Rapid molecular diagnostics for detecting multidrug-resistant and extensively drug-resistant tuberculosis (M/XDR-TB) primarily identify mutations in Mycobacterium tuberculosis (Mtb) genes associated ...with drug resistance. Their accuracy, however, is dependent largely on the strength of the association between a specific mutation and the phenotypic resistance of the isolate with that mutation, which is not always 100%. While this relationship is well established and reliable for first-line anti-TB drugs, rifampin and isoniazid, it is less well-studied and understood for second-line, injectable drugs, amikacin (AMK), kanamycin (KAN) and capreomycin (CAP).
We conducted a systematic review of all published studies evaluating Mtb mutations associated with resistance to AMK, KAN, CAP in order to characterize the diversity and frequency of mutations as well as describe the strength of the association between specific mutations and phenotypic resistance in global populations. Our objective was to determine the potential utility and reliability of these mutations as diagnostic markers for detecting AMK, KAN and CAP resistance. Mutation data was reviewed for 1,585 unique clinical isolates from four continents and over 18 countries. Mutations in the rrs, tlyA, eis promoter and gidB genes were associated with AMK, KAN and/or CAP resistance.
The rrs A1401G mutation was present in the majority of AMK, KAN and CAP resistant Mtb strains reviewed, but was also found in 7% of CAP susceptible strains. The 1401 mutation alone, however, was not found with sufficient frequency to detect more than 70-80% of global Mtb strains resistant to AMK and CAP, and 60% of strains resistant to KAN. Additional mutations in the rrs, eis promoter, tlyA and gidB genes appear to be associated with resistance and could improve sensitivity and specificity of future diagnostics.
Despite the advent of new diagnostics, drugs and regimens, tuberculosis (TB) remains a global public health threat. A significant challenge for TB control efforts has been the monitoring of TB ...therapy and determination of TB treatment success. Current recommendations for TB treatment monitoring rely on sputum and culture conversion, which have low sensitivity and long turnaround times, present biohazard risk, and are prone to contamination, undermining their usefulness as clinical treatment monitoring tools and for drug development. We review the pipeline of molecular technologies and assays that serve as suitable substitutes for current culture-based readouts for treatment response and outcome with the potential to change TB therapy monitoring and accelerate drug development.
A laboratory validation study was conducted to assess the equivalence of Xpert MTB/RIF Ultra testing on the GeneXpert System and the GeneXpert Omni System ('Omni') for tuberculosis and rifampicin ...resistance. High concordance of the two devices was demonstrated for well-characterized clinical samples as well as control materials, with controls tested on Omni at normal and challenging environmental conditions (i.e. 35°C, 90% relative humidity). Equivalence of the Cts for all probes was also shown. Equivalence was demonstrated for the Omni and GeneXpert devices for tuberculosis and rifampicin resistance detection for a diverse range of clinical specimens and environmental conditions.
The WHO End TB Strategy requires drug susceptibility testing and treatment of all people with tuberculosis, but second-line diagnostic testing with line-probe assays needs to be done in experienced ...laboratories with advanced infrastructure. Fewer than half of people with drug-resistant tuberculosis receive appropriate treatment. We assessed the diagnostic accuracy of the rapid Xpert MTB/XDR automated molecular assay (Cepheid, Sunnyvale, CA, USA) to overcome these limitations.
We did a prospective study involving individuals presenting with pulmonary tuberculosis symptoms and at least one risk factor for drug resistance in four sites in India (New Delhi and Mumbai), Moldova, and South Africa between July 31, 2019, and March 21, 2020. The Xpert MTB/XDR assay was used as a reflex test to detect resistance to isoniazid, fluoroquinolones, ethionamide, amikacin, kanamycin, and capreomycin in adults with positive results for Mycobacterium tuberculosis complex on Xpert MTB/RIF or Ultra (Cepheid). Diagnostic performance was assessed against a composite reference standard of phenotypic drug-susceptibility testing and whole-genome sequencing. This study is registered with ClinicalTrials.gov, number NCT03728725.
Of 710 participants, 611 (86%) had results from both Xpert MTB/XDR and the reference standard for any drug and were included in analysis. Sensitivity for Xpert MTB/XDR detection of resistance was 94% (460 of 488, 95% CI 92–96) for isoniazid, 94% (222 of 235, 90–96%) for fluoroquinolones, 54% (178 of 328, 50–61) for ethionamide, 73% (60 of 82, 62–81) for amikacin, 86% (181 of 210, 81–91) for kanamycin, and 61% (53 of 87, 49–70) for capreomycin. Specificity was 98–100% for all drugs. Performance was equivalent to that of line-probe assays. The non-determinate rate of Xpert MTB/XDR (ie, invalid M tuberculosis complex detection) was 2·96%.
The Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment.
German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.
