djgo_W49jCeny8j4ubLny-h8HKsuETFiSbG5/s200/Neisseria+gonorrhoeae.png" width="200" /></a></td></tr>
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<b>Photo 1:</b> Neisseria gonorrhoeae.</div>
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<a href="http://www.medical-labs.net/neisseria-gonorrhoeae-smear-from-urethral-discharge-2466/">Source</a></div>
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<i>Neisseria gonorrhoeae</i> or what is commonly known as the gonococcus is sounding serious alarms all over the globe. In a <a href="http://varuncnmicro.blogspot.in/2016/08/the-rise-of-superbug-gonococcus.html">post </a>almost an year ago, I talked about how gonococcus is slowly rising to the status of true superbug. Gonococcus is responsible for a sexually transmitted disease called as <a href="https://en.wikipedia.org/wiki/Gonorrhea">Gonorrhea</a>. Decades ago, this was absolutely treatable with a simple penicillin. However, they have now acquired several genes that makes it more untreatable.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuBaKN4-2VFsXNE26pjNVWFlepnLlKr9y28Wpdr_KlfN1hgzZBaYzMdZxYwQfZCT61kYvVZwfutpvEj3X2IMozmdY0xXFP2rXU3cTd2J-RUMzumOOhJk_cTnQfrgZyV-6k5qqS6hKMyC6q/s1600/journal.pmed.1002344.t001.png" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="180" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuBaKN4-2VFsXNE26pjNVWFlepnLlKr9y28Wpdr_KlfN1hgzZBaYzMdZxYwQfZCT61kYvVZwfutpvEj3X2IMozmdY0xXFP2rXU3cTd2J-RUMzumOOhJk_cTnQfrgZyV-6k5qqS6hKMyC6q/s320/journal.pmed.1002344.t001.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Table 1:</b> Resistance pattern of Gonococcus. <a href="http://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1002344">Source</a></td></tr>
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Here is the summary of current issue based on WHO reports in June. WHO has estimated that nearly 78 million people are infected with gonococcus. There are countries that are reporting their annual statistics and some countries that dont. The current recommended regimen for gonorrhea treatment is a combination of azithromycin and ceftriaxone. Table 1 shows a summary of number of countries in different WHO regions reporting gonococcal isolates with resistance to azithromycin and ciprofloxacin, and decreased susceptibility or resistance to extended-spectrum cephalosporin (Cefixime and/or ceftriaxone) for at least 1 year from 2009 to 2014. It should be noted that the report is based on data from 77 countries. Most African nations where the percentage of gonorrhea is expected to be higher has not reported. Interestingly, WHO has reported 3 confirmed cases of gonococcus that are resistant to all the anitbiotics tested. </div>
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“These are cases that can infect others. It can be transmitted. And these cases may just be the tip of the iceberg, since systems to diagnose and report untreatable infections are lacking in lower-income countries where gonorrhea is a ctually more common.”</div>
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<span style="text-align: justify;">-<a href="http://globalnews.ca/news/3581063/untreatable-superbug-gonorrhea-who/">Teodora Wi</a> (Department of Reproductive Health and Research, WHO)</span></div>
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“To control gonorrhoea, we need new tools and systems for better prevention, treatment, earlier diagnosis, and more complete tracking and reporting of new infections, antibiotic use, resistance and treatment failures. Specifically, we need new antibiotics, as well as rapid, accurate, point-of-care diagnostic tests – ideally, ones that can predict which antibiotics will work on that particular infection – and longer term, a vaccine to prevent gonorrhoea.”</div>
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-<a href="https://www.theguardian.com/society/2017/jul/07/untreatable-gonorrhoea-superbug-spreading-around-world-who-warns">Marc Sprenger</a>, (Director of antimicrobial resistance, WHO)</div>
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By now you understand that gonorrhea is a serious issue and needs to be addressed qucikly. Vaccine is an excellent choice. But vaccine research in gonorrhea isn't so great and there are no candidates in sight that are ready to be launched. Howewer, there is a silver lining.</div>
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A study was recently published which retrospectivly looked into vaccine effectiveness of outer membrane vesicle meningococcal B vaccine (MeNZB) in a case-control study of patients at sexual health clinics aged 15–30 years. They found that the gonorrhea rate among teens and young adults who had received a meningitis B vaccine during an emergency campaign in the early 2000s was significantly lower than the rate seen in people of the same age who weren’t vaccinated. The estimated vaccine effectiveness of MeNZB against gonorrhoea was about 31%. Of course its a chance observation and some form of clinical trial needs to be done to have more credibitily for the claim and get a more realistic estimate. There are also similar reports of decline in gonorrhea rates follwing meningococcal vaccine in Cuba, and Norway.</div>
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There is a lot of effort currently in trying to come up with new antibiotics especially ones that can target such superbugs. A compound referred to as closthioamide has been shown to have promising results against drug resistant gonococcus.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: justify;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhDfSTZmNP7kCLxC6w4DNpPQB8YjRAahdVSXtHi0I0ls3RAzi1kpQnqvhZfktHvQbU-Fvxtp1o7rTuiUSY6CKXYipcviU4GmTYfCpQCRzDRXRJc_TH4-n8GUPzx1L7I1DInnZFgj6EA_xQg/s1600/Fig+1.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="362" data-original-width="1600" height="72" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhDfSTZmNP7kCLxC6w4DNpPQB8YjRAahdVSXtHi0I0ls3RAzi1kpQnqvhZfktHvQbU-Fvxtp1o7rTuiUSY6CKXYipcviU4GmTYfCpQCRzDRXRJc_TH4-n8GUPzx1L7I1DInnZFgj6EA_xQg/s320/Fig+1.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Structure of Clostioamide.<br />
<a href="http://www.google.com/patents/US8673980">Source</a></td></tr>
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Closthioamide (CTA) was first discovered and isolated from Anaerobic Bacterium Clostridium cellulolyticum in 2010 which was initially tested as a possible compound against multi drug resistant Staphylococcus. It functions through attacking DNA gyrase. In brief, the researchers tested 149 strains isolated from patients, 8 WHO reference strains of of N. gonorrhoeae and 4 commensal Neisseria strains were tested. CTA performed really well against 146/149 (98%) of clinical gonococcal strains at ≤0.125mg/L. The study also noted that<span style="font-family: "arial"; font-size: 9pt; text-align: left;"> two</span> <i>N. perflava</i> strains had the highest CTA MIC (>1 mg/L).</div>
