Wednesday, April 24, 2013

Controlling malaria- Perspectives


      The last 2 weeks have been the show of H7N9. The eyes of the whole world is concentrated on China. An extra ordinary effort is put into studying whatever is possible. Various teams have visited and in the process of scientific search. In that event let me ask you a question. What would be the most difficult and challenging infections globally? Someone would argue, most infectious diseases are important and challenging. But considering the best of the best, what bothers the most? I would say Tuberculosis, Influenza, HIV and Malaria. Decades of research is available in all these areas, yet we have failed to beat these, most of the times at least.

Photo 1: Female Anopheles.
    Malaria is a very interesting disease. Seen mainly in the tropics, caused by Plasmodium species and a vector borne problem. I often had wondered, if you should consider humans as the incubators for malaria so that we could infect the mosquito. We are the intermediate hosts and Mosquito is the definitive host. I can trash the argument by saying that humans suffer strong clinical symptoms from infection and fatal in many cases, but not so with mosquito. A second basic question is why only female anopheles? The answer is both males and females suck on plant and fruit saps for nutrition, but for the purpose of ovulation, materials are derived from mammalian blood. And since only females ovulate, only females need blood.

     Speaking about the Plasmodium, 4 different species are seen to infect humans- P vivax, P. falciparum, P. ovale, P. malariae. More recently a 5th species referred as P. knowlesi (Known to cause primate malaria), is seen to cause infections in humans, especially in South East Asian regions.

Table 1: Important features of Plasmodium
     Malaria control was once attempted by using DDT (1,1,1-trichloro-2,2-di (4-chlorophenyl) ethane). Though there was a heavy reduction, the whole problem bounced back, anopheles developed resistance to DDT. Since, we have tried variety of approaches. The best of all that has worked to date without problem is the use of bed nets. However, you cannot get a person inside a bed net all over the time and hence we need more methods.

       I have some good news. In the past few years the global malaria eradication problems have been successful, at least to a reasonable extent. The number of cases reported annually in most of the endemic countries has reduced by almost 80%. Armenia, Morocco, Turkmenistan, and the United Arab Emirates, which was known for a good number of malaria cases has been now convincingly certified as malaria free. I feel optimistic here, as it shows that we can achieve it. A review article in lancet by Cotter etal, discuss in detail how and what we have achieved in malaria control.

Fig 1: Malaria Control Strategies.
       There are different possible methods to control malaria. I have summarized the most well known methods in Fig 1 (Shown to the right). The physical methods are very promising if used right. They are often inexpensive. Use of mosquito repellants and Insect nets are the most common methods under this. More recently scientist at colombia university has developed a laser shield that can repel the mosquito. But the technique hasn't become famous enough yet. My most obvious argument is that if the person can afford laser based machines. they better invest in a simple net. But, it has an advantage that it can be installed in windows in such a way that the window is open for everything except mosquito. Read here

      The second method is vaccination. A very great deal of expectation was put forward on the RTS,S/AS01 vaccine targeting the CSP (circumsporozoite protein). RTS is a hybrid polypeptide consisting of a portion of the circumsporozoite protein (CS), a sporozoite surface antigen of the malaria parasite P. falciparum strain NF54, fused to the amino-terminal end of the hepatitis B virus S protein.  AS01 Adjuvant System consists of a liquid suspension of liposomes with two immuno-stimulant components- 3’-O-desacyl-4’-monophosphoryl lipid A (MPL) and Quillaja saponaria 21 (QS21). The recent publication in NEJM, cast some doubts on the efficacy of the vaccine (has been calculated to be modest only). However the final conclusive results are yet to be published.

Photo 2: Wolbachia in egg. Source
     The third method, that is gaining popularity is the Vector Control Programs. Vector control programs against malaria has some additional set of advantages. Once successful, they are calculated to be self sustaining (At least theoretically) and can also confer protection against other vector borne infections (caused by the same vector) over the long run. Impressive.

