Madhuri Thakar

Nevirapine Resistance and Breast-Milk HIV Transmission: Effects of Single and Extended-Dose Nevirapine Prophylaxis in Subtype C HIV-Infected Infants

Background Daily nevirapine (NVP) prophylaxis to HIV-exposed infants significantly reduces breast-milk HIV transmission. We assessed NVP-resistance in Indian infants enrolled in the “six-week extended-dose nevirapine” (SWEN) trial who received single-dose NVP (SD-NVP) or SWEN for prevention of breast-milk HIV transmission but who also acquired subtype C HIV infection during the first year of life. Methods/Findings Standard population sequencing and cloning for viral subpopulations present at ≥5% frequency were used to determine HIV genotypes from 94% of the 79 infected Indian infants studied. Timing of infection was defined based on when an infants blood sample first tested positive for HIV DNA. SWEN-exposed infants diagnosed with HIV by six weeks of age had a significantly higher prevalence of NVP-resistance than those who received SD-NVP, by both standard population sequencing (92% of 12 vs. 38% of 29; p = 0.002) and low frequency clonal analysis (92% of 12 vs. 59% of 29; p = 0.06). Likelihood of infection with NVP-resistant HIV through breast-milk among infants infected after age six weeks was substantial, but prevalence of NVP-resistance did not differ among SWEN or SD-NVP exposed infants by standard population sequencing (15% of 13 vs. 15% of 20; p = 1.00) and clonal analysis (31% of 13 vs. 40% of 20; p = 0.72). Types of NVP-resistance mutations and patterns of persistence at one year of age were similar between the two groups. NVP-resistance mutations did differ by timing of HIV infection; the Y181C variant was predominant among infants diagnosed in the first six weeks of life, compared to Y188C/H during late breast-milk transmission. Conclusions/Significance Use of SWEN to prevent breast-milk HIV transmission carries a high likelihood of resistance if infection occurs in the first six weeks of life. Moreover, there was a continued risk of transmission of NVP-resistant HIV through breastfeeding during the first year of life, but did not differ between SD-NVP and SWEN groups. As with SD-NVP, the value of preventing HIV infection in a large number of infants should be considered alongside the high risk of resistance associated with extended NVP prophylaxis. Trial Registration ClinicalTrials.gov NCT00061321

Incidence of common opportunistic infections in HIV-infected individuals in Pune, India: analysis by stages of immunosuppression represented by CD4 counts

BACKGROUND Opportunistic infections (OIs) influence the morbidity and mortality due to HIV infections. Data from India on the incidence of OIs among HIV-infected individuals by stages of immunodeficiency are scarce. METHODS Between September 2002 and November 2004, HIV-infected individuals were enrolled in a prospective study in Pune. They were clinically and immunologically evaluated quarterly. Incidence rates of specific OIs were calculated. RESULTS Median CD4 counts in HIV-infected male and female patients at baseline were 197/mm(3) and 413/mm(3), respectively. Tuberculosis was the most common OI with an incidence of 15.4 (95% CI 12.2-19.2) per 100 person-years, followed by oral candidiasis 11.3 (95% CI 8.6-14.5), herpes zoster 10.1 (95% CI 7.6-13.1), and cryptococcal meningitis 1.7 (95% CI 0.8-3.1) per 100 person-years. Patients with baseline CD4 counts of <200/mm(3) were six times more likely to develop OIs compared to those with CD4 counts of >350/mm(3) (p<0.001). CONCLUSIONS The high incidence of commonly reported OIs in Indian HIV-infected individuals highlights the need for early screening and also the need to increase awareness in healthcare providers, in order to improve decisions regarding prophylaxis for prevention and appropriate therapeutic intervention. Emphasis needs to be given to the early diagnosis and management of tuberculosis in HIV-infected individuals.

A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine.

A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman primates. In this randomized, dose escalation phase I trial, HIV-uninfected healthy volunteers (50 in Europe, 30 in India) received a single intramuscular injection of tgAAC09 at 3 x 10(9) DNase resistant particles (DRP) (n = 16), 3 x 10(10) DRP (n = 23), 3 x 10(11) DRP (n = 25), or placebo (n = 16). Twenty-one participants in Europe received a second (boost) dose of 3 x 10(11) DRP tgAAC09 or placebo at least 24 weeks after the first injection. The vaccine was safe and well-tolerated after initial and boost vaccination. Local and systemic reactogenicity was experienced by 13-25% of participants and was not dose related. No vaccine-related serious adverse events were reported. Modest HIV-specific T cell responses were detected in 7/64 vaccinees (40-385 SFC/10(6) PBMC), with 16% (4/25) responders in the highest dose group. All responses were to Gag epitopes. tgAAC09 appears to be safe, well-tolerated, and modestly immunogenic. Further evaluation of higher doses of tgAAC09 and boost injections is ongoing in Africa.

