Soham Gupta

Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

Background India has the third largest HIV-1 epidemic with 2.4 million infected individuals. Molecular epidemiological analysis has identified the predominance of HIV-1 subtype C (HIV-1C). However, the previous reports have been limited by sample size, and uneven geographical distribution. The introduction of HIV-1C in India remains uncertain due to this lack of structured studies. To fill the gap, we characterised the distribution pattern of HIV-1 subtypes in India based on data collection from nationwide clinical cohorts between 2007 and 2011. We also reconstructed the time to the most recent common ancestor (tMRCA) of the predominant HIV-1C strains. Methodology/Principal Findings Blood samples were collected from 168 HIV-1 seropositive subjects from 7 different states. HIV-1 subtypes were determined using two or three genes, gag, pol, and env using several methods. Bayesian coalescent-based approach was used to reconstruct the time of introduction and population growth patterns of the Indian HIV-1C. For the first time, a high prevalence (10%) of unique recombinant forms (BC and A1C) was observed when two or three genes were used instead of one gene (p<0.01; p = 0.02, respectively). The tMRCA of Indian HIV-1C was estimated using the three viral genes, ranged from 1967 (gag) to 1974 (env). Pol-gene analysis was considered to provide the most reliable estimate [1971, (95% CI: 1965–1976)]. The population growth pattern revealed an initial slow growth phase in the mid-1970s, an exponential phase through the 1980s, and a stationary phase since the early 1990s. Conclusions/Significance The Indian HIV-1C epidemic originated around 40 years ago from a single or few genetically related African lineages, and since then largely evolved independently. The effective population size in the country has been broadly stable since the 1990s. The evolving viral epidemic, as indicated by the increase of recombinant strains, warrants a need for continued molecular surveillance to guide efficient disease intervention strategies.

Diabetes mellitus and HIV as co-morbidities in tuberculosis patients of rural south India.

OBJECTIVES Incidence of tuberculosis (TB) is greatest among patients with impaired immunity. India is experiencing a double epidemic of HIV and diabetes mellitus (DM), both of which are strongly associated with immuno-suppression. This study aimed to discover the prevalence of HIV and DM in both the pulmonary and extra-pulmonary TB patients of rural south India, retrospectively. METHODS Medical records of 192 microbiologically diagnosed pulmonary TB and 37 extra-pulmonary TB patients were thoroughly studied and data were extracted. The frequency distribution of HIV and DM was evaluated along with other demographic details such as age, sex and occupation in both groups. RESULTS The mean age of the pulmonary TB patients was 41.11±15.7 years, with significantly higher (p<0.0001) preponderance of DM (31.8%) over HIV (8.9%). 72.13% of the diabetic patients belonged to the age group of 41-60 years. Extra-pulmonary TB patients had a mean age of 34.62±12.9, years with a significantly higher (p<0.006) HIV prevalence of 32.43% over DM (5.4%). 75% of the HIV patients belonged to the age group of 41-60 years. Occupationally, the majority of the pulmonary TB patients were agricultural labourers (25.2%) while the majority of the extra-pulmonary TB patients were housewives or self employed (18.92%). CONCLUSION Though more importance is being given to HIV-TB coinfection, we cannot overlook DM, which showed a significantly higher prevalence in pulmonary TB patients compared to HIV. The rising prevalence of DM in high TB burden countries may adversely affect TB control.

Role of risk factors and socio-economic status in pulmonary tuberculosis: a search for the root cause in patients in a tertiary care hospital, South India.

Objective  To determine the frequency of underlying risk factors and the socio‐economic impact based on occupation in the development of tuberculosis.

Community prevalence of methicillin and vancomycin resistant Staphylococcus aureus in and around Bangalore, southern India

