POLYMERASE CHAIN-REACTION FOR THE DETECTION OF PSEUDOMONAS-AERUGINOSA, STENOTROPHOMONAS-MALTOPHILIA AND BURKHOLDERIA-CEPACIA IN SPUTUM OF PATIENTS WITH CYSTIC-FIBROSIS

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