Vol. 28, No. 5
OF CLINICAL MICROBIOLOGY, May 1990, p. 975-979 0095-1137/90/050975-05$02.00/0 Copyright © 1990, American Society for Microbiology
JOURNAL
Evaluation of an Indirect Fluorescent-Antibody Stain for Detection of Pneumocystis carinii in Respiratory Specimens VALERIE L. NGl 2.3* DAVID M. YAJKO,"3 LARON W. McPHAUL,"3 IRIS GARTNER,"3 BARRY BYFORD,"3 CAROLE D. GOODMAN,"3 PATRICIA S. NASSOS,"3 CYNTHIA A. SANDERS,"3'4 EDWARD L. HOWES,5'6 GIFFORD LEOUNG,2'4'7 PHILIP C. HOPEWELL,2'4 AND W. KEITH HADLEY"3 Departments of Laboratory Medicine,' Anatomic Pathology,5 and Medicine/ University of California, San Francisco, San Francisco, California 94143, and Clinical Laboratories, Division of Microbiology,3* Department of Anatomic Pathology,6 Medical and Chest Service,4 and Divisions of infectious Diseases and of AIDS Activities, Medical Service,7 San Francisco General Hospital, San Francisco, California 94110 Received 9 August 1989/Accepted
5
January 1990
Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue 0, and Gomori methenamine silver stains for detecting P. caring in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P < 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy. It is possible to diagnose Pneumocystis carinii pneumonia by examining a variety of respiratory specimens, including open lung or transbronchial biopsy (TBBx) samples, bronchoalveolar lavages (BALs), and induced sputa (IS) (1-4, 9, 15, 17, 20, 23, 25). In patients at risk for or with the acquired immunodeficiency syndrome (AIDS), P. carinii pneumonia has been diagnosed from IS specimens with a high degree of sensitivity (1, 15, 20, 25). A variety of stains have been used to detect P. carinii. The three most commonly used stains are Diff-Quik (DQ; a rapid Giemsa-like stain) (1, 20), modified toluidine blue O (TBO) (9), and Gomori methenamine silver (GMS) (10). Recently, mouse monoclonal antibodies, primarily directed against P. carinii cyst wall antigens, have been adapted for examining IS and BALs by using an indirect fluorescent-antibody (IFA) stain (8, 14, 15). This IFA stain was shown to be more sensitive in detecting P. carinii in IS than was either the DQ stain or the TBO stain in a population at risk for or with AIDS (15). However, there was no comparison in that study with the GMS stain, which is used in many microbiology and pathology laboratories for the detection of P. carinii (10). The original IFA procedure (8, 14, 15) has since been modified with the intent of adapting it to general clinical laboratory use (Merifluor-pneumocystis; Meridian Diagnostics, Inc., Cincinnati, Ohio). Here we report the results of a study in which we compared the performance of the modified IFA stain with those of DQ, TBO, and GMS stains for detecting P. carinii in IS, BALs, and TBBx touch preparations obtained from individuals at risk for or with AIDS.
MATERIALS AND METHODS Study design. Respiratory specimens from patients in two independent studies were examined by various stains for the presence of P. carinii. The first study evaluated all IS obtained from asymptomatic patients with AIDS who were entered into a study to examine the efficacy of prophylactic aerosolized pentamidine. Further details of the clinical aspects of this study will be reported elsewhere (G. Leoung, A. B. Montgomery, D. J. Abrams, L. Wardlaw, K. Corkery, D. Busch, S. Gordon, D. W. Feigal, and the San Francisco County Community Consortium, unpublished data). All patients enrolled in this study had survived one episode of P. carinii pneumonia, had completed specific anti-P. carinii therapy at least 15 days and not more than 6 months prior to the beginning of the study, and were asymptomatic at the time of enrollment in the study. Patient enrollment in the study began on 25 April 1988 and was scheduled to continue until 100 patients were enrolled; however, the study was terminated early because of the Food and Drug Administration announcement of its approval of aerosolized pentamidine as an investigational new drug. All 78 patients entered in this study were scheduled to have IS obtained at the time of entry into the study and at week 24. The second study was a prospective evaluation of respiratory specimens obtained from patients with respiratory symptoms who were assumed to have risk factors for or who had AIDS and were referred to the Chest Service at San Francisco General Hospital for further evaluation. Patient selection and evaluation and specimen acquisition were done
reported previously (1, 15, 20). Specimen processing and staining. Mucolysis and the concentration of IS by centrifugation were performed as deas
*
Corresponding author. 975
976
NG ET AL.
scribed previously (1, 15, 20). IS lacking alveolar macrophages were not examined for P. carinii. BALs and TBBxs were processed as described previously (2). Two smears (for the first study) and eight smears (for the second study) were made from each specimen and stored at -20°C until staining was performed. DQ and IFA staining were performed according to the instructions of the manufacturers (20); TBO and GMS staining were performed as described previously (9, 10). For the first study, one smear from each specimen was stained with DQ and the remaining smear was stained with IFA. For the second study, two each of the eight smears were stained with DQ, TBO, GMS, and IFA. The stained smears were coded and examined microscopically for P. carinii by an experienced reader (for IFA, V.L.N., D.M.Y., C.A.S., P.S.N., W.K.H.; for DQ, I.G., C.D.G., V.L.N., C.A.S., P.S.N., W.K.H.; for TBO, B.B., V.L.N., W.K.H.; for GMS, L.W.M., E.L.H., V.L.N., W.K.H.). The readers did not know the results of the other stains at the time of examination. Smears stained with DQ were screened at x500 magnification (xlO ocular, x50 oil immersion objective). Smears stained with GMS or TBO were screened at x100 magnification (x 10 ocular, x 10 objective). Any material resembling P. carinii was further examined at x 1,000 magnification (x 10 ocular, x 100 oil immersion objective). Smears stained with IFA were screened at x 200 magnification (x 12.5 ocular, x 16 objective). Any fluorescent material that was observed was further examined at x 500 magnification (x 12.5 ocular, x40 objective). Interpretation of stained smears. A DQ-stained smear was interpreted as positive if cysts, trophozoites, or both were present (Fig. 1A and B). The observation of a single morphologically unequivocal clump of trophozoites was sufficient for interpreting a smear as positive. The TBO- and GMS-stained smears were interpreted as positive if cysts with characteristic morphology (Fig. 1C and D) were observed. The IFA-stained smears were interpreted as positive if at least three well-defined cysts, usually within a clump of P. carinii organisms, were present. Clumps of P. carinii organisms detected by the IFA stain had two distinct morphological appearances: the unequivocal classic honeycomblike appearance (Fig. 1E) or clumps of cysts morphologically consistent with P. carinii but lacking the characteristic honeycomb appearance (Fig. 1F). A smear containing fluorescent material resembling trophozoites in the absence of cysts was not interpreted as positive. Statistical analysis. For the purposes of this study, the identification of P. carinii by DQ, TBO, or GMS stains was considered to be a true-positive result. In contrast, specimens in which P. carinii was detected only by the IFA stain were subjected to a more rigorous set of interpretive criteria in which results from subsequent bronchoscopic specimens, a review of medical records, or both were also considered. Chi-square analysis (6) and sensitivity, specificity, and positive and negative predictive values (7) were calculated as described previously. Follow-up of specimens positive by IFA staining only. Medical records were reviewed for those patients whose specimens had P. carinii, as detected by IFA staining only. The result was considered to be truly positive if (i) the subsequent clinical course was consistent with P. carinji pneumonia, (ii) the patient responded to anti-P. carinii therapy, or (iii) a subsequent BAL specimen obtained within 7 days of the original IS specimen contained P. carinii which was detected by any of the traditional stains. The result was
