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Background: Mycobacterium abscessus complex (MabC) has emerged as an important cause of human infections, including meningitis. In the absence of correct microbiological identification, cases of MabC meningitis are treated with conventional anti-tubercular therapy, thereby worsening the outcome., Objective: The current study was conducted to determine the clinical features, antimicrobial susceptibility, and outcome of patients with MabC meningitis., Material and Methods: Cerebrospinal fluid specimens processed between 2011 and 2021 were subjected to smear, culture, MALDI TOF identification, hsp65 gene sequencing, and susceptibility testing using Sensititre™ RAPMYCOI plates along with a literature review., Results: 12 cases of MabC meningitis were identified between 2011 and 2021, 11 of which were M. abscessus subspecies abscessus on hsp65 gene sequencing. A pioneer case of meningitis with M. abscessus subspecies bolletii was also identified. The common predispositions were TB elsewhere, HIV positivity, and head injury. Two patients had dual infections, both MabC and TB. Ten patients succumbed to infection with a mean survival of 11 months. All isolates were susceptible to amikacin and tigecycline and subspecies bolletii had a higher minimum inhibitory concentration (MIC) than subspecies abscessus. A combined analysis with the available literature, reporting 19 more cases, revealed that the overall mortality of MabC meningitis was 61.3% (19/31) and that of shunt-associated/neurosurgical intervention-related MabC meningitis was 66.7% (12/20). To date, out of 20 MabC meningitis isolates in which subspecies identification was carried, 13 were M. abscessus, six were M. massiliense, and one was M. bolletii., Conclusion: MabC is an important differential diagnosis of chronic meningitis. Prompt identification and speciation are imperative for targeted therapy, thus improving the overall patient outcome., Competing Interests: None

Background: Xpert MTB/RIF Ultra (Ultra) was evaluated for the first time on Ocular tuberculosis (OTB) samples and compared with Xpert., Methods: Seventy five vitreous fluid samples (3 confirmed OTB, 47 clinically suspected OTB, and 25 controls) were subjected to Ultra, Xpert and Multiplex-PCR and compared against culture, composite reference standard (CRS), and gene sequencing., Results: The sensitivity of Ultra was 50% in diagnosing OTB (100% against culture and 46.8% against CRS). The overall sensitivity of Xpert and MPCR was 16% and 72%, respectively. Xpert missed three culture-positive cases and MPCR detected additional 11. Ultra and Xpert missed two and four cases of RifR, respectively. A total of 13(59%) cases were reported 'trace' by Ultra in which RifR could not be evaluated., Conclusion: Ultra outperformed Xpert in diagnosing OTB. The advantage of Ultra's simultaneous RifR detection is lost since the trace bacterial loads in the specimens cause indeterminate results of RifR testing. Abbreviations: OTB: Ocular tuberculosis; Ultra: Xpert MTB/RIF Ultra; Xpert: Xpert MTB/RIF, MPCR: multiplex polymerase chain reaction; NAATs: Nucleic acid amplification tests; MLAMP: multitargeted loop-mediated isothermal amplification; PPV: positive predictive value; NPV: negative predictive value; EPTB: extrapulmonary tuberculosis; VF: vitreous fluid; DNA: deoxyribonucleic acid; ATT: antitubercular therapy; RifR: Rifampicin resistance; RifS: Rifampicin susceptible; RifI: Rifampicin indeterminate.

