Bleach method in comparison with NALC-NaOH specimen processing method for the detection of mycobacterium in sputum specimen

Amar Kumar G., S. Chandrasekaran, Visalasree J., Vignesh Kanna B.


Background: Tuberculosis is an infectious disease still remains to be a foremost public health risk worldwide. Even though there is an availability of many highly sensitive diagnostic tools, early case detection plays a significant role in control of the disease which relies specially on the detection of acid-fast bacilli in clinical samples which is low sensitive. Earlier decontamination of sputum sample may improve the detection of mycobacterium.

Methods: A prospective study was conducted with a total of (N=464) clinically suspected TB patient sputum samples which were collected and processed directly for AFB. From which AFB +ve samples were excluded. The direct AFB -ve (N=279) samples were processed using concentration methods (NALC, Bleach). After concentration, smears were examined under oil immersion for acid fast bacilli by ZN staining.

Results: Out of 464 sputum samples, 185 direct ZN stain +ve were excluded. Among the ZN-ve (N=279) samples, n=44 (15.7%) were positive using bleach method. The positivity has improved to n=48 (17.2%) using NALC-NaOH.

Conclusions: Bleach method of sputum decontamination is comparable to standard NALC-NaOH method. Therefore, we conclude that the identification of AFB using bleach concentration method can be used in resource limited health care setups especially in laboratories where mycobacterial culture is not performed which might help us in early diagnosis of tuberculosis.


Acid fast bacilli, Bleach, NALC-NaOH-N-acetyl-L-cysteine sodium hydroxide, Ziehl-neelsen staining

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Uddin MKM, Chowdhury MR, Ahmed S, Rahman MT, Khatun R, van Leth F, et al. Comparison of direct versus concentrated smear microscopy in detection of pulmonary tuberculosis. BMC Research Notes. 2013;6(1):291.

Mycobacteriology Laboratory Manual April; 2014

Daniel TM. Toman's tuberculosis. Case detection, treatment and monitoring: questions and answers. Toman's tuberculosis. Case detection, treatment and monitoring: questions and answers. 2004;

Verma S, Dhole TN, Kumar M, Kashyap S. Novel approach for improving sensitivity of microscopic detection of acid-fast bacilli (AFB) by use of the ReaSLR method. J Clin Microbiol. 2013;51(11):3597-601.

Laserson KF, Yen NTN, Thornton CG, Mai VTC, Jones W, An DQ, et al. Improved sensitivity of sputum smear microscopy after processing specimens with C18-carboxypropylbetaine to detect acid-fast bacilli: a study of United States-bound immigrants from Vietnam. J Clin Microbiol. 2005;43(7):3460-2.

Morcillo N, Imperiale B, Palomino JC. New simple decontamination method improves microscopic detection and culture of mycobacteria in clinical practice. Infect Drug Resist. 2008;1:21.

Mindolli PB, Salmani MP, Parandekar PK. Improved diagnosis of pulmonary tuberculosis using bleach microscopy method. J Clin Diagnost Res. JCDR. 2013;7(7):1336.

Sharath BN, Shastri S. India's new TB diagnostic algorithm-far from reality?. Public Health Action. 2016;6(3):206-6.

Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture and Drug susceptibility testing; 2009

Peres RL, Maciel EL, Morais CG, Ribeiro FCK, Vinhas SA, Pinheiro C, et al. Comparison of two concentrations of NALC-NaOH for decontamination of sputum for mycobacterial culture. Int J Tuberculosis Lung Dis. 20096;13(12):1572-5.

Steingart KR, Ng V, Henry M, Hopewell PC, Ramsay A, Cunningham J, et al. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis. 2006;6(10):664-74.

Lawrence MD, Baveja CP, Kumar S, Khanna A, Sapriina J. Comparative Evaluation of Flourescent Staining with Ziehl-Neelsen and Kinyoun Staining in the Diagnosis of Clinically Suspected Cases of Pulmonary Tuberculosis. Int J Contem Med Res. 2016;3(7):1970-4.

Cattamanchi A, Dowdy DW, Davis JL, Worodria W, Yoo S, Joloba M, et al. Sensitivity of direct versus concentrated sputum smear microscopy in HIV-infected patients suspected of having pulmonary tuberculosis. BMC Infect Dis. 2009;9(1):53.

Kang H, Sung N, Lee S, Kim D, Jeon D, Hwang S, et al. Comparison of smear and culture positivity using NaOH method and NALC-NaOH method for sputum treatment. Tuberculosis Respirat Dis. 2008;65(5):379-84.

Ongkhammy S, Amstutz V, Barennes H, Buisson Y. The bleach method improves the detection of pulmonary tuberculosis in Laos. Int J Tuberculosis Lung Dis. 2009;13(9):1124-9.

Ă„ngeby KAK, Alvarado-Galvez C, Pineda-Garcia L, Hoffner SE. Improved sputum microscopy for a more sensitive diagnosis of pulmonary tuberculosis. Int J Tuberculosis Lung Dis. 2000;4(7):684-7.

Muhammad KU, Bello M, Manga BS, Isa MA. Comparison between bleach concentration method and conventional ziehl neelsen technique of determination of mycobacterium tuberculosis in sokoto metropolis. Int J Res. 2014;1(11):504-11.

James A, Ochei K, Emenyonu N, Lawson L. Improved sensitivity, safety and laboratory turnaround time in the diagnosis of pulmonary tuberculosis by use of bleach sedimentation. African J Lab Med. 2015;4(1):1-8.

Chandrasekhar B, Prayaga AK. Utility of concentration method by modified bleach technique for the demonstration of acid-fast bacilli in the diagnosis of tuberculous lymphadenopathy. J Cytol/Ind Acad Cytol. 2002;29(3):165.

Khar M, Kaushal M. Utility of modified bleach method technique for the demonstration of acid fast bacilli in the diagnosis of Tuberculous lymphadenopathy in comparison to routine Ziehl-neelsen staining. Ann Pathol Lab Med. 2016;3(4):A329-332.