Value o f Immunodiffusion Tests in the Diagnosis o f Systemic M ycotic Diseases

Transcription

1 A n n a l s of C l i n i c a l L a b o r a t o r y S c i e n c e, Vol. 3, N o. 2 Copyright , Institute for Clinical Science Value o f Immunodiffusion Tests in the Diagnosis o f Systemic M ycotic Diseases LEO KAUFMAN, Ph.D. Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, U. S. Department of Health, Education and Welfare, Atlanta, GA ABSTRACT Properly evaluated and standardized immunodiffusion procedures useful for the diagnosis of aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, histoplasmosis, and paracoccidioidomycosis are available for everyday use. It is believed that the widespread use of the immunodiffusion tests will contribute significantly to the rapid and accurate detection of these diseases and to their proper treatment. The techniques and antigens used in the procedures and their sensitivity and specificity are reviewed. In trod u ction Because the symptoms of aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, histoplasmosis and paracoccidioidomycosis are not pathognomonic, these diseases cannot always be clinically diagnosed with certainty. Neither can a definitive diagnosis always be made by culture or histology, despite repeated efforts to isolate a fungus from severely ill patients or to demonstrate its presence in biopsy material. Clinicians have found that immunological tests frequently provide the earliest evidence of fungus infection. The tests may also provide information on the effects of therapy. The complement-fixation (C F) test is used in the diagnosis of human cases of aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, histoplasmosis and paracoccidioidomycosis. Unfortunately, the antigens presently used in these tests are known to cross-react in varying degrees with antibodies to a variety of fungus pathogens. Consequently, laboratories performing only CF tests for the diagnosis of systemic mycotic infections may expect to encounter cross-reactions that may lead to incorrect diagnoses. In contrast to the CF test, the immunodiffusion (ID ) test is simple to perform and yields results that are highly specific and accurate indicators of infection. Diagnostic ID tests have been standardized and evaluated for several mycotic infections. It is the author s intention, in this presentation, to describe briefly some of these procedures, to appraise their diagnostic and prognostic value and to point out their usefulness in monitoring the effects of therapy. M ycotic D iseases A s p e r g i l l o s i s Numerous workers have demonstrated that the ID test is a useful adjunct in estab 1 4 1

2 142 KAUFM AN lishing a diagnosis of aspergillosis Stallybrass22 in 1963 reported that the manifestation of precipitins in a patient s serum is presumptive evidence for aspergillosis and is of confirmatory value when radiological, clinical and mycological evidence of pulmonary aspergillosis exists. In recent ID studies, Coleman and Kaufman5 confirmed these observations. It has been shown in our laboratory that the greatest number of aspergillosis cases were revealed by the concurrent use of Aspergillus fumigatus, A. flavus and A. niger precipitinogens. Standardized and reproducible aspergillus antigens that do not react with C reactive protein were prepared by acetone extractions of five-week-old Sabouraud dextrose broth cultures. Only sera that produced a line or lines of identity with a reference serum from a proven human aspergillosis cases were considered positive. Demonstration of one or more precipitins in a patient s serum was taken to be indicative of infection, colonization or allergy due to an Aspergillus species. Using the ID test, serological diagnosis was performed upon 28 of 30 ( 93 percent) fungus ball cases, 14 of 16 (88 percent) cases of invasive aspergillosis and 7 of 14 (50 percent) cases of allergic bronchopulmonary aspergillosis. Although one or two precipitins could occur with any clinical form of aspergillosis, the presence of three or more precipitins without exception was associated either with a fungus ball or invasive disease. The contentions of Murray16 and Henderson et al10 that the ID test could be helpful in diagnosing systemic aspergillosis were supported by the studies of Coleman and Kaufman5 but are contrary to the recent report of Young and Bennett.27 The ID test when used with reference sera is 100 percent specific.5 Although aberrant lines may occur with sera from persons with aspergillosis, a specific diagnosis cannot be made in the absence of lines of identity with reference bands, since sera from persons with candidiasis and histoplasmosis may contain precipitins reactive with Aspergillus precipitinogens. In aspergillosis, the number of precipitins in a series of sera or the precipitin titers obtained provide clues to the course of the disease. A decline in the number of precipitins or in the titer reflects recovery. B l a s t o m y c o s i s Using the micro-immunodiffusion template described by Busey and Hinton3 with a concentrated yeast-form filtrate antigen of B. dermatitidis, a simple and specific ID test for blastomycosis has been developed at the Center for Disease Control (CD C). The test has a sensitivity of approximately 80 percent. Sera, containing A and B precipitins, from patients with proven blastomycosis are used as references. Only sera that produce lines of identity with either the A or B or both reference bands are considered positive for blastomycosis. With this system, no false positives were found with sera from patients with a variety of other methods. Although the disappearance of precipitin lines has been noted in sera from treated blastomycosis patients, recovery may occur without the concomitant disappearance of precipitins. C a n d i d i a s i s The most common fungus infection affecting the compromised host is candidiasis Despite the awareness of the factors which contribute to nosocomial systemic candidiasis, the majority of diagnoses are still made postmortem. In recent years, immunological procedures have been introduced that permit the rapid and accurate detection of systemic candidiasis and enable the course of the disease to be monitored. Stallybrass23 and Taschdjian et al,25 using the ID procedure, were the first to demonstrate precipitins to Candida albicans in sera from patients with systemic or

