The Value of Pneumocystis carinii Antibody and Antigen Detection for Diagnosis of Pneumocystis carinii Pneumonia after Marrow Transplantation'-3 JOEL D. MEYERS,4 LINDA L. PIFER,s GEORGE E. SALE,6 and E. DONNALL THOMAS7
SUMMARY ________________________________________________________ Thirty-three marrow transplant patients with Pneumocystis carinii pneumonia were studied to determine the usefulness of antibody and antigen detection in the diagnosis of pneumocystis infection. Antibody against P. carinii was present in one half of all patients tested, and changes in antibody titer were not helpful diagnostically. P. carinii antigen was detected by counterimmunoelectrophoresis in the serum of 22 of 28 patients tested. Fifteen of 28 patients had antigen detected before or within 72 h after diagnosis. However, antigen was also present in 35 of 52 marrow transplant patients with viral or idiopathic pneumonia, in 11 of 25 transplant patients with no pneumonia, and in 22 of 28 other patients with pulmonary infiltrates. Only 1 of 50 normal marrow donors had detectable antigenemia. Detection of this antigen does not appear to establish the diagnosis of P. carinii pneumonia in the absence of other clinical or histologic data. The data may suggest that subclinical infection with this agent is more common than was previously recognized.
Pneumocystis carinii is a less common cause of nonbacterial pneumonia after marrow transplantation than is either cytomegalovirus (CMV) or idiopathic pneumonia (1-4). However, 11% (28 of 248) of patients who received (Received in original form May 29, 1979 and in revised form August 16, 1979) 1 From the Divisions of Infectious Diseases and Oncology, Department of Medicine, and the Department of Pathology, University of Washington School of Medicine, Seattle, Wa.; the Fred Hutchinson Cancer Research Center, Seattle, Wa.; and the Infectious Diseases Service, St. Jude Children's Research Hospital, Memphis, Tenn. 2 Supported by Grant No. CA 15704 and No. CA 18029 From the National Cancer Institute, U.S. Department of Health, Education, and Welfare. 3 Presented in part at the 18th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, Ga., October 1978. 4 Requests for reprints should be addressed to Dr. Joel D. Meyers, Division of Infectious Diseases,
allogeneic marrow transplants before the use of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis developed P. carinii pneumonia, with mortality of 76 %. Determination of the cause of pneumonia is difficult in immunosuppressed populations without invasive procedures (5-8). Newer techniques that detect both pneumocystis antibody and antigen have been proposed as alternatives to invasive procedures for the diagnosis of P. carinii infection (9-11). We examined the usefulness of these techniques in the diagnosis of P. carinii pneumonia after marrow transplantation. Fred Hutchinson Cancer Research Center, 1124 Columbia St., Seattle, Wa. 98104. 5 Present address: Department of Pediatrics, University of Tennessee Center for the Health Sciences, Memphis, Tenn. 6 Supported in part by a Junior Faculty Clinical Fellowship from the American Cancer Society. 7 Recipient of Research Career Award No. AI 02425 from the National Institute of Allergy and Infectious Diseases.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 120, 1979
MEYERS, PIFER, SALE, AND THOMAS
Methods Patients were admitted to the study if they had clinical pneumonia and had P. carinii cysts demonstrated in pulmonary tissue. Tissue specimens were examined by the Grocott-Gomori methenamine silver technique (12) and by toluidine blue 0 staining (13). In addition, all histologic material from patients who received transplants between 1969 and mid-1973 was reviewed in late 1973 by independent observers as part of a separate study of interstitial pneumonia (3). Of 33 patients, five were studied prospectively and 28 retrospectively. All serums were studied retrospectively. Survival was considered in relation to recovery from the studied episode of penumocystis pneumonia. Several survivors died subsequently with relapse of their malignancy and/or