Wednesday, August 31, 2016

The Rise of Superbug Gonococcus

Greetings,

The world is talking about the emergence of MCR-1 and every single time a new strain is detected it is a news. Brazil recently reported its first case. A case was also reported from New Jersey of a strain that carried MCR-1 and NDM-5. I have already talked about MCR-1 gene multiple times and there is nothing new (except reporting of more new cases). MCR-1 has taken over so much of the news space that one another phenomenon is just not getting the right coverage. I intend to draw some attention on the increasingly reported resistance to antimicrobials by Gonococcus.

Fig 1: Antibiotic resistance in Gonococcus. Source
Gonococcus or Neisseria gonorrhoeae is a gram-negative diplococcus (GNC), one of the important etiological agent of STI (Sexually transmitted infections). They are identified in clinical laboratory based on their staining, oxidase positive and inability to utilise Maltose, lactose and sucrose. Other more confirmatory test including γ-Glutamyl aminopeptidase test (negative), Propyl aminopeptidase test (positive) and Butyrate esterase (negative). Gonococcal infection is considered as a surrogate to increased risk for HIV infection. Until a couple of decades ago, most gonococci was easy to treat infection.

Gonococci is kind of special member in the Neisseria group by their ability to accumulate genes. Until early 1980's most of the strains were sensitive to penicillin. Resistance began emerging rapidly and by 2010, resistant strains have been circulating for most of the antibiotics in the panel. The last 2 antibiotics in the resort have been ceftriaxone and azithromycin. Over the period since, most strains have acquired resistance to azithromycin and all chemicals of quinolone class.

Quinolone resistance has some interesting consequences. Quinolones attack DNA gyrase and DNA topoisomerase which is essentially involved in keeping the DNA structure in right shape. It has been hypothesised that mutations in these which help overcome quinolone resistance indirectly can influence the mutation rate and thus increase resistance to other drugs. This has also shown to be the case in a couple of experiments though it has been heavily debated across the community. Whatever is the case, currently at least in the UK gonococcal resistance has reached a new peak and most are resistant to almost everything except for ceftriaxone. WHO has expressed its concern over the issue. With roughly 78 million new cases globally reported every year, a sudden superbug phenomenon is projected. WHO has released its new guidelines recommending complete elimination of quinolone from the treatment panel. This is the first recommendation change coming after 2003. There is a catch. Though WHO recommends cephalosporins, 46 countries have reported gonorrhoea strains with decreased susceptibility to ceftriaxone, and 10 have reported patients for whom none of the usual antibiotics was effective.

The following statements by Zenilman capture the whole story

"Gonorrhea has been plaguing humanity for centuries. But ever since penicillin came along a dose of antibiotics would usually take care of the disease. Gonorrhea used to be susceptible to penicillin, ampicillin, tetracycline and doxycycline very commonly used drugs. But one by one, each of those antibiotics and almost every new one that has come along since eventually stopped working. One reason is that the bacterium that causes gonorrhoea can mutate quickly to defend itself. If this was a person, this person would be incredibly creative. The bug has an incredible ability to adapt and just develop new mechanisms of resisting the impact of these drugs."
Fig 2: Cases of highly azithromycin-resistant
Neisseria gonorrhoeae 2014 to
February 2016 in the  UK. Source 
To have a look at how bad is the situation in the UK, I traced a literature describing Azithromycin resistance in gonorrhoea cases. Fig 2, summarised here is a clear layout of the problem. A strain can be called as High-level Azithromycin-resistant if MIC >256 mg/L. These strains also possess multiple genes for resistance and is widely resistant to almost every possible drug. They are sometimes referred in the literature as XDR-Gonococcus.

Kai Kupferschmidt wrote an interesting article in science, suggesting that there is a reasonably good possibility that gonococci are going to be soon resistant to all the antibiotics that can target the bacteria, and this will be the first true superbug.


Tuesday, August 23, 2016

Indigenously developed Leprosy vaccine

Greetings

Recent buzz of talk is revolving around the leprosy vaccine. An indigenously developed vaccine by G.P Talwar, (Founder-director of National Institute of Immunology) which uses a strain Mycobacterium indicus pranii (MIP). Let us look into some details.

