Search

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: Tuberculous meningitis (TBM) is the most common form of neurotuberculosis and the fifth most common form of extrapulmonary TB. Early diagnosis and prompt treatment are the cornerstones of effective disease management. The accurate diagnosis of TBM poses a challenge due to an extensive differential diagnosis, low bacterial load and paucity of cerebrospinal fluid (CSF) especially in children. Methodology/Principal Findings: We describe the utility of ELISA and qPCR for the detection of Mycobacterium tuberculosis (M. tb) proteins (GlcB, HspX, MPT51, Ag85B and PstS1) and DNA for the rapid diagnosis of TBM. CSF filtrates (n = 532) derived from children were classified as 'Definite'TBM (M. tb culture positive, n = 29), 'Probable and Possible' TBM (n = 165) and 'Not- TBM' including other cases of meningitis or neurological disorders (n = 338). ROC curves were generated from ELISA and qPCR data of 'Definite' TBM and Non-Tuberculous infectious meningitis (NTIM) samples and cut-off values were derived to provide ≥95% specificity. devR qPCR, GlcB, HspX and PstS1 ELISAs showed 100% (88;100) sensitivity and 96-97% specificity in 'Definite' TBM samples. The application of these cut-offs to 'Probable and Possible' TBM groups yielded excellent sensitivity (98%, 94;99) and specificity (98%, 96;99) for qPCR and for GlcB, HspX and MPT51 antigen ELISAs (sensitivity 92- 95% and specificity 93-96%). A test combination of qPCR with GlcB and HspX ELISAs accurately detected all TBM samples at a specificity of ~90%. Logistic regression analysis indicated that these tests significantly added value to the currently used algorithms for TBM diagnosis. Conclusions: The detection of M. tb GlcB/HspX antigens/devR DNA in CSF is likely to improve the utility of existing algorithms for TBM diagnosis and also hasten the speed of diagnosis. [ABSTRACT FROM AUTHOR]

This study was designed to assess the vital issue of gene target length and PCR assay performance in relation to the detection of Mycobacterium tuberculosis in clinical specimens. Two PCR assays that amplify fragments of varying lengths from the devR gene of M. tuberculosis were evaluated. Using M. tuberculosis DNA the ‘short-length’ PCR assay detected 250–500 genome equivalents vs. 500–1000 genome equivalents by the ‘long-length’ assay. In comparison to a highly sensitive smear microscopy test (universal sample processing smear), the sensitivity of the ‘short-length’ assay was 97.8% vs. 69.9% of the ‘long-length’ assay in sputum specimens ( n=506) from patients being evaluated for a possible diagnosis of tuberculosis. The 27.9% absolute increase in sensitivity was statistically significant ( P<0.001). Our results indicate that in a clinical setting when all other conditions are equal, the amplification of a shorter gene fragment of devR increases the sensitivity and efficiency of the PCR assay in spite of using a single copy gene as target. [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.)