A protocol for feasibility of plasma based GeneXpert platform and Dried Blood Spot (DBS) based Abbott platform for HIV-1 viral load testing among the people living with HIV attending ART centers in India.

BACKGROUND: HIV-1 Viral load (VL) measures efficiency of the antiretroviral therapy (ART) after treatment initiation and helps to diagnose virological failures at an early stage. Current VL assays require sophisticated laboratory facilities. As well as there are other challenges pertaining to insufficient laboratory access, cold-chain management and sample transportation. Hence the number of HIV-1 VL testing laboratories is inadequate in the resource limited settings. The revised national tuberculosis elimination programme (NTEP) in India has developed a vast network of point of care (PoC) testing facilities for diagnosis of tuberculosis and several GeneXpert platforms are functional under this programme. Both the GeneXpert HIV-1 assay and HIV-1 Abbott real time assay are comparable and GeneXpert HIV-1 assay can be used as PoC for HIV-1 Viral load testing. Also, the dried blood spot (DBS) as a sample type has been considered as a good option for HIV-1 VL testing in hard to reach areas. This protocol is therefore developed to assess the feasibility of integrating HIV-1 VL testing among people living with HIV (PLHIV) attending ART centres using the two public health models under the current programme: 1. HIV-1 VL testing using GeneXpert platform and plasma as a sample type, and 2. HIV-1 VL testing using Abbott m2000 platform and DBS as a sample type. METHODS: This ethically approved feasibility study will be implemented at two moderate to high burden ART centres where VL testing facility is not available in the town. Under Model-1, arrangements will be made to carry out VL testing on the adjacent GeneXpert facility and under Model-2, DBS will be prepared on site and couriered to identified viral load testing laboratories. In order to assess the feasibility, data will be collected on pretested questionnaire pertaining to number of samples tested for VL testing, number of samples tested for tuberculosis (TB) diagnosis and the turnaround time (TAT). In-depth interviews will be conducted among the service providers at ART centre and different laboratories for addressing any issues regarding the model implementation. RESULTS: The proportion of PLHIV tested for VL at ART centres, total TAT for both models including TAT for sample transportation, sample testing and receipt of results as well as proportion of sample rejections and reasons for the same, correlation coefficient between DBS based and plasma based VL testing will be estimated using various statistical tools. CONCLUSION: If found promising, these public health approaches will be helpful for the policy makers and program implementation in scaling up HIV-1 viral load testing within India.

Access, timeliness and retention for HIV testing under early infant diagnosis (EID) program, India.

Early Infant Diagnosis of HIV infection services are crucial for managing the perinatally acquired HIV infection. Assessing the performance of the EID services and its underlying determinants is important for the National AIDS Control Program, India. The objectives of this study were to find out access to HIV testing, the timeliness of the testing cascade, and the proportion of HIV exposed infants who are followed up to 18 months for a definitive diagnosis of HIV. The study design was a mixed method. A total of 11 states accounting for 80% of HIV-positive pregnant women were selected. Program records from a total of 62 Integrated counselling and testing centres (ICTCs) served as the source of information. The qualitative component included interviews of program managers at the state and district level, service providers at the ICTC level, and caregivers of HIV exposed infants. In the sampled 62 ICTCs, 78% of the HIV exposed infants had at least one HIV test. Of the infants who had HIV tests, 50% had at first sample collected by 8 weeks of age. The median turnaround time from sample collection to DNA PCR testing was 36 (IQR 19-70) days and that to next sample collection in case of detection of virus in the first sample was 66 (IQR 55-116) days. At 18 months of age, 544 (62%) HIV exposed infants were retained in the EID testing cascade. A total of 30 infants were diagnosed with HIV at a median age of 421 (IQR 149-650) days. More than three fourth of the HIV exposed infants had access to early infant diagnosis (EID) services. Both demand and supply-side factors contribute to access, timeliness and retention and there is a need to address these factors.

Loss to follow-up of HIV-exposed infants for confirmatory HIV test under Early Infant Diagnosis program in India: analysis of national-level data from reference laboratories.