3 IM M UNODIFFUSION TESTS IN DIAGNOSIS OF SYSTEM IC MYCOTIC DISEASES 143 visceral Candida sp. infections. In a later study by Taschdjian et al,26 the test was shown to be free of cross-reactions, except among the several species of the genus Candida. More recently, Stickle et al24 evaluated a modified ID test with a homogenate antigen of C. albicans. The ID test permitted the detection of 38 of 43 ( 88 percent) proven candidiasis cases. The ID test was highly specific. Extra generic cross-reactions occurred only with Torulopsis glabrata antiserum. Six distinct precipitins were detected with the C. albicans homogenates. The occurrence of four or more precipitin lines was suggestive of severe candidiasis. Unfortunately, a decline in the number of ID bands did not invariably accompany or indicate disease regression after successful treatment. C o c c id i o i d o m y c o s i s Coccidioides immitis elicits an immunological response in the host. Conversion from a negative to a positive skin test with coccidioidin is usually the earliest immunologic response to infection. Because nearly every infected individual will develop a hypersensitivity to coccidioidin, the skin test is considered a valuable screen for serological testing. Smith and his coworkers,21 for example, never noted positive serologic results in patients with primary coccidioidal infections who had negative skin tests. The CF and tube precipitin tests are valuable diagnostic and prognostic aids in coccidioidomycosis.21 The precipitin test is most effective in detecting early primary infections or persons undergoing an exacerbation of an existing disease. The CF test may also be positive with sera taken early in the course of disease, but its reacting antibodies persist for longer periods of time than those reactive in the tube precipitin test. The CF titer generally parallels the severity of the infection.21 It rises as the patient s condition deteriorates and declines as the patient improves. These tests are very specific and do not cross-react with sera from viral, rickettsial, bacterial or most other mycotic infections. Smith et al21 found that the combination of the CF and tube precipitin tests yielded positive results in over 90 percent of the primary symptomatic cases of coccidioidomycosis. Screening procedures that yield results comparable to those of the tube precipitin or CF tests are available for use by laboratories that are not able to carry out the conventional tests. One of them, the latex particle agglutination test, is more sensitive than the tube precipitin test and yields a higher percentage of positive responses. It has the additional advantage of speed since results can be obtained in a few minutes.13 Another procedure is the ID test11 12 which gives results that qualitatively parallel those of the CF test. The antigen is a heat-labile toluene extract of the mycelial growth. The combined use of the latex particle and ID tests has permitted the detection of 93 percent of the serum specimens from persons with proven coccidioidomycosis. Huppert et al13 recommend that sera found to be positive by either of these procedures be further analyzed with the standard CF and tube precipitin tests in order to determine their clinical significance. H i s t o p l a s m o s i s Hypersensitivity to histoplasmin may be demonstrated in man within two weeks after exposure to infection by Histoplasma capsulatum? The skin test is most useful in delineating endemic areas of histoplasmosis. However, it has limited diagnostic value, since it does not distinguish between past or present infections. In general, a positive reaction is of diagnostic significance only if a skin test was negative before the onset of clinical symptoms.

