A New Enzyme Immunoassay Specific for Blastomycosis1 •2
CHING Y. LO and ROBERT H. NOTENBOOM
Introduction The serodiagnosis of blastomycosis has been met with pessimism. "Serodiagnosis of histoplasmosis and blastomycosis remains stalled . . . ultrasensitive assays improved sensitivity but sacrifices specificity" (1). Indeed, sera from all patients with activehistoplasmosis exhibited falsepositive reactions in an enzyme immunoassay (EIA) using blastomyces yeast antigen (2). In fact, these false-positive EIA antibody titers were often as high as or higher than their EIA titers to histoplasma yeast and histoplasmin antigens. Wehave designed a new version ofEIA that appears to break through the obstacle of cross-reactions with histoplasmosis. In the first part of this study, the familiar problem of cross-reactions using commercial antigen in a conventional EIA is illustrated. In the second part, a specific EIA based on the principle of antigen-capture is described. Methods Sera Sera selected for this study were among clinical specimens submitted for the differential diagnosis of mycotic diseases. Relevant clinical histories and/or diagnoses were provided by the attending physicians. The sera were characterized according to the criteria as stated by the American Thoracic Society on laboratory diagnosis of mycotic infections (3). Rheumatoid factors (RF) were measured in international units (IV) by a Beckman ICS laser nephelometer (Beckman Instruments, Fullerton, CA). RF under 60 IU/ml wereconsidered negative. Normal sera were obtained from 23 of our staff. Immunodiffusion Tests Commercial immunodiffusion (10) kits for the detection of antibodies to the A antigen of Blastomyces dermatitidis, or to the H and M antigens of Histoplasma capsulatum, or to the F antigen of Coccidioides immitis, including reference rabbit antisera, were purchased from Nolan/Scott Biological Laboratories (Atlanta, OA). ID tests were conducted according to the manufacturer's instructions. Complement Fixation 'Tests Yeast-phase antigens of B. dermatitidis and H. capsulatum and coccidioidin were pre84
SUMMARY A highly .peclflc .nzym. Immunoassay (EIA) _s davaloped for the serodiagnosis of blastomycosis. Among the .arll.st sera avallabl. from eight active c.... of bl..tomycosls, seven were reectIve by EIA, th.... by Immunodiffusion, and th.... by complement fixation. Th. one seronegative case __ assocIated with acquired Immunod.flcl.ncy syndrome (AIDS). No c~reactlon __ observed with sera from 12 patl.nts with active hlstoplasmo.ls or from five patl.nts with active coccidioidomycosis or from 23 healthy p.l'lIOn •. In contrast, all patl.nts' sera c~ In conventional EIAs based on comm.rclallmmunodlffuslon antlg.ns. The blastomycosis-specific EIA seems to offer sensitivity without compromising specificity. Rh.umatold factora, which could cause falss-posltlve raactlons, _ra controlled for. AM REV RESPIR DIS 1910; 141:84-88
pared and used in standard complement fixation (CF) test (4). CF titers, defined as the reciprocal of the highest serum dilution giving no hemolysis, were considered insignificant below 8.
