Comparative evaluation of a two-reagent cold stain method with Ziehl-Nelseen method for pulmonary tuberculosis diagnosis

BACKGROUND: Bacteriological examination of sputum is the cornerstone in diagnosis of pulmonary tuberculosis in developing world, which is usually done using a Ziehl-Nelseen (ZN) method. However, due to limited laboratory facilities that can satisfy the procedure, applicability of this procedure appears to be adversely affected in field conditions and at peripheral health institutions. Hence, it has become necessary to look for a procedure which can be used as alternative in such conditions. In a cross-sectional study, using convenient sampling technique 362 pulmonary tuberculosis suspected patients who attended at Mekelle University Hospital (MUH) between November 2011 and February 2012 were included. After obtaining an informed consent, spot- morning-spot sputum samples were collected from suspected patients. Then a set of duplicate slides, of which one was allocated to a two-reagent cold method (a method of staining which requires carbol fuchsine as a primary stain and Gabbet’s methylene blue both as a decolorizer and counter stain) and the other to the Zeihl-Nelseen method were smeared evenly from representative portion of each specimen using the protocol for duplicate smear preparation. Stained smears were read blindly by two technologists at different occasions. Finally to assure quality, all positive smears and 25% of the negative smears were cross checked by senior experienced examiner. FINDINGS: Overall concordance between the two methods was 99.7% (kappa (κ) = 0.98; 95%, confidence interval 0.93-1.00), and the observed agreement was statistically significant (p<0.001). When evaluated against Ziehl-Nelseen method, sensitivity and specificity of the two-reagent cold staining method were 95.8% (95% confidence interval 93.7-97.9) and 100% respectively. Positive and negative predictive values of the two-reagent cold staining method were respectively 100% and 99.7%. Positive and negative agreements between the two techniques were respectively 97.9% and 99.9%. CONCLUSION: The two-reagent cold staining method was found to be a suitable alternative to the conventional Ziehl-Nelseen method; it was at least as specific as Ziehl-Neelsen method although somewhat less sensitive. However, large scale multicentric studies need to be performed for further evaluation of this cold staining method.