HIV-1 low-level viraemia predicts virological failure in first-line and second-line ART-experienced individuals in India: A retrospective longitudinal study.

OBJECTIVE: To study the prevalence of low-level viraemia (LLV) and its association with virological failure (VF). METHODS: We conducted a retrospective analysis of 3498 participants at YRG CARE, Chennai, India (2013-2018) on antiretroviral therapy (ART) for ≥6 months with two or more plasma viral load (pVL) measurements. Results were stratified for those with pVL <1000 copies/mL: fully suppressed (FS) (pVL <40), low-LLV (pVL 40-199), mid-LLV (pVL 200-399), and high-LLV (pVL 400-999). The study assessed the association with VF (pVL >1000 copies/mL) using Cox proportional hazard model. RESULTS: Among 3498 participants, 2965 (84.8%) were FS and 533 (15.2%) were LLV. During the follow-up, 348 (10%) experienced VF, with 222 (6.3%) experienced after LLV (42% of LLV) and 126 (3.6%) experienced after FS (4.3% of FS). When compared with FS, those with LLV had a greater risk of VF [adjusted hazard ratio (aHR) = 12.7; 95% confidence interval (CI): 10.2-15.9]. First-line participants had a higher VF incidence (aHR = 15.8, 95% CI: 11.4-21.9) than second-line participants (aHR = 5.6, 95% CI: 4.1-7.7). Those with high-LLV had the highest VF risk (aHR = 22.856, 95% CI: 15.204-34.359 vs. aHR = 8.186, 95% CI: 5.564-12.043, for first-line vs. second-line participants, respectively), followed by those with mid-LLV (aHR = 13.375, 95% CI: 8.327-21.483 vs. aHR = 6.261, 95% CI: 4.044-9.695) and low-LLV (aHR = 12.976, 95% CI: 7.974-21.118 vs. aHR = 4.158, 95% CI: 2.826-6.119). CONCLUSIONS: The prevalence of LLV was intermediate in our study population. There was a higher risk of VF among individuals with LLV, and this risk increased with the increasing levels of LLV. Close monitoring of individuals experiencing LLV could help in the early identification of VF.

Performance characteristics of two new rapid HIV diagnostic assays and use of test band reader.

PURPOSE: Accurate HIV diagnosis is essential for appropriate patient care. This present study evaluated the performance of two different new rapid HIV diagnostic tests; 1) TRUSTline HIV-1/2 Ab rapid test (Athenese-DxPvt. Ltd, Chennai, India)and 2) OnSite HIV 1/2 Ab Plus Combo Rapid Test (CTK Biotech Inc., San Diego, USA) and also validated ALTA Rapid Test Reader (RTR-1) (CTK Biotech Inc., San Diego, USA), the device is a user-friendly and image-analysis based qualitative/semi-quantitative tabletop reader. METHODS: A total of n â€‹= â€‹500 characterized specimens were used for this evaluation and the results of the new test kits (TRUSTline and OnSite) were also compared with 4th generation ELISA kit (Genescreen™Ultra HIV Ag-Ab ELISA) and 3 other commercially available rapid tests that were in the market; 1)SD Bioline™ HIV 1/2 3.0, 2) Aspen® HIV 1/2 Rapid Ab Test and 3) Diagnostic enterprises HIVTRI-DOT. The test band intensities of the TRUSTline and OnSite tests were measured in an ALTA rapid test reader and compared with the naked eye reading. RESULTS: The sensitivity, specificity, positive predictive value, negative predictive value and efficacy of TRUSTline and OnSite were 100%, 99.6%, 99.5%, 100% and 99.8% and 100%, 100%, 100%, 100% and 100% respectively. CONCLUSIONS: The 'TRUSTline HIV-1/2' and 'OnSite HIV 1/2' kits are suitable to use in the HIV testing algorithm. Use of the ALTA rapid test reader could be user's friendly in the field level testing in resource-limited settings".

The Evolution of Regulatory Elements in the Emerging Promoter-Variant Strains of HIV-1 Subtype C.