      The Biological Vector control Program against female anopheles, that has gained a very strong popularity is the use of Wolbachia. Wolbachia (alpha-proteobacteria member) are gram negative, intracellular, endosymbiontic bacteria, known to infect arthropods, insects and some nematodes. They are referred sometimes as gonad tampering bacteria. They have the ability to changing the sex of host and kill their offspring. This can be achieved either by distorting the host sex ratio and (or) by inducing cytoplasmic incompatibility. Moreover they are known to maternally transmit through the egg cytoplasm. W pipentis, is the potential candidate. The bacteria accumulates at the end of the egg that is destined to develop into the reproductive organs (See Photo 2). This induce the eggs of this wasp to develop into female offspring without fertilization. Source. The idea of using such a biological system to control the mosquito was proposed as early as 1967 and a few trials were conducted in India in 1970s with some field testing. However it had not received much interest and now there is a revived interest owing to its possible potential.

    Though many different mosquito varieties including Culex pipiens, C. quinquefasciatus, Aedes fluviatilis and A. albopictus are naturally infected, the most important vector A. aegypti and Anopheles species are not. To create a stable infection, embryonic microinjection of Wolbachia near the pole cells in pre-blastoderm embryosis is used, to incorporate Wolbachia into the developing germline and favor the transmission of Wolbachia. In addition to cytoplasmic incompatibility, Wolbachia is also shown to directly interfere with variety of virus such as Dengue and West Nile. The bacteria also confers a reduced life span for the insect and thus reduces transmission (Remember, it takes at least 2 blood meals ;2 weeks apart for the mosquito to successfully transmit plasmodium).

       The Second bacterium that has gained popularity is a bio-engineered strain of Pantoea agglomerans (previously known as Enterobacter agglomerans). In a recent blog post (Link), I had talked about how one bacteria can be engineered to sense another bacteria and secrete toxic molecules to destroy the pathogen. The approach has been used in many scenario, and the same tactic is employed here. A variety of secretable compounds such as dodecapeptide SM1, anti-Pbs21 single-chain variable-fragment (scFv) antibody and phospholipase A2 had been engineered into the bacteria. This can be secreted through a pelB or hlyA protein secretion pathways from P. agglomerans (Paratransgenic P. agglomerans strain). The studies show that when the plasmodium enters the mosquito gut, the bacteria secrete engineered compounds inhibiting the plasmodium life cycle

     The main hurdles in introducing such Biological control programs is to stabilize. The bacteria needs to have a fitness in transmission (Refer to Bartonian wave; Link)and not effect evolution. Our main aim in these new strategies is to inhibit the plasmodium life cycle (Or virus) and not destroy the mosquito. This gets around the problem of evolution and resistance emergence. A field trial (Started in 2011) is currently evaluated against Dengue in Australia, with awaited scientific results. A field trial is also scheduled in Brazil in May 2014. The trial will answer many questions. Link
Iturbe-Ormaetxe, I., Walker, T., & O' Neill, S. (2011). Wolbachia and the biological control of mosquito-borne disease EMBO reports, 12 (6), 508-518 DOI: 10.1038/embor.2011.84

Walker, T., & Moreira, L. (2011). Can Wolbachia be used to control malaria? Memórias do Instituto Oswaldo Cruz, 106, 212-217 DOI: 10.1590/S0074-02762011000900026

Bisi DC, & Lampe DJ (2011). Secretion of anti-Plasmodium effector proteins from a natural Pantoea agglomerans isolate by using PelB and HlyA secretion signals. Applied and environmental microbiology, 77 (13), 4669-75 PMID: 21602368

Wang, S., Ghosh, A., Bongio, N., Stebbings, K., Lampe, D., & Jacobs-Lorena, M. (2012). Fighting malaria with engineered symbiotic bacteria from vector mosquitoes Proceedings of the National Academy of Sciences, 109 (31), 12734-12739 DOI: 10.1073/pnas.1204158109

Tuesday, April 16, 2013

H7N9- Follw up post

     The last weeks block buster news on H7N9 is still the most important news. Its everywhere on the web. For Scientific publishers like Nature news and Science news, that's the most important flash news. Even news papers, magazines, everywhere it is up. (Oh, by the way microbiology blogs too!!!). The sudden data build up, and opinions in publication, in the last week around the globe has made me give a thought again. Perhaps, I need to write some follow up.
Fig 1: Schematic diagram of novel
influenza A(H7N9) virus generation.
    For people who haven't yet read my previous post (Link), need to read it up first. As of on 15 April 2013, WHO data (Link), 60 patients have been laboratory-confirmed with influenza A(H7N9) virus in China which includes 13 deaths. Almost all possible contacts are being strictly quarantined. There are no new cases confirmed, with officials reporting a controlled situation. A heavy inspection of 1,127 restaurants, 95 poultry farms, 16 supermarkets and 30 other related companies has been evaluated with not much of a finding. Source.

