DIRECT-DETECTION AND IDENTIFICATION OF PSEUDOMONAS-AERUGINOSA IN CLINICAL-SAMPLES SUCH AS SKIN BIOPSY SPECIMENS AND EXPECTORATIONS BY MULTIPLEX PCR BASED ON 2 OUTER-MEMBRANE LIPOPROTEIN GENES, OPRI AND OPRL

A multiplex PCR test based on the simultaneous amplification of two li poprotein genes, oprI and oprL, was designed and evaluated for its abi lity to directly detect fluorescent pseudomonads (amplification of opr I open reading frame, 249 bp) and Pseudomonas aeruginosa (amplificatio n of oprL open reading frame, 504 bp) in clinical material, A collecti on of reference strains including 20 different species of fluorescent pseudomonads was tested, Positive PCR results for both genes were obse rved only for P. aeruginosa isolates (n = 150), including strains of c linical and environmental origin, while only one gene, oprI, was ampli fied from the other fluorescent pseudomonads. All other bacteria teste d (n = 15) were negative by the amplification test, The lower detectio n level for P. aeruginosa was estimated to be 10(2) cells/ml. Prelimin ary evaluation on testing skin biopsy specimens from patients with bur ns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificit y in comparison with culture, This multiplex PCR assay appears promisi ng for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clini cal studies is warranted.