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.

Keywords: Ag detection; Ag/Ab combo; HIV; analytic sensitivity; diagnostics; ultrasensitive Ag; viral load.

FIG 1

Comparison and confirmation of viral load. (Left) Correlation between Abbott RealTime HIV viral load assay results (performed by the University of Washington) and the Hologic Aptima HIV-1 Quant Dx VL assay results (Aptima viral load). (Right) The difference in viral loads between the Aptima and Abbott assays. Each dot represents a sample, with the total number being 300. The color denotes the HIV subtype of the sample. Pearson's r correlation was determined to be 0.98.

FIG 2

Model-based proportion detected by assay for subtype C and by viral subtype detected by the Bio-Rad GS HIV combo Ag/Ab EIA. (Left) Predicted proportion of samples detectable by each of the four FDA-approved Ag/Ab combo assays and the one CE-marked p24 Ag-alone assay utilizing HIV subtype C to illustrate the relative rank order of performance with reactive S/CO by VL. (Right) Predicted proportion of viral subtypes detectable by the Bio-Rad GS HIV combo Ag/Ab EIA, which was used as a representative of the relative rank order performance with reactive S/CO by VL.

FIG 3

All S/CO data by viral load. S/CO data by viral load were based on viral subtypes (left) and assay platform (right).

FIG 4

S/CO relative to viral load for the BioPlex 2200 HIV Ag-Ab assays and the Ortho Clinical Diagnostics Vitros HIV combo assay. (Left) Results of the Bio-Rad 2200 HIV BioPlex Ag-Ab assay run at Public Health England (PHE); (right) results of the Vitros HIV combo assay run at Ortho Clinical Diagnostics (OCD).

FIG 5

Comparing assays run in both manufacturer and clinical labs. (Left) The Abbott Architect HIV Ag/Ab combo assay was performed at the University of Washington (UW) and at Abbott (ABT; the manufacturer's lab). (Middle) The Bio-Rad GS HIV combo Ag/Ab EIA was performed at Bio-Rad (the manufacturer's lab) and at WRAIR (WR). (Right) The Bio-Rad Genscreen HIV-1 p24 Ag assay was performed at WRAIR and at Bio-Rad (the manufacturer's lab). Blue, the manufacturer's lab; red, the clinical lab.

FIG 6

Ultrasensitive p24 Ag assay signal intensity compared to viral load. (Left) MSD ultrasensitive p24 assay; (right) Quanterix Ultrasensitive Simoa assay.