Fig 1: Leprosy pathogenesis. Source
Leprosy is caused by M leprae, one of the least understood Mycobacterium species. The bacilli are acquired by inhalation though it is not clearly defined how do the bacilli reach back to the respiratory system and discharged. Another rare mode of transmission involves direct skin contact (Though not common such cases have been reported).

The bacilli basically infect the peripheral nervous system. Though the details are not clear, current evidence suggests that the bacilli attack Schwann cells. The immune system comes into play and cellular response (CD4+ T cell response) leads to granulomatous lesions and intense intraneural oedema. The subsequent destruction of schwann cells and axons leads to nerve function impairment, a hallmark feature of leprosy. Most often the diagnostic is quite straightforward with a deep skin smear. Clinically, five forms of leprosy are defined which includes lepromatous leprosy (LL), borderline lepromatous (BL), mid-borderline (BB), borderline tuberculoid (BT), and tuberculoid leprosy (TT).

WHO estimates the global prevalence of leprosy at about 0.24 cases per 10 000 people (Data doesn't include Europe). This is well within the WHO's aim of a prevalence rate of less than 1 case per 10000 persons. But, India stands out in global statistics of Leprosy. As per 2013-14 statistics, nearly 59% of new leprosy cases in the world are from India. 2013-14 alone registered 1.27 lakhs new cases in India.

When I searched the literature for Leprae vaccines, I find that people have tried a variety of stuff. This includes- BCG with killed M leprae, BCG with M vaccae etc. The first significant publication of GP Talwar regarding Leprae vaccine based on MIP has come in 2005. The study was a double-blind study and the vaccination schedule of the Healthy household contacts comprised two doses at 6-month intervals. The first dose of vaccine consisted of 1x109 autoclaved MIP bacilli in 0.1 ml physiological saline (0.85% NaCl), while the subsequent doses contained half the number of bacilli. The study showed an overall prophylactic efficacy of 65–70%. The idea that MIP could serve as a surrogate comes from a publication in 1991 showing that MIP shares numbers of common B and T cell determinants with M leprae.

Photo: GP Talwar. Source
MIP (earlier known as Mw) is a cultivable, non-pathogenic and rapidly growing saprophyte classifiable in Runyon's group IV- Mycobacterium avium complex. Its use as an adjunct in TB therapy has been shown to be useful and studies are investigating its potential against TB cases. 

Dr Soumya Swaminathan comments, "It is the first vaccine for leprosy, and India will be the first to have a large-scale vaccination programme. Trials have shown that if the vaccine is given to people in close contact with the affected, cases can be brought down by 60% in three years. It expedites cure rate if given to people with skin lesions".

Currently, a clinical trial is aimed to be started. About 7.5 crore people have been screened across the country (from 50 high-risk regions), of which 5000 have been confirmed with leprosy. The idea is to treat the diagnosed cases and prophylactically give rifampicin to close contacts. It is not clear to me about the vaccine trial design. But I think the idea would be to administer the vaccine in these high-risk areas and look for a reduction in number of cases.

Yadava A et al. T-Cell Responses to Fractionated Antigens of Mycobacterium w, a Candidate Anti-Leprosy Vaccine, in Leprosy Patients. Scand J Immunol. 1991;34(1):23-31. 

Sharma et al. Immunoprophylactic effects of the anti-leprosy Mw vaccine in household contacts of leprosy patients: clinical field trials with a follow up of 8-10 years. Lepr Rev. 2005 Jun;76(2):127-43.

Gupta A et al. Efficacy of Mycobacterium indicus pranii Immunotherapy as an Adjunct to Chemotherapy for Tuberculosis and Underlying Immune Responses in the Lung. PLoS ONE. 2012;7(7):e39215. 

Wednesday, August 10, 2016

SER109- Phase 2 trial doesn't look good

Greetings

A really long time back, I had written about FMT (Fecal Microbiota Transplant). There have been a large number of studies for more than 2-3 years showing that FMT is beneficial and restoring a healthy microbiome is the way to look forward to conditions related to the gut. It has also been predicted that if we could make a synthetic microbiome of right combination, that would be helpful to people such as one's who are on antibiotics for a long time.