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As the senior author of the study <a href="https://www.sciencedaily.com/releases/2017/08/170807133129.htm">John Heap comments</a>, "The imminent threat of untreatable antibiotic-resistant infectious diseases, including gonorrhoea, is a global problem, for which we urgently need new antibiotics. This new finding might help us take the lead in the arms race against antimicrobial resistance. We believe there are many undiscovered antibiotics out there in nature, but they are difficult to find and test. For example, the bacteria which produce closthioamide naturally make only tiny amounts that are not enough to test or use, so we had to chemically manufacture it ourselves by mimicking its natural structure. The next step will be to continue lab research to further assess the drug's safety and effectiveness. Despite showing tremendous promise, it will be a number of years before, and if, we can use the drug in real life human cases."</div>
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As of now there is no clear answer as to how to tackle the globally spreading true superbug "gonococci" is to be controlled. Perhaps the best method is the same as standard STD prevention methods.</div>
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<b>References:</b><br />
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1. Wi T, Lahra M, Ndowa F, Bala M, Dillon J, Ramon-Pardo P et al. Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action. <i>PLOS Medicine</i>. 2017;14(7):e1002344. </div>
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2. Petousis-Harris H, Paynter J, Morgan J, Saxton P, McArdle B, Goodyear-Smith F et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. <i>The Lancet</i>. 2017; doi: 10.1016/S0140-6736(17)31449-6. [Epub ahead of print]</div>
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3. Miari V, Solanki P, Hleba Y, Stabler R, Heap J. In vitro susceptibility to closthioamide among clinical and reference strains of Neisseria gonorrhoeae. <i>Antimicrobial Agents and Chemotherapy</i>. 2017;:AAC.00929-17.<br />
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4. Lincke T, Behnken S, Ishida K, Roth M, Hertweck C. Closthioamide: An Unprecedented Polythioamide Antibiotic from the Strictly Anaerobic Bacterium Clostridium cellulolyticum. <i>Angewandte Chemie</i>. 2010;122(11):2055-2057.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-53881260827451801832017-08-09T13:01:00.004+05:302017-08-09T13:01:55.937+05:30Scientists find a quick method to get Monoclonal Antibodies of interest.<div dir="ltr" style="text-align: left;" trbidi="on">
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A lot of new emerging infectious diseases are no on global radar and that highlights how unprepared we are in fighting it. Most of these are geographically limited and terminate with a few countable number of cases. The one's like Influenza variants, Zika and Ebola have been more rampant. Though a short term solution is to administer antibiotics or quarantine, the best approach is to vaccinate. The centre of this whole problem lies in B cells.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: justify;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgeE54Zwquv_XXvZ4dQL5CBW2RAF9PDex_Ni7tlYXQ3uanBQxT-izXpsq5fEVwabDIZ3OkLRGDg7V0JuwYM2BT23VxaXuG0C1BvdsfbJh4dVwzuCyj7e0xW1-i1ep1KguJx7VuJq0O457s4/s1600/Fig+1.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="358" data-original-width="441" height="259" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgeE54Zwquv_XXvZ4dQL5CBW2RAF9PDex_Ni7tlYXQ3uanBQxT-izXpsq5fEVwabDIZ3OkLRGDg7V0JuwYM2BT23VxaXuG0C1BvdsfbJh4dVwzuCyj7e0xW1-i1ep1KguJx7VuJq0O457s4/s320/Fig+1.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> B cell activation. Source: Kuby Textbook; 5e</td></tr>
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I need to visit back the B cell activation pathway. B cells are a type of lymphocytes that is involved in making antibodies. B cells by nature are inactive and have to be activated specifically. The individual lineage of B cells can make antibodies against a specific antigen. These inactive B cells which are competent enough to start making antibodies, provided they have the right signal, is called as immunocompetent B cells.</div>
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Depending on the nature of the antigen, there are two modes of B-cell activation. TH cells dependent (TD) and TH cell independent (TI). There are 2 types of signals that are required as membrane events, to activate a B cell. The first activation signal is an antigen binding to B cell receptors (BCRs). Once bound, the antigen is internalized by receptor-mediated endocytosis, digested, and complexed with MHC II molecules on the B cell surface. The second activation signal (also referred as the costimulatory signal) is CD40/CD40L interaction. See Fig 1. Once activated they go on to mature and convert to B cells which start making antibodies. There is some evidence that this is not completely true and there are more signals in the interaction. For example, <a href="http://varuncnmicro.blogspot.com/2015/03/greetings-cells-are-amazing-bag-of-self.html">TLR</a> is possibly the third signal. </div>
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There have been several previous attempts in the laboratory to replicate this process in the laboratory condition. Previous studies have shown that patient-derived B cells when treated with CpG oligonucleotides, they stimulate every B cell in the population. <a href="https://en.wikipedia.org/wiki/CpG_Oligodeoxynucleotide">CpG oligonucleotides</a> are short single-stranded synthetic DNA molecules that contain a cytosine triphosphate deoxynucleotide followed by a guanine triphosphate deoxynucleotide. The CpG is mainly recognised by TLR 9, which is expressed in B cells and Plasmacytoid dendritic cells and is thus an excellent immunostimulant. Antigen-dependent activation of B cells in-vitro is difficult to achieve result because the wide haplotype variation of MHC IIs necessitates the use of unique T cells specific to a particular MHC II to activate B cells in vitro. This problem was solved by the team led by Facundo Batista, from the Francis Crick Institute in London based on which the current paper is built.<br />
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The researchers started with coated streptavidin polystyrene nanoparticles containing a mixture of biotinylated anti-κ antibody and the TLR ligand CpG. The team showed that by treating the patient derived B cells with the coated nanoparticles and the appropriate antigen. In short, CpG oligonucleotides are only internalized into those B cells that recognize the specific antigen coated, and these cells are therefore the only ones in which TLR9 is activated to induce their proliferation and development into antibody-secreting plasma cells. </div>
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The team has shown that the results are replicable when done with different bacterial and viral antigens (such as tetanus toxoid and proteins from several strains of influenza A, HIV gp120). The studied showed specifically that in vitro stimulation of memory B cells with particulate antigen-CpG selectively enriched the frequency of CD27hi/CD38hi antigen-specific plasma cells irrespective of the nature of the antigen that was chosen. So technically in proof, this method can be applied to any antigen. Further, it was achieved in a very short time, almost a week.</div>
