AIDS Malignancy Consortium 054: Safety and Immunogenicity of the Quadrivalent Vaccine in Indian Women Living With HIV.

BACKGROUND: Human papillomavirus (HPV)-associated cervical cancer is a leading cause of death among Indian women. Indian women living with HIV (WLWH) may be at especially high risk. The quadrivalent HPV (qHPV) vaccine is effective in prevention of initial infection with HPV-6/11/16/18 in HIV-negative women. Little is known about previous exposure to HPV-6/11/16/18, safety, and immunogenicity of qHPV in Indian WLWH. METHODOLOGY: One hundred fifty WLWH with different CD4 levels and HIV viral load (VL) were vaccinated at 0/2/6 months at CART-CRS-IDMC, Chennai, India. Serology was performed at weeks 0, 28, and 52 for HPV-6/11/16/18 using a competitive Luminex immunoassay and for HPV-16/18 using a pseudovirion-based neutralization assay. RESULTS: Mean age was 30.8 years (range, 19-44 years). 71/87/73/81% of women were naive (sero-negative and DNA-negative) to HPV-6/11/16/18 at baseline, respectively. Among per-protocol women naive to HPV-6/11/16/18 at baseline, 100/99/99/90%, respectively, seroconverted at week 28 and 95/96/98/71% were sero-positive at week 52, respectively. Pseudovirion-based neutralization assay identified more seroconversion to HPV-18 than competitive Luminex immunoassay. There were no significant differences in the proportion seroconverting by baseline or nadir CD4 or HIV VL; however, there was a trend for increased proportion seroconverting to HPV-18 among women with higher baseline CD4 level (P = 0.052). There were no qHPV-related serious adverse events and no change in CD4 level or HIV VL among women on ART. CONCLUSIONS: qHPV vaccine was safe and immunogenic in Indian WLWH. A high proportion were naive to HPV-6/11/16/18 and may benefit from vaccination although many were married and several years post-initiation of sexual activity.

Brief Report: The Anti-HIV-1 ADCC-Mediating Antibodies From Cervicovaginal Secretions of HIV-Infected Women Have an Ability to Mediate Lysing of Autologous CD4+ HIV-Infected Cells.

BACKGROUND: Fragment crystallizable region of antibody-mediated mechanism such as antibody-dependent cellular cytotoxicity (ADCC) has been identified as an important component of immune protection against HIV. We assessed whether the anti-HIV antibodies mediating ADCC from cervicovaginal lavages (CVLs) of HIV-infected women have an ability to mediate lysing of autologous CD4 HIV-infected cells. METHODOLOGY: The CVLs of 62 HIV-infected (37 long-term slow progressors and 25 with progressive HIV infection: progressors) and 20 HIV-uninfected Indian women with high risk of HIV acquisition were tested for the presence of ADCC-mediating anti-HIV antibodies against HIV-1 C Env in a fluorometric assay. Furthermore, we tested the ability of these antibodies to mediate ADCC-dependent killing of the autologous HIV-infected CD4 T cells using paired peripheral blood mononuclear cells containing target and effector cells. RESULTS: The numbers of ADCC responders were significantly higher in long-term slow progressors (34/37) as compared to the progressor group (9/25) with no significant difference in the magnitude. The magnitude of response was inversely associated with detectable CVL viral load (P < 0.003). The lysis of target cells was significantly higher in enriched IgG fraction as compared to the respective non-IgG fraction. The ADCC antibodies from CVLs significantly reduced the frequency of HIV-1 Env-activated autologous CD4 T cells in the presence of autologous effector cells. CONCLUSIONS: The presence of ADCC antibodies in CVLs with an ability to mediate lysing of HIV-infected autologous CD4 T cells provides evidence of their promising contribution to mucosal defense against HIV-1 and has implications in designing prophylactic and immunotherapeutic strategies.

Occult HBV infection in HIV-infected adults and evaluation of pooled NAT for HBV.

