Reduced detection by Ziehl-Neelsen method of acid-fast bacilli in sputum samples preserved in cetylpyridinium chloride solution

Abstract

Setting: Twelve health facilities implementing the DOTS strategy, and the Tuberculosis Research Centre (TRC), Chennai, India. Objective: To determine the detection rates using Ziehl-Neelsen (ZN) and auramine-phenol to stain acid-fast bacilli (AFB) in sputum samples stored in cetylpyridinium chloride (CPC) solution. Methods: Two smears were prepared from each of 988 sputum samples collected in CPC and randomly allocated, one to ZN and the other to auramine-phenol staining. All samples were processed for culture of Mycobacterium tuberculosis. Results: A significantly higher proportion of samples were negative using the ZN method compared to the auramine-phenol method (74.5% vs. 61.8%, McNamara's paired χ² test; P < 0.001). Among 377 samples that were positive using auramine-phenol, 44% were negative using ZN. There were more culture-positive, smear-negative samples in ZN (52.7%) than in auramine-phenol (30%); the difference attained statistical significance (McNemar's paired χ² test; P < 0.00004). Using ZN, of the 104 smears made immediately after collection, 52 were positive for AFB, of which only 35 (67.3%) were positive after storage in CPC; the reduction in the number of positive smears attained statistical significance (McNemar's paired χ² test; P = 0.004). Conclusion: Detection of AFB in sputum samples preserved in CPC is significantly reduced using ZN staining.