other infections. Adequate treatment was considered to be administration of pentamidine isethionate at a dose of 4 mg/kg/day given intramuscularly or TMP-SMX at a dose equal to or greater than 10 mg/kg/day of the trimethoprim component. Two patients received pyrimethamine at the recommended dose instead of trimethoprim. Antibody against P. carinii was measured by indirect immunofluorescence at the Center for Disease Control, Atlanta, Ga.; a titer of 1: 16 or greater was considered positive (14). Circulating P. carinii antigen was detected by counterimmunoelectrophoresis (CIE) using rabbit antiserums prepared against both human and murine pneumocystis strains (11). Results were equivalent with both antiserums. Absorption experiments showed that antibody activity could be removed by absorption with P. cariniiinfected human or rat lungs, cultured and purified P. carinii organisms, infected chick embryo lung (CEL) cells, and soluble P. carinii antigen. Activity was not affected by absorption with normal human lung, normal murine lung, uninfected CEL cells, mouse liver powder, Candida albicans, Escherichia coli, or Staphylococcus aureus (11). This antibody forms precipitin bands by CIE when reacted with soluble P. carinii antigen and serum from humans and rats with proved P. carinii pneumonia, but not when reacted with CEL cell homogenates or normal human lung homogenates. For both of these procedures, patients' serums were stored at -70 0 C until testing. Results
Clinical data. Thirty-three cases of histologically proved pneumocystis pneumonia occurred in 424 allogeneic transplant patients. Incidence increased from no cases in 1969 to 1971 (27 transplants) to 11 cases in 1976 (69 transplants) (table 1). The absence of cases before 1972 is unexplained, but may have been related in part to shorter survival in the early years of marrow transplantation. Prophylaxis with oral adminis-
TABLE 1 YEARLY INCIDENCE OF PNEUMOCYSTIS CAR/Nil PNEUMONIA AFTER ALLOGENEIC MARROW TRANSPLANTATION
1969 1970 1971 1972 1973 1974 1975 1976 1977" 1978t Total
Total no. of Transplants
Patients with P. carinii Pneumonia (no.) Incidence (%)
5 7 15 32 36 53 58 69 99 50
0 0 0 3 3 5 6 11 5 0
0 0 0 9 8 9 10 16 5 0
" Prophylaxis with trimethoprlm·sulfamethoxazole (TMp·SMX) was begun In mid·1977. t First 6 months.
tration of TMP-SMX was begun in mid-1977, and only 1 case has occurred among patients receiving TMP-SMX. Twenty-nine of the cases occurred between 5 and 14 wk after transplantation, with median onset at 9 wk. Eight patients survived their infection. Four of these had onset after week 14 (figure 1). Average and median age of patients was 22 yr (range, 3 to 56 yr), and 23 of 33 patients were male. Age, although not sex predominance, was similar to the marrow transplant population in general. Twenty-five patients received transplants for hematologic malignancy (5 survivors), and eight received them for aplastic anemia (3 survivors). Twenty-five patients underwent diagnostic biopsy procedures. Twenty-two had open-lung biopsy, 17 as the sole procedure and five with simultaneous bronchoscopic biopsy. Three patients had bronchoscopic biopsy alone. Twentyfour of these procedures were diagnostic for P. carinii, including 21 of 22 open biopsies and the 3 bronchoscopic biopsies that were performed alone. Nine cases were diagnosed at autopsy, including the one in a patient with a negative open-lung biopsy. Among the 5 patients with simultaneous open and bronchoscopic procedures (forceps biopsy in 4 cases and needle biopsy in one), only 3 cases were diagnosed by bronchoscopy. Fifteen of 33 patients had mixed infections, 13 with CMV and two with Aspergillus. Fourteen of these 15 died. Eighteen patients had P. carinii infection alone; seven of 16 who received appropriate treatment survived their infection.
PNEUMOCYSTIS CAR/NIl PNEUMONIA
D Survivi ng case ~ Fatal case
Postmortem findings: P =Pneumocystis C=CMV A= Aspergillus
N = No Autopsy
* Pneumocystis relapse
2 Number of weeks after transplantation Fig. 1. Occurrence of Pneumocystis carinii pneumonia by week after marrow transplant. CMV = cytomegalovirus.
