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The fact that almost half of the 1 million cases of childhood tuberculosis (TB) globally remain undiagnosed jeopardizes the TB elimination goal. Fortunately, there are new advances in this field which have the potential to bridge this diagnostic gap. Advances in imaging include computer assisted interpretation of chest X-rays (CXRs), point of care ultrasound (POCUS) and faster and superior computed tomography/ magnetic resonance imaging (CT/ MRI) protocols. The urine lipoarabinomannan test has proved to be a good point of care test for diagnosing TB in Human immunodeficiency virus (HIV) infected children. Stool and nasopharyngeal aspirates are emerging as acceptable alternatives for gastric lavage and induced sputum for diagnosing intrathoracic tuberculosis. Xpert MTB/RIF Ultra has improved sensitivity compared to Xpert MTB/RIF for diagnosing both pulmonary/ extrapulmonary TB. Xpert XDR is another commercially available accurate point of care test for detecting resistance to drugs other than rifampicin in smear positive samples. Other molecular methods including new line probe assays, pyrosequencing, whole genome sequencing, and targeted next generation sequencing are extremely promising but not available commercially at present. The C-Tb skin test is an acceptable alternative to the tuberculin skin test and interferon gamma release assays for diagnosis of latent infection. There is an urgent need to incorporate some of these advances in the existing diagnostic algorithms of childhood TB., (© 2023. The Author(s), under exclusive licence to Dr. K C Chaudhuri Foundation.)

Background: 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., Methods: 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., Findings: 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%., Interpretation: 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., Funding: German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade., Competing Interests: Declaration of interests APN, MR, CMD, SBG, and SGS are employed by FIND, Geneva, Switzerland, a not-for-profit foundation that supports the evaluation of publicly prioritised tuberculosis assays and the implementation of WHO-approved (guidance and prequalification) assays using donor grants. FIND has product evaluation agreements with several private sector companies that design diagnostics for tuberculosis and other diseases. These agreements strictly define FIND's independence and neutrality with regards to these private sector companies. LS declares she is an inventor of the Dried Culture Spot technology (USP 8,709,712), which is used to measure the quality of molecular diagnostic M tuberculosis assays, and has received royalties from the University of the Witwatersrand, Johannesburg, South Africa. These assays are used within the context of this study but had no influence on the outcomes. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.)

Background: Hemophagocytic lymphohistiocytosis is a rare, potentially fatal syndrome of immune hyperactivation. Here we describe a ganglionar tuberculosis evolving to hemophagocytic lymphohistiocytosis following adjuvant immunotherapy in a melanoma patient., Case Presentation: A 76-year-old Caucasian male with melanoma started with fever, diffuse petechiae, splenomegaly, anemia, thrombocytopenia, hypofibrinogenemia, and hyperferritinemia 2 months following completion of adjuvant treatment with nivolumab. Positron emission tomography scan showed significant hypermetabolism in cervical, supraclavicular, mediastinal, and abdominal lymph nodes. Bone marrow aspiration demonstrated no alterations, except for a hypercellular pattern. Dexamethasone and intravenous immunoglobulin were started owing to suspicion of hemophagocytic lymphohistiocytosis. Core biopsy of the infracarinal lymph node revealed a chronic granulomatous inflammation and caseous necrosis, with positivity for Mycobacterium tuberculosis by polymerase chain reaction, and treatment for ganglionar tuberculosis was started., Conclusion: This case highlights the challenges involving programmed cell death 1 blockade in high-risk melanoma, in which infections, lymphoproliferative disorders, and sarcoidosis can mimic disease progression and trigger immune-related adverse events.

We present a case of a 59-year-old man, who on being evaluated for abdominal pain and headache, was found to have a pancreatic head mass and inflammatory hypophysitis. Xpert MTB/Rif of the pancreatic mass biopsy showed the presence of tuberculosis (TB) with a very low load, and rifampicin resistance was detected with absence of probes A and B. Pyrosequencing (a novel genotypic test for TB) of the Xpert MTB/Rif isolate detected a single, rare, high-confidence mutation (S512T) in the rpoB region (rifampicin resistance determining region in the MTB genome). The TB mycobacteria growth indicator tube (TBMGIT) phenotypic drug susceptibility test (DST), however, showed rifampicin susceptibility. Incidentally, he was unable to tolerate rifampicin and responded well to a non-rifampicin-based regimen. We discuss a possible hypothesis of the Xpert-DST discordance in accordance with a recent literature review on phenotypic DST methods. We also discuss the utility of pyrosequencing in clinical practice for the diagnosis of TB and its resistance patterns., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear., Methods: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance., Results: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted., Conclusions: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Paradoxical response (PR) in patients on anti-tuberculosis drugs and immune reconstitution inflammatory syndrome (IRIS) in patients started on antiretroviral therapy are well known phenomenon. We encountered a case of a paradoxical response in cerebral nocardiosis in a renal transplant recipient. To our knowledge this phenomenon in cerebral nocardiosis has not been reported earlier in literature., (© Journal of the Association of Physicians of India 2011.)