The study evaluated real-time quantitative polymerase chain reaction (qPCR) and high-resolution melt-curve analysis (HRM) for simultaneous diagnosis of osteo-articular tuberculosis (OATB) and drug resistance. Two hundred and fifty synovial fluid and pus specimens (20 confirmed OATB by culture, 130 suspected OATB, and 100 controls) processed in the Department of Medical Microbiology, PGIMER were subjected to qPCR using rpoB, MPB64, and IS6110 genes. All OATB positive specimens were subjected to HRM for detecting resistance to rifampicin and isoniazid. qPCR detected 129/150 OATB cases with a sensitivity of 86% (95% for confirmed and 84.6% for suspected OATB cases) and specificity of 100%. rpoB and MPB64 genes had higher sensitivity than IS6110 (86% vs. 74.6%). HRM reported eight multidrug resistant (MDR), two mono-rifampicin, and five mono-isoniazid resistant cases, all were concordant with gene sequencing. qPCR followed by HRM analysis offer a simple, accurate, and rapid platform for simultaneous detection of OATB and MDR., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Tuberculosis (TB) is a serious zoonotic threat, impacting the human-livestock-wildlife interface globally. Here, we evaluated the status and histomorphological differentiation of TB lesions in 89 morbid cases of wild animals (bovids, cervids, carnivores, non-human primates, and pachyderms) in India. Histomorphological and molecular studies were done using Ziehl-Neelsen (ZN) staining, immunohistochemistry, and polymerase chain reaction (PCR), whereas cultural isolation was performed on selected samples. A total of 32 (35.95%) cases were confirmed as TB, comprising of 12 carnivores, 09 bovids, 06 cervids, 04 non-human primates, and a pachyderm. The TB lesions in the lungs, liver, and lymph nodes varied from the large-sized caseous nodules filled with dry cheesy material in bovids and cervids to variable-sized cavitations containing liquefied caseum in carnivores' lungs. The lungs, livers, and spleens of non-human primates exhibited small to medium-sized nodules. Histologically, lesions were divided into four categories (Types I, II, III, and IV) based on the extent of necrosis, the presence of mineralization, giant cells, and fibrous encapsulation. Extensive caseous necrosis with calcification, abundant giant cells, and thick fibroblastic encapsulation were consistent findings in the lungs, livers, and lymph nodes of bovids and cervids, whereas airway impaction with cellular exudate containing a teeming number of acid-fast bacilli and, at times, alveolar rupture leading to cavity formation was present in the lungs of carnivores. Absence of calcification and fibrous encapsulation was recorded in lungs of non-human primates. Immunohistochemical labelling with anti-early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antibodies showed mild, moderate, and intense positive reactions in type II and III, type I, and type IV granulomatous lesions, respectively. Molecular detection by PCR revealed Mycobacterium tuberculosis (12 carnivores, 02 non-human primates and 01 pachyderm), M. bovis (02 cervids and 01 bovid) and M. orygis (02 cervids and 01 bovid). Cultural isolation confirmed M. tuberculosis in 03 carnivores and M. orygis in 02 cervids and 01 bovid. Our findings imply that TB is quite prevalent in the wildlife of India and there are considerable differences in the histomorphological lesions induced by distinct Mycobacterium species in different wild animals. The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated to prevent spillover and spillback incidences at the human-livestock-wildlife interface., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: Intra ocular tuberculosis (IOTB) can affect nearly any part of the eye and delayed diagnosis can result in permanent loss of vision. mRNA, with the ability to detect viable bacilli just like culture, could serve as an important tool to differentiate whether ocular manifestations of IOTB are due to active infection or hypersensitivity response to dead mycobacteria., Methods: A total of 19 patients with suspected IOTB and 20 controls (10 patients with active inflammation but no evidence of IOTB and 10 patients with no intraocular inflammation) were included. Vitreous fluid samples were collected from all patients and subjected to DNA and mRNA extraction followed by multiplex polymerase chain reaction (MPCR) using three specific targets for M. tuberculosis, i.e. IS6110, MPB64 and protein b. All samples were also subjected to Gene Xpert MTB/RIF assay., Results: Among the 19 patients in group 1, the highest test positivity was noted for DNA-MPCR (73.7%) followed by mRNA-MPCR (42.1%) and GeneXpert MTB/RIF assay (10.5%). All the three tests were negative in the 20 control samples demonstrating a 100% specificity. The negative predictive value was highest for DNA-MPCR (80%), followed by mRNA-PCR (64.5%) and Xpert MTB/RIF assay (48.8%). All patients with any one test positive were treated with anti-tubercular therapy (ATT) and there was good clinical response noted in all., Conclusion: mRNA-based MPCR can be used as a marker for detecting viable M. tuberculosis in IOTB cases., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Background: Tuberculosis infection accounts for more annual deaths than any other