In a recent report of a systematic review of critical concentrations (CCs), the World Health Organization (WHO) lowered the rifampin (RIF) CC for antimicrobial susceptibility testing (AST) of the
...complex using Middlebrook 7H10 medium and the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system from 1 to 0.5 μg/ml. The previous RIF CC for 7H10 had been in use for over half a century. Because it had served as the
reference standard, it contributed to the endorsement of inappropriately high CCs for other AST methods, including the U.S. Food and Drug Administration (FDA)-approved MGIT system. Moreover, this resulted in confusion about the interpretation of seven borderline resistance mutations in
(i.e., L430P, D435Y, H445L, H445N, H445S, L452P, and I491F). In this issue of the
, Shea et al. (J Clin Microbiol 59:e01885-20, 2021, https://doi.org/10.1128/JCM.01885-20) provide evidence that the CC endorsed by the Clinical and Laboratory Standards Institute for the Sensititre MYCOTB system, which is not FDA approved but is CE-IVD marked in the European Union, is likely also too high. These findings underscore the importance of calibrating AST methods against a rigorously defined reference standard, as recently proposed by the European Committee on Antimicrobial Susceptibility Testing, as well as the value of routine next-generation sequencing for investigating discordant AST results.
Universal drug susceptibility testing (DST) for tuberculosis is a major goal of the END TB strategy. PCR-based molecular diagnostic tests have been instrumental in increasing DST globally and several ...assays have now been endorsed by the World Health Organization (WHO) for use in the diagnosis of drug resistance. These endorsed assays, however, each interrogate a limited number of mutations associated with resistance, potentially limiting their sensitivity compared to sequencing-based methods. We applied an in silico method to compare the sensitivity and specificity of WHO-endorsed molecular based diagnostics to the mutation set identified by the WHO mutations catalogue using phenotypic DST as the reference. We found that, in silico, the mutation sets used by probe-based molecular diagnostic tests to identify rifampicin, isoniazid, pyrazinamide, levofloxacin, moxifloxacin, amikacin, capreomycin and kanamycin resistance produced similar sensitivities and specificities to the WHO mutation catalogue. PCR-based diagnostic tests were most sensitive for drugs where mechanisms of resistance are well established and localised to small genetic regions or a few prevalent mutations. Approaches using sequencing technologies can provide advantages for drugs where our knowledge of resistance is limited, or where complex resistance signatures exist.
Drug-resistant tuberculosis (DR-TB) threatens to derail tuberculosis control efforts, particularly in Africa where the disease remains out of control. The dogma that DR-TB epidemics are fueled by ...unchecked rates of acquired resistance in inadequately treated or non-adherent individuals is no longer valid in most high DR-TB burden settings, where community transmission is now widespread. A large burden of DR-TB in Africa remains undiagnosed due to inadequate access to diagnostic tools that simultaneously detect tuberculosis and screen for resistance. Furthermore, acquisition of drug resistance to new and repurposed drugs, for which diagnostic solutions are not yet available, presents a major challenge for the implementation of novel, all-oral, shortened (6–9 months) treatment. Structural challenges including poverty, stigma, and social distress disrupt engagement in care, promote poor treatment outcomes, and reduce the quality of life for people with DR-TB. We reflect on the lessons learnt from the South African experience in implementing state-of-the-art advances in diagnostic solutions, deploying recent innovations in pharmacotherapeutic approaches for rapid cure, understanding local transmission dynamics and implementing interventions to curtail DR-TB transmission, and in mitigating the catastrophic socioeconomic costs of DR-TB. We also highlight globally relevant and locally responsive research priorities for achieving DR-TB control in South Africa.
The aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational ...field trial.
Samples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa.
Specificity for all three assays was excellent: 97-100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99-100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94-100% for INH, RIF, MOX and OFX, and 84-90% for AMK and CAP, but only 48-62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT.
All three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.
Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid ...characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.
Despite recent diagnostic advances, the majority of multidrug-resistant tuberculosis (MDR-TB) cases remain undiagnosed. Line probes assays (LiPAs) hold great promise to curb the spread of MDR-TB as ...they can rapidly detect MDR-TB even when laboratory infrastructure is limited, yet few of these assays are currently widely available or supported by World Health Organization (WHO) policy.
The aim of this prospective, blinded, non-inferiority study was to compare the performance of YD Diagnostics REBA MTB MDR LiPA (YD) to the WHO-endorsed Hain MTBDRplus V1 LiPA (Hain V1) for the detection of rifampicin and isoniazid resistance. In phase 1, YD and Hain V1 diagnostic performance was assessed with selected culture isolates and results were compared to phenotypic drug susceptibility testing (DST) results and targeted sequencing data. In phase 2, both assays were tested on processed sputum samples and results were compared to phenotypic DST results.
In phase 1, YD did not achieve non-inferiority to Hain V1. For isoniazid resistance detection, Hain V1 had a sensitivity of 89% (95%CI 83.8-93%) and specificity of 99.4% (95%CI 96.9-100%). While YD had a similar sensitivity of 92% (95%CI 87.3-95.4%), the specificity was inferior at 92.6% (95%CI 87.6-96%). For rifampicin resistance detection, Hain V1 had a sensitivity of 90.2% (95%CI 84.8-94.2%) and specificity of 98.5% (95%CI 95.7-99.7%) while YD had an inferior sensitivity of 72.4% (95%CI 65.1-78.9%) and a comparable specificity of 98% (95%CI 95-99.5%). Similar results were observed in phase 2. For MDR-TB detection, the sensitivity and specificity of Hain V1 was 93.4% (95%CI 88.2-96.2%) and 96.2% (95%CI 88.2-96.8%), respectively, compared to 75.7% (95%CI 68-82.2%) and 92% (95%CI 88.2-94.9%) for YD.
YD did not achieve non-inferiority with Hain V1. Further improvements and repeat evaluation of YD is necessary prior to recommending its use for clinical settings.
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