Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C.

INTRODUCTION: HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C. METHODS: Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict  the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability. RESULTS: The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2-6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region. CONCLUSIONS: We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.

Declining HIV prevalence and risk behaviours in Zambia: evidence from surveillance and population-based surveys.

OBJECTIVE: To examine trends in HIV prevalence and behaviours in Zambia during the 1990s. METHODS: The core Zambian system for epidemiological surveillance and research has two major components: (i) HIV sentinel surveillance at selected antenatal clinics (ANC) in all provinces; and (ii) population-based HIV surveys in selected sentinel populations (1996 and 1999). The former was refined in 1994 to improve the monitoring of prevalence trends, whereas the latter was designed to validate ANC-based data, to study change in prevalence and behaviour concomitantly and to assess demographic impacts. RESULTS: The ANC-based data showed a dominant trend of significant declines in HIV prevalence in the 15--19 years age-group, and for urban sites also in age-group 20--24 years and overall when rates were adjusted for over-representation of women with low education. In the general population prevalence declined significantly in urban women aged 15--29 years whereas it showed a tendency to decline among rural women aged 15-24 years. Prominent decline in prevalence was associated with higher education, stable or rising prevalence with low education. There was evidence in urban populations of increased condom use, decline in multiple sexual partners and, among younger women, delayed age at first birth. CONCLUSIONS: The results suggested a dominant declining trend in HIV prevalence that corresponds to declines in incidence since the early 1990s attributable to behavioural changes. Efforts to sustain the ongoing process of change in the well-educated segments of the population should not be undervalued, but the modest change in behaviour identified among the most deprived groups represents the major preventive challenge.

Studying dynamics of the HIV epidemic: population-based data compared with sentinel surveillance in Zambia.

OBJECTIVES: To establish population-based HIV survey data in selected populations, and to assess the validity of extrapolation from HIV sentinel surveillance amongst antenatal clinic attenders (ANC) to the general population. METHODS: In a population survey, adults aged > or = 15 years were selected by stratified random cluster sampling (n = 4195). The survey was carried out in catchment populations of clinics used for national HIV surveillance. The methodology allows detailed comparisons of HIV infection patterns to be made in two areas (urban and rural). Whereas the sentinel surveillance used serum-based HIV testing, the population survey used saliva (93.5% consented to provide a saliva sample). RESULTS: Surveillance of ANC tended to underestimate the overall HIV prevalence of the general population, but differences were not statistically significant. In the urban area, the adjusted overall HIV prevalence rate of ANC (aged 15-39 years) was 24.4% [95% confidence interval (CI), 20.9-28.0] compared with 26.0% (95% CI, 23.4-28.6) in the general population. The respective rural estimates were 12.5% (95% CI, 9.3-15.6) versus 16.4% (95% CI, 12.1-20.6). Age-specific prevalence rates showed ANC to overestimate infection in teenagers (aged 15-19 years), whereas in the reverse direction of those aged > or = 30 years. Teenagers analysed by single year of age revealed both ANC and women in the general population with about the same steep increase in prevalence by age, but the former at consistently higher rates. Extrapolations might be biased substantially due to the higher pregnancy rates amongst uninfected individuals. CONCLUSIONS: ANC-based data might draw a rather distorted picture of current dynamics of the HIV epidemic. Even though representing an obvious oversimplification, extrapolations of overall prevalence rates may correlate with that of the general population.

PIP: Assessments of the dynamics of the HIV/AIDS epidemic in Africa are based largely on sentinel surveillance of antenatal clinic (ANC) attenders. The validity of use of this key sentinel group in terms of HIV prevalence estimation was evaluated in a population-based survey of 4195 Zambian adults recruited through stratified random cluster sampling in urban Lusaka and rural Mposhi district in 1995-96. The survey was carried out in catchment populations of clinics used for national HIV surveillance. Overall, ANC surveillance tended to underestimate HIV prevalence in the general population, but differences were not statistically significant in either the urban or the rural area. In Lusaka, the adjusted overall HIV prevalence among ANC attenders was 24.4% compared with 26.0% in the population-based survey. The rural estimates were 12.5% and 16.4%, respectively. Comparison of age-specific prevalences indicated ANC overestimated infection in teenagers and underestimated HIV in those aged 30 years and over. Prevalence rates increased steeply by age, at comparable increment levels among women in both samples, while men had stable prevalence rates by age. These findings suggest that extrapolations from surveillance data may indeed produce a distorted picture of the current dynamics of the HIV epidemic in the population at large. Women with higher educational attainment are underrepresented in the ANC surveys. The higher pregnancy and birth rates among HIV-negative compared to HIV-positive women is another potential source of bias, as is the high rate of ANC clients aged 18-19 years (66%, compared with 34% in the general population of women). Information on single year of age, educational level, and residence should be collected in sentinel surveillance systems to allow appropriate stratification. Since HIV prevalence in young people may be the most reliable marker of changes in incidence, sample size increases in the 15-19 year age group should be considered.

The HIV epidemic in Zambia: socio-demographic prevalence patterns and indications of trends among childbearing women.

OBJECTIVE: To examine socio-demographic HIV prevalence patterns and trends among childbearing women in Zambia. DESIGN: Repeated cross-sectional surveys. METHODS: Personal interviews and unlinked anonymous testing of blood samples of women attending antenatal care in selected areas. RESULTS: The 1994 data includes information from 27 areas and a total of 11,517 women. The HIV prevalence among urban residents appeared with moderate variation at a very high level (range 25-32%, comparing provinces). The geographical variation was more prominent in rural populations (range 8-16%) and was approximately half the prevalence level of the urban populations. With the exception of the 15-19 years age-group, HIV infection was found to rise sharply with increasing educational attainment (odds ratio, 3.1; confidence interval, 2.6-3.8) when contrasting extreme educational levels. Although the assessment of trends is somewhat restricted, the available information indicates stable prevalence levels in most populations over the last 2-4 years. CONCLUSIONS: The data showed extremely high HIV prevalence levels among childbearing women. Longer time-intervals between surveys are needed, however, in order to verify the stability in prevalence identified by this study. The tendency to changing differentials by social status is suggested as a possible sign of an ongoing process of significant behavioural change.

PIP: A series of cross-sectional surveys conducted in Zambia revealed extremely high HIV prevalence among childbearing women. The analysis was based on data derived from an HIV sentinel surveillance system established among childbearing-age women in Zambia in 1990 and expanded in 1994 to improve geographic coverage and obtain data on socio-demographic characteristics. In 1994, when complete data on HIV status was obtained on 11,517 women from 27 areas, overall HIV prevalence was 19.8%. This rate ranged from 22% to 35% in Lusaka and provincial headquarters, from 25% to 32% in urban areas, and from 8% to 16% in rural areas. In both urban and rural areas, HIV prevalence was highest in the 25-29 year age group (34% and 17%, respectively). With the exception of the 15-19 year group, seroprevalence also rose significantly with increasing educational attainment; the odds ratio was 3.13 for the most educated compared to the least educated women. The lack of HIV prevalence socioeconomic differentials among 15-19 year olds, who became sexually active at a time when information about AIDS prevention was available, may be indicative of a process of behavioral change. Only 1 area (Kalabo) showed a trend of increase in the past 2-3 years; in other areas, HIV infection rates appear to have stabilized.