single infectious disease, except the Corona Virus infection. It is a significant global issue and India is one of the most affected countries. Religious mass gatherings congregate millions of pilgrims at one place. Over-crowding is a high-risk factor for the transmission of tuberculosis. Knowledge and awareness of the disease are proven prerequisites for the spread-prevention and early diagnosis of tuberculosis. The present study was designed to explore the knowledge of tuberculosis and awareness of disease transmission risk among pilgrims attending a religious mass gathering, the Kumbh Mela (2016) in Ujjain, India., Methods: Self-reported data on the pilgrims' tuberculosis-knowledge were collected using a cross-sectional study design. A contextual, pre-tested questionnaire was used, using the convenience sampling method. In addition to the anonymous descriptive analyses, a composite knowledge-score was developed to enable comparisons between demographic groups., Results: In total, 1665 pilgrims participated in the study with 38.8 years of mean age and 59% literacy rate. The most recognized symptoms of tuberculosis were "Cough for more than 15 days with sputum" (94%) and "Blood in sputum" (81%). Most participants knew that tuberculosis is an infectious disease (93%) and not hereditary (91%). Additionally, 84% considered it is a potentially lethal disease that requires extensive treatment. However, vaccine awareness was poor (4%). "Direct contact with tuberculosis patient" (78%) and "To be in a crowded area" (4%) were considered as the most important risk factors for tuberculosis transmission. For the composite knowledge-score, a significantly higher mean score was seen among those with at least ten years of schooling compared with illiterates (p <  0.001). Occupation and residency also affected the mean score., Conclusion: Most pilgrims attending the Kumbh Mela had basic knowledge of the characteristics of tuberculosis, but some important knowledge gaps concerning the transmission risks associated with crowded situations existed. These gaps need to be addressed in future policies to enable safer mass gatherings and to end the TB epidemic, globally., (© 2021. The Author(s).)

Aims: The present study was conceived to evaluate multi-targeted loop mediated amplification (MLAMP) for the rapid diagnosis of extrapulmonary tuberculosis (EPTB)., Methods and Results: A total of 700 patients were included who were classified into 2 groups: Group 1 (n = 400) included a 100 culture confirmed EPTB patients and 300 culture negative, suspected EPTB patients. Group 2 (n = 300) included negative controls from non-tubercular patients. All samples were subjected to Ziehl-Neelsen microscopy, solid culture on Lowenstein Jensen media, Polymerase chain reaction (PCR) targeting IS6110 gene and LAMP targeting both IS6110 and MPB64 individually and as MLAMP. The overall sensitivity of microscopy, culture, IS6110 PCR, IS6110 LAMP, MPB64 LAMP and the MLAMP assay were 12%, 25%, 72.5%, 80% and 86.6% respectively and the specificity of all the tests was 100%., Conclusion: MLAMP is a rapid robust tool for the diagnosis of EPTB and utilizing two targets for M. tuberculosis can improve the overall sensitivity and increase the yield of detection from extrapulmonary samples. The rapidity, ease of performance and low cost make MLAMP an excellent alternative in low-income, resource limited settings., Competing Interests: Conflicts of interest All authors have none to declare., (Copyright © 2020 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide. Extrapulmonary tuberculosis (EPTB) constitutes around 15-20% of TB cases in immunocompetent individuals. Extrapulmonary sites that are affected by TB include bones, lymph nodes, meningitis, pleura, and genitourinary tract. Whole genome sequencing has emerged as a powerful tool to map genetic diversity among Mycobacterium tuberculosis (MTB) isolates and identify the genomic signatures associated with drug resistance, pathogenesis, and disease transmission. Several pulmonary isolates of MTB have been sequenced over the years. However, availability of whole genome sequences of MTB isolates from extrapulmonary sites is limited. Some studies suggest that genetic variations in MTB might contribute to disease presentation in extrapulmonary sites. This can be addressed if whole genome sequence data from large number of extrapulmonary isolates becomes available. In this study, we have performed whole genome sequencing of five MTB clinical isolates derived from EPTB sites using next-generation sequencing platform. We identified 1434 nonsynonymous single nucleotide variations (SNVs), 143 insertions and 105 deletions. This includes 279 SNVs that were not reported before in publicly available datasets. We found several mutations that are known to confer resistance to drugs. All the five isolates belonged to East-African-Indian lineage (lineage 3). We identified 9 putative prophage DNA integrations and 14 predicted clustered regularly interspaced short palindromic repeats (CRISPR) in MTB genome. Our analysis indicates that more work is needed to map the genetic diversity of MTB. Whole genome sequencing in conjunction with comprehensive drug susceptibility testing can reveal clinically relevant mutations associated with drug resistance.