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This novel method is much superior to several other methods such as phage display, EBV immortalization, yeast display, and humanized animal models since it doesn't rely on a large scale screening and identification. Basically, this method allows for selective stimulation of memory B cells from healthy donors, leading to proliferation and differentiation into plasma cells that produce antigen-specific antibodies, even in antigen-naive donors (They demonstrated by showing you could develop antibodies against HIV from cells derived from HIV negative donors), which means making therapeutic vaccines (Antibodies) could be very fast.</div>
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As <a href="http://www.medicalnewstoday.com/articles/318569.php">Facundo Batista explains</a>, "Specifically, it should allow the production of these antibodies within a shorter time frame in vitro and without the need for vaccination or blood/serum donation from recently infected or vaccinated individuals. In addition, our method offers the potential to accelerate the development of new vaccines by allowing the efficient evaluation of candidate target antigens."</div>
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<b>References:</b></div>
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Irene Sanjuan Nandin, Carol Fong, Cecilia Deantonio, Juan A. Torreno-Pina,Simone Pecetta, Paula Maldonado, Francesca Gasparrini, Jose Ordovas-Montanes, Samuel W. Kazer, Svend Kjaer, Daryl W. Borley, Usha Nair, Julia A. Coleman, Daniel Lingwood, Alex K. Shalek Eric Meffre, Pascal Poignard, Dennis R. Burton, and Facundo D. Batista. Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies. <i>The Journal of Experimental Medicine</i>, 2017 DOI: 10.1084/jem.20170633.</div>
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Eckl-Dorna J, Batista F. BCR-mediated uptake of an antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation. <i>Blood</i>. 2009;113(17):3969-3977.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-82602156106449076022017-07-25T17:15:00.000+05:302017-07-25T17:15:08.778+05:30Dopamine says "Make antibodies"<div dir="ltr" style="text-align: left;" trbidi="on">
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjcWK07GSvBrCGGawt8JHRXQBVOgg4Sf1wcYj_iFtLzpzTGX3Ua9d1Yru-8A5mThapGVAslWPFbbrNKk6nNpDRWQtVmmMUcwgQFz3dJczKtDGA2a2D3HNvnCjp8fA5R9nD2INjEMSxT4Rhw/s1600/Screen+Shot+2017-07-23+at+11.48.23+AM.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="521" data-original-width="454" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjcWK07GSvBrCGGawt8JHRXQBVOgg4Sf1wcYj_iFtLzpzTGX3Ua9d1Yru-8A5mThapGVAslWPFbbrNKk6nNpDRWQtVmmMUcwgQFz3dJczKtDGA2a2D3HNvnCjp8fA5R9nD2INjEMSxT4Rhw/s200/Screen+Shot+2017-07-23+at+11.48.23+AM.png" width="172" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Photo 1: </b>Lymphatic system<br />
in brain. <a href="http://www.nature.com/nature/journal/v523/n7560/full/nature14432.html">Source</a></td></tr>
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For a long number of years nervous system and immunological system have been seen as two separate systems. In the last couple of decades this idea has been strongly questioned. Central nervous system which has been assumed to be devoid of immune activity is now known to harbour immune system of its own (<a href="http://varuncnmicro.blogspot.com/2016/06/btb7-immune-cells-of-central-nervous.html">Link</a>). Their location was quite close to prominent blood vessels. There has been some proof that neural system could in part regulate neural activity also (<a href="http://varuncnmicro.blogspot.com/2014/12/greetings-lot-of-time-have-i-talked.html">Link</a>). In 2015, <a href="http://www.nature.com/nature/journal/v523/n7560/full/nature14432.html">Louveau et al</a> reported that the brain has a lymphatic system of its own. The anatomical discovery was surprising since the vessels’ were hidden in a location deep within the brain. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), is a marker of lymphatic system. LYVE1 immunostaining of whole-mount meninges is shown in Photo 1 showing distribution of brain lymphatics.</div>
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There are several papers that have established that neuroimmune interactions are 2 way. Classical nervous system molecules such as dopmaine can act on immune cells. For example, T cells express several dopamine receptors (DARs). It has been established that stimulation of specific DARs on Dendritic cells and T cells, influence CD4+ T cell differentiation into Th1 or Th17 inflammatory cells. Dopamine receptors are universally expressed in T cells, dendritic cells (DCs), B cells, NK cells, neutrophils, eosinophils, and monocytes. Fig 1, shows an example of established pathways on how neurotransmitters affect T cell response. <span style="text-align: left;">Ofcourse, the reverse is also true, though the phenomenon is less well studied. A really good example is cytokines. IL-6 is now highly implicated as a Neuropoetin (Stimulate Neuronal growth), though mechanism is not clearly defined.</span></div>
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEitqz8PG1kYFY6IHj4UzM4Y7gQ_fLtAXDABvAN6imk5swzPoG3kpv8LWPX4OJ03VdMozc4TqnokF4vqy2KkK_jBmWlnk5boTKu-SXXc9EPrZnfnFxa0F54NAfO6h9DAokSjN0NyZLVQefR9/s1600/Fig+1.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="434" data-original-width="995" height="276" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEitqz8PG1kYFY6IHj4UzM4Y7gQ_fLtAXDABvAN6imk5swzPoG3kpv8LWPX4OJ03VdMozc4TqnokF4vqy2KkK_jBmWlnk5boTKu-SXXc9EPrZnfnFxa0F54NAfO6h9DAokSjN0NyZLVQefR9/s640/Fig+1.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Neurotransmitter-mediated regulation of T cell response. <a href="https://www.intechopen.com/books/cell-interaction/cells-molecules-and-mechanisms-involved-in-the-neuro-immune-interaction">Source</a></td></tr>
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So far, so interesting. There is convincing evidence that dopamine receptor is important. But having a receptor is one thing. There has been some research earlier suggesting that the immune cells can themselves also make dopamine and it has not been clear as to what is the role. And thats the new story.</div>
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Making an antibody is a very tightly regulated process. When the immune system encounters a foriegn molecules the T cells delivers signal to B cells in a complicated molecular process. In germinal centres, highly mobile T cells and B cells specific for the same pathogen can directly interact with each other through the formation of dynamic specialized surface structures called T–B immunological synapses. In a new study by Papa etal, showed that the <span style="text-align: start;">Follicular helper T cells or TFH (They are also known as Follicular B helper T cells)</span> use dopamine as cargo loader for immune molecules which mediates a T-B synapse which triggers B cell maturation.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnCpb1fpUIyy2F0yjMeK0WjiuQt2Z3DsFXeFbT6ggyuqPcexGrLr0sR-w9S_Vey2Pz-vehFk31wsKjspq3m7g-E2g4wcCaGLtKcVfjb5x2A4jD5pgI_vx56A5uWjlAH3_teNAwKubz_3ld/s1600/Fig+2.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="346" data-original-width="534" height="207" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnCpb1fpUIyy2F0yjMeK0WjiuQt2Z3DsFXeFbT6ggyuqPcexGrLr0sR-w9S_Vey2Pz-vehFk31wsKjspq3m7g-E2g4wcCaGLtKcVfjb5x2A4jD5pgI_vx56A5uWjlAH3_teNAwKubz_3ld/s320/Fig+2.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2:</b> Graphic model of the proposed positive feedback<br />