4 144 KAUFM AN It has been adequately demonstrated that the level of complement-fixing antibodies, precipitins and agglutinins to H. capsulatum antigens may be significantly increased in histoplasmin sensitized subjects after a single histoplasmin skin test Clearly, the laboratory worker and clinician should be fully cognizant of any factor or factors that might influence serologic results. In a recent study of 114 histoplasmin-sensitized but clinically well subjects, approximately 15 percent developed precipitating antibodies after a single histoplasmin skin test. In most cases, precipitating antibody responses were detected in sera drawn 15 days after testing. Preferably, blood for serological studies should be drawn before skin testing, but the patient can usually be bled within two or three days after the skin test without induced antibodies being detected in the serum. A single histoplasmin skin test produces no serological response in nonsensitized individuals. Serological data frequently lead to intensive histologic and cultural studies that permit a definitive diagnosis of histoplasmosis. Serologic evidence of infection is usually obtained through the CF and ID tests, used either alone or in combination. Generally, CF titers of 1:8 or greater are considered to be presumptive evidence of histoplasmosis. Although high titers are of more diagnostic significance than those in the 1:8 to 1:16 range, they cannot be relied upon as the sole means of diagnosis.14 Because a serologic reaction is not always indicative of active infection, any interpretation of CF titers must take into account the total clinical picture and any radiological findings. Changes in titer are of diagn o stic significance, and fourfold titer fluctuations in either direction are usually of prognostic value. The absence of a specific immunological response does not exclude histoplasmosis, particularly when only a single specimen has been tested and when the clinical picture strongly suggests the existence of a pulmonary mycotic disease. In such situations, it is recommended that serial serum specimens taken three to four weeks apart be tested for antibodies. The ID test, in which concentrated histoplasmin is used, may be employed as an adjunct or screening procedure in the diagnosis of histoplasmosis. Of 220 serum specimens from persons with proven histoplasmosis, 180 (82 percent) were positive by the ID test in a recent study at CDC. Two precipitin bands have diagnostic value.9 One, the H band, is rarely influenced by skin tests. Patients with active and progressive histoplasmosis usually demonstrate H antibody. The H precipitin may be detectable one to two years after apparent clinical recovery. The second, or M precipitin band, is found in sera of patients with acute and chronic histoplasmosis and may appear in sensitized normal individuals after skin testing with histoplasmin. The demonstration of only an M band may be indicative of active infection, past infection or a recent skin test. Proper interpretation of the ID test requires that the physician know whether or not the patient had a skin test recently. The presence of M antibody, when there has been no recent skin test, may indicate an early infection, since this antibody appears before the H precipitin. The disappearance of the H precipitin is of prognostic value. The M precipitin will eventually disappear, but at a slower rate than the H precipitin. In our laboratory, the ID test was found to be useful in interpreting the cross-reactions so often encountered with the CF test and also in examining anticomplementary sera. P a r a c o c c i d io i d o m y c o s i s Restrepo18 reported on a very practical, sensitive and effective ID test performed with a filtrate antigen of Paracoccidioides brasiliensis. The procedure gave negative results in tests with 50 sera from normal