J. CLIN. MICROBIOL.
A
B
c
D f
b1
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lomm -,**
-w'À
-à
ig,
E
F
FIG. 1. Morphological appearance of P. carinii by various stains. (A) A clump of Pneumocystis cysts stained with the DQ. (B) A clump of Pneumocystis trophozoites stained with DQ. (C) A clump of Pneumocystis cysts stained with TBO. (D) A clump of Pneumocystis cysts stained with GMS. (E) A clump of P. carinii stained with IFA exhibiting the classic honeycomb appearance. (F) A clump of P. carinfi stained with IFA but lacking the classic honeycomb appearance. Magnifications: x2,000 (A to D); x 1,000 (E and F).
considered to be falsely positive if (i) a respiratory pathogen other than P. carinii was identified, (ii) the patient spontaneously improved, or (iii) the patient responded to appropriate therapy which did not include anti-P. carinii therapy. The result was considered to be indeterminate if (i) P. carinii could not be eliminated as the cause of disease or (ii) there was no clinical evaluation within 30 days of specimen
acquisition. Follow-up evaluation of specimens lacking P. carinii (negative specimens). The medical records of patients who had a single IS or BAL specimen that lacked P. carinii were reviewed for a 30-day period after the specimen was obtained. The test results were considered to be truly negative
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FLUORESCENT-ANTIBODY STAIN FOR P. CARINII DETECTION
if (i) P. carinii was not found by any stain in a BAL specimen obtained within 7 days of the original IS specimen, (ii) the respiratory process responded to treatment that did not include anti-P. carinii agents, or (iii) the respiratory process resolved spontaneously and the patient had no new respiratory symptoms. The test results were considered to be falsely negative if (i) the patient had a subsequent bronchoscopy sample which contained P. carinii or (ii) the patient had a progressive or persistent respiratory illness which responded to anti-P. carinii treatment. The test results were considered indeterminate if (i) P. carinii could not be eliminated as the cause of the respiratory disease or (ii) there was no clinical evaluation within 30 days of specimen acquisition. RESULTS For the first study, 95 IS were obtained from 77 patients who were prophylactically administered aerosolized pentamidine. The IS were examined by the DQ and IFA stains. P. carinii was detected in only one specimen and was detected by both stains. Prophylactic aerosolized pentamidine treatment did not alter the morphology of P. carinii as detected by either stain. The patient in whom P. carinii was detected remained clinically well until 16 weeks later, when he developed respiratory symptoms which resolved with standard anti-P. carinii therapy. Of the remaining 76 patients enrolled in this study, 5 patients had a second episode of P. carinii pneumonia during the course of this study. Although P. carinii was detected in IS obtained when these patients became symptomatic, P. carinii was not detected in the IS which were routinely collected as part of this study. The interval between the time that IS was collected as part of the examination for participation in this study and the time of P. carinii infection relapse ranged from 2 to 16 weeks. For the second study, 182 specimens from 130 symptomatic patients were examined by the DQ, TBO, GMS, and IFA stains. These 182 specimens consisted of 124 IS, 56 BALs, 1 TBBx, and 1 lung tissue specimen that was obtained at the time of autopsy. P. carinii was detected by one or more stains in 105 (57.7%) of the 182 specimens. These 105 specimens consisted of 79 IS, 25 BALs, and 1 touch preparation from a lung tissue specimen that was obtained at the time of autopsy. In only 58 (55%) of the 105 positive specimens was P. carinji detected by all four stains. P. carinii was detected in 15 (14%) specimens by three stains and in 11 (10%) specimens by two stains. P. carinii was detected in 1 sample by DQ only, in 1 sample by GMS only, in 2 samples by TBO only, and in 17 samples by IFA only. Thus, P. carinii was detected by one or more of the traditional stains in 88 of 105 P. carinii-containing specimens. The overall performance of each of the stains was as follows. P. carinii was detected in 73 (70%) specimens by DQ, in 75 (71%) by TBO, in 76 (72%) by GMS, and in 95 (90%) by IFA. The increased sensitivity of P. carinii detection with the IFA stain was statistically significant (P < 0.01), whereas there was no statistical difference in the sensitivity of P. carinii detection by the other three stains (0.975 > P > 0.950). There were 17 specimens in which P. carinii was detected by the IFA stain only. Nine of these specimens (from eight patients) contained clumps of P. carinii organisms that had the classic honeycomb appearance. Three of these specimens were true positives based on subsequent BAL speci-
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TABLE 1. Sensitivity, specificity, and positive and negative predictive values for detecting P. carinii in respiratory specimens with each staina No. of results
SanTPb DQ GMS TBO IFA
73 76 75 81
FN FP TN
28 25 26 20
0 0 0 6
63 63 63 57
Sensitivity Specificity (%)b (% 72 75 74 80
100 100 100 90
+
(%)b
+Vm
100 100 100 93
-Pv (%) P
69 72 71 74
a Specimens from patients who had indeterminate clinical courses were not included in these calculations. Abbreviations: TP, true positives; TN, true negatives; FP, false positives; FN, false negatives; +PV, positive predictive value; -PV, negative predictive value. TP was the presence of P. carinii in the smear and the presence of P. carinii pneumonia. FP was the presence of P. carinii in the smear and the absence of P. carinii pneumonia. FN was the absence of P. carinii in the smear and the presence of P. carinji pneumonia. TN was the absence of P. carinii in the smear and the absence of P. carinii pneumonia. Sensitivity was calculated as TP/TP + FN. Specificity was calculated as TN/TN + FP. +PV was calculated as TP/TP + FP. -PV was calculated as TN/TN + FN. b p. carinii organisms detected by DQ, TBO, or GMS stains were considered to be true positives for these calculations. Thus, the specificity was 100% and the positive predictive value was 100%t for these stains.