BACKGROUND: Early Infant Diagnosis was launched in India in 2010 and its effect on the diagnosis of HIV-exposed infants needs to be assessed. The present study was done to find out the median age at DBS sample collection for early infant diagnosis and its trend over years, the median age at diagnosis of HIV among the HIV-exposed infants with DNA PCR tests, and the proportion of infants who completed testing cascades after detection of HIV-1 in a sample. METHODS: DNA PCR data (from 2013 to 2017) maintained at all regional reference laboratories in India was collated with each infant identified by a unique code. Cohort analysis of the infant data was used to find the median age at sample collection and diagnosis. The outcomes of testing in each cascade and the overall outcomes of testing for infants were prepared. RESULTS: The median age at sample collection for the four years combined at all India level was 60 days (48-110 days). The median age at diagnosis of HIV was 285 days (174-418 days). HIV-1 was detected in samples of 1897 (6.3%) infants out of 30,216 infants who had a DNA PCR test, out of whom 1070 (56.4%) completed the testing cascade and the rest were lost to follow-up. CONCLUSION: The data highlights delay in diagnosis; both due to delay in sample collection and turn-around-times. Loss to follow-up of HIV-exposed infants with virus detection is a significant concern to the Early Infant Diagnosis and tracking systems need to be strengthened.

Reference Ranges of Different Lymphocyte Subsets in Indian Children: A Multi-Centric Study.

OBJECTIVE: To determine the reference ranges of various lymphocyte subsets in healthy Indian children. DESIGN: Descriptive cross-sectional study. SETTING: Four centers in India representing four geographical regions. PARTICIPANTS: 1104 children from neonatal age to 18 years of age. MEASUREMENT: One time measurement of absolute count and percentages of different lymphocyte subsets i.e. T lymphocytes (CD3+T, CD4+T, CD8+T cells), B lymphocytes (CD19+B cells) and Natural Killer lymphocytes (CD15/16+NK cells) in whole blood using multicolor flow cytometry. RESULTS: The absolute cell counts of various lymphocytes were found to increase from newborn to 10 months of age, followed by gradual decline until 18 years; however, the proportion of immune cells remained largely similar. Gender did not have a significant impact on the reference ranges, whereas counts were found to vary as per the geographical locations. CONCLUSIONS: These reference ranges will be useful to monitor and predict the immune status in pediatric population. The variation in region wise ranges could be confirmed by testing more number of samples in the specific age groups.

Persons living with HIV continue to present late for care: A trend analysis from 2011 to 2015.

CONTEXT: The clinical and prevention benefits of early initiation of antiretroviral therapy (ART) have led to the adoption of test and treat policy for HIV. Early diagnosis of HIV is crucial for maximal benefits from ART. AIMS: This study aims to assess trends in CD4 cell counts at diagnosis and determinants of late presentation. SETTINGS AND DESIGN: We analyzed 5-year data from a free HIV/sexually transmitted infection referral clinic immune. SUBJECTS AND METHODS: Persons presenting for HIV testing from January 2011 to December 2015, for whom CD4 cell count results were available within 3 months of HIV diagnosis, were included in the analysis. Persons on ART were excluded from the study. STATISTICAL ANALYSIS: The predictors of CD4 cell count at presentation were assessed using univariate and multivariate linear regression. RESULTS: Of 1001 persons diagnosed HIV-1 positive, 659 had received CD4 test within 3 months of diagnosis. The median CD4 count at presentation ranged from 212 to 352 cells/cmm in these 5 years and did not show any significant change with time. Nearly 40% had CD4 cell counts below 200 cells/cmm (AIDS); additionally, 23% presented below 350 cells/cmm. Older age (beta: -5.78; P = 0.001), education above matriculation (beta: -123.72; P = 0.014), having current opportunistic infections (beta: -173.58; P = 0.037), and being symptomatic (beta: -101.8; P = 0.002) were predictors of presenting at lower CD4 counts. CONCLUSION: Between 2011 and 2015, persons with HIV continued to present late in spite of changes in ART access program. Education focused on the benefits of early diagnosis and availability of free immediate treatment in the public sector, are crucial to the achievement of the India's 90-90-90 goals.

