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.

Fig. 1.

ACHI_EV_2E primers and probes mapped to the M15390 HIV-2 ROD genome. The figure depicts the relevant portion of the HIV-2 ROD genome (GenBank accession number M15390) with nucleotide positions indicated. The nucleotide positions of all primers and their binding orientation (>>>, forward strand; <<<, reverse strand) are indicated. Note that the group B primers used by the Swiss group (HIV-2TMFPRB, HIV-2TMRPRB, TMPROBEB) are not shown but map to the comparable positions in HIV-2 group viruses as TMFPR1, TMRPR1, and TMPROBE1, respectively. Some of the LTR-targeted primers have a second target site in the 3′ LTR.

Fig. 2.

Accuracy of HIV-2 group A RNA quantification assays evaluated by the ACHI_EV_2E collaboration in 2009. Quantification results are reported for each participating laboratory. The accuracy interval is represented by the white area for each of the three theoretical viral loads used. (A) Theoretical viral load of 2.7 log₁₀ copies/ml; (B) theoretical viral load of 3.7 log₁₀ copies/ml.

Fig. 3.

Accuracy of HIV-2 group B RNA quantification assays evaluated by the ACHI_EV_2E collaboration in 2009. Quantification results are reported for each participating laboratory. The accuracy interval is represented by the white area for each of the three theoretical viral loads used. (A) Theoretical viral load of 2.7 log₁₀ copies/ml; (B) theoretical viral load of 3.7 log₁₀ copies/ml.