Conventional EIA A conventional version of EIA was set up by coating 100 III of commercial antigens on polystyrene microtiter plates with flat-bottomed wells (Immulon114 II; Dynatech Laboratories, Alexandria, VA). The assay wasempirically optimized by comparing the various coating parameters such as antigen concentrations, coating temperatures, and coating buffers (see RESULTS). In the optimization procedure, the commercial rabbit antiserum, diluted 1:100, was used in conjunction with a peroxidase-labeledgoat antirabbit IgO (H and L chains) (Cappel Laboratories, Cochranville, PA) diluted 1:2,000. Then the assay was applied to the detection of human antibodies by substituting the antirabbit conjugate with an antihuman conjugate. The conventionalEIA wasperformed as follows. The antigen-coated plates were washed four times with 0.01 M sodium phosphatebuffered saline (pH, 7.2) containing 0.05"70 (vol/vol) 1\veen-20(pBSIT). Th block anyexcess binding sites on the plastic, 100 III of a 1% (wt/vol) bovine serum albumin (BSA) in 0.1 M sodium carbonate buffer at pH 9.6 were added. The plates were incubated at 37° C for 1 h and then washed three times with PBS/To Human sera were preheated at 56° C for 30 min. 1\vofold dilution series were prepared in PBS/T/BSA, and 100IIIof the diluted samples were added to the coated plates. The plates were incubated at 37° C for 1 h, and then washed four times. Peroxidase-labeled goat antihuman IgO (H and L chains) (MilesYeda, Kankakee, IL) was added at I:2,000dilution in PBS/T/BSA containing 1% (vol/vol) normal goat serum. After incubation at 37° C
for I h, the plates were washed four times, followed by the addition of chromogenic substrate solutions. The chromogen of choice was 5-aminosalicylic acid (5AS) for conventional EIA because of two reasons. First, conventional EIAs were highly nonspecific (see RESULTS) and the use of a chromogen that had a weaker signal helped to scale down the magnitude of the cross-reactions. Second, the chromogen with a weaker signal was useful in titrations to determine the optimal concentration of antigen for coating (figure 1). Substrate solutions were prepared as follows: 80 mg of 5AS (K & K Labs, Plainview, NY) was dissolved in 100ml of hot water (65 to 70° C) and stored at 4°C for a maximum of 1 wk. The 5AS solution was brought to room temperature, and immediately before use the pH was adjusted to 6.0 with 1 N NaOH. To 9.9 ml of the 5AS, 0.1 ml of 0.5 % H 202 was added. After 1 h of color development with 200 III of 5AS in the dark, absorbance was read at 450 nm in a Titertek Multiskan (Eflab Oy, Helsinki, Finland). Titer of the conventional EIA was defined as the reciprocal ofthe highest serum dilution that gave optical density units of 0.1 (± 0.02).
Production of Specific Anti-A Antibodies in Rabbits Immune precipitates were obtained by mix-
(Received in original form October 7, 1988 and in revised form June 5, 1989) 1 From the Serology Section, Ontario Ministry of Health, Laboratory Services Branch, Toronto, Ontario, Canada. 2 Correspondence and requests for reprints should be addressed to Ching Y. Lo, Serology Section, Ontario Ministry of Health, Laboratory Services Branch, P.O. Box 9000, Terminal A, Toronto, Ontario, Canada M5W IR5.
A NEW ENZYME IMMUNOASSAY SPECIFIC FOR BLASTOMYCOSIS
ing commercial A antigen and referenceantiA antiserum (Nolan/Scott BiologicalLaboratories, Atlanta, GA). The supernatant wasdiscarded after centrifugation (1,000x g for 30 min at 4° C) and the precipitates were vortexed in sterilePBS. The precipitates wereleft standing at 4° C for 1wk to reform, and then they were collected by centrifugation. A New Zealand white rabbit was immunized subcutaneously with 0.5 mg of the immune precipitatesmixedin 0.2 ml of Freund's incomplete adjuvant, followedby three intravenous boosters without adjuvant over the next 4.5 months. During the fifth month, the rabbit wasbled,and the immune serawasfractionated three times with ammonium sulfate (330/0 vol/vol final concentration). The immune rabbit IgG was redissolved in PBS, dialyzed, preservedwith 0.01% merthiolate, aliquoted, and stored at -70° C until use.
Blastomycosis-specific Enzyme Immunoassay Microtiter wells were coated with 100 j.11 of immune rabbit IgG (200ng per well)in 0.1M sodium bicarbonate-carbonate buffer (pH, 9.6) at 4° C for 2 days.After four washeswith PBS/T, the plates were blocked with 100 j.11 of diluent (PBS/T/BSA containing 1% normal rabbit serum and 1% normal goat serum) for 1hat 37° C. This blocking step wasnecessary in order to reduce nonspecific binding. After threewasheswithPBS/T, 100j.11 of commercial A antigen, diluted 1:320with the diluent and preincubated at 37° C for 1 h, were added. The plates were incubated at 37° C for 1 h and washed four times. Human sera were diluted 1:400with the diluent and heated at 56° C for 30 min. After cooling to room temperature, the heated samples wereadded to the plates containing captured A antigens and incubated at 37° C for 1 h. After five washings with PBS/T, 100 j.11 of peroxidaselabeled goat antihuman IgG were added at 1:2,000dilution. This reagent waspreincubated in the diluent at 37° C for 1 h before use. After 1 h of incubation at 37° C, the plates were washed five times before the substrate solutions wereadded. Because the blastomycosis-specificEIA wasfree of cross-reactions (see RESULTS), a chromogen such as orthophenylenediamine (OPD) that gave a much higher signal than 5AS (figure I) was employed. OPD (ICN Pharmaceuticals Inc., Plainview, NY) substrate solutions were prepared immediately before use (5). Reactions with 180 j.11 of OPD wereallowed to proceed for 20 min, then stopped with 20 j.11 of IO M H 2S04 and read at 492 nm. The Multiskan Reader (Titertek)wasblanked with wells identical to the test except for the omission of antisera. All tests and serum controls were done in duplicate. The assay was repeated on any serum that exhibited a coefficient of variation exceeding 15% betweenduplicates. Contents in the serum control wells were identical to the test wells except for the omission of the antigen. The assay was considered reactive
when the mean absorbance of the test wells was at least 0.2 optical density units above the mean absorbance of the serum control wellsand the ratio of the former to the latter was at least 2.5 times.
