Comparison of Ziehl Neelsen and Auramine O staining methods on direct and concentrated smears in clinical specimens.

Background: In developing countries like ours with a large number of tuberculosis (TB) cases and limited resources, the diagnosis of TB relies primarily on smear microscopy for Acid Fast Bacilli (AFB) but its sensitivity is limited in paucibacillary cases. Aim: To evaluate the increase in efficacy of smear microscopy when smears are prepared from clinical samples after concentration by Petroff’s method and stained by Auramine O (AO) fluorescent dye as against Ziehl Neelsen (ZN) staining of similar taking culture as the gold standard. Methods: Smears were prepared from 393 clinical samples both by direct and after Petroff’s concentration and examined by fluorescent microscopy and Ziehl Neelsen method .The concentrated material was also cultured on Lowenstein Jensen media and the results of the two microscopy methods were compared with the culture results taken as the gold standard. Results: Mycobacterial growth was detected in 137(35.77%) specimens, out of which three were non-tubercular mycobacteria. Using culture as the reference method, the sensitivity of direct staining was 55.55% for ZN and 71.85% for AO. Direct fluorescent microscopy detected 9.29% paucibacillary sputum samples that were missed on ZN staining. On concentration, the sensitivity increased by 6.67% for ZN and 11.11% for AO. The sensitivity of AFB smear microscopy increased by 27.41% and was statistically significant (p=<.001) when both methods were combined. The specificity was 99.19% for both ZN and AO. Conclusion: Fluorescent microscopy has higher sensitivity and comparable specificity which is further enhanced by concentration. Now with the advent of newer inexpensive Light Emitting Diode (LED) based fluorescent microscopes (FM), which are easier to use, fluorescent microscopy can be widely used even in peripheral laboratories where culture facilities are not available.

Comparison of MGIT 960 & pyrazinamidase activity assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis.

Background & objectives: Pyrazinamide is an important front line antimycobacterial drug, which is also being used in the treatment of multi drug resistant tuberculosis along with second line drugs in DOTS plus programme. Conventional testing of pyrazinamide on solid medium is difficult as it is active at acidic pH. Therefore, there is a need for a rapid and simple method for susceptibility testing of pyrazinamide. This study was carried out to compare pyrazinamide susceptibility testing by MGIT 960 and two rapid pyrazinamidase activity tests. Methods: Pyrazinamide susceptibility was tested in 136 clinical isolates of Mycobacterium tuberculosis by MGIT 960 and pyrazinamidase activity was tested by classical Wayne’s method and modified PZase agar method. Results: There was 88.9 per cent concordance between MGIT 960 and classical Wayne’s method and 93.38 per cent with modified method for pyrazinamidase activity. Using MGIT 960 results as gold standard the sensitivity and specificity of Wayne’s method was 88.15 and 90 per cent respectively and that of modified method was 89.4 and 98.3 per cent. Interpretation & conclusions: Our study demonstrates that the modified pyrazinamidase activity test can be used as a screening test to detect resistance to pyrazinamide specially in resource limited settings but confirmation of susceptibility should be done by standard methods like MGIT 960.

Transport and storage of sputum specimen by using cetylpyridinium chloride for isolation of mycobacteria.

Of the 191 sputum specimens that were collected from pulmonary tuberculosis patients, 78.65% (140/178) specimens were culture positive when processed within 48 h by the NaOH method. The culture positivity in the same specimen that were stored with cetylpyridinium chloride (CPC) and processed after 7-8 days was 70.22% (125/178), whereas those stored without CPC and processed by the NaOH method was 46.62% (83/178). The difference in number of positive cultures obtained before storage and after storage (without CPC) was statistically significant (P = 0.001). Culture positivity by the CPC method was comparable with that of NaOH method before storage and the difference was not statistically significant (P = 0.35).