Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compare to AFB staining in pathological sections.

BACKGROUND: Polymerase chain reaction (PCR) is a recent, rapid and reliable method in the detection of causative organism. The authors tried to determine the possibility of using PCR technique as an alternative way to detect mycobacterial DNA from paraffin-embedded tissue to avoid repeated biopsy from the patient. MATERIAL AND METHOD: Paraffin-embedded tissue blocks, the corresponding histopathologic slides, and cultural results were retrospectively searched for according to the patient's records, the granuloma clinic, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand from 1994-2000. One hundred and thirty-one tissue blocks and slides were found but only 120 cultural results were retrieved Histologic sections were reviewed for AFB findings and PCR was done using 16S rRNA sequences to detect M. tuberculosis by one-tube nested technique and multiplex PCR for M. marinum and M. fortuitum complex. RESULTS: The causative organisms were identified by AFB staining in pathologic sections 31.29%, by PCR 35.87%, and by culture 30.00% of tested samples. The sensitivity of PCR when compared to AFB result was 29.26%, specificity 61.11% but when compared to cultural results, the sensitivity of PCR was 66.67% and AFB sensitivity was 41.66% with specificity 76.19% and 72.61% respectively. CONCLUSION: The low sensitivity of the PCR method may be due to formalin fixation, deparaffinization process, DNA extraction method, the use of 16S rRNA-based primers and the length of the expected product, and the tissue type that may have Taq polymerase inhibitor. Therefore, PCR should be used to augment the information of the conventional method in the diagnosis of mycobacterial infection.

Mycobacterial skin infections: comparison between histopathologic features and detection of acid fast bacilli in pathologic section.

BACKGROUND: Detection of acid fast bacilli (AFB) in chronic granulomatous inflammation is an important clue for mycobacterial infection. DESIGN: A retrospective review of 104 pathologic sections (from 1994 to 2001) of suspected cases of mycobacterial (tuberculous and nontuberculous) skin infections to study histopathologic features and the correlation with the presence of AFB in the section was performed. RESULTS: All cases showed granulomatous inflammations that can be categorized into 4 types: mixed cell, suppurative, tuberculoid and palisading granuloma. AFB was found in 32 sections (30.77%). Ninety five specimens from 104 specimens were simultaneously cultured. AFB positive cases yielded higher positive cultural results, 17 from 29 cases (58.62%) compared to the AFB negative group, 23 from 66 cases, (34.85%). Mixed cell granuloma was the most common histologic feature, but suppurative granuloma was the most common histological feature (56.25%) in which AFB could be found, which was statistically significantly different from other types of granuloma. Tuberculoid granuloma was more common in the AFB negative group (20.83%) compared to the AFB positive group (9.37%) but the difference was not statistically significant. In cases that AFB could not be found, the inflammation tended to be located in the upper half of the dermis. CONCLUSION: AFB can be more frequently detected in suppurative granuloma that might be located in any portion of the dermis. This finding was not species specific.

Nontuberculous mycobacterial skin infections: clinical and bacteriological studies.

OBJECTIVE: Nontuberculous mycobacterial (NTM) skin infections were analysed in terms of clinical manifestation in different species to provide clues for the clinical diagnosis and sensitivity patterns of these species were studied for planning appropriate therapy. DESIGN: A retrospective study was performed in 123 suspected cases of NTM infections from January 1994 to December 2000. NTM infection was documented by culture result of the infected tissue obtained by skin biopsy. Drug susceptibility test was done as requested. RESULT: Rapid growers (M. fortuitum-chelonae) were found in 26 cases (65%) and M. marinum was responsible for 12 cases (30%) and caused only localized skin lesions on arms or legs as indurated plaque, Disseminated skin infections manifested as multiple abscesses were found in 2 cases caused by M. avium in an HIV-infected male patient and mixed infection of M. szulgai and M. terrae in an immunocompetent female patient after a dental procedure. Both sexes were affected equally in overall number but male predominated in M. marinum infection and females predominated in rapid growers. All ages can be affected but most cases were middle aged. Scrofuloderma-like cervical lymphadenitis and cutaneous abscesses were the common manifestation of rapid grower infections. Hyperkeratotic verrucous plaques (tuberculosis verrucosa cutis-like) and sporotrichoid lesions were the common manifestations of M. marinum infection. M. marinum is sensitive to minocyclin, clarithromycin, amikacin, rifampicin and ethambutol and a good clinical response was obtained with doxycyclin 100 mg orally twice a day for 3 months. Clarithromycin and amikacin showed in vitro activity against the same strain of M. fortuitum but most strains of rapid growers resisted antituberculous drugs and also various antibiotics. CONCLUSION: Clinical manifestations can be used as clues for diagnosis. Medical therapy is recommended for M. marinum infection and surgical treatment is recommended for rapid growers.