Papanicolaou induced fluorescence, Ziehl-Neelsen and Auramine O stains on lymph node fine needle aspiration biopsy specimens from children: A comparative study.

BACKGROUND: The rapid diagnosis of extrapulmonary tuberculosis in children remains challenging. The presence of enlarged lymph nodes provides an opportunity to obtain diagnostic material through fine needle aspiration biopsy (FNAB). Mycobacterial culture, traditionally the reference standard, has a slow turnaround time and PCR-based methods are not widely available in developing countries. Direct visualization of mycobacteria on microscopy can be a rapid method to confirm the diagnosis. This study compared three staining methods to visualize mycobacteria. METHODS: Hundred FNAB specimens from persistently enlarged lymph nodes in children, clinically suspicious for tuberculosis, were evaluated for the presence of mycobacteria by three staining methods: Papanicolaou induced fluorescence (PIF) and Auramine O staining using fluorescence microscopy and Ziehl-Neelsen (ZN) staining using conventional light microscopy. These methods were evaluated against mycobacterial culture. RESULTS: PIF positivity was 30%, with 38% and 48% for Auramine O and ZN respectively. The combined ZN/PIF positivity was 56%. The highest diagnostic accuracy (73%) was demonstrated by ZN alone and in combination with PIF, with PIF alone showing the lowest (49%) accuracy. Although the combined test showed the highest sensitivity, it had the lowest specificity, while ZN was significantly more sensitive than both other staining modalities. No statistical difference in specificity was seen among the tests. CONCLUSION: This study suggests that Auramine O staining on previously ZN stained slides does not significantly improve diagnostic accuracy. While currently widely available methods of direct visualization of mycobacteria suffer from low sensitivity, the ZN stain remains a useful diagnostic test, particularly in resource-constrained countries.

Lessons from a proficiency testing event for acid-fast microscopy.

OBJECTIVES: To evaluate the routine performance and the technical parameters of different acid-fast staining methods: Kinyoun, Ziehl-Neelsen (ZN), auramine, and auramine-rhodamine. DESIGN AND PARTICIPANTS: The performance of 167 laboratories was analyzed using prestained and unstained slides. SETTING: Laboratories holding New York State permits. RESULTS: The results revealed that Kinyoun's cold carbol fuchsin method is inferior to both the ZN and fluorochrome (auramine and/or auramine-rhodamine) methods. Even though 91% of the participants used commercial staining kits, the study identified unexpected errors concerning the concentration of carbol fuchsin, time for staining and counterstaining, and the concentration of acid alcohol for decolorization, which may significantly influence the sensitivity. Besides these findings, the present study showed that the examination of < 300 view fields may also decrease the sensitivity of acid-fast microscopy. In addition, we found that the sensitivity and specificity of the ZN and fluorochrome methods are comparable if the procedural standards are followed. CONCLUSIONS: The strict and ongoing quality control of the "simple to perform" acid-fast microscopy and the immediate review of commercially available staining kits are necessary. Because of the rapidity of the fluorochrome method, laboratories with large specimen numbers should use this technique. In all other cases, the ZN method should be used. Moreover, all clinicians should be aware of the method of acid-fast microscopy used and the proficiency of the laboratory in performing the assay.