Antibody studies. Twenty-five patients had pre transplant serum available, and 13 had detectable antibody. Pretransplant serums from 15 patients without pneumonia and 11 patients with idiopathic interstitial pneumonia diagnosed by open-lung biopsy were also tested. Seven in each group had detectable titers. The geometric mean titer in each group was I: 16. "Acute" serums were obtained from the 25 patients with P. carinii a median of 4 days before clinical diagnosis (range, 39 days before to 4 days afterwards). Twelve had detectable antibody. "Convalescent" serums were taken a median of 16 days after clinical diagnosis (range, 6 to 82 days). Thirteen had detectable antibody. Five patients (3 survivors) had a 4-fold or greater increase in antibody titer. Ten patients with idiopathic interstitial pneumonia also had acute and convalescent serums tested. These were drawn a median of 3 days before clinical diagnosis (range, 0 to 9 days before) and 31 days after clinical diagnosis (range, 10 to 63 days), respectively. Three of these 10 patients had a 4-fold or greater increase in antibody titer even though P. carinii had not been found at open-lung biopsy. Antigen studies. Twenty-eight patients had serums tested for P. carinii antigen. An average of 2.1 sllecimens (range, 1 to 6) obtained from 12 days before to 17 days after diagnosis was tested; 22 of 28 patients had serums available within 3 days of diagnosis. Twenty-two patients (79%) had detectable antigenemia (table 2). Eighteen of 25 positive serum samples were
taken an average of 5 days after diagnosis (range, to 17 days), whereas seven were taken an average of 5 days before clinical diagnosis. In 15 of 22 patients with positive results, the first positive sample was taken either before diagnosis (7 patients) or within 72 h after diagnosis (8 patients). Five of the 6 patients without detectable antigen had at least 1 serum sample available during this interval. Fifty-two "control" patients with either viral (25 CMV and I herpes simplex virus) or idiopathic pneumonia diagnosed by open-lung biopsy or autopsy examination had an average of 3.7 serum samples tested for pneumocystis antigen. These specimens were taken from 28 days before to 28 days after diagnosis. Forty-four of 52 patients had samples available within 3 days of diagnosis of pneumonia. Thirty-five patients (67 %) had detectable antigen, 24 of them either before or within 72 h after the diagnosis of pneumonia. Twenty-five transplant patients with no clinical evidence of pneumonia had serums taken I, 4, 7, and 10 wk after transplant. Eleven patients (44 %) had detectable antigen: eight were positive once, I patient on 2 occasions, and 2 patients had detectable antigen in all specimens tested. Eight of these II patients had detectable antigen the first wk after transplant. These 77 "control" patients were similar in age (median, 18 yr; range, 2 to 55 yr) and underlying illness (hematologic malignancy in 60, aplastic anemia in 17) to the study group. However, there was less male predominance (57%).
MEYERS, PIFER, SALE, AND THOMAS
PNEUMOCYSTIS CARINfI ANTIGENEMIA AFTER MARROW TRANSPLANTATION
No. with Detectable Antigen (%)
Marrow transplant patients P. carinii pneumonia Viral pneumonia Idiopathic pneumonia No pneumonia
28 26 26 25
22 18 17 11
Other patients Patients with pulmonary infiltrates· Normal marrow donors
22 (79) 1 (2)
(79) (69) (65) (44)
No. Detectable before or within 72 h after Diagnosis (%)
15 (54) 12 (46) 12 (46)
Average no. of Specimens Tested
2.1 3.9 3.5 4 1.1
• Patients with undiagnosed pulmonary infiltrates whose serums were submitted for antibody testing to Legionnaires' bacillus.
Additional specimens from 50 normal marrow donors and from 28 patients with pulmonary infiltrates, only four of whom were immunosuppressed, whose serums were submitted for measurement of antibody to Legionnaires' bacillus were tested. Twenty-two (79 %) of the latter group, but only I normal marrow donor, had detectable antigen (table 2). Discussion P. carinii pneumonia is similar to other non-
bacterial pneumonias that occur after marrow transplant in time of onset and eventual outcome (2-4). There were no features that distinguished these patients from marrow transplant patients with other pneumonias or from those without pneumonia. The survival rate was only 44 % among those with pure P. carinii infection despite appropriate treatment. Openlung biopsy is our diagnostic procedure of choice. In only 3 of 5 patients with simultaneous open and bronchoscopic biopsies were cases diagnosed by the bronchoscopic procedure (8). A similar proportion of patients with P. carinii pneumonia, idiopathic interstitial pneumonia, or no pneumonia had antibody against P. carinii detectable before transplant. This suggests that those with P. carinii pneumonia after transplant were no more likely to have had recent infection before transplant than were those without subsequent P. carinii pneumonia. Furthermore, an equal proportion of patients with P. carinii and idiopathic pneumonia had increases in antibody in convalescent serums. Thus, detection of antibody was not diagnostically useful in this population, similar to studies in other populations (15, 16). Twenty-two of 28 patients (79%) with P. carinii pneumonia had detectable P. carinii anti-
gen during the acute stages of illness. This prevalence was comparable to that found in the original description of P. carinii antigenemia (62 %) (ll). One half of our patients (15 of 28) had antigenemia preceding or within the first 72 h after diagnosis, suggesting a potential diagnostic role for this test. This conclusion must be modified by the prevalence of antigenemia observed in the other groups: a substantial proportion of marrow transplant patients with other pneumonias, other patients with pulmonary infiltrates, and marrow transplant patients without pneumonia had detectable P. carinii antigen. The apparent common denominator among these patients was that all had recent pulmonary insults, including total-body irradiation and/ or cyclophosphamide for pre transplant conditioning and posttransplant methotrexate among the recipients of marrow transplants. The significance of detecting P. carinii antigen in marrow transplant patients with other pneumonias, and in those with no pneumonia, is unclear. These patients did not have P. carinii pneumonia by standard criteria, and several patients with idiopathic pneumonia recovered with minimal or no specific antipneumocystis therapy. Several explanations may be considered: (1) Antigen detection is nonspecific. However, only one of 50 normal marrow donors, and none of 120 normal children (ll) had detectable antigen. (2) Immunosuppressed patients have subclinical P. carinii infection and antigenemia without clinical pneumonia. Fifteen of 100 leukemic children without pneumonia had detectable antigen in the study of Pifer and associates (ll). This may represent spontaneous reactivation of P. carinii or release of pneumocystis antigen from the lung by other pulmonary insults. The frequent occurrence of antigenemia in
PNEUMOCYSTIS CARINII PNEUMONIA
our patients may have been due to pulmonary damage associated with marrow transplantation. (3) The antigen is associated with pulmonary pathology, but is not specific for P. carinii. However, absorption experiments failed to show activity of this antibody against normal human lung homogenates or uninfected CEL cell homogenates by CIE. These possibilities will require further study. At this time, it does not appear that detection of this antigen alone, in the absence of adjunctive clinical or histologic data, is adequate to establish the diagnosis of P. carinii pneumonia. However, the possibility that antigenemia may represent colonization or "subclinical" infection with P. carin ii, perhaps in the "trophozoite" form (17), has not been excluded.