The study aimed to determine the prevalence of occult hepatitis B virus infection among HIV-infected persons and to evaluate the use of a pooling strategy to detect occult HBV infection in the setting of HIV infection. Five hundred and two HIV-positive individuals were tested for HBV, occult HBV and hepatitis C and D with serologic and nucleic acid testing (NAT). We also evaluated a pooled NAT strategy for screening occult HBV infection among the HIV-positive individuals. The prevalence of HBV infection among HIV-positive individuals was 32 (6.4%), and occult HBV prevalence was 10%. The pooling HBV NAT had a sensitivity of 66.7% and specificity of 100%, compared to HBV DNA NAT of individual samples. In conclusion, this study found a high prevalence of occult HBV infection among our HIV-infected population. We also demonstrated that pooled HBV NAT is highly specific, moderately sensitive and cost-effective. As conventional HBV viral load assays are expensive in resource-limited settings such as India, pooled HBV DNA NAT might be a good way for detecting occult HBV infection and will reduce HBV-associated complications.

Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library.

More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 µg/mL to 100 µg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design.

A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India.

BACKGROUND: The converging epidemics of HIV and tuberculosis (TB) pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. OBJECTIVE: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS) assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB) directly from sputum specimens, in the Indian setting. MATERIALS AND METHODS: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. RESULTS: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P<0.001). CONCLUSION: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.

Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India.

PURPOSE: Human immunodeficiency virus (HIV) diagnostic tests are being used extensively in India. However, the evaluation data on these assays are very limited. The present study evaluates indigenous HIV test kits manufactured in India. MATERIALS AND METHODS: A total of 200 characterised specimens were assayed with Comb AIDS - RS Advantage HIV 1+2 Immunodot Test, Enzaids HIV 1+2 ELISA test, Enzaids Duet HIV Antigen+antibody ELISA test and Signal HIV Flow Through HIV 1+2 test kits. Performance characteristics of these assays were calculated. RESULTS: Sensitivity, specificity, positive predictive value, negative predictive value and efficiency of all the assays were 100% except for Signal HIV Flow Through HIV 1+2 test kit. The specificity, positive predictive value and efficiency of the Signal HIV Flow Through HIV 1+2 test kit were 98.9%, 98.9% and 99.4%, respectively. The Enzaids Duet HIV kit was found to be extremely sensitive in detecting p24 Ag with the sensitivity of 1.5 pg/mL. CONCLUSIONS: To conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. All these assays under evaluation in this report have got excellent performance characteristics and much suitable to use in serial testing algorithms in use for resources limited settings.

Couples at risk for HIV infection in Southern India: characteristics of HIV-infected patients in concordant and discordant heterosexual relationships.

The aim of the article is to compare the clinical and behavioural characteristics of HIV-infected South Indian patients in concordant and discordant heterosexual relationships. A cross-sectional analysis of married couples in concordant and discordant relationships was carried out. Demographic and clinical characteristics, sexual behaviours, CD4 cell count and plasma HIV-1 RNA loads were assessed. A total of 839 concordant patients and 996 discordant patients were included in this analysis. Significantly more men were in discordant than concordant relationships (97% versus 59%; P = 0.002). More discordant patients had never initiated highly active antiretroviral treatment (HAART) than concordant patients (14.1% versus 8.5%; P = 0.004). Concordant patients had significantly higher CD4 cell counts than discordant patients at the time of enrolling to care (205 versus 139 cells/microL; P = 0.001). Discordant patients had significantly higher plasma viral loads than concordant patients (100,000 copies/mL versus 89,154 copies/mL; P = 0.002). Discordant patients were more likely to use condoms with their spouses than concordant patients (49% versus 28.8%; P = 0.01). In conclusion, couples-based interventions and the provision of HAART could substantially decrease behavioural and clinical correlates of HIV transmission among discordant South Indian married couples. The spouses of HIV-infected index patients are at increased risk for HIV infection, and further preventive measures are needed.

Establishment of T-lymphocyte subset reference intervals in a healthy adult population in Chennai, India.