    A very detailed genetic analysis has been reported by Kageyama etal, and by comparing as many possible from the database. As I mentioned in my previous post, an important mutation is T160A substitution in H3 loop, leading to a loss of glycosylation site, results in increased virus binding to human-type receptors. The second point is all studied genomes shows deletion at positions 69–73 of the NA stalk region. Based on the data, it is most likely that, this new assortent is possibly a mix, as shown by the author in Fig 1.

     So, with that background, I want to revisist  a question that i asked last time. So the main question is, "Is this the pandemic strain?".

     The most important feature that could be attributed to a pandemic strain in influenza is almost unchallengably, the agility to transmit and move around too fast. First case being reported on March and its now more than a 40 days have passed since, with a few confirmed cases. Data is still lacking. If there's a data on seroconversion we would have better projection. If we are to see a large number of people with antibodies, but no apparent infection that means this virus is spreading, but the clinical apparency is low. If we are to find low seroconversion, then arguably, we are talking of a possible pandemic strain. To date, there is no documented evidence of any sustained human-to-human spread of the H7N9 virus. Lipsitch says, "It's too soon to say how big a threat H7N9 poses because we don't know how many animals of which species have it, how genetically diverse it is, or what the geographic extent is, It looks as though it will be at least as challenging as H5N1."

     Whatever the case maybe, it is not as bad as the H1N1 was. And the most important question currently faced is what is the source and reservoir. Taej Mundkur comments “There is very little specific information on the source of this particular virus strain, its ecology or reservoir, and it is premature to be hypothesizing on the vectors”. Source

    That brings me to an important point. Most people are worried of eating poultry meat. Though, I personally am a pure vegetarian and dont recommend eating meat, I dont see it to be unsafe. As far as the facts are known, hygeinc and good cooking practices can kill the viral particles if present and hence shouldnt be problem. And for people who are working with poultry industry, (especially imported meat), possibly need to be watched.

ResearchBlogging.orgKageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, Neumann G, Saito T, Kawaoka Y, Tashiro M (2013). Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013 Eurosurveillance, 18 (15)

Friday, April 12, 2013

H7N9 in News


       Microbes come up with a lot of surprises. Many new pathogens are discovered, some potentially pathogenic and some are not. We study a lot of microbes in every category and every time there is a new infection with some mortality there is a global alert. People watch out for it and sometimes also anticipate. Sometimes a non human pathogen, shifts to human population and zoonotic disease ensues. One such virus that is always in news, is now all over the news. The H7N9 is at the top of infectious disease literature at this time. And probably the topic attracts a bit of discussion here.

     In this blog, I have talked about various aspects of Influenza multiple times. So for people who need a background discussion, refer to my earlier posts. Avian influenza is a well studied entity. From a wide range of studies it is known to us that poultry infections by avian influenza is a common feature. And such a virus making a leap to humans is also common. It is a well accepted theory that the influenza can undergo a genetic shift or drift to create a new assortent virus. Often during such process two strains recombine, to form a brand new reassortent. Almost once in 3-5 years we see a new influenza making a emergence, and takes advantage of lack of herd immunity. The virus enters population causing infections. There is a pandemic sweep, and then appears the herd immunity. Again the cycle repeats.

       So what is it that makes the news this time? Its the H7N9 reassortent virus. H stands for Haemagglutinin and Neuraminidase. There are several varieties of this, and based on the type present it is numbered. A few cases have been described for lower respiratory tract infections with varieties such as H7N2, H7N3, H9N2, or H10N7. Almost all these are case reports and has not been seen as threat. Influenza is transmitted primarily by droplets or respiratory secretions of infected persons. Influenza occurs all over the world, with an annual global attack rate estimated at 5 – 10% in adults and 20 – 30% in children. Most of the cases, It doesn't have the high fatality rate as media often hypes it.

Photo 1: H7N9 in news. Source
     In the first week of March three urban residents of Shanghai appeared with respiratory infections, it was a great deal. Cause we were expecting the H5 version to make an entry. Transmission of H7 viruses is rarely seen and these are the first cases of documented N9 cases in the world. So when the researchers saw this immediately laid hands on everything they got- PCR, sequencing and characterization. They found mutations at various points. The most interesting to me was the T160A mutation at the H3 region (150 bp loop) of HA gene and Five amino acids were deleted in the stalk region of NA residue 69 to 73. This probably has allowed the species jump. Rest of the mutations seen in other parts of the gene has possible conferred some additional fitness.