mens or the clinical course of infection, five specimens were from four patients who had an indeterminate clinical course, and one specimen was a false positive based on clinical response to antibiotics. The remaining eight specimens (from seven patients) contained P. carinii organisms that lacked the characteristic honeycomb appearance but that had a morphology consistent with clumps of P. carinii (Fig. 1F). Three specimens were from two patients who had indeterminate clinical courses, and five specimens were judged to be false positives: one specimen was from a patient who had resolution of his pulmonary symptoms with antimicrobial therapy other than anti-P. carinii therapy; the remaining four specimens came from four patients diagnosed with (i) pulmonary tuberculosis, (ii) disseminated tuberculosis, (iii) Strongyloides stercoralis hyperinfection syndrome, and (iv) disseminated coccidioidomycosis. Thus, of the 17 specimens determined to be positive by the IFA stain only, three were judged to be true positives, eight were judged to be indeterminate, and six were judged to be false positives based on clinical criteria. Of the 95 specimens in which P. carinii was detected by the IFA stain, only 81 were judged to be true
positives.
A total of 77 (42%; 45 IS, 31 BALs, and 1 TBBx touch preparation) of the 182 specimens did not contain P. carinii
when examined by any of the four stains. Of these 77, 57 were considered to be true negatives based on the inability to find P. carinii in subsequent BAL specimens or by resolution of clinical disease without the use of specific anti-P. carinji therapy. Ten specimens were judged to be false negatives based on the clinical response to anti-P. carinii therapy, and 10 specimens were from patients who had indeterminate clinical courses. The sensitivities, specificities, and positive and negative predictive values were calculated for each of the stains (Table 1). Specimens from patients who had indeterminate clinical courses were not included in the calculations. The sensitivity of each of the four stains for the detection of P. carinii ranged from 72 to 80%, the specificity ranged from 90 to 100%, the positive predictive value ranged from 93 to 100%, and the negative predictive value ranged from 69 to 74%.
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DISCUSSION We demonstrated that the commercially available modified IFA stain is more sensitive than the traditional staining methods for detecting P. carinii in respiratory specimens from patients at risk for or with AIDS. This increased sensitivity of P. carinii detection by the IFA stain is in agreement with results obtained by using the original staining procedure (14, 15), indicating that the modifications made by the manufacturer did not significantly alter the performance of the test. An added advantage of the IFA stain was that evaluation of smears, especially those lacking P. carinii organisms, required significantly less time (usually less than 5 min) than the time required for any of the traditional stains. The sensitivities for P. carinii detection of each of the traditional stains ranged from 72 to 75%, in accordance with previously reported sensitivities (1, 9, 15, 20, 25). Viewed in an alternate manner, the false-negative rate for the traditional stains ranged from 25 to 28%. There were three different groups of specimens contributing to this falsenegative rate. The first group (three IS, one BAL) was obtained from individuals who had a clinical response to anti-P. carinii therapy in the absence of a laboratory diagnosis of P. carinii pneumonia. The interpretation of this group of specimens as false negatives is debatable, in that respiratory processes other than P. carinii pneumonia can respond clinically to anti-P. carinii therapy. The second group (six IS) was from individuals in whom P. carinii was detected in a subsequent BAL specimen. Many factors probably contributed to the inability to find P. carinii in the original IS, including the overall burden of P. carinii in the patient as well as the ability of sputum induction to provide a sample from the infected area. The third group (40 IS, 16 BALs) was from individuals in whom P. carinii was detected by one or two, but not all three, of the traditional stains. In fact, it is noteworthy that only 55% of all specimens containing P. carinii in the second study were positive by all four stains. The discrepancies most often occurred with specimens which contained few to moderate numbers of organisms and were most likely attributable to an uneven distribution of a limited number of organisms among slides. It is of interest that although the IFA stain demonstrated a statistically significant (P < 0.01) increased sensitivity over the traditional stains for P. carinii detection in the second study, the presence of P. carinii as detected only by the IFA stain did not always correlate with clinical P. carinii pneumonia. Clinical evaluation revealed that six of these specimens that were positive by IFA staining only from six patients were in fact false positives by clinical criteria. Although P. carinii was detected in respiratory specimens, therapy directed against the other pathogens that were recovered (i.e., Mycobacterium tuberculosis, Coccidioides immitis, and S. stercoralis) resulted in clinical recovery. However, it must be emphasized that of the other 11 specimens from nine patients in which P. carinii was detected only by the IFA stain, P. carinii could not be eliminated as the cause of the respiratory illness in six patients, and P. carinii was presumed to be the cause of the respiratory illness in the other three patients, as evidenced by a clinical response to anti-P. carinii therapy. We reevaluated our data using more stringent morphological criteria to determine whether these false-positive results might have been avoided. We stipulated that a classic honeycomb clump of P. carinii organisms, in addition to at least three well-defined cysts, had to be present before a
