Histopathological Detection of Mycobacterium Tuberculosis and Mycobacterium Leprae using a Modified Acid-Fast Technique

Estatera Cabic, Alpha Grace Cabic, Sheila Marie Esposo, Florencio Dizon, Gloria June Quinones, Arnel Guia


Introduction. Mycobacterium tuberculosis and Mycobacterium leprae are acid-fast organisms with lipid-rich cell walls that resist decolorization with acidified alcohol after application of a dye with heat. The Ziehl-Neelsen and Fite Faraco staining technique, which are diagnostic tools for identification of acid-fast bacilli (AFB) found in histopathologic samples, are based on this principle. A modification of the Ziehl-Neelsen technique is described as an alternative rapid and reliable method of diagnosis for prompt detection and treatment.

Methodology. One hundred and seven (107) archived tissue specimens from autopsy and dermatology cases interpreted as positive for M. tuberculosis and M. leprae were stained using the proposed modified acid-fast (MAF) technique compared with Fite Faraco (FF) staining method as reference standard. Each specimen was read by two independent evaluators.

Results. The degree of diagnostic agreement of the MAF with FF was calculated. For autopsy (n=16) and dermatology (n=91) samples, the Cohen’s kappas are 0.765 (substantial) and 0.397 (fair), respectively. Overall, the Cohen’s kappa is 0.458 (moderate).

Conclusion. The proposed modified Acid-Fast staining method may be considered as an alternative to the conventional Ziehl-Neelsen method and the Fite Faraco method in identifying positive acid-fast bacilli in tissue samples taken from clinical cases of M. tuberculosis and M. leprae.


acid-fast bacilli, Mycobacterium tuberculosis, Mycobacterium leprae, tuberculosis, leprosy, Ziehl-Neelsen, Fite Faraco

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Rose AH. Chemical microbiology: an introduction to microbial physiology. USA: Elsevier, 1976.

World Health Organization. Tuberculosis: WHO Fact Sheet 104. Accessed November 25, 2016.

World Health Organization. Leprosy: WHO Fact Sheet 101. Accessed November 25, 2016.

Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C. Tuberculosis. Lancet. 2003; 13;362(9387):887-99. PMID: 13678977. https://doi.org/10.1016/S0140-6736(03)14333-4.

WHO-Western Pacific Region. Tuberculosis. http://www.wpro.who.int/philippines/areas/communicable_diseases/tb/story_continuation_tb_area_page/en/. Accessed November 25, 2016.

World Health Organization. Epidemiology: leprosy. Accessed November 25, 2016.

WHO-Western Pacific Region. Leprosy control and the burden of leprosy in the Philippines: 2006-2010. Available at: http://www.wpro.who.int/philippines/areas/communicable_diseases/leprosy/who_leprosy_control_burden_.pdf.

Schlossberg D, ed. Clinical infectious disease. New York: Cambridge University Press, 2008.

Acharya T. Ziehl-Neelsen technique (AFB staining): principle, procedure and reporting. Bacteriology, laboratory diagnosis of bacterial disease staining techniques in microbiology, 2013. Retrieved from Microbe Online: http://microbeonline.com/ziehl-neelsen-technique-principle-procedure-reporting/.

Watson PF, Petrie A. Method agreement analysis: a review of correct methodology. Theriogenology. 2010;73(9):1167-79. https://doi.org/10.1016/j.theriogenology.2010.01.003.

Landis JR., Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;3(1):159-74. PMID: 843571.

Balakrishna J, Shahapur P, Chakradhar P, Saheb H. Comparative study of different staining techniques -Ziehlneelsen stain, Gabbet’s stain, Fluorochrom stain- for detecting of mycobacterium tuberculosis in the sputum. J Pharm Sci Res. 2013;5(4):89-92.

Karadağ A, Usta E, Bilgin K, etc. Comparison of culture, real-time DNA amplification assay and Ehrlich-Ziehl-Neelsen for detection of mycobacterium tuberculosis. Balkan Med J. 2013;30:13-5. https://doi.org/10.5152/balkanmedj.2012.061.

Murray SJ, Barrett A, Magee JG, Freeman R. Optimisation of acid-fast smears for the direct detection of mycobacteria in clinical samples. J Clin Pathol. 2003;56(7):613-5. PMID: 12890813. PMCID: PMC1770033.

Tille P. Mycobacteria and other bacteria with unsual growth requirements. In: Bailey and Scott's Diagnositc Microbiology, 13th ed. St. Louis, Missiouri: Mosby, Inc., 2014.

Reija AH, Biswas N, Biswas S, et al. Fite-faraco staining in combination with multiplex polymerase chain reaction: A new approach to leprosy diagnosis. Indian J Dermatol Venereol Leprol. 2013;79(5):693-700. PMID: 23974586. https://doi.org/10.4103/0378-6323.116740.

Sandhika AW, Adriaty D, Agusni I. Detection of mycobacterium leprae in formalin-fixed paraffin-embedded sample by fite-faraco staining and polymerase chain reaction. Elsevier Procedia Chemistry. 2016;18:231-6. https://doi.org/10.1016/j.proche.2016.01.036.

Lydard P, Cole M, Holton J, et al. Case studies in infectious disease: Mycobacterium leprae. New York: Garland Science, Taylor and Francis Group, LLC, 2009.

Murray SJ, Barrett A, Magee JG, Freeman R. Optimisation of acid-fast smears for the direct detection of mycobacteria in clinical samples. J CLin Pathol. 2003;56(8):613-5. PMID: 12890813. PMCID: PMC1770033.

Dalynn Biologicals. Carbol fuchsin stain (Ziehl-Neelsen), 2004. http://www.dalynn.com/dyn/ck_assets/files/tech/SC25.pdf. Accessed November 25, 2016.

Zhao D, Yang XM, Chen QY, Zhang XS, Guo CJ, Che XY. A modified acid-fast staining method for rapid detection of mycobacterium tuberculosis. J Microbiol Methods. 2012;91(1):128-32. PMID: 22902528. https://doi.org/10.1016/j.mimet.2012.07.024.

Chandra TJ, Raj RS, Sharma YV. Hydrochloric acid vs. sulfuric acid - an economical destaining reagent for Ziehl Neelsen staining to detect acid-fast bacilli in sputum smears. Int J.Bioassays. 2014;3(8):3241-3.

Sekar MG, Rehman F, Kumar V, Selvakumar N. Equivalence of acid alone or acid-alcohol as decolourizing agent in Ziehl-Neelsen method. Indian J Tuberc. 2012;59(4):219-23. PMID: 23342542.

Chandra T, Raj R, Sharma YV. Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry. Indian J Med Microbiol 2014;32(2):153-6. PMID: 24713901. https://doi.org/10.4103/0255-0857.129801.

Mokhtari Z, Larbaoui D. Corrélation entre les résultats d‘unexamen direct très faiblement positif et ceux de la culture—résultats préliminaires. Bull Union Int Tuberc. 1973;48:88-89.

Aung KJ, Nandi P, Hamid Salim A, Hossain A, Van Deun A. Hydrochloric vs. sulphuric acid in water for Ziehl-Neelsen staining of acid-fast bacilli. Int J Tuberc Lung Dis. 2011; 15(7):955-8. PMID: 21682971. https://doi.org/10.5588/ijtld.10.0722.

Nayak SV, Shivarudrappa AS, Nagarajappa AH, Sacchidanand S, Ahmed SM. Role of modified rapid AFB method in histopathological sections of Hansen's disease. Indian J Dermatol Venereol Leprol. 2003;69(2):173-4. PMID: 17642871.


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