POLYMERASE CHAIN-REACTION FOR THE DETECTION OF PSEUDOMONAS-AERUGINOSA, STENOTROPHOMONAS-MALTOPHILIA AND BURKHOLDERIA-CEPACIA IN SPUTUM OF PATIENTS WITH CYSTIC-FIBROSIS
F. Karpati et J. Jonasson, POLYMERASE CHAIN-REACTION FOR THE DETECTION OF PSEUDOMONAS-AERUGINOSA, STENOTROPHOMONAS-MALTOPHILIA AND BURKHOLDERIA-CEPACIA IN SPUTUM OF PATIENTS WITH CYSTIC-FIBROSIS, Molecular and cellular probes, 10(6), 1996, pp. 397-403
Citations number
33
Categorie Soggetti
Cell Biology",Biology,"Biochemical Research Methods
Occurrence of Pseudomonas aeruginosa, Stenotrophomonas (Xanthomonas) m
altophilia and Burkholderia (Pseudomonas) cepacia in sputum of cystic
fibrosis (CF) patients was demonstrated with a simple and rapid polyme
rase chain reaction (PCR) technique. The PCR was performed with a set
of three primer pairs based on 16S rRNA sequences after sputum prepara
tion with dithiothreitol and NaOH lysis. All three pathogens could be
individually detected by the use of this technique. To prevent carry-o
ver contamination, dUTP and uracil-N-glycosylase were included in the
reaction. The amplicons were visualized by agarose gel electrophoresis
. Sputum culture was performed on all samples. Ninety specimens from C
F patients were analysed. The sensitivity for the detection of P. aeru
ginosa was 37/40 (93%) compared to culture. Bacterial growth of P. aer
uginosa was found in three cases, where PCR amplicons were not detecte
d, while PCR was positive in five cases, where culture did not reveal
the presence of this bacterium. For this reason, the specificity was 4
5/50 (90%). For S. maltophilia, the PCR was less sensitive than cultur
e (positive in three of six cases). In our series, B. cepacia was dete
cted by culture in one case and this was also detected by PCR. There w
ere no false-positive PCR results regarding S. maltophilia or B. cepac
ia. Thus, combined PCR-based detection of these three clinically relev
ant bacteria in sputum samples from CF patients can be performed by a
reliable technique in a relatively simple manner. The present data ind
icate a high sensitivity and specificity for P. aeruginosa. The lower
sensitivity observed for the detection of S. maltophilia in sputum and
B. cepacia, as estimated from laboratory strains, may depend on PCR c
onditions and genetic heterogeneity, respectively. The greatest gains
with this method can be made when it is used for the early detection o
f P. aeruginosa in sputum-producing CF patients. (C) 1996 Academic Pre
ss Limited