An International Collaboration To Standardize HIV-2 Viral Load Assays: Results from the 2009 ACHI _E V _2E Quality Control Study

Abstract

Accurate HIV-2 plasma viral load quantification is crucial for adequate HIV-2 patient management and for the proper conduct of clinical trials and international cohort collaborations. This study compared the homogeneity of HIV-2 RNA quantification when using HIV-2 assays from ACHI E V 2E study sites and either in-house PCR calibration standards or common viral load standards supplied to all collaborators. Each of the 12 participating laboratories quantified blinded HIV-2 samples, using its own HIV-2 viral load assay and standard as well as centrally validated and distributed common HIV-2 group A and B standards ( http://www.hiv.lanl.gov/content/sequence/HelpDocs/subtypes-more.html ). Aliquots of HIV-2 group A and B strains, each at 2 theoretical concentrations (2.7 and 3.7 log 10 copies/ml), were tested. Intralaboratory, interlaboratory, and overall variances of quantification results obtained with both standards were compared using F tests. For HIV-2 group A quantifications, overall and interlaboratory and/or intralaboratory variances were significantly lower when using the common standard than when using in-house standards at the concentration levels of 2.7 log 10 copies/ml and 3.7 log 10 copies/ml, respectively. For HIV-2 group B, a high heterogeneity was observed and the variances did not differ according to the type of standard used. In this international collaboration, the use of a common standard improved the homogeneity of HIV-2 group A RNA quantification only. The diversity of HIV-2 group B, particularly in PCR primer-binding regions, may explain the heterogeneity in quantification of this strain. Development of a validated HIV-2 viral load assay that accurately quantifies distinct circulating strains is needed.

New Sensitive One-Step Real-Time Duplex PCR Method for Group A and B HIV-2 RNA Load

“…Specific molecular methods are therefore necessary for diagnosis and patient monitoring. Current assays, mainly consisting of in-house methods or unvalidated derivatives of commercial kits, suffer from major limitations in terms of their sensitivity, accuracy, and coverage of HIV-2 genetic diversity (19,20).…”

“…The aim of this work was to develop a quantitative assay that takes into account both the low viral load seen in most HIV-2-infected patients and the broad genetic diversity of HIV-2, especially group B (13,19,20). In addition, as most cases of HIV-2 infection occur in West Africa, such a test must be easy to implement in developing countries, as previously achieved with the generic HIV-1 viral load assay marketed by Biocentric.…”

“…We chose to optimize the assay currently used to monitor the French HIV-2 cohort based on amplification of the HIV-2 gag region, which gave accurate results in the ACHI E V 2E study (19,20), but with a lower limit of quantification of 100 cp/ml (13) and not completely adapted to some group B strains. We also used the same operating conditions as those of the Biocentric HIV-1 assay kit, in order to facilitate its use either for HIV-2 alone or jointly for HIV-1 and HIV-2.…”

“…The high genetic diversity of HIV-2, with 9 groups designated A to I, of which only groups A and B are epidemic, also represents an obstacle to accurate viral load quantification (21)(22)(23)(24)(25). Previous studies showed that group B viruses are particularly difficult to quantify (13,19,20).…”

“…In-house assays are therefore widely used (12)(13)(14)(15)(16)(17)(18). The ACHI E V 2E international collaboration on HIV-2 infection showed that plasma HIV-2 RNA values vary considerably between laboratories (19,20). The high genetic diversity of HIV-2, with 9 groups designated A to I, of which only groups A and B are epidemic, also represents an obstacle to accurate viral load quantification (21)(22)(23)(24)(25).…”