Performance of Three Commercial Viral Load Assays, Versant Human Immunodeficiency Virus Type 1 (HIV-1) RNA bDNA v3.0, Cobas AmpliPrep/Cobas TaqMan HIV-1, and NucliSens HIV-1 EasyQ v1.2, Testing HIV-1 Non-B Subtypes and Recombinant Variants

Abstract

Monitoring antiretroviral therapy requires that human immunodeficiency virus type 1 (HIV-1) viremia assays are applicable to all distinct variants. This study evaluates the performance of three commercial viral load assays-Versant HIV-1 RNA bDNA v3.0, Cobas AmpliPrep/Cobas TaqMan HIV-1, and NucliSens HIV-1 EasyQ v1.2-in testing 83 plasma specimens from patients carrying HIV-1 non-B subtypes and recombinants previously defined by phylogenetic analysis of the pol gene. All 28 specimens from patients under treatment presented viremia values below the detection limit with the three methods. In the remaining 55 specimens from naive individuals viremia could not be detected in 32.7, 20, and 14.6% using the NucliSens, Versant, or TaqMan tests, respectively, suggesting potential viral load underestimation of some samples by all techniques. Only 32 (58.2%) samples from naive subjects were quantified by the three methods; the NucliSens test provided the highest HIV RNA values (mean, 4.87 log copies/ml), and the Versant test provided the lowest (mean, 4.16 log copies/ml). Viremia differences of greater than 1 log were seen in 8 (14.5%) of 55 specimens, occurring in 10.9, 7.3, and 5.4%, respectively, of the specimens in comparisons of Versant versus NucliSens, Versant versus TaqMan, and TaqMan versus NucliSens. Differences greater than 0.5 log, considered significant for clinicians, occurred in 45.5, 27.3, and 29% when the same assays were compared. Some HIV-1 strains, such as subtype G and CRF02_AG, showed more discrepancies in distinct quantification methods than others. In summary, an adequate design of primers and probes is needed for optimal quantitation of plasma HIV-RNA in non-B subtypes. Our data emphasize the need to use the same method for monitoring patients on therapy and also the convenience of HIV-1 subtyping.

Cross-Platform Analysis of HIV-1 RNA Data Generated by a Multicenter Assay Validation Study with Wide Geographic Representation

“…A good assay validation plan must include the use of a well-characterized control material for purposes of verifying assay specifications as well as use of locally collected clinical samples for purposes of clinical correlation studies. A number of investigators have reported significantly discordant virus load results in samples containing non-B HIV-1 subtypes across the new HIV-1 RNA real-time PCR assays (4,11,13,23,26,31). Such correlation studies are extremely important and actually resulted in the modification from the RTv1 to the RTv2 assay (6,7,12).…”

“…assay. An understanding of how the results generated from the different HIV-1 RNA assays compare is critical for all HIV-1 subtypes, especially since the genetic diversity of HIV-1 continues to evolve and underquantitation of HIV-1 continues to be documented (4,11,13,23). Surveillance and comparative studies worldwide (2,17,22,25,30,31) are extremely important for documenting potential problems and forcing manufacturers to improve their assays in response to deficiencies (6,7,14).…”

Changing patterns in HIV‐1 non‐B clade prevalence and diversity in Italy over three decades*

“…HIV-1 diversity has an impact on many aspects of HIV infection, including molecular diagnostics [31], cellular tropism [32,33], pathways to drug resistance [34], fitness [35], disease progression [36] and mother-to-child transmission [37]. Despite some limitations, subtype assignment derived from routine drug resistance testing is a fundamental tool for basic surveillance of the spread of HIV-1 clades, with the potential to improve understanding of the biological and clinical features of HIV infection and to enhance prevention strategies.…”

Evaluation of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test and Identification of Rare Polymorphisms Potentially Affecting Assay Performance

“…However, the challenge posed by the continued diversification of the highly variable HIV genome in clinical isolates worldwide and its impact on testing is critically important and has increasingly become the focus of study. While most reports have emphasized underquantification of non-B subtypes (2,3,5,6,7,9), the discrepant samples in this study and others (2,10) showed that underquantification can also occur in subtype B and is not restricted to specific subtypes.…”