positive result was assigned. However, by these criteria, only 51% of the P. carinii-containing specimens in this study would have been interpreted as positive and we would have erroneously interpreted 49% of P. carinii-containing specimens as negative. In addition, one of the false-positive specimens also had a classic honeycomb clump present. Thus, strict adherence to rigid morphological criteria not only would dramatically decrease the sensitivity of the IFA stain but would not eliminate the problem of false-positive results. The false-positive rate of the traditional stains is not known, because it has been accepted that the detection of P. carinii by one of these stains is diagnostic of P. carinii pneumonia and necessitates appropriate anti-P. carinii therapy. Although P. carinii has not been found in BALs from asymptomatic human immunodeficiency virus type 1-infected individuals (15, 22; J. D. Lundgren, M. Orholm, T. L. Nielsen, J. Iversen, J. Hertz, and J. O. Nielsen, IV Int. Conf. AIDS, abstr. no. 7144, 1988), it has been well documented that in a subset of patients with AIDS and P. carinii pneumonia, the pulmonary burden of P. carinii persists virtually unchanged for at least 35 days after appropriate therapy against P. carinii pneumonia has been given, even with clinical resolution of symptoms (12, 23, 26). Thus, false-positive results would be expected to occur most frequently in individuals with a history of P. carinii pneumonia. Our one DQ- and IFA-positive result in the first study suggests that this is not a frequent occurrence; however, this one positive result demonstrated that P. carinii could still be detected in IS by both the IFA and DQ stains for at least 18 weeks after appropriate anti-P. carinii treatment in an asymptomatic individual receiving prophylactic therapy. Most individuals in the general U.S. population have serological evidence of exposure to P. carinii by adulthood (16, 24), and it is assumed that most adults harbor P. carinii in small numbers. Studies (5, 11, 27) of lungs obtained at the time of autopsy from patients who were not clinically suspected of having died from P. carinii pneumonia have demonstrated that 2 to 15% of patients have detectable P. carinii organisms; most of these cases occurred in patients who were presumed to be immunosuppressed by virtue of underlying malignancies. It has also been well documented in a rat model system that immunosuppression with steroids allows multiplication of the latent organism to detectable levels (13). Although the frequency of P. carinii organisms present concomitantly with another pulmonary pathogen in an immunosuppressed individual with a respiratory illness is not known, there have been reports of patients with AIDS in whom P. carinii pneumonia was coexistent with Legionella sp. (19), Chlamydia trachomatis (18), or Bordetella pertussis (21). These data suggest that P. carinii can be detected at a low frequency in individuals in whom P. carinii pneumonia is not suspected and that activation from the latent state may occur with a variety of conditions. All of the patients from whom false-positive specimens were obtained fit one or more of the categories described above. In summary, the IFA stain was more sensitive than the DQ, TBO, or GMS stains for detecting P. carinii. An added advantage was that less time was required to examine an IFA-stained smear. The ability of this more sensitive stain to detect P. carinii in asymptomatic individuals or in individuals with subclinical disease makes it essential that laboratory findings be carefully considered in conjunction with clinical evaluation of the patient.
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FLUORESCENT-ANTIBODY STAIN FOR P. CARINII DETECTION
ACKNOWLEDGMENTS We gratefully acknowledge the helpful comments and suggestions of Joseph Kovacs, Vee Gill, Allen Nickol, and Bruce A. Clinton; the assistance with statistical analysis of Lewis B. Sheiner and Stuart Beal; the clinical assistance of Erin McGinty; the expert technical assistance of Histopathology Laboratory technologists Cel Rutledge, Rose Nunez, Martha Glenn, and Norma Swain and the pulmonary function technologists Joe Viana, Althanette Johnson, and Miriam Bryant; the cooperation of the pulmonary fellows in the Chest Service; the input of Chris Jacques; the administrative assistance of Dennis Dunlop; and the reagents and materials supplied by Meridian Diagnostics, Inc. We also gratefully acknowledge the partial financial support for this study provided by Meridian Diagnostics, Inc., and Lyphomed, Inc. (Melrose Park, 111.). LITERATURE CITED 1. Bigby, T. D., D. Margolskee, J. L. Curtis, P. F. Michael, D. Sheppard, W. K. Hadley, and P. C. Hopewell. 1986. The usefulness of induced sputum in the diagnosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Am. Rev. Respir. Dis. 133:515-518. 2. Blumenfeld, W., E. Wagar, and W. K. Hadley. 1984. Use of the transbronchial biopsy for diagnosis of opportunistic pulmonary infections in acquired immunodeficiency syndrome (AIDS). Am. J. Clin. Pathol. 81:1-5. 3. Broaddus, C., M. D. Dake, M. S. Stulbarg, W. Blumenfeld, W. K. Hadley, J. A. Golden, and P. C. Hopewell. 1985. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann. Intern. Med. 102:747-752. 4. Coleman, D. L., P. M. Dodek, J. M. Luce, J. A. Golden, W. M. Gold, and J. F. Murray. 1983. Diagnostic utility of fiberoptic bronchoscopy in patients with Pneumocystis carinii pneumonia and the acquired immune deficiency syndrome. Am. Rev. Respir. Dis. 128:795-799. 5. Esterly, J. A. 1968. Pneumocystis carinii in lungs of adults at autopsy. Am. Rev. Respir. Dis. 97:935-937. 6. Fleiss, J. L. 1973. The comparison of proportions from many samples, p. 92-108. In Statistical methods for rates and proportions. John Wiley & Sons, Inc., New York. 7. Galen, M. F., and S. R. Gambino. 1975. Beyond normality: the predictive value and efficiency of medical diagnoses. John Wiley & Sons, Inc., New York. 8. Gill, V. J., G. Evans, F. Stock, J. E. Parrillo, H. Masur, and J. A. Kovacs. 1987. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J. Clin. Microbiol. 25:1837-1840. 9. Gosey, L. L., R. M. Howard, and F. G. Witebsky. 1985. Advantages of a modified toluidine blue O stain and bronchoalveolar lavage for the diagnosis of Pneumocystis carinii pneumonia. J. Clin. Microbiol. 22:803-807. 10. Grocott, R. G. 1955. A stain for fungi in tissue sections and smears using Gomori's methenamine-silver nitrate technic. Am. J. Clin. Pathol. 25:975-979. 11. Hamlin, W. B. 1%8. Pneumocystis carinii. J. Am. Med. Assoc.
