An evaluation of 5% Naoci microscopy method for the laboratory diagnosis of pulmonary tuberculosis

Abstract

Direct sputum smear microscopy forms the mainstay for pulmonary tuberculosis case-finding in the resource-poor countries like Nepal. However, this method is hampered by the lack of sensitivity. Bleaching of sputum with sodium hypochlorite and concentration of mycobacteria prior to Ziehl-Neelsen staining method is a possible means of improving the sensitivity of direct microscopy. Therefore, this study was performed in National Tuberculosis Centre, Thimi, Bhakatapur, Nepal with an objective to evaluate 5% NaOCl microscopy method for the primary diagnosis of pulmonary tuberculosis. A total 475 sputum were collected from 159 suspected pulmonary tuberculosis patients. Direct smears were prepared from all sputum. ‘On-spot’ sputum was divided equally into three tubes (A, B and C). Tube A and B were treated by NaOCl-centrifugation method and NaOCl-sedimentation method respectively, and ZN smears were prepared. Tube C was treated by Modified Petroff’s method for culture. NaOCl-centrifuged and NaOCl-sedimented ‘on-spot’ sputum showing positive direct smears were also used for culture to ascertain the sterilizing activity of NaOCl. Culture method was employed as a gold standard for pulmonary tuberculosis diagnosis. Direct ‘on-spot’ sputum Ziehl-Neelsen smears, NaOCl-centrifuged Ziehl-Neelsen smears and NaOCl-sedimented Ziehl-Neelsen smears had 17.7%, 16.3% and 10.1% positivity respectively compared to 14.5% of the standard three smears strategy. There was a statistically significant increase in the number of acid-fast bacilli positive sputum samples by NaOCl methods (P<0.05). In addition, there was no statistical significant between two NaOCl methods (P>0.05). With reference to culture, sensitivity of direct ‘on-spot’ sputum ZiehlNeelsen smears, NaOCl-centrifuged Ziehl-Neelsen smears and NaOCl-sedimented ZiehlNeelsen smears were found to be 35.1%, 67.5% and 62.1% compared to 54% of standard three smears strategy. The specificity was found to be same i.e. 97.4% for all methods. The positive and negative predictive values were found to be 81.2%/82.3% for direct ‘on-spot’ sputum Ziehl-Neelsen smears, 89.2%/90.4% for NaOCl-centrifuged Ziehl-Neelsen smears, 88.4%/89% for NaOCl-sedimented Ziehl-Neelsen smears and 87%/87% for standard three smears strategy. No growth of Mycobacteria tuberculosis was seen in culture media (Lowenstein-Jensen) inoculated with NaOCl-centrifuged and NaOCl-sedimented ‘on-spot’ sputum showing the biocidal activity of NaOCl. This study indicates that 5% NaOCl microscopy method i.e. NaOCl-centrifuged ZiehlNeelsen smears andNaOCl-sedimented Ziehl-Neelsen smears could be alternative to direct microscopy and the application of these methods would make a positive impact on the effectiveness of TB control programs. Keywords: Tuberculosis, M. tuberculosis, Sputum smear microscopy, Bleach digestion, Ziehl-Neelsen.