There are a lot of reasons to why FMT in its current raw form is not ideal. The most challenging reason is the requirement for donor screening and potential ability to transmit infections. Since there is no practical test that can screen for every possible pathogen, there is always a given risk in FMT directly from a donor. An idealistic approach would be to synthetically make the right combination in the pharmacy and give it as a pill. The problem is we really have no idea about what is the right combination.

Fig 1: Concept of Ecobiotic drugs. Source
In a study sponsored by Seres Therapeutics for recurrent CDI (Clostridium difficile infection) efficacy of SER-109 was evaluated. SER-109 was prepared from seven adult donors of stool specimens which were collected and treated with Ethanol to remove vegetative forms, spores washed and used. The preparation mainly contains about 50 species of Firmicutes spores. A vial administered orally as a single dose contains 1 X 108 bacterial spores. The initial report of the study showed that Per-protocol efficacy was about 86% over 8 weeks of follow-up. The company is also developing a product called SER-262 which targets primary CDI. The formulation contains spores from 12 strains. The company calls this concept of re-establishing a healthy microbiome as Ecobiotics.

On 29th July 2016; Seres announced interim results of Phase 2 trial of 89 people, but results look less promising. The study was conducted at 36 centres across the United States. Quoting from Business Wire Eight-week study data shows that the pill simply failed.

In subjects <65 years old, CDI recurrence occurred in 43% of subjects who received SER-109 (12 of 28) and in 27% of subjects who received placebo (4 of 15). In subjects ≥65 years old, CDI recurrence occurred in 45% of subjects who received SER-109 (14 of 31), and in 80% of those who received placebo (12 of 15).

There are a lot of questions that scientists are asking with this finding. The very first question is what was different between Phase 1 and 2 which explains the failure. I have some questions here but I would want to reserve myself till I see a publication about it with details. I also would like to have a sequencing data of SER-109 and know what exactly is in there.

For the time being this press release shows that we are not yet into microbiome pill, partly because we don't completely understand the details.

Reference

Khanna S, Pardi D, Kelly C, Kraft C, Dhere T, Henn M et al. A Novel Microbiome Therapeutic Increases Gut Microbial Diversity and Prevents Recurrent Clostridium difficile Infection. Journal of Infectious Diseases. 2016;214(2):173-181. Link

Friday, August 05, 2016

Zika infection- Updates II

Greetings

Zika has been the focus of talk worldwide. I have a couple of times talked about Zika virus previously. In the past 2-3 months, research has shot up on Zika. From a skeptic perspective of Zika's role in microcephaly, there is nearly a convincing evidence to show that Zika is neurotropic possibly in reduced immune context. Most of the questions are unanswered at this point of time.

Fig 1: Current Zika transmission. Data as of on 2nd August 2016. Source: ECDC

I don't have an exact information of global statistics in terms of numbers. Fig 1, obtained from ECDC shows that Zika is potentially spreading into more parts of the world. Brazil seems to be the most affected though the trend shows a decline in a number of cases. As of 28 July 2016, WHO has reported 64 countries and territories with a mosquito-borne transmission. of these 14 countries have reported microcephaly and other CNS malformations potentially associated with Zika virus infection in a congenital context. Colombia had approximately 1,00,000 people suspected of having contracted Zika, with roughly 18,000 pregnant women constantly being medically monitored. More than 20 cases of microcephaly were reported. Colombia declared the end of the Zika epidemic on 26 July 2016.

The latest entry into the list is China. There are reports of at least 3 persons with a travel history with no reported incidents of local transmission. Though India has not reported any case to date, it is a hotspot with the possibility of acquiring Zika infections. According to a recent paper published by Messina et al, Indian tropical conditions have a good setting for Zika transmission. factors that support Zika-bearing mosquitoes include temperature, precipitation, humidity, and population density. Quoting from the article,

"Much of sub-Saharan Africa (where several sporadic cases have been reported since the 1950s) also presents an environment that is highly suitable for the Zika virus. While no cases have yet been reported in India, a large portion of the subcontinent is also suitable for Zika transmission."

