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1.  Rapid detection of Pneumocystis carinii using a direct fluorescent monoclonal antibody stain. 
Journal of Clinical Microbiology  1990;28(10):2228-2233.
A collaborative study was undertaken at two institutions to assess the performance of a direct fluorescent-antibody stain for the detection of Pneumocystis carinii in respiratory specimens from patients with known or suspected human immunodeficiency virus type 1 infections. A total of 163 specimens (125 induced sputa, 37 bronchoalveolar lavage fluids, and 1 tracheal aspirate) from 124 patients were examined by using modified Giemsa (Diff-Quik; Baxter American Scientific Products, Chicago, Ill.) and direct fluorescent-antibody stains. A total of 73 specimens contained P. carinii, which was detected in 66 (92%) of the specimens by using the modified Giemsa and in 71 (97%) of the specimens by using the fluorescent-antibody stain. One bronchoalveolar lavage fluid specimen in which P. carinii was detected only with the fluorescent-antibody stain was determined to be a false-positive based on subsequent clinical evaluation of the patient. Although the overall time for processing and examining specimens stained with either stain was not significantly different for those specimens containing P. carinii, considerably less time was required for microscopic examination of those fluorescent-antibody-stained specimens lacking P. carinii.
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PMCID: PMC268153  PMID: 1699968
2.  Quantitative and qualitative comparison of DNA amplification by PCR with immunofluorescence staining for diagnosis of Pneumocystis carinii pneumonia. 
Journal of Clinical Pathology  1993;46(2):140-144.
AIM: To compare the results of DNA amplification by the polymerase chain reaction (PCR) with immunofluorescence staining for detecting Pneumocystis carinii in bronchoalveolar lavage specimens taken from symptomatic HIV seropositive patients with suspected P carinii pneumonia (PCP). METHODS: Bronchoalveolar lavage specimens were obtained from 28 symptomatic HIV seropositive patients. Specimens were examined for P carinii using immunofluorescence, and by DNA amplification with PCR to obtain results on gel electrophoresis (gel) and a more sensitive Southern hybridisation (blot) technique. Specimens positive by immunofluorescence and gel electrophoresis were serially diluted to a 10(-6) concentration and each dilution strength tested for P carinii using PCR to compare quantitatively immunofluorescence with PCR. RESULTS: Of the 28 specimens analysed, 18 were negative for P carinii by both immunofluorescence and PCR, two were positive only by the blot technique of PCR, four were equivocally positive and four unequivocally positive by immunofluorescence. Three of the four equivocally positive patients tested by immunofluorescence were negative for P carinii by PCR, although one was positive by PCR (blot) technique. This patient had clinically confirmed PCP. Of the four unequivocally positive patients tested by immunofluorescence, three were gel and blot positive by PCR and had PCP clinically, but one was negative by both gel and blot techniques, although the patient certainly had PCP on clinical grounds. This patient had received nine days of treatment with high dose co-trimoxazole before bronchoalveolar lavage specimens were obtained. The three specimens positive by gel and blot techniques remained gel positive down to dilutions of between 10(-4) and 10(-6). CONCLUSIONS: PCR results may become negative soon after starting treatment for PCP. Specimens should therefore be taken before, or soon after, starting treatment. PCR seems to be between 10(4) and 10(6) times more sensitive than immunofluorescence.
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PMCID: PMC501145  PMID: 8459034
3.  Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups. 
Journal of Clinical Microbiology  1997;35(6):1445-1449.
Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.
PMCID: PMC229764  PMID: 9163459
4.  The role of a nested polymerase chain reaction in the diagnosis of Pneumocystis carinii pneumonia 
Clinical Molecular Pathology  1995;48(6):M347-M350.
Aim—To compare the techniques and results of a nested PCR and an immunofluorescence assay (IFA) for the detection of Pneumocystis carinii infection; to consider the role of the nested PCR in the diagnosis of P carinii pneumonia (PCP).
Methods—Serial dilutions of two known P carinii positive samples were tested by IFA and PCR to determine their relative sensitivities. Seventy eight respiratory samples (15 from 11 patients with HIV infection/acquired immunodeficiency syndrome (AIDS) and 63 from 42 patients with other forms of immunodeficiency) were tested using both assays, and the costs and technical requirements of each assay were assessed.
