Development, evaluation and multi-centric validation of a novel bio-safe filter paper-based kit for sputum microscopy to directly detect Mycobacterium tuberculosis
Divya Anthwal1,2, Surabhi Lavania3, Rakesh Kumar Gupta1,2, Ajoy Verma4, Manpreet Bhalla4, Ritu Singhal4, Prem Prakash Sharma4, Khalid Umar Khayyam4, Vithal Prasad Myneedu4, Rohit Sarin4, Hitesh Verma5, Viveksheel Malhotra5, Narayan Sivaramakrishnan Gomathi6, Srikanth Prasad Tripathy6, Dasarathi Das7, Sanghamitra Pati7, Nikita Panwalkar8, Prabha Desikan8, Kiran Bala9, Urvashi B. Singh9, Sandeep Sharma10, Avi Kumar Bansal10, Umesh D. Gupta10, Sripad A. Patil10, Abhinav Goyal11, Ashawant Gupta11, Nalini Kant Gupta11, Manjula Singh12, Jaya Sivaswami Tyagi2,3*, Sagarika Haldar1,2*.
Affiliations: ¹Department of Experimental Medicine and Biotechnology, Post Graduate Institute Of Medical Education and Research, Chandigarh, India; 2Translational Health Science and Technology Institute, Faridabad, India;3Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India; ⁴Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India; ⁵TB Hospital, Ambala, India; 6Department of Bacteriology, National Institute for Research in Tuberculosis, Chennai, India; 7Regional Medical Research Center, Bhubaneshwar, India; 8Bhopal Memorial Hospital and Research Centre, Bhopal, India; 9Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India; 10National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra, India; 11Advanced Microdevices Pvt Ltd, Industrial Area, Ambala Cantt, India; 12India Tuberculosis Research Consortium (ITRC), Indian Council of Medical Research, New Delhi, India.
India has the highest burden of Tuberculosis (TB) and innovative technology is the need of the hour to identify cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. To overcome this challenge, we developed a novel filter paper-based technology namely, ‘TBDetect’ kit, which enables sputum concentration through filtration using Bio-safe fluorescent microscopy filter (BioFM-Filter) device for improved and bio-safe smear microscopy at primary healthcare centres. In a 2-site evaluation study on presumptive TB patients (n=1190 sputum specimens), BioFM-Filter smear microscopy exhibited a significant increase in positivity of 7% and 4% over Ziehl Neelsen (ZN) smear microscopy and Light emitting diode-fluorescence microscopy (LED-FM, 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.
In a 6 site multi-centric validation study on sputum collected from 2086 presumptive TB patients in India, the combined positivity of ‘TBDetect’ microscopy was 20% vs. 16.1% LED-FM and 16% of ZN smear microscopy. The increment in positivity of ‘TBDetect’ over both LED-FM and ZN smear microscopy was significant (p<0.001). The overall sensitivity of ‘TBDetect’ for 6 sites was ~55% (95% Confidence interval [CI]: 50, 60%) vs. 52% (95% CI: 47, 57%) for LED-FM (p=0.14) and 50.9% (95% CI: 46, 56%) for ZN smear (p<0.05), using Mycobacterium Growth Indicator Tube culture as the reference standard. A bio-safety evaluation at 6 sites confirmed efficient sputum disinfection by ‘TBDetect’; 99.95% samples (1873/1874) were sterile after 42 days of incubation. Scientists and technicians at the study sites indicated the ease of use and convenience of ‘TBDetect’ microscopy during feedback evaluation. In conclusions, the ‘TBDetect’ based microscopy added value to the sputum smear microscopy test due to its improved performance, convenience and user safety. These findings indicate that equipment-free ‘TBDetect’ technology has the potential to improve TB diagnosis in basic laboratory settings by leveraging on the existing nationwide network of Designated Microscopy Centres (DMCs) and Primary Healthcare Centres (PHCs). The ‘TBDetect’ smear microscopy holds promise for integration into TB programmes for enabling prompt initiation of treatment and thereby aid in TB control in resource-limited countries. Our findings pave the way for larger feasibility study in different point-of-care field settings, including high-density urban areas and remote geographical locations.