Safety and immunogenicity of DNA and MVA HIV-1 subtype C vaccine prime-boost regimens: a phase I randomised Trial in HIV-uninfected Indian volunteers.

Study Design A randomized, double-blind, placebo controlled phase I trial. Methods The trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos. Results Both vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1st and 2nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination. Conclusions Although DNA priming resulted in enhancement of immune responses after 1st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting. Trial Registration Clinical Trial Registry CTRI/2009/091/000051

HIV infection in India: epidemiology, molecular epidemiology and pathogenesis.

The year 1986 saw first case of HIV infection as well as first report of AIDS case in India. Since then the epidemic has spread throughout the country. In the recent years there is evidence of epidemic being stabilized with decrease in new infections reported from some parts of the country. The absolute number of HIV infections in the country is expected to be close to 2.5 million and National AIDS Control Programme, phase III is geared to contain the epidemic. HIV viruses circulating in India predominantly belong to HIV-1 subtype C. However, there have been occasional reports of HIV-1 subtype A and B. Matter of concern is reports of A/C and B/C mosaic viruses that are being reported from different parts of the country. The data on HIV drug resistance from India is rather limited. Most of the studies have shown that the virus strains from drug naïve patients do not show significant level of drug resistance mutations. The few immunological studies in Indian patients show that the Indian HIV infected patients show both HIV-specific CTL responses as well as neutralizing antibody response. Mapping of CTL epitopes showed that while Indian patients identify same regions of Gag antigen as recognized by South African subtype C infected patients, some regions are uniquely recognized by Indian patients. There are very few studies on host genetic factors in India in context with HIV infection. However there are evidences reported of association of host genetic factors such as HLA types and haplotypes and HIV disease.

Cytolytic T Lymphocytes (CTLs) from HIV-1 Subtype C–Infected Indian Patients Recognize CTL Epitopes from a Conserved Immunodominant Region of HIV-1 Gag and Nef

Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes--15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)--were predicted using a combination of peptide matrix ELISPOT assay and CTL epitope-prediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype C-affected regions.

Utility of the point of care CD4 analyzer, PIMA, to enumerate CD4 counts in the field settings in India

BackgroundIn resource limited settings non-availability of CD4 count facility at the site could adversely affect the ART roll out programme. Point of care CD4 enumerating equipments can make the CD4 count available at the site of care and improve the patients’ management considerably. This study is aimed at determining the utility of a Point of Care PIMA CD4 analyzer (Alere, Germany) in the field settings in India.MethodThe blood samples were collected from 1790 participants at 21 ART centers from different parts of the country and tested using PIMA and the reference methods (FACSCalibur, FACSCount and CyFlow SL3). The paired finger prick and venous blood samples from 175 participants were tested by the PIMA CD4 Analyzer and then by FACSCalibur.ResultThe CD4 counts obtained by PIMA CD4 analyzer showed excellent correlation with the counts obtained by the reference methods; for venous blood the Pearson’s r was 0.921, p < 0.001 and the relative bias was 0.2% (range: -42 to 42%) and for finger prick samples, the Pearson’s r was 0.856 and the relative bias was −9.1% (range: -46% to 27%). For CD4 ranges; <250, 251–350, 351–500 and >500 cells/mm3, the differences in the median CD4 counts obtained by the reference method and the PIMA analyzer were not significant (P > 0.05) and the relative bias were low (−7 to 5.1%). The Intermachine comparison showed variation within the acceptable limit of%CV of 10%.ConclusionIn the field settings, the POC PIMA CD4 analyzer gave CD4 counts comparable to the reference methods for all CD4 ranges. The POC equipment could identify the patients eligible for ART in 91% cases. Adequate training is necessary for finger prick sample collection for optimum results. Decentralization of CD4 testing by making the CD4 counts available at primary health centers, especially in remote areas with minimum or no infrastructure would reduce the missed visits and improve adherence of the patients.

Variations in autologous neutralization and CD4 dependence of b12 resistant HIV-1 clade C env clones obtained at different time points from antiretroviral naïve Indian patients with recent infection.

