D irect Fluorescent Antibody Tests for the Diagnosis of M ycotic Diseases

Transcription

1 A n n a l s o p 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, No. 1 Copyright 1973, Institute for Clinical Science D irect Fluorescent Antibody Tests for the Diagnosis of M ycotic Diseases WILLIAM KAPLAN, D.V.M. Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, U. S. Department of Health, Education and Welfare, Atlanta, GA ABSTRACT The current status of the direct fluorescent antibody procedure for the diagnosis of fungus diseases is reviewed. Emphasis is placed upon those diagnostic applications that have been developed to a practical level. The versatility of the fluorescent antibody ( FA ) technique as a diagnostic tool in medical mycology has been firmly established. It can be used for the rapid detection and identification of fungi, both viable and nonviable, in culture as well as in clinical materials. In modified form, immunofluorescence can also be used to detect and measure antibodies in sera and in other body fluids. The direct FA procedure, which is the subject of the present report, is the simplest form of the fluorescent antibody technique. It entails the application of solutions of known labeled antibody on smears of cultures or clinical materials or on sections of tissue. After an appropriate period of incubation, previously determined for each system under study, excess labeled antibody is removed by washing, and the preparation is mounted and examined for stained organisms under a fluorescence microscope. In diagnostic testing, the direct FA procedure is primarily used to detect and identify organisms or antigens. The direct FA procedure can be used for the rapid detection and identification of fungi in culture and in most types of clinical materials including paraffin sections of formalin-fixed tissues. 11 It is even possible to use this technique to demonstrate and identify fungi in tissue sections that have been stained previously with hematoxylin and eosin, the Brown and Brenn, and the Giemsa stains. It has not been possible, however, to stain fungi by FA in histological sections previously stained by the PAS, Gridley, or Gomori procedures. Prolonged storage of formalin-fixed tissues, either wet or in paraffin blocks, does not cause observable effect on the antigenicity of fungi. Therefore, this procedure can be used as a histological tool in making a retrospective diagnosis as well as a diagnosis of a current mycotic infection. A number of workers have been aware of the potentialities of this versatile technique and have investigated the possibility of applying the direct FA procedure to the diagnosis of mycotic diseases. Their studies have dealt with the development of reliable re

2 2 6 K A PLA N agents and effective methods for detecting and identifying mycotic disease agents in cultures and in clinical materials. A review of what has been accomplished indicates that the technique has been developed to a practical level for the diagnosis of a number of fungus diseases. For other mycoses, much more developmental work is needed. The present paper covers those applications considered to be of practical diagnostic value. Aspergillosis. The direct FA procedure can be used to advantage in the histological diagnosis of infections caused by members of the genus Aspergillus. Conjugates have been produced for demonstrating aspergilli in sections of frozen tissue5 as well as in paraffin sections o f formalin-fixed tissues Morphologically typical as well as atypical Aspergillus species elements which would be difficult to identify by conventional staining procedures can be readily stained by FA and, when the need arises, differentiated from other fungi such as Candida species and zygomycetes (phycomycetes) that also occur in tissue in the form of mycelium. 2 0 As yet, FA reagents have not been developed for the differential identification of species of Aspergillus in culture or in tissue. Blastomycosis. The direct FA procedure is useful for the diagnosis of blastomycosis. An FA reagent specific for the yeast form of Blastomyces dermatitidis has been developed, 10 and its efficacy in the detection and identification of most strains of this fungus in culture and in various types of clinical materials has been confirmed. This specific reagent does not, however, stain the mycelial form of B. dermatitidis. As yet, an FA reagent has not been developed for the specific identification of the mycelial form of this fungus. Candidiasis. Various workers have investigated the possibility of using immunofluorescene for the identification of Candida albicans and other Candida species of medical importance. In all cases, the FA reagents that have been produced brightly stained the homologous species in culture and in clinical materials. However, these products cross-stained other members of the genus and in some cases members of other genera. Attempts, to date, to produce species-specific conjugates for the identification of the Candidas have been unsuccessful. As yet, no single species-specific reagent of practical diagnostic value has been developed. It is possible, however, to identify some of the Candida species with a combination of reagents. 4 Although species-specific FA reagents have not been produced, some of the available conjugates can be used to advantage for the diagnosis of Candida infections. FA reagents for Candida sp. that show broad intrageneric cross-reactivity can be used for the rapid screening of clinical materials for the presence of Candida sp. elements. Extrageneric cross-reactivity that can cause diagnostic problems can be removed by diluting or absorbing the conjugates; the resulting genus-specific products can be used for the demonstration of Candida elements in tissue sections and for the differentiation of Candida from other morphologically similar fungi in tissue. There is little doubt that the accuracy of a histological diagnosis of candidiasis can be increased by using such conjugates. Coccidioidomycosis. The direct FA technique can play a useful role in the diagnosis of infections caused by Cocciodioides immitis. An FA reagent for the specific detection and identification of the tissue form of C. immitis has been produced. 7 This product has been successfully used for the demonstration of this fungus in many types of clinical materials. It is noteworthy, from a diagnostic standpoint, that this reagent generally stains the walls of endospores and the contents of spherules. The walls of mature spherules that have been produced in vivo often are not stained. In contrast, the

