Gulnaz BASHİR, Tufail AHMED, Abiroo JAN, Bashir1, FOMDA, Insha ALTAF, Sofia ZAFFAR

Colorimetric Assays as the Diagnostic Modality of Pulmonary and Extra-pulmonary Tuberculosis in Resource-limited Settings

Objectives: Diagnosis of tuberculosis (TB) is challenging, especially in a resource-limited setting. Non-commercial alternatives such as colorimetric assays must be used for effective diagnosis and prompt treatment. In this study we aimed to determine the diagnostic performance, time to detection, cost, contamination rate, and ease of performance of the oxidation-reduction assays; Resazurin tube assay (RETA) and Malachite Green decolorization assay (MGDA) and their modifications using para-nitro benzoic acid (PNB) for detection of TB against the results on Lowenstein-Jensen medium (LJ) medium. Methods: Two-hundred-seventeen samples were subjected to colorimetric assays by incubating the inoculated media at 370C. Dyes were added on days 10, 14, 18, 28, and 42 days of incubation, and color change was noted after 24 hours and was compared with results on the LJ medium. Results: Diagnostic performance of colorimetric assays increased from day 10 to day 28 (maximum at day 28), while it was the same on days 28 and 42. The sensitivity, specificity, and diagnostic accuracy of RETA were 87.6%, 95.7%, and 88.6%, while that of MGDA were 81.7%, 95.5%, and 86.5%, respectively. On day 18, more than 51% of the samples were positive by colorimetric assays, whereas only 20.4% were positive by LJ culture. In addition, the colorimetric assays were as economical as LJ culture. Conclusion: Colorimetric methods can potentially become non-commercial alternatives for rapidly detecting Mycobacterium. tuberculosis, especially in TB laboratories with limited resources. The ease of interpreting the results and its cost-effectiveness is an additional advantage. J Microbiol Infect Dis 2022; 12(3):108-115.

Keywords:

Mycobacterium tuberculosis, colorimetric assays, LJ culture, malachite green, resazurin,