were based on the arithmetic mean of 18 replicate tests for each combination of coating conditions, and the coefficient of variation was below 70/0 in all cases (data not shown). The amount of antigen bound was found to increase with temperature in both buffer systems. At 4° or 23° C, no significant difference in coating was observed between the two buffers. However, coating in PBS at 37° C generated the highest signals. Therefore, the optimal conditions for coating were chosen to be 37° C overnight in PBS. Similarly, conventional EIAs were developed for histoplasmosis and coccidioidomycosis using commercial immunodiffusion antigens. In the histoplasmosis group (table 1), the conventional EIA titer correlated very well with the CF titer (r = 0.80, p < 0.01). In the coccidioidomycosis group (table 2), the correlation between CF titer and EIA titer was significant (r = 0.87, P < 0.05) except for one patient (EP). Nonspecificities ofconventional EfA. When the sera from the 12 patients with . histoplasmosis were tested by EIAs using plates coated with commercial immunodiffusion antigens, cross-reactions were evident (table 1). These results agreed with the study by Lambert and George (2) that patients with histoplasmosis had EIA antibodies that cross-reacted with commercial blastomyces yeast antigen (Meridian Diagnostics, Cincinnati, OH) with titers as high or higher than their EIA titers to Histoplasma antigens. Simi-
Conventional EfA Optimization ofconventional EfA. The optimal dilution of commercial antigen (lot BDAD2) for coating was found to be 1:320 using 5AS as the substrate (figure 1). The 5AS curve exhibited a decrease in signal as the coating concentration increased. This phenomenon was interpreted as the "high-dose hook effect" (7). The 5AS titration curves were similar for two other lots of the antigen (Lots 1-80 and 3-84). Thus, the optimal coating titer (l :320) and the "high-dose hook" phenomenon were consistently reproduced. Although the OPD signal was about 64fold higher than 5AS, the latter was useful in revealing the optimal coating concentration of the antigen, a crucial EIA parameter (figure 1). The effects of temperature and buffer on antigen coating were evaluated as follows: the commercial antigen was diluted 1:320 in two different buffers (PBS or 0.1 M carbonate buffer at pH 9.6) and coated overnight at three temperatures (4°,23°, and 37° C, respectively). Bound antigen was measured using commercial rabbit anti-A antiserum (1:8,000) followed by peroxidase conjugate and OPD substrate solution. The following results
, •..•..•..•..• _.....
' t. 't
". • ' t"
RECIPROCAL OF ANTIGEN DILUTIONS Fig. 1. Titration curves for the coating concentration of antigen. Each data point represents the mean absorbance of triplicate tests. The coeffecient of variations for all the data points was below 10%. Absorbance was at 492 nm with OPD substrate solutions (closed circles) or at 450 nm with 5AS substrate solutions (open circles).