Timely diagnosis of tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is only achieved for ~58% cases. An improved, accurate, time- and cost-effective method for bacteriological confirmation of MTB is necessary. We evaluated Mycotube, a new variant of Lowenstein-Jensen (LJ) culture medium, by comparing it with Mycobacterium Growth Indicator Tube (MGIT) 960 (gold standard), local LJ, and bioMérieux LJ-T in terms of isolation rate and time-to-growth. Pulmonary and extra-pulmonary samples from treatment-naïve suspects (n = 207) were decontaminated by the N-acetyl-L-cysteine-sodium hydroxide method and used to inoculate the four media. Subjective and objective parameters were used for evaluation. Mycotube yielded 140 positive results, compared to 162, 69, and 141 from MGIT, local LJ, and LJ-T, respectively. Of these, 139 (67%) were true-positive results and 1 (0.5%) was false-positive. The mean time-to-growth detection was 17.4 days for Mycotube, compared to 14.5, 28.1, and 16.5 days for MGIT, local LJ, and LJ-T, respectively. The mean time-to-growth for local LJ significantly differed from that for MGIT, but not those for LJ-T and Mycotube. No contamination was observed. Mycotube had a sensitivity of 85.8% and a specificity of 97.8% as compared to MGIT. Mycotube offers good results, comparable with those observed for conventional LJ. It requires only basic laboratory infrastructure. The overall cost of the test should be nearly three times lower than that of MGIT. Mycotube helps with TB diagnosis and generates pure isolates for drug susceptibility testing.

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R., (Copyright © 2017 Chakravorty et al.)

Background: By using whole genome sequencing (WGS), researchers are beginning to understand the genetic diversity of Mycobacterium tuberculosis (MTB) and its consequences for the diagnosis of multidrug-resistant tuberculosis (MDR-TB) on a genomic scale. The Global Consortium for Drug-resistant TB Diagnostics (GCDD) conducted a genome scale variant analyses of 366 clinical MTB genomes (mostly MDR/XDR [extensively drug resistant]) from four countries in order to inform the development of rapid molecular diagnostics. This project has been extended by performing an evolutionary analysis of isoniazid (INH)-resistant isolates for prognostic purposes., Methods: 151 (130 INH R , 21 INH S ) clinical MTB isolates from India (19: 17 INH R , 2 INH S ), Moldova (48: 42 INH R , 6 INH S ), the Philippines (26: 20 INH R , 6 INH S ), and South Africa (58: 51 INH R , 7 INH S ) were included in this study. INH drug susceptibility was determined by using MGIT 960 and WHO (World Health Organization)-recommended critical concentration of 0.1 mg/L. Isolates were sequenced using PacBio RS WGS platform. A genome-wide variant analysis was conducted using a proprietary pipeline (PacDAP) developed at San Diego State University. To infer the amino acid changes in katG that confer resistance, PAML was utilized to detect sites in silico that are under positive selection. The d N /d S method was used in combination with Bayes empirical Bayes to determine sites under positive selection and Chi-Squared analysis to determine the significance of the selected sites., Results: PacDAP variant analysis revealed 22 novel catalase-peroxidase ( katG product) mutations. Of these, 14 were single nucleotide polymorphisms, while 8 novel mutations appeared in combination with katG S315T and/or with inhA promoter C-15T. These SNPs have not been previously reported. Additionally, 11 previously observed, but uncommon, katG mutations were also observed in these clinical isolates. These results suggest that 17 amino acids in the enzyme are under positive selective pressure; most significantly in South Africa and the Philippines. No selective pressure on codons other than 315 was observed in isolates from Moldova. Due to the low number of isolates from India, the significance of the sites under positive selection was low and no prediction for India could be made based on this study., Conclusions: Eleven of the 14 SNPs are resistance conferring, and it is believed that the remaining 8 combinatorial mutations are either compensatory in nature or, in combination with known SNPs, could increase resistance levels. Positive selection results indicate a diversifying evolutionary path to resistance more in line with long tail statistics and therefore indicate a departure from the traditional point mutation (or "hotspot") model that current molecular diagnostics are based on. Positive selection pressures indicate a future with elevated diagnostic and prognostic significance of the "long tail" (i.e., alternative mechanisms of resistance) and potentially diminishing significance of the canonical mutations (especially in South Africa and the Philippines), which could have significant future implications on narrowly targeting molecular diagnostics.

Background: Drug-resistant tuberculosis (DR-TB) is a looming threat to tuberculosis control in India. However, no countrywide prevalence data are available. The burden of DR-TB in HIV-co-infected patients is likewise unknown. Undiagnosed and untreated DR-TB among HIV-infected patients is a major cause of mortality and morbidity. We aimed to assess the prevalence of DR-TB (defined as resistance to any anti-TB drug) in patients attending public antiretroviral treatment (ART) centers in greater metropolitan Mumbai, India., Methods: A cross-sectional survey was conducted among adults and children ART-center attendees. Smear microscopy, culture and drug-susceptibility-testing (DST) against all first and second-line TB-drugs using phenotypic liquid culture (MGIT) were conducted on all presumptive tuberculosis patients. Analyses were performed to determine DR-TB prevalence and resistance patterns separately for new and previously treated, culture-positive TB-cases., Results: Between March 2013 and January 2014, ART-center attendees were screened during 14135 visits, of whom 1724 had presumptive TB. Of 1724 attendees, 72 (4%) were smear-positive and 202 (12%) had a positive culture for Mycobacterium tuberculosis. Overall DR-TB was diagnosed in 68 (34%, 95% CI: 27%-40%) TB-patients. The proportions of DR-TB were 25% (29/114) and 44% (39/88) among new and previously treated cases respectively. The patterns of DR-TB were: 21% mono-resistant, 12% poly-resistant, 38% multidrug-resistant (MDR-TB), 21% pre-extensively-drug-resistant (MDR-TB plus resistance to either a fluoroquinolone or second-line injectable), 6% extensively drug-resistant (XDR-TB) and 2% extremely drug-resistant TB (XDR-TB plus resistance to any group-IV/V drug). Only previous history of TB was significantly associated with the diagnosis of DR-TB in multivariate models., Conclusion: The burden of DR-TB among HIV-infected patients attending public ART-centers in Mumbai was alarmingly high, likely representing ongoing transmission in the community and health facilities. These data highlight the need to promptly diagnose drug-resistance among all HIV-infected patients by systematically offering access to first and second-line DST to all patients with 'presumptive TB' rather than 'presumptive DR-TB' and tailor the treatment regimen based on the resistance patterns.

