Simultaneous detection and strain differentiation of Mycobacterium tuberculosis complex in paraffin wax embedded tissues and in stained microscopic preparations.
OBJECTIVE
To detect and differentiate Mycobacterium tuberculosis simultaneously by polymerase chain reaction (PCR) in clinical samples prepared for histopathological analysis and for microscopic detection of acid fast bacteria.


METHODS
Paraffin wax embedded tissue samples and Ziehl-Neelsen (ZN) and auramine stained microscopic preparations from culture positive tuberculosis patients were subjected to DNA extraction and amplification by PCR. PCR was performed with primers specific for direct repeats and the product was detected by hybridisation to a set of 43 different oligonucleotides, a procedure designated as "spoligotyping".


RESULTS
Mycobacterium tuberculosis complex DNA was detected in all of the 23 paraffin wax embedded tissues analysed. Strain differentiation was possible in 20 of the 23 paraffin wax embedded tissues. Mycobacterium tuberculosis complex DNA was also detected and typed in eight of 10 ZN stained microscopic preparations. The hybridisation patterns obtained from virtually all of these samples were identical to those obtained from DNA extracted from cultures.


CONCLUSIONS
Simultaneous detection and strain differentiation of M. tuberculosis complex bacteria is possible in clinical samples prepared by current methods for microscopic and histopathological analysis, without the need to culture. The methodology described opens the way to rapid disclosure of outbreaks in high risk settings, such as hospitals and prisons, where dissemination of tuberculosis might be very fast as a result of a high prevalence of human immunodeficiency virus infected patients.
van der Zanden
A G
AG
Regional Institute for Public Health, Deventer, The Netherlands. baclab@pi.net
Hoentjen
A H
AH
Heilmann
F G
FG
Weltevreden
E F
EF
Schouls
L M
LM
van Embden
J D
JD
D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000302:isolation & purification; D006801:Humans; D008297:Male; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000302:isolation & purification*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
D004269:DNA, Bacterial