Results—The PCR had a greater relative sensitivity over the IFA of 2 × 101 to 2 × 103 fold in a postmortem lung sample and 2 × 105 to 2 × 106 fold in a bronchoalveolar lavage sample from a patient with PCP. P carinii was detected in all 15 samples from the patients with HIV/AIDS by both IFA and PCR. Of the 63 samples from the patients with immunodeficiencies other than HIV/AIDS, the PCR was more sensitive than IFA.
Conclusions—The nested PCR is a more sensitive assay than the IFA. It may be useful in the diagnosis of PCP in patients with immunodeficiencies other than HIV/AIDS. Similarly, PCR may be of benefit for this patient group as less invasive specimens are needed. PCR has an increasing role to play in the diagnosis of PCP in the routine laboratory.
PMCID: PMC408003  PMID: 16696036
Pneumocystis carinii; PCR; immunofluorescence assay; acquired immunodeficiency syndrome
5.  Pneumocystis carinii pneumonia: detection of parasites in sputum and bronchoalveolar lavage fluid by monoclonal antibodies. 
BMJ : British Medical Journal  1988;297(6645):381-384.
Diagnosis of pneumocystis pneumonia is based on identifying Pneumocystis carinii cytochemically in material from the lung. The silver methenamine staining methods most commonly used are technically difficult and lack specificity. The diagnostic value of immunocytological identification of the parasite was evaluated by using mouse monoclonal antibody 3F6, specific for human pneumocystis, to identify P carinii in bronchoalveolar lavage fluid and sputum by immunofluorescence and was compared with that of other variables. Bronchoalveolar lavage was performed on 25 patients positive for HIV antibody with clinically suspected pneumocystis pneumonia and 40 patients negative for HIV antibody who presented with interstitial disorders of the lung. Lavage fluid showed pneumocystis only in the patients positive for antibody, the parasite being detected in 19 by immunofluorescence and in 17 by a modified silver methenamine staining method. Chest x ray films obtained at the time of bronchoscopy showed interstitial or alveolar shadowing in 17 of the 19 patients, but clinical symptoms and the presence of antibodies to pneumocystis did not seem to be predictive. Sputum samples were collected during 43 episodes of clinically suspected pneumocystis pneumonia in patients positive for HIV antibody. Pneumocystis was detected consistently more commonly by immunofluorescence than the silver strain in sputum collected routinely and induced by inhalation of saline. In 17 patients bronchoalveolar lavage followed sputum collection, and the sensitivity of detection of pneumocystis in immunofluorescence in sputum compared with lavage fluid was 57% (8/14). Immunofluorescence was suitable for specimens fixed in ethanol and seemed highly specific and more sensitive than the standard cytochemical methods for identifying pneumocystis.
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PMCID: PMC1834294  PMID: 3044514
6.  Granulomatous Pneumocystis carinii pneumonia: DNA amplification studies on bronchoscopic alveolar lavage samples. 
Journal of Clinical Pathology  1994;47(7):664-666.
Three HIV positive subjects presented with symptoms and radiographic changes suggestive of Pneumocystis carinii pneumonia. Methenamine silver staining of bronchoscopic alveolar lavage (BAL) fluid was negative (from one sample in one patient and two samples in the other two patients). Open lung biopsy was performed because of uncertain clinical progress and diagnosis; all three patients were found to have multiple pulmonary granulomata encasing numerous P carinii organisms. DNA amplification, using P carinii specific oligonucleotides, was performed on stored bronchoscopic BAL samples. P carinii specific amplification product was detected by ethidium bromide staining after electrophoretic separation on agarose gel in one case, and by the more sensitive technique of oligohybridisation in all three cases. In granulomatous P carinii pneumonia organisms are rarely identified in bronchoscopic alveolar lavage samples using histochemical staining, but are detectable by DNA amplification, although not at levels which can be readily distinguished from low, subclinical infection.
PMCID: PMC502116  PMID: 8089227
7.  Comparison of PCR and standard cytological staining for detection of Pneumocystis carinii from respiratory specimens from patients with or at high risk for infection by human immunodeficiency virus. 
Journal of Clinical Microbiology  1995;33(11):3004-3007.
