JOURNAL OF CLINICAL MICROBIOLOGY, June 1997, p. 1445–1449 0095-1137/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 35, No. 6
Usefulness of PCR for Diagnosis of Pneumocystis carinii Pneumonia in Different Patient Groups ¨ GNER,1 ANGELIKA MEIER,1 MICHAEL WEIG,1* HARTWIG KLINKER,2 BARBARA H. BO 1 AND UWE GROSS Institute for Hygiene and Microbiology1 and Department of Internal Medicine,2 University of Wu ¨rzburg, Wu ¨rzburg, Germany Received 4 December 1996/Returned for modification 24 January 1997/Accepted 20 March 1997
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.
In addition, P. carinii is a ubiquitous pathogen (3, 35); serological data suggest that almost every individual experiences P. carinii infection in early childhood (22). Therefore, it is important to know whether P. carinii persists throughout life and whether PCP might result from reactivation of a persistent infection. Low positive predictive values might result for a PCR-based diagnosis of PCP if respiratory samples of latently infected persons contain detectable amounts of P. carinii DNA. In order to evaluate the usefulness of P. carinii DNA amplification for the diagnosis of PCP, we studied groups of patients with different forms of immunosuppression exhibiting acute respiratory symptoms, such as a dry, nonproductive cough, fever, weight loss, shortness of breath, and hypoxemia. For nested-PCR analysis, external primers were used as described by Wakefield et al. (36). These primers are complementary to sequences of the mitochondrial large subunit rRNA gene of P. carinii and have been found to be highly specific for P. carinii DNA amplification (21). To increase the sensitivity of the PCR, we constructed a set of nested primers. These internal primers were designed by matching sequences of the gene encoding the large subunits of mitochondrial rRNAs of human P. carinii isolates (19, 28) to highly homologous sequences of other organisms. The chosen oligonucleotides were efficient in amplification of DNA from human P. carinii isolates but did not amplify DNA of other organisms, including potential pulmonary pathogens. The resulting data were correlated to the clinical diagnosis of PCP.
The incidence of Pneumocystis carinii pneumonia (PCP) has sharply increased since the emergence of AIDS in the early 1980s (4, 13). Samples from the respiratory tract are the mainstay for laboratory diagnosis of PCP. As P. carinii organisms cannot yet be successfully cultured from human specimens, microbiological confirmation of PCP is based on morphological detection of the organisms by using different staining techniques, such as Grocott-Gomori silver, toluidine blue O, or calcofluor staining, to identify the cyst stage of P. carinii or Giemsa or Wright staining to detect trophozoites and intracystic bodies (6). Immunofluorescence assays utilizing either monoclonal or polyclonal antibodies specific for the trophozoite and/or the cyst stage of P. carinii have been developed recently (2, 6, 9, 10, 20). However, all these conventional methods face the obstacle of low sensitivity (14, 20, 24). As a consequence, only material obtained through invasive procedures, such as bronchoalveolar lavage, is considered appropriate for these methods. The alternative method of nested PCR offers a sensitive and specific tool for detecting P. carinii DNA in clinical specimen (1, 11, 17, 31, 36). However, the possible role of the PCR technique in the laboratory diagnosis of PCP has not yet been satisfactorily defined. To date, little information exists about the correlation of a positive P. carinii PCR result and the clinical manifestation of PCP in patient groups with different causes of immunosuppression. * Corresponding author. Mailing address: Institute for Hygiene and Microbiology, Josef-Schneider-Str. 2, D-97080 Wu ¨rzburg, Germany. Phone: 49-931-2015160. Fax: 49-931-2013445. E-mail: [email protected]
MATERIALS AND METHODS Patients and clinical specimens. Samples were obtained from patients who exhibited acute episodes of respiratory symptoms and were derived from (i) 43 episodes in 25 human immunodeficiency virus (HIV)-infected patients with
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TABLE 1. Numbers of clinical specimens from different patient groups No. of specimens in patient group Specimen type
Sputum Tracheal-bronchial aspirate BAL Lung biopsy Total
Immunosuppressed HIV negative
Total no. of specimens