India is already testing for Zika. Soumya Swaminathan says, "India, at the moment, for Zika, what we are doing is surveillance. Making sure that we equip our labs to be able to test for Zika. We want to find out if there is any Zika circulating. So, we are testing people who present with fever, just like a dengue but a dengue negative. We have not found any case so far, but we will continue this exercise." I'm a little skeptic about this approach. Worldwide people have shown that Zika is serologically cross-reactive with dengue and other flaviviruses. In a country where serology is mainly the mode of diagnostics, it would make more sense to me to perform molecular Zika tests, in cases where dengue serology is positive. There is also a great need for field testing of trapped mosquitoes for Zika which would signal an alarm.

Fig 2: Dengue case statistics in India.
Data as on 28th June 2016
Source: NVBDCP
I just looked into the number of dengue cases reported in India. If there is a sudden increase in dengue cases reported and we argue that some of them are actually Zika that we are misdiagnosing then we would still see some increase in dengue cases. But that is not what I see based on statistics data. But of course, you could argue that Zika doesn't become clinically relevant in most of the cases and hence my argument may not be totally valid.

There is almost nothing known about Zika molecular life cycle. The cellular receptor is not known. There is some research evidence to suggest that DC- SIGN, AXL, Tyro, and TIM-1 entry/adhesion factors permit the entry of ZIKV. ZIKV replication also activates an antiviral immune response and the production of type I interferon in infected cells. Mouse and macaque models show that indeed ZIKV significantly affects fetal development leading to microcephaly.

There is a lot of debate on if ZIKV is sexually transmitted. There are 4 reports that suggest that ZIKV is sexually transmitted. But the results and data are not quite convincing and there is a lot of debate on what are the chances. There are also a couple of reports suggesting blood transfusion as another possible risk. The only universally agreed upon mode of transmission is mosquito borne. 

Another discussed point is the Zika transmission with reference to Olympics. Rio de Janeiro and the five sites outside the city are in areas where Zika-bearing mosquitoes can thrive. According to recent opinion, the risk of global transmission of ZIKV from Rio due to people attending Olympics is low. Though the estimate is not null, the number of people that would travel to the Olympics is going to be really a low number. Also, statistics show that the number of cases in Rio has come down significantly. As Paulo Gadelha comments, “Several simulation studies show that the expectation of contamination is 1.8 cases per one million people. This is a very low risk". The authorities are taking steps to minimize mosquito bites.

Fig 3: Model of ADE in dengue infection.
Source
The greatest challenge in ZIKV is making a vaccine. ZIKV, like other flaviviral members, exhibit a property called as ADE (Antibody dependent enhancement). This is a phenomenon where viral entry occurs when Fc receptors on the cell surface bind to the antibody that is attached to virus particles. The result is higher levels of virus replication and more severe disease. This phenomenon is called antibody-dependent enhancement, or ADE. This phenomenon is well known in Dengue where infection for the second time causes severe problems. This is why making a vaccine against dengue a problem. Interestingly, due to cross-reactivity presence of Dengue antibodies increases the susceptibility to Zika (Link). I recommend you read Vincent's blog post here and here which has a great explanation of details.

Though there are several candidate vaccines proposed as a possibility, there isn't any approved vaccine to date. As discussed above the vaccine will have to circumvent the problem of ADE and also not cross-reactive enhance other infections especially Dengue. Barouch et al; has published a DNA vaccine candidate. DNA vaccine expresses full-length ZIKV pre-membrane and envelope (prM-Env). The full-length prM-Env DNA vaccine has shown complete protection and protective efficacy correlates with Env-specific antibody titers. A single immunization with a plasmid DNA vaccine provided a complete protection in susceptible mice against challenge with a ZIKV outbreak strain from northeast Brazil. NIH will soon be leading a clinical trial. Another vaccine that has received approval for phase I study is the Inovio DNA vaccine. Earlier studies have shown that their vaccine induced robust antibody and T cell responses in animal models. The phase I, open-label, will study 40 healthy subjects for GLS-5700 (Inovio ZIKV DNA vaccine) planned to be administered intradermally using Inovio’s proprietary DNA delivery device system- Cellectra.

The laboratory diagnostic capabilities have tremendously increased for ZIKV. There is RT-PCR using specific primers, and an RNA detection platform on filter paper. Serological testing methods such as ELISA is also available. PRNT (Plaque reduction neuralization test) is currently the gold standard.

