1. ¹Institute of Pathology, Technical University of Munich, Klinikum rechts der Isar, Munich, Germany
2. ²Department of Medicine, Division of Bone Marrow Transplantation, Stanford University Medical Center, Stanford, CA, USA
3. ³Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Klinikum rechts der Isar, Munich, Germany
4. ⁴Institute of Pathology, Krhs. d. Barmherzigen Schwestern, Austria
5. ⁵Institute of Pathology, Universitätsklinikum Freiburg, Freiburg, Germany

Correspondence: Dr med. S Schulz, MD, Department of Medicine, Division of Bone Marrow Transplantation, Stanford University Medical Center, 300 Pasteur Drive, H1353, MC:5623, Palo Alto, Stanford, CA 94305-5623, USA. E-mail: stschulz@stanford.edu

Received 3 September 2004; Revised 13 July 2004; Accepted 15 July 2004; Published online 8 October 2004.

Abstract

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium–intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.