between human TFH and germinal centre B cells.<br />
<a href="https://www.nature.com/nature/journal/v547/n7663/full/nature23013.html">Source</a></td></tr>
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The first experiment goes on to show that Chromogranin B (CGB) is present in human tonsils, spleens, and lymph nodes and determined that the T cells isolated from these samples icontained granules filled with dopamine. Using a method called live-cell RNA detection CGB was shown to be present in high quantities of human germinal centre TFH cells but not so much in other T cells. Subsequent experiments showed that dopamine was not really there in detectable amounts in cells other than TFH cells. Next set of experiments showed that human TFH cells released dopamine on stimulation by germinal-centre B cells and it upregulated the ICOSL on the cell surface of germinal-centre B cells. This process was also shown to enhance accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. The tests also showed that the process could be blocked by haloperidol and a DRD1 specific antagonist SKF83566, thus narrowing down the receptor to DRD1. Based on the experimental findings, the authors proposed an interaction model, shown in Figure 2. According to an explanatory accompanying paper, it has been hypothesised that TFH cells have a very stringent requirement for efficiency and specificity at the immunological synapse, which explains the finding that dopamine is used by human TFH cells, but not by human T cells of other subclasses.</div>
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So now I have some questions. Is there a possible mechanism where dopamine from a neuron stimulates B cells? Does this finding explain why in certain dopamine related disorders such as schizophrenia (where there is presumably an increased dopamine actvity) have an increased autoimmune phenomenon. As Hai Qi speculates, "When disease characteristics or treatment options are associated with changes in dopamine, the possible involvement of, and implications for, antibody- mediated immunity should be considered".<br />
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As Papa the <a href="http://neurosciencenews.com/immune-system-brain-activity-7074/">lead author comments</a>, “These particles were previously thought to only exist in neurons in the brain and we think they are, potentially, an excellent target for therapies to speed up or dampen the body’s immune response, depending on the disease you’re dealing with. Like neurons, specialised T cells transfer dopamine to B cells that provides additional ‘motivation’ for B cells to produce the best antibodies they can to help to clear up an infection. The human body has developed an advanced form of protection against bacteria, viruses and other foreign bodies that relies on the immune system".</div>
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<b>References:</b><br />
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Papa I, Saliba D, Ponzoni M, Bustamante S, Canete P, Gonzalez-Figueroa P et al. TFH-derived dopamine accelerates productive synapses in germinal centres. <i>Nature</i>. 2017;547(7663):318-323.</div>
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Qi H. Immunology: Nervous crosstalk to make antibodies. <i>Nature</i>. 2017;547(7663):288-290.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-38479709453974571372017-07-21T10:44:00.000+05:302017-07-22T10:01:16.264+05:30nCD64 as a marker of Sepsis<div dir="ltr" style="text-align: left;" trbidi="on">
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Several times in my blogs, I have talked about how important it is to make a diagnosis at the fastest turn around time possible. In an attempt to miniaturise the testing platform and obtaining faster results, several technologies have been tested. In context with infections, genome detection and sequencing based technologies are increasingly becoming better and more accessible. Another example is pathogen specific molecular marker detection method on which a good lot of R&D is invested. MALDI-TOF is an excellent example.</div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg-jK8-qeJsT8NPCl2G8126XOp4OXXsmbkT18OI2mE-jSeF9CEYj_xlXXAuvFV7jejVpblWYQmEAigcMXMz0LhP6nffvk-SKd3pU1q0FD3EQZJZ05PDopZM74c0OEzOjzfNfMthorL1Y8S0/s1600/Fig+1.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="646" data-original-width="960" height="215" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg-jK8-qeJsT8NPCl2G8126XOp4OXXsmbkT18OI2mE-jSeF9CEYj_xlXXAuvFV7jejVpblWYQmEAigcMXMz0LhP6nffvk-SKd3pU1q0FD3EQZJZ05PDopZM74c0OEzOjzfNfMthorL1Y8S0/s320/Fig+1.png" width="320" /></a></td></tr>
<tr><td class="tr-caption"><div style="text-align: center;">
<b>Fig 1:</b> Hospitalisation rates for sepsis or septicemia.</div>
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<a href="https://www.cdc.gov/nchs/products/databriefs/db62.htm">Source</a></div>
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Sepsis is a serious issue. Any clinical microbiologist who works in association with the hospital knows the seriousness of sepsis. The terms "Sepsis" and "Septicemia" both refer to a bloodstream infection. Though in a strictly technical sense they mean two different things, they have been interchangeably used in literature and was widely accepted as similar. The earlier definition of sepsis was based on the idea that it is a systemic response and was thus assessed using a systemic inflammatory response syndrome (SIRS) criteria. To date, there is no clear definition of what sepsis is though it is generally agreed that it means circulating pathogen in blood. The diagnosis is based on evidence of fever, respiratory rate and abnormal total WBC count followed by bacterial identification from blood culture. There are no global estimates of sepsis prevalence. Available estimates suggest a range of <1% in a population. However, there is a significant trend observed everywhere as shown in Fig 1.</div>
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In most parts of the globe, a prediction of sepsis is made based on markers such as C reactive protein and procalcitonin levels. Many studies have attempted to come up with a marker. Some of the well-researched markers of sepsis include <span style="background-color: white; font-family: "times new roman" , "stixgeneral" , serif; font-size: 15.999099731445313px; text-align: left;">triggering receptor expressed on myeloid cells-1 (TREM-1), </span><span style="background-color: white; font-family: "times new roman" , "stixgeneral" , serif; font-size: 15.999099731445313px; text-align: left;">azurocidin, </span><span style="background-color: white; font-family: "times new roman" , "stixgeneral" , serif; font-size: 15.999099731445313px; text-align: left;">CD64, CD11b etc.</span></div>
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<tr><td class="tr-caption"><b>Fig 2:</b> Process schematic of the differential expression-based<br />
cell-counting technology. <a href="https://www.nature.com/articles/ncomms15949">Source</a></td></tr>