PDF

___

1. Global Tuberculosis Report 2021 [Internet]. [cited 2022 Sep 5]. Available from: https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2021.
2. Liu KT, Su WJ, Perng RP. Clinical utility of polymerase chain reaction for diagnosis of smear-negative pleural tuberculosis. J Chin Med Assoc 2007; 70(4):148-151; discussion 146-147.
3. Haldar S, Sharma N, Gupta VK, Tyagi JS. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR. J Med Microbiol 2009; 58:616-624.
4. Rouillon A, Perdrizet S, Parrot R. Transmission of tubercle bacilli: The effects of chemotherapy. Tubercle 1976; 57(4):275-299.
5. TB/HIV research priorities in resource-limited settings: report of an expert consultation [Internet]. [cited 2022 Sep 5]. Available from: https://www.who.int/publications/i/item/WHO-HTM-TB-2005.355.
6. The Stop TB strategy: building on and enhancing DOTS to meet the TB-related Millennium Development Goals [Internet]. [cited 2022 Sep 5]. Available from: https://apps.who.int/iris/handle/10665/69241.
7. Leung E, Minion J, Benedetti A, Pai M, Menzies D. Microcolony culture techniques for tuberculosis diagnosis: a systematic review. Int J Tuberc Lung Dis 2012; 16(1):16-23, i-iii.
8. Moore DA, Evans CA, Gilman RH, et al. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 2006; 12;355(15):1539-1550.
9. De Souza AO, Santos Júnior RR, et al. Antimycobacterial activity of 4'-bromo-[1,1'-biphenyl]-4-yl 4-x-phenylmethanone derivatives, and their acute toxicity and cytotoxicity. Pharmazie 2001; 56(11):871-874.
10. Affolabi D, Odoun M, Sanoussi N, et al. Rapid and inexpensive detection of multidrug-resistant Mycobacterium tuberculosis with the nitrate reductase assay using liquid medium and direct application to sputum samples. J Clin Microbiol 2008; 46(10):3243-3245.
11. Martin A, Portaels F, Palomino JC. Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Antimicrob Chemother 2007; 59(2):175-83.
12. Palomino JC, Martin A, Portaels F. Rapid drug resistance detection in Mycobacterium tuberculosis: a review of colourimetric methods. Clin Microbiol Infect 2007; 13(8):754-762.
13. Angeby KA, Klintz L, Hoffner SE. Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol 2002; 40(2):553-555.
14. Primm T, Franzblau S. Recent Advances in Methodologies for the Discovery of Antimycobacterial Drugs. Curr Bioact Compd 2007; 30;3(3):201-208.
15. Franzblau S. A rapid, microplate-based assay for evaluating the activity of drugs against Mycobacterium leprae, employing the reduction of Alamar Blue. Lepr Rev 2000; 71(Supplement).
16. Laszlo A, Siddiqi SH. Evaluation of a rapid radiometric differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone. J Clin Microbiol 1984, 19:694-698.
17. Rastogi N, Goh KS, David HL. Selective inhibition of the Mycobacterium tuberculosis complex by p-nitro-alpha-acetylamino-beta-hydroxypropio phenone (NAP) and p-nitrobenzoic acid (PNB) used in 7H11 agar medium. Res Microbiol 1989; 140(6):419-423.
18. Martins M C, Ueki S Y M, Palhares M C A, et al. An alternative biphasic culture system for recovery of mycobacteria and for differentiation of species other than M. tuberculosis complex from blood specimens. Rev Microbiol 1997; 183-189.
19. Koneman EW. Color Atlas and Textbook of Diagnostic Microbiology 1997 [cited 2022 Sep5]; 63. Available from: https://books.google.com/books/about/Color_Atlas_and_Textbook_of_Diagnostic_M.html?hl=fr&id=OHhhQgAACAAJ.
20. Tripathi K, Tripathi PC, Nema S, Shrivastava AK, Dwiwedi K, Dhanvijay AK. Modified Petroff’s method: an excellent simplified decontamination technique in comparison with Petroff’s method. Int J Recent Trends Sci Technol. 2014; 10(3):461.
21. Khalifa RA, Nasser MS, Gomaa AA, Osman NM, Salem HM. Resazurin Microtiter Assay Plate method for detection of susceptibility of multidrug resistant Mycobacterium tuberculosis to second-line anti-tuberculous drugs. Egypt J Chest Dis Tuberc 2013; 62(2): 241-247.
22. Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug Susceptibility testing. Central TB division, Directorate General of Health Services, Ministry of Health and Family Welfare, New Delhi, India, 2009. https://tbcindia.gov.in/WriteReadData/l892s/6995271860Training%20manual%20M%20tuberculosis%20C%20DST. pdf.
23. Tsukamura M. [P-nitrobenzoic acid medium (PNB medium) as an aid to differentiate tubercle bacilli from other mycobacteria. (A review)]. Kekkaku 1984; 59(6):361-368.
24. Boum Y, Orikiriza P, Rojas-Ponce G, et al. Use of colorimetric culture methods for detection of Mycobacterium tuberculosis complex isolates from sputum samples in resource-limited settings. J Clin Microbiol 2013; 51: 2273-2239. 25. Farnia P, Mohammadi F, Mirsaedi M, et al. Application of Oxidation-Reduction Assay for Monitoring Treatment of Patients with Pulmonary Tuberculosis. J Clin Microbiol 2004; 42 :3324-3325.
26. Harrell FE Jr, Califf RM, Pryor DB, Lee KL, Rosati RA. Evaluating the yield of medical tests. JAMA 1982; 247(18):2543-2546.
27. Walusimbi S, Kwesiga B, Rodrigues R, et al. Cost-effectiveness analysis of microscopic observation drug susceptibility test versus Xpert MTB/Rif test for diagnosis of pulmonary tuberculosis in HIV patients in Uganda. BMC Health Serv Res 2016; 16: 563.
28. Chihota VN, Grant AD, Fielding K, et al. Liquid vs. solid culture for tuberculosis: performance and cost in a resource-constrained setting. Int J Tuberc Lung Dis 2010; 14(8): 1024-1031.
29. Smaoui S, Kammoun S, Marouane C, Slim L, Messadi-Akrout F. Evaluation of the BACTEC MGIT 960 TB with Solid Media for Recovery of Mycobacteria from Extrapulmonary Specimens in South Tunisia. J Med Diagn Meth 2015; 4: 171. doi: 10.4172/2168-9784.1000.171.