HIV was diagnosed according to national testing guidelines in each country, including algorithms based on HIV rapid tests and/or immunoassays. CD4 count was enumerated using standard clinical flow cytometric methods at each site. HIV-1 RNA was measured via nucleic acid amplification methods on one of several testing platforms with lower limit of quantification 20–48 copies/mL, including the Cobas® Ampliprep/Cobas® TaqMan HIV-1 Test, v2.0 (Roche Diagnostics), High Pure/COBAS® TaqMan® HIV-1 Test v2.0 (Roche Diagnostics,), COBAS® AmpliPrep/COBAS® TaqMan® 48 HIV-1 Test (Roche Diagnostics), or Real Time HIV-1 Viral Load assay (Abbott). All testing was performed according to package inserts.
Real time hiv 1 viral load assay
Manufactured by Abbott
Sourced in United States
The Real Time HIV-1 Viral Load assay is a quantitative in vitro diagnostic test used for the detection of HIV-1 RNA in human plasma. The assay provides a measurement of the amount of HIV-1 virus present in a patient's blood sample.
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Lab products found in correlation
Facscount, by BD (2 mentions)
Liaison instrument, by DiaSorin (1 mentions)
Gs hiv combo ag ab eia, by Bio-Rad (1 mentions)
Facspresto, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Multispot hiv 1 2 rapid test, by Bio-Rad (1 mentions)
Architect hiv ag ab combo assay, by Bio-Rad (1 mentions)
Aptima hiv 1 rna qualitative assay, by Hologic (1 mentions)
16 protocols using real time hiv 1 viral load assay
1
HIV Diagnosis, CD4 Enumeration, and Viral Load Measurement
2
Quantifying HIV-1 Viral Load and Mutations
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HIV-RNA load in plasma was measured using a COBAS AmpliPrep/COBAS Taqman HIV-1 assay or the Abbott RealTime HIV-1 Viral Load assay depending on the participating site. The primary endpoint at week 24 was defined as a short-term virological response and categorized by responders (VL < 50 cp/mL) or non-responders (VL > 50 cp/mL). If no data at week 24 were available, the closest time point to the primary endpoint at week 24 was taken to assign the short-term response category. In a post hoc analysis, additional follow-up data were used to assign a long-term virological response category of persistent responders (VL < 50 cp/mL in all measurements), individuals with persistent LLV (VL > 50 cp/mL in more than two measurements, >25% of measurements) or individuals with 1–2 blips. Plasma RNA was isolated according to an LLV protocol using higher plasma inputs varying from 1.0 to 6.0 mL depending on the VL, followed by ultracentrifugation, then RT-PCR and population-based Sanger sequencing of reverse transcriptase and protease (pol). RNA sequences and the presence of any type of mutation was evaluated using the Stanford HIVdb-algorithm version 8.5. The evolution of the viral population was evaluated using MEGA-X version 10.0.1 and using the dN/dS ratio with the Nei-Gojobori model when two or more pol sequences from one individual were available.
3
Galectin-9 Levels in HIV-Infected Patients
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The study was conducted at the ICMR-National AIDS Research Institute (ICMR-NARI), India, after the approval of the designed protocol by the ICMR-National AIDS Research Institute Ethics Committee. Written informed consent was obtained from the study participants before enrolling them in the study. Both viremic (>1000 copies/mL) and aviremic PLHIV on antiretroviral therapy were enrolled from the institute’s clinic. Viremic and aviremic HIV PLHIV were categorized based on VL monitoring by Abbott real-time HIV-1 viral load assay (Abbott, Chicago, IL, USA) with a lower limit of detection of 40 RNA copies/mL. Participants with a VL of less than 40 copies/mL or target not detected (TND) were selected as aviremic, whereas those with a VL of more than 1000 copies/mL were selected as viremic PLHIV. Assessment of serum creatinine, blood urea nitrogen, blood pressure, and inflammatory markers was completed on a subset of aviremic individuals (n = 70). HIV-uninfected individuals (n = 69) were also enrolled to assess total galectin-9 levels in them.
4
HIV Viral Load and CD4/CD8 Assessment
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Blood samples were collected for the described laboratory analyses that were conducted at the International Clinical Laboratory (ICL), in Addis Ababa, Ethiopia. HIV testing was performed according to the national guidelines, while HIV-1 RNA levels in plasma were quantified using the Abbott RealTime HIV-1 Viral Load assay (Abbott Laboratories, Chicago, IL, USA). The CD4+/CD8+ T cell counts was determined using BD FACSCount (BD Biosciences, San Jose, NJ, USA). Levels of 25(OH)D3 in plasma were analyzed at the Department of Clinical Chemistry, Karolinska University Hospital in Stockholm, Sweden using a chemiluminescence immunoassay (CLIA) on a LIAISON-instrument (DiaSorin Inc., Stillwater, MN, USA), detectable range 7.5–175 nmol/L, CV 2–5%. Safety assessments to monitor AEs included clinical examinations (week 4, 8, 16, and 24) and blood chemistry analysis (week 0, 4, 8, and 16) to measure liver and kidney function, and calcium/phosphate homeostasis.
