Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction.
A polymerase chain reaction (PCR)-based assay was developed for the detection of Pneumocystis carinii DNA in induced sputum and bronchoscopic alveolar lavage samples. The primer pair was selected from the published sequence of the thymidylate synthase gene of P. carinii derived from infected rats. The amplified DNA fragment of 403 bp was detected by agarose gel electrophoresis and by Southern and slot blot hybridization. No positive reaction was seen with DNA from different microorganisms typically found in the respiratory tract. P. carinii DNA was demonstrated in 30 of 42 sputum samples from immunosuppressed patients, whereas 21 of 42 sputum samples were positive by indirect immunofluorescence (IFL). Among the 42 patients, 14 were receiving prophylactic chemotherapy. In that group, PCR detected P. carinii in nine sputum samples, whereas IFL detected P. carinii in only four sputum samples. A positive PCR result was also seen in 5 of 43 IFL-negative bronchoscopic alveolar lavage samples from patients with respiratory symptoms. The PCR assay detected 10 copies of the target DNA, which corresponds to 10(-18) g of the specific P. carinii sequence. The results indicate that PCR amplification in combination with DNA hybridization is specific and is a more sensitive diagnostic method than IFL for the detection of P. carinii.
Olsson
M
M
Department of Parasitology, National Bacteriological Laboratory, Stockholm, Sweden.
Elvin
K
K
Löfdahl
S
S
Linder
E
E
D001483:Base Sequence; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D005455:Fluorescent Antibody Technique; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D011010:Pneumocystis / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013940:Thymidylate Synthase / Q000235:genetics
D004271:DNA, Fungal; D013940:Thymidylate Synthase