204:171-172. 12. Hartman, B., M. Koss, A. Hui, W. Baumann, L. Athos, and T. Boylen. 1985. Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome (AIDS). Chest 87:603-607. 13. Hughes, W. T. 1987. Pneumocystis carinii pneumonitis, vol. I
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JOURNAL
Evaluation of an Indirect Fluorescent-Antibody Stain for Detection of Pneumocystis carinii in Respiratory Specimens VALERIE L. NGl 2.3* DAVID M. YAJKO,"3 LARON W. McPHAUL,"3 IRIS GARTNER,"3 BARRY BYFORD,"3 CAROLE D. GOODMAN,"3 PATRICIA S. NASSOS,"3 CYNTHIA A. SANDERS,"3'4 EDWARD L. HOWES,5'6 GIFFORD LEOUNG,2'4'7 PHILIP C. HOPEWELL,2'4 AND W. KEITH HADLEY"3 Departments of Laboratory Medicine,' Anatomic Pathology,5 and Medicine/ University of California, San Francisco, San Francisco, California 94143, and Clinical Laboratories, Division of Microbiology,3* Department of Anatomic Pathology,6 Medical and Chest Service,4 and Divisions of infectious Diseases and of AIDS Activities, Medical Service,7 San Francisco General Hospital, San Francisco, California 94110 Received 9 August 1989/Accepted
5
January 1990
Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue 0, and Gomori methenamine silver stains for detecting P. caring in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P < 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy. It is possible to diagnose Pneumocystis carinii pneumonia by examining a variety of respiratory specimens, including open lung or transbronchial biopsy (TBBx) samples, bronchoalveolar lavages (BALs), and induced sputa (IS) (1-4, 9, 15, 17, 20, 23, 25). In patients at risk for or with the acquired immunodeficiency syndrome (AIDS), P. carinii pneumonia has been diagnosed from IS specimens with a high degree of sensitivity (1, 15, 20, 25). A variety of stains have been used to detect P. carinii. The three most commonly used stains are Diff-Quik (DQ; a rapid Giemsa-like stain) (1, 20), modified toluidine blue O (TBO) (9), and Gomori methenamine silver (GMS) (10). Recently, mouse monoclonal antibodies, primarily directed against P. carinii cyst wall antigens, have been adapted for examining IS and BALs by using an indirect fluorescent-antibody (IFA) stain (8, 14, 15). This IFA stain was shown to be more sensitive in detecting P. carinii in IS than was either the DQ stain or the TBO stain in a population at risk for or with AIDS (15). However, there was no comparison in that study with the GMS stain, which is used in many microbiology and pathology laboratories for the detection of P. carinii (10). The original IFA procedure (8, 14, 15) has since been modified with the intent of adapting it to general clinical laboratory use (Merifluor-pneumocystis; Meridian Diagnostics, Inc., Cincinnati, Ohio). Here we report the results of a study in which we compared the performance of the modified IFA stain with those of DQ, TBO, and GMS stains for detecting P. carinii in IS, BALs, and TBBx touch preparations obtained from individuals at risk for or with AIDS.
MATERIALS AND METHODS Study design. Respiratory specimens from patients in two independent studies were examined by various stains for the presence of P. carinii. The first study evaluated all IS obtained from asymptomatic patients with AIDS who were entered into a study to examine the efficacy of prophylactic aerosolized pentamidine. Further details of the clinical aspects of this study will be reported elsewhere (G. Leoung, A. B. Montgomery, D. J. Abrams, L. Wardlaw, K. Corkery, D. Busch, S. Gordon, D. W. Feigal, and the San Francisco County Community Consortium, unpublished data). All patients enrolled in this study had survived one episode of P. carinii pneumonia, had completed specific anti-P. carinii therapy at least 15 days and not more than 6 months prior to the beginning of the study, and were asymptomatic at the time of enrollment in the study. Patient enrollment in the study began on 25 April 1988 and was scheduled to continue until 100 patients were enrolled; however, the study was terminated early because of the Food and Drug Administration announcement of its approval of aerosolized pentamidine as an investigational new drug. All 78 patients entered in this study were scheduled to have IS obtained at the time of entry into the study and at week 24. The second study was a prospective evaluation of respiratory specimens obtained from patients with respiratory symptoms who were assumed to have risk factors for or who had AIDS and were referred to the Chest Service at San Francisco General Hospital for further evaluation. Patient selection and evaluation and specimen acquisition were done
reported previously (1, 15, 20). Specimen processing and staining. Mucolysis and the concentration of IS by centrifugation were performed as deas
*
Corresponding author. 975
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NG ET AL.
scribed previously (1, 15, 20). IS lacking alveolar macrophages were not examined for P. carinii. BALs and TBBxs were processed as described previously (2). Two smears (for the first study) and eight smears (for the second study) were made from each specimen and stored at -20°C until staining was performed. DQ and IFA staining were performed according to the instructions of the manufacturers (20); TBO and GMS staining were performed as described previously (9, 10). For the first study, one smear from each specimen was stained with DQ and the remaining smear was stained with IFA. For the second study, two each of the eight smears were stained with DQ, TBO, GMS, and IFA. The stained smears were coded and examined microscopically for P. carinii by an experienced reader (for IFA, V.L.N., D.M.Y., C.A.S., P.S.N., W.K.H.; for DQ, I.G., C.D.G., V.L.N., C.A.S., P.S.N., W.K.H.; for TBO, B.B., V.L.N., W.K.H.; for GMS, L.W.M., E.L.H., V.L.N., W.K.H.). The readers did not know the results of the other stains at the time of examination. Smears stained with DQ were screened at x500 magnification (xlO ocular, x50 oil immersion objective). Smears stained with GMS or TBO were screened at x100 magnification (x 10 ocular, x 10 objective). Any material resembling P. carinii was further examined at x 1,000 magnification (x 10 ocular, x 100 oil immersion objective). Smears stained with IFA were screened at x 200 magnification (x 12.5 ocular, x 16 objective). Any fluorescent material that was observed was further examined at x 500 magnification (x 12.5 ocular, x40 objective). Interpretation of stained smears. A DQ-stained smear was interpreted as positive if cysts, trophozoites, or both were present (Fig. 1A and B). The observation of a single morphologically unequivocal clump of trophozoites was sufficient for interpreting a smear as positive. The TBO- and GMS-stained smears were interpreted as positive if cysts with characteristic morphology (Fig. 1C and D) were observed. The IFA-stained smears were interpreted as positive if at least three well-defined cysts, usually within a clump of P. carinii organisms, were present. Clumps of P. carinii organisms detected by the IFA stain had two distinct morphological appearances: the unequivocal classic honeycomblike appearance (Fig. 1E) or clumps of cysts morphologically consistent with P. carinii but lacking the characteristic honeycomb appearance (Fig. 1F). A smear containing fluorescent material resembling trophozoites in the absence of cysts was not interpreted as positive. Statistical analysis. For the purposes of this study, the identification of P. carinii by DQ, TBO, or GMS stains was considered to be a true-positive result. In contrast, specimens in which P. carinii was detected only by the IFA stain were subjected to a more rigorous set of interpretive criteria in which results from subsequent bronchoscopic specimens, a review of medical records, or both were also considered. Chi-square analysis (6) and sensitivity, specificity, and positive and negative predictive values (7) were calculated as described previously. Follow-up of specimens positive by IFA staining only. Medical records were reviewed for those patients whose specimens had P. carinii, as detected by IFA staining only. The result was considered to be truly positive if (i) the subsequent clinical course was consistent with P. carinji pneumonia, (ii) the patient responded to anti-P. carinii therapy, or (iii) a subsequent BAL specimen obtained within 7 days of the original IS specimen contained P. carinii which was detected by any of the traditional stains. The result was