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not the coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but no significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a coinfection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.

Occurrence of extended-spectrum ß-lactamase, AmpC, and carbapenemase-producing genes in gram-negative bacterial isolates from human immunodeficiency virus infected patients.

BACKGROUND: Progressive decline of immune response in HIV patients makes them susceptible to frequent bacterial infections. High usage of antibiotics influences the emergence of multidrug-resistant bacteria and worsens the clinical outcomes. In this study, the occurrence of drug-resistant genes in Gram-negative bacterial isolates from HIV patients in South India was analyzed. METHODS: A total of 173 Gram-negative bacterial (GNB) isolates from HIV patients were screened for antibiotic susceptibility profile using the Kirby-Bauer diskdiffusion method. Positivity of drug-resistant genes was analyzed using polymerase chain reaction method. RESULTS: In this study, 72.8% of bacterial isolates were obtained from urine specimens, and Escherichia coli (47.4%) was the predominantly isolated bacterium. Overall, 87.3% and 83.2% of GNB were resistant to 3rd generation cephalosporin antibiotics such as cefotaxime and ceftazidime, respectively, 56.6% were resistant to cephamycin (cefoxitin) and 43% to carbapenem (imipenem) antibiotics. Extended-spectrum ß-lactamases (ESBL) production was noted among 79.5% of GNB isolates, followed by AmpC (57.1%) and Metallo ß-lactamases (37.3%). Molecular analysis revealed that ESBL genes such as blaTEM (94.1%), blaCTX-M (89.2%), and blaSHV (24.2%) were detected at higher levels among GNB isolates. Carbapenemase-producing genes such as blaOXA-48 (20%), blaOXA-23 (2.6%), and both blaOXA-23 and blaOXA-51 like genes (2.6%) and AmpC producing genes such as blaCIT (26.7%), blaDHA (3.6%), and blaACC (1.8%) were detected at low-level. CONCLUSIONS: This study concludes that ESBL producing genes are detected at high level among gram-negative bacterial isolates from HIV patients in South India.

Awareness of and willingness to use pre-exposure prophylaxis (PrEP) among people who inject drugs and men who have sex with men in India: Results from a multi-city cross-sectional survey.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) is effective in reducing HIV transmission among key populations. In India, where PrEP is not currently part of the national HIV program, little is known about PrEP awareness, willingness to use PrEP, and barriers to uptake among people who inject drugs (PWID) and men who have sex with men (MSM). METHODS: We used respondent-driven sampling to accrue PWID and MSM in 22 sites from August 2016 to May 2017. Participants were asked about awareness of PrEP, willingness to use PrEP (following a brief description) and reasons why they might not be willing to use PrEP. Participants were also queried on preferences for PrEP delivery modality (oral vs. injectable). Multi-level logistic regression models were used to determine participant correlates of willingness to use PrEP. Estimates were weighted for the sampling method. RESULTS: A total of 10,538 PWID and 8,621 MSM who self-reported being HIV-negative were included in the analysis. Only 6.1% (95% confidence interval [CI]: 5.9, 6.3) of PWID and 8.0% of MSM (95% CI: 7.7, 8.4) were aware of PrEP. However, willingness to use PrEP was substantially higher in both groups: 52.4% of PWID and 67.6% of MSM. Participants commonly cited a perceived low risk for acquiring HIV infection, being perceived by others as being HIV-positive, and side effects as reasons why they would be unwilling to use PrEP. Among PWID, sharing needles and hazardous alcohol use were associated with increased willingness to use PrEP. Among MSM, having a main male partner and injection drug use were associated with increased willingness to use PrEP. Preference for daily oral or monthly injectable PrEP was similar among MSM (39.6%% vs. 41.7%,), while PWID were more likely to prefer oral to injectable administration routes (56.3% vs. 31.1%). CONCLUSIONS: As India plans to roll-out of PrEP in the public sector, our multi-city survey of PWID and MSM highlights the need for key population-focused education campaigns about PrEP and self-assessment of risk.

Performance characteristics of an instrument-free point-of-care CD4 test (VISITECT®CD4) for use in resource-limited settings.