Background: The aim of this multi-centric prospective study in India was to assess the accuracy of a serological test as an additional tool for diagnosing active tuberculosis (ATB). In particular, an assay based on ELISA using a phenolic glycolipid (PGL-Tb1) or a fusion protein (ESAT-6/CFP10) was compared to the tuberculin skin test (TST) and the microbiological results according to HIV status., Methods: Individuals with and without ATB and HIV infection were enrolled. Serology and TST results were analyzed per se and in combination with the microbiological data., Results: Among the 778 ATB patients, 102 were HIV-infected, 316 HIV-uninfected and 360 had an HIV-unknown status. Of the 945 non-ATB subjects, 559 were at low risk (community adults) and 386 at high risk of M. tuberculosis exposure. Among those with ATB, the sensitivity of ELISA-PGL-Tb1 for ATB was higher than that of ELISA-ESAT-6/CFP10, both in HIV-infected (72.3% versus 63.7%, p = 0.29) and HIV-uninfected/HIV-unknown groups (40.5% versus 28.6%; p<0.0001), whereas the specificity was around 91% for both tests. Sensitivity for ATB increased when the results of the two ELISA were combined, reaching 75.5% in the HIV-infected and 50.9% in the group of HIV-uninfected/HIV-unknown ATB, with a significant decrease of the global specificity (83.9%). Analyzing the ELISA results with the microbiological results, we observed that the sensitivity of both serology tests was independent of the ATB patients' smear microscopy (SM) status and grade. Combining the results of SM with both ELISA, the detection of ATB patients significantly increased (p<0.0001), particularly in those with extrapulmonary TB (up to 45.1%) or HIV infection (up to 83.3%). No significant association was observed between TST and serology results., Conclusions: In this prospective multi-centric study, the combination of two rapid tests, such as SM and serology, might be useful in detecting ATB, especially in HIV-infected patients.

Background: The aim of this multicentric prospective study in India was to assess the performance of the QuantiFERON TB-Gold in tube (QFT-GIT), Tuberculin Skin Test (TST) and microbiological results as additional tools for diagnosing active tuberculosis (TB) and latent infection (LTBI) according to Human Immunodeficiency Virus (HIV) status., Methods: Individuals with and without active TB and HIV infection were enrolled between 2006-2008. QFT-GIT and TST results were analyzed per se and in combination with microbiological data., Results: Among the 276 individuals (96 active pulmonary TB and 180 no active TB) tested by QFT-GIT, 18 indeterminate results (6.5%) were found, more significantly numerous in the HIV-infected (15/92; 16.3%) than the HIV-uninfected (3/184; 1.6%)(p<0.0001). QFT-GIT sensitivity for active TB was 82.3% and 92.9% respectively after including or excluding indeterminate results. Clinical sensitivity was significantly lower in the HIV-infected (68.4%) than the HIV-uninfected (91.4%) patients (p = 0.0059). LTBI was detected in 49.3% of subjects without active TB but varied according to TB exposure. When the TST and QFT-GIT were concomitantly performed, the respective sensitivity for active TB diagnosis was 95.0% and 85.0% in the HIV-uninfected (p = 0.60), and 66.7% and 51.5% in the HIV-infected patients (p = 0.32). QFT-GIT and TST respective specificity for active TB in the HIV-uninfected was 25.0% and 57.1% (p = 0.028), and 64.8% and 83.3% in the HIV-infected (p = 0.047). In those with active TB, QFT-GIT results were not associated with microbiological parameters (smear grade, liquid culture status, time-to-positivity of culture) or clinical suspicion of active TB score (provided by the clinicians at enrollment). Combining microbiological tests with both immunological tests significantly increased sensitivity for active TB diagnosis (p = 0.0002), especially in the HIV-infected individuals (p = 0.0016)., Conclusion: QFT-GIT and TST have similar diagnostic value for active TB diagnosis. In HIV-infected patients, combining microbiological tests with both immunological tests significantly increases the sensitivity for active TB diagnosis.

In this study, we aimed to correlate the analytical performance of SD BIOLINE TB Ag MPT64 Rapid Test kit (MPT64 assay) with the mycobacterial growth unit (GU) reported by the BACTEC MGIT 960 (MGIT 960) instrument. A total of 394 culture isolates reported positive by MGIT 960 were processed daily (until 'day 4') with the MPT64 assay until a positive MPT64 result was obtained and their GU values were noted daily before MPT64 testing. Based on this correlation of MPT64 positivity and corresponding GU values, a GU cut-off was determined. In the validation phase, with the experimentally determined GU cut-off value, 99.1% (576/581) of culture isolates were correctly identified as MTB within 2 days from instrument positivity. All results were available using a single-MPT64 assay strip, making the assay cost-effective. Thus, systematic implementation of the MPT64 assay proved to be cost-effective in a high-throughput laboratory without any delay in patient reporting., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Delay in laboratory diagnosis of tuberculosis (TB) is a major obstacle in TB control programs. There is an imperative need for scale-up of peripheral health care laboratories with conventional and molecular technologies for rapid and reliable diagnosis of TB. A cost-effective diagnostic algorithm for rapid diagnosis of TB should be implemented and followed, thereby reducing cost burden on patients.

