Method Description

HIV-1 RNA quantification:

The cobas HIV-1 assay is a US Food and Drug Administration (FDA)-approved, in vitro nucleic acid amplification test for the quantification of HIV-1 RNA in human plasma using the cobas 5800/6800/8800 systems for fully automated viral nucleic acid extraction (generic silica-based capture technique) and automated amplification and detection of the viral nucleic acid sequence. This polymerase chain reaction (PCR) assay amplifies sequences within the gag gene and long terminal repeat (LTR) region of the HIV-1 genome and generates amplification products that are detected and quantified in real-time with 2 sequence-specific TaqMan probes. A non-HIV armored RNA quantitation standard (RNA-QS) is introduced into each specimen during sample preparation to serve as internal control for nucleic acid extraction and PCR amplification/detection processes. Fluorescent reporter dye-labeled TaqMan probes hybridized to the complementary HIV-1 target sequences and RNA-QS sequence undergo hydrolysis during PCR amplification step to generate fluorescent signal detected in 2 different dye channels. Concentration of the HIV-1 RNA in a patient's plasma sample is determined by a ratio of the intensity of the fluorescent dye from the cleaved HIV-1 target sequence probes and that from the RNA-QS target probe detected throughout the PCR process.(Package insert: HIV-1-Quantitative nucleic acid test for use on the cobas 5800/6800/8800 Systems. Roche Molecular Systems Inc; Doc rev. 4.0, 11/2022)

HIV-1 genotypic drug resistance:

This test utilizes the FDA-approved, commercially available Sentosa SQ HIV-1 Genotyping Assay, a next-generation sequencing assay based on a "sequencing by synthesis" method. The assay is designed to generate 2 amplicons (approximately 1500 base pairs [bp] and approximately 1000 bp in length) spanning the PR / RT- and INT-coding regions, respectively, of the HIV-1 genome for sequencing. Codon positions 1 to 99, 1 to 387, and 1 to 288 in the PR-, RT-, and INT-coding regions, respectively, are subsequently analyzed by the assay software for clinically relevant codon substitutions.

Clinical plasma specimens are subjected to automated HIV-1 RNA extraction and purification, followed by reverse-transcription (RT)-PCR of HIV-1 target sequences, with both a system control and a positive control included in each assay run for quality control purposes. Automated DNA library preparation is performed using the amplified products, including enzymatic shearing, adapter ligation, purification, and normalization, prior to DNA template preparation and sequencing. Sequencing reactions are conducted with the Sentosa SQ301 sequencer, and the assembled sequence data are analyzed using proprietary analysis and interpretive software applications. HIV-1 antiviral drug-resistance interpretations are based on algorithms implemented in the most current version of the Stanford University HIV Drug Resistance Database (HIVdb; Stanford University) using a 5% variant detection cutoff threshold set by the assay manufacturer.(Instruction manual: Sentosa SQ HIV-1 Genotyping Assay User Manual. Vela Diagnostics; version 1.0, 10/2019)