Comparison of Detection Limits of Fourth- and Fifth-Generation Combination HIV Antigen-Antibody, p24 Antigen, and Viral Load Assays on Diverse HIV Isolates

Abstract

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical fourth-generation antigen (Ag)/antibody (Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab-alone assays but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening, and next-generation assays. Three-hundred-member panels of 20 serially diluted well-characterized antibody-negative HIV isolates for which the researchers were blind to the results (blind panels) were distributed to manufacturers and end-user labs to assess the relative analytic sensitivity of currently approved and preapproved clinical HIV fourth-generation Ag/Ab combo or p24 Ag-alone immunoassays for the detection of diverse subtypes. The limits of detection (LODs) of virus were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blind panel. On the basis of the proportion of positive results on 300 observations, all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half-log LODs, illustrating the similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo assays performed poorly. The similar performance of the different commercially available fourth-generation assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next-generation preclinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while rapid fourth-generation assays performed poorly for p24 Ag detection.

Decreased Seroreactivity in Individuals Initiating Antiretroviral Therapy during Acute HIV Infection

“…The limit of detection of the p24 Ag by these assays ranged from 2 to 7 pg, which corresponds to 4.4 to 4.9 log copies of RNA per ml. The assays detect HIV infection from 7 to 11 days earlier than the anti-HIV IgM/IgG immunoassays (32)(33)(34)(35). In our study, the results of the BRC, ARC, and BPX assays from samples collected at 24 weeks after the initiation of ART in AHI also showed generally good agreement at S/CO results below 10.…”

Current and historic HIV-1 molecular epidemiology in paediatric and adult population from Kinshasa in the Democratic Republic of Congo

“…Although two previous studies reported the full HIV-1 genome (including pol ) from a vertically infected 12-month-old baby 54 or gag sequences from 15 children in Kimpese, rural DRC 53 , our study presents the most extensive data regarding HIV-infected children and adolescents in the country. The updated high genetic diversity observed in the DRC also represents a real challenge for future vaccine development and for efficiency of antiretroviral treatment, diagnostic and monitoring tests of HIV infection 69 .…”

Lessons in diagnostic virology: expected and unexpected sources of error

“…Although viral diversity could affect the limit of detection of serological assays this is more of a concern with molecular assays, as peptides (amino acid sequences) are more conserved than nucleic acid sequences. Primer or probe mismatches with template sequences may result in under‐ quantification or non‐detection in the case of quantitative or qualitative assays, respectively, or in case of genotypic drug resistance assays in failure of amplification or sequencing reactions.…”

“…In these cases both the screening and confirmation assay could be performed on the wrong patient, unless a separate, independent sample is obtained. Fourth generation HIV‐1 serology assays, in addition to being able to detect antigen, albeit only at relatively high concentration, are often highly sensitive for low level antibody, which enables the detection of early stage (Fiebig 3) antibody positive cases . However, as antibody levels in patients with chronic HIV infection can be very high, microscopic carryover from positive samples could result in false positive results.…”