AIDS, (ii) 26 episodes in 24 HIV-negative immunosuppressed patients with leukemia or lymphoma, and (iii) 68 episodes in 61 immunocompetent patients. Nested PCR was performed on 136 induced and expectorated sputum samples (confirmed by Gram staining), 27 tracheal and bronchial aspirate samples, 35 bronchoalveolar lavage (BAL) samples, and 11 specimens obtained by lung biopsy (Table 1). Symptoms were judged as being indicative of PCP when a combination of the following parameters could be observed: typical radiography of the lungs; typical clinical signs and symptoms, such as a dry, nonproductive cough, shortness of breath, fever, weight loss, and hypoxemia; an elevated lactate dehydrogenase level; and clinical and/or radiological improvement upon treatment with high doses of co-trimoxazole. Diagnosis of PCP was confirmed by Grocott-Gomori staining, which was performed in duplicate on all specimens tested by PCR. DNA amplification and detection. To show that the chosen primers were specific for P. carinii, DNA from human P. carinii isolates and DNA from other organisms, including potential pulmonary pathogens such as Aspergillus fumigatus, Aspergillus nidulans, Rhizomucor sp., Candida albicans, Candida glabrata, Toxoplasma gondii, Encephalitozoon cuniculi, Saccharomyces cerevisiae, and Escherichia coli, and from human fibroblasts were investigated by nested PCR. Sputum samples, tracheal-bronchial aspirate samples, and BAL samples were diluted 1:5 in lysis buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 0.5% [vol/vol] Tween 20, 2.5 mM MgCl2, 0.5% [vol/vol] Nonidet P-40) and treated with proteinase K (2 mg/ml) for 1 h at 56°C. Following proteinase K digestion, the biopsy specimen DNA was prepared by using a Gene Clean II kit (BIO 101, Vista, Calif.) in accordance with the suggestions of the manufacturer. A two-step protocol was applied for nested PCR: external primers pAZ 102E and pAZ102 H were first used to yield a 340-bp fragment (36), after which was performed a second round of amplification with the nested primers pLE1 (59-TCGGACTA GGATATAGCTGG-39) and pLE2 (59-CCCTTTCGACTATCTACC-39) to yield a final product of 193 bp. Several controls were included in the PCR experiments. All amplifications were performed in parallel with a negative control (autoclaved water) in order to exclude spurious results due to trace contamination. The precautions used to prevent contamination were those described by Kwok and Higuchi (18). To exclude amplicon contamination in the sensitive nested PCR, Biomaster pipets (Eppendorf, Hamburg, Germany) and Safe Seal Tips (Biozym, Oldendorf, Germany) were used. Primary PCR was performed on 10 ml of proteinase-treated specimen that had been added to 40 ml of PCR reagent mixture (20 mM [each] dATP, dCTP, dGTP, and dTTP; 25 pmol of each primer; 2.5 U of Taq polymerase [Amersham, Braunschweig, Germany]; 5 ml of 103 Taq buffer [Amersham]; 3 ml of MgCl2 [25 mM]; and 28.5 ml of sterile water). Amplification was per-
formed in an Omni-Gene temperature cycler (Hybaid, Teddington, United Kingdom). For a hot start, the PCR reagent mixture was preheated to 94°C before DNA was added. Thirty-five cycles were carried out, consisting of (i) denaturation for 1 min at 94°C, (ii) annealing for 1 min at 55°C, and (iii) extension for 90 s at 72°C. A final extension was performed for 10 min at 72°C after the last cycle. One microliter of the first PCR product was used for the second round of PCR (hot start; 30 cycles with the following parameters: 1 min at 94°C, 1 min at 58°C, and 90 s at 72°C; final extension for 10 min at 72°C). Samples were stored at 220°C until further analysis was performed. All PCR products were investigated by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. DNA sequencing. The amplified gene products of three BAL specimens with corresponding positive Grocott-Gomori staining results and of two BAL samples with negative Grocott-Gomori staining results were purified with a QIA Quickspin PCR purification kit (Quiagen, Chatsworth, Calif.) in accordance with the suggestions of the manufacturer, and 0.3 mg of the purified double-stranded DNA was subjected to sequencing with a Prism Dye Terminator Cycle Sequencing Ready Reaction DNA sequencing kit (Applied Biosystems, Darmstadt, Germany). Briefly, 9.5 ml of terminator premix (containing 4 ml of reaction buffer, 1 ml of deoxynucleoside triphosphate mix [10 mM], 1 ml of DyeDeoxy A [900 mM], 1 ml of DyeDeoxy T [450 mM], 1 ml of DyeDeoxy G [15 mM], 1 ml of DyeDeoxy C [4 ml], and 0.5 ml [4 U] of AmpliTaq DNA polymerase), 0.3 mg of template DNA, and 5 pmol of primer pLE1 were mixed and adjusted to a final volume of 20 ml by the addition of sterile water. The samples were placed in a thermal cycler preheated to 96°C and were subjected to 25 cycles with the following parameters: 96°C for 15 s, 59°C for 15 s, and 60°C for 4 min. Afterward, the sequencing products were ethanol precipitated and the resulting DNA pellets were dissolved in 4 ml of a 5:1 mixture of formamide and 50 mM EDTA (pH 8.0). Separation of sequencing products was performed on 7% denaturing polyacrylamide gels in an automated sequencer (Model A737; Applied Biosystems, Weiterstadt, Germany). Sequence analyses were carried out with the DNAsis program, version 2.0 (Pharmacia LKB, Freiburg, Germany). The obtained sequence was compared with known sequences from the EMBL gene bank by using the FASTA program (R. Pearson, University of Virginia, Charlottesville).