To summarise, Zika is not evident in most cases which may serve as a carrier. The problem is evident in a subset of pregnant mothers where fetus is affected for which we dont know how and why. There are no vaccines approved to date and tropical countries are at risk. The strain is slowly spreading thanks to global travel.

Correction/Update (5th Aug 2016):

I missed to state that there is another vaccine being developed by Walter Reed Army Institute of Research in association with NIAID (National Institute of Allergy and Infectious Diseases). The vaccine is a purified, inactivated Zika virus from Puerto Rico. Preliminary results are encouraging.

References

1. Larocca R, Abbink P, Peron J, Zanotto P, Iampietro M, Badamchi-Zadeh A et al. Vaccine protection against Zika virus from Brazil. Nature. 2016

2. Messina J, Kraemer M, Brady O, Pigott D, Shearer F, Weiss D et al. Mapping global environmental suitability for Zika virus. eLife. 2016;5.

Tuesday, August 02, 2016

S Typhimurium Persistence model

Greetings

In my previous blog post, I had explained about how resistance can play out without acquiring any resistance factor or mutation in particular. I also mentioned that persistence can be achieved by simply shifting the cellular state to dormancy. Recently, an article published in Molecular cell by Cheverton et al; explored the mechanism of persistence in Salmonella typhimurium which makes an interesting case in point.

Fig 1: Salmonella classification.
Source
Salmonella is a gram-negative bacteria of Enterobacteriaceae group. There are something like 3000 members of in Salmonella. As per the previous classification, Salmonella had a single species Salmonella enterica and it was then typable into multiple subspecies. Subsequent DNA analysis showed that one of them Salmonella bongori is a different species and hence the current classification shows 2 species. See Fig 1, for classification details. Salmonella Typhimurium is not a species. Originally, it is Salmonella enterica subsp enterica serovar Typhimurium. In order to avoid the lengthy name, it is simply called S Typhimurium . It Infects humans, cattle, swine, sheep, horses, rodents and galliformes.

The Toxin- Antitoxin system (TAT) is a bi-component system. The two components include a toxin that has a long half-life and an antitoxin with a short half-life. The point is the toxin will be produced by the bacteria and is lethal to itself until there is an antitoxin expressed somewhere in the genome to save itself. This provides a strong control over a variety of factors. Refer to my earlier blog post for details. TAT provides a mechanism of self-control. 

Fig 2: Graphical abstract of the study. Source
In the study, researchers showed that TacT is actually part of a TAT system and the protein is functionally related to GNAT (Gcn5 N-acetyltransferases) superfamily with known acetylation capabilities. They basically catalyse acetyl group transfer from acetyl-coenzyme A (Ac-CoA) to a wide range of substrates. Further cell free assays showed that TacT acetylates the amino acid of aminoacyl-tRNAs to inhibit translation, using acetyl-CoA as a cofactor. TacT acetylates the primary amine group of the amino acid on the charged tRNA molecules. Once acetylated, the amine group of the A-site amino acid would be unavailable to the carboxyl group and protein synthesis would be blocked. This merely leads to a prolonged lag phase a feature of being persistent.

Most of the times, TAT involves a antitoxin mechanism to come out of locking effect. In this case, translation inhibition is not easy to overcome and hence researchers explored to see if they could turn out any Antitoxin independent mechanism. By screening for genes that suppress TacT toxicity the team hit upon peptidyl-tRNA hydrolase (Pth) as a possibility. Pth can deacetylate an already acetylated aminoacyl-tRNA thus reversing the effect.

It is interesting to note that GNAT toxins are present in many pathogenic species  such as Salmonella, Shigella, Vibrio etc. Typhimurium is notoriously known for its ability to persist inside macrophages and the same team had earlier reported that TacT is specifically activated upon entry of Salmonella in macrophages contributing to persister formation.

This study provides a great model to look into how persistence can be achieved. It would be interesting to know what actually triggers the TacT.

1. Cheverton A, Gollan B, Przydacz M, Wong C, Mylona A, Hare S et al. A Salmonella Toxin Promotes Persister Formation through Acetylation of tRNA. Molecular Cell. 2016;63(1):86-96.

2. Attar N. Bacterial toxins: An antidote to persistence. Nature Reviews Microbiology. 2016;14(8):473-473.