3 FLU O RESCEN T ANTIBODY TESTS FO R DIAGNOSIS OF M YCOTIC DISEASES 2 7 walls of spherules produced in. vitro are regularly stained by this reagent. An FA reagent has not been produced for the specific identification of the mycelial form of C. immitis. The reagent that is specific for the tissue form of this fungus does not, as a rule, stain the mycelial form. However, the specific reagent can be used indirectly for the identification of mycelialform cultures of C. immitis. It is generally not possible to definitively identify C. immitis cultures by means of morphological criteria alone. An accurate identification of this fungus requires tests that demonstrate conversion to the tissue form. Laboratory animals are often used for this purpose. The reagent that is specific for the tissue form of C. immitis can be used to detect and identify endospores and spherules of C. immitis in clinical materials from these test animals. It is particularly valuable when the identification of such tissue-form elements is not possible by conventional methods. Cryptococcosis. The direct FA procedure can play a very useful role in the diagnosis of this disease. Among the early reports on the application of immunofluorescence to medical mycology is one by Eveland et al. 2 These workers successfully used fluoresceinlabeled C. neoformans antiglobulins to study the distribution of C. neoformans and its polysaccharide breakdown products in formalin-fixed tissues. Subsequently, Marshall et al1 6 examined sections of tissue from human cases of cryptococcosis and compared the findings with those obtained with Mayer s mucicarmine stain. They reported that their labeled C. neoformans antiglobulins stained this fungus more intensely and more rapidly than did mucicarmine. Unabsorbed fluorescein-labeled C. neoformans antiglobulins generally cross-stain other Cryptococcus species and also yeastlike fungi belonging to other genera. Such nonspecific reagents can be used for screening purposes, but the morphological characteristics of the stained organisms are of great importance in making a tentative identification. A specific conjugate is needed, however, for the definitive identification of C. neoformans. Such a reagent has been developed18 and found to be effective for the detection and identification of C. neoformans in culture and in clinical materials. Histoplasmosis. The direct FA technique can be used to great advantage in the diagnosis of this disease. It can be used for the detection and identification of the tissue form of Histoplasma capsulatum in culture and in clinical materials. Unabsorbed fluorescein-labeled antiglobulins against the yeast form of H. capsulatum may brightly stain the homologous organism, but they usually cross-react with B. dermatitidis and also with other heterologous fungi. Such cross-reactive reagents can be of value for screening purposes, and they may be useful for differentiating tissueform cells of H. capsulatum from morphologically similar but not antigenically related organisms. Specific reagents are required, however, for a definitive identification of this fungus. Kaufman and Kaplan14 prepared such a product by adsorbing fluorescein-labeled H. capsulatum antiglobulins with yeast-form cells of B. dermatitidis. This specific reagent proved to be useful for the identification of tissueform cells of H. capsulatum in culture and in impression smears of infected tissue. In an extensive evaluation of this conjugate, however, a number of H. caspulatum isolates were encountered that failed to stain or stained poorly These isolates were shown to belong to one of the five recognized serotypes of H. capsulatum, designated type 1, 4. This lack of sensitivity severely limits this reagent s value for diagnostic purposes. A partially specific reagent prepared by adsorbing fluorescein-labeled H. capsulatum anti globulins with C. albicans cells is preferable for diagnostic purposes. 13 This C. albicans-adsorbed reagent

