c.i.-fluorescent-brightening-agent-28 and Pneumonia--Pneumocystis

Pneumocystis carinii (P. carinii) remains a major pulmonary pathogen for the immunocompromised patient. In HIV infected patients, P. carinii represents the most commonly diagnosed cause of pneumonia. In the AIDS patient, empiric therapy based on clinical presentation has its proponents. However, this approach has been associated with a worse overall prognosis for the at risk patient. Because P. carinii can not be cultured, specific identification relies on examining respiratory specimens ranging from expectorated sputum to bronchoscopy with bronchoalveolar lavage (BAL). The low sensitivity of conventional stains has led to the search for antibodies to P. carinii and the use of immunofluorescent techniques. In addition, the polymerase chain reaction (PCR) is successfully being used in the diagnosis of P. carinii. Overall, these techniques allow the clinician to tailor the diagnostic testing for the individual patient.

Topics: Acquired Immunodeficiency Syndrome; Azure Stains; Benzenesulfonates; Biopsy; Bronchoalveolar Lavage Fluid; Bronchoscopy; CD4 Lymphocyte Count; Coloring Agents; Diagnostic Errors; Fluorescent Antibody Technique; Humans; L-Lactate Dehydrogenase; Methenamine; Pneumonia, Pneumocystis; Polymerase Chain Reaction; Radiography; Sputum; Tolonium Chloride

We compared the FXG™: RESP (Asp +) real-time PCR assay (Myconostica Ltd) with two microscopic staining methods (direct immunofluorescence [IFA] and calcofluor white) for the detection of Pneumocystis jirovecii in 411 respiratory specimens submitted for P. jirovecii examination. We considered the specimen to be microscopically positive if the organism could be visualized through the use of either IFA or calcofluor white. A second, published real-time PCR assay targeting the cdc2 gene of P. jirovecii was used to adjudicate those specimens that were microscopically negative but Myconostica PCR positive. The Myconostica PCR positive samples were deemed to be true positives if they were concordant with microscopically positive results or if they were positive by the second PCR assay. As a result, the Myconostica PCR assay was found to be more sensitive than the two microscopy methods in detecting P. jirovecii (10.5% true positivity rate by PCR, 8.0% by immunofluorescence, and 7.1% by calcofluor white). The Myconostica PCR assay showed 93.5% sensitivity, 95.1% specificity, 70.5% positive predictive value, and 99.1% negative predictive value. Its high negative predictive value suggests a role of the Myconostica PCR assay in ruling out Pneumocystis pneumonia.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Benzenesulfonates; Child; Child, Preschool; Female; Fluorescent Antibody Technique, Direct; Humans; Infant; Male; Microbiological Techniques; Microscopy; Middle Aged; Mycology; Pneumocystis carinii; Pneumonia, Pneumocystis; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Staining and Labeling; Young Adult

Pneumocystis jirovecii is an opportunistic pathogen that causes pneumonia, particularly in immunodeficient hosts.. We retrospectively compared the results obtained by two staining methods (toluidine blue and calcofluor white) and two quantitative (q) real time PCR assays for the detection of P. jirovecii in bronchoalveolar lavage (BAL) specimens. For the qPCR assays, we used newly selected probes and primers targeting the Kex-1 gene, which codes for a serine endoprotease, and compared the results to those from the published assay targeting the beta-tubulin gene.. A total of 1,843 BAL specimens were analyzed microscopically in parallel, and 74 (4.0%) were found to be positive with both stains, 23 (1.2%) were positive only with the toluidine blue stain, and six (0.3%) only with the calcofluor stain (p = 0.003). Of these, a selection of 186 consecutive BAL fluid samples were tested by qPCR using the respective different primer pairs. 21 of the 186 samples (11.3%) were microscopically positive with both stains as well as qPCR positive after 18-31 cycles (corresponding to 5.24 x 10(6) copies/ml to 640 copies/ml of native BAL) using the Kex-1 primer pair and between 21-33 cycles using the beta-tubulin assay. A good correlation between semi-quantitative microscopy and the number of PCR cycles needed for a positive signal was noted. Of the remaining 165 samples, 153 (82%) were both microscopically and PCR negative (PCR with the two sets of primers); the remaining 12 samples (7%) were Kex-1-based PCR positive (from cycles 33 to 41, corresponding to 160 copies/ml of BAL or less) but microscopically negative. Of these latter samples, ten (6%) were also positive (from cycles 34 to 38) with the primers targeting the beta-tubulin gene. Taking microscopy as a reference, the sensitivity of qPCR targeting the Kex-1 gene was 100%, and the specificity was 92.4%.. The sensitive qPCR analysis proved to be a rapid and reliable method to detect P. jirovecii in BAL.