Background and Aim: Emergence of drug resistance, especially to second‐line drugs, hampers tuberculosis elimination efforts. The present study aimed to evaluate MTBDRplus and MTBDRsl assays for detecting first‐line and second‐line drug resistance, respectively, in gastrointestinal tuberculosis (GITB). Methods: Thirty ileocecal biopsy specimens, processed in the Department of Microbiology between 2012 and 2022, that showed growth of Mycobacterium tuberculosis on culture were included in the study. DNA, extracted from culture, was subjected to MTBDRplus and MTBDRsl (Hain Lifescience GmbH, Nehren, Germany), following manufacturer's instructions. Their performance was compared against phenotypic drug susceptibility testing (pDST) and gene sequencing. Results: Out of the 30 specimens, 4 (13.33%) were mono‐isoniazid resistant, 4 (13.33%) were multidrug resistant (MDR), 2 (6.67%) were pre‐extensively drug resistant (pre‐XDR), and 2 (6.67%) were mono‐fluoroquinolone resistant. The results were 100% concordant with pDST and gene sequencing. Conclusions: In the wake of growing drug resistance in all forms of extrapulmonary tuberculosis, including GITB, MTBDRplus and MTBDRsl are reliable tools for screening of resistance to both first‐line and second‐line drugs. [ABSTRACT FROM AUTHOR]

Background and Aim: Prompt and accurate diagnosis of gastrointestinal tuberculosis (GITB) along with simultaneous detection of drug resistance is inevitable for tuberculosis elimination. Truenat MTB Plus (TruPlus), a chip‐based real‐time polymerase chain reaction assay, was evaluated for the first time for diagnosing GITB and detecting rifampicin resistance. Methods: Fifty ileocecal biopsy specimens (5 microbiologically confirmed GITB [culture‐positive], 25 clinically confirmed GITB [culture‐negative], and 20 control patients) processed in the Department of Microbiology between 2011 and 2021 were subjected to TruPlus assay, Xpert MTB RIF assay multiplex polymerase chain reaction. Their performance was evaluated against both culture and composite reference standard. Results: The overall sensitivity and specificity of TruPlus in diagnosing GITB was 70% (21/30) and 100%, respectively. The sensitivity was 60% (3/5) for microbiologically confirmed cases and 72% (18/25) for clinically confirmed cases. Performance of TruPlus was superior to Xpert (sensitivity = 30%; P = 0.001) and comparable with MPCR (sensitivity = 83.33%; P = 0.13). Both TruPlus and MPCR had moderate agreement with reference standards, and MPCR detected additional three cases. Both TruPlus and Xpert correctly reported Rifampicin resistance in three cases. Conclusions: TruPlus, with its greater portability and higher sensitivity than Xpert, could serve as an important tool for diagnosing GITB and rifampicin resistance at outreach endemic areas. [ABSTRACT FROM AUTHOR]

Objective Infections due to nontuberculous mycobacteria (NTM) is increasing globally and may present as drug-resistant tuberculosis (DRTB). In India, data on NTM prevalence and species diversity is limited. Present study was conducted to detect the prevalence and profile of NTM among patients suspected of DRTB using paraffin slide culture (PSC)and mycobacteria growth indicator tube (MGIT) culture methods for isolation of NTM. Material and Method A total of 2,938 samples suspected of TB/DRTB were cultured on PSC and MGIT960. Species identification of mycobacterial isolate was done by sequencing of 16s ribosomal RNA gene. Result Among 2938 samples, 35 (1.19%) were found positive for NTM by PSC and 9 (0.30%) were found positive by MGIT. The diversity of NTM species was high (13 species). Out of 35 NTM isolates by PSC, maximum 34.29% (12) isolates were found to be Mycobacterium fortuitum , followed by 11.43% (4) Mycobacterium abscessus and Mycobacterium chelonae , and 42.85% (15) were other species viz. 8.57% (3) were Mycobacterium intracellulare and Mycobacterium kansasii , 5.71% (2) were Mycobacterium peregrinum , and 2.85% (1) were Mycobacterium flavescens, Mycobacterium farcinogenes, Mycobacterium moriokanese, Mycobacterium wolinskyi, Mycobacterium simiae, Mycobacterium goodii , and Mycobacterium terrae each. Coinfection of Mycobacterium tuberculosis (MTB) and NTM was found in 60% (21) samples. Conclusion Prevalence of NTM was low among multidrug resistant tuberculosis/TB suspected patients, similar to other studies done in India. PSC was found better than MGIT for the isolation of NTM, though poor separation of NTM and MTB on subculture may have led to false negativity in cases of coinfection. About 13 species were isolated; M. fortuitum was the most common of all. Since coinfection of NTM and TB can also occur, samples of patients suspected of NTM should be cultured on PSC even if positive for MTB. [ABSTRACT FROM AUTHOR]