INTRODUCTION Staphylococcus aureus is a known colonizer in humans and has been implicated in community acquired soft tissue infections. However emergence of methicillin resistant S. aureus(MRSA) has aroused great concern worldwide. This study aimed to determine the prevalence of MRSA in the community of Bangalore, southern India. METHODS Swabs were collected from anterior nares, forearm, dorsum and palm of the hands of 1,000 healthy individuals residing in and around Bangalore, belonging to different socioeconomic strata and age groups. RESULTS Analysis verified that 22.5% and 16.6% of the individuals presented Staphylococcus aureus and MRSA, respectively, at any of the three sites. Vancomycin resistance was observed in 1.4% of the S. aureus isolates, which was confirmed by detection of the vanA gene. It was interesting to note that 58.8% of the children in the age group 1-5 years-old presented MRSA, the highest percentage compared to other age groups of < 1 (44.4%) year-old, 5-20 (21.7%) years-old, > 40(11%) years-old and 20-40 (9.9%) years-old. Among the population of various socioeconomic strata, maximum MRSA colonization was observed among doctors (22.2%), followed by upper economic class (18.8%), lower economic class (17.7%), apparently healthy hospital in-patients (16.5%), nurses (16%) and middle economic class (12.5%). Most of the MRSA isolates were capsular polysaccharide antigen type 8 (57.1%). CONCLUSIONS There is a need for continuous surveillance and monitoring of the presence of MRSA in the community and a clearer understanding of the dynamics of the spread of MRSA will assist in controlling its dissemination.

Naturally Occurring Polymorphisms and Primary Drug Resistance Profile Among Antiretroviral-Naive Individuals in Bangalore, India

Although India has a large burden of HIV infection, good access to first-line antiretroviral therapy is widely available. However, understanding HIV resistance-associated mutations and polymorphisms is critical for continued success. The RT region of the HIV-1 pol gene was studied among 21 ART-naive HIV-1-infected individuals from South India. In addition, 421 published Indian HIV-1 subtype C sequences were analyzed for time trends in polymorphism frequency. Among primary isolates, all HIV-1 isolates were subtype C, and drug-resistant mutations were identified among two (9.56%) subjects. Mutations included E138A (etravirine resistance associated) and L210LS (thymidine analog mutation). The overall frequency of specific polymorphisms was similar to frequencies reported from different regions of India. Novel mutations were observed at positions Q23P/H and A129AG among isolates from our clinical cohort. Over a span of 10 years, the median polymorphism frequency among ART-naive subjects has remained unchanged, suggesting the slow evolution of HIV-1 subtype C in India.

High concordance of genotypic coreceptor prediction in plasma-viral RNA and proviral DNA of HIV-1 subtype C: implications for use of whole blood DNA in resource-limited settings

OBJECTIVES Genotypic tropism testing (GTT) of HIV is increasingly used prior to the initiation of CCR5 antagonist therapy in HIV-infected individuals. Normally performed on plasma-derived virus, the test is challenging when performed in patients with suppressed viraemia. We aimed to evaluate the performance of cell-associated proviral DNA against plasma-derived viral RNA as the genetic material for GTT in an Indian clinical setting. METHODS From 52 HIV-1-infected individuals, the env V3 region was successfully amplified and sequenced from both proviral DNA and plasma RNA paired samples having a viral load >2500 copies/mL (n = 42) and from proviral DNA only in 10 antiretroviral therapy (ART)-experienced patients with a viral load <500 copies/mL. GTT was performed using the Geno2Pheno algorithm with the interpretative false positive rate (FPR) cut-off of 10%. RESULTS Among paired samples, 40 of 42 patients harboured subtype C strains. Plasma RNA tropism prediction revealed X4 tropism in 4 of 42 (9.5%). A high concordance of 97.6% in tropism prediction was noted in simultaneous RNA/DNA samples (38 R5 and 3 X4). Discordance was observed in one sample showing R5 tropism in proviral DNA and X4 tropism in plasma RNA. Comparison of Geno2Pheno FPRs in both the plasma and proviral compartments showed good correlation (overall, r = 0.87; ART-naive patients, r = 0.79; ART-failing patients, r = 0.97). GTT was successfully performed in all 10 whole blood DNA samples having a viral load <500 copies/mL, all showing R5 tropism. CONCLUSIONS High concordance in tropism prediction from proviral DNA and plasma-viral RNA suggests that prediction of viral tropism using proviral DNA is accurate and feasible in resource-limited clinical settings, particularly in patients with low or suppressed viraemia.

Limited evolution but increasing trends of primary non-nucleoside reverse transcriptase inhibitor resistance mutations in therapy-naive HIV-1-infected individuals in India.