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Studying these markers in the laboratory is not the big deal, since instruments such as Flow cytometers and other sophisticated equipments can do it. But they are not ideal for POCT (Point of care testing). In 2015, this problem was addressed by developing a POCT equipment based on <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761450/">microfluidics</a>. The same group has now come up with improvements in design. The microfluidic biochip is capable of enumerating leukocytes and quantify neutrophil CD64 (nCD64) levels from 10 ml of whole blood without any manual processing. The tech uses whole blood (10ml) which is pumped into the biochip along with lysing and quenching buffers, to lyse erythrocytes. Cells are electrically counted and differentiated based on size using microfabricated electrodes. The CD64+ cells get captured based on their CD64 expression level. The difference in the cell counts is used to calculate nCD64 expression level. See Fig 2.<br />
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The authors claim that this technology can have profound results since the assay takes about 30 min and has scope for further improvement. That would be something really usefull to clinicians as a bedside tool for identifying sepsis.</div>
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<b>References:</b><br />
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Mervyn Singer et al. The Third International ConsensusDefinitions for Sepsis and Septic Shock (Sepsis-3). <i>JAMA</i>. 2016;315(8):801-810. doi:10.1001/jama.2016.0287</div>
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Mayr F, Yende S, Angus D. Epidemiology of severe sepsis. <i>Virulence</i>. 2013;5(1):4-11.<br />
<br />Wang X, Li ZY, Zeng L, Zhang AQ, Pan W, Gu W, Jiang JX. Neutrophil CD64 expression as a diagnostic marker for sepsis in adult patients: a meta-analysis. <i>Crit Care</i>. 2015 Jun 10;19:245. doi: 10.1186/s13054-015-0972-z.</div>
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Hassan U, Reddy B Jr, Damhorst G, Sonoiki O, Ghonge T, Yang C, Bashir R. A microfluidic biochip for complete blood cell counts at the point-of-care. <i>Technology (Singap World Sci)</i>. 2015 Dec;3 (4):201-213. DOI: 10.1142/S2339547815500090</div>
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Hassan U et al. A point-of-care microfluidic biochip for quantification of CD64 expression from whole blood for sepsis stratification. <i>Nat Commun</i>. 2017 Jul 3;8:15949. doi: 10.1038/ncomms15949.</div>
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Varun C Nhttp://www.blogger.com/profile/16780683018907433029noreply@blogger.com0tag:blogger.com,1999:blog-5697433480702397522.post-70463520548403501942017-07-03T10:20:00.000+05:302017-07-03T10:20:43.851+05:30Lab series #17: Labelling methods for Quantitative Proteomics by MS<div dir="ltr" style="text-align: left;" trbidi="on">
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In an earlier post, I have talked about the principle of how a mass spectrometry works (<a href="http://varuncnmicro.blogspot.com/2016/06/lab-series-13-mass-spectrometry.html">Link</a>) and how proteomics by sequencing is done using MS (<a href="http://varuncnmicro.blogspot.in/2016/07/lab-series-14-ms-based-proteomics.html">Link</a>). I had a few readers who suggested the idea that I have talked about MS-based shotgun sequencing but proteomics could be done even without sequencing. For example, MALDI-TOF analysis can tell about the protein identity which doesn't involve sequencing. This is absolutely true. However, such assays are now nearly outdated and sequence information can give us a lot more insight than just predicting protein based on the m/z values. In the earlier post, I ended with a note saying that I will revert back to the topic and talk about proteogenomics, targeted proteomics and quantitative proteomics. In this post, I will talk about labelling methods for quantitative proteomics or sometimes referred to as differential proteomics. If you have not read my earlier posts on MS, I strongly recommend that you read them first.</div>
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Let us build an example scenario. You want to learn what are the changes that occur in the cell after a virus infection. The most likely scenario in terms of proteome would be certain proteins will have increased expression and certain will have decreased expression, as a result of interaction with a virus. If you could find out what those proteins are, then there is a good chance that you could predict the pathways that have been disrupted. But for identifying what is the fold change, we have to quantify each protein. In a traditional assay like quantitative ELISA, the protein is directly estimated using a set of standards and then plot a graph. In proteomics, several thousand proteins are estimated in a single run and hence it is not practical to have several standards for every individual protein. MS technique is originally designed to be a detection methodology and not a quantitative technique.</div>
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MS is a very sensitive technique, and there is a statistical chance that certain ions are more easily picked up than others which mean that the peak height or area in a mass spectrum in itself does not accurately reflect the abundance of a peptide in the sample. The main reasons for this are the differences in ionisation efficiency and detectability of peptides. Mathematically the equation would look something like this (I will not get into the actual mathematics since that is not relevant here).</div>
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Protein concentration= MS abundance value x Error factor</div>
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The error factor depends on each run and will vary from experiment to experiment. Consider this experiment. If you have a cell lysate you run it 10 times in LC-MS/MS analysis the final result will be varied from experiment to experiment. In fact, the number of proteins identified will also significantly change and you can expect a variation of at least 30% between any two runs as shown by multiple studies. If you run 2 independent batches of LC-MS/MS for comparison then the final result will consist only of error for purposes of direct comparison. The best idea would be to compare proteins from test and control in the same run so that the error will be constant. Since the error factor is the same in both cases (which is unknown), relative fold change can be accurately calculated by comparing the abundance value of m/z peak from the experiment. </div>
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So what is required for comparison is to run all the protein preparation that has to be compared in a single mass spec run. Now you need a method to tell which peptide came from whom. That is why we label the peptide library obtained from each case. Let us say you want to run 5 biological test cases against 5 biological control case that would be a 10 plex labelling experiment with each condition being labelled with a different label. The label will tell MS where the peptide originally came from and how much of it is there in t.</div>
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<tr><td class="tr-caption" style="text-align: center;"><b>Fig 1:</b> Hypothetical example of m/z abundance<br />
as an indicator of fold change.</td></tr>
