Comparison of an in-house ‘home-brew’ and commercial ViroSeq integrase genotyping assays on HIV-1 subtype C samples

Abstract

BackgroundRoll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals.MethodsWe used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time.ResultsAmong 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C.ConclusionsThe IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

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

“…Because of our assay design, 10 μL of 25 μL of TNA from 200 μL of plasma or one DBS spot may be used to perform HIVDR genotyping for both PRRT and INT which saves sample and workflow. This contrasts with the ViroSeq HIV-1 Integrase Genotyping kit and ABL, which both require 500 μL ( 8 , 9 ) of plasma for PRRT genotyping and an additional 500 μL for INT genotyping. Sentosa is a great multiplexed and fully automated HIVDR assay using NGS technology.…”

“…For the HIVDR assay application, the most valuable feature of this HIVDR assay is that both plasma and DBS samples may be used for HIVDR genotyping. The Abbott ViroSeq HIV-1 Integrase Genotyping kit ( 8 ), ABL DeepCheck ( 9 ), and Sentosa ( 10 ) HIV-1 genotyping assay have not demonstrated data on the performance of DBS. The DBS application of this assay is very important in resource-limited settings or remote areas where plasma samples may not be feasible to collect or transport for VL testing and genotyping ( 15 – 17 ).…”

“…Currently, there are a few HIVDR kits commercially available to monitor HIVDR for both PRRT and INT. Both Abbott ( 8 ) and Advanced Biological Laboratories (ABL) ( 9 ) have a single-plex HIV genotyping assay for either PRRT or INT using Sanger technology. Recently, Vela deployed a Federal Drug Administration (FDA)-approved multiplexed HIV genotyping assay, Sentosa, for PRRT and INT genotyping using next-generation sequencing (NGS) ( 10 ).…”

Limited HIV-1 Subtype C nef 3′PPT Variation in Combination Antiretroviral Therapy Naïve and Experienced People Living with HIV in Botswana

“…VF was defined as two or more consecutive plasma HIV-1 RNA levels (viral loads (VL)) > 400 copies/mL as per standard of care guidelines in Botswana. The HIV-1 integrase region was amplified using nested reverse transcription-polymerase chain reactions (RT-PCRs) where necessary and sequenced using a BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) on a 3130xl Genetic Analyser (Life Technologies Corporation, Applied Biosystems, Carlsbad, CA, USA) as previously described [ 7 , 20 ]. Sequencing of the nef gene was attempted from the same HIV-1 extracts that integrase sequences were successfully generated from.…”

Affordable drug resistance genotyping of HIV-1 reverse transcriptase, protease and integrase genes, for resource limited settings

“…With more studies reporting cases of DTG resistance [17][18][19] and with increased access to integrase strand transfer inhibitors (INSTIs), the demand for INSTI resistance testing has increased and there exists a risk of emergence of increasing INSTI resistance-associated mutations in RLS [20]. Most available HIVDR genotypic methods in RLS entail two separate assays for the detection of HIVDR mutations in the (i) reverse transcriptase (RT) and protease (PR) genes, and the (ii) integrase (IN) gene alone, such as the Applied Biosystems HIV-1 Genotyping kit [21].…”