Estimation of CD4+ T-lymphocytes continues to be an important aspect for monitoring HIV disease progression and response to antiretroviral therapy. Most of the diagnostic laboratories often rely on western text books for CD4+ T-lymphocyte reference values, which could, often be unreliable for usage in local settings. Therefore, we attempted to establish the reference values for T-lymphocyte subsets among healthy adults in a cross-sectional study carried out at the YRG Centre for AIDS Research and Education (YRG CARE) in Chennai, south India, in 213 (84 female and 129 male) healthy, HIV-1/2 seronegative adults as volunteers. Whole blood specimens were processed for CD4+, CD8+ T-lymphocyte estimation and haematological parameters. The established range of CD4+ T-lymphocyte counts for men and women were 383-1347 cells/microl (mean 865 and median 845 cells/microl) and 448-1593 cells/microl (mean 1021 and median 954 cells/microl), respectively. Women had significantly higher absolute CD4+ Tlymphocyte counts (P<0.001) and CD4+:CD8+ T-lymphocyte ratio as compared to men. The established normal range of CD4+ T-lymphocyte % was 21-59 (mean 40.2 and median 40.1). The influence of age was not observed in any of the parameters except CD4+/CD8+ T-lymphocyte ratio with the >45 yr age group. Further studies with greater sample size may be required to define the staging of HIV disease in relation to the normal CD4 T-lymphocyte count in the general population.

Performance characteristics of a new rapid immunochromatographic test for the detection of antibodies to human immunodeficiency virus (HIV) types 1 and 2.

Diagnostic kits for the detection of human immunodeficiency virus (HIV) antibodies have reached an unprecedented number. But choice of an ideal, cost-effective, and rapid test for HIV infection is of immense value for use in developing countries like India, where resources are limited. In this study we have evaluated the performance characteristics of the rapid immunochromatographic HIV test kit First Response HIV 1-2.O. First, the laboratory archived 450 characterized plasma/serum specimens, which were tested on First Response HIV 1-2.O. Second, a total of 134 consecutive voluntary counseling and testing (VCT) specimens were also tested and positive specimens were further confirmed with HIV TRI-DOT. All these VCT specimens were cross-checked with HIV double-enzyme-linked immunosorbent assay (ELISA) (Murex and Vironostika), and the results were matched. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency of First Response HIV 1-2.O with the 450 characterized specimens was 100% for HIV-1 with reference assay. The results in the VCT algorithm were correlating with double-ELISA. In the HIV-2 analysis, five HIV-2-positive specimens in First Response HIV 1-2.O were found to be HIV-2-indeterminate on Western blot. HIV TRI-DOT was unable to pick up two HIV-2 Western blot-positive specimens. First Response HIV 1-2.O has several advantages: low-cost (U.S. $0.70); only 10 microL of specimen; involves only two steps; room temperature storage; ability to differentiate HIV-1 and 2; and use of whole blood specimen. Hence this test kit could be suitable for initial screening in the HIV testing algorithm in resource-limited settings. J. Clin. Lab. Anal. 22:178-185, 2008. (c) 2008 Wiley-Liss, Inc.

Ethnic variation in certain hematological and biochemical reference intervals in a south Indian healthy adult population.

BACKGROUND: We established the biochemical and hematological reference intervals among a south Indian healthy adult population attending an HIV referral centre in Chennai, southern India. METHODS: In a cross sectional study, 213 study subjects (129 male and 84 female) were studied between March and August 2005. All of the parameters were analyzed using standard hematological and biochemical techniques. RESULTS: Certain biochemical (viz. total bilirubin, alanine transaminase, albumin, creatinine, total protein, lipid profile, creatine phosphokinase, uric acid and lactate) and hematological (mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and lymphocyte levels) parameters presented higher upper limits. In addition, the upper limits of white blood cell count, platelet count, hematocrit, red blood cell count and hemoglobin level were low in comparison to the currently reported ranges. CONCLUSION: Ethnic variation in reference intervals was observed in certain biochemical and hematological analytes in a south Indian adult population.

Use of an HIV-1 reverse-transcriptase enzyme-activity assay to measure HIV-1 viral load as a potential alternative to nucleic acid-based assay for monitoring antiretroviral therapy in resource-limited settings.

