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Background: Microbiological confirmation of tuberculous meningitis (TBM) remains problematic. We assessed the diagnostic performance of a modified Ziehl-Neelsen (MZN) staining method that showed promise in earlier studies. Methods: Patients evaluated for TBM in Shaanxi province, China, were prospectively enrolled from May, 2011 to April, 2013. Cerebrospinal fluid (CSF) specimens were evaluated using the Xpert MTB/RIF® assay, MZN staining, and standard biochemical and microbiological tests, together with detailed clinical and radiological assessment. Results: Among 316 patients included in the study, 38 had definite TBM, 66 probable TBM, 163 possible TBM and 49 "no TBM," using consensus uniform research case definition criteria. Comparing "definite or probable TBM" to "no TBM" MZN staining had higher sensitivity than Xpert MTB/RIF® (88.5 vs. 36.5%), but greatly reduced specificity (71.4 vs. 100.0%); 14/49 (28.6%) cases with "no TBM" tested positive on MZN. Mycobacterium tuberculosis culture was performed in 104/179 (58.1%) of MZN positive samples; 12.5% (13/104) were positive. Using Xpert MTB/RIF® as the reference standard, MZN had a sensitivity of 92.1% (95% CI 79.2-97.3) and specificity of 71.4% (95% CI 57.6-82.2). Conclusion: Xpert MTB/RIF® offered a rapid and specific TBM diagnosis, but sensitivity was poor. MZN was mainly hampered by false positives. Strategies to enhance the sensitivity of Xpert MTB/RIF® or improve the diagnostic accuracy of MZN should be explored. [ABSTRACT FROM AUTHOR]

Background: India relies primarily on direct smear microscopy for tuberculosis (TB) diagnosis. However, the low sensitivity of smear microscopy emphasizes the need to improve its performance. We recently described the development of 'TB Detect ' kit which showed improved performance over direct smear microscopy at National Reference Laboratories (NRLs) in India., Methods: The present study was aimed to assess the operational feasibility of 'TB Detect ' microscopy in field settings. This was evaluated by (i) assessing the performance of 'TB Detect ' microscopy vs. LED-fluorescence microscopy (LED-FM) on consecutive presumptive pulmonary TB patients ( n  = 5300) who attended Designated Microscopy Centres (DMCs, n  = 13) under 4 NRLs at Bhubaneswar, Bhopal, Chennai, and New Delhi, and (ii) obtaining feedback from Scientists ( n  = 10) and laboratory technicians ( n  = 42) using semi-structured questionnaires under the following parameters: feasibility of initiation of 'TB Detect' microscopy in DMCs, sample preparation and testing, training, time-to-result, logistics, and troubleshooting. A scoring questionnaire was also used to assess 'TB Detect ' microscopy vs . LED-FM and statistical significance of the scores was calculated using paired t -test., Results: The overall positivity of 'TB Detect ' microscopy was 10.32% (547/5300) vs. 8.96% (475/5300) of LED-FM at all sites and the increment in positivity was significant ( p  = 0.019). In addition, 'TB Detect ' microscopy yielded an increment in smear grade status over LED-FM ( p  = 0.043). The feedback from the study-in-charge and kit users indicated that 'TB Detect ' microscopy was easily adapted in point-of-care settings. An analysis of scoring feedback suggested that it was easy to perform and observe in comparison to LED-FM ( p  < 0.005)., Conclusions: This study established the feasibility of 'TB Detect ' microscopy in field settings.

Multidrug-resistant tuberculosis (MDR-TB) requires treatment with fluoroquinolone (FLQ) drugs, however, the excessive use of FLQ has led to the rise of extensively drug-resistant TB. In 2019, ~ 20% of total MDR-TB cases were estimated to be resistant to FLQ drugs. In the present study, we developed and evaluated the utility of high-resolution melt curve analysis (HRM) for the rapid detection of FLQ-resistant Mycobacterium tuberculosis for the first time directly from sputum samples. A reference plasmid library was generated for the most frequently observed mutations of gyrA gene and was used to discriminate between mutant and wild-type samples in the FLQ-HRM assay. The developed assay was evaluated on n = 25 MDR M. tuberculosis clinical isolates followed by validation on archived sputum DNA (n = 88) using DNA sequencing as a gold standard. The FLQ-HRM assay showed a 100% sensitivity [95% Confidence Interval (CI): 71.5 to 100] and specificity (95% CI: 39.7 to 100) in smear-positive category, and a sensitivity of 88.9% (95% CI: 77.3 to 95.8) with 84.2% (95% CI: 60.4 to 96.6) specificity in smear-negative category. The assay showed a high level of concordance of ~ 90% (κ = 0.74) with DNA sequencing, however, we were limited by the absence of phenotypic drug susceptibility testing data. In conclusion, HRM is a rapid, cost-effective (INR 150/USD 1.83) and closed-tube method for direct detection of FLQ resistance in sputum samples including direct smear-negative samples., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)