J. CLIN. MICROBIOL.
A
B
c
D f
b1
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lomm -,**
-w'À
-à
ig,
E
F
FIG. 1. Morphological appearance of P. carinii by various stains. (A) A clump of Pneumocystis cysts stained with the DQ. (B) A clump of Pneumocystis trophozoites stained with DQ. (C) A clump of Pneumocystis cysts stained with TBO. (D) A clump of Pneumocystis cysts stained with GMS. (E) A clump of P. carinii stained with IFA exhibiting the classic honeycomb appearance. (F) A clump of P. carinfi stained with IFA but lacking the classic honeycomb appearance. Magnifications: x2,000 (A to D); x 1,000 (E and F).
considered to be falsely positive if (i) a respiratory pathogen other than P. carinii was identified, (ii) the patient spontaneously improved, or (iii) the patient responded to appropriate therapy which did not include anti-P. carinii therapy. The result was considered to be indeterminate if (i) P. carinii could not be eliminated as the cause of disease or (ii) there was no clinical evaluation within 30 days of specimen
acquisition. Follow-up evaluation of specimens lacking P. carinii (negative specimens). The medical records of patients who had a single IS or BAL specimen that lacked P. carinii were reviewed for a 30-day period after the specimen was obtained. The test results were considered to be truly negative
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FLUORESCENT-ANTIBODY STAIN FOR P. CARINII DETECTION
if (i) P. carinii was not found by any stain in a BAL specimen obtained within 7 days of the original IS specimen, (ii) the respiratory process responded to treatment that did not include anti-P. carinii agents, or (iii) the respiratory process resolved spontaneously and the patient had no new respiratory symptoms. The test results were considered to be falsely negative if (i) the patient had a subsequent bronchoscopy sample which contained P. carinii or (ii) the patient had a progressive or persistent respiratory illness which responded to anti-P. carinii treatment. The test results were considered indeterminate if (i) P. carinii could not be eliminated as the cause of the respiratory disease or (ii) there was no clinical evaluation within 30 days of specimen acquisition. RESULTS For the first study, 95 IS were obtained from 77 patients who were prophylactically administered aerosolized pentamidine. The IS were examined by the DQ and IFA stains. P. carinii was detected in only one specimen and was detected by both stains. Prophylactic aerosolized pentamidine treatment did not alter the morphology of P. carinii as detected by either stain. The patient in whom P. carinii was detected remained clinically well until 16 weeks later, when he developed respiratory symptoms which resolved with standard anti-P. carinii therapy. Of the remaining 76 patients enrolled in this study, 5 patients had a second episode of P. carinii pneumonia during the course of this study. Although P. carinii was detected in IS obtained when these patients became symptomatic, P. carinii was not detected in the IS which were routinely collected as part of this study. The interval between the time that IS was collected as part of the examination for participation in this study and the time of P. carinii infection relapse ranged from 2 to 16 weeks. For the second study, 182 specimens from 130 symptomatic patients were examined by the DQ, TBO, GMS, and IFA stains. These 182 specimens consisted of 124 IS, 56 BALs, 1 TBBx, and 1 lung tissue specimen that was obtained at the time of autopsy. P. carinii was detected by one or more stains in 105 (57.7%) of the 182 specimens. These 105 specimens consisted of 79 IS, 25 BALs, and 1 touch preparation from a lung tissue specimen that was obtained at the time of autopsy. In only 58 (55%) of the 105 positive specimens was P. carinji detected by all four stains. P. carinii was detected in 15 (14%) specimens by three stains and in 11 (10%) specimens by two stains. P. carinii was detected in 1 sample by DQ only, in 1 sample by GMS only, in 2 samples by TBO only, and in 17 samples by IFA only. Thus, P. carinii was detected by one or more of the traditional stains in 88 of 105 P. carinii-containing specimens. The overall performance of each of the stains was as follows. P. carinii was detected in 73 (70%) specimens by DQ, in 75 (71%) by TBO, in 76 (72%) by GMS, and in 95 (90%) by IFA. The increased sensitivity of P. carinii detection with the IFA stain was statistically significant (P < 0.01), whereas there was no statistical difference in the sensitivity of P. carinii detection by the other three stains (0.975 > P > 0.950). There were 17 specimens in which P. carinii was detected by the IFA stain only. Nine of these specimens (from eight patients) contained clumps of P. carinii organisms that had the classic honeycomb appearance. Three of these specimens were true positives based on subsequent BAL speci-
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TABLE 1. Sensitivity, specificity, and positive and negative predictive values for detecting P. carinii in respiratory specimens with each staina No. of results
SanTPb DQ GMS TBO IFA
73 76 75 81
FN FP TN
28 25 26 20
0 0 0 6
63 63 63 57
Sensitivity Specificity (%)b (% 72 75 74 80
100 100 100 90
+
(%)b
+Vm
100 100 100 93
-Pv (%) P
69 72 71 74
a Specimens from patients who had indeterminate clinical courses were not included in these calculations. Abbreviations: TP, true positives; TN, true negatives; FP, false positives; FN, false negatives; +PV, positive predictive value; -PV, negative predictive value. TP was the presence of P. carinii in the smear and the presence of P. carinii pneumonia. FP was the presence of P. carinii in the smear and the absence of P. carinii pneumonia. FN was the absence of P. carinii in the smear and the presence of P. carinji pneumonia. TN was the absence of P. carinii in the smear and the absence of P. carinii pneumonia. Sensitivity was calculated as TP/TP + FN. Specificity was calculated as TN/TN + FP. +PV was calculated as TP/TP + FP. -PV was calculated as TN/TN + FN. b p. carinii organisms detected by DQ, TBO, or GMS stains were considered to be true positives for these calculations. Thus, the specificity was 100% and the positive predictive value was 100%t for these stains.