OBJECTIVE: CD4+ T lymphocyte count remains the most common biomarker of immune status and disease progression in human immunodeficiency virus (HIV)-positive individuals. VISITECT®CD4 is an instrument-free, low-cost point-of-care CD4 test with a cut-off of 350 CD4 cells/µL. This study aimed to evaluate VISITECT®CD4 test's diagnostic accuracy. METHODS: Two hundred HIV-positive patients attending a tertiary HIV centre in South India were recruited. Patients provided venous blood for reference and VISITECT®CD4 tests. An additional finger-prick blood sample was obtained for VISITECT®CD4. VISITECT®CD4's diagnostic performance in identifying individuals with CD4 counts ≤350 cells/µL was assessed by calculating sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) taking flow cytometry as the reference. RESULTS: The overall agreement between VISITECT®CD4 and flow cytometry was 89.5% using venous blood and 81.5% using finger-prick blood. VISITECT®CD4 showed better performance using venous blood [sensitivity: 96.6% (95% confidence interval: 92.1%-98.9%), specificity: 70.9% (57.1%-82.4%), PPV: 89.7% (83.9%-94.0%) and NPV: 88.6% (75.4%-96.2%)] than using finger-prick blood [sensitivity: 84.8% (77.9%-90.2%), specificity: 72.7% (59.0%-83.9%), PPV: 89.1% (82.7%-93.8%) and NPV: 64.5% (51.3%-76.3%)]. CONCLUSION: VISITECT®CD4 performed well using venous blood, demonstrating its potential utility in decentralization of CD4 testing services in resource-constrained settings.

Integrating HCV testing with HIV programs improves hepatitis C outcomes in people who inject drugs: A cluster-randomized trial.

BACKGROUND & AIMS: There have been calls to integrate HCV testing into existing services, including harm reduction and HIV prevention and treatment, but there are few empirical trials to date. We evaluated the impact of integrating HCV testing/education into integrated care centers (ICCs) delivering HIV services to people who inject drugs (PWID) across India, using a cluster-randomized trial. METHODS: We compared ICCs with usual care in the PWID stratum (12 sites) of a 22-site cluster-randomized trial. In 6 sites, ICCs delivering HIV testing, harm reduction, other preventive services and linkage to HIV treatment were scaled from opioid agonist therapy centers and operated for 2 years. On-site rapid HCV antibody testing was integrated after 1 year. To assess impact, we conducted baseline and evaluation surveys using respondent-driven sampling (RDS) across the 12 sites (n = 11,993 recruited at baseline; n = 11,721 recruited at evaluation). The primary outcome was population-level self-reported HCV testing history. RESULTS: At evaluation, HCV antibody prevalence ranged from 7.2-76.6%. Across 6 ICCs, 5,263 ICC clients underwent HCV testing, of whom 2,278 were newly diagnosed. At evaluation, PWID in ICC clusters were 4-fold more likely to report being tested for HCV than in usual care clusters, adjusting for baseline testing (adjusted prevalence ratio [aPR] 3.69; 95% CI 1.34-10.2). PWID in ICC clusters were also 7-fold more likely to be aware of their HCV status (aPR 7.11; 95% CI 1.14-44.3) and significantly more likely to initiate treatment (aPR 9.86; 95% CI 1.52-63.8). CONCLUSIONS: We provide among the first empirical data supporting the integration of HCV testing into HIV/harm reduction services. To achieve elimination targets, programs will need to scale-up such venues to deliver comprehensive HCV services. CLINICALTRIALS. GOV IDENTIFIER: NCT01686750. LAY SUMMARY: Delivering hepatitis C virus (HCV) testing to people who inject drugs (PWID) in places where they also have access to HIV prevention and treatment services is an effective way to improve uptake of HCV testing among communities of PWID. To achieve the World Health Organization's ambitious elimination targets, integrated programs will need to be scaled up to deliver comprehensive HCV services.

Correction: Tuberculosis patients in an Indian mega-city: Where do they live and where are they diagnosed?