The detection of Pneumocystis carinii DNA by PCR was compared with routine cytologic staining techniques (CYT). A total of 284 clinical respiratory specimens, including 137 bronchoalveolar lavage (BAL), 63 bronchoalveolar washing, 63 sputum, and 21 induced sputum samples, obtained from patients with or at high risk for human immunodeficiency virus infection were evaluated. Eighty specimens were positive by PCR, and 69 were positive by CYT. PCR was able to detect P. carinii in more bronchoalveolar washing specimens (15 versus 11) and in comparable BAL specimens (53 versus 54) compared with CYT. PCR was particularly more sensitive than CYT in detecting P. carinii in expectorated sputum (12 versus 4 samples). Of the 19 patients whose respiratory specimens were positive for P. carinii by PCR but negative by CYT, 5 had P. carinii pneumonia (PCP) confirmed by subsequent BAL and transbronchial or mediastinal lymph node biopsy and 9 had a clinical course highly suggestive of acute PCP. Eleven (58%) of the 19 patients with discordant PCR and CYT results had received prior anti-PCP prophylaxis. In this clinical setting in particular and in the evaluation of sputum specimens, the ability of PCR to detect a low parasitic load suggests that this technique may become an important additional tool, along with current cytological methods, for the detection of P. carinii.
PMCID: PMC228623  PMID: 8576362
8.  Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. 
Journal of Clinical Microbiology  1987;25(10):1837-1840.
A combination of three monoclonal antibodies, two prepared against human and one against rat Pneumocystis carinii, was used in an indirect fluorescent-antibody stain (IFA) to diagnose P. carinii in both bronchoalveolar lavage and lung biopsy specimens. This combination of monoclonal antibodies was specific for P. carinii and yielded bright fluorescence of both P. carinii cysts and trophozoites. A total of 126 specimens from 93 patients were stained for P. carinii by a toluidine blue O stain and the monoclonal IFA. Forty-five (35.7%) of these were positive for P. carinii by toluidine blue O, and 43 (34.1%) were positive by IFA. There was 98.4% agreement between both methods, with no false-positive and two false-negative occurrences by IFA. An IFA procedure with monoclonal antibodies such as those used in these studies can provide a simple, fast, and sensitive method for diagnosing P. carinii pneumonia by microbiology laboratories.
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PMCID: PMC269351  PMID: 3312285
9.  Evaluation of Diagnostic Value and Epidemiological Implications of PCR for Pneumocystis carinii in Different Immunosuppressed and Immunocompetent Patient Groups 
Journal of Clinical Microbiology  2000;38(4):1461-1467.
To evaluate the value of single and nested PCRs for diagnosis of Pneumocystis carinii pneumonia (PCP) in a variety of respiratorily distressed patient groups, 574 respiratory samples from 334 patients (89 human immunodeficiency virus [HIV]-positive patients, 61 transplant recipients, 66 malignancy patients, 34 otherwise immunosuppressed patients, and 84 immunocompetent patients) were prospectively examined by microscopy and single and nested PCRs. The resulting data were correlated with clinical evidence of PCP. Microscopy and single PCR of bronchoalveolar lavage (BAL) specimens from HIV patients were 100% sensitive and specific in detecting PCP, whereas nested PCR, although being 100% sensitive, reached a specificity of only 97.5%. In the three non-HIV immunosuppressed patient groups, both single and nested PCR invariably produced lower positive predictive values than microscopy. Among immunocompetent patients, the positive predictive values of both PCRs were 0%. Therefore, the diagnostic values of the PCR methods tested do not seem to offer any additional advantage compared to that of conventional microscopy for these patient groups. However, nested PCR identified a significant percentage of clinically silent P. carinii colonizations in about 17 to 20% of immunocompetent and immunosuppressed non-HIV patients.
PMCID: PMC86465  PMID: 10747126
10.  Application of immunoblotting to detect soluble Pneumocystis carinii antigen(s) in bronchoalveolar lavage of patients with Pneumocystis pneumonia and AIDS. 
Journal of Clinical Pathology  1990;43(7):584-586.
