B-266 Analytical Performance Comparison of Two Genotyping Assays for HIV-1 Drug Resistance Using Sanger Sequencing
Authors: Marinho F L , Silva J d , Alvim L B , Zauli D A 
Published: September 2023

Abstract

Background
The first case of Acquired Immunodeficiency Syndrome (AIDS) was registered in the United States in early 1980. Two years later, was identified the Human Immunodeficiency Virus (HIV-1), which causes AIDS. Compared to other diseases, the treatment of AIDS achieved an important advance in just over 30 years, in which several drugs were used and replaced. However, resistance to antiretrovirals (ARVs) remains a problem in clinical practice. There are about 20 approved HIV-1 treatment drugs belonging to four different classes: Protease Inhibitors (PI), Nucleoside Reverse Transcriptase Inhibitors (NRTI), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI), and Integrase Strand Transfer Inhibitor (INSTI). ARVs resistance is caused by mutations in molecular targets of these drugs: protease, reverse transcriptase, and integrase enzymes. Virus genotyping that includes these regions is extremely important to identify the resistance mutations to ARVs therapies, being able to direct treatments. Thus, this study aimed to evaluate the analytical performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase compared to in-house Sanger genotyping method.

Methods
Viral RNA isolation from 20 individuals was performed by QIAamp Viral RNA Kit (Qiagen), according to the manufacturer’s instructions. HIV-1 genotyping was performed using both methods: in-house Sanger genotyping and Applied Biosystems HIV-1 Genotyping Kit with Integrase (Thermo Fisher), according to the manufacturer’s instructions. All results were submitted to SeqScape software to evaluate sequencing metrics. The consensus sequence was generated for each sample and analyzed at the online Stanford HIVdb Program (version 9.4) for drug resistance evaluation.

Results
The 54 mutations analyzed were distributed among the targets: PI (27.8%; 15/54), NRTI (24.1%; 13/54), NNRTI (22.2%; 12/54), and INSTI (25.9%; 14/54). The in-house method evaluated the susceptibility to PI drugs (ATV, DRV, FPV, IDV, LPV, NFV, SQV, and TPV), NRTI (ABC, AZT, D4T, DDI, FTC, 3TC, and TDF), and NNRTI (DOR, EFV, ETR, NVP, and RPV). In addition to protease and reverse transcriptase targets, the commercial kit also evaluated INSTI (BIC, CAB, DTG, EVG, and RAL) drugs. Four of 20 individuals evaluated had viral loads below 1000 copies/mL and have no results in both methods, as expected due to the detection limit of both assays. Furthermore, due to sequencing limitations using the in-house methodology, three samples were not included in the analysis as it didn't show any results. It was observed 92.3% agreement (n = 12/13) in the analyzed samples. A sensitivity of 100% and precision of 99% were found in the commercial kit using the in-house kit as a reference. The evaluation of the intra-assay (repeatability) and inter-assay (reproducibility) indicated 100% of agreement.

Conclusions
The Applied Biosystems HIV-1 Genotyping Kit with Integrase showed satisfactory performance in the detection of the main antiretroviral resistance mutations.