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Background: Bringing reliable and accurate tuberculosis (TB) diagnosis closer to patients is a key priority for global TB control. Molbio Diagnostics have developed the Truenat point-of-care molecular assays for detection of TB and rifampicin (RIF) resistance., Methods: We conducted a prospective multicentre diagnostic accuracy study at 19 primary healthcare centres and seven reference laboratories in Peru, India, Ethiopia and Papua New Guinea to estimate the diagnostic accuracy of the point-of-care Truenat MTB, MTB Plus and MTB-RIF Dx assays for pulmonary TB using culture and phenotypic drug susceptibility testing as the reference standard, compared with Xpert MTB/RIF or Ultra., Results: Of 1807 enrolled participants with TB signs/symptoms, 24% were culture-positive for Mycobacterium tuberculosis , of which 15% were RIF-resistant. In microscopy centres, the pooled sensitivity of Truenat MTB and Truenat MTB Plus was 73% (95% CI 67-78%) and 80% (95% CI 75-84%), respectively. Among smear-negative specimens, sensitivities were 36% (95% CI 27-47%) and 47% (95% CI 37-58%), respectively. Sensitivity of Truenat MTB-RIF was 84% (95% CI 62-95%). Truenat assays showed high specificity. Head-to-head comparison in the central reference laboratories suggested that the Truenat assays have similar performance to Xpert MTB/RIF., Conclusion: We found the performance of Molbio's Truenat MTB, MTB Plus and MTB-RIF Dx assays to be comparable to that of the Xpert MTB/RIF assay. Performing the Truenat tests in primary healthcare centres with very limited infrastructure was feasible. These data supported the development of a World Health Organization policy recommendation of the Molbio assays., Competing Interests: Conflict of interest: A. Penn-Nicholson reports grants from Bill and Melinda Gates Foundation, during the conduct of the study; reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: S.N. Gomathi reports grants from Indian Council of Medical Research - India TB Research Consortium, during the conduct of the study. Conflict of interest: C. Ugarte-Gil has nothing to disclose. Conflict of interest: A. Meaza has nothing to disclose. Conflict of interest: E. Lavu has nothing to disclose. Conflict of interest: P. Patel reports grants from the Indian Council of Medical Research through the National Institute for Research in Tuberculosis Chennai, during the conduct of the study. Conflict of interest: B. Choudhury reports grants from Indian Council of Medical Research – India TB Research Consortium through the National Institute for Research in Tuberculosis Chennai, during the conduct of the study. Conflict of interest: C. Rodrigues has nothing to disclose. Conflict of interest: S. Chadha reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: M. Kazi has nothing to disclose. Conflict of interest: A. Macé reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: P. Nabeta reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: C. Boehme reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: R.R. Gangakhedkar has nothing to disclose. Conflict of interest: S. Sarin reports grants from Bill and Melinda Gates Foundation, during the conduct of the study; and reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: E. Tesfaye has nothing to disclose. Conflict of interest: E. Gotuzzo has nothing to disclose. Conflict of interest: P. du Cros reports grants from FIND and the Australian Dept of Foreign Affairs and Trade during the conduct of the study; other (consultancy) from TB Alliance, outside the submitted work; and is a member of the steering committee for the MSF Sponsored TB PRACTECAL Clinical Trial for novel regimens for MDR-TB treatment. Conflict of interest: S. Tripathy reports grants from ICMR-India TB Research Consortium (ICMR-ITRC), during the conduct of the study. Conflict of interest: M. Ruhwald reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Conflict of interest: M. Singh has nothing to disclose. Conflict of interest: C.M. Denkinger reports grants from the Bill and Melinda Gates Foundation, during the conduct of the study; and reports working for FIND until April 2019. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access. Since leaving FIND, C.M. Denkinger continues to hold a collaborative agreement with FIND. Conflict of interest: S.G. Schumacher reports working for FIND. FIND conducts multiple clinical research projects to evaluate multiple new diagnostic tests against published target product profiles that have been defined through consensus processes. These include studies of diagnostic products developed by private sector companies who provide access to know-how, equipment/reagents, and may contribute through unrestricted donations according to FIND policies and in line with guidance from the organisation's external scientific advisory council. FIND does not attribute any financial value to such access., (Copyright ©The authors 2021.)

