Sensitivity of two-stage PCR amplification for detection of Mycobacterium tuberculosis in paraffin-embedded tissues

In order to improve the sensitivity of polymerase chain reaction (PCR) for the detection of mycobacterial DNA in paraffin-embedded tissues, a new approach with two sets of specific primers in two-stage PCR was employed in specimens obtained from tuberculosis patients and controls. Thirty-nine paraffin blocks selected from patients who had been diagnosed as having tuberculosis by radiological evaluations, histopathological findings, and clinical symptoms and signs including response to antituberculous treatment were examined. The control group consisted of 10 specimens from individuals that were proved to be negative for tuberculosis. After deparaffinization, lysis, phenol–chloroform extraction, and ethanol precipitation, the isolated DNA was amplified by PCR. Initially, all specimens were examined by the one-stage PCR using specific primers for 123-base pair (bp) fragment in IS6110 of mycobacterial DNA which yielded positive results only in 3 out of 39 (7.7%). In the two-stage PCR technique, 245-bp fragment of mycobacterial DNA was amplified at the first-step, then the PCR products were reamplified using the second specific primer pairs for 123-bp fragment. The true positivity of the two-stage PCR was 84.6% (33/39). The results indicate that two-stage PCR is more sensitive than one-stage (84.6% vs. 7.7%). All control specimens were negative by both PCR amplification methods, indicating that specificity of both methods was high. When the two-stage amplification was used, PCR positivity in the specimens obtained from different tissues was as follows: peritoneal and omental biopsies, 4/4; bone biopsies, 3/3; lymph node biopsies, 12/14; genito–urinary biopsies, 7/9; skin biopsies, 4/6; and one from each lung, breast, and pleural biopsies. PCR showed a good correlation with the granulomatous tissue reaction resulting in a 83.8% (31/37) positivity. The results indicate that the two-stage PCR amplification can be used for detection of M. tuberculosis in paraffin-embedded tissues and is a useful technique in confirming tuberculosis in patients with clinically suspected disease who have acid-fast stain-negative.