Nano-RECall provides an integrated pipeline for HIV-1 drug resistance testing from Oxford Nanopore sequence data.

OBJECTIVES: Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing. METHODS: We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n = 46) and (b) homebrew integrase (IN) (n = 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only. RESULTS: The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both. CONCLUSIONS: The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings.

Low lopinavir plasma or hair concentrations explain second-line protease inhibitor failures in a resource-limited setting.

BACKGROUND: In resource-limited settings, many patients, with no prior protease inhibitor (PI) treatment on a second-line, high genetic barrier, ritonavir-boosted PI-containing regimen have virologic failure. METHODS: We conducted a cross-sectional survey to investigate the aetiology of virologic failure in 2 public health antiretroviral clinics in South Africa documenting the prevalence of virologic failure (HIV RNA load >500 copies/mL) and genotypic antiretroviral resistance; and lopinavir hair and plasma concentrations in a nested case-control study. RESULTS: Ninety-three patients treated with a second-line regimen including lopinavir boosted with ritonavir were included, of whom 50 (25 cases, with virologic failure and 25 controls) were included in a nested case control study. Of 93 patients, 37 (40%) had virological failure, only 2 of them had had major PI mutations. The negative predictive values: probability of failure with lopinavir plasma concentration >1 µg/mL or hair concentrations >3.63 ng/mg for virologic failure were 86% and 89%, and positive predictive values of low concentrations 73% and 79%, respectively, whereas all virologic failures with HIV RNA loads above 1000 copies per milliliter, of patients without PI resistance, could be explained by either having a low lopinavir concentration in plasma or hair. CONCLUSIONS: Most patients who fail a lopinavir/ritonavir regimen, in our setting, have poor lopinavir exposure. A threshold plasma lopinavir concentration (indicating recent lopinavir/ritonavir use) and/or hair concentration (indicating longer term lopinavir exposure) are valuable in determining the aetiology of virologic failure and identifying patients in need of adherence counselling or resistance testing.