The technique of immunoblotting for detecting soluble Pneumocystis carinii antigen(s) in bronchoalveolar lavage fluid specimens from patients with AIDS and Pneumocystis pneumonia was evaluated. A soluble 67 kilodalton polypeptide that was immunoreactive with an anti-P carinii monoclonal antibody (2G2) was found in the supernatants of 26 lavage samples from patients with pneumocystosis. Intact organisms in lavage sediments were detected by methenamine silver or immunofluorescence staining procedures. The diagnostic use of this technique was shown in four cases in which lavage sediments proved negative for intact Pneumocystis carinii organisms on first examination; 2G2 reactive soluble antigen, however, was identified in the immunoblots of the supernatants from the same samples. It is concluded that immunoblotting of bronchoalveolar lavage specimens using 2G2 monoclonal antibody as a detection probe may be a useful adjunct to the morphological demonstration of organisms by special staining procedures.
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PMCID: PMC502586  PMID: 2380406
11.  EB9, a new antibody for the detection of trophozoites of Pneumocystis carinii in bronchoalveolar lavage specimens in AIDS. 
Journal of Clinical Pathology  1994;47(12):1108-1111.
AIM--To prepare a monoclonal antibody (EB9) against the trophozoite form of Pneumocystis carinii and to test its efficacy for detecting infection with this organism. METHOD--The sensitivity and specificity of the EB9 antibody were assessed by comparing it with other conventional stains (Papanicolaou, Giemsa and Grocott) and 3F6 antibody in 33 bronchoalveolar lavage specimens from HIV positive patients suspected of having P carinii pneumonia. RESULTS--P carinii infection was detected in 15 of 33 patients. In 14 cases the organism was detected by two or more of the staining methods used; however, EB9 failed to detect infection in two cases which were positive by other staining techniques. In one case P carinii infection was detected by EB9 only. CONCLUSION--The results of this study suggest that P carinii infection in the lung may occur in two forms: the cyst form and the trophozoite form, which may explain the observed variation in response to treatment.
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PMCID: PMC502204  PMID: 7876385
12.  Study of internal transcribed spacer and mitochondrial large-subunit genes of Pneumocystis carinii hominis isolated by repeated bronchoalveolar lavage from human immunodeficiency virus-infected patients during one or several episodes of pneumonia. 
Journal of Clinical Microbiology  1997;35(7):1687-1690.
The objective of this study was to type, analyze, and compare Pneumocystis carinii hominis strains obtained from different samples during a given or recurrent episodes of P. carinii pneumonia (PCP) for epidemiologic purposes. We studied 36 bronchoalveolar lavage (BAL) or induced sputum (IS) samples from 16 human immunodeficiency virus-infected patients with one or several episodes of PCP. PCR amplification and direct sequencing were performed on the two internal transcribed spacers (ITS1 and ITS2) of P. carinii hominis rRNA genes by using DNA extracted from BAL or IS samples, and the sequences were compared to the mitochondrial large-subunit (mt LSU) gene sequence determined in a previous study in our laboratory. The studies of the mt LSU and ITS sequences showed that some patients (n = 10) were infected with the same strains of P. carinii hominis during a given episode of PCP. In one patient infected with strains with identical sequences in several episodes, the recurrence could have been due to reactivation of organisms not eliminated by treatment during the first episode or to de novo infection by an identical strain. In five patients infected with strains with different sequences in each episode, recurrence was due to de novo infection. Sequence analysis of these two P. carinii hominis gene regions showed that de novo infection can occur in AIDS patients with recurrent PCP.
PMCID: PMC229822  PMID: 9196174
13.  Simplified exercise test for the initial differential diagnosis of Pneumocystis carinii pneumonia in HIV antibody positive patients. 
Thorax  1994;49(2):112-114.
BACKGROUND--This study was designed to evaluate the usefulness of a simplified exercise test in the differential diagnosis of Pneumocystis carinii pneumonia (PCP). METHODS--Forty five subjects with antibodies against the human immunodeficiency virus (HIV) and pneumonia were included and divided into two groups: those with PCP and those with "other pneumonias" (non-PCP). The test involved pedalling for two minutes on a stretcher bed and was considered positive if SaO2 decreased by at least 3%. RESULTS--During the exercise the mean(SE) SaO2 fell in patients with PCP from 88(4)% to 84(3)%, p < 0.01, whilst it improved slightly in subjects with non-PCP from 91(1)% to 93(3)%, p < 0.05. Sensitivity was 77% and specificity 91%. CONCLUSIONS--This simple test seems potentially useful for the initial investigation of HIV antibody positive patients with pneumonia.