[This corrects the article DOI: 10.1371/journal.pone.0183240.].

Association of circulatory Tfh-like cells with neutralizing antibody responses among chronic HIV-1 subtype C infected long-term nonprogressors and progressors.

HIV-1 vaccine functioning relies on successful induction of broadly neutralizing antibodies (bNAbs). CXCR3- circulatory T-follicular helper (cTfh) cells are necessary for inducing B-cells for generating bNAbs. Recent studies have suggested that CXCR3+ Tfh cells might also influence bNAb production. Plasma samples from 34 ART-Naïve HIV-1 infected individuals [long-term nonprogressors (LTNP)-19; Progressors-13] were tested against a heterologous virus panel (n = 11) from subtypes A, B, C, G, AC, BC and AE. Frequencies of CXCR3+ and CXCR3- cTfh-like cells in peripheral circulation were studied using flow cytometry. LTNP showed significantly lower CXCR3+ and higher CXCR3- cTfh-like cell frequencies, while neutralization breadth was observed to be broader in progressors. A positive correlation was observed between bNAb breadth and potency with CXCR3+PD-1+ cTfh-like cells in LTNP. Based on neutralization breadth, 9 HIV-1 infected individuals were classified as 'top neutralizers' and 23 as 'low neutralizers' and they did not show any correlations with CXCR3+ and CXCR3- cTfh-like cells. These preliminary data suggest that CXCR3+ similar to CXCR3- might possess significant functional properties for driving B-cells to produce bNAbs. Hence, an HIV vaccine which is capable of optimal induction of CXCR3+ cTfh cells at germinal centers might confer superior protection against HIV.

Changing drug resistance profile in Pseudomonas aeruginosa infection among HIV patients from 2010-2017: A retrospective study.

OBJECTIVES: Pseudomonas aeruginosa is an important aetiological agent causing pneumonia, urinary tract infections and bacteraemia. High antibiotic use in nosocomial settings and for immunocompromised conditions results in increasing multidrug resistance. This study analysed the antimicrobial resistance profile of P. aeruginosa isolates in an HIV setting. METHODS: A total of 7386 clinical specimens were collected from HIV patients attending YRG CARE from 2010-2017. P. aeruginosa isolated from clinical specimens were identified conventionally, and antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method. RESULTS: A total of 260 P. aeruginosa strains were isolated, with 165 P. aeruginosa (63.5%) being isolated from hospitalised patients. A higher incidence of P. aeruginosa infection (25.8%) was observed in 2017, and most of the P. aeruginosa were isolated from sputum specimens (57.3%). A high level of resistance was noted to ceftazidime (49.6%), followed by ticarcillin (41.5%). Imipenem and meropenem resistance was observed in 15.0% and 16.9% of P. aeruginosa isolates, respectively. A high rate of imipenem resistance was noted in 2016 (46.2%) and a high rate of meropenem resistance was noted in 2017 (20.5%). An increasing resistance rate of P. aeruginosa was observed against aztreonam, cefepime, levofloxacin, meropenem, piperacillin, piperacillin/tazobactam, ticarcillin and tobramycin from 2010 to 2017. CONCLUSION: A constant increase in drug-resistant P. aeruginosa isolates from HIV patients was observed from 2010 to 2017. Findings from this study urge the need for periodical monitoring and surveillance of the P. aeruginosa resistance profile, especially in hospitalised and immunocompromised patients in resource-limited settings.

HIV-specific T-cell Responses and Generalized Activation in HIV-1 Infected Long-term Non-progressors and Progressors from South India.