RESULTS In order to show the specificity of the reamplified 193-bp PCR product for P. carinii, the amplified gene products of selected BAL specimens were sequenced. Comparison of the resulting data with sequences obtained from the EMBL gene bank showed that the PCR products were 100% homologous to positions 26 to 219 of sequences from the mitochondrial large subunit rRNA genes of human P. carinii isolates which were published by Latouche et al. (19) (EMBL sequence no. S77852). On the other hand, no DNA was amplified from other organisms, including potential pulmonary pathogens. By the nested-PCR technique, no P. carinii DNA could be detected in the 77 specimens from immunocompetent patients (Table 2). These specimens included nine BAL and three lung biopsy samples (Table 1). In contrast to this, 30 of 77 samples from HIV-infected patients with AIDS were P. carinii DNA positive. Conventional methods do not allow reliable morphological detection of P. carinii in materials such as sputum and tracheal or bronchial aspirates, although PCP is obviously present. Therefore, we included (i) samples from patients with morphologically and/or
TABLE 2. Results of P. carinii PCR and correlation with clinical evidence in patients with acute episodes of respiratory symptoms No. (%) of patients who were: Patient group (n)
P. carinii PCR positive
P. carinii PCR positive, clinically proven PCP
P. carinii PCR positive, no clinical evidence of PCP
P. carinii PCR negative
P. carinii PCR negative, clinically proven PCP
P. carinii PCR negative, no clinical evidence of PCP
AIDS (77) Immunosuppressed HIV negative (55) Immunocompetent (77)
26 (86.7) 4 (18.2)
4 (13.3) 18 (81.8)
21 (44.7) 6 (18.2)
26 (55.3) 27 (81.8)
a b c
n, total number of samples tested. A total of 39.0% of the respiratory samples from HIV patients with AIDS (n 5 77) that were examined were P. carinii DNA positive. A total of 40.0% of the respiratory samples from patients with leukemia or lymphoma (n 5 55) that were examined were P. carinii DNA positive.
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TABLE 3. Correlation of PCR and Grocott-Gomori staining results for specimens from different patient groups No. of positive specimens/total no. of specimens by test type for patient group: AIDS
Lung biopsy BAL Tracheal-bronchial aspirate Sputum Total
By Grocott-Gomori staining
6/8 3/4 9/15 12/50 30/77
By Grocott-Gomori staining
By Grocott-Gomori staining
4/8 3/4 2/15 2/50
0/0 11/22 4/6 7/27
0/0 0/22 0/6 0/27
0/3 0/9 0/6 0/59
0/3 0/9 0/6 0/59
histologically proven PCP (n 5 11) and (ii) samples from patients with defined clinical parameters indicative of PCP but without morphological demonstration of the organism (n 5 15) in our study (Table 3). In every case, the final diagnosis of PCP was made by the clinician using the criteria mentioned above. All together, PCP was present in 26 of the 30 DNApositive cases (87%). The calculated positive predictive value for all respiratory samples obtained in the AIDS group was 0.86. It is important to stress that the kind of respiratory specimen is critical for PCR diagnosis of PCP. P. carinii PCR performed with BAL samples and tissue biopsy specimens from AIDS patients (n 5 12) had positive and negative predictive values of 1 and a sensitivity and specificity of 100% (Table 4). For sputum and tracheal-bronchial aspirate samples, the positive predictive value was 0.81, the negative predictive value was 0.57, the specificity was 86.2%, and the sensitivity was 47.2%. For these materials, 21 false-negative PCR results (as determined by correlation with the clinical evidence of PCP) were found in this group of patients. A good correlation of Grocott-Gomori staining and PCR results was found for BAL and biopsy specimens from AIDS patients (Table 3). Positive Grocott-Gomori staining could be obtained in only 2 of 9 PCR-positive tracheal-bronchial aspirate samples and in only 2 of 12 PCR-positive sputum samples. All samples with positive Grocott-Gomori staining except one correlated with clinical evidence of PCP.