PMCID: PMC474319  PMID: 8128398
14.  Bronchoalveolar lavage cell analysis in patients with human immunodeficiency virus related diseases. 
Thorax  1989;44(4):289-291.
The value of differential cell counts in bronchoalveolar lavage fluid in patients who were serologically positive for the human immunodeficiency virus (HIV) was studied in 30 patients with classified into four groups according to the severity of illness: (1) seven subjects with the AIDS related complex without clinical or radiological evidence of pulmonary infection; (2) eight patients with the AIDS related complex and pulmonary tuberculosis; (3) eight patients with AIDS and Pneumocystis carinii pneumonia; and (4) seven patients with AIDS, Pneumocystis carinii pneumonia, and severe respiratory failure. All four groups had a similar percentage of lymphocytes, significantly higher than that of a control group of 15 healthy volunteers. A significant increase in the percentage of neutrophils was observed in groups 2, 3, and 4. The lavage fluid differential cell count does not therefore appear to help in the differential diagnosis of pulmonary infections in HIV positive patients. The abnormal percentage of lymphocytes observed in some patients with the AIDS related complex without clinical evidence of pulmonary infection suggests that lung injury may exist before clinical or radiological abnormalities develop. This might be related to an immunological mechanism or might be caused by an undetected subclinical infection.
PMCID: PMC461795  PMID: 2788319
15.  Pneumocystis carinii antigenemia in acquired immunodeficiency syndrome. 
Journal of Clinical Microbiology  1988;26(7):1357-1361.
The present study was conducted to determine the prevalence and significance of Pneumocystis carinii antigenemia in patients with acquired immunodeficiency syndrome (AIDS) and clinically or invasively diagnosed P. carinii pneumonitis. Single serum specimens from 20 AIDS patients invasively examined for P. carinii organisms and 106 AIDS patients with a clinical diagnosis only of P. carinii pneumonitis were blindly tested for P. carinii antigenemia by a counterimmunoelectrophoresis assay. In the 20 specimen-documented cases, the antigen test demonstrated a sensitivity of 75% and a specificity of 90%. The positive predictive value of the test was 90%, while the negative predictive value was 70%. In AIDS patients with specimen-documented P. carinii pneumonitis, the prevalence of P. carinii antigenemia coincided almost exactly with the prevalence of positive invasively obtained specimens (60 and 59%, respectively). In patients with a clinical diagnosis only of P. carinii pneumonitis, half as many (30%) were found to exhibit antigenemia. Sequential P. carinii antigen titers determined by a new latex agglutination technique on three AIDS patients with specimen-documented P. carinii pneumonitis demonstrated the influence of specific therapy upon P. carinii antigenemia and its potential prognostic application.
PMCID: PMC266609  PMID: 3261737
16.  Evidence for depressed humoral immunity to Pneumocystis carinii in homosexual males, commercial plasma donors, and patients with acquired immunodeficiency syndrome. 
Journal of Clinical Microbiology  1987;25(6):991-995.
Heterosexual controls were found to have significantly higher titers of immunoglobulin G antibody to Pneumocystis carinii than did patients with the acquired immunodeficiency syndrome (AIDS) and P. carinii pneumonitis, human immunodeficiency virus (HIV) antibody-positive or -negative homosexual male "gay bar" patrons, and HIV antibody-positive or -negative commercial plasma donors. The T-helper/T-suppressor lymphocyte ratios of HIV antibody-negative homosexual male gay bar patrons were slightly depressed (mean = 1.31 +/- 0.54) compared with those of heterosexual controls (mean = 1.79 +/- 0.32). In addition to other recognized factors, preexisting humoral as well as cell-mediated immune deficits before infection with HIV may help to explain the prevalence of and morbidity and mortality associated with P. carinii pneumonitis in AIDS patients.
PMCID: PMC269122  PMID: 2954997
17.  Identification of Pneumocystis carinii f. sp. hominis Gene Sequences in Filtered Air in Hospital Environments 
Journal of Clinical Microbiology  1998;36(6):1737-1740.
