Pot method for storage & detection of acid-fast bacilli from sputum samples.

BACKGROUND & OBJECTIVES: Sputum acid-fast bacilli (AFB) microscopy services are not available in all health facilities. Alternative procedures are needed to transport sputum samples to the diagnostic centres for detection of AFB. The objective of the present study was to evaluate sputum smears made by pot-method with the direct smears made immediately after sputum collection by Ziehl-Neelsen (ZN) method. METHODS: Ninety three sputum samples from 49 pulmonary tuberculosis suspects were studied. Their direct smears (ZN smears) were stained by hot ZN method. The samples were then mixed with phenol ammonium sulphate basic fuchsin solution and stored at ambient conditions. The smears (pot smears), made on day 7, were then, decolourized and counter-stained for detection of AFB (pot method). The ZN and pot smears were read blind. After excluding 18 samples for various reasons, the results of pot and ZN smears of 63 samples from smear positive (2 of 3 direct smears were positive) and 12 from smear negative (3 of 3 direct smears were negative) patients were analysed. ZN method was the gold standard. RESULTS: Pot and ZN smears were positive in 61 of 63 samples from smear-positive patients and negative in 11 of 12 smear-negative patients (kappa = 0.87). The sensitivity and specificity of pot method were 96.8 and 91.7 per cent respectively. INTERPRETATION & CONCLUSION: Sputum samples can be stored for up to seven days in the sputum container with phenol ammonium sulphate basic fuchsin solution. However, a comprehensive study needs to be done confirm the accuracy of the pot method for storage and transportation of sputum to microscopy centres for detection of AFB.

Phenol ammonium sulphate basic fuchsin staining of sputum in pot for the detection of acid-fast bacilli.

BACKGROUND & OBJECTIVES: Improper practices of making direct smears of sputum for detection of acid-fast bacilli (AFB) and of disposing sputum cups are hazardous. The present study was undertaken with the objective to stain sputum samples in their containers by 'phenol (10%) ammonium sulphate (4%) basic fuchsin (2%) solution' and to decolourize and counterstain their smears for detection of AFB- (henceforth called pot method) and to compare the smear results of pot method with the standard Ziehl-Neelsen (ZN) method. METHODS: A total of 575 selected sputum samples from pulmonary tuberculosis patients were stained by the standard ZN and pot methods and the proportions of AFB positive smears were compared. RESULTS: Of the 575 samples, 126 were AFB positive for both the staining methods and the difference was not statistically significant. Pot method missed 9 ZN positive smears (8 scanty and one 1+) and ZN method missed 9 pot positive smears (9 scanty) and the difference was not significant. High grade smears (3+) were seen more in pot method (42) than in ZN method (25) and the difference was significant. INTERPRETATION & CONCLUSION: Our findings showed that pot method was comparable to standard ZN method and had many advantages. Pot method can be explored further for the detection of AFB in sputum samples obtained from pulmonary tuberculosis suspects.

A novel method of staining acid-fast bacilli in sputum containers.

BACKGROUND & OBJECTIVES: Making centrifuged deposit smears from sputum to detect acid-fast bacilli (AFB) is considered hazardous. We carried out this study to stain the centrifuged deposits with carbol-fuchsin in sputum containers and to decolourize and counterstain their smears made on glass slides. METHODS: The centrifuged deposits of 180 sputum samples from pulmonary tuberculosis patients were used for making smears (initial deposit smears) and staining by Ziehl-Neelsen (ZN) method for the detection of AFB. Each of the sputum deposit was then treated with one ml of 1 per cent carbol-fuchsin and a smear made between 2 to 3 h was then decolourized and counterstained by the same procedures followed in ZN method (2 h stained deposit smear). The coded initial deposit smears and the corresponding 2 h stained deposit smears were read by the same readers and the results compared. RESULTS: One hundred and fifty (70 positive and 80 negative) 2 h stained deposit smears were compared with initial deposit smears and the difference was not statistically significant. INTERPRETATION & CONCLUSION: Centrifuged deposits of sputum in sputum containers can be stained by carbol-fuchsin within 2-3 h and their smears made subsequently on glass slides can then be decolourized and counterstained by the procedures followed in ZN method for detection of AFB by light microscopy.

Proficiency to read sputum AFB smears by senior tuberculosis laboratory supervisors under training at a reference laboratory in India.

Background: A national reference laboratory imparting training on sputum AFB smear microscopy to fresh Senior Tuberculosis Laboratory Supervisors (STLS). Aim: To assess the proficiency of STLSs under training to read sputum AFB smears. Methods: Each of 342 trainees read the same set of 15 to 20 Ziehl Neelsen stained smears in a blinded fashion on day– 1 and on day-15 of the training programme. The smear results were matched with the original results. Observations: The sensitivity, specificity, positive predictive value and negative predictive value of smear reading were 75%, 88%, 93%, 63% and 94%, 99%, 99%, 89% respectively on day-1 and day –15. Conclusion: The sensitivity to read sputum AFB smears by fresh STLSs with little or no experience increased from 75% to 94% during the carefully planned training programme; the specificity increased from 88% to 99%. The study highlights the importance of training in improving the microscopy results.