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Fig 1, is a hypothetical example of m/z abundance as an indicator of fold change. Consider you are comparing 3 cases against a control sample. The height of the peak represents the peptide abundance. In comparison to control, the case 1 is slightly elevated, case 2 is drastically down and case 3 is unchanged. This kind of comparison is available for all the peptides that have been detected in MS. The overall finding is then curated by the software and presented as a protein expression data with reference to the control.</div>
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<tr><td class="tr-caption" style="text-align: center;"><b>Fig 2:</b> Labelling methods for quantification of proteins in Mass Spectrometry.</td></tr>
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There are wide varieties of labelling methods available and different literature have a different classification and there is an overlap in some cases. For simplicity, labelling methods can be broadly classified into 3 subtypes- Metabolic, Enzymatic and chemical labelling.See Fig 2 for a summarised classification. It is not possible to talk about all the methods and intricate details of every method, which would make this post too long. I will stick to explaining a few methods that are more famous in biological practice which will give an idea of what exactly is happening. Chemical labelling is much similar to metabolic labelling except that the label is chemically attached to a particular peptide after extraction unlike doing it metabolically. Enzymatic labelling is almost a chemical labelling except that it is done using an enzymatic process.</div>
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<b>Stable isotope labelling by amino acids in cell culture (SILAC)</b></div>
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<b>Fig 3:</b> Example light and heavy amino acids for SILAC.</div>
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<a href="https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/metabolic-labeling-chemoselective-ligation.html">Source</a></div>
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SILAC labelling was first demonstrated from Matthias Mann lab; 2002. The method is a metabolic labelling method. The core idea is that cells are given essential amino acids that carry heavy stable isotopes continuously, which gets converted into proteins in the cell. This process run for sufficient time, a great majority of the cell proteins contain heavy labelled isotopes which can be picked in the mass spec. In a typical SILAC labelling experiment, lysine and arginine residues are isotope labelled. Since trypsin digestion is commonly used for obtaining peptides this results in labelling of every peptide in the mixture. The labels are available as N terminal and C terminal labelled lysine or arginine. See Fig 3. In addition, leucine, tyrosine and methionine amino acids with incorporated isotopes have also been used as labels. SILAC method has a high efficiency but comes with inherent limitations. Other than the facts that it is time consuming and expensive it requires that the method uses a culture system only those that can be cultured are available to work with this method.</div>
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<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Signature"/>
<w:LsdException Locked="false" Priority="1" SemiHidden="true"
UnhideWhenUsed="true" Name="Default Paragraph Font"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text Indent"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="List Continue"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="List Continue 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="List Continue 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="List Continue 4"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="List Continue 5"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Message Header"/>
<w:LsdException Locked="false" Priority="11" QFormat="true" Name="Subtitle"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Salutation"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Date"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text First Indent"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text First Indent 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Heading"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text Indent 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Body Text Indent 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Block Text"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Hyperlink"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="FollowedHyperlink"/>
<w:LsdException Locked="false" Priority="22" QFormat="true" Name="Strong"/>
<w:LsdException Locked="false" Priority="20" QFormat="true" Name="Emphasis"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Document Map"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Plain Text"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="E-mail Signature"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Top of Form"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Bottom of Form"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Normal (Web)"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Acronym"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Address"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Cite"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Code"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Definition"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Keyboard"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Preformatted"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Sample"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Typewriter"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="HTML Variable"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Normal Table"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="annotation subject"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="No List"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Outline List 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Outline List 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Outline List 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Simple 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Simple 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Simple 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Classic 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Classic 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Classic 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Classic 4"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Colorful 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Colorful 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Colorful 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Columns 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Columns 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Columns 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Columns 4"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Columns 5"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 4"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 