Topics: Base Sequence; Benzenesulfonates; Bronchoalveolar Lavage Fluid; DNA, Fungal; Female; Humans; Male; Pneumocystis carinii; Pneumonia, Pneumocystis; Polymerase Chain Reaction; Retrospective Studies; Sensitivity and Specificity; Serine Endopeptidases; Staining and Labeling; Tolonium Chloride; Tubulin

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis carinii in clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage 1(BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

Topics: Aged; Benzenesulfonates; Bronchoalveolar Lavage Fluid; DNA, Mitochondrial; Female; Genes, rRNA; Humans; Male; Middle Aged; Pneumocystis; Pneumonia, Pneumocystis; Polymerase Chain Reaction; Reproducibility of Results; RNA, Fungal; RNA, Ribosomal, 28S; RNA, Ribosomal, 5S; Sensitivity and Specificity; Staining and Labeling

Topics: Benzenesulfonates; Biopsy; Fluorescent Dyes; Humans; Pneumocystis; Pneumonia, Pneumocystis

To compare the detection rate of Pneumocystis carinii in endotracheal aspirates with that rate in bronchoalveolar lavage fluid, using calcofluor-white (Fungi-Fluor) and immunofluorescence antibody (Genetic Systems) staining methods.. Prospective, consecutive cases.. Medical intensive care unit at Ben Taub General Hospital.. Thirty-one intubated patients admitted with respiratory failure and suspected P. carinii pneumonia.. An endotracheal aspirate specimen was obtained after maximally advancing a closed-system suction catheter, instilling aliquot portions of saline, and suctioning the lavage fluid. This procedure was followed within 30 mins by fiberoptic bronchoscopy and bronchoalveolar lavage.. Endotracheal aspirate and bronchoalveolar lavage specimens from each patient were mixed with Saccomano's fixative, blended, and centrifuged. Using a modified method for P. carinii cysts, the sediment was stained with the test calcofluor-white stain Solution A and the test antibody stain. The test antibody stain on the bronchoalveolar lavage specimens was positive for P. carinii for 13 patients and was used as the standard for comparison. In the endotracheal aspirate specimens, the test antibody stain detected 12 (92%) P. carinii-positive patients while the test calcofluor-white stain detected ten (77%) P. carinii-positive patients.. We described a simple method for obtaining, processing, and staining endotracheal aspirate specimens for P. carinii. Obtaining an endotracheal aspirate specimen did not require specially trained personnel or a specialized and more expensive catheter, and was not associated with any complications.

Topics: Benzenesulfonates; Contrast Media; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Intubation, Intratracheal; Pneumocystis; Pneumonia, Pneumocystis; Prospective Studies; Respiratory Insufficiency; Suction; Trachea

Calcofluor white (CFW), a chemofluorescent agent, has proven effective in the recognition of Pneumocystis carinii cysts in respiratory fluids and secretions. However, its usefulness in the recognition of P. carinii cysts in tissue preparations has not been established. We studied 68 formalin-fixed, paraffin-embedded, transbronchial tissue biopsy specimens from individuals seropositive for the human immunodeficiency virus and stained them with the CFW stain and the conventional Gomori methenamine silver (GMS) stain to determine the concordance rate of the two stains. CFW-positive specimens showed light peripheral staining and a unique double parenthesis-like structure near the center of the cysts. Thirty-six (52.9%) of the 68 specimens were CFW and GMS positive, whereas 27 (39.7%) of the specimens were negative by both techniques, yielding a concordance rate of 92.6%. Five (7.4%) of the 68 specimens showed disparate results, and, of these, four (5.9%) were CFW positive and GMS negative, whereas one (1.5%) was CFW negative and GMS positive. We conclude that the CFW stain is suitable and useful for the demonstration of P. carinii cysts in tissue preparations.

Topics: AIDS-Related Opportunistic Infections; Benzenesulfonates; Biopsy; Fluorescent Dyes; Humans; Lung; Paraffin Embedding; Pneumocystis; Pneumonia, Pneumocystis; Silver Staining; Tissue Fixation

Three monoclonal antibody staining kits, from Genetic Systems, Disease Detection International, and Meridian Diagnostics, were compared with calcofluor white for the direct detection of Pneumocystis carinii in respiratory specimens. Of the 150 specimens tested, 23 were found positive for P. carinii by any of the four stains; 13 were bronchoalveolar lavage, 7 were induced sputum and 3 were expectorated sputum specimens. All stains detected the positive bronchoalveolar lavage specimens, the Genetic Systems stain detected six induced sputum specimens, and the other stains each detected four induced sputum specimens. The monoclonal antibody stains detected all three expectorated specimens, while the calcofluor stain detected only one. Overall, the sensitivities of the stains were 96% for Genetic Systems, 87% for both Disease Detection International and Meridian Diagnostics, and 78% for calcofluor.

Topics: Antibodies, Fungal; Antibodies, Monoclonal; Benzenesulfonates; Bronchoalveolar Lavage Fluid; Evaluation Studies as Topic; Humans; Pneumocystis; Pneumonia, Pneumocystis; Staining and Labeling