Comparison of capillary based microflurometric assay for CD4+ T cell count estimation with dual platform Flow cytometry

The CD4+ T cell count estimation is an important monitoring tool for HIV disease progression and efficacy of anti-retroviral treatment (ART). Due to availability of ART at low cost in developing countries, quest for reliable cost effective alternative methods for CD4+ T cell count estimation has gained importance. A simple capillary-based microflurometric assay (EasyCD4 System, Guava Technology) was compared with the conventional flow cytometric assay for estimation of CD4+ T cell counts in 79 HIV infected individuals. CD4+ T cell count estimation by both the assays showed strong correlation (r = 0.938, p < 0.001, 95% CI 0.90 to 0.96). The Bland Altman plot analysis showed that the limits of variation were within agreeable limits of ± 2SD (-161 to 129 cells/mm(3)). The Easy CD4 assay showed 100% sensitivity for estimating the CD4+ T cell counts < 200 cells/mm(3 )and < 350 cells/mm(3 )and 97% sensitivity to estimate CD4+ T cell count < 500 cells/mm(3). The specificity ranged from 82 to 100%. The Kappa factor ranged from 0.735 for the CD4+ T cell counts < 350 cells/mm(3 )to 0.771 for < 500 cells/mm(3 )CD4+ T cell counts. The system works with a simple protocol, is easy to maintain and has low running cost. The system is compact and generates minimum amount of waste. Hence the EasyCD4 System could be applied for estimation of CD4+ T cell counts in resource poor settings

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

<p>Abstract</p> <p>Background</p> <p>CD4+ 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.</p> <p>Methods</p> <p>A 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.</p> <p>Results</p> <p>The 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.</p> <p>Conclusion</p> <p>The 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.</p

QASI: A collaboration for implementation of an independent quality assessment programme in India

Objective: The HIV pandemic remains a significant global health concern. Accurate determination of CD4+ T-cells in patient samples relies on reliable CD4 enumeration. The Quality Assessment and Standardization programme for Immunological measures relevant to HIV/AIDS (QASI) programme of the Public Health Agency of Canada provides clinical laboratories from resource-limited countries with a mechanism to evaluate the quality of CD4 testing and develop the implementation of an independent national External Quality Assessment (EQA) programme. This study describes how QASI helped develop the capacity for managing a sustainable national CD4 EQA programme in India. Design: Supported by the Public Health Agency of Canada and Clinton Foundation HIV/AIDS Initiative, QASI engaged with the National AIDS Control Organization and the Indian National AIDS Research Institute to assist in technology transfer in preparation for the implementation/ management of an independent CD4 EQA programme. Technology transfer training was provided to support corrective actions and to improve the quality of CD4 testing. Inter- laboratory variation of EQA surveys between pre- and post-skill development was compared. Results: Prior to training, coefficient of variation values were 14.7% (mid-level CD4 count controls) and 39.0% (low-level). Following training, variation was reduced to 10.3% for mid- level controls and 20.0% for low-level controls. Conclusion: This training assisted the National AIDS Control Organization and the Indian National AIDS Research Institute in identifying the information necessary for management of an EQA programme, and developed the foundation for India to provide corrective actions for sites with challenges in achieving reliable results for CD4 enumeration. This led to a demonstrable improvement in CD4 testing quality and illustrates how country-specific training significantly improved CD4 enumeration performance for better clinical management of HIV care in India

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 1(st) and 2(nd) 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 1(st) 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/2009/091/00005

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

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.Standard population sequencing and cloning for viral subpopulations present at > or =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 infant's 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.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.ClinicalTrials.gov NCT00061321