Discrepant amplification results during the development of an assay leads to reclassification of two AIDS reagent repository HIV-2 isolates as HIV-1.

The development and verification of HIV-2 assays depends in part on the availability of well-characterized samples, including those from reagent repositories. During the development of an HIV-2 RNA quantification assay, two HIV-2 viral isolates (CDC 301340 and CDC 301342) obtained from the NIAID AIDS Reagent and Reference Repository were not detected leading to an investigation. Two HIV-2 primers/probe sets of known performance in real-time viral RNA quantification assays, targeting different regions of the virus, also failed to generate RT-PCR products for these two isolates. These isolates were tested in the HIV-1 specific COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (Roche Molecular Diagnostics) and were quantified at high copy number. Other HIV-2 isolates tested were not amplified in the COBAS HIV-1 TaqMan assay. Furthermore, the discrepant isolates were highly reactive in an HIV-1 p24 antigen test while the other HIV-2 isolates showed very weak, if any, cross-reactivity with the HIV-1 p24 assay. Phylogenetic tree analysis of sequences from the protease-reverse transcriptase regions of the discrepant HIV-2 isolates mapped with HIV-1 Group M, Subtype CRF02_AG confirming these isolates were of HIV-1 origin and had been misclassified as HIV-2. The use of misclassified isolates in the verification of molecular and immunological assays can lead to misinterpretation of test results, misdirection of efforts into assay redesign and increased development costs. The results of this study were shared with the NIAID AIDS Reagent Program, leading to the reclassification of the two discrepant isolates as HIV-1

ART Adherence and Viral Suppression are High Among Most Non-Pregnant Individuals with Early-stage, Asymptomatic HIV Infection: an Observational Study from Uganda and South Africa

Introduction The success of universal antiretroviral therapy (ART) access and aspirations for an AIDS‐free generation depend on high adherence in individuals initiating ART during early‐stage HIV infection; however, adherence may be difficult in the absence of illness and associated support. Methods From March 2015 to October 2017, we prospectively observed three groups initiating ART in routine care in Uganda and South Africa: men and non‐pregnant women with early‐stage HIV infection (CD4 \u3e 350 cells/μL), pregnant women with early‐stage HIV infection and men and non‐pregnant women with late‐stage HIV infection (CD4 \u3c 200 cells/μL). Socio‐behavioural questionnaires were administered and viral loads were performed at 0, 6 and 12 months. Adherence was monitored electronically. Results Adherence data were available for 869 participants: 322 (37%) early/non‐pregnant, 199 (23%) early/pregnant and 348 (40%) late/non‐pregnant participants. In Uganda, median adherence was 89% (interquartile range 74 to 96) and viral suppression was 90% at 12 months; neither differed among groups (p \u3e 0.72). In South Africa, median adherence was higher in early/non‐pregnant versus early/pregnant or late/non‐pregnant participants (76%, 37%, 52%; p \u3c 0.001), with similar trends in viral suppression (86%, 51%, 79%; p \u3c 0.001). Among early/non‐pregnant individuals in Uganda, adherence was higher with increasing age and lower with structural barriers; whereas in South Africa, adherence was higher with regular income, higher perceived stigma and use of other medications, but lower with maladaptive coping and cigarette smoking. Discussion ART adherence among non‐pregnant individuals with early‐stage infection is as high or higher than with late‐stage initiation, supporting universal access to ART. Challenges remain for some pregnant women and individuals with late‐stage infection in South Africa and highlight the need for differentiated care delivery

Performance of two HIV-2 primers/probe sets in real-time RT-qPCR assays.

<p>Two HIV-2 primers/probe sets were assessed for amplification of serial dilutions of an HIV-2 isolate, NIH-Z, under standard ampification conditions that were not optimized for each primers/probe set. Primers/probe sets, designated as PD  =  Primer Design LTD HIV-2 PCR Kit and SM  =  Delarue et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096554#pone.0096554-Delarue1" target="_blank">[12]</a>, showed linear amplification profiles that were parallel. PD  =  •. SM  =  ▴.</p

Performance of HIV-1/2 viral stocks in real-time RT-qPCR and HIV-1 p24 antigen tests.

<p>Individual viral stocks identified as HIV-2 were obtained from the NIAID AIDS Reagent and Reference Repository and SeraCare, Inc. The HIV-1 subtype and HIV-2 group identification is based upon data sheets provided by NIAID and from publications. An HIV-1 Subtype B isolate from the United States (91US_4) was used as an HIV-1 control. Viral RNA was extracted and tested in HIV-2 real-time RT-qPCR assays. Viral isolates selected for testing in the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v2.0 quantitative RNA assay and the Perkin-Elmer p24 Antigen test included the two viral isolates that were not amplified in the HIV-2 real-time RT-qPCR assays, three HIV-2 viral isolates that amplified well and the HIV-1 91US_4 isolate. Although the HIV-2 RT-qPCR assays were not optimized, the HIV-2 amplifications were robust for the majority of the isolates tested as reflected in the HIV-2 RNA concentrations reported based upon relative values extrapolated from the NIH-Z standard for each primers/probe set. The CDC 310340 and CDC 310342 viral stocks were not amplified in HIV-2 RT-qPCR assays but were quantified and reactive in HIV-1 specific tests. PD  =  Primer Design LTD HIV-2 PCR Kit primers/probe. SM  =  Delarue et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096554#pone.0096554-Delarue1" target="_blank">[12]</a> primers/probe set. TND  =  Target Not Detected. UNK is unknown group. Viral isolate was not tested (−).</p

Phylogenetic Tree Analysis of Protease/Reverse Transcriptase Sequences for CDC 310340 and CDC 310342 Viral Stocks.

<p>HIV-1 sequences were obtained using the Siemens' TRUGENE HIV-1 Genotype Test, analyzed using MegAlign version 9.0.4 (DNASTAR, Inc) and subtyped against the HIV Sequence Database using BLAST (<a href="http://www.hiv.lanl.gov/content/sequence/BASIC_BLAST.html" target="_blank">www.hiv.lanl.gov/content/sequence/BASIC_BLAST.html</a>). Both sequences were found to align with HIV-1 Group M, subtype CRF02_AG. The homology between the two sequences was 98% with 0.3% divergence. Both sequences are designated in the tree with a •. The dotted line indicates a negative branch length, which is a result of averaging.</p