To evaluate the risk of a nosocomial spread of Pneumocystis carinii f. sp. hominis (P. carinii hominis), air filter samples from rooms of P. carinii pneumonia (PCP) patients, adjacent corridors, and other hospital environments have been investigated for the presence of P. carinii hominis. Amplified DNA from air filters and sputum or bronchoalveolar lavage samples from the PCP patients have been genotyped with the P. carinii hominis genes of the mitochondrial large-subunit (mtLSU) rRNA and the internal transcribed spacers (ITS1 and ITS2) of the rRNA. Genotypes of the two loci were identified by direct sequencing, and for site 85 of the mtLSU locus, three allele-specific PCR assays were used. P. carinii hominis DNA was identified in the air of five of seven PCP patient rooms and in the air of two of four air filtrations from the ward corridors. The P. carinii hominis genotypes were the same in four of the five room air samples as those in the corresponding patients, suggesting a risk of person-to-person transmission of P. carinii hominis from PCP patients. Three of 16 air samples collected in infectious disease wards without the presence of PCP patients and one sample from a cardiology unit in a separate hospital building were also positive, which further strengthens the possibility of acquisition of P. carinii hominis from the environment.
PMCID: PMC104910  PMID: 9620410
18.  Transmission of Pneumocystis carinii from patients to hospital staff 
Thorax  1997;52(5):422-424.
BACKGROUND: An extrahuman reservoir of human pathogenic Pneumocystis carinii remains unknown. Host to host transmission has been described in animal studies and in cluster cases among immunodeficient patients. P carinii DNA has recently been detected in air filters from inpatient and outpatient rooms in departments of infectious diseases managing patients with P carinii pneumonia (PCP), suggesting the airborne route of transmission. Exposure of staff to P carinii may occur in hospital departments treating patients with PCP. METHODS: Exposure to P carinii was detected by serological responses to human P carinii by ELISA, Western blotting, and indirect immunofluorescence in 64 hospital staff with and 79 staff without exposure to patients with PCP from Denmark and Sweden. DNA amplification of oropharyngeal washings was performed on 20 Danish staff with and 20 staff without exposure to patients with PCP. RESULTS: There was no significant difference in the frequency or level of antibodies to P carinii between staff exposed and those unexposed to patients with PCP. None of the hospital staff had detectable P carinii DNA in oropharyngeal washings. CONCLUSIONS: There is no difference in antibodies and no detectable P carinii DNA in oropharyngeal washings, which suggests that immunocompetent staff treating patients with PCP are not a potentially infectious source of P carinii for immunocompromised patients. 



PMCID: PMC1758557  PMID: 9176532
19.  Blinded comparison of a direct immunofluorescent monoclonal antibody staining method and a Giemsa staining method for identification of Pneumocystis carinii in induced sputum and bronchoalveolar lavage specimens of patients infected with human immunodeficiency virus. 
Journal of Clinical Microbiology  1990;28(9):2136-2138.
A new direct immunofluorescence monoclonal antibody (DFA) method (Genetic Systems, Inc., Seattle, Wash.) for identification of Pneumocystis carinii in induced sputum and bronchoalveolar lavage specimens was compared in a blinded study with an established Giemsa stain method. We evaluated 148 consecutive clinical specimens from 104 patients with the following results. For the 67 patients (64%) infected with the human immunodeficiency virus (HIV), 49 were initially negative by both the DFA and the Giemsa methods, none were negative by DFA and positive by Giemsa, 6 were positive by DFA and negative by Giemsa, and 12 were positive by both methods, for a sensitivity and a negative predictive value of greater than 99%. For the six patients positive by DFA and negative by Giemsa, all were positive by both methods on evaluation of subsequently obtained clinical specimens, suggesting a specificity of greater than 99% and a false-positive rate of less than 1%. For 37 patients whose HIV status was negative or unknown, 35 were negative by both methods and 2 were positive by DFA and negative by Giemsa. The DFA method was simple to perform and required less time for scoring of stained slides than the Giemsa method, but care had to be taken to avoid false-positive readings due to extraneous fluorescence. This study indicates that the DFA method represents an advance as a sensitive, simple, and rapid way to identify P. carinii in induced sputum and bronchoalveolar lavage specimens from HIV-infected patients and suggests greater sensitivity of the DFA than the Giemsa method in this patient population.
PMCID: PMC268123  PMID: 1699970
20.  Localization of host immunoglobulin G to the surface of Pneumocystis carinii. 
Infection and Immunity  1990;58(2):456-463.
