Detection of Mycobacterium tuberculosis DNA in Ethanol-Fixed and Papanicolaou Stained Archival Materials

ABSTRACT

Granuloma is a chronic inflammatory process associated with non-infectious agents or infectious diseases such as tuberculosis. It is well known that AFB staining, which has been used to determine the etiology of the granulomatous inflammation, lacks both sensitivity and specificity. Due to the slow growth rate of most pathogenic mycobacteria, culturing of organisms can take up to eight weeks. It is not uncommon for specific therapy to be delayed, or for an inappropriate treatment be given to patients without mycobacterial infections or with infections caused by atypical mycobacteria. Determination of the causative agent in Papanicolaou stained cytology specimens gives pathologists even more difficulties when only necrotic material has been aspirated from the center of the granuloma. In recent years, the use of a polymerase chain reaction for the amplification of DNA has appeared promising in terms of speed, efficiency, sensitivity, and specificity. Since a polymerase chain reaction permits the sensitive genetic analysis of small amounts of tissue, it is ideally suited to the genetic analysis of cytologic specimens. A polymerase chain reaction is easily performed on unfixed and unstained cells, however, an analysis of ethanol fixed and Papanicolaou-stained archival smears has also been described. We have recently established a method to detect Mycobacterium tuberculosis organism by a nested polymerase chain reaction with primers in the insertion sequence IS 6110, using cellular digests of ethanol-fixed and Papanicolaou-stained archival specimens aspirated from the lymph nodes, lungs, thyroid, etc. Inhibitors present in Papanicolaou stained material was removed by destaining the slides with 0.5% HCl solution for 10-30 minutes. Eight out of ten cases which have shown the epithelioid granulomas revealed a positive reaction and four out of ten cases which have shown lymphohistiocytic cells in a necrotic background without any evidence of granuloma revealed a positive reaction. This study showed that it was possible to employ a polymerase chain reaction to detect Mycobacterium tuberculosis in Papanicolaou stained archival cytology specimens.