Differing HIV vulnerability among female sex workers in a high HIV burden Indian state.

INTRODUCTION: The HIV sentinel surveillance [HSS] conducted in 2010-11 among female sex workers [FSW] in the state of Maharashtra, India provided an opportunity to assess characteristics of different types of FSWs and their HIV risk. It is important for India's National AIDS Control Program, to understand the differences in vulnerability among these FSW, in order to define more specific and effective risk reduction intervention strategies. Therefore, we analyzed data from HSS with the objective of understanding the HIV vulnerability among different types of FSW in Maharashtra. MATERIAL AND METHODS: Cross sectional data collected as a part of HSS among FSWs in year 2010-11 from 21 sentinel sites in the state of Maharashtra were analyzed to understand the vulnerability and characteristics of different types of female sex workers based on their place of solicitation using multinomial logistic regression. RESULTS: While the HIV prevalence was 6.6% among all FSWs, it was 9.9% among brothel based [BB], 9% among street based [SB] and 3.1% and 3.7% among home based [HB], and bar based [Bar-B] sex workers respectively. SB FSWs were least likely to be located in HIV low burden districts [ANC] [ARRR: 0.61[95% CI: 0.49, 0.77]], but were 6 times more likely to be recently [<1 year] involved in sex work [ARRR: 6.15 [95% CI: 3.15, 12.0]]. The number of clients of SB FSWs in the preceding week were lower than 11% [ARRR: 0.89 [95%CI: 0.87, 0.90]] as compared to the BB FSWs denoting lesser client load. The duration since last paid sex was shorter [ARRR: 0.94[95%CI: 0.91, 0.96]] as compared to the BB FSWs. CONCLUSION: Street based FSWs have emerged as one of the most vulnerable types of FSW with a high HIV prevalence similar to BB FSWs. Our study reveals that they have more frequent sex acts despite lower client loads, and are more likely to be located in districts highly affected by HIV (ANC prevalence >1%). We identify them as a group to be focused on for prevention interventions and it is likely that they would be easily amenable to novel interventions due to their higher literacy rate as compared to other typologies.

Characterization of vaginal lactobacilli from HIV-negative and HIV-positive Indian women and their association with genital HIV-1 shedding.

One of the crucial determinants for successful administration of lactobacilli to the vaginal niche is the use of appropriate Lactobacillus species. In this cross-sectional study 54 human immunodeficiency virus (HIV)-negative and 76 HIV-positive antiretroviral treatment-naïve women were evaluated for culturable vaginal lactobacilli and their association with genital HIV-1 shedding. Lactobacillus species were identified by 16S rDNA sequencing while cervical and plasma HIV-1 viral load was determined by Abbott real-time PCR. Lactobacilli were isolated in 77.8 % HIV-negative and 73.7 % HIV-positive women. The mean log10 plasma and cervical HIV-1 viral loads (RNA copies ml-1) were 3.73±1.02 and 2.85±0.32 respectively. We observed that presence of L. crispatus, L. gasseri or L. jensenii species was associated with undetectable cervical HIV-1 (P=0.046) and reduced genital HIV-1 shedding (P=0.048) compared to other species. Our findings endorse using Lactobacillus-based strategies to aid the prevention of HIV-1 transmission among Indian women, however confirmation by future prospective studies is indeed warranted.

Fitting HIV Prevalence 1981 Onwards for Three Indian States Using the Goals Model and the Estimation and Projection Package.

Models are designed to provide evidence for strategic program planning by examining the impact of different interventions on projected HIV incidence. We employed the Goals Model to fit the HIV epidemic curves in Andhra Pradesh, Maharashtra and Tamil Nadu states of India where HIV epidemic is considered to have matured and in a declining phase. Input data in the Goals Model consisted of demographic, epidemiological, transmission-related and risk group wise behavioral parameters. The HIV prevalence curves generated in the Goals Model for each risk group in the three states were compared with the epidemic curves generated by the Estimation and Projection Package (EPP) that the national program is routinely using. In all the three states, the HIV prevalence trends for high-risk populations simulated by the Goals Model matched well with those derived using state-level HIV surveillance data in the EPP. However, trends for the low- and medium-risk populations differed between the two models. This highlights the need to generate more representative and robust data in these sub-populations and consider some structural changes in the modeling equation and parameters in the Goals Model to effectively use it to assess the impact of future strategies of HIV control in various sub-populations in India at the sub-national level.