In immunoblotting studies of Pneumocystis carinii surface proteins, we found that a secondary antibody, anti-human immunoglobulin G (IgG), recognized a 52-kilodalton (kDa) band in homogenates of P. carinii purified from human autopsy lungs and bronchoalveolar lavage fluids, even when serum as a source of primary antibody was omitted. The electrophoretic mobility of the 52-kDa band is identical to that of IgG heavy chains. In addition to affinity-purified, anti-human IgG, monoclonal antibodies specific for the Fab and Fc regions of human IgG recognized the 52-kDa band. To determine whether the 52-kDa band represents IgG bound to the surface of P. carinii, we treated intact organisms with Triton X-100 and acid in order to elute immunoglobulin from the surface of P. carinii. After purification over a protein G column, the eluate comigrated with human IgG, was recognized by anti-IgG, and bound to discrete bands with molecular sizes of 65 to 70, 60, 50, and 35 kDa in purified, rat-derived P. carinii. To confirm the presence of human IgG on the surface of P. carinii, we performed immunocytochemical and immunoelectron microscopic studies. Staining of intact P. carinii aggregates by anti-human IgG was pronounced and was abolished by acid treatment. IgA was also present. Ultrastructural studies showed the presence of IgG on the cyst wall and on fine membranous structures and vesicles adjacent to cysts. We conclude that the surface of P. carinii is coated with human IgG. The close association of human IgG with P. carinii may have implications for the pathogenesis of P. carinii pneumonia in acquired immunodeficiency syndrome.
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PMCID: PMC258479  PMID: 2298486
21.  Inhibition of Pneumocystis carinii dihydropteroate synthetase by para-acetamidobenzoic acid: possible mechanism of action of isoprinosine in human immunodeficiency virus infection. 
Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown. para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis. This pathway is known to be important for P. carinii because sulfonamides, which are effective anti-P. carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS). Inhibition of P. carinii DHPS by PAcBA was investigated by using two assays. In short-term cultures of P. carinii from rats, [3H]PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration [IC50], 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM). The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM). Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM). PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae. In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug. Prevention of PCP by PaCBA through inhibition of P. carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients.
PMCID: PMC187944  PMID: 7687120
22.  Interleukin-6 production in a murine model of Pneumocystis carinii pneumonia: relation to resistance and inflammatory response. 
Infection and Immunity  1993;61(1):97-102.
The production of interleukin-6 (IL-6) and its possible relationship to host resistance and inflammatory response to Pneumocystis carinii infection were examined in mice with severe combined immunodeficiency (SCID mice). IL-6 activity was detected in the serum and lungs of P. carinii-infected mice but not in mice free of P. carinii. Moreover, the IL-6 levels in P. carinii-infected mice increased markedly after spleen cell reconstitution but then decreased to an undetectable level after the clearance of P. carinii. However, neutralization of IL-6 activity in spleen cell-reconstituted SCID mice by treatment with anti-IL-6 immunoglobulin G (IgG) resulted in no significant effect on the clearance of P. carinii (P > 0.05). Both the serum and lungs of treated mice contained an excess amount of anti-IL-6 IgG and lacked detectable IL-6. These results suggest that failure to inhibit the P. carinii clearance by anti-IL-6 treatment was not due to insufficient administration of antibody or incomplete neutralization of IL-6 activity. However, compared with mice receiving rat control IgG, mice treated with anti-IL-6 IgG had significantly higher numbers of neutrophils and lymphocytes (particularly CD8+ cells) in the lung lavage fluids (P < 0.05 for both) at day 19 after reconstitution. In addition, the levels of both total IgG (P < 0.001) and P. carinii-specific antibodies (P < 0.05) in the serum of mice treated with anti-IL-6 were significantly higher than those in control mice. These results indicate that although P. carinii infection causes both local and systemic production of IL-6 in SCID mice, IL-6 does not appear to play a crucial role in the clearance of P. carinii. However, it appears that during resolution of P. carinii pneumonia, IL-6 plays a role in the regulation of pulmonary inflammation and antibody responses.
PMCID: PMC302692  PMID: 8418070
23.  Cellular and humoral immune responses of mice subclinically infected with Pneumocystis carinii. 
Infection and Immunity  1985;47(2):544-548.