Various diagnostic companies have developed high throughput molecular assays for tuberculosis (TB) and resistance detection for rifampicin and isoniazid. We performed a systematic review and meta-analyses to assess the diagnostic accuracy of five of these tests for pulmonary specimens. The tests included were Abbott RealTime MTB, Abbott RealTime RIF/INH, FluoroType MTB, FluoroType MTDBR and BD Max MDR-TB assay.A comprehensive search of six databases for relevant citations was performed. Cross-sectional, case-control, cohort studies, and randomised controlled trials of any of the index tests were included. Respiratory specimens (such as sputum, bronchoalveolar lavage, tracheal aspirate, etc ) or their culture isolates.A total of 21 included studies contributed 26 datasets. We could only meta-analyse data for three of the five assays identified, as data were limited for the remaining two. For TB detection, the included assays had a sensitivity of 91% or more and the specificity ranged from 97% to 100%. For rifampicin resistance detection, all the included assays had a sensitivity of more than 92%, with a specificity of 99-100%. Sensitivity for isoniazid resistance detection varied from 70 to 91%, with higher specificity of 99-100% across all index tests. Studies that included head-to-head comparisons of these assays with Xpert MTB/RIF for detection of TB and rifampicin resistance suggested comparable diagnostic accuracy.In people with symptoms of pulmonary TB, the centralised molecular assays demonstrate comparable diagnostic accuracy for detection of TB, rifampicin and isoniazid resistance to Xpert MTB/RIF assay, a WHO recommended molecular test., Competing Interests: Conflict of interest: E. MacLean has nothing to disclose. Conflict of interest: M. Pai has nothing to disclose. Conflict of interest: S.G. Schumacher has nothing to disclose. Conflict of interest: C.M. Denkinger has nothing to disclose. Conflict of interest: M. Kohli has nothing to disclose., (Copyright ©ERS 2021.)

Antibiotic treatment of tuberculosis takes ≥6 months, putting a major burden on patients and health systems in large parts of the world. Treatment beyond 2 months is needed to prevent tuberculosis relapse by clearing remaining, drug-tolerant Mycobacterium tuberculosis bacilli. However, the majority of patients treated for only 2-3 months will cure without relapse and do not need prolonged treatment. Assays that can identify these patients at an early stage of treatment may significantly help reduce the treatment burden, while a test to identify those patients who will fail treatment may help target host-directed therapies.In this review we summarise the state of the art with regard to discovery of biomarkers that predict relapse-free cure for pulmonary tuberculosis. Positron emission tomography/computed tomography scanning to measure pulmonary inflammation enhances our understanding of "cure". Several microbiological and immunological markers seem promising; however, they still need a formal validation. In parallel, new research strategies are needed to generate reliable tests., Competing Interests: Conflict of interest: D. Goletti reports personal fees from Qiagen, Quidel and Janssen, outside the submitted work. Conflict of interest: C.S. Lindestam Arlehamn has nothing to disclose. Conflict of interest: T.J. Scriba reports two pending patents of blood transcriptomic signatures of risk. Conflict of interest: R. Anthony reports grants from FIND to study the utility of IP-10 for treatment monitoring, outside the submitted work. Conflict of interest: D.M. Cirillo reports an unrestricted grant from QIAGEN to research the use of Quantiferon plus to predict outcome, and grants from JANSSEN on the establishment of Bedaquiline DST as SRL, outside the submitted work. Conflict of interest: T. Alonzi has nothing to disclose. Conflict of interest: C.M. Denkinger reports that FIND is a non-for-profit foundation, whose mission is to find diagnostic solutions to overcome diseases of poverty in LMICs. It works closely with the private and public sectors and receives funding from some of its industry partners. It has organisational firewalls to protect it against any undue influences in its work or the publication of its findings. All industry partnerships are subject to review by an independent scientific advisory committee or another independent review body, based on due diligence, TTPs and public sector requirements. FIND catalyses product development, leads evaluations, takes positions, and accelerates access to tools identified as serving its mission. It provides indirect support to industry (e.g. access to open specimen banks, a clinical trial platform, technical support, expertise, laboratory capacity strengthening in LMICs) to facilitate the development and use of products in these areas. FIND also supports the evaluation of prioritised assays and the early stages of implementation of WHO-approved (guidance and PQ) assays using donor grants. In order to carry out test validations and evaluations, has product evaluation agreements with several private sector companies for the diseases FIND works in which strictly define its independence and neutrality vis-a-vis the companies whose products get evaluated, and describes roles and responsibilities. Conflict of interest: F. Cobelens has nothing to disclose., (Copyright ©ERS 2018.)