Although 22 of the 55 samples collected from immunosuppressed HIV-negative patients gave P. carinii DNA signals, a positive correlation with PCP was found in only four of these cases (82% false-positive results, if correlated to the clinical situation [Table 2]). No sample exhibited a corresponding positive Grocott-Gomori staining result (Table 3). The positive predictive value for all respiratory samples obtained from this group was 0.18. In 10 of 11 BAL samples resulting in positive PCR results, no clinical correlation to PCP could be found. The positive predictive value for BAL and biopsy samples was calculated to be 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. For sputum and tracheal-bronchial aspirate samples, the positive predictive value was determined to be 0.27. These findings are in contrast to the positive predictive value of 0.81 obtained for the same materials in the AIDS group. Sensitivities, specificities, and positive and negative predictive values of the nested P. carinii PCR with different specimens from the patient groups examined are summarized in Table 4. For two AIDS patients, specimens could be obtained over a period including 7 to 10 months after their presentation of clinically overt PCP, allowing us to follow the time course of detectability of P. carinii DNA in sputum samples. In both cases, no P. carinii DNA could be amplified 3 months after the onset of acute symptoms of PCP (Table 5; Fig. 1).
TABLE 4. Sensitivity, specificity, and positive and negative predictive values of P. carinii PCR for different specimens in the patient groups examined PCR parameters Patient group
BAL and lung biopsy Sputum and tracheal-bronchial aspirate Total
BAL and lung biopsy Sputum and tracheal-bronchial aspirate Total
BAL and lung biopsy Sputum and tracheal-bronchial aspirate Total
ND, not defined.
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TABLE 5. Time course of PCR-based P. carinii DNA detection in sputum samples after onset of PCP in a patient with AIDS Time (mo)
Clinical evidence of PCP
0 1 3 10
1 1a 2 2
1 1b 2 2
Patient showed improvement in clinical parameters. Positive result also obtained for BAL specimen.
DISCUSSION Using the nested-PCR technique, no P. carinii DNA could be detected in the 77 respiratory samples obtained from immunocompetent persons, confirming the results of previous studies. Peters et al. could not demonstrate any P. carinii DNA in 45 PCR investigations on postmortem-obtained lungs of 15 immunocompetent individuals aged 15 to 75 years (25). Millard and Heryet used a monoclonal antibody technique to show that persistence of P. carinii in the lungs of immunocompetent individuals is time limited (23). We found a high correlation between positive P. carinii PCR results and clinical evidence of PCP in HIV patients with AIDS. For the respiratory samples obtained in this group, the positive predictive value was 0.86. For BAL and lung biopsy samples, specimens considered to be the “gold standard” for PCP diagnosis (32), the positive predictive value was 1 and the sensitivity and specificity of the nested PCR were 100%. For sputum samples and trachealbronchial aspirate samples, we determined a positive predictive value of 0.81, a specificity of 86%, and a sensitivity of 47%. Because of the high positive predictive value and specificity associated with nested PCR, this technique could improve PCP diagnosis from respiratory materials obtained in a noninvasive manner from AIDS patients. Repetitive sampling and different procedures (e.g., chloroform-phenol treatment) for extraction of DNA from sputum and tracheal-bronchial-aspirate samples might increase the sensitivity of the PCR.
FIG. 1. PCR results of specimens from a patient suffering from AIDS (see also Table 5). PCR products were subjected to agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Kb, molecular weight markers. Lane 1, positive control; lane 2, negative control; lane 3, reagent control; lane 4, sputum, month 0 (acute PCP); lane 5, BAL, month 1; lane 6, sputum, month 1; lane 7, sputum, month 3; lane 8, sputum, month 10.
