Development and genomic characterization of a diverse HIV-1 variant reference panel for nucleic acid-based testing

BACKGROUND: The high worldwide genetic diversity of HIV poses significant challenges for its detection and diagnosis by nucleic acid testing (NAT). Well-characterized reference panels are important for evaluating the analytical performance of HIV tests. OBJECTIVE: To develop a reference panel for HIV NAT that reflects the genetic diversity of circulating strains. STUDY DESIGN: HIV was cultured from blood specimens collected from blood donor and clinical sites in Cameroon. Metagenomic next-generation sequencing in combination with spiked primer enrichment along with Sanger sequencing were used to sequence 101 cultured HIV-1 samples representing 59 strains. To establish an HIV-1 variant reference panel, a diverse subset of cultured viruses was analyzed in multiple laboratories with different assays to determine consensus viral loads. RESULTS: Near full-length HIV-1 genomes, with an average of 9589 base pairs (bp), were recovered from 37 (62.7%) of the 59 strains. The whole genome sequences of 28 strains exhibited more than 95% similarity to our previously reported genomes obtained by Sanger sequencing. An HIV variant reference panel for NAT comprising 18 diverse HIV-1 strains was developed. The panel included four subtypes, four circulating recombinant forms, and eight unique recombinant forms. Strains were prepared at low (n = 18, 2.53 log10 copies/mL), medium (n = 18, 3.61 log10 copies/mL), and high viral loads (n = 15, 4.66 log10 copies/mL), yielding 51 panel members in total. CONCLUSION: This diverse HIV reference panel can be used to evaluate the performance of HIV NAT and is available upon request to developers and manufacturers of HIV tests.