5
Comprehensive HIV Monitoring Protocol
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Laboratory assessment at each visit included CD4 T-cell count, plasma HIV viral load, full heamogram, serum chemistry, serum cryptococcal antigen, and QuantiFERON for TB exposure. HIV RNA viral load was assessed every 6 months using several different platforms; Roche Cobas Ampliprep/Cobas TaqMan HIV-1 Test, v2.0 (linear range 20–10,0000,000 copies/mL), Roche High Pure/COBAS TaqMan HIV-1 Test v2·0 (linear range 34–10,000,000 copies/mL), Roche COBAS AmpliPrep/COBAS TaqMan 48 HIV-1 Test (linear range 48–10,000,000 copies/mL) or Abbott Real Time HIV-1 Viral Load assay (linear range 40–10,000,000 copies/mL). Adherence was assessed by self-report.
6
Rapid HIV and STI Screening Protocol
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Rapid screening tests were used in the clinics to determine HIV‐1 infection status, as per Kenyan HIV testing guidelines. HIV‐positive results were confirmed via GS HIV Combo Ab/Ab EIA (Bio‐Rad, Hercules) and Avioq HIV‐q Microelisa System (Avioq Inc.) at the JC Wilt lab in Winnipeg, Manitoba. HIV viral loads were also quantified using the RealTime HIV‐1 viral load assay (Abbott) on an automated m2000 RealTime system (Promega). STIs including Neisseria gonorrhea were tested using the Seegene Anyplex 6‐plex kit from pre‐enema rectal swabs.
7
Comprehensive HIV Status Determination
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Five assays were used to determine HIV status (Supplemental File 1 ). Testing was performed at in‐country laboratories using a 4th‐generation HIV test (the CE marked ARCHITECT HIV Ag/Ab COMBO test [Architect]). The LC performed the Architect test (cleared by the United States FDA); a second 4th‐generation test (the GS HIV Combo Ag/Ab EIA [BioRad], performed during PC0, PC12, and PC24); a 5th‐generation test (the BioPlex 2200 HIV Ag/Ab assay [BioPlex], performed during PC36); a discriminatory HIV test (Geenius HIV‐1/2 Supplemental assay [Geenius]); and an HIV RNA test (Abbott RealTime HIV‐1 Viral Load assay [HIV RNA]; validated dilution method, limit of quantification: 400 copies/mL). Selected samples were tested with both the BioRad and BioPlex assays. To streamline testing, samples were tested once with the Architect test at all three laboratories.
8
Multiplatform HIV Viral Load Monitoring
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HIV RNA was quantified assessed using several different platforms over the course of the study: Roche Cobas Ampliprep/Cobas TaqMan HIV Type 1 (HIV-1) Test, v2.0 (linear range 20–10 0000 000 or 48–10 000 000 copies/mL); Roche High Pure/COBAS TaqMan HIV-1 Test, v2.0 (linear range 34–10 000 000 copies/mL); COBAS® AmpliPrep/COBAS® TaqMan® 48 HIV-1 Test (linear range 48–10 000 000 copies/mL); or Abbott Real Time HIV-1 Viral Load assay (linear range 40–10 000 000 copies/mL). CD4 T-cell count was assessed using BD FACSCount, BD FACSCalibur, BD FACSCanto II, or BD FACSPresto. Full hemogram was assessed using Beckman Coulter AcT5 Diff and Sysmex ×1000i.
9
Comprehensive HIV Screening Protocol
10
Retrospective Testing for HIV Infection
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Retrospective testing was performed at the HPTN Laboratory Center to confirm HIV infection and determine the timing of infection (see File S1 in the supplemental material) (3 (link)). This testing included qualitative RNA testing, VL testing, HIV genotyping, and measurements of drug concentrations (3 (link)). Qualitative RNA testing was performed using the Aptima HIV-1 RNA qualitative assay (Hologic, Inc., San Diego, CA). VL testing was performed using the RealTime HIV-1 viral load assay (Abbott Molecular, Des Plaines, IL) (LLOQ of 40 copies/mL); selected samples were analyzed using a single-copy RNA assay (3 (link)). Drug concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (3 (link), 15 (link), 16 (link)). HIV genotyping was performed using the GenoSure PRIme assay (Monogram Biosciences, South San Francisco, CA) for samples with VLs of ≥500 copies/mL. Samples with VLs of <500 copies/mL were analyzed using a low-VL single-genome sequencing INSTI genotyping assay (low-VL SGS-IN) (CAB arm only) (4 (link)). Resistance-associated mutations (RAMs) were classified as major or accessory mutations using the Stanford University HIV Drug Resistance Database (17 (link), 18 ). HIV subtyping was performed at Monogram Biosciences (3 (link)).
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