The time course of P. carinii DNA detection after the onset of PCP in two HIV-infected patients is suggestive of the limited persistence and subsequent elimination of P. carinii after treatment of PCP. Although these kinetic results were obtained with sputum samples, they are consistent with data from Vargas and coworkers, who were able to show clearance of P. carinii in more than 75% of the lungs of immunosuppressed rats within 1 year after an episode of PCP, implying that P. carinii organisms do not persist in the lungs (34). It is not clear whether persistence of P. carinii is possible at an extrapulmonary site. Chen and coworkers failed to reveal extrapulmonary foci in an animal model using SCID mice (7). In the group consisting of leukemia and lymphoma patients, a low correlation between positive P. carinii PCR results and clinical evidence of PCP was observed (positive predictive value, 0.18). By correlation to clinical presentation data, it was determined that we obtained 82% false-positive PCR results. None of these patients developed PCP later on, as defined by clinical and radiological parameters. The precise mechanism by which changes in humoral and cell-mediated immunity lead to clinically overt pneumonia after P. carinii infection still has to be determined (12, 30, 33). In contrast to leukemia and lymphoma patients, modifications in the immune systems of AIDS patients, such the lack of a T-cell blastogenic response to P. carinii organisms (15), low CD41 cell counts (27), reduced gamma interferon production by activated T cells (5), and reduced interleukin-1 secretion by macrophages (8), may contribute to PCP. In summary, our data indicate that PCP might not result from reactivation of latent infection during immunosuppression, as no single sample from any immunocompetent person contained detectable amounts of P. carinii DNA. With a rat model, Hughes confirmed the airborne route of P. carinii infection (16). In addition to the fact that nosocomial clusters of PCP do occur (3, 13, 26, 29), our findings propose that infection in man is similarly acquired. Since many of the specimens from immunocompetent persons studied were sputum samples and tracheal-bronchial aspirates, we cannot completely rule out the possibility that the kind of material analyzed was not adequate and/or that the technique used was not sensitive enough to detect the small numbers of P. carinii organisms expected in latently infected persons. Similarly, the lack of P. carinii DNA found in postmortem lungs of immunocompetent persons (25) may be explained by inhibition or organism deterioration. In leukemia and lymphoma patients, PCR had a low positive predictive value for PCP diagnosis. In this patient group, a PCR-based laboratory diagnosis of PCP should be viewed with caution because detection of P. carinii DNA might be due to subclinical manifestation of P. carinii infection, a reflection of transient carriage of the organism, or a result of persistently dormant foci. We do not know whether a conversion to negative PCR results of respiratory samples in these patients does occur at a later time when immunosuppression eventually resolves. On the other hand, our data affirm the usefulness of P. carinii DNA detection for PCP diagnosis in HIV-infected patients with AIDS. A high correlation between positive PCR results and clinical evidence of PCP was found. In addition, P. carinii DNA could not be detected in respiratory samples from AIDS patients after PCP resolution. Because of the superior sensitivity of the nested-PCR technique compared to conventional morphological detection of P. carinii (20), we propose the use of nested PCR for improved laboratory diagnosis of PCP in AIDS patients. This could be of predominant importance (i) in cases when P. carinii burdens in BAL or biopsy
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PCR FOR DIAGNOSIS OF PCP
