title: Detection of Pneumocystis carinii in induced sputa from immunocompromised patients using a repetitive DNA probe.
abstract: A hybridization assay for the detection of Pneumocystis carinii was developed using a repetitive DNA fragment of P.c. hominis. The assay was specific as different micro-organisms typically found in the respiratory tract, normal human lung DNA (A 549 cell line) and normal rat lung DNA did not react with the repetitive probe. In a slot blot (SB) hybridization assay, the repetitive probe was able to detect as few as 100 P.c. hominis organisms with no false-positives. The results of the SB hybridization assay were compared with an immunofluorescence (IFA) assay for the detection of P.c. hominis in 84 induced sputum (IS) samples obtained from 52 human immunodeficiency virus (HIV)-seropositive patients, 22 HIV-seronegative patients and 10 healthy individuals. Samples from 24 patients clinically diagnosed with P. carinii pneumonia (PCP) were positive for P.c. hominis by both assays. In addition, the SB assay detected P.c. hominis in 14 patients (10 HIV-positive and four HIV-negative) who were negative by IFA. All 14 samples showed a positive PCR signal for the P.c. hominis dihydrofolate reductase gene, further confirming the presence of P.c. hominis in these specimens. Twelve of these patients had a clinical course highly suggestive of PCP and were either on P. carinii prophylaxis or P. carinii chemotherapy. The other two samples were from HIV-positive patients who had respiratory illness due to causes other than P.c. hominis (disseminated histoplasmosis and fatal Bordetella pneumonia). Detection of P.c. hominis in these samples suggests that these patients may have subclinical colonization by P.c. hominis. Furthermore, P.c. hominis was detected in all 12 sequential IS samples from six AIDS patients who had primary episodes of PCR using the SB assay, while P.c. hominis was detected only in eight samples by IFA (66.6%). All six patients developed recurrent PCP within 6 months from the time the assays were performed, further illustrating the potential of the SB hybridization assay in monitoring PCP recurrence. Thus, the ability of the SB hybridization assay to detect a low parasite load suggests that this assay may become an important supplemental tool, along with current cytological methods, for detecting P.c. hominis in patient populations with lower burdens of the organism and in identifying asymptomatic carriers of the parasite in healthy and immunosuppressed individuals.
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Graves
authors.forename: S
authors.initials: S
authors.lastname: Chary-Reddy
authors.forename: M
authors.initials: M
authors.lastname: Becker-Hapak
mesh_terms: D017088:AIDS-Related Opportunistic Infections / Q000175:diagnosis*; D015139:Blotting, Southern; D015342:DNA Probes; D015200:Fluoroimmunoassay; D018023:HIV Seronegativity; D006679:HIV Seropositivity; D006801:Humans; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D011010:Pneumocystis / Q000235:genetics / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis*; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*
chemical_list: D015342:DNA Probes