Genotyping of Mycobacterium tuberculosis isolates is a useful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. There is a lack of information on the discriminatory power of standard 24-locus mycobacterial interspersed repetitive unit (MIRU) - variable number tandem repeats (VNTR) in India, which has the highest tuberculosis (TB) burden worldwide. Therefore, we assessed its utility on 69 M. tuberculosis (MTB) isolates from patients with extrapulmonary tuberculosis, in comparison to standard insertion sequence (IS) 6110-Restriction fragment length polymorphism (RFLP) fingerprinting and spoligotyping. IS6110-RFLP (HGDI, 0.9987) identified a single cluster of 3 (4.3%) single-copy IS6110 isolates. Spoligotyping showed 69.5% clustering (HGDI, 0.8857). In contrast, MIRU-VNTR analysis identified 69 (100%) unique strains (HGDI, 1.0000). Within the study limits, this observed high discriminatory power suggests that 24-locus MIRU-VNTR genotyping could potentially be used to study long-term transmission of MTB infection in Mumbai. Moreover, high congruence between the MIRU-VNTR-based and spoligotyping-based strain groupings suggests that CAS, EAI and Beijing are the predominant strain lineages in the Mumbai TB patient population. The Beijing lineage isolates were found to be more significantly associated with multi-drug resistance (p < 0.01) than CAS and EAI lineages., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Setting: A tertiary care centre in Mumbai with a referral bias towards treatment failures., Objective: To standardise and evaluate a novel single tube multiplex nested polymerase chain reaction (PCR) targeting insertion sequence (IS) 6110, mpb64, rrs and rpoB genes for rapid diagnosis of extra-pulmonary tuberculosis (EPTB)., Methods: The PCR assay was evaluated among 489 consecutive consenting patients, and results were compared against a composite reference standard comprising smear microscopy, culture, clinical symptoms, radiological scan and histology., Results: PCR assay reported a pooled sensitivity of 94.5% (242/256, 95%CI 91-97): 91.9% (125/136, 95%CI 86-96) for smear-negative composite reference standard (CRS) positive cases and 97.5% (117/120, 95%CI 93-99) for smear-positive CRS-positive cases. The PCR positivity rate increased from 91.7% (235/256, 95%CI 88-95) when presence of IS6110 was considered alone for reporting a test as positive to 94.5% (242/256, 95%CI 91-97) when used in combination with other three gene targets, with a specificity of 96.4% (212/220, 95%CI 93-98). A positive likelihood ratio of 26 (95%CI 13-51) and a negative likelihood ratio of 0.06 (95%CI 0.03-0.09) makes the test useful for ruling out and ruling in the disease., Conclusion: Culture should not be replaced by PCR as a gold standard; however, PCR can be used as a rapid, accurate tool in the diagnosis of EPTB.

Background: The aim of this multicentric prospective study in India was to assess the value of several microbiological tools that contribute to the diagnosis of tuberculosis (TB) according to HIV status., Methods: Standard microbiological tools on individual specimens were analyzed., Results: Among the 807 patients with active TB, 131 were HIV-infected, 316 HIV-uninfected and 360 had HIV-unknown status. Among the 980 non-active TB subjects, 559 were at low risk and 421 were at high risk of M. tuberculosis (Mtb) exposure. Sensitivity of smear microscopy (SM) was significantly lower in HIV-infected (42.2%) than HIV-uninfected (75.9%) (p = 0.0001) and HIV-unknown pulmonary TB patients (61.4%) (p = 0.004). Specificity was 94.5% in non-TB patients and 100% in health care workers (HCW) and healthy family contacts. Automated liquid culture has significantly higher diagnostic performances than solid culture, measured by sensitivity (74.7% vs. 55.9%) (p = 0.0001) and shorter median time to detection (TTD) (12.0 vs. 34.0 days) (p = 0.0001). Specificity was 100% in HCW and cured-TB patients, but was lower in non-TB patients (89%) due to isolation of Mycobacteria other than tuberculosis (MOTT). TTD by both methods was related to AFB score. Contamination rate was low (1.4%). AccuProbe hybridization technique detected Mtb in almost all culture-positive specimens, but MOTT were found in 4.7% with a significantly higher frequency in HIV-infected (15%) than HIV-uninfected TB patients (0.5%) (p = 0.0007). Pre-test classification significantly increased the diagnostic value of all microbiological tests in pulmonary TB patients (p<0.0001) but to a lesser degree in extrapulmonary TB patients., Conclusions: Conventional microbiological tools led to results similar to those already described in India special features for HIV-infected TB patients included lower detection by SM and culture. New microbiological assays, such as the automated liquid culture system, showed increased accuracy and speed of detection.

