Operationalising targeted next-generation sequencing for routine diagnosis of drug-resistant TB.

BACKGROUND: Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India. METHODS: Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below. RESULTS: Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119. CONCLUSIONS: Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.

CONTEXTE: Les tests phénotypiques de sensibilité aux médicaments (pDST) pour Mycobacterium tuberculosis peuvent prendre jusqu'à 8 semaines, tandis que les tests moléculaires conventionnels identifient un ensemble limité de mutations de résistance. Le séquençage ciblé de la prochaine génération (tNGS) offre des résultats rapides pour prédire la résistance globale aux médicaments, et cette étude avait pour objectif d'explorer sa faisabilité opérationnelle au sein d'un laboratoire de santé publique à Mumbai, en Inde. MÉTHODES: Des échantillons pulmonaires de patients consentants testés positifs au Xpert MTB ont été testés pour la résistance aux médicaments par des méthodes conventionnelles et en utilisant le tNGS. Les expériences des membres de l'équipe de l'étude en matière de fonctionnement du laboratoire et de mise en œuvre logistique sont présentées ci-dessous. RÉSULTATS: Sur le nombre total de patients testés, 70% (113/161) n'avaient pas d'antécédents de TB ou de traitement ; cependant, 88,2% (n = 142) présentaient une TB résistante à la rifampicine/multirésistante aux médicaments (RR/MDR-TB). La concordance entre les prédictions de résistance de la tNGS et de la pDST était élevée pour la plupart des médicaments, la tNGS identifiant globalement la résistance avec plus de précision. La tNGS a été intégrée et adaptée au flux de travail du laboratoire ; toutefois, la mise en lots des échantillons a entraîné un délai d'obtention des résultats beaucoup plus long, le plus rapide étant de 24 jours. L'extraction manuelle de l'ADN a été source d'inefficacité ; le protocole a donc été optimisé. L'analyse des mutations non caractérisées et l'interprétation des modèles de rapport ont nécessité une expertise technique. Le coût du tNGS par échantillon s'élevait à US$230, contre US$119 pour le pDST. CONCLUSIONS: La mise en œuvre de la tNGS est possible dans les laboratoires de référence. Elle permet d'identifier rapidement la résistance aux médicaments et devrait être considérée comme une alternative potentielle à la pDST.

Clinical utility of target-based next-generation sequencing for drug-resistant TB.

BACKGROUND: In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).METHODS: A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDRplus and MTBDRsl).RESULTS: There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n = 13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.CONCLUSIONS: Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.

Scaling up HIV viral load monitoring in Manicaland, Zimbabwe: challenges and opportunities from the field.

BACKGROUND: Demand for viral load (VL) monitoring is expected to increase; however, implementation of the multifaceted VL testing poses numerous challenges. We report experiences from Médecins Sans Frontiéres (MSF) and partners in the scale-up of HIV VL in collaboration with the Ministry of Health and Child Care (MoHCC) of Zimbabwe. METHODS: A retrospective data review of routine reports from MSF-supported health facilities in Manicaland Province (Zimbabwe) was conducted. These secondary aggregate data were triangulated, and emerging themes of lessons learnt from VL monitoring were shared. RESULTS: A VL testing coverage of 63% (5966/9456) was achieved among the 40 health facilities, together with a switch rate to second-line antiretroviral therapy (ART) of 46.4% (108/233). The key enablers to scaling-up the VL monitoring were well-equipped and supported VL laboratories, the operationalisation of the on-the-job clinical mentoring and systematic weaning off of better performing health facilities. Concerted efforts from different implementing partners and funders in the HIV programme, and close collaboration with MoHCC were pivotal. CONCLUSION: Our experience indicates that clinical mentoring is effective, and resulted in high VL testing coverage and up-skilling primary health care workers in VL monitoring. Attention must be focused on innovations for improving VL result utilisation, especially the identification and management of patients who fail ART.