mens or the clinical course of infection, five specimens were from four patients who had an indeterminate clinical course, and one specimen was a false positive based on clinical response to antibiotics. The remaining eight specimens (from seven patients) contained P. carinii organisms that lacked the characteristic honeycomb appearance but that had a morphology consistent with clumps of P. carinii (Fig. 1F). Three specimens were from two patients who had indeterminate clinical courses, and five specimens were judged to be false positives: one specimen was from a patient who had resolution of his pulmonary symptoms with antimicrobial therapy other than anti-P. carinii therapy; the remaining four specimens came from four patients diagnosed with (i) pulmonary tuberculosis, (ii) disseminated tuberculosis, (iii) Strongyloides stercoralis hyperinfection syndrome, and (iv) disseminated coccidioidomycosis. Thus, of the 17 specimens determined to be positive by the IFA stain only, three were judged to be true positives, eight were judged to be indeterminate, and six were judged to be false positives based on clinical criteria. Of the 95 specimens in which P. carinii was detected by the IFA stain, only 81 were judged to be true
positives.
A total of 77 (42%; 45 IS, 31 BALs, and 1 TBBx touch preparation) of the 182 specimens did not contain P. carinii
when examined by any of the four stains. Of these 77, 57 were considered to be true negatives based on the inability to find P. carinii in subsequent BAL specimens or by resolution of clinical disease without the use of specific anti-P. carinji therapy. Ten specimens were judged to be false negatives based on the clinical response to anti-P. carinii therapy, and 10 specimens were from patients who had indeterminate clinical courses. The sensitivities, specificities, and positive and negative predictive values were calculated for each of the stains (Table 1). Specimens from patients who had indeterminate clinical courses were not included in the calculations. The sensitivity of each of the four stains for the detection of P. carinii ranged from 72 to 80%, the specificity ranged from 90 to 100%, the positive predictive value ranged from 93 to 100%, and the negative predictive value ranged from 69 to 74%.
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DISCUSSION We demonstrated that the commercially available modified IFA stain is more sensitive than the traditional staining methods for detecting P. carinii in respiratory specimens from patients at risk for or with AIDS. This increased sensitivity of P. carinii detection by the IFA stain is in agreement with results obtained by using the original staining procedure (14, 15), indicating that the modifications made by the manufacturer did not significantly alter the performance of the test. An added advantage of the IFA stain was that evaluation of smears, especially those lacking P. carinii organisms, required significantly less time (usually less than 5 min) than the time required for any of the traditional stains. The sensitivities for P. carinii detection of each of the traditional stains ranged from 72 to 75%, in accordance with previously reported sensitivities (1, 9, 15, 20, 25). Viewed in an alternate manner, the false-negative rate for the traditional stains ranged from 25 to 28%. There were three different groups of specimens contributing to this falsenegative rate. The first group (three IS, one BAL) was obtained from individuals who had a clinical response to anti-P. carinii therapy in the absence of a laboratory diagnosis of P. carinii pneumonia. The interpretation of this group of specimens as false negatives is debatable, in that respiratory processes other than P. carinii pneumonia can respond clinically to anti-P. carinii therapy. The second group (six IS) was from individuals in whom P. carinii was detected in a subsequent BAL specimen. Many factors probably contributed to the inability to find P. carinii in the original IS, including the overall burden of P. carinii in the patient as well as the ability of sputum induction to provide a sample from the infected area. The third group (40 IS, 16 BALs) was from individuals in whom P. carinii was detected by one or two, but not all three, of the traditional stains. In fact, it is noteworthy that only 55% of all specimens containing P. carinii in the second study were positive by all four stains. The discrepancies most often occurred with specimens which contained few to moderate numbers of organisms and were most likely attributable to an uneven distribution of a limited number of organisms among slides. It is of interest that although the IFA stain demonstrated a statistically significant (P < 0.01) increased sensitivity over the traditional stains for P. carinii detection in the second study, the presence of P. carinii as detected only by the IFA stain did not always correlate with clinical P. carinii pneumonia. Clinical evaluation revealed that six of these specimens that were positive by IFA staining only from six patients were in fact false positives by clinical criteria. Although P. carinii was detected in respiratory specimens, therapy directed against the other pathogens that were recovered (i.e., Mycobacterium tuberculosis, Coccidioides immitis, and S. stercoralis) resulted in clinical recovery. However, it must be emphasized that of the other 11 specimens from nine patients in which P. carinii was detected only by the IFA stain, P. carinii could not be eliminated as the cause of the respiratory illness in six patients, and P. carinii was presumed to be the cause of the respiratory illness in the other three patients, as evidenced by a clinical response to anti-P. carinii therapy. We reevaluated our data using more stringent morphological criteria to determine whether these false-positive results might have been avoided. We stipulated that a classic honeycomb clump of P. carinii organisms, in addition to at least three well-defined cysts, had to be present before a