BACKGROUND: Anti-viral cytokine expressions by cytotoxic T-cells and lower activation rates have been reported to correlate with suppressed HIV replication in long-term non-progressors (LTNP). Immune mechanisms underlying disease non-progression in LTNP might vary with HIV-1 subtype and geographical locations. OBJECTIVE: This study evaluates cytokine expression and T-cells activation in relation to disease non-progression in LTNP. METHODS: HIV-1 Subtype C infected LTNP (n=20) and progressors (n=15) were enrolled and flowcytometry assays were performed to study HIV-specific CD8 T-cells expressing IL-2, IFN-γ, TNF-α and MIP-1ß against gag and env peptides. CD4+ T-cell activation was evaluated by surface expression of HLADR and CD38. RESULTS: Proportions of cytokines studied did not differ significantly between LTNP and progressors, while contrasting correlations with disease progression markers were observed in LTNP. CD4+ T-cell activation rates were significantly lower in LTNP compared to progressors which indicate the potential role of T-cell activation rates in disease non-progression in LTNP. CONCLUSION: LTNP and progressors showed similar CD8+ T-cell responses, but final conclusions can be drawn only by comparing multiple immune factors in larger LTNP cohort with HIV-1 infected individuals at various levels of disease progression. A possible role of HIV-1 subtype variation and ethnic differences in addition to host-genetic and viral factors cannot be ruled out.

Mitochondrial DNA content of peripheral blood mononuclear cells in ART untreated & stavudine/zidovudine treated HIV-1-infected patients.

BACKGROUND & OBJECTIVES: Nucleoside reverse transcriptase inhibitors (NRTIs) are known to cause mitochondrial toxicity. This study was done to estimate mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) among human immunodeficiency virus (HIV) infected, NRTI treated and antiretroviral therapy (ART)-naïve patients and evaluate the utility of mtDNA content as a biomarker of mitochondrial toxicity. METHODS: mtDNA content in PBMCs of 57 HIV-infected ART untreated and 30 ART treated with stavudine (d4T) or zidovudine (AZT) containing regimen were compared against 24 low-risk healthy controls (LoRHC). RESULTS: There was a significant (P=0.01) reduction in mtDNA content among HIV-infected (104; 80-135) compared to LoRHC (127; 110-167), and it was the same in both the treated (104.8; 88-130) and untreated patients (104.7; 78-142). mtDNA significantly (P=0.014) declined in ART treated patients symptomatic for toxicity (97; 74-111) than the asymptomatic patients (128; 103- 153). INTERPRETATION & CONCLUSIONS: mtDNA depletion in PBMCs was evident among HIV-infected individuals on ART. Moreover, as mtDNA content was reduced among the patients symptomatic for toxicity than the asymptomatic in both the HIV-infected groups, the current study supports mtDNA content of PBMCs to serve as a biomarker of mitochondrial dysfunction induced by NRTI and HIV. Longitudinal studies with a large sample need to be done to confirm these findings.

Broad neutralization response in a subset of HIV-1 subtype C-infected viraemic non-progressors from southern India.

Broadly neutralizing antibodies (bnAbs) have been considered to be potent therapeutic tools and potential vaccine candidates to enable protection against various clades of human immunodeficiency virus (HIV). The generation of bnAbs has been associated with enhanced exposure to antigen, high viral load and low CD4+ T cell counts, among other factors. However, only limited data are available on the generation of bnAbs in viraemic non-progressors that demonstrate moderate to high viraemia. Further, since HIV-1 subtype C viruses account for more than 50 % of global HIV infections, the identification of bnAbs with novel specificities is crucial to enable the development of potent tools to aid in HIV therapy and prevention. In the present study, we analysed and compared the neutralization potential of responses in 70 plasma samples isolated from ART-naïve HIV-1 subtype C-infected individuals with various disease progression profiles against a panel of 30 pseudoviruses. Among the seven samples that exhibited a neutralization breadth of ≥70 %, four were identified as 'elite neutralizers', and three of these were from viraemic non-progressors while the fourth was from a typical progressor. Analysis of the neutralization specificities revealed that none of the four elite neutralizers were reactive to epitopes in the membrane proximal external region (MPER), CD4-binding site and V1V2 or V3 glycan. However, two of the four elite neutralizers exhibited enhanced sensitivity towards viruses lacking N332 glycan, indicating high neutralization potency. Overall, our findings indicate that the identification of potent neutralization responses with distinct epitope specificities is possible from the as yet unexplored Indian population, which has a high prevalence of HIV-1 subtype C infection.