specimens are too low for morphological detection, e.g., as a consequence of pentamidine prophylaxis; or (ii) when only materials obtained in a noninvasive manner, such as sputum samples and tracheal-bronchial aspirates, are available as a result of a life-threatening clinical situation. ACKNOWLEDGMENTS We thank Karin Lemmer from the Max von Pettenkofer Institut in Munich for helpful discussions, Martin Wilhelm from the outpatient department of the University of Wu ¨rzburg for providing some of the clinical data, and Marion Patzke for excellent technical assistance. REFERENCES 1. Adachi, N. 1994. Polymerase chain reaction assay for the diagnosis of Pneumocystis carinii and cytomegalovirus pneumonia. Nippon Kyobu Shikkan Gakkai Zasshi 32:433–440. 2. Armbruster, C., L. Pokieser, and A. Hassl. 1995. Diagnosis of Pneumocystis carinii pneumonia by bronchoalveolar lavage in AIDS patients. Comparison of Diff-Quik, Fungifluor stain, direct immunofluorescence test and polymerase chain reaction. Acta Cytol. 39:1089–1093. 3. Bartlett, M. S., C. H. Lee, J. J. Lu, N. L. Bauer, J. F. Bettz, G. L. McLaughlin, and J. W. Smith. 1994. Pneumocystis carinii detected in air. J. Eukaryot. Microbiol. 41:75S. 4. Bartlett, M. S., and J. W. Smith. 1991. Pneumocystis carinii, an opportunist in immunocompromised patients. Clin. Microbiol. Rev. 4:137–149. 5. Beck, J. M., H. D. Liggitt, E. N. Brunette, H. J. Fuchs, J. E. Shellito, and R. J. Debs. 1991. Reduction in intensity of Pneumocystis carinii pneumonia in mice by aerosol administration of gamma interferon. Infect. Immun. 59:3859– 3862. 6. Blumenfeld, W., O. McCook, M. Holodniy, and D. A. Katzenstein. 1992. Correlation of morphologic diagnosis of Pneumocystis carinii with the presence of pneumocystis DNA amplified by the polymerase chain reaction. Mod. Pathol. 5:103–106. 7. Chen, W., F. Gigliotti, and A. G. Harmsen. 1993. Latency is not an inevitable outcome of infection with Pneumocystis carinii. Infect. Immun. 61:5406– 5409. 8. Chen, W., E. A. Havell, L. L. Moldawer, K. W. McIntyre, R. A. Chizzonite, and A. G. Harmsen. 1992. Interleukin 1: an important mediator of host resistance against Pneumocystis carinii. J. Exp. Med. 176:713–718. 9. Eisen, D., B. C. Ross, J. Fairbairn, R. J. Warren, R. W. Baird, and B. Dwyer. 1994. Comparison of Pneumocystis carinii detection by toluidine blue O staining, direct immunofluorescence and DNA amplification in sputum specimens from HIV positive patients. Pathology 26:198–200. 10. Elvin, K. 1994. Laboratory diagnosis and occurrence of Pneumocystis carinii. Scand. J. Infect. Dis. Suppl. 94:1–34. 11. Evans, R., A. W. Joss, T. H. Pennington, and D. O. Ho Yen. 1995. The use of a nested polymerase chain reaction for detecting Pneumocystis carinii from lung and blood in rat and human infection. J. Med. Microbiol. 42:209–213. 12. Ezekowitz, R. A., D. J. Williams, H. Koziel, M. Y. Armstrong, A. Warner, F. F. Richards, and R. M. Rose. 1991. Uptake of Pneumocystis carinii mediated by the macrophage mannose receptor. Nature 351:155–158. 13. Follansbee, S. E., D. F. Busch, C. B. Wofsy, D. L. Coleman, J. Gullet, G. P. Aurigemma, T. Ross, W. K. Hadley, and W. L. Drew. 1982. An outbreak of Pneumocystis carinii pneumonia in homosexual men. Ann. Intern. Med. 96:705–713. 14. Galan, F., J. L. Oliver, P. Roux, J. L. Poirot, and G. Bereziat. 1991. Detection of Pneumocystis carinii DNA by polymerase chain reaction compared to direct microscopy and immunofluorescence. J. Protozool. 38:199S–200S. 15. Hagler, D. N., G. S. Deepe, C. L. Pogue, and P. D. Walzer. 1988. Blastogenic responses to Pneumocystis carinii among patients with human immunodeficiency (HIV) infection. Clin. Exp. Immunol. 74:7–13. 16. Hughes, W. T. 1982. Natural mode of acquisition for de novo infection with