Failure to Use and Sustain Male Condom Usage: Lessons Learned from a Prospective Study among Men Attending STI Clinic in Pune, India.

BACKGROUND: Sustained or consistent use of condoms by men remains a challenge. A study was carried out to identify factors associated with failure to use condoms consistently by men attending STD clinics in Pune, India. METHOD: Among 14137 STI clinic attendees, 8360 HIV sero-negative men were enrolled in a cohort study. The changes in condom usage behavior were studied among 1284 men who returned for first scheduled quarterly follow up, 309 reported consistent condom use at the time of enrollment in the cohort. Data pertaining to heterosexual men practicing high risk behavior were analyzed to identify factors associated with change in condom use behavior using logistic regression model. Demographic, behavioral and biological factors observed to be associated with condom use were fitted in five Cox proportional hazards models to calculate hazard ratios and their 95% confidence intervals to identify independent predictors of failure to sustain condom use behavior. RESULTS: The univariate analysis showed that men who were 30 years or older in age (p = 0.002) and those who did not have contact female sex worker (FSW) were more likely to fail to sustain consistent condom use. However both these factors did not show significant association in multivariable analysis. Marital status and contact with Hijra (eunuch) in lifetime were associated with failure to change in their condom use behavior [AOR 0.33 (CI 0.13-0.82; p = 0.017)]. During the follow up of 2 years, 61 events (15.5 per 100 person years, 95% CI 12.3-19.5 years) of 'failure of condom use' were recorded despite counseling. Older age, contact with non CSW partner and presence of genital ulcer disease / discharge syndrome were significant predictors of failure to sustain condom use. DISCUSSION: Married monogamous older men, who report contact with sex worker and present with genital ulcer disease are at risk of failure to use condom after first exposure to voluntary HIV counseling and testing. This is a scenario of primary prevention program. Condom promotion and counseling needs to be reinforced through follow up counseling among this population.

Use of first line antiretroviral therapy from a free ART programme clinic in Pune, India - a preliminary report.

BACKGROUND & OBJECTIVES: The treatment outcomes under national antiretroviral therapy (ART) programme are being evaluated in some ART centres in the country. We carried out this study to analyze the impact of first line antiretroviral therapy in HIV infected patients attending a free ART roll out national programme clinic in Pune, India. METHODS: Antiretroviral naive HIV infected patients attending the clinic between December 2005 and April 2008 and followed up till March 31, 2011 were included in the analysis. The enrolment and follow up of these patients were done as per the national guidelines. Viral load estimations were done in a subset of patients. results: One hundred and forty two patients with median CD4 count of 109 cells/µl (IQR: 60-160) were initiated on treatment. The median follow up was 44 months (IQR: 37-53.3 months). Survival analysis showed that the probability of being alive at the end of 5 years was 85 per cent. Overall increase in the median CD4 count was statistically significant (P<0.001). It was significant in patients with >95 per cent adherence (P<0.001). In 14 per cent patients, the absolute CD4 count did not increase by 100 or more cells/µl at the end of 12 months. Viral load estimation in a subset of 68 patients showed undetectable levels in 61 (89.7%) patients after a median duration of 46 months (IQR: 38.3-54.8). INTERPRETATION & CONCLUSIONS: The first line treatment was effective in patients attending the programme clinic. The adherence level influenced immunological and virological outcomes of patients.

Increased expression of virulence attributes in oral Candida albicans isolates from human immunodeficiency virus-positive individuals.