Rationale: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy., Objectives: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods., Methods: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results., Measurements and Main Results: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy., Conclusions: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

Approximately 10 to 15% of tuberculosis (TB) cases in India are estimated to have extrapulmonary disease, and due to a lack of diagnostic means, they often remain untreated. The early detection of Mycobacterium tuberculosis and multidrug resistance is a priority in TB diagnosis to improve the successful treatment rate of TB and reduce transmission. The Xpert MTB/RIF (Xpert) test, recently endorsed by the World Health Organization for the detection of pulmonary TB, was evaluated to test its utility in 547 patients with suspected extrapulmonary tuberculosis. Five hundred forty-seven extrapulmonary specimens were split and processed simultaneously for both culture (solid and liquid) and Xpert testing. For culture, the sensitivity was low, 53% (150/283 specimens). Xpert sensitivity and specificity results were assessed in comparison to a composite reference standard made up of smear and culture results and clinical, radiological, and histological findings. The sensitivity of the Xpert assay was 81% (228/283 specimens) (64% [89/138] for smear-negative cases and 96% [139/145] for smear-positive cases), with a specificity of 99.6%. The sensitivity was found to be high for the majority of specimen types (63 to 100%) except for cerebrospinal fluid, the sensitivity of which was 29% (2/7 specimens). The Xpert test correctly identified 98% of phenotypic rifampin (RIF)-resistant cases and 94% of phenotypic RIF-susceptible cases. Sequencing of the 6 discrepant samples resolved 3 of them, resulting in an increased specificity of 98%. In conclusion, the results of this study suggest that the Xpert test also shows good potential for the diagnosis of extrapulmonary TB and that its ease of use makes it applicable for countries where TB is endemic.

Objectives: To evaluate fluorescence microscopy (FM) using light emitting diode (LED) technology for the detection of acid-fast bacilli at a tertiary referral centre in Mumbai, India, a tuberculosis-endemic country., Design: LED FM was introduced into a laboratory experienced with Ziehl-Neelsen (ZN) microscopy but unfamiliar with FM. It was evaluated in parallel with routine ZN microscopy services and compared with mycobacterial culture as a reference standard., Results: A total of 1357 pulmonary and 917 extra-pulmonary specimens were examined during the study. LED FM had 78.3% sensitivity and 92.0% specificity against mycobacterial culture when using pulmonary specimens, and 34.0% sensitivity and 88.8% specificity for extra-pulmonary specimens. The mean time per smear examination was 2.48 min for ZN vs. 1.41 min for LED FM. Several biases in study design and operation identified during analysis, which are likely to lead to underestimates of LED FM accuracy, are discussed in the context of future LED FM evaluations., Conclusions: Although LED FM has significant benefits over both ZN microscopy and conventional FM, its implementation and validation may be prone to difficulties which could hamper evaluation of its performance. Adequate training and detailed standard operating procedures are important to maximise accuracy.

Background: Traditionally, the Lowenstein Jensen (LJ) medium has been used for culturing Mycobacterium tuberculosis. In abdominal tuberculosis (TB), the reported yield from tissue culture is between 20% and 60%. Liquid cultures are reported to give a higher yield but there is little data available in abdominal TB., Aim: To compare the yield of TB culture with BACTEC 460TB liquid medium and LJ medium for patients with suspected abdominal TB and determine cost effectiveness., Methods: This prospective study was done in consecutive cases with clinical, radiological, endoscopic/surgical, and histological suspicion of abdominal TB. Tissue biopsies obtained at colonoscopy or surgery were processed and plated on LJ medium as well as the BACTEC 460TB system. NAP (ρ-nitro-α-acetylamino-β-hydroxy-propiophenone) differentiation was carried out to determine species. The cost of each method and cost per yield were calculated., Results: Of the 29 cases, 22 cases (76%) were positive on BACTEC 460TB culture while 14 (48%) were positive on LJ medium giving a 64% increment in yield. However, the culture of one patient grew on LJ medium, where the BACTEC 460TB was negative. The additional cost of BACTEC 460TB is Rs. 460 and LJ is Rs. 40., Conclusions: Samples from patients with abdominal TB should be processed on both liquid and LJ medium. For high yield, the use of a liquid culture medium system is essential.

Background: Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect., Methods: We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test., Results: Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay., Conclusions: The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Background: The suboptimal sensitivity of Interferon (IFN)-γ-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis (TB). The objective of this study was to evaluate whether interferon-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-2 and interleukin (IL)-2 can be useful biomarkers for evaluating a specific response to RD1 antigens associated to active TB disease in HIV-infected individuals., Methodology/principal Findings: The study was carried out in India, the country with the highest TB burden in the world. Sixty-six HIV-infected individuals were prospectively enrolled, 28 with active-pulmonary-TB and 38 without. The whole blood assay based on RD1-selected peptides (experimental test) and QuantiFERON-TB Gold In tube (QFT-IT) was performed. Plasma was harvested at day-1-post-culture and soluble factors were evaluated by ELISA. The results indicate that by detecting IP-10, the sensitivity of the experimental test and QFT-antigen (75% and 85.7% respectively) for active TB was higher compared to the same assays based on IFN-γ (42.9% and 60.7% respectively) and was not influenced by the ability to respond to the mitogen. By detecting IP-10, the specificity of the experimental test and QFT-antigen (57.9% and 13.2% respectively) for active TB was lower than what was reported for the same assays using IFN-γ-detection (78.9% and 68.4% respectively). On the other side, in vitro IL-2 and MCP-2 responses were not significantly associated with active TB., Conclusions: HIV infection does not impair RD1-specific response detected by IP-10, while it significantly decreases IFN-γ-mediated responses. At the moment it is unclear whether higher detection is related to higher sensitivity or lower specificity of the assay. Further studies in high and low TB endemic countries are needed to elucidate this.

