Recombinant HIV-1 Gag polyprotein Pr55 (HXB2)

Protease inhibitors (Pis) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Target enrichment and next-generation sequencing (NGS) with the Illumina MiSeq system were followed by haplotype reconstruction. Full-length Gag-protease gene regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced, and used to construct gag-pro-pseudotyped viruses. Phylogenetic analysis was performed using maximum-likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) was measured using a single-cycle replication assay. Western blotting was used to analyze Gag cleavage. In one of six participants (subtype CRF02_AG), we found 4-fold-lower LPV susceptibility in viral clones during failure of second-line treatment. A combination of four mutations (S126del, H127del, T122A, and G123E) in the p17 matrix of baseline virus generated a similar 4-fold decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B Gag-protease backbone. Western blotting demonstrated significant Gag cleavage differences between sensitive and resistant isolates in the presence of drug. Resistant viruses had around 2-fold-lower infectivity than sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed that resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. We used a multipronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV-1 matrix, revealing the interplay between Gag-associated resistance and fitness.

Background Deep learning-based radiological image analysis could facilitate use of chest x-rays as triage tests for pulmonary tuberculosis in resource-limited settings. We sought to determine whether commercially available chest x-ray analysis software meet WHO recommendations for minimal sensitivity and specificity as pulmonary tuberculosis triage tests. Methods We recruited symptomatic adults at the Indus Hospital, Karachi, Pakistan. We compared two software, qXR version 2.0 (qXRv2) and CAD4TB version 6.0 (CAD4TBv6), with a reference of mycobacterial culture of two sputa. We assessed qXRv2 using its manufacturer prespecified threshold score for chest x-ray classification as tuberculosis present versus not present. For CAD4TBv6, we used a data-derived threshold, because it does not have a prespecified one. We tested for non-inferiority to preset WHO recommendations (0.90 for sensitivity, 0.70 for specificity) using a non-inferiority limit of 0 .05. We identified factors associated with accuracy by stratification and logistic regression. Findings We included 2198 (92.7%) of 2370 enrolled participants. 2187 (99.5%) of 2198 were HIV-negative, and 272 (12.4%) had culture-confirmed pulmonary tuberculosis. For both software, accuracy was non-inferior to WHO-recommended minimum values (qXRv2 sensitivity 0.93 [95% CI 0.89-0.95], non-inferiority p=0.0002; CAD4TBv6 sensitivity 0.93 [0.90-0.96], p<0.0001; qXRv2 specificity 0.75 [0.73-0.77], p<0.0001; CAD4TBv6 specificity 0.69 [0.67-0.71], p=0.0003). Sensitivity was lower in smear-negative pulmonary tuberculosis for both software, and in women for CAD4TBv6. Specificity was lower in men and in those with previous tuberculosis, and reduced with increasing age and decreasing body mass index. Smoking and diabetes did not affect accuracy. Interpretation In an HIV-negative population, these software met WHO-recommended minimal accuracy for pulmonary tuberculosis triage tests. Sensitivity will be lower when smear-negative pulmonary tuberculosis is more prevalent. Funding Canadian Institutes of Health Research. Copyright (c) The Author(s). Published by Elsevier Ltd.