LO AND NOTENBOOM
TABLE 1 CROSS·REACTIONS OF HISTOPLASMOSIS SERA IN CONVENTIONAL EIA Tests Using Homologous Antigen
Tests Using Heterologous Antigens Conventional EIA Titer in Plates Coated with 10 Antigens of
line of Nonidentity in 10 with Antigens of
Histoplasma Hand M
+ + +
128 64 16
1,280 320 80
320 320 80
SM1314 RR1057 SD1134 TG945 FF911 KJ1447 SM23n
+ + + + + + +
64 64 16 16 16 16 16
320 320 640 160 160 40 20
640 320 320 160 40 40 80
320 320 2,560 320 640 160 160
Histoplasma H M
BJ1215 RR1009 CM1899
+ + +
DeNnlt10n of abbreviations: EIA
= enzyme immunoassay;
larly, sixsera from four patients with coccidioidomycosis cross-reacted in BIAs, although the cross-reactive titers werealways lessthan the anti-Coccidioides titers (table 2). The cross-reactions of the histoplasmosis group of sera in conventional EIA were further investigated using A and F antigens in ID tests. Commercial anti-A and anti-F antisera were used to create a reference line adjacent to each specimen. Lines of nonidentity, usually exhibiting a diffused pattern, were repeatedly observed in six of the 12 sera. These six sera generally had relatively high CF titers and anti-Histoplasma EIA titers (table 1). However, no significant correlation existed between the conventional anti-Histoplasma EIA titer and their cross-reactive anti-Blastomyces EIA titer (r = 0.49, p > 0.1) or their cross-
+ + + +
+ + + +
= immunodiffusion; CF = complement fixation;
reactive anti-Coccidioides EIA titer (r = 0.31, p > 0.1). In brief, EIAs based on commercial immunodiffusion antigens are of questionable value for differential serodiagnosis in these three fungal infections, even under optimal assay conditions.
Blastomycosis-specific EIA Optimization of blastomycosis-specific EIA. Ocasionally, some sera from healthy humans exhibit nonspecific reactions in the assay. These false-positive reactions were prevented by the inclusion of a 1070 (vol/vol) each of normal rabbit serum and normal goat serum in the diluent, and the blocking of anti-A-coated plates prior to the addition of patient's serum (see METHODS). Specific positive reactions with sera from human blastomy-
AC = anticomplementary.
cosis were not affected by the blocking procedure (data not shown). Rheumatoid factors (RF), if present in the patient's serum, would be expected to bind to the Fc portion of the immune rabbit IgO coated on the microwells. A panel of seven sera with RF values ranging from zero to 8000 IU/ml were selected. Their false-positive signals are depicted in figure 2. An attempt to reduce the RF by absorbing these sera in another rabbit IgO-coated plate (4 h, 37° C) was conducted in parallel. No significant reduction of the falsereactions was achieved (figure 2). Because the potential falsepositive signals caused by RF could not be eliminated by blocking or preadsorption in plates, they were controlled by the serum control wells as described in METHODS.
Performance ofBlastomycosis-specific EIA. A total of 18 sera from eight cases TABLE 2 CROSS-REACTIONS OF COCCIDIOIDOMYCOSIS SERA IN CONVENTIONAL EIA EIA Titer in Plates Coated with Antigens of
Histoplasma Hand M
640 640 320
DeNnition of abbreviations: INS = insuffICient quantity for testing. For other definitions, see table 1.
of blastomycosis wereavailable for study (table 3). Among these, 15sera from seven patients (all with pulmonary involvements) were all reactive by this EIA. Particularly noteworthy is that in four of these seven cases, the early acute phase sera (CD199, BA372, CH1696, CHI744, and AE1089) were negative by ID, and two (BA372 and AE1089) were negative by CF. The remaining case (ChR) was a patient with AIDS in whom all three serologic tests were negative during his two microbiologically confirmed episodes of active blastomycosis. The first episode was pulmonary, whereas the second was disseminated. The negative serologic results in this case were confirmed by the
A NEW ENZYME IMMUNOASSAY SPECIRC FOR BLASTOMYCOSIS
SPECIFIC REACTIONS IN THE NEW EIA FOR BLASTOMYCOSIS
+ + +
8 8 8 8
R R R R
Blastomycosis group Jan 19/82 CDl99 Feb 17/82 CD564 Feb 19/82 CD589 Feb 22/82 CD619
• 200 100
RF( I U/ml ) Fig. 2. False positive reactions caused by rheumatoid factors. Sera were heated at 56" C for 30 min and tested at 1:400 dilutions. The regression lines through the six RF-positive sera before adsorption (closed circles) and after adsorption (opensquares) did not differ significantly in their regression coefficient at the 0.05 level (6).