5"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 6"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 7"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Grid 8"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 4"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 5"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 6"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 7"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table List 8"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table 3D effects 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table 3D effects 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table 3D effects 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Contemporary"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Elegant"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Professional"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Subtle 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Subtle 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Web 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Web 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Web 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Balloon Text"/>
<w:LsdException Locked="false" Priority="39" Name="Table Grid"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Table Theme"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 1"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 2"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 3"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 4"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 5"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 6"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 7"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 8"/>
<w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
Name="Note Level 9"/>
<w:LsdException Locked="false" SemiHidden="true" Name="Placeholder Text"/>
<w:LsdException Locked="false" Priority="1" QFormat="true" Name="No Spacing"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading"/>
<w:LsdException Locked="false" Priority="61" Name="Light List"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 1"/>
<w:LsdException Locked="false" Priority="61" Name="Light List Accent 1"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 1"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 1"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 1"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 1"/>
<w:LsdException Locked="false" SemiHidden="true" Name="Revision"/>
<w:LsdException Locked="false" Priority="34" QFormat="true"
Name="List Paragraph"/>
<w:LsdException Locked="false" Priority="29" QFormat="true" Name="Quote"/>
<w:LsdException Locked="false" Priority="30" QFormat="true"
Name="Intense Quote"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 1"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 1"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 1"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 1"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List Accent 1"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 1"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 1"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 1"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 2"/>
<w:LsdException Locked="false" Priority="61" Name="Light List Accent 2"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 2"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 2"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 2"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 2"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 2"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 2"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 2"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 2"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List Accent 2"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 2"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 2"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 2"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 3"/>
<w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/>
<w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 4"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 4"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 4"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List Accent 4"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 4"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 4"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 4"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 5"/>
<w:LsdException Locked="false" Priority="61" Name="Light List Accent 5"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 5"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 5"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 5"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 5"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 5"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 5"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 5"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 5"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List Accent 5"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 5"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 5"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 5"/>
<w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 6"/>
<w:LsdException Locked="false" Priority="61" Name="Light List Accent 6"/>
<w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 6"/>
<w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 6"/>
<w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 6"/>
<w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 6"/>
<w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 6"/>
<w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 6"/>
<w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 6"/>
<w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 6"/>
<w:LsdException Locked="false" Priority="70" Name="Dark List Accent 6"/>
<w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 6"/>
<w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 6"/>
<w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 6"/>
<w:LsdException Locked="false" Priority="19" QFormat="true"
Name="Subtle Emphasis"/>
<w:LsdException Locked="false" Priority="21" QFormat="true"
Name="Intense Emphasis"/>
<w:LsdException Locked="false" Priority="31" QFormat="true"
Name="Subtle Reference"/>
<w:LsdException Locked="false" Priority="32" QFormat="true"
Name="Intense Reference"/>
<w:LsdException Locked="false" Priority="33" QFormat="true" Name="Book Title"/>
<w:LsdException Locked="false" Priority="37" SemiHidden="true"
UnhideWhenUsed="true" Name="Bibliography"/>
<w:LsdException Locked="false" Priority="39" SemiHidden="true"
UnhideWhenUsed="true" QFormat="true" Name="TOC Heading"/>
<w:LsdException Locked="false" Priority="41" Name="Plain Table 1"/>
<w:LsdException Locked="false" Priority="42" Name="Plain Table 2"/>
<w:LsdException Locked="false" Priority="43" Name="Plain Table 3"/>
<w:LsdException Locked="false" Priority="44" Name="Plain Table 4"/>
<w:LsdException Locked="false" Priority="45" Name="Plain Table 5"/>
<w:LsdException Locked="false" Priority="40" Name="Grid Table Light"/>