Novel accurate tests are needed that identify individuals infected with Mycobacterium tuberculosis who have incipient disease and are likely to develop clinical tuberculosis (TB) in the near future to allow for targeted preventive treatment beyond the current risk groups. Recently, a target product profile was developed that outlines the minimal and optimal characteristics for such an incipient TB test. We describe an evaluation framework for generating evidence to inform the development of policy guidance for the use of such a new test by the World Health Organization. Two research objectives are addressed. 1) The predictive ability of an incipient TB test should be assessed in clinical evaluation studies that include the intended target population and follow-up of sufficient duration to observe whether individuals do or do not progress to clinical TB disease. 2) Studies are needed to evaluate the test under routine programmatic conditions and measure its impact on patient- or health-system-important outcomes. For both research objectives, study designs, methods and analysis are described, with the intent to inform the clinical development plans of test manufacturers, researchers and funders., Competing Interests: Conflict of interest: C.M. Denkinger: FIND (Foundation for Innovative New Diagnostics) is a not-for-profit foundation, whose mission is to find diagnostic solutions to overcome diseases of poverty in low- and middle-income countries (LMICs). It works closely with the private and public sectors and receives funding from some of its industry partners. It has organisational firewalls to protect it against any undue influences in its work or the publication of its findings. All industry partnerships are subject to review by an independent Scientific Advisory Committee or another independent review body, based on due diligence, target product profiles and public sector requirements. FIND catalyses product development, leads evaluations, takes positions and accelerates access to tools identified as serving its mission. It provides indirect support to industry (e.g. access to open specimen banks, a clinical trial platform, technical support, expertise, laboratory capacity strengthening in LMICs) to facilitate the development and use of products in these areas. FIND also supports the evaluation of prioritised assays and the early stages of implementation of WHO-approved (guidance and prequalification) assays using donor grants. In order to carry out test validations and evaluations, FIND has product evaluation agreements with several private sector companies, which strictly define its independence and neutrality vis-a-vis the companies whose products get evaluated, and describe roles and responsibilities., (The content of this work is copyright of the authors or their employers. Design and branding are copyright ©ERS 2018.)

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Only 25% of multidrug-resistant tuberculosis (MDR-TB) cases are currently diagnosed. Line probe assays (LPAs) enable rapid drug-susceptibility testing for rifampicin (RIF) and isoniazid (INH) resistance and Mycobacterium tuberculosis detection. Genotype MTBDRplusV1 was WHO-endorsed in 2008 but newer LPAs have since been developed.This systematic review evaluated three LPAs: Hain Genotype MTBDRplusV1, MTBDRplusV2 and Nipro NTM+MDRTB. Study quality was assessed with QUADAS-2. Bivariate random-effects meta-analyses were performed for direct and indirect testing. Results for RIF and INH resistance were compared to phenotypic and composite (incorporating sequencing) reference standards. M. tuberculosis detection results were compared to culture.74 unique studies were included. For RIF resistance (21 225 samples), pooled sensitivity and specificity (with 95% confidence intervals) were 96.7% (95.6-97.5%) and 98.8% (98.2-99.2%). For INH resistance (20 954 samples), pooled sensitivity and specificity were 90.2% (88.2-91.9%) and 99.2% (98.7-99.5%). Results were similar for direct and indirect testing and across LPAs. Using a composite reference standard, specificity increased marginally. For M. tuberculosis detection (3451 samples), pooled sensitivity was 94% (89.4-99.4%) for smear-positive specimens and 44% (20.2-71.7%) for smear-negative specimens.In patients with pulmonary TB, LPAs have high sensitivity and specificity for RIF resistance and high specificity and good sensitivity for INH resistance. This meta-analysis provides evidence for policy and practice., (Copyright ©ERS 2017.)

