Multiplex polymerase chain reaction for the detection of mycobacterial DNAin cases of tuberculosis and sarcoidosis

The aims of the present study were: (1) to design a sensitive and specific polymerase chain reaction-based method that would allow detection of most c ommon human typical and atypical mycobacterial strains and (2) to apply the method on formalin-fixed paraffin-embedded (FFPE) tissue and sputum sample s from patients with clinicopathological evidence of tuberculosis and sarco idosis. Three sets of primers were selected. The first detects specifically members of the Mycobacterium tuberculosis (M. tuberculosis) complex, ampli fying a 243 Ipp fragment of the gene encoding the immunogenic protein MPB 6 4, whereas the second traces members of the Mycobacterium avium (M; avium) complex producing a 91 bp fragment of the IS1110 element. The third pair of primers is specific for slow-growing mycobacteria, amplifying a 383 bp reg ion of the 65 kDa mycobacterial antigen gene. Our multiplex polymerase chai n reaction assay identified mycobacterial DNA of 10(-3) colony-forming unit s (CFU)/mL from sputum samples, 10(-5) CFU/mL from FFPE tissue samples and 10(-6) CFU/mL from pure broth cultures. By performing the method on 75 FFPE tissue samples with histological and clinical evidence of tuberculosis and 300 sputum specimens from patients suspected of tuberculosis, we found 38 M. tuberculosis complex, 7 M. avium complex, and 14 slow-growing mycobacter ia positive samples in the first case and in the second we found 95 M. tube rculosis complex, 21 M. avium complex, and 35 slow-growing mycobacteria pos itive samples. The sensitivity of the assay was significantly higher than t hat of Ziehl-Neelsen and in some cases higher than culture, especially when applied on atypical mycobacteria. In addition, 25 cases histologically and clinically characterized as sarcoidosis were investigated for mycobacteria l DNA sequences and in nine of these, DNA corresponding to M. tuberculosis complex was detected. The method described can be applied directly on FFPE and sputum samples and allows not only the detection of mycobacterial DNA, but also an assessment concerning the species involved.