Utility of universal sample processing methodology, combining smear microscopy, culture, and PCR, for diagnosis of pulmonary tuberculosis

Abstract

The universal sample processing (USP) multipurpose methodology was developed for the diagnosis of tuberculosis (TB) and other mycobacterial diseases by using smear microscopy, culture, and PCR (S. Chakravorty and J. S. Tyagi, J. Clin. Microbiol. 43:2697-2702, 2005). Its performance was evaluated in a blinded study of 571 sputa and compared with that of the direct and N-acetyl L-cysteine (NALC)-NaOH methods of smear microscopy and culture. With culture used as the gold standard, USP smear microscopy demonstrated a sensitivity and specificity of 98.2% and 91.4%, respectively, compared to 68.6% and 92.6%, respectively, for the direct method. For a subset of 325 specimens, the USP method recorded a 97.1% sensitivity and 83.2% specificity compared to the NALC-NaOH method, which had a sensitivity and specificity of 80.0% and 89.7%, respectively, with culture used as the gold standard. Thus, the USP method exhibited a highly significant enhancement in sensitivity (P < 0.0001) compared to the direct and NALC-NaOH methods of smear microscopy. The USP culture sensitivity was 50.1% and was not significantly different from that of conventional methods (53.6%). The sensitivity and specificity of IS6110 PCR were 99.1% and 71.2%, respectively, with culture used as the gold standard, and increased to 99.7% and 78.8%, respectively, when compared with USP smear microscopy. Thus, the USP methodology was highly efficacious in diagnosing TB by smear microscopy, culture, and PCR in a clinical setting.

FIG. 1.

Physical appearance of smears. Smears were prepared from equal aliquots of a sputum sample processed by the CDC and USP smear methods. (i) A loopful from the purulent portion of sputum was taken in a 3-mm sterile wire loop and smeared onto a glass slide (direct). (ii) Two loopfuls of specimen processed by the CDC method were smeared onto a glass slide. (iii) Ten percent of the USP sputum was smeared onto a glass slide.

FIG. 2.

Enhancement of smear grade status of sputum specimens. (A) Percent distribution of different grades of smear positivity by the direct and USP methods of smear microscopy (n = 243). (B). Percent distribution of the different grades of smear positivity by the CDC and USP methods of smear microscopy (n = 152).