An inexpensive and technically less-demanding methodology to quantify HIV-1 viral load would be of great value for resource-limited settings, where the nucleic-acid amplification technique (NAAT) is impractical and/or resource-prohibitive. In this study, an HIV-1 reverse-transcriptase enzyme-activity assay (ExaVir Load assay, version 1) was compared with the gold standard RT-PCR assay, Roche HIV-1 Amplicor Monitor, version 1.5. A total of 121 plasma specimens were used for the evaluation. ExaVir Load had a sensitivity of 97 % and a specificity of 71 % in identifying specimens with <400 copies ml(-1) in the Roche RT-PCR assay as being less than the detection limit of the assay (5500 copies ml(-1)). The mean difference (95 % limits of agreement) between Roche RT-PCR and ExaVir Load was -0.23 (-1.59 to 1.13) log(10)(copies ml(-1)) by Bland-Altman analysis. Significant negative correlations were seen between CD4(+) T-cell counts and the ExaVir Load assay (r=-0.32, P<0.05), and between CD4(+) T-cell counts and the Roche RT-PCR (r=-0.38, P<0.01). The present study with HIV-1 showed a strong correlation between the ExaVir Load assay and the RT-PCR assay. Hence, the ExaVir Load assay could be considered for use in resource-limited settings as an alternative viral-load assay to the standard NAAT-based assay after further evaluation with prospective specimens.

High proportion of isosporiasis among HIV-infected patients with diarrhea in southern India.

We investigated 245 diarrheal stool specimens from HIV-positive subjects between January 2003 and December 2006 to determine the etiological role of coproparasites. Parasitic etiology was observed in 91 (37.1%) cases. Isospora belli (26.1%) was the most common parasite followed by Entameba histolytica/dispar (3.3%), Cryptosporidium spp. (2.9%), Giardia intestinalis (1.6%), and Strongyloides stercoralis (1.2%). Interesting trends of significant increase in the number of cases of I. belli and decline in Cryptosporidium spp. were observed during the study period.

Hydrothorax in association with Scopulariopsis brumptii in an AIDS patient in Chennai, India.

Over the past decade, an increasing number of opportunistic fungal infections have been reported in immunocompromised subjects. Microascus spp. and their anamorphs Scopulariopsis spp. have been recovered from a wide geographical range. We report a case of Scopulariopsis brumptii in a 27-year-old man with AIDS presenting with breathlessness, pericardial effusion and hydrothorax in Chennai, southern India, in February 2007. Because of respiratory arrest, the patient was intubated. However, the patient developed obstructive shock and died due to cardiac dysfunctions. This report underlines the need for a direct, intensive approach to investigations in immunocompromised patients, especially those with AIDS.

Prevalence of aflatoxin B1 in liver biopsies of proven hepatocellular carcinoma in India determined by an in-house immunoperoxidase test.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death in the world. The incidence of HCC in India is reportedly low and varies from 0.2 to 1.9 %. Aflatoxins, secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, are potent human carcinogens implicated in HCC. The prevalence of aflatoxin B1 (AFB1) as co-carcinogen was analysed using an in-house immunoperoxidase test in 31 liver biopsies and 7 liver-resection specimens from histopathologically proven HCC, and in 15 liver biopsies from cirrhosis patients (control group). Serum was tested for hepatitis B and C serological markers using commercial assays, and for AFB1 using an in-house ELISA with a sensitivity of approximately 1 ng ml(-1) for AFB1. In spite of positive AFB1 immunostaining in HCC cases, all serum specimens, from both HCC and the control groups, were AFB1-negative. There were 18 (58.1 %) HCC cases that revealed AFB1 in liver biopsies; 68.8 % (n=11) of non-B non-C hepatitis cases with HCC and 46.1 % (n=6) of the hepatitis B surface-antigen-positive subjects were positive for AFB1. Out of the two hepatitis B/hepatitis C virus co-infected cases, one was positive for AFB1. Of seven tumour-resection samples, six were positive for AFB1. Only one case revealed AFB1 in the non-tumour area of the resected material. Thus AFB1 staining was significantly associated with tumour tissue (P=0.03). Aflatoxins proved to have a significant association with HCC in this peninsular part of the subcontinent. The impact seems to be a cumulative process, as revealed by the AFB1 deposits in HCC liver tissue, even though the serum levels were undetectable.

Detection of pulmonary Mycoplasma pneumoniae infections in HIV-infected subjects using culture and serology.

