This study evaluates the performance of three commercial viral load assays (the Versant HIV-1 RNA bDNA v3.0, Cobas AmpliPrep/Cobas TaqMan HIV-1 test, and NucliSens HIV-1 QT) testing a panel of 83 plasma specimens infected with 27 HIV-1 non-B subtypes and 56 recombinants, previously defined by phylogenetic analysis of the pol gene. All subjects were in regular follow-up in a reference hospital in Madrid, Spain. Twenty-eight specimens were collected from HAART patients, which explains the viremia values below the detection limit obtained by the three tests in all of them. Since viremia is not controlled without antiretroviral treatment, all 55 plasma specimens from naive subjects should have had high HIV-1 viremia viral loads, theoretically susceptible to be quantified by the tests. However, only 32 of these 55 specimens (58.2%) presented viremia values above the detection limit in all three assays, and 23 could not be quantified by one or two techniques, indicating an underestimation of HIV-1 viral load in some non-B specimens. In fact, from 10 to 40% of the HIV-1 non-B subtypes and from 17.5 to 28.6% of recombinants infecting naive subjects could not be quantified for some of the three tests (Table ).
Previous studies quantifying 15 different non-B HIV-1 variants revealed that viremia values comparing the Cobas AmpliPrep/Cobas TaqMan HIV-1 test and bDNA v3.0 yielded results within the accuracy limits of ±0.3 log, being lower (±0.1 log) when only subtype B specimens were analyzed (27
). Another report quantifying 10 non-B subtypes observed that the mean difference between viremia detected by Cobas AmpliPrep/Cobas TaqMan HIV-1 test and bDNA v3.0 was 0.360-log RNA copies (24
), increasing to 0.553 log when Cobas and NucliSens were compared (24
). Other authors published that the coefficients of variance in testing NucliSens EasyQ in 35 clade B specimens ranged from 2.3 to 10.4% (21
), increasing to differences of 0.32 viremia log at low-end HIV-1 RNA concentrations (13
). In our study, 14.5 and 60% of the 55 plasma samples from naive subjects carrying non-B and recombinant variants provided viral load differences above ≥1 log and ≥0.5 log, respectively, when two of those techniques were compared. In fact, the subtype D showed ≥2-log viremia differences since it could not be quantified by the Versant assay (Table ). We strongly believe that the observed viremia differences among assays in some of our non-B specimens could have clinical relevance during antiretroviral treatment monitoring of these patients.
Our data revealed that some strains, such as subtype G and CRF02_AG, showed more viremia discrepancies than others. In agreement with our findings, a recent evaluation testing the Roche Cobas TaqMan and Abbott RealTime extraction-quantification systems for HIV-1 subtype quantification also reported large viremia differences in non-B subtypes, particularly in CRF02_AG variants (14
). As for other HIV-1 variants, a recent study showed the versatility of both the NucliSens EasyQ v1.1 and the Cobas Amplicor tests in monitoring subtype B and recombinant CRF01_AE viruses prevalent in Southeast Asia (21
). It should be noted that neither of these variants was included in our study.
Although TaqMan and NucliSens assays target the gag
coding region, significant quantification differences between the two assays were shown in 29% (16/55) of samples from naive subjects, increasing to 37.5% (12/32) in those samples with viremia values above the detection limit by the three methods. Our results revealed that NucliSens EasyQ HIV-1 v1.2 assay was more sensitive than Versant for HIV-1 non-B subtypes quantification in plasma, according to a previous multicenter evaluation using the NucliSens EasyQ HIV-1 v1.1 versus Versant assay (8
New versions of HIV-1 quantification assays are continuously being developed to improve virus quantification of all viral variants. Several studies have been conducted to test the agreement among different versions of the same assay, but mainly testing clade B specimens. In fact, less-significant viremia differences among tests occurred when only clade B viruses were tested, as was observed in comparisons of the Cobas AmpliPrep/Cobas Amplicor HIV-1 Monitor Ultrasensitive Test and the Cobas Amplicor HIV-1 Monitor test using manual specimen preparation (1
). However, disagreement in viremia quantification between two versions of the same assay has been reported testing non-B subtypes (2
). According to all of these data, we strongly suggest measuring all HIV-1 viral load determinations in each HIV-1-infected patient during follow-up by the same quantification technique and version.
In summary, an adequate design of primers and probes is needed for optimal quantitation of plasma HIV-RNA in non-B subtypes. Our data reinforce the need for monitoring patients on therapy with the same method and the convenience of HIV-1 subtyping.