Objective: To identify and characterise disease caused by non-tuberculous mycobacteria (NTM) in a tuberculosis-endemic region., Material and Methods: A total of 133 NTM isolates cultivated using Mycobacteria Growth Indicator Tube 960 (MGIT 960) plus Löwenstein-Jensen (LJ) and identified using the NAP and PNBA tests were screened to species level using molecular methods. The 360-base pair (bp) region of the rpoB gene was amplified and analysed using an in-house RLBH assay, PCR-RE and sequencing. The clinical significance of all isolated NTM was determined as per published guidelines., Results and Discussion: Of the 133 isolates, 127 were confirmed as NTM and six as Mycobacterium tuberculosis complex by molecular methods; 81% of the NTM were recovered from pulmonary and 19% from extra-pulmonary specimens. Among the rapidly growing NTM, M. fortuitum (41%) and M. abscessus (59%) were predominant. Among the slow growing NTM, 40% were identified as M. intracellulare, followed by M. simiae (35%), M. kansasii (6%), M. gordonae (4%) M. szulgai (2%) and M. avium (1%). Mixed infections were noted in 10 (12%) cases. In all, 58 (46%) NTM met clinical, radiological and microbiological criteria and were considered definite NTM infections, 33 (26%) were highly probable disease and 36 (28%) were possible disease or colonisation., Conclusion: Overall, 72% of isolated NTM were found to be clinically significant. Accurate species identification and adherence to published guidelines help to outline pathogenicity.

Objectives: To evaluate whether in vitro response to Mycobacterium tuberculosis RD1 peptides selected by computational analysis, measured by IFN-gamma, IP-10, MCP-2 or IL-2 production, is associated with active tuberculosis (TB) in a country with a high incidence of TB., Methods: 129 individuals were prospectively enrolled, 41 with active-pulmonary TB and 88 without (household contacts and community controls). A whole blood assay based on RD1 selected peptides was performed. Soluble factors were evaluated by ELISA in plasma harvested at day1-post-culture. Enrolled individuals were also tested by QuantiFERON TB-Gold In tube (QFT-IT) and tuberculin skin tests (TST)., Results: IFN-gamma response to RD1 selected peptides was significantly higher in active TB patients than in household contacts and community controls. IP-10 and MCP-2 response did not differ between active TB patients and household contacts, although it was higher in these groups compared to community controls; conversely IL-2 response did not differ among the three groups. When IFN-gamma response to RD1 selected peptides was scored based on receiver-operator-characteristic analysis, active TB was predicted with 68% sensitivity and 86% specificity. QFT-IT and TST showed a sensitivity for active TB of 90% and 68% and a specificity of 58% and 59%, respectively., Conclusions: IFN-gamma (but not IP-10, MCP-2 and IL-2) response to RD1 selected peptides is associated with active TB with a higher specificity than QFT-IT and TST.

Background: Serpentine cord formation in BACTEC MGIT 960 medium was evaluated as a rapid method for the presumptive identification of M. tuberculosis complex (MTBC)., Material & Methods: Total 2527 samples were processed for AFB culture using MGIT 960 TB system over a period of three months. AFB smears were prepared from 1000 MGIT tubes flagged positive by the MGIT instrument and stained by ZN method to examine presence or absence of serpentine cording. The cord formation was compared with PNBA [p-nitro benzoic acid] test on MGIT system and all controversial cases were further evaluated by NAP [p-nitro-a-acetylamino-phydroxypropiophenone] test on BACTEC 460 TB system., Results & Discussion: Of the 1000 culture positives, 904 (90.4%) were identified as mycobacteria, of which 869 (96%) showed cording by smear microscopy. One (0.1%) was identified as nocardia. In the remaining 95 (9.5%) cases, primary smear made from MGIT vial was negative. Of 869 cultures showing serpentine cord formation, 842 were confirmed as MTBC and 27 as NTM by PNBA assay on MGIT 960 TB system. The sensitivity, specificity, positive and negative predictive values are found to be 99.6%, 54%, 96% and 91% respectively. An average detection time for PNBA assay was found to be eight days whereas cording results were available on the same day of culture positivity., Conclusion: Though highly sensitive it is not very specific and hence cannot be the only test for presumptive diagnosis of MTBC.

Aim: To evaluate the performance of an automated BACTEC MGIT 960, a non-radioactive, non-invasive liquid culture system for cultivation of M. tuberculosis complex in terms of recovery rate and time., Materials and Methods: From March 2005 to December 2007, 14,597 specimens were processed using the MGIT 960 system and the results were compared with conventional L.J medium. We standardised r-nitro benzoic acid (PNBA) assay on MGIT 960 TB system for identification of M. tuberculosis complex and evaluated its usefulness by comparing the results with an in-house molecular assay and sequencing., Results and Discussion: Of the total 6143 (42%) isolates positive for M. tuberculosis complex, 6015 (41%) were positive by MGIT 960 TB system. In contrast, 3526 (24%) M. tuberculosis complex isolates grew on the conventional L.J medium. The mean turn around time for mycobacterial growth in smear-positive specimens was nine days for MGIT 960, and 38 days for L.J. medium whereas in smear negative specimens it was 16 days by MGIT vs. 48 days by L.J., Conclusion: MGIT 960 system with PNBA assay for identification of M. tuberculosis complex is a rapid and useful method in laboratories processing a large number of specimens.