positive result was assigned. However, by these criteria, only 51% of the P. carinii-containing specimens in this study would have been interpreted as positive and we would have erroneously interpreted 49% of P. carinii-containing specimens as negative. In addition, one of the false-positive specimens also had a classic honeycomb clump present. Thus, strict adherence to rigid morphological criteria not only would dramatically decrease the sensitivity of the IFA stain but would not eliminate the problem of false-positive results. The false-positive rate of the traditional stains is not known, because it has been accepted that the detection of P. carinii by one of these stains is diagnostic of P. carinii pneumonia and necessitates appropriate anti-P. carinii therapy. Although P. carinii has not been found in BALs from asymptomatic human immunodeficiency virus type 1-infected individuals (15, 22; J. D. Lundgren, M. Orholm, T. L. Nielsen, J. Iversen, J. Hertz, and J. O. Nielsen, IV Int. Conf. AIDS, abstr. no. 7144, 1988), it has been well documented that in a subset of patients with AIDS and P. carinii pneumonia, the pulmonary burden of P. carinii persists virtually unchanged for at least 35 days after appropriate therapy against P. carinii pneumonia has been given, even with clinical resolution of symptoms (12, 23, 26). Thus, false-positive results would be expected to occur most frequently in individuals with a history of P. carinii pneumonia. Our one DQ- and IFA-positive result in the first study suggests that this is not a frequent occurrence; however, this one positive result demonstrated that P. carinii could still be detected in IS by both the IFA and DQ stains for at least 18 weeks after appropriate anti-P. carinii treatment in an asymptomatic individual receiving prophylactic therapy. Most individuals in the general U.S. population have serological evidence of exposure to P. carinii by adulthood (16, 24), and it is assumed that most adults harbor P. carinii in small numbers. Studies (5, 11, 27) of lungs obtained at the time of autopsy from patients who were not clinically suspected of having died from P. carinii pneumonia have demonstrated that 2 to 15% of patients have detectable P. carinii organisms; most of these cases occurred in patients who were presumed to be immunosuppressed by virtue of underlying malignancies. It has also been well documented in a rat model system that immunosuppression with steroids allows multiplication of the latent organism to detectable levels (13). Although the frequency of P. carinii organisms present concomitantly with another pulmonary pathogen in an immunosuppressed individual with a respiratory illness is not known, there have been reports of patients with AIDS in whom P. carinii pneumonia was coexistent with Legionella sp. (19), Chlamydia trachomatis (18), or Bordetella pertussis (21). These data suggest that P. carinii can be detected at a low frequency in individuals in whom P. carinii pneumonia is not suspected and that activation from the latent state may occur with a variety of conditions. All of the patients from whom false-positive specimens were obtained fit one or more of the categories described above. In summary, the IFA stain was more sensitive than the DQ, TBO, or GMS stains for detecting P. carinii. An added advantage was that less time was required to examine an IFA-stained smear. The ability of this more sensitive stain to detect P. carinii in asymptomatic individuals or in individuals with subclinical disease makes it essential that laboratory findings be carefully considered in conjunction with clinical evaluation of the patient.
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FLUORESCENT-ANTIBODY STAIN FOR P. CARINII DETECTION
ACKNOWLEDGMENTS We gratefully acknowledge the helpful comments and suggestions of Joseph Kovacs, Vee Gill, Allen Nickol, and Bruce A. Clinton; the assistance with statistical analysis of Lewis B. Sheiner and Stuart Beal; the clinical assistance of Erin McGinty; the expert technical assistance of Histopathology Laboratory technologists Cel Rutledge, Rose Nunez, Martha Glenn, and Norma Swain and the pulmonary function technologists Joe Viana, Althanette Johnson, and Miriam Bryant; the cooperation of the pulmonary fellows in the Chest Service; the input of Chris Jacques; the administrative assistance of Dennis Dunlop; and the reagents and materials supplied by Meridian Diagnostics, Inc. We also gratefully acknowledge the partial financial support for this study provided by Meridian Diagnostics, Inc., and Lyphomed, Inc. (Melrose Park, 111.). LITERATURE CITED 1. Bigby, T. D., D. Margolskee, J. L. Curtis, P. F. Michael, D. Sheppard, W. K. Hadley, and P. C. Hopewell. 1986. The usefulness of induced sputum in the diagnosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Am. Rev. Respir. Dis. 133:515-518. 2. Blumenfeld, W., E. Wagar, and W. K. Hadley. 1984. Use of the transbronchial biopsy for diagnosis of opportunistic pulmonary infections in acquired immunodeficiency syndrome (AIDS). Am. J. Clin. Pathol. 81:1-5. 3. Broaddus, C., M. D. Dake, M. S. Stulbarg, W. Blumenfeld, W. K. Hadley, J. A. Golden, and P. C. Hopewell. 1985. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann. Intern. Med. 102:747-752. 4. Coleman, D. L., P. M. Dodek, J. M. Luce, J. A. Golden, W. M. Gold, and J. F. Murray. 1983. Diagnostic utility of fiberoptic bronchoscopy in patients with Pneumocystis carinii pneumonia and the acquired immune deficiency syndrome. Am. Rev. Respir. Dis. 128:795-799. 5. Esterly, J. A. 1968. Pneumocystis carinii in lungs of adults at autopsy. Am. Rev. Respir. Dis. 97:935-937. 6. Fleiss, J. L. 1973. The comparison of proportions from many samples, p. 92-108. In Statistical methods for rates and proportions. John Wiley & Sons, Inc., New York. 7. Galen, M. F., and S. R. Gambino. 1975. Beyond normality: the predictive value and efficiency of medical diagnoses. John Wiley & Sons, Inc., New York. 8. Gill, V. J., G. Evans, F. Stock, J. E. Parrillo, H. Masur, and J. A. Kovacs. 1987. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J. Clin. Microbiol. 25:1837-1840. 9. Gosey, L. L., R. M. Howard, and F. G. Witebsky. 1985. Advantages of a modified toluidine blue O stain and bronchoalveolar lavage for the diagnosis of Pneumocystis carinii pneumonia. J. Clin. Microbiol. 22:803-807. 10. Grocott, R. G. 1955. A stain for fungi in tissue sections and smears using Gomori's methenamine-silver nitrate technic. Am. J. Clin. Pathol. 25:975-979. 11. Hamlin, W. B. 1%8. Pneumocystis carinii. J. Am. Med. Assoc.
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