Development and evaluation of novel bio-safe filter paper-based kits for sputum microscopy and transport to directly detect Mycobacterium tuberculosis and associated drug resistance

Abstract

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely ‘TB Detect’ (containing BioFM-Filter device), ‘TB Concentration and Transport’ (containing Trans -Filter device) and ‘TB DNA Extraction’ kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p<0.05), respectively and an increment in smear grade status (1+ or 2+ to 3+) of 16% over ZN smear and 20% over LED-FM smear. The sensitivity of Mol-DST in presumptive MDR-TB and XDR-TB cases (n = 148) was 90% for Rifampicin (95% confidence interval [CI], 78–96%), 84% for Isoniazid (95% CI, 72–92%), 83% for Fluoroquinolones (95% CI, 66–93%) and 75% for Aminoglycosides (95% CI, 35–97%), using phenotypic DST as the reference standard. Test specificity was 88–93% and concordance was ~89–92% (κ value 0.8–0.9). The patient-friendly kits described here address several of the existing challenges and are designed to provide ‘Universal Access’ to rapid TB diagnosis, including drug-resistant disease. Their utility was demonstrated by application to sputum at 2 sites in India. Our findings pave the way for larger studies in different point-of-care settings, including high-density urban areas and remote geographical locations.

Covid-19 and Tuberculosis Classification Based on Chest X-Ray Using Convolutional Neural Network

“…Also, DSS can be used as a support system to anticipate the pandemic Covid-19 in the future. According to WHO the research on DSS TB is one of the most urgent implementation studies required today [3][4][5]. Several studies based on image processing [6][7][8][9][10][11][12] have been carried out to detect MTB and so far the CNN method [13][14][15] shows the best performance among other methods [14,16,17].…”

Evaluation of ‘TBDetect’ sputum microscopy kit for improved detection of Mycobacterium tuberculosis: a multi-centric validation study

“…Samples were collected at five National Reference Laboratory sites (NIRT (n ¼ 358), RMRC (n ¼ 353), BMHRC (n ¼ 341), NITRD (n ¼ 348) and NJILOMD (n ¼ 341)) and one Intermediate Reference Laboratory site (AIIMS (n ¼ 345)). Sample size was estimated based on 85% power, alpha of 5% and pooled positivity of 26% and 22% of TBDetect and LED-FM, respectively (data from primary validation study for TBDetect kit [9]).…”

“…Briefly, 400 mL of 'Dissolving solution' from the TBDetect kit was added to 100 mL of sputum and incubated for 30 min [9]. Then 300 mL of liquefied sputum was filtered and stained in situ [9] and the fluorescent BioFM-Filter was viewed at 40 Â magnification as described above.…”

“…We recently developed the TBDetect kit (Supplementary Fig. S1) which enables improved and bio-safe smear microscopy [9]. The kit technology enables retention of bacteria present in sputum through filtration by gravity and absorbent pad-based capillary action without the use of any equipment to create a vacuum or pressure.…”

“…The kit technology enables retention of bacteria present in sputum through filtration by gravity and absorbent pad-based capillary action without the use of any equipment to create a vacuum or pressure. A black non-fluorescent membrane and Bio-safe Fluorescent Microscopy Filter (BioFM-Filter) device provide good contrast for rapid visualization of in situ fluorescent-stained bacteria [9]. Here, we evaluated the performance of TBDetect microscopy in comparison with direct smear microscopy and culture for the detection of pulmonary TB at six different sites in India.…”