Pneumocystis carinii. J. Infect. Dis. 145:842–848. 17. Ishimine, T., A. Nakamoto, F. Higa, M. Koide, J. Inadome, K. Kawakami, H. Fukuhara, N. Kusano, K. Kitsukawa, and A. Saito. 1994. Usefulness of polymerase chain reaction method in the early diagnosis of Pneumocystis carinii pneumonia. Kansenshogaku Zasshi 68:751–758. 18. Kwok, S., and R. Higuchi. 1989. Avoiding false positives with PCR. Nature 339:237–238. (Erratum, 339:490.) 19. Latouche, S., P. Roux, J. L. Poirot, I. Lavrard, B. Hermelin, and V. Bertrand. 1994. Preliminary results of Pneumocystis carinii strain differentiation by using molecular biology. J. Clin. Microbiol. 32:3052–3053. 20. Leigh, T. R., A. E. Wakefield, S. E. Peters, J. M. Hopkin, and J. V. Collins. 1992. Comparison of DNA amplification and immunofluorescence for detecting Pneumocystis carinii in patients receiving immunosuppressive therapy. Transplantation 54:468–470. 21. Lu, J.-J., C.-H. Chen, M. S. Bartlett, J. W. Smith, and C.-H. Lee. 1995. Comparison of six different PCR methods for detection of Pneumocystis carinii. J. Clin. Microbiol. 33:2785–2788. 22. Meuwissen, J. H., I. Tauber, A. D. Leeuwenberg, P. J. Beckers, and M. Sieben. 1977. Parasitologic and serologic observations of infection with Pneumocystis in humans. J. Infect. Dis. 136:43–49. 23. Millard, P. R., and A. R. Heryet. 1988. Observations favouring Pneumocystis carinii pneumonia as a primary infection: a monoclonal antibody study on paraffin sections. J. Pathol. 154:365–370. 24. Nogues, A., M. Garcia, C. Rivas, M. Falguera, T. Puig, and A. Ruiz. 1993. Detection of Pneumocystis carinii in clinical samples by the polymerase chain reaction. Comparison with the methenamine silver technique. Enferm. Infecc. Microbiol. Clin. 11:143–146. 25. Peters, S. E., A. E. Wakefield, K. Sinclair, P. R. Millard, and J. M. Hopkin. 1992. A search for Pneumocystis carinii in post-mortem lungs by DNA amplification. J. Pathol. 166:195–198. 26. Santiago Delpin, E. A., E. Mora, Z. A. Gonzalez, L. A. Morales Otero, and R. Bermudez. 1988. Factors in an outbreak of Pneumocystis carinii in a transplant unit. Transplant. Proc. 20:462–465. 27. Shellito, J., V. V. Suzara, W. Blumenfeld, J. M. Beck, H. J. Steger, and T. H. Ermak. 1990. A new model of Pneumocystis carinii infection in mice selectively depleted of helper T lymphocytes. J. Clin. Invest. 85:1686–1693. 28. Sinclair, K., A. E. Wakefield, S. Banerji, and J. M. Hopkin. 1991. Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct. Mol. Biochem. Parasitol. 45:183–184. 29. Singer, C., D. Armstrong, P. P. Rosen, and D. Schottenfeld. 1975. Pneumocystis carinii pneumonia: a cluster of eleven cases. Ann. Intern. Med. 82:772– 777. 30. Smulian, A. G., S. A. Theus, N. Denko, P. D. Walzer, and J. R. Stringer. 1993. A 55 kDa antigen of Pneumocystis carinii: analysis of the cellular immune response and characterization of the gene. Mol. Microbiol. 7:745–753. 31. Tamburrini, E., P. Mencarini, A. De Luca, A. Antinori, E. Visconti, A. Ammassari, L. Ortona, E. Ortona, A. Siracusano, and G. Vicari. 1993. Simple and rapid two-step polymerase chain reaction for diagnosis of Pneumocystis carinii infection. J. Clin. Microbiol. 31:2788–2789. 32. Tamburrini, E., P. Mencarini, A. De Luca, G. Maiuro, G. Ventura, A. Antinori, A. Ammassari, E. Visconti, L. Ortona, A. Siracusano, et al. 1993. Diagnosis of Pneumocystis carinii pneumonia: specificity and sensitivity of polymerase chain reaction in comparison with immunofluorescence in bronchoalveolar lavage specimens. J. Med. Microbiol. 38:449–453. 33. Theus, S. A., D. W. Sullivan, P. D. Walzer, and A. G. Smulian. 1994. Cellular responses to a 55-kilodalton recombinant Pneumocystis carinii antigen. Infect. Immun. 62:3479–3484. 34. Vargas, S. L., W. T. Hughes, A. E. Wakefield, and H. S. Oz. 1995. Limited persistence in and subsequent elimination of Pneumocystis carinii from the lungs after P. carinii pneumonia. J. Infect. Dis. 172:506–510. 35. Wakefield, A. E. 1994. Detection of DNA sequences identical to Pneumocystis carinii in samples of ambient air. J. Eukaryot. Microbiol. 41:116S. 36. Wakefield, A. E., F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin. 1990. Detection of Pneumocystis carinii with DNA amplification. Lancet 336:451–453.