Design and evaluation of a human immunodeficiency virus type 1 RNA assay using nucleic acid sequence-based amplification technology able to quantify both group M and O viruses by using the long terminal repeat as target

Abstract

Currently available human immunodeficiency virus type 1 (HIV-1) RNA quantification assays can detect most viruses of the group M subtypes, but a substantial number are missed or not quantified reliably. Viruses of HIV-1 group O cannot be detected by any commercially available assay. We developed and evaluated a quantitative assay based on nucleic acid sequence-based amplification (NASBA) technology, with primers and probes located in the conserved long terminal repeat (LTR) region of the HIV-1 genome. In 68 of 72 serum samples from individuals infected with HIV-1 subtypes A to H of group M, viruses could be detected and quantified. In serum samples from two patients infected with HIV-1 group O viruses, these viruses as well could be detected and quantified. In contrast, the currently used gag-based assay underestimated the presence of subtype A viruses and could not detect subtype G and group O viruses. The discrepancy between the results of the two assays may be explained by the number of mismatches found within and among the probe and primer regions of the subtype isolates. These data indicate that LTR-based assays, including the NASBA format chosen here, are better suited to monitoring HIV-1 therapy than are gag-based assays in an era in which multiple HIV-1 subtypes and groups are spreading worldwide.

FIG. 1

Assessed viral RNA levels versus given viral RNA levels in serial dilutions of HIV-1 HXB3 (19) in plasma as determined with the LTR-based (○) and the gag-based (□) NASBAs. The results shown are the means of at least three independently performed experiments.

FIG. 2

Assessed viral RNA levels versus dilution factor of supernatant from cultures of HIV-1 group M subtypes A (○), B (□), C (▵), D (●), E (■), F (▴), and G (◊) (A) and from four different HIV-1 group O viruses (B).

FIG. 3

Scatter diagrams of log₁₀ RNA levels as assessed by the LTR-based NASBA versus the log₁₀ RNA levels as assessed by the gag-based NASBA for HIV-1 subtypes B (○), C (□), and D (▵) (A) and subtypes A (○), E (□), and G (▵) (B). The arrow in panel B indicates a sample positive for subtype E in both assays. LDL, lower detection limit.

FIG. 4

(A) Mean log₁₀ RNA level with standard deviation (error bars) for each HIV-1 subtype as assessed by the LTR-based NASBA (L) and the gag-based NASBA (G). LDL, lower detection limit. (B) Mean number of mismatches with standard deviation (error bars) for each subtype for the primers and probes of the LTR-based assay (L) and the gag-based assay (G).