Detection of Mycobacterium tuberculosis in paraffin embedded intestinal tissue specimens by polymerase chain reaction: characterization of IS6110 element negative strains

Tuberculosis is still one of the most widespread infection known to mankind. Although lung is the predominant site of disease, a sizeable population in Pakistan gets intestinal disease. Clinical presentation, radiologic and endoscopic examination provide clues to the diagnosis. However, a definitive diagnosis requires biopsy material with granulomas and/or caseation complemented by acid fast staining and culture. There are many occasions when biopsy material is scanty and even in some intestinal resection cases histologic evaluation fails to confirm or rule out tuberculosis. Therefore, an investigation was conducted to assess the efficacy of PCR in the detection of mycobacterial DNA in paraffin embedded intestinal tissue. In this study 12 histologically confirmed cases of intestinal tuberculosis and 2 cases with non specific inflammation but clinically suspected for abdominal tuberculosis were selected. One case of rectal polyp was included to serve as a negative control. M. tuberculosis DNA was amplified in 8 out of 12 histologically confirmed cases and in 2 cases diagnosed with non specific inflammation. Amplified products were obtained in 6 out of 10 PCR positive specimens with IS6110 region specific primers while 4 samples were negative, suggesting the absence of insertion sequence 6110 in these strains. However, amplification was obtained in these negative specimens with a second primer pair confirming them as M. tuberculosis complex species. On the basis of this study we conclude that; (1) Processed and paraffin embedded tissue material is suitable for PCR analysis, (2) PCR assay can be used to complement the diagnosis of intestinal tuberculosis especially in situations where a definite conclusion can not be drawn by conventional methods, (3) M. tuberculosis species lacking insertion sequence 6110 element are present in our population. Therefore, several primer pair sets should be included when applying PCR for the detection of mycobacterial DNA.