Oral candidiasis caused by Candida albicans is recognized as one of the most frequent opportunistic infections in human immunodeficiency virus (HIV)-infected individuals. The overall severity and chronicity of oral candidiasis has been attributed exclusively to the HIV-induced immune deficiency of the affected individuals but not to the virulence factors of the pathogen, i.e. C. albicans. However, genotypic and phenotypic studies have suggested that HIV infection might be associated with preferential selection of C. albicans strains with altered virulence determinants, leading to colonization with Candida populations that are better able to cause disease in these immunologically compromised hosts. If this process of selection is indeed related to pathogenicity, it may be possible to measure alterations in different virulence factors produced by C. albicans in HIV-infected patients. To evaluate this hypothesis, the present work was undertaken to determine simultaneously the expression of five virulence factors in oral C. albicans isolates colonizing and infecting HIV-positive and -negative individuals. The significance of genotypes in the pathogenesis of oral candidiasis was also elucidated. Oral swabs were collected from 335 consecutive individuals (210 HIV-positive and 125 HIV-negative). Virulence factors and genotypes were determined for all the C. albicans strains isolated. The results showed significantly increased expression of proteinase, phospholipase and haemolytic activities, as well as a greater ability to adhere, in isolates from HIV-positive compared with HIV-negative individuals (P<0.05). However, no significant differences in virulence factor expression in isolates colonizing or infecting HIV-positive individuals were seen. Genotype A was the predominant type (71.3 %); however, a relationship could not be established between the genotypes and the virulence factors, or with clinical infection. These data support the concept of preferential C. albicans strain selection with altered virulence determinants in HIV-infected individuals and emphasize the need for further molecular genetic linkage studies that could be helpful in dissecting the molecular causes of preferential strain selection, which may lead to new approaches for therapeutic intervention.

Predictors of retention among men attending STI clinics in HIV prevention programs and research: a case control study in Pune, India.

BACKGROUND: Retention is critical in HIV prevention programs and clinical research. We studied retention in the three modeled scenarios of primary prevention programs, cohort studies and clinical trials to identify predictors of retention. METHODOLOGY/PRINCIPAL FINDINGS: Men attending Sexually Transmitted Infection (STI) clinics (n = 10, 801) were followed in a cohort study spanning over a ten year period (1993-2002) in Pune, India. Using pre-set definitions, cases with optimal retention in prevention program (n = 1286), cohort study (n = 940) and clinical trial (n = 896) were identified from this cohort. Equal number of controls matched for age and period of enrollment were selected. A case control analysis using conditional logistic regression was performed. Being employed was a predictor of lower retention in all the three modeled scenarios. Presence of genital ulcer disease (GUD), history of commercial sex work and living away from the family were predictors of lower retention in primary prevention, cohort study and clinical trial models respectively. Alcohol consumption predicted lower retention in cohort study and clinical trial models. Married monogamous men were less likely to be retained in the primary prevention and cohort study models. CONCLUSIONS/SIGNIFICANCE: Predicting potential drop-outs among the beneficiaries or research participants at entry point in the prevention programs and research respectively is possible. Suitable interventions might help in optimizing retention. Customized counseling to prepare the clients properly may help in their retention.

Adherence to buccal epithelial cells, enzymatic and hemolytic activities of Candida isolates from HIV-infected individuals.

Candida spp., along with other microorganisms are usually found in the normal flora of the human oral mucosa. However in HIV-infected individuals these yeasts can become opportunistic pathogens and lead to mucosal infections. The transformation from a harmless commensal to a virulent pathogen under conditions of dysfunctional host defense system is attributable to an extensive repertoire of selectively expressed virulence determinants. The present study was undertaken to analyze the production of four important virulence factors viz. adherence to buccal epithelial cells (BEC), proteinase activity, phospholipase activity and hemolysis, and to evaluate the correlation between these virulence factors in 65 Candida isolates from HIV-infected individuals with oral candidiasis. A total of 95.3%, 67.7%, 41.5% and 100% of the Candida isolates showed adherence to BEC, and proteinase, phospholipase and hemolytic activities, respectively. Production of proteinase and phospholipase enzymes was seen in 89.7% and 59.0% of C. albicans isolates and 34.6% and 15.4% of non-Candida albicans Candida (NAC) isolates, respectively. C. albicans showed significantly greater level of virulence factor expression with regards to adherence to BEC (P < 0.001), phospholipase production (P < 0.044) and hemolysis (P = 0.037) as compared to NAC. A correlative relationship between proteinase activity and adherence to BEC, as well as phospholipase production was noted.