     As on April 11th 2013, i saw a report on Forbes News (Link), that reports a total of 33 cases indentified and 9 of them have died. If you are about to make a conclusion that the fatality rate is more than 25%, the hold on. We don't have a clear estimate of how many are actually infected. 33 is the identified case (not the actual number, which will be much more). so don't panic. Uyeki comments on the issue "Some of them had multi-organ failure, This is very, very severe disease, and rapidly progressive. And it's similar to H5N1 [bird flu], but it's a little hard to make comparisons based upon three cases and limited data". Source

      That brings me to the most important point. Is this the next pandemic strain? Predicting that is much difficult when compared to our previous pandemic strain H1N1. Cause this is the first time this type has been encountered in humans. We virtually know nothing about the dynamics of this new reassortent. Right now various health authorities including the WHO is keeping a close watch. By using a Global Alert and Response (GAR) (Link) strategy, we all are keeping a close watch. Vaccine represents the best option. But easily said than done, we currently don't have any.

    Probably we need to wait for some more data, but the search is on.
Uyeki, T., & Cox, N. (2013). Global Concerns Regarding Novel Influenza A (H7N9) Virus Infections New England Journal of Medicine DOI: 10.1056/NEJMp1304661

Gao, R., Cao, B., Hu, Y., Feng, Z., Wang, D., Hu, W., Chen, J., Jie, Z., Qiu, H., Xu, K., Xu, X., Lu, H., Zhu, W., Gao, Z., Xiang, N., Shen, Y., He, Z., Gu, Y., Zhang, Z., Yang, Y., Zhao, X., Zhou, L., Li, X., Zou, S., Zhang, Y., Li, X., Yang, L., Guo, J., Dong, J., Li, Q., Dong, L., Zhu, Y., Bai, T., Wang, S., Hao, P., Yang, W., Zhang, Y., Han, J., Yu, H., Li, D., Gao, G., Wu, G., Wang, Y., Yuan, Z., & Shu, Y. (2013). Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus New England Journal of Medicine DOI: 10.1056/NEJMoa1304459

Monday, April 08, 2013

Resistance Detection- Faster the better


Fig 1: ESBL E-test. Source
     Time and again, we take clinical microbiology seriously. If I had to say from a rough statistics, the most discussed topic in microbiology is the antibiotic resistance. I have written a few posts on the same. Needless to say, testing for resistance has been very important. A number of research has been dedicated to this field. Classic laboratory techniques, such as ESBL detection by disc synergy test are time consuming (turn around time of at least 18 hr). The most commonly suggested solution is often a molecular technique- PCR, detection of resistance enzymes by using HPLC, MALDI etc etc. Though not rocket science, the cost of molecular work, has not encouraged its use, especially in finance limited countries.

Fig 2: Pros and cons- use of molecular techniques for detecting Antibiotic resistance. 
      So the most important problem appears to be the work cost and time. Now what if I told you that, you can get your valid clinical data about antibiogram in few hours at a very low cost (When I say "Low", I mean reasonably low cost). There are many such tests and more are in development. My aim here is to introduce you to a few.

Fig 3: Carba NP test. Source
     Carbapenems, are the latest addition of wide range antibiotics, and its use is common. The commonly named drugs under this category include- Imipenem, Meropenem, Ertapenem and Doripenem. Resistance can be mediated by multiple factors, but the most common would be a carbapenemase or efflux pumping of drug. There are multiple studies that imply carbapenemase based resistance as the more prevalent cause and my experience also dictates the same. They are metallo-beta-lactamases, detected by double disc synergy, Combined disc diffusion, Hodge test or E-test. Currently there is no CLSI guidelines for the afore mentioned methods. And yes the turn around time is at least overnight.

    That brings me to the first test gaining popularity. The Carba NP Test. The test uses a simple idea. Imipenem on hydrolysis, produces a acidic condition, coupled with tazobactam and EDTA as inhibitors which can be detected using a indicator dye. That's all. The total turn around time for this test is not more than 2 hrs (in comparison to molecular tests, which takes 4-6 hrs). And what's better? It has perfect score (Sensitivity and specificity of 100%).