OBJECTIVE: The true prevalence of Mycoplasma pneumoniae infections involving the respiratory tracts of HIV-infected individuals is still unclear. This study examined the prevalence of M. pneumoniae in 100 HIV-infected individuals at an AIDS care center in Chennai, India, using conventional laboratory techniques and interpretation criteria. METHODS: Diagnosis was based on culture, cold agglutination test, and commercial enzyme-linked immunosorbent assay (ELISA) for the qualitative determination of IgM antibodies against M. pneumoniae. The efficacies of the different diagnostic procedures used in the study were analyzed. RESULTS: The prevalence of M. pneumoniae was 31% by culture and 21% by IgM ELISA. Cough (p=0.03, OR 3.8, 95% CI 1-17.8), myalgia (p=0.04, OR 2.5, 95% CI 1-6.6), rales (p=0.04, OR 2.4, 95% CI 1-6.6), and cervical adenopathy (p=0.03, OR 2.7, 95% CI 1-7.1) were the symptoms that significantly corroborated culture positivity. Patients positive for M. pneumoniae by culture or IgM antibody had significantly greater CD4+ T-cell depletion and anemia than those without any evidence of infection. CONCLUSIONS: This study provides the means to diagnose M. pneumoniae infection and information on the prevalence of the pathogen in HIV-infected individuals in resource constrained settings. Although modern molecular techniques may provide more insight into the prevalence of M. pneumoniae in HIV-infected individuals, conventional methods can still be used in diagnosis.

Evaluation and diagnostic usefulness of domestic and imported enzyme-linked immunosorbent assays for detection of human immunodeficiency virus type 1 antibody in India.

Diagnosis of human immunodeficiency virus (HIV) infection is important for patient management and prevention of new infections. The number of test kits available for the detection of HIV antibodies is unprecedented. In order to identify appropriate test kits, we evaluated a variety of commercial kits manufactured abroad as well as in India. The plasma and serum specimens (n=264) were collected from individuals attending the Voluntary Counseling and Testing Centre at the YRG Centre for AIDS and Education. The specimens were used to evaluate six commercially available HIV test kits: Enzaids HIV 1+2, HIV-CheX, Murex HIV-1.2.0, Genscreen HIV 1/2 version 2, Vironostika HIV Uni-Form II Ag/Ab, and CombAids RS Advantage. High sensitivities and specificities (>or=99%) were observed for the Enzaids, Murex, Vironostika, and CombAids assays. HIV-CheX showed the highest number of false-positive and false-negative results. The Genscreen test also gave many false positives. The study indicated that the Enzaids, Murex, and Vironostika enzyme-linked immunosorbent assay kits and the CombAids RS Advantage rapid assay could be used to achieve acceptable results for the detection of HIV antibodies. A combination of two tests is recommended to optimize the efficiency of HIV antibody testing algorithms, especially when evaluation with an HIV Western blot confirmatory test is not possible.

HIV-1 western blot assay: What determines an indeterminate status?

BACKGROUND: The Western blot assay is the gold standard for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate Western blot reactivity to HIV-1 proteins may occur in individuals, who may not be infected with HIV. AIM: This retrospective study was aimed to determine the diagnostic value of the interpretation criteria in relation to commercial kits for HIV-1 diagnosis. METHODS AND MATERIALS: A total of 556 serum/plasma specimens collected from high-risk population attending our HIV clinic from 2000-2004 were tested by three different western blot kits: NEW LAV BLOT I (n=244), HIV BLOT 2.2; (n=112), Genetic Systems HIV-1 (n=237). And the results of western blot strips were analyzed using the various interpretation criteria: WHO/NACO, CDC/ ASTPHLD, ARC, FDA, CRSS and JHU. Some specimens were run on more than one kit. RT-PCR assay was performed on 5 specimens, which were indeterminate with LAV BLOT I. RESULTS: The discrepancy in LAV BLOT I positive results were between 157(64)-176(72), and indeterminate results were between 44(18) to 63(25). No such variations were observed in genetic systems. There are some HIV negative (by PCR) specimens were indeterminate in LAV BLOT I revealing the kit more sensitive and less effective for diagnostic purpose. CONCLUSION: The genetic systems kit is superior to other kits we analyzed and its results are concordant with HIV-1 PCR results. To report, the choice of western blot commercial kit is paramount important than the use of particular interpretation criteria for the diagnosis of HIV-1.