Comparison of Multiplex PCR and Acid fast and Auramine-Rhodamine staining for detection of Mycobacterium tuberculosis and nontuberculosis Mycobacteria in Paraffin- Embedded pleural and bronchial tissues with granulomatous inflammation and caseous necrosis

摘要

Aim: The aim of this study was to compare the sensitivity and specificity of Acid fast and Auramine-Rhodamine staining and Multiplex PCR for the detection of Mycobacterium Tuberculosis complex and nontuberculosis Mycobacteria on formalin fixed paraffin embedded tissues (FFPE) Material and Method: 40 cases of FFPE pleural and bronchial tissue with chronic granulomatous inflammation and caseous necrosis and 10 cases with bronchogenic carcinoma as controls were investigated. We designed a Muliplex PCR DNA amplification Method with two targets: 123bp DNA fragment from IS6110, which is present only in mycobactrum tuberculosis complex and 162bp DNA encoding Ag 85complex which is present in all of mycobacteria. The FFPE also stained by Acid fast and Rhodamine-Auramine staining method. Results: In 26 samples (%65) 123bp and 162bp DNA fragments were detected together .The 162bp fragment didn't detect alone. The sensitivity of PCR was %65 and the specificity was %100.11 cases were positive for Acid fast staining. There was %27/5 sensitivity and %100 specificity.13 cases were positive for Rhodamine- Auramine staining (R-A-S) there was %32/5 sensitivity and %100 specificity.All of the 10 controls were negative for 123bp and 162bp DNA fragments and for Acid fast and Rhodamine- Aauramine staining.Conclusion: Multiplex PCR is a sensitive, specific and rapid method for detection of mycobacterium tuberculosis in FFPE tissues. Introduction Despite longstanding efforts to conquer tuberculosis, this disease remains an expanding global health crisis with 1/86 billion infected people (1,2). Methods for the diagnosis of tuberculosis improved in recent years and several molecular techniques for its diagnosis have been introduced for clinical use. Molecular methods provide several advantages, including confirmation of the presence of M.tuberculosis within 1 to 3 day (2,3).The use of DNA amplification for detection of M. tuberculosis in formalin. Fixed paraffin embedded tissue samples would be useful for patient's in whom diagnosis depends on tissue examination, rather than detection of M. tuberculosis in body secretion. (1,4)The diagnosis of tuberculosis is largely based on conventional approaches, which rely on clinical features and the result of microscopy and culture. Culture methods are sensitive and specific but they are slow and take 2-6 wks (3,4). Unfortunately, there are frequent occasions when tissue obtained by biopsy is not sent for culture because the diagnosis was not a clinical consideration before the report of findings on microscopic examination of the tissues. (6,13)Acid fast and Rhodamine- Auramine staining are rapid and inexpensive methods for diagnosis tuberculosis but their sensitivity is low. The number of bacilli in tissue section stained with Acid- fast and Rhodamine- Auramine staining seems to be much lower than that expected from the sputum smear data or the patients condition. (3,5 ,7)Infection caused by nontuberculosis mycobacteria (NTM) are increasing in immunocompromised individuals. Effective therapeutic regiments are different for patients infected with M.tuberculosis or NTM.Therefore it is necessary to establish and evaluate PCR assay to differentiate between these two groups of mycobacteria.In order to establish sensitive Multiplex PCR, we selected two DNA targets : insertion sequences 6110 (IS6110) genes which present in multiple copies in the M.tuberculosis genome and the gene encoding Ag 85 complex.We also used Acid fast and Rhodamine- Auramine staining to evaluate the sensitivity of these methods and compare the results with Multiplex PCR. Material and Methods Forty formalin-fixed, paraffin-embedded (FFPE) specimens(20 pleural, 20 bronchial samples) with granulomatous inflammation and caseous necrosis were obtained from lung biopsy files at the Department of Pathology of Ghaem Hospital from January 2002 to June 2005. 2 pathologists confirmed that every section included granuloma with caseous necrosis. The patients