<w:LsdException Locked="false" Priority="46" Name="Grid Table 1 Light"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark"/>
<w:LsdException Locked="false" Priority="51" Name="Grid Table 6 Colorful"/>
<w:LsdException Locked="false" Priority="52" Name="Grid Table 7 Colorful"/>
<w:LsdException Locked="false" Priority="46"
Name="Grid Table 1 Light Accent 1"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 1"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 1"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 1"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 1"/>
<w:LsdException Locked="false" Priority="51"
Name="Grid Table 6 Colorful Accent 1"/>
<w:LsdException Locked="false" Priority="52"
Name="Grid Table 7 Colorful Accent 1"/>
<w:LsdException Locked="false" Priority="46"
Name="Grid Table 1 Light Accent 2"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 2"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 2"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 2"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 2"/>
<w:LsdException Locked="false" Priority="51"
Name="Grid Table 6 Colorful Accent 2"/>
<w:LsdException Locked="false" Priority="52"
Name="Grid Table 7 Colorful Accent 2"/>
<w:LsdException Locked="false" Priority="46"
Name="Grid Table 1 Light Accent 3"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 3"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 3"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 3"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 3"/>
<w:LsdException Locked="false" Priority="51"
Name="Grid Table 6 Colorful Accent 3"/>
<w:LsdException Locked="false" Priority="52"
Name="Grid Table 7 Colorful Accent 3"/>
<w:LsdException Locked="false" Priority="46"
Name="Grid Table 1 Light Accent 4"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 4"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 4"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 4"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 4"/>
<w:LsdException Locked="false" Priority="51"
Name="Grid Table 6 Colorful Accent 4"/>
<w:LsdException Locked="false" Priority="52"
Name="Grid Table 7 Colorful Accent 4"/>
<w:LsdException Locked="false" Priority="46"
Name="Grid Table 1 Light Accent 5"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 5"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 5"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 5"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 5"/>
<w:LsdException Locked="false" Priority="51"
Name="Grid Table 6 Colorful Accent 5"/>
<w:LsdException Locked="false" Priority="52"
Name="Grid Table 7 Colorful Accent 5"/>
<w:LsdException Locked="false" Priority="46"
Name="Grid Table 1 Light Accent 6"/>
<w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 6"/>
<w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 6"/>
<w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 6"/>
<w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 6"/>
<w:LsdException Locked="false" Priority="51"
Name="Grid Table 6 Colorful Accent 6"/>
<w:LsdException Locked="false" Priority="52"
Name="Grid Table 7 Colorful Accent 6"/>
<w:LsdException Locked="false" Priority="46" Name="List Table 1 Light"/>
<w:LsdException Locked="false" Priority="47" Name="List Table 2"/>
<w:LsdException Locked="false" Priority="48" Name="List Table 3"/>
<w:LsdException Locked="false" Priority="49" Name="List Table 4"/>
<w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark"/>
<w:LsdException Locked="false" Priority="51" Name="List Table 6 Colorful"/>
<w:LsdException Locked="false" Priority="52" Name="List Table 7 Colorful"/>
<w:LsdException Locked="false" Priority="46"
Name="List Table 1 Light Accent 1"/>
<w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 1"/>
<w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 1"/>
<w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 1"/>
<w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 1"/>
<w:LsdException Locked="false" Priority="51"
Name="List Table 6 Colorful Accent 1"/>
<w:LsdException Locked="false" Priority="52"
Name="List Table 7 Colorful Accent 1"/>
<w:LsdException Locked="false" Priority="46"
Name="List Table 1 Light Accent 2"/>
<w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 2"/>
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The methodology considers the idea of class-2 proteases, such as trypsin, to catalyse the exchange of two<span style="font-family: , serif;"> <sup>16 </sup></span>O atoms for two <span style="font-family: , serif;"><sup>18 </sup></span>O atoms at the C-terminal carboxyl group of proteolytic peptides. Hydrolysis of a protein in <span style="font-family: , serif;">H<sub>2</sub><sup>18</sup></span>O by a protease results in the incorporation of one<span style="font-family: , serif;"> <sup>18 </sup></span>O atom into the carboxyl terminus of each proteolytically generated peptide. Despite its simplicity, the method is not in regular use owing to the difficulty in attaining a high labelling accuracy.</div>
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<b>Labelling using Isobaric tags</b></div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjQxrJdKb4gfJSpAjj-sIVt1LYNju23n_b3m5jNsmvEdKwo3-u4rS-6SIRQbuIo8bfwZUfi2KseIgBNquScir8IEWm7T9Qlo1Zq12IhWa4ZZg-RnjnoUHYno3kudJMGPjZ1yOC8sZQen_eB/s1600/Fig+4.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" data-original-height="225" data-original-width="550" height="130" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjQxrJdKb4gfJSpAjj-sIVt1LYNju23n_b3m5jNsmvEdKwo3-u4rS-6SIRQbuIo8bfwZUfi2KseIgBNquScir8IEWm7T9Qlo1Zq12IhWa4ZZg-RnjnoUHYno3kudJMGPjZ1yOC8sZQen_eB/s320/Fig+4.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Fig 4:</b> Structure of TMT tags. <a href="https://www.thermofisher.com/order/catalog/product/90063">Source</a></td></tr>
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This is probably one of the most common labelling methods to be used. Let us take the example of TMT (Tandem mass tags). Labels are basically isobaric compounds (They have same net mass) with a peptide binding site.</div>
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Each chemical tag contains a different number of heavy isotopes in the mass reporter region, which gives a unique reporter mass during tandem MS/MS for sample identification and relative quantitation, a mass normaliser which adjusts for the mass and a reactive group.</div>
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I have limited the discussion on labelling methods to the basic essence to give you an idea of how the system works. I recommend you read the references to have a detailed picture of the process.<br />
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<b>References:</b></div>
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Tabb et al. Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-Tandem Mass Spectrometry. <i>J Proteome Res</i>. 2010 Feb 5; 9(2): 761. doi: 10.1021/pr9006365</div>
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Ong S, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). <i>Nature Protocols</i>. 2007;1(6):2650-2660.</div>
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Rauniyar N, Yates J. Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics. <i>Journal of Proteome Research</i>. 2014;13(12):5293-5309.</div>
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