Background:  There is currently no “gold standard” for the quantification of plasma HIV-2 RNA. At the patient level, difficulties arise for the interpretation of results and may yield inappropriate clinical decision; at the population level, this may explain controversial evaluation of response to treatment among different cohorts. We studied the validity of HIV-2 subtype A RNA quantification assays performed in reference centers in 9 countries (Belgium, France, Gambia, Germany, Portugal, Spain, Sweden, Switzerland, and the United Kingdom) within the ACHI_eV₂E (a collaboration on HIV-2 infection) study group.

Methods:   In a double-blind experimental design, each of the 9 centers quantified 3 series of 10 aliquots of NIHZ HIV-2 subtype A strain (reference count obtained by electronic microscopy), each at a different viral load (1.7, 2.3, and 3 log copies/mL), and 5 HIV-2-negative aliquots. Centers used mainly real-time polymerase chain reaction (RT- PCR) assays, with primers and probes located in various regions of HIV-2 genome and different HIV-2 standards. Aliquots were prepared in 1 center and sent in a randomized order. Accuracy and precision of assays were estimated for the 3 concentration levels. A quantification assay was defined as accurate if at least 9 of 10 of the quantifications were in the expected interval:  ([theoretical viral load]/3 – [theoretical viral load] x 3). Precision of assays was evaluated using the intra-laboratory coherence coefficient (ILCC) and the coefficient of variation (CV).

Results:  No false positive result was reported. At the concentration level of 3 log copies/mL, 5 centers reported accurate results and 7 reported precise results (CV = 3 to 6%; ILCC = 0.5 to 0.9). At the concentration level of 2.3 log copies/mL, 5 centers reported accurate results, and 6 reported precise estimations (CV = 5 to 8%; ILCC = 0.5 to 1.1). At the concentration level of 1.7 log copies/mL, 2 centers reported accurate results, but with lack of precision (CV = 24% and 33%; ILCC = 1.3 and 1.5, respectively), and 3 reported precise results (CV = 5% to 11%; ILCC = 0.4 to 0.8).

Conclusions:  HIV-2 subtype A viral load quantifications varied widely among centers. Only 2 centers reported accurate and precise results at the theoretical viral loads of both 3 and 2.3 log copies/mL. Standardization of quantification assays is required to understand the response to treatment in HIV-2-infected patients and implement future international clinical trials.