Objectives: To evaluate Pyrazinamide (PZA) susceptibility results obtained by phenotypic MGIT 960 TB system against enzymatic Pyrazinamidase assay and genotypic pncA gene sequencing. To find the prevalence of infections caused by M. bovis in PZA resistant M. tuberculosis complex isolates., Methods: 33 consecutive PZA resistant and 30 consecutive PZA susceptible isolates reported for PZA susceptibility testing by MGIT 960 TB system were included in this study. Presence of active pyrazinamidase enzyme was sought by using the Wayne assay. The pncA gene was amplified by PCR and then sequenced to screen mutations. All the PZA resistant isolates were further spoligotyped to identify M. bovis, if present., Results: Of 33 PZA resistant strains by MGIT 960, 31 were Wayne assay negative and two were positive. Of the 30 susceptible PZA strains six were Wayne assay negative reporting false resistance. PncA gene sequencing revealed that 32 of the 33 MGIT PZA resistant isolates had diverse nucleotide changes scattered throughout the pncA gene (one isolate did not show any mutation). Of the 30 phenotypically susceptible isolates, 21 were wild types whilst nine isolates showed the presence of a silent mutation C-T at codon 195. Fifteen mutations found in this study has not been described earlier. Not a single isolate of M. bovis was detected among PZA resistant M. tuberculosis complex isolates., Conclusion: MGIT 960 showed better concordance with sequencing results in comparison with Wayne assay. In present study, a high proportion (85%) of MDR-TB isolates from patients receiving anti-TB treatment were found to be resistant to PZA.

Objective: To design and standardize an in-house reverse line blot hybridization (RLBH) assay for the accurate identification of 15 clinically relevant species of mycobacteria and for the detection of drug resistance to rifampin (RIF), isoniazid (INH), and streptomycin (STR) in Mycobacterium tuberculosis complex (MTB)., Material and Methods: Oligonucleotides specific for 15 different species of mycobacteria and wild type and mutant alleles of selected codons in the rpobeta, inhA, katG, rpsL, and rrs genes were designed and immobilized on a membrane. A multiplex PCR was standardized to amplify all target genes. The assay was optimized using ATCC and known mutant strains. Three hundred MTB isolates, 85 non-tuberculous mycobacteria (NTM) isolates, and 48 smear-positive specimens were analyzed. Results were confirmed by PCR restriction enzyme assay and sequencing., Results: Upon RLBH analysis, among the NTM, 14% were identified as Mycobacterium fortuitum, 16% were identified as Mycobacterium abscessus, 20% showed 99% homology with Mycobacterium intracellulare, and 31% showed 98% homology with Mycobacterium simiae. Of the 300 MTB isolates analyzed, 75% RIF-resistant isolates had Ser531Leu mutation in the rpobeta gene. Of the INH-resistant isolates, 89% showed Ser315Thr mutation in the katG gene, whereas 16% showed -15 C-->T mutation in the promoter region of the inhA gene. Among STR-resistant isolates, 75% had A-->G mutation in the rpsL gene at codon 43. RLBH results showed 96-99% concordance with phenotypic culture results., Conclusion: This is a first attempt at combining speciation with detection of drug resistance to RIF, INH, and STR in MTB for accurate and rapid management of mycobacterial infections as well as for compiling genotypic epidemiological data.

Purpose: To evaluate, the efficacy of BACTEC 460 TB system for the diagnosis of tuberculosis in a tertiary care hospital in Mumbai, India., Methods: We compared 12,726 clinical specimens using BACTEC 460 TB system and conventional method for detection of Mycobacterium tuberculosis over a period of six years., Result: The overall recovery rate was 39% by BACTEC technique and 29% using Lowenstein-Jensen (LJ) medium. An average detection time for B actec0 460 TB system was found to be 13.3 days and 15.3 days as against 31.2 days and 35.3 days by LJ method for respiratory and nonrespiratory specimens respectively. The average reporting time for drug susceptibility results ranged from 6-10 days for the BACTEC 460 TB system., Conclusions: The BACTEC system is a good system for level II laboratories, especially in the diagnosis of extrapulmonary and smear negative tuberculosis.

We report a high frequency (35%) of the Beijing genotype among multidrug-resistant isolates recovered in and around Mumbai, India. Further restriction fragment-length polymorphism typing showed that these strains were closely related. We also report a high frequency of the Delhi genotype (31% of isolates). Our data indicate considerable ongoing transmission of multidrug-resistant Mycobacterium tuberculosis strains of the Beijing genotype in Mumbai.

Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin. The M. tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M. bovis bacille Calmette-Guérin (BCG) and most environmental mycobacteria. We recently identified circulating ESAT-6-specific T cells as an accurate marker of M. tuberculosis infection. Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India. Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides. In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen. This study suggests an 80% prevalence of latent M. tuberculosis infection in urban India.

