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

Linda L Jagodzinski,Mark M Manak,Ying Liu,Sheila A Peel,Catherine Kibirige,Holly R Hack

doi:10.1371/journal.pone.0096554

Linda L Jagodzinski, Mark M Manak + Show 4 more

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https://doi.org/10.1371/journal.pone.0096554

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Abstract

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.

Highlights

Infection with Human Immunodeficiency Virus Type 2 (HIV2), as in the case of HIV-1, is associated with opportunistic infections and AIDS
This study reports an investigation of discrepant results obtained in reverse transcription HIV-2 quantitative real-time PCR assays (RT-qPCR) for two HIV-2 viral stocks received from the NIAID AIDS Reagent and Reference Repository
Two HIV-2 primers/probe sets developed for use in quantification of HIV-2 RNA, one by Delarue et al 2013 (SM) and a commercially available set from Primer Design (PD) were selected for amplification of HIV-2 RNA in real-time RT-qPCR assays

Summary

Introduction

Infection with Human Immunodeficiency Virus Type 2 (HIV2), as in the case of HIV-1, is associated with opportunistic infections and AIDS. Serological diagnosis and differentiation of HIV-2 infection is difficult due to cross reactivity with HIV-1 antibodies [8]. The development of commercial HIV-2 plasma RNA assays has been hampered by low HIV-2 prevalence, slower progression to AIDS, lower viral load set points in HIV-2 infected individuals (as compared to HIV-1) and limited sequence data from wellcharacterized isolates. The availability of highly sensitive and specific HIV-2 RNA assays would be useful to confirm or rule-out HIV-2, assist in therapeutic management of infection, or, as recommended by the proposed CDC algorithm, to confirm or rule-out HIV-1/HIV-2 dual infection [3,9]

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