Direct and Rapid Diagnosis of Extra-Pulmonary Tuberculosis in Archival Tissue by Nested and Touchdown PCR

Abstract

Aims: To identify and differentiate mycobacterium spp. in archival formalin-fixed, paraffin-embedded tissue by PCR to supply additional differential diagnostic method for Mycobacterium infections, where tuberculosis had been tested for by histopathological methods but without culturing.  Place and Duration of Study: Department of Medical Laboratory Sciences, Department of Pathology and Microbiology (Faculty of Medicine) and King Abdulla University Hospital, between January 2004 and July 2010. Methodology: Fifty-six extra-pulmonary specimens of formalin-fixed, paraffin-embedded tissue obtained from patients showing granulomatus inflammation and/or other histopathologic features. Specimens were analyzed by a hemi-nested PCR assay targeting the gene encoding for 16S ribosomal RNA, which is common to all mycobacteria spp. The PCR positive specimens were amplified by a touch-down PCR targeting a fragment in the insertion sequence IS6110 specific to Mycobacterium tuberculosis complex. Results were compared to acid fast bacilli stain and with histopathology of each specimen. Results: M. tuberculosis complex DNA was detected in 27 (48.2%) specimens, and non- tuberculous mycobacteria in four specimens, compared to 10 (18%) specimens that were positive by acid fast staining. The positive cases were observed more in the lymph nodes, and pleural specimens. Conclusions: This study is among few studies to use the touch-down PCR assay as a promising auxiliary tool for the diagnosis of extra-pulmonary tuberculosis in archival tissue specimens. It could be used in conjunction with routine laboratory tests (e.g., cultures, acid fast staining) and clinical criteria of the patient to increase the accurate diagnosis of such cases. It is also recommended that culture be routinely done for all tuberculosis suspected cases.