Operational feasibility and multi-centric evaluation of 'TBDetect sputum microscopy kit' for the direct detection of Mycobacterium tuberculosis in field settings.

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 'TBDetect' 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 'TBDetect' microscopy in field settings. This was evaluated by (i) assessing the performance of 'TBDetect' 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 'TBDetect' microscopy in DMCs, sample preparation and testing, training, time-to-result, logistics, and troubleshooting. A scoring questionnaire was also used to assess 'TBDetect' microscopy vs. LED-FM and statistical significance of the scores was calculated using paired t-test. RESULTS: The overall positivity of 'TBDetect' 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, 'TBDetect' 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 'TBDetect' 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 'TBDetect' microscopy in field settings.

Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe.

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.

Evaluation of Diagnostic Performance of Line Probe Assay on Smear-Negative Samples of Pulmonary Tuberculosis.

BACKGROUND: This study aims to compare the performance of line probe assay (LPA) on smear-negative samples with that of smear-positive samples for diagnosing pulmonary tuberculosis (PTB) and first-line drug sensitivity testing (FL DST). METHODS: A total of 196 sputum samples including both smear-positive (112) and negative (84) samples of patients suspected of PTB were subjected to LPA for TB detection and FL DST. TB culture followed by MPT 64 Ag was done and conventional FL DST was performed on all culture-positive isolates. Results of LPA on smear-negative were compared with smear-positive samples. RESULTS: The LPA confirmed the diagnosis of PTB in 104/112 smear-positive cases but in only 36/84 smear-negative cases. The assay had 47.36%, 72.72%, and 88.88% sensitivity and 86.96%, 95.23%, and 95.65% specificity in smear-negative cases compared to 89.09%, 95.83%, and 98.07% sensitivity and 100%, 98.36%, and 98.24% specificity in smear-positive cases for detecting Mycobacterium tuberculosis (MTB), rifampicin (RMP) resistance, and isoniazid (INH) resistance, respectively. CONCLUSION: LPA performance was better on smear-positive than smear-negative sputum samples. Further larger studies are needed to justify the use of LPA on smear-negative pulmonary samples for diagnosis.