Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay

Abstract

The ability of commercially available PCR-based assays to accurately detect or quantitate human immunodeficiency virus type 1 (HIV-1) DNA or RNA in individuals predominantly infected with HIV-1 subtypes A and D is not known. Therefore, peripheral leukocytes from 43 individuals in Kampala, Uganda, positive for HIV by the Western blot (immunoblot) assay were tested by using the Roche AMPLICOR HIV-1 assay for the detection of DNA gag sequences. Plasma from these same individuals was tested by using the Roche HIV-1 AMPLICOR MONITOR HIV-1 assay for the quantitation of HIV-1 RNA gag sequences. In addition, peripheral leukocytes were tested for HIV-1 DNA by using a lower annealing temperature or a different primer pair for the HIV-1 pol region. The proportions of individuals with detectable HIV-1 DNA and RNA gag sequences by the Roche assays were 74 and 90%, respectively. The proportions positive for HIV-1 DNA sequences by using a 50؇C annealing temperature or the pol primer pair were 71 and 98%, respectively. In summary, the standard Roche assay did not detect HIV-1 DNA sequences in a significant number of HIV-1-infected individuals in Uganda. However, use of a pol primer pair increased the sensitivity of the assay to 98%. The sensitivity of the Roche AMPLICOR MONITOR assay for the detection and quantitation of HIV-1 RNA sequences was significantly higher than that of the DNA-based assay, but the efficiency of the assay, and hence, the accuracy of the values obtained with RNA, is not known. Modifications to existing assays are needed to enhance the sensitivities and accuracies of these commercially available assays for use in developing countries where non-B HIV-1 subtypes predominate.

Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

“…It was reported that a decrease in amplification efficiency occurs with five to six mutations in the primerbinding region (10,24), and this amount of divergence occurs most frequently with subtypes A and E. Similar results were reported when samples from Uganda, where subtypes A and D predominate, were tested with the Amplicor HIV-1 DNA test; the sensitivity for the samples from Uganda was 74% (22). Replacement of the primer-probe set selected from the gag gene with a primer-probe set from the better conserved pol gene increased the sensitivity of the DNA detection assay to 98% (22). Similarly, Respess et al (35) reported 100% sensitivity for detection of subtypes A and E with pol-based primers but only 87 and 67% sensitivities, respectively, when gag-based primers were used.…”

“…In the multiplex HIV-1/2 qualitative RNA assay described here, these difficulties were overcome by the meticulous choice of primers and cycling conditions, without compromising sensitivity for HIV-1 detection. The multiplex assay for HIV-1 and HIV-2 detection has a sensitivity of 20 to 50 copies per ml for HIV-1, which is similar to the sensitivities of ultrasensitive versions of assays that detect only HIV-1 (22,30,37). Sensitivity is also critical for detection of HIV-2, especially since lower HIV-2 RNA levels in plasma have been demonstrated (29,41).…”

“…All the nucleic acid detection assays developed so far have been based primarily on subtype B strains from North America and Europe. The sensitivities of these tests for detection of divergent strains were evaluated in several studies (3,14,22,35,44). Evaluation of the HIV-Monitor, version 1.0, and the nucleic acid sequence-based amplification (NASBA) assays revealed that 56 and 44% of subtype A-containing samples tested were negative by these assays, respectively (3).…”

Development of an Ultrasensitive HIV-1 DNA Detection Assay Based on an Automated πCode End-Point PCR System

“…The πCode assay platform is based on the IntelliPlex1000 πCode Processor, an automated hybridizing and washing machine, and the PlexBio100 Analyzer for the fluorescent signal detection of the πCode MicroDiscs. Despite the high detection sensitivity achieved with the sk145 and skcc1b primer set, further optimization can accomplish a much higher sensitive HIV-1 DNA assay using different sets of primers, considering the limitation of the sk145 and skcc1b primer set to detect non-B clade HIV-1 subtypes [45,[78][79][80]. In future studies, we will use a different primer set that can potentially achieve a successful detection of all HIV-1 subtypes plus maximum sensitivity for HIV-1 DNA detection.…”

Determinations of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma: Reassessment of Parameters Affecting Assay Outcome

“…In a study evaluating a limited number of RNA transcripts representing sev-eral different clades, the Chiron bDNA assay appeared to quantify the subtypes similarly (22). Modification of the procedures and/or the primers of other assays may improve detection of different clades of HIV-1 by these assays (20). A comprehensive set of reagents representing the various clades will be needed to provide standards for appropriate comparability studies with kits from various manufacturers.…”