Acknowledgment Pneumocystis antibody studies were arranged through Alexander J. Sulzer, Ph.D., Bureau of Laboratories, Center for Disease Control, Atlanta, Ga. Serums originally submitted for detection of antibody to Legionnaires' bacillus were obtained from James J. Plorde, M.D., Chief, Infectious Diseases and Clinical Microbiology, Seattle Veterans Administration Hospital, Seattle, Wa. The writers acknowledge with gratitude the laboratory assistance of Linda Freeman, Diane Woods, Pamela Hanks, and Lila Day; the manuscript assistance of Janis Korbol; and the editorial assistance of Arnold L. Smith, M.D., Chief, Division of Infectious Disease, Children's Orthopedic Hospital and Medical Center, Seattle, Wa.
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allogeneic bone marrow grafts. J Infect Dis 1977; 136:754-67. Rosen P, Armstrong D, Ramos C. Pneumocystis carinii pneumonia: a clinicopathologic study of twenty patients with neoplastic diseases. Am J Med 1972; 53:428-36. Greenman RL, Goodall PT, King D. Lung biopsy in immunocompromised hosts. Am J Med 1975; 59:488-96. Pennington JE, Feldman NT. Pulmonary infiltrates and fever in patients wit1l. hematologic malignancy. Assessment of transbronchial biopsy. Am J Med 1977; 62:581-87. Petersen DL, Sale GE, Silvestri Re, Huseby JS, Hudson LD. Open lung biopsy is superior to transbronchial lung biopsy in immunosuppressed patients with interstitial pneumonia (abstract). Am Rev Respir Dis 1978; 117 (Suppl: 164). Meuwissen JHET, Tauber I, Leeuwenberg AD EM, Beckers PJA, Sieben M. Parasitologic and serologic observations of infection with Pneumocystis in humans. J Infect Dis 1977; 136:43-49. Lau WK, Young LS. Immunofluorescent antibodies against Pneumocystis carinii in patients with and without pulmonary infiltrates (abstract). Clin Res 1977; 25:379A. Pifer LL, Hughes WT, Stagno S, Woods D. Pneumocystis carinii infection: evidence for high prevalence in normal and immunosuppressed children. Pediatrics 1978; 61 :35-41. Grocott RG. A stain for fungi in tissue sections and smears. Am J Clin Pathol 1955; 25:975-79. Chalvardjian AM, Grawe LA. New procedure for the identification of Pneumocystis carinii cysts in tissue sections and smears. J Clin Pathol 1963; 16:383-84. Norman L, Kagan IG. A preliminary report of an indirect fluorescent antibody test for detecting antibodies to cysts of Pneumocystis carinii in human sera. Am J Clin Pathol 1972; 58: 170-76. Singer C, Armstrong D, Rosen PP, Schottenfeld D. Pneumocystis carinii pneumonia: a cluster of eleven cases. Ann Intern Med 1975; 82: 772-77. Singer C, Armstrong D, Rosen PP, Walzer PD, Yu B. Diffuse pulmonary infiltrates in immunosuppressed patients. Prospective study of 80 cases. Am J Med 1979; 66: 110-20. Pifer LL, Hughes WT, Murphy MJ Jr. Propagation of Pneumocystis carinii in vitro. Pediatr Res 1977; 11:305-16.