BACKGROUND After the rapid scale-up of antiretroviral therapy (ART) in resource-limited settings, surveillance of primary drug resistance mutations (DRMs) among ART-naive individuals has important public health benefits. Although a highly successful national ART programme initiated by the Government of India exists, data on the prevalence of primary DRMs is scarce. The objective of the study is to estimate the prevalence, pattern and spectrum of population-based primary DRMs in therapy-naive HIV-1-infected individuals using clinical strains and database sequences from seven HIV prevalent states of India. METHODS Drug resistance genotyping was performed on either plasma RNA or whole-blood genomic DNA using a validated in-house method on 170 HIV-1-positive therapy-naive individuals. An additional 679 database-derived sequences from four other states were included in the analysis. The WHO-recommended list of mutations (SDRM_2009) for nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were used for interpretation of DRMs. Trends of primary DRMs before and after the ART rollout were studied. RESULTS The overall prevalence of primary DRMs was 2.6% in the selected states of India when clinical isolates as well as database-derived sequences were combined. Common mutations included T69D and D67N (NRTI mutations), and L100I, K101E, K103N and Y181C (NNRTI mutations). There was a significant increase in NNRTI mutations over time. CONCLUSIONS The overall DRM prevalence in this study was low. However, an increasing trend in primary NNRTI resistance has been observed during the past decade. Establishment of HIV drug resistance threshold surveillance will be useful in understanding further trends of transmitted resistance.

Ceftriaxone resistant Shigella Flexneri, an emerging problem

Shigellosis is a disease of public health importance in developing countries. It may cause self-limited diarrhea to severe dysentery. Emergence of multi drug resistant (MDR) strains is a growing concern globally. Ceftriaxone and ciprofloxacin are the drugs of choice for MDR cases. Here, we report a case of MDR Shigella flexneri from an immunocompromised patient. The strain was resistant to ceftriaxone [minimum inhibitory concentration (MIC) ≥ 64 μg/ml], limiting the treatment option. Simultaneously, the strain was also found to be resistant to ciprofloxacin (MIC ≥ 4 μg/ml). However, it was susceptible to ceftazidime (MIC 4 μg/ml). This is the first case of ceftriaxone resistant Shigella spp. reported from our hospital.

In Vitro Evaluation of the Antimicrobial Efficacy of Four Endodontic Biomaterials against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus

Root canal sealers that possess good antimicrobial property can prevent residual and recurrent infection and contribute to successful endodontic therapy. This study evaluated the antimicrobial activity of four endodontic sealers, AH Plus, Tubliseal EWT, EndoRez, and iRoot SP, against three different microorganisms, E. faecalis, C. albicans, and S. aureus, by direct contact test. 10 μL microbial suspensions were allowed to directly contact the four endodontic sealers for 1 hr at 37°C. Subsequently microbial growth was measured spectrophotometrically every 30 min for 18 hours. The microbial suspensions were simultaneously tested to determine the antimicrobial effect of components which are capable of diffusing into the medium. The results revealed that AH Plus and iRootSP had significantly higher antimicrobial activity against E. faecalis. AH Plus and Tubliseal EWT showed significantly higher antimicrobial activity against C. albicans and S. aureus compared to iRoot SP and EndoRez. EndoRez showed the least antimicrobial activity against all the three microorganisms. Inhibition of microbial growth is related to the direct contact of microorganisms with the sealers. In conclusion AH Plus had significantly higher antimicrobial activity against E. faecalis, C. albicans, and S. aureus.

Performance of Genotypic Tools for Prediction of Tropism in HIV-1 Subtype C V3 Loop Sequences

Currently, there is no consensus on the genotypic tools to be used for tropism analysis in HIV-1 subtype C strains. Thus, the aim of the study was to evaluate the performance of the different V3 loop-based genotypic algorithms available. We compiled a dataset of 645 HIV-1 subtype C V3 loop sequences of known coreceptor phenotypes (531 R5-tropic/non-syncytium-inducing and 114 X4-tropic/R5X4-tropic/syncytium-inducing sequences) from the Los Alamos database (http://www.hiv.lanl.gov/) and previously published literature. Coreceptor usage was predicted based on this dataset using different software-based machine-learning algorithms as well as simple classical rules. All the sophisticated machine-learning methods showed a good concordance of above 85%. Geno2Pheno (false-positive rate cutoff of 5-15%) and CoRSeqV3-C were found to have a high predicting capability in determining both HIV-1 subtype C X4-tropic and R5-tropic strains. The current sophisticated genotypic tropism tools based on V3 loop perform well for tropism prediction in HIV-1 subtype C strains and can be used in clinical settings.