A Partially Multiplexed HIV Drug Resistance (HIVDR) Assay for Monitoring HIVDR Mutations of the Protease, Reverse-Transcriptase (PRRT), and Integrase (INT)

Microbiol Spectr. 2022 Jun 29;10(3):e0177621. doi: 10.1128/spectrum.01776-21. Epub 2022 May 5.


As dolutegravir (DTG)-containing HIV regimens are scaled up globally, monitoring for HIV drug resistance (HIVDR) will become increasingly important. We designed a partially multiplexed HIVDR assay using Sanger sequencing technology to monitor HIVDR mutations in the protease, reverse-transcriptase (PRRT), and integrase (INT). A total of 213 clinical and analytical plasma and dried blood spot (DBS) samples were used in the evaluation. The assay detected a wide range of known HIV-1 subtypes and circulating recombinant forms (CRFs) of group M from 139 samples. INT accuracy showed that the average nucleotide (nt) sequence concordance was 99.8% for 75 plasma samples and 99.5% for 11 DBS samples compared with the reference sequences. The PRRT accuracy also demonstrated the average nucleotide sequence concordance was 99.5% for 57 plasma samples and 99.2% for 33 DBS samples. The major PRRT and INT DR mutations of all samples tested were concordant with those of the reference sequences using the Stanford HIV database (db). Amplification sensitivity of samples with viral load (VL) >5000 copies/mL showed plasma exceeded 95% of positivity, and DBS exceeded 90% for PRRT and INT. Samples with VL (1000 to 5000 copies/mL) showed plasma exceeded 90%, and DBS reached 88% positivity for PRRT and INT. Assay precision and reproducibility showed >99% nucleotide sequence concordance in each set of replicates for PRRT and INT. In conclusion, this HIVDR assay met WHO HIVDR assay performance criteria for surveillance, worked for plasma and DBS, used minimal sample volume, was sensitive, and was a potentially cost-effective tool to monitor HIVDR mutations in PRRT and INT. IMPORTANCE This HIVDR genotyping assay works for both plasma and DBS samples, requires low sample input, and is sensitive. This assay has the potential to be a user-friendly and cost-effective HIVDR assay because of its partially multiplexed design. Application of this genotyping assay will help HIVDR monitoring in HIV high-burdened countries using a DGT-based HIV drug regimen recommended by the U.S. President's Emergency Plan for AIDS Relief and the WHO.

Keywords: drug resistance; human immunodeficiency virus; integrase.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anti-HIV Agents* / therapeutic use
  • DNA-Directed RNA Polymerases
  • Drug Resistance, Viral* / genetics
  • Genotype
  • HIV Infections* / drug therapy
  • HIV-1* / genetics
  • Humans
  • Integrases / genetics
  • Mutation
  • Peptide Hydrolases / genetics
  • RNA-Directed DNA Polymerase / genetics
  • Reproducibility of Results
  • Viral Load


  • Anti-HIV Agents
  • Integrases
  • RNA-Directed DNA Polymerase
  • DNA-Directed RNA Polymerases
  • Peptide Hydrolases