Cellular and humoral immune responses to Pneumocystis carinii were investigated. ICR and DDD mice were intranasally infected with 10(4) mouse lung-derived P. carinii, and the delayed-type hypersensitivity (DTH) reaction and antibody titers to P. carinii were measured along with the number of P. carinii cysts in the lungs after infection. The number of P. carinii cysts in the lungs peaked at 2 weeks after infection and then decreased to barely detectable levels by 4 weeks. Serum antibody (immunoglobulin G) titers measured by indirect immunofluorescence increased up to 4 weeks. The DTH footpad reaction was most prominent at 2 weeks postinfection and declined thereafter. Thus, the decline in the number of P. carinii cysts in the lungs corresponded well with the time of the peak of the DTH reaction but not with the serum antibody response. Spleen T cells from infected mice mediated the DTH reaction when transferred intravenously into normal recipients and reduced the number of P. carinii cysts in the lungs when transferred intravenously into P. carinii-infected mice. The results indicated that cellular immunity is important for protection from subclinical P. carinii infections.
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PMCID: PMC263206  PMID: 3871426
24.  Diagnosis of Pneumocystis carinii pneumonia in HIV antibody positive patients by simple outpatient assessments. 
Thorax  1992;47(12):1005-1009.
BACKGROUND: As increasing numbers of patients with immunosuppression induced by the human immunodeficiency virus (HIV) present with respiratory symptoms it is important to differentiate Pneumocystis carinii pneumonia from other chest diseases rapidly and start treatment early. The management of pneumocystis pneumonia could be improved if clinicians could diagnose this condition confidently on the basis of simple clinical assessments. METHODS: Three hundred and eighteen patients with evidence of immunosuppression due to HIV infection and suspected pneumocystis pneumonia were investigated. A clinical history was taken and arterial blood gas analysis, chest radiography, oximetry during exercise, and sputum induction or bronchoscopy (or both) were performed. RESULTS: Pneumocystis pneumonia was confirmed microscopically from induced sputum or bronchoalveolar lavage fluid in 154 patients; 118 had other chest disease. The remaining 46 patients had no definitive diagnosis. The best single independent predictors of a diagnosis of pneumocystis pneumonia were exercise induced oxygen desaturation and obvious interstitial infiltrates on the chest radiograph (odds ratios of 4.88 and 5.44 respectively). The symptom triad of exertional dyspnoea, cough, and fevers; the absence of pneumocystis pneumonia prophylaxis; and resting arterial hypoxaemia were less predictive (odds ratio 2.07, 3.72, and 0.69). An algorithm was developed that gave a positive predictive value for confirmed pneumocystis pneumonia of 95% and also identified those patients with a very small chance of having pneumocystis pneumonia (negative predictive value 85%). CONCLUSIONS: The diagnosis of an initial episode of pneumocystis pneumonia can be confidently made in a large proportion of immunosuppressed patients with respiratory symptoms on the basis of clinical symptoms, the absence of prophylaxis, chest radiographic appearances, and oxygen desaturation during exercise as shown by oximetry. Using these simple features clinicians can rapidly assign patients to the appropriate type of management at presentation.
PMCID: PMC1021090  PMID: 1494761
25.  Alveolar permeability in HIV antibody positive patients with Pneumocystis carinii pneumonia. 
Genitourinary Medicine  1987;63(4):268-270.
Pulmonary permeability was assessed using the technique of 99mTc (technetium-99m) diethylene triamene pentacetic acid (DTPA) aerosol transfer in 10 patients who had antibodies to human immunodeficiency virus (HIV) and were non-smokers and in 20 HIV antibody positive smokers. Five patients had Pneumocystis carinii pneumonia (PCP) proved by transbronchial lung biopsy; four were non-smokers and one a smoker. Two findings emerged: patients with PCP had greater epithelial permeability than non-smokers and smokers; and the permeability curves were monophasic in smokers and non-smokers, but biphasic in patients with PCP. The biphasic curve observed is indicative of diffuse alveolar damage and might be useful to predict PCP in patients with antibodies to HIV who have normal chest radiographs. As the study was of only five patients with PCP, however, further studies are necessary to confirm this observation.
PMCID: PMC1194081  PMID: 3308684

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