Background: Delay in diagnosis is one of the most important factors for the control of tuberculosis (TB) in endemic countries like India. As laboratory diagnosis is the mainstay for identification of active disease, we aim to explore and understand the opinions of medical doctors about the laboratory diagnosis of TB in Ujjain, India. Methods: Sixteen qualified specialist medical doctors from Ujjain were purposefully selected for the study. Individual interviews with the doctors (13 men and 3 women), were conducted. As one interview could not be completed, data from15 interviews were analyzed using manifest and latent content analysis. Results: Based on perception of the doctors, the theme; 'challenges and need for the laboratory diagnosis of TB' emerged from the following subthemes: (i) Relationship between basic element of the TB diseases process such as 'Symptoms prior to diagnoses' and 'Clinical characteristics of TB', which were not specific enough to diagnose TB (ii) The prevailing conditions such as lack of explicit diagnostic tools, lead to the doctors using the 'multiple tests' or 'empiric treatment' approach (iii) The doctors proposed that there is a need for access to a rapid, single and simple diagnostic test, and a need for awareness and knowledge of the practitioners regarding specific TB investigations, and early referral to improve the situation at resource-limited settings. Conclusion: The medical specialists use a 'multiple test' or 'empiric treatment' approach to diagnose TB. According to the participants, there is a low dependence and uptake of the available laboratory TB investigations by medical practitioners. There is an urgent need to have a specific, simple and reliable test, and a protocol, to improve diagnosis of TB and to prevent development of resistant TB. [ABSTRACT FROM AUTHOR]

Background: Delay in diagnosis is one of the most important factors for the control of tuberculosis (TB) in endemic countries like India. As laboratory diagnosis is the mainstay for identification of active disease, we aim to explore and understand the opinions of medical doctors about the laboratory diagnosis of TB in Ujjain, India. Methods: Sixteen qualified specialist medical doctors from Ujjain were purposefully selected for the study. Individual interviews with the doctors (13 men and 3 women), were conducted. As one interview could not be completed, data from15 interviews were analyzed using manifest and latent content analysis. Results: Based on perception of the doctors, the theme; ‘challenges and need for the laboratory diagnosis of TB’ emerged from the following subthemes: (i) Relationship between basic element of the TB diseases process such as ‘Symptoms prior to diagnoses’ and ‘Clinical characteristics of TB’, which were not specific enough to diagnose TB (ii) The prevailing conditions such as lack of explicit diagnostic tools, lead to the doctors using the ‘multiple tests’ or ‘empiric treatment’ approach (iii) The doctors proposed that there is a need for access to a rapid, single and simple diagnostic test, and a need for awareness and knowledge of the practitioners regarding specific TB investigations, and early referral to improve the situation at resource-limited settings. Conclusion: The medical specialists use a ‘multiple test’ or ‘empiric treatment’ approach to diagnose TB. According to the participants, there is a low dependence and uptake of the available laboratory TB investigations by medical practitioners. There is an urgent need to have a specific, simple and reliable test, and a protocol, to improve diagnosis of TB and to prevent development of resistant TB. [ABSTRACT FROM AUTHOR]