Fungus Immunology Laboratory, CDC, Atlanta, Georgia. No cross-reaction was observed in any of the sera from the histoplasmosis group, the coccidioidomycosis group, or the normal group (table 3). Discussion The coating of antigens directly onto microwells has been the conventional design of EIA for blastomycosis, but sera of patients with histoplasmosis often cross-reacted. These notorious cross-reactions in conventional EIA were reported by several teams of investigators (table 4). The sample size in each report is too small for meaningful comparisons, but these reports may be pooled into two categories depending on the type of antigen used. Thus, studies using the ion-ex- . change, chromatographed antigen added up to 19.7% cross-reactivity among 61 hisotoplasmosis sera, whereas studies using the Meridan ID antigen amounted to 42.9% cross-reactivity among 42 histoplasmosis sera. The difference in crossreactivities between the two categories is 23.20/0 (standard error = 9.2%), and the 95% confidence intervals of the difference are 5.2 to 41.2%. This difference is significant by chi-square test (p < 0.02). Therefore, fewer cross-reactions were reported in conventional EIA based on ion-exchange, chromatographed antigen when compared with commercial ID antigen (Meridian). This may imply that the purity of the antigen is important in de-
Feb 4/85 Apr 2/85
Oct 24/88 Nov 3/88 Nov 17/88 Dec 22/88 Jan 6189
CH1696 CH1744 CHl838 CH20n CH28
INC P + +
INS 64 N N N
R R R R R
Mar 26/80 Apr 7/80
Aug/86 ChR273 Aug 1/87 ChR Histoplasmosis group 12 sera from 12 patients (table 1) Coccidioidomycosis group 8 sera from 5 patients (table 2) Normal group
N N all 12 N all 8 N all 23 N
Definition of BbbIevla/ions: R = reactive; N = nonreactive; INS = insufficient for testing; INC = inconclusive; P = partialidentity. For otherdefinitions, see table1.
TABLE 4 REPORTS OF HISTOPLASMOSIS SERA THAT CROSS-REACTED IN CONVENTIONAL BLASTOMYCOSIS EIA RESULTING IN FALSE-POSITIVE REACTIONS False + Antigen Used Recycled lon-exchange Chromatography
False +/Total Sera
(8) (9) (10) (11)
2/12 3/24 3/6 4/19 12/61
17 13 50 21 19.7
20 100 42.9
Pooled results Commercial 10 antigen (Meridian) Pooled results
This study Commercial 10 antigen (Nolan)
Conventional EIA Blastomycosis-specific EIA
For definition of abbreviations, see table 1.
termining the extent of the cross-reaction. Our detection of lines of nonidentity with the two heterologous fungal antigens substantiated the presence of contaminating systems that may have contributed to the cross-reactions (table 1). Our experience of 100% cross-reactivity in conventional EIA based on commercial ID antigen (Nolan/Scott) is consistent with the recent report by Lambert and George (2). Furthermore, we have extended the
observation by including a group of coccidioidomycosis sera. Perhaps an ultra-pure A antigen could resolve the cross-reactive problem. However, such a purification method has not yet been developed. Instead of devising an elaborate purification procedure, we proposed a relatively simpler serologic solution. In this report, we described a blastomycosis-specific EIA based on the principle of antigen-capture. As a result,
cross-reactions with sera from the histoplasmosis and coccidioidomycosisgroups have been overcome. Although none of the mycotic sera in this study was RF positive, the potential problem with RF was considered. A double algorithm was developed to control RF by comparing the test signal to the noise signal. This eliminated the need for RF testing prior to EIA. If RF is present, the assay is invalid. We have not yet investigated the feasibility of removing RF in our system by other methods such as absorption with IgG-coated latex particles (13). The superior sensitivity of the blastomycosis-specific EIA was indicated by the early acute phase sera, which were falsenegative by ID and CF. However, a reliable determination of sensitivity, specificity, and predictive values of the new test (14) would require further studies with much larger sample sizes. The seronegativity in the patient with AIDS was not surprising in view of the immunodeficiency state. Conceivably, human immunodeficiency virus infection could suppress the humoral response to a second pathogen (15-18), thereby rendering serodiagnoses of new infections unreliable (19). The proclivity for dissemination of blastomycosis in immunocompromised hosts was noted earlier by others (20) and thought to be due to defective cell-mediated immunity (21). In conclusion, a new EIA based on the principle of A-antigen capture was devel-