BackgroundLimited information is available on HIV-1 Indian clade C sensitivities to autologous antibodies during the course of natural infection. In the present study, a total of 37 complete envelope clones (Env) were amplified at different time points predominantly from the plasma of five Indian patients with recent HIV-1 infection and envelope-pseudotyped viruses were examined for their magnitude of sensitivity to autologous plasma antibodies during natural course of infection.ResultsVariable low levels of neutralization were consistently detected with contemporaneous autologous plasma. In contrast to clade B and African clade C HIV-1 envelopes, Env clones obtained from four patients were found to be resistant to IgG1b12. The majority of the Env clones were resistant to 2G12 and 2F5 due to the absence of the minimal motifs required for antibody recognition, but were sensitive to 4E10. Nonetheless, Env clones from one patient were found to be sensitive to 2G12, atypical for clade C, and one Env clone exhibited unusual sensitivity to 17b, suggesting spontaneous exposure of CD4i epitopes. Phylogenetic analysis revealed that Env clones were closely clustered within patients. Variation in the potential N-linked glycosylation pattern also appeared to be different in patients over the course of infection. Interestingly, we found that the sensitivity of Envs to contemporaneous autologous NAbs correlated positively with increased sensitivity to soluble CD4 and inversely with anti-CD4 antibody and Envs with increased NAb sensitivity were able to efficiently infect HeLa cells expressing low CD4.ConclusionOur data showed considerable variations in autologous neutralization of these early HIV-1 clade C Envs in each of these patients and indicate greater exposure to CD4 of Envs that showed increased autologous neutralization. Interestingly, Env clones obtained from a single patient at different time points were found to retain sensitivity to b12 antibody that binds to CD4 binding site in Env in contrast to Envs obtained from other patients. However, we did not find any association between increased b12 sensitivity of Envs obtained from this particular patient with their degree of exposure to CD4.

Pediatric Tuberculosis in Young Children in India: A Prospective Study

Background. India has one of the highest tuberculosis (TB) burdens globally. However, few studies have focused on TB in young children, a vulnerable population, where lack of early diagnosis results in poor outcomes. Methods. Young children (≤5 years) with suspected TB were prospectively enrolled at a tertiary hospital in Pune, India. Detailed clinical evaluation, HIV testing, mycobacterial cultures, and drug susceptibility testing were performed. Results. 223 children with suspected TB were enrolled. The median age was 31 months, 46% were female, 86% had received BCG, 57% were malnourished, and 10% were HIV positive. 12% had TB disease (definite or probable), 35% did not have TB, while TB could not be ruled out in 53%. Extrapulmonary disease was noted in 46%, which was predominantly meningeal. Tuberculin skin test (TST) was positive in 20% of children with TB. Four of 7 (57%) children with culture-confirmed TB harbored drug-resistant (DR) strains of whom 2 (50%) were multi-DR (MDR). In adjusted analyses, HIV infection, positive TST, and exposure to household smoke were found to be significantly associated with children with TB (P ≤ 0.04). Mortality (at 1 year) was 3 of 26 (12%) and 1 of 79 (1%), respectively, in children with TB and those without TB (P < 0.05). Conclusions. Diagnosis of TB is challenging in young children, with high rates of extra-pulmonary and meningeal disease. While the data on DR-TB are limited by the small sample size, they are however concerning, and additional studies are needed to more accurately define the prevalence of DR strains in this vulnerable population.

Establishment of reference CD4+ T cell values for adult Indian population

BackgroundCD4+ T lymphocyte counts are the most important indicator of disease progression and success of antiretroviral treatment in HIV infection in resource limited settings. The nationwide reference range of CD4+ T lymphocytes was not available in India. This study was conducted to determine reference values of absolute CD4+ T cell counts and percentages for adult Indian population.MethodsA multicentric study was conducted involving eight sites across the country. A total of 1206 (approximately 150 per/centre) healthy participants were enrolled in the study. The ratio of male (N = 645) to female (N = 561) of 1.14:1. The healthy status of the participants was assessed by a pre-decided questionnaire. At all centers the CD4+ T cell count, percentages and absolute CD3+ T cell count and percentages were estimated using a single platform strategy and lyse no wash technique. The data was analyzed using the Statistical Package for the Social Scientist (SPSS), version 15) and Prism software version 5.ResultsThe absolute CD4+ T cell counts and percentages in female participants were significantly higher than the values obtained in male participants indicating the true difference in the CD4+ T cell subsets. The reference range for absolute CD4 count for Indian male population was 381-1565 cells/μL and for female population was 447-1846 cells/μL. The reference range for CD4% was 25-49% for male and 27-54% for female population. The reference values for CD3 counts were 776-2785 cells/μL for Indian male population and 826-2997 cells/μL for female population.ConclusionThe study used stringent procedures for controlling the technical variation in the CD4 counts across the sites and thus could establish the robust national reference ranges for CD4 counts and percentages. These ranges will be helpful in staging the disease progression and monitoring antiretroviral therapy in HIV infection in India.