title: Variable efficiency of three primer pairs for the diagnosis of Pneumocystis carinii pneumonia by the polymerase chain reaction.
abstract: The efficiency of three different primer pairs, complementary to different Pneumocystis carinii DNA regions, was compared in the polymerase chain reaction (PCR) for the diagnosis of Pneumocystis carinii pneumonia (PCP) on bronchoalveolar fluid (BALF) from patients with AIDS. PCR coupled with dot-blot hybridization (BLOT) using primers and probe from the mitochondrial 23SrDNA region showed the highest sensitivity, with a lower detection limit of 0.5-1 organisms microliter-1. When testing 47 BALF, PCR plus BLOT of the mitochondrial 23SrDNA region showed also the best diagnostic efficiency (97% sensitivity, 100% specificity). Sensitivity was significantly higher than with PCR and BLOT of the 5SrDNA region (81.5% sensitivity; P = 0.025, McNemar test); and of the dehydrofolate reductase (DHFR) gene region (75.6% sensitivity; P = 0.019). Sensitivity was also significantly higher than indirect immunofluorescence (75.8% sensitivity; P = 0.008). Using DHFR primers and probe, specificity was also reduced. The diagnostic sensitivity in clinical specimens paralleled the detection limit in the standard dilutions. The use of repeated DNA sequences of proven specificity as target of PCR amplification favourably influences sensitivity and specificity. This comparative study demonstrates that primer selection plays a significant role in the diagnosis of PCP by PCR.
authors.affiliation: Institute of Clinical Infectious Diseases, Catholic University, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: De Luca
authors.forename: E
authors.initials: E
authors.lastname: Tamburrini
authors.forename: E
authors.initials: E
authors.lastname: Ortona
authors.forename: P
authors.initials: P
authors.lastname: Mencarini
authors.forename: P
authors.initials: P
authors.lastname: Margutti
authors.forename: A
authors.initials: A
authors.lastname: Antinori
authors.forename: E
authors.initials: E
authors.lastname: Visconti
authors.forename: A
authors.initials: A
authors.lastname: Siracusano
mesh_terms: D017088:AIDS-Related Opportunistic Infections / Q000175:diagnosis*; D001483:Base Sequence; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011010:Pneumocystis / Q000235:genetics / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D015203:Reproducibility of Results; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D013762:Tetrahydrofolate Dehydrogenase