LO AND NOTENBOOM
oped. Preliminary data suggest that the assay is more sensitive and specific than ID and CF tests and may improvethe serodiagnosis of blastomycosis. Therefore, further developmental work is justified. References 1. Davies SF, Sarosi GA. Serodiagnosis of histoplasmosis and blastomycosis. Am Rev Respir Dis 1987; 136:254-5. 2. Lambert RS, George RB. Evaluation of enzyme immunoassay as a rapid screeningtest for histoplasmosis and blastomycosis. Am Rev Respir Dis 1987; 136:316-9. 3. Sarosi GA, Armstrong D, Davis SF, et al. Laboratory diagnosis of mycotic and specific fungal infections. Am Rev Respir Dis 1985; 132:1373-9. 4. Kaufman L, Reiss E. Serodiagnosis of fungal diseases. In: Lennette EH, Balows A, Hausler WJ Jr, Shadomy HJ, eds. Manual of clinical microbiology. 4th ed. Washington, DC: American Society for Microbiology, 1985; 927-8. 5. Voller A, Bidwell DE, Bartlett A. The enzyme linked immunosorbent assay. Alexandria, VA:Dynatech, 1979. 6. Sokal RR, Rohlf FJ. Biometry. Philadephia: WH Freeman and Co., 1%9; 457. 7. RodbardD, Feldman Y,JaffeML, MilesLEM. Kineticsof two-siteimmunoradiometric ('sandwich') assays. II. Studies on the nature of the 'high-dose hook effect.' Immunochemistry 1978; 15:77-82. 8. Green JH, Harrel WK, Johnson JE, Benson R. Isolation of an antigen from Blastomycesdermatitidisthat is specific for the diagnosis of biastomycosis. Curr Microbiol 1980; 4:292-6. 9. Thrner S, Kaufman L, Jalbert M. Diagnostic assessment of an enzyme-linked immunosorbent assay for human and canine blastomycosis. J Clin Microbiol 1986; 23:294-7. 10. Klein BS, Kuritsky IN, Chappel WA, et al. Comparison of enzyme immunoassay, immunodiffusion and complement fixation tests in detecting antibody in human serum to the A antigen of
Blastomyces dermatitidis. Am Rev Respir Dis 1986; 133:144-8. 11. Klein BS, Vergeront JM, Kaufman L, et al. Serologicaltests for blastomycosis:assessments during a large point-source outbreak in Wisconsin. J Infect Dis 1987; 155:262-8. 12. Scalarone GM. Use of a urease-antibody conjugate in an enzyme immunoassay for the detection of blastomycosis. J Med Vet Mycol 1987; 25: 215-22. 13. Chantler S, Devries E, Allen PR, Hurn BAL. A rapid immunofluorescent procedure for the detection of specific IgG and IgM antibodies in sera using Staphylococcus aureusand latex-IgG as absorbents. J Immunol Methods 1976; 13:367-80. 14. Schachter J. Rapid diagnosis of sexually transmitted diseases: speed has a price. Diagn Microbi01 Infect Dis 1986; 4:185-9. 15. Luft BJ, Conley F, Remington JS. Outbreak of central-nervous-system toxoplasmosis in Western Europe and North America. Lancet 1983; 1: 781-3. 16. Roberts CJ. Coccidioidomycosis in acquired immune deficiency syndrome. Depressed humoral as well as cellular immunity. Am J Med 1984; 76: 734-6. 17. Hicks CB, Benson PM, Lupton GP, Tramont EC. Seronegative secondary syphilis in a patient infected with the human immunodeficiency virus (HIV) with Kaposi's sarcoma. Ann Intern Med 1987; 107:492-5. 18. Radolf JD, Kaplan RP. Unusual manifestations of secondary syphilis and abnormal humoral immune response to Treponema pallidum antigens in a homosexual man with asymptomatic human immunodeficiency virus infection. J Am Acad Dermatol 1988; 18:423-8. 19. Bowen DL, Lane HC, Fauci AS. Immunopathogenesis of the acquired immunodeficiency syndrome. Ann Intern Med 1985; 103:704-9. 20. Sarosi GA, Davies SF. Blastomycosis.Am Rev Respir Dis 1979; 120:911-38. 21. Varkey B, Lohaus G, Rose HD, Sohnle PG. Blastomycosis: clinical and immunologic aspects. Chest 1980; 77:789-95.