A Partially Multiplexed HIV Drug Resistance (HIVDR) Assay for Monitoring HIVDR Mutations of the Protease,...

Area of Science:

• Virology
• Molecular Diagnostics
• Public Health

Background:

• Global scale-up of dolutegravir (DTG)-containing HIV regimens necessitates robust monitoring for HIV drug resistance (HIVDR).
• Accurate and accessible HIVDR surveillance is crucial for effective treatment strategies and preventing resistance spread.

Purpose of the Study:

• To develop and evaluate a partially multiplexed HIV drug resistance (HIVDR) assay utilizing Sanger sequencing.
• To assess the assay's performance in detecting HIVDR mutations in protease, reverse-transcriptase (PRRT), and integrase (INT) using both plasma and dried blood spot (DBS) samples.

Main Methods:

• A partially multiplexed HIVDR assay was designed based on Sanger sequencing technology.
• The assay was validated using 213 clinical and analytical plasma and DBS samples, covering diverse HIV-1 subtypes and CRFs.
• Performance was evaluated based on accuracy, sensitivity across various viral loads, precision, and reproducibility compared to reference sequences and databases.

Main Results:

• The assay demonstrated high nucleotide sequence concordance: 99.8% for plasma INT, 99.5% for DBS INT, 99.5% for plasma PRRT, and 99.2% for DBS PRRT.
• High positivity rates were observed for samples with viral loads >5000 copies/mL (plasma >95%, DBS >90%) and 1000-5000 copies/mL (plasma >90%, DBS ~88%).
• Assay precision and reproducibility consistently exceeded 99% nucleotide sequence concordance.

Conclusions:

• The developed HIVDR assay meets WHO performance criteria for surveillance, demonstrating effectiveness for both plasma and DBS samples.
• The assay is sensitive, requires minimal sample volume, and offers a potentially cost-effective and user-friendly solution for monitoring PRRT and INT mutations.
• This genotyping assay can significantly aid HIVDR monitoring in high-burden countries utilizing DTG-based regimens.