New approaches to control the spread of tuberculosis (TB) are needed, including tools to predict development of active TB from latent TB infection (LTBI). Recent studies have described potential correlates of risk, in order to inform the development of prognostic tests for TB disease progression. These efforts have included unbiased approaches employing "omics" technologies, as well as more directed, hypothesis-driven approaches assessing a small set or even individual selected markers as candidate correlates of TB risk. Unbiased high-throughput screening of blood RNAseq profiles identified signatures of active TB risk in individuals with LTBI, ≥1 year before diagnosis. A recent infant vaccination study identified enhanced expression of T-cell activation markers as a correlate of risk prior to developing TB; conversely, high levels of Ag85A antibodies and high frequencies of interferon (IFN)-γ specific T-cells were associated with reduced risk of disease. Others have described CD27 - IFN-γ + CD4 + T-cells as possibly predictive markers of TB disease. T-cell responses to TB latency antigens, including heparin-binding haemagglutinin and DosR-regulon-encoded antigens have also been correlated with protection.Further studies are needed to determine whether correlates of risk can be used to prevent active TB through targeted prophylactic treatment, or to allow targeted enrolment into efficacy trials of new TB vaccines and therapeutic drugs., (Copyright ©ERS 2016.)

The global roll-out of Xpert MTB/RIF (Cepheid Inc., Sunnyvale, CA, USA) has changed the diagnostic landscape of tuberculosis (TB). More than 16 million tests have been performed in 122 countries since 2011, and detection of multidrug-resistant TB has increased three- to eight-fold compared to conventional testing. The roll-out has galvanised stakeholders, from donors to civil society, and paved the way for universal drug susceptibility testing. It has attracted new product developers to TB, resulting in a robust molecular diagnostics pipeline. However, the roll-out has also highlighted gaps that have constrained scale-up and limited impact on patient outcomes. The roll-out has been hampered by high costs for under-funded programmes, unavailability of a complete solution package (notably comprehensive training, quality assurance, implementation plans, inadequate service and maintenance support) and lack of impact assessment. Insufficient focus has been afforded to effective linkage to care of diagnosed patients, and clinical impact has been blunted by weak health systems. In many countries the private sector plays a dominant role in TB control, yet this sector has limited access to subsidised pricing. In light of these lessons, we advocate for a comprehensive diagnostics implementation approach, including increased engagement of in-country stakeholders for product launch and roll-out, broader systems strengthening in preparation for new technologies, as well as quality impact data from programmatic settings., (Copyright ©ERS 2016.)

Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) is endorsed for the detection of pulmonary tuberculosis (TB). We performed a systematic review and meta-analysis to assess the accuracy of Xpert for the detection of extrapulmonary TB. We searched multiple databases to October 15, 2013. We determined the accuracy of Xpert compared with culture and a composite reference standard (CRS). We grouped data by sample type and performed meta-analyses using a bivariate random-effects model. We assessed sources of heterogeneity using meta-regression for predefined covariates. We identified 18 studies involving 4461 samples. Sample processing varied greatly among the studies. Xpert sensitivity differed substantially between sample types. In lymph node tissues or aspirates, Xpert pooled sensitivity was 83.1% (95% CI 71.4-90.7%) versus culture and 81.2% (95% CI 72.4-87.7%) versus CRS. In cerebrospinal fluid, Xpert pooled sensitivity was 80.5% (95% CI 59.0-92.2%) against culture and 62.8% (95% CI 47.7-75.8%) against CRS. In pleural fluid, pooled sensitivity was 46.4% (95% CI 26.3-67.8%) against culture and 21.4% (95% CI 8.8-33.9%) against CRS. Xpert pooled specificity was consistently >98.7% against CRS across different sample types. Based on this systematic review, the World Health Organization now recommends Xpert over conventional tests for diagnosis of TB in lymph nodes and other tissues, and as the preferred initial test for diagnosis of TB meningitis., (©ERS 2014.)