The quantification of HIV-1 RNA levels in blood plasma is an important tool for determining the risk of progression of infection and monitoring the response of HIV-infected patients to antiretroviral therapy (ART). HIV diversity is a critical point to be considered for the development of genomic amplification techniques, particularly for plasma viral load assays. On the one hand, from an epidemiological point of view, in the United States and Europe there is an increasing number of patients newly infected by non-B subtypes, especially by CRF02_AG strains (21
). In France, the proportion of non-B subtypes increased dramatically between 1995 and 2002 and has remained stable since 2003. Indeed, almost 40% of the newly diagnosed patients are infected by a non-B subtype. The increasing diversity of HIV-1 viruses in France, even in Caucasian patients diagnosed at the time of primary infection, was recently described in the French ANRS CO06 primo cohort study (6
). Moreover, in resource-limited countries, where 90% of new infections occur, there is increasing access to ART for HIV-infected people, thanks especially to action plans decided on in 2000 during the International AIDS Conference of Durban, South Africa, and to the “3 by 5′” initiative and thus an increasing need to quantify HIV viral loads. For these reasons it is of high importance to use appropriate viral load assays able to span this genetic diversity for the management of naïve and also ARV-treated patients. To verify this critical point, there is a need of comparative studies for the determination of the sensitivity of existing assays and their ability to amplify RNA, especially for non-B subtypes.
In our study, whatever the HIV-1 subtype amplified, the CAP/CTM v2.0 assay exhibited the lowest SDEVL, the best precision, and mean values significantly higher than those of the 3 other assays. This overquantification could be due to the performance of the automated extractors but more probably to the amplification step and viral load calculation since the same extractor is used for the 2.0 and 1.0 versions of the Roche test. Our findings confirm the results found by Scott et al. (17
) that, for a group of predominantly HIV-1 C subtype patients, CAP/CTM v2.0 presented a positive bias of 0.33 and 0.48 log copies/ml over the m
e and CAP/CTM v1.0 assays, respectively. In our study, an improved sensitivity of the 2.0 over the 1.0 version was also observed, in agreement with previous studies (13
); Scott et al. also described more quantifiable results down to 20 HIV RNA copies/ml for ARV-treated patients (17
); since our samples originated from newly diagnosed and untreated patients, we were unable to confirm this point. In our study, the NucliSens HIV-1 EasyQ v1.2 assay was the least favorable, exhibiting significantly lower viral loads and wider discrepancies. This observation was also reported in two other studies (18
) using the former version 1.1. Holguin et al. (9
) found that the new version of NucliSens HIV-1 EasyQ compares favorably with RNA bDNA v3.0 and Cobas AmpliPrep/Cobas TaqMan v1.0, but false-positive results obtained with the NucliSens HIV-1 EasyQ v1.2, due to human plasma, were also recently reported (22
). It is nevertheless fair to mention that positive results for patients having a low viral load are stronger evidence of good sensitivity than a high mean value which might be a biased estimate. Surprisingly, B subtypes were “overquantified” by the CAP/CTM v2.0 compared to the m
e test. The explanation is unclear since this phenomenon was not observed for CRF02_AG strains, suggesting possible bias due to the amplification of two targets per genome for CAP/CTM v2.0, with inherent calculations. An increased number of detectable viral loads at the critical threshold of 50 copies per ml with the CAP/CTM v1.0 versus the Amplicor assay was recently reported, suggesting that caution is required in the interpretation of low viral loads obtained with this assay (11
Although only 23 strains were tested, CRF02_AG subtype samples yielded significantly higher results when viral loads were measured with the CAP/CTM HIV-1 v2.0 and m
e assays than with the CAP/CTM HIV-1 v1.0 and NucliSens EasyQ ones. Similar results were previously obtained for the subtype C and CRF02_AG strains (13
). The m
e assay also compared favorably with NucliSens EasyQ and CAP/CTM v1.0 for the G and CRF02_AG strains in another study (18
). It is interesting to notice that the means of the viral loads assessed by the m
e and CAP/CTM v2.0 assays were comparable for the CRF02_AG subtype, whereas the mean of viral loads estimated by m
e was significantly lower than the mean estimated with CAP/CTM v2.0 for B subtypes. This could be due, at least in part, to genetic differences between integrase and gag
regions. The poor recognition by CAP/CTM v1.0 of non-B subtypes was previously described by others (8
), and De Bel et al. recently also reported an underquantification with some B subtype strains (3
). Indeed, the ability of the CAP/CTM assay to recognize non-B subtypes was dramatically improved between v1.0 and v2.0 (17
; this study). Recently, the ability of another real-time assay, namely, the generic HIV viral load assay, compared to the Amplicor HIV-1 Monitor v1.5 and NucliSens EasyQ v1.2 to better quantify non-B strains was established by Rouet et al. (15
). Until now, no comparison of this generic assay with the CAP/CTM v2.0 and the m
e tests had been done.
Statistically significant wider discrepancies were observed for CRF02_AG than for B strains when comparing results obtained with the four assays tested in the present study, suggesting a weaker ability of these assays to quantify these non-B strains. Holguin et al. (9
) also showed differences or discrepancies among non-B HIV-1 subtypes when comparing the performances of the following three assays: Versant HIV-1 RNA bDNA v3.0, CAP/CTM v2.0, and NucliSens EasyQ v1.2. This observation emphasizes the need to improve the design of primers and probes and could be linked, at least in part, to differences of genetic variability among strains belonging to the same subtype.
Our study is focused on samples taken from a French cohort, including B- and CRF02_AG-infected patients. Globally, NucliSens EasyQ v1.2 seems to differ from CAP/CTM v1.0, CAP/CTM v2.0, and m2000 RealTime. However, our results also underline the limitation of automated real-time protocols for non-B HIV-1 subtypes, despite a recent improvement of the sensitivity of these assays with these strains, with the possibility of wide discrepancies and the misestimating of the viral load. As a whole, these observations speak in favor of using the same assay for monitoring treatment of HIV-1-infected patients, for resistance studies, and for clinical trials, eventually, after testing for the most appropriate assay for non-B subtypes.