Realtime hiv 1 assay

The testing described in this report was performed at the HPTN Laboratory Center (Baltimore, MD). Recency was assessed using four MAAs that included two or more assays (Table 1). These four MAAs were developed for population-level cross-sectional HIV incidence estimation. The HIV-1 Limiting Antigen (LAg)-Avidity EIA (Sedia Biosciences Corporation, Beaverton, OR; LAg-Avidity assay) was performed according to manufacturer’s instructions. The JHU BioRad-Avidity assay was performed as previously described [35 (link)]; this assay is based on the Genetic Systems 1/2 + O ELISA (Bio-Rad Laboratories, Redmond, WA). The rapid LAg assay (Asanté HIV-1 Rapid Recency assay, Sedia Biosciences Corporation, Beaverton, OR) was performed according to manufacturer’s instructions; results for all samples were read manually by a single laboratory technician. Samples where the long-term infected band was not detected were categorized as recent. Viral load testing was performed previously using the RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL) with a validated dilution procedure (limit of quantification: 400 copies/mL) [32 (link)]. Antiretroviral (ARV) drug testing was performed using an assay that detects 22 drugs in five drug classes [36 (link)]. Detection of one or more ARV drugs was considered to be indicative of ART.

At each visit, a laboratory‐based HIV antigen/antibody test was performed at centralized laboratories in Zambia and South Africa. Additional testing was performed retrospectively at the HPTN Laboratory Center (Johns Hopkins Univ., Baltimore, MD, USA) [6 (link)], including confirmation of seroconversion events and determination of the timing of HIV acquisition. Viral load testing was performed using the RealTime HIV‐1 assay (Abbott Molecular; validated dilution method, limit of quantification: 400 copies/ml). Further details of laboratory testing are described elsewhere [6 (link)].

Participants were enrolled at the clinic of the National Tuberculosis Programme (NTP) in Manhiça village after informed consent and data were collected through specific questionnaires. Blood samples were obtained for viral load testing using the automated RealTime HIV-1 Assay (Abbott Molecular). If participants had no recent CD4 results (documented within the last 3 months), TruCount blood tubes (Becton Dickinson Biosciences, San Jose, CA) were also collected to determine T-cells counts by flow cytometry.
As part of the TB diagnostic workup, participants provided two sputum samples, which were received the day following enrolment at the CISM laboratory. Sputum induction was performed for individuals that were unable to provide sputum spontaneously. Clinically diagnosed or laboratory-confirmed TB patients were managed according to the National Tuberculosis Programme (NTP) guidelines and were started on TB treatment. In case of discordant results between RT-MTB (positive) and the standard of care (culture and Xpert negative), patients were contacted for re-evaluation and were requested to provide a third sputum sample to aid the decision of treatment iniciation. All participants were scheduled for a follow-up visit 2 months after enrolment. Those who did not attend the clinic on day 60 were contacted by telephone and interviewed.