Validation of a Low-cost in-house Hiv Integrase Inhibitor Drug-resistance Assay and Characterization of the Pol- Integrase Region From Patients Failing Treatment at Kenyatta National Hospital Comprehensive Care Centre

Abstract

Background: HIV drug resistance testing (DRT) plays an essential role in the surveillance of HIV drug resistance (HIVDR) and informs on the effectiveness of treatment regimens for people living with HIV (PLHIV). HIV DRT is costly and its access is limited especially in poor-resourced settings such as Kenya. With the recent inclusion of integrase strand transfer inhibitors (INSTIs) as part of the first-line regimen for all PLHIV, there arises a need for an affordable and effective INSTI drug resistance test. Further, there is a knowledge gap on the prevalence of INSTI resistance-associated mutations (RAMs) that may affect the utility of INSTIs. This study aims to validate an affordable in-house INSTI drug resistance test and characterize the pol-integrase region of INSTI-naïve treatment-experienced patients. Broad objective: To validate an in-house INSTI HIV drug resistance test and characterize the HIV-1 pol-integrase region of INSTI-naïve patients experiencing first-line or second-line treatment failure at the Kenyatta National Hospital Comprehensive Care Centre (KNH CCC) Study design: This was a laboratory-based cross-sectional study done to validate an in-house INSTI HIV drug resistance test and characterize the pol-integrase region of HIV-1 isolates from INSTI-naïve patients failing treatment Methodology: The performance characteristics (accuracy, precision, reproducibility and amplification sensitivity) of an in-house HIV INSTI drug resistance assay were assessed using 36 plasma samples. Genetic analysis was performed on 87 plasma-derived samples from INSTI-naïve HIV-1 infected patients for characterization of the pol-integrase region. REGA HIV-1 subtyping tool was used for subtype analysis and Stanford HIV database for analysis of resistance-associated loci. The cost per test was estimated using an ingredient costing approach. 1

Results: The mean nucleotide and amino acid sequence identity between the in-house and reference assay was 99.5%, CI [99.21- 99.77] and 99.0%, CI [98.58, 99.42] respectively. The mean nucleotide sequence identity between replicates intra-assay (precision) and inter-assay (reproducibility) was 100% and 99.1% respectively. Amplification sensitivity for samples with VL> 1000 copies/mL was 100% and 50% for samples with VL <1000 copies/mL. Characterization of the pol-integrase region revealed genetic variability with 76% of the samples belonging to subtype A, 13% subtype D, 9% subtype C, 1% CRF10_CD and 1% subtype G. Drug resistance mutation analyses revealed the absence of major mutations and identified four accessory mutations (T97A, Q146QR, D232N, and T97TA) and two APOBEC mutations (E198K and R224Q). The estimated cost of providing the in-house INSTI-HIVDR test to a patient was $50.31 (reagents and consumables). Conclusion: The in-house HIV-Integrase assay satisfied the validation criteria for an in-house genotyping assay and proved affordable, making it an attractive alternative to the costly commercially available INSTI drug resistance tests, especially for resource-poor settings. In INSTI-naïve patients, the occurrence of major mutations was uncommon and accessory mutations with no influence on drug susceptibility occurred more frequently indicating that INSTI treatment is likely to be effective and underscoring the need for continued genetic surveillance for HIV drug resistance.