Molecular diagnostic assays, with their ability to rapidly detect resistance-associated mutations in bacterial genes, are promising technologies to control the spread of drug-resistant tuberculosis (DR-TB). Sequencing assays provide detailed information for specific gene regions and can help diagnostic assay developers prioritize mutations for inclusion in their assays. We performed pyrosequencing of seven Mycobacterium tuberculosis gene regions (katG, inhA, ahpC, rpoB, gyrA, rrs, and eis) for 1,128 clinical specimens from India, Moldova, and South Africa. We determined the frequencies of each mutation among drug-resistant and -susceptible specimens based on phenotypic drug susceptibility testing results and examined mutation distributions by country. The most common mutation among isoniazid-resistant (INH(r)) specimens was the katG 315ACC mutation (87%). However, in the Eastern Cape, INH(r) specimens had a lower frequency of katG mutations (44%) and higher frequencies of inhA (47%) and ahpC (10%) promoter mutations. The most common mutation among rifampin-resistant (RIF(r)) specimens was the rpoB 531TTG mutation (80%). The mutation was common in RIF(r) specimens in Mumbai (83%) and Moldova (84%) but not the Eastern Cape (17%), where the 516GTC mutation appeared more frequently (57%). The most common mutation among fluoroquinolone-resistant specimens was the gyrA 94GGC mutation (44%). The rrs 1401G mutation was found in 84%, 84%, and 50% of amikacin-resistant, capreomycin-resistant, and kanamycin (KAN)-resistant (KAN(r)) specimens, respectively. The eis promoter mutation -12T was found in 26% of KAN(r) and 4% of KAN-susceptible (KAN(s)) specimens. Inclusion of the ahpC and eis promoter gene regions was critical for optimal test sensitivity for the detection of INH resistance in the Eastern Cape and KAN resistance in Moldova. (This study has been registered at ClinicalTrials.gov under registration number NCT02170441.).

ObjectiveTo evaluate the performance of a recently updated rapid molecular diagnostic test, GenoType® MTBDRplus version 2, designed to detect drug resistance in both acid-fast bacilli (AFB) smear-negative and -positive specimens.DesignSputum samples from 1128 patients at risk for multidrug-resistant tuberculosis (MDR-TB) were tested using MTBDRplus v2 and compared with reference standard MGIT™ 960™ drug susceptibility testing. The relationship of participant human immunodeficiency virus (HIV) status, diabetic status, previous treatment, and smear gradation to the likelihood of obtaining an interpretable result was assessed using logistic regression.ResultsThe sensitivity and specificity of MTBDRplus v2 for detecting MDR-TB, when compared to a reference standard, were respectively 96.0% (95%CI 93.5-97.6) and 99.2% (95%CI 97.0-99.9) in AFB smear-positive specimens and 82.8% (95%CI 63.5-93.5) and 98.3% (95%CI 89.9-99.9) in AFB smear-negative specimens. A dose-response relationship was observed between the proportion of interpretable test results and AFB smear bacterial load after adjusting for age, sex, body mass index, HIV status, previous treatment and diabetic status.ConclusionWhile MTBDRplus v2 performs well among both AFB smear-positive and -negative specimens, smear gradation appears to influence both the probability of obtaining an interpretable result and test sensitivity, indicating a significant association between bacillary load and test performance.

ObjectiveTo develop and evaluate rapid, molecular-based drug susceptibility testing (DST) for extensively drug-resistant tuberculosis (XDR-TB), we assembled a phenotypically and genotypically diverse collection of Mycobacterium tuberculosis isolates from patients evaluated for drug resistance in four high-burden countries.MethodsM. tuberculosis isolates from India (n = 111), Moldova (n = 90), the Philippines (n = 96), and South Africa (n = 103) were selected from existing regional and national repositories to maximize phenotypic diversity for resistance to isoniazid, rifampin (RMP), moxifloxacin, ofloxacin, amikacin, kanamycin, and capreomycin. MGIT™ 960 was performed on viable isolates in one laboratory using standardized procedures and drug concentrations. Genetic diversity within drug resistance phenotypes was assessed.ResultsNineteen distinct phenotypes were observed among 400 isolates with complete DST results. Diversity was greatest in the Philippines (14 phenotypes), and least in South Africa (9 phenotypes). Nearly all phenotypes included multiple genotypes. All sites provided isolates resistant to injectables but susceptible to fluoroquinolones. Many patients were taking drugs to which their disease was resistant.DiscussionDiverse phenotypes for XDR-TB-defining drugs, including resistance to fluoroquinolones and/or injectable drugs in RMP-susceptible isolates, indicate that RMP susceptibility does not ensure effectiveness of a standard four-drug regimen. Rapid, low-cost DST assays for first- and second-line drugs are thus needed.

ObjectiveTo establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay.SettingA multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa.DesignIn the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined. In the second phase, MODS second-line drug susceptibility testing (DST) as an indirect test was compared to MGIT™ DST (n = 89). In the third (n = 30) and fourth (n = 156) phases, we determined the reproducibility and concordance of MODS second-line DST directly from sputum.ResultsBreakpoints for MFX (0.5 μg/ml), OFX (1 μg/ml), AMK (2 μg/ml), KM (5 μg/ml) and CPM (2.5 μg/ml) were determined. In all phases, MODS results were highly concordant with MGIT DST. The few discrepancies suggest that the MODS breakpoint concentrations for some drugs may be too low.ConclusionMODS second-line DST yielded comparable results to MGIT second-line DST, and is thus a promising alternative. Further studies are needed to confirm the accuracy of the drug breakpoints and the reliability of MODS second-line DST as a direct test.

Sensitivity, specificity, PPV and NPV of CSF pyrosequencing to diagnose TB meningitis was 96.7%, 100%, 100% and 94.4% respectively. B Background: b We evaluated the performance of pyrosequencing, a rapid genotypic test which detects TB and resistance to isoniazid, rifampicin, aminoglycosides and fluoroquinolones within 6h, directly on CSF samples in the diagnosis of TB meningitis. [Extracted from the article]