5 IM M U N O D IFFU SIO N T E S T S IN DIAGNOSIS O F S Y S T E M IC M Y C O T IC DISEASES individuals and 21 sera from patients with either histoplasmosis, blastomycosis, coccidioidomycosis or sporotrichosis. In a later study with sera from 20 patients with paracoccidioidomycosis, Restrepo19 reported that antibodies were detected in 80 percent of the cases with the CF test and in 90 percent of the cases with the ID test. In the ID test, the number of precipitin bands varies from 1 to 3; the highest number of bands is found in the sera of patients with lung involvement or disseminated disease. Precipitins may remain for long periods after recovery and do not appear to be of prognostic value. Sum m ary Properly evaluated and standardized immunodiffusion procedures useful for the diagnosis of aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, histoplasmosis and paracoccidioidomycosis are available for everyday use. It is believed that the widespread use of the immunodiffusion tests will contribute significantly to the rapid and accurate detection of these diseases and to their proper treatment. The techniques and antigens used in the procedures and their sensitivity and specificity are reviewed. R eferences 1. B e n n e t t, D. E.: The histoplasmin latex agglutination test; clinical evaluation and a review of the literature. Amer. J. Med. Sci. 251: , B o d e y, G. P.: Fungal infections complicating acute leukemia. J. Chronic Dis. 19: , B u s e y, J. F. a n d H i n t o n, P. F.: Precipitins in histoplasmosis. Amer. Rev. Resp. Dis. 92: , C a m p b e l l, C. C. a n d H i l l, G. B.: Further studies on the development of complementfixing antibodies and precipitins in healthy histoplasmin-sensitive persons following a single histoplasmin skin test. Amer. Rev. Resp. Dis. 90: , C o l e m a n, R. M. a n d K a u f m a n, L.: Use of the immunodiffusion test in the serodiagnosis of aspergillosis. Appl. Microbiol. 23: , E n g l is h, M. P. a n d H e n d e r s o n, A. H.: Significance and interpretation of laboratory tests in pulmonary aspergillosis. J. Clin. Path. 20: , F u r c o l o w, M. L.: Tests of immunity in histoplasmosis. New Eng. J. Med. 268: , H a r t, P. D., R u s s e l l, E., J r. a n d R e m i n g t o n, J. S.: The compromised host and infection. II. Deep fungal infection. J. Infect. Dis. 120: , H e i n e r, D. C.: Diagnosis of histoplasmosis using precipitin reactions in agar gel. Pediatrics 22: , H e n d e r s o n, A. H., E n g l is h, M. P., a n d S t f.w - a r t -S m i t h, G.: Fungal infections. Lancet 1: 502, H u p p e r t, M. a n d B a i l e y, J. W.: The accuracy and reproducibility of the immunodiffusion test which correlates with complement fixation. Amer. J. Clin. Path. 44: , H u p p e r t, M. a n d B a i l e y, J. W.: An immunodiffusion test as a substitute for the tube precipitin test. Amer. J. Clin. Path. 44: , H u p p e r t, M., P e t e r s o n, E. T., S u n, S. H., C h i t j i a n, P., a n d D e r r e v e r e, W.: Evaluation of a latex particle agglutination test for coccidioidomycosis. Amer. J. Clin. Path. 49:96-102, K a u f m a n, L.: Serology of systemic fungus diseases. Public Health Rep. 82: , K a u f m a n, L., T e r r y, R. T., S c h u b e r t, J. H., a n d M c L a u g h l i n, D.: Effects of a single histoplasmin skin test on the serological diagnosis of histoplasmosis. J. Bact. 94: , M u r r a y, I. G.: Aspergillosis. Lancet 1 :1373, P e p y s, J. : Hypersensitivity Diseases of Lungs Due to Fungi and Organic Dusts. S. Kerger, New York, p. 21, R e s t r e p o, M. A.: La prueba de immunodiffusion en el diagnostic de la paracoccidioidomycosis. Sabouraudia 4: , R e s t r e p o, M. A.: Comportamiento immunologico de zo pacientes con paracoccidioidomycosis. Antioquia Med. 17: , R i f k in d, D., M a r c h io r o, T. L., S c h n e c k, S. A., a n d H i l l, R. B.: Systemic fungal infection complicating renal transplantation and immunosuppressive therapy. Amer. J. Med. 43: 28-38, Sm i t h, C. E., S a it o, M. T., B e a r d, R. R., K e p p, R. M., C l a r k, R. W., a n d E d d ie, B. U.: Serologic tests in the diagnosis and prognosis of coccidioidomycosis. Amer. J. Hyg. 52:1-21, S t a l l y b r a s s, F. C.: The precipitin test in human aspergillosis. Mycopath. Mycol. Appl. 21: , 1963.

6 1 4 6 KAUFM AN 23. S t a l l yb r a s s, F. C.: Candida precipitins. J. Path. Bact. 87:89-97, S t i c k l e, D., K a u f m a n, L., B l u m e r, S. O., a n d M c L a u g h l i n, D. W.: Comparison of a newly developed latex agglutination test and an immunodiffusion test in the diagnosis of systemic candidiasis. Appl. Microbiol. 23: , T a s c h d j i a n, C. L., K o z i n n, P. J., a n d C a r o l i n e, L. : Immune studies in candidiasis. III. Precipitating antibodies in systemic candidiasis. Sabouraudia 3: , T a s c h d j i a n, C. L., K o z i n n, P. J., O k a s, A., C a r o l i n e, L., a n d H a i a e, M. A.: Serodiagnosis of systemic candidiasis. J. Infect. Dis. 117: , Y o u n g, R. C. a n d B e n n e t t, J. E.: Invasive aspergillosis Observance of detectable antibody response. Amer. Rev. Resp. Dis. 104: , SPRING MEETING of the ASSOCIATION OF CLINICAL SCIENTISTS Topic: R ecen t Advances in Clinical Science Tam pa, Florida May 3-5, 1973