      Another test that uses a similar principle of acidification is the ESBL NDP (Nordmann/ Dortet/ Poirel) test. The test is based on hydrolysis of the beta -lactam ring of a cephalosporin (cefotaxime in this case), which generates a carboxyl group, by acidifying the culture medium. The acidity resulting from this hydrolysis is identified by the color change generated using a pH indicator (red phenol). The test can be done in a microwell or tube format. The technique can be applied directly to a culture plate. In a study by Nordmann et al The sensitivity and specificity was found to be 92.6% and 100%, respectively. The turn around time again is less than 2 hrs costing not more than 4 euros a test. "We can hope, in particular in many Western countries where the situation has not yet reached endemic proportions multi-resistances (France, in particular), to be able to preserve to a certain extent the efficiency of wide-spectrum cephalosporins and carbapenems, antibiotics used as a "last resource", says Patrice Normann. Source

       That brings me to the second method of analysis. Imagine this. An antibiotic is thrown at a organism. If it is sensitive, the organism is killed and degenerates. Now throw a nucleic acid specific dye (such as SYBR green I, PicoGreen, and YOYO-1). Since the cell is more permeable the dye binds to nuclear material and can be detected by using flow cytometry method. The method has been of extensive value in detection of resistance in plasmodium. Oops, but I just said, not to make use of a complicated machinery (That means no Flow cytometry). So, I recall having read a paper (For which am unable to find the reference), where you can do the same in a gel. Put some gel containing SYBR green on a slide and add anitibiotic and organism. After about 4 hours if the cell has lysed or killed, DNA will bind SYBR green and can be seen in a fluorescence microscope.

    By now, you will realize that we actually don't need too much sophisticated technology to get antibiogram results quickly. But then you would also realize that these techniques are applied to vibrant organisms (One's who are very active and replicate quickly). They are of excellent use when it comes to organisms such as Enterobacteriaceae members, Staphylocococcus etc. There are more sophisticated technologies like the eFluxx-ID screening technique (Link) and Anopore method (Link), slowly coming into practice. But, would it be of any use in say tubercle bacilli? Not much I would say.

     That brings me to the next technique. Perhaps, this is the one which has the maximum potential. Vital dyes are chemicals that can stain a cell living component. It is known that vital dyes staining correlates inversely with the multidrug resistant phenotypes. One of the old and best paper on this issue can be found here. Fluorescein diacetate (FDA) is a very well known vital dye in microbiology. It was of great use in determining variety of bacterial activity such as predicting counts. The dye was also used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli (Reference).

     Let's combine the two above ideas. After treatment with a particular drug the efficacy of drug can be estimated by checking viability using FDA, say in a sputum sample. Simple. By using a little tweak the same idea could be applied in principle in the laboratory also, I would say.

Fig 4: Result of a FDA-test under the microscope: the fluorescent lines are living tuberculosis bacilli, on a background of cellular debris from human sputum. Source
       Labmedica reports (Source) as follows. "If after treatment the FDA-test was negative, in 95% of cases more elaborate tests did not find active bacilli in the patient's sputum. And if the test was positive, a resistant bacillus had been found". That summarizes whatever I want to say.

     I want to end here with a note. The use of vital dye can be extended to other bacteria testing. And non expensive techniques can be equally good in detecting resistance as the expensive molecular techniques at least in majority of the cases.
Nordmann P, Poirel L, & Dortet L (2012). Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerging infectious diseases, 18 (9), 1503-7 PMID: 22932472

Nordmann, P., Dortet, L., & Poirel, L. (2012). Rapid Detection of Extended-Spectrum- -Lactamase-Producing Enterobacteriaceae Journal of Clinical Microbiology, 50 (9), 3016-3022 DOI: 10.1128/JCM.00859-12

Johnson JD, Dennull RA, Gerena L, Lopez-Sanchez M, Roncal NE, & Waters NC (2007). Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrobial agents and chemotherapy, 51 (6), 1926-33 PMID: 17371812

Schramm, B., Hewison, C., Bonte, L., Jones, W., Camelique, O., Ruangweerayut, R., Swaddiwudhipong, W., & Bonnet, M. (2012). Field Evaluation of a Simple Fluorescence Method for Detection of Viable Mycobacterium tuberculosis in Sputum Specimens during Treatment Follow-Up Journal of Clinical Microbiology, 50 (8), 2788-2790 DOI: 10.1128/JCM.01232-12