Performance of variations of carbolfuchsin staining of sputum smears for AFB under field conditions.

SETTING: A field project in Bangladesh. OBJECTIVE: To compare the effectiveness of commonly used carbolfuchsin staining variations. DESIGN: Routine hot Ziehl-Neelsen (ZN) 1% basic fuchsin staining for 15 min in 75 field clinics. Blind reading of duplicate smears stained by ZN 1% vs. 0.3% basic fuchsin applied for 5 min, or by ZN 1% 5 min vs. Kinyoun cold staining. Rechecking of discordant series. RESULTS: For comparable numbers of false positives, sensitivity was significantly lower with Kinyoun than with ZN 1% 5 min (85.6% vs. 93.0%, P < 0.001). Sensitivity with ZN 1% 5 min was not significantly higher than with 0.3% 5 min staining (89.9% vs. 86.5%). Routine examination using 1% 15 min ZN identified more positives than any of the study techniques. CONCLUSIONS: Kinyoun cold staining sensitivity was unsatisfactory in field clinics. The sensitivity of the WHO/IUATLD recommended 0.3% fuchsin for 5 min was not significantly different from the original 1% ZN for 5 min, but 1% 15 min hot staining might be superior. A reduced fuchsin concentration together with a short staining time may leave too narrow a margin for error. TB programmes using hot ZN with a concentrated stain or longer staining time should not be urged to change.

Optimal tuberculosis case detection by direct sputum smear microscopy: how much better is more?

SETTING: A tuberculosis control project in Bangladesh. OBJECTIVE: To define the efficiency of numbers of microscopic fields screened and the sputum collection scheme used for diagnostic smear examination. DESIGN: Quality controllers noted cumulative numbers of acid-fast bacilli per 100 fields screened. The incremental diagnostic yield of different sputum sampling strategies was determined. Doubtful series were re-checked and/or further samples examined. RESULTS: Acid-fast bacilli were found in 99.6% of 1412 positive and in 79.3% of 576 scanty slides in the first 100 fields. Examination of a third specimen yielded a maximum of 2.7% positives incrementally. The most efficient strategy, using three morning specimens, yielded 94.2% positives on the first and 1.0% on the third sputum; although 10% of suspects did not return, only 1.5% of the positives were among them and more cases were confirmed and treated. The positive predictive value of a single positive or scanty smear was very high (99.2%). CONCLUSIONS: Reading more than 100 fields per smear or examining a third sputum has insufficient marginal returns to justify the workload. Examining morning samples only is more efficient, and their collection does not necessarily inconvenience patients. Treatment can be started on the basis of one positive smear. Provided that a well functioning system of smear-microscopy quality control is in place, we propose a strategy based on examination of two morning sputum samples for negative suspects, with the diagnosis based on a single positive result.

Reproducibility of sputum smear examination for acid-fast bacilli: practical problems met during cross-checking.

SETTING: A TB control programme in Bangladesh with proficiency testing of sputum smears for acid-fast bacilli (AFB) using cross-checking of routine smears. OBJECTIVE: To document factors that may adversely affect repeatability of the AFB smear, and which should be taken into account for interpretation of cross-checking. DESIGN: A number of simple experiments falling within the scope of small routine laboratories in a developing country. RESULTS: Fuchsin staining is not stable, fading quickly in direct sunlight and combinations of high temperature and humidity. Diffuse daylight, immersion oil or xylene did not have the same effect. Contamination of smears by saprophytic mycobacteria may occur during staining or rinsing, with contaminants becoming visible only after restaining and cross-checking. Finally, AFB may be dislodged from smears during soaking in xylene or restaining, especially if the smears are made from thin, liquefied sputum. CONCLUSIONS: These possible interfering factors should be taken into account when organizing proficiency testing and interpreting its results.