4 2 8 KAPLA N stains the yeast form of all known serotypes of H. capsulatum. Furthermore, with the exception of B. dermatitidis and H. capsulatum var. duboisii, it does not cross-stain other pathogenic fungi. Although it is not fully specific for H. capsulatum, it is very useful for diagnostic purposes. In most instances the tissue form of H. capsulatum can be differentiated from that of B. dermatitidis on the basis of morphology. When necessary, these two fungi can be differentiated by FA by using a conjugate specific for the tissue form of B. dermatitidis. 10 The latter reagent stains B. dermatitidis, but not H. capsulatum. When necessary, H. capsulatum can be differentiated from the duboisii variety by conventional mycological methods. It is noteworthy that unadsorbed and also absorbed fluorescein-labeled H. caspulatum antiglobulins regularly stain H. capsulatum in sections of fixed tissue with active disease processes. Therefore, the direct FA technique can be used in the histological diagnosis of active histoplasmosis. However, H. capsulatum cells in healed, calcified lesions are not regularly stained by FA. 6 This fact limits the value of FA for detecting and identifying H. capsulatum in such foci. As yet, FA reagents have not been developed for the specific identification of the mycelial form of H. capsulatum. Therefore, immunofluorescence cannot be used for this purpose. Sporotrichosis. The direct FA technique is an excellent procedure for the rapid diagnosis of sporotrichosis. Reagents of high quality have been developed for the specific detection and identification of the yeast form of Sporothrix schenckii in cultures and in clinical specimens. 8 9 Although, as a rule, few S. schenckii cells are found in human clinical material, they can usually be detected when stained by FA. Conjugates that are specific for the yeast form of S. schenckii also stain the mycelial form of this fungus. Additional evaluation is necessary, however, before the reagents can be relied upon for the identification of the mycelial form. Actinomycosis. The direct FA procedure is an excellent method for the detection and identification of the principal etiologic agents of actinomycosis in man. FA reagents have been produced for the specific staining of Actinomyces israelii, A naeslundii, and Arachnia (Actinomyces) propionica both in culture and in clinical materials The development of such conjugates has greatly simplified the identification of these organisms, a process which by conventional methods is time-consuming and often difficult. C om m ents It is evident that the direct FA technique can play a very useful role in the diagnosis of fungus diseases. Reagents and methods have now been developed for the detection and identification of many of the important mycotic disease agents. For the identification of others, much more developmental work remains to be done. The FA procedure is currently in routine use in some medical mycological centers, and the overall results have been very satisfactory. Immunofluorescence has still not received the widespread use that it merits. This is due in part to a shortage of personnel trained to use the FA technique in medical mycology, and in part to the fact that very few conjugates for the identification of fungi are available commercially. In time, these deficiencies will, undoubtedly, be corrected, and the technique should be used more widely. R eferences 1. B l a n k, C. H. a n d G e o r g, L. K.: The use of fluorescent antibody methods for the detection and identification of Actinomyces species in clinical material. J. Lab. Clin. Med. 71: , E v e l a n d, W. C., M a r s h a l l, J. D., S il v e r - s t e i n, A. M., J o h n s o n, F. V., I v e r s o n, L.,

5 FLU O RESCEN T ANTIBODY TESTS FO R DIAGNOSIS O F M YCOTIC DISEASES 2 9 a n d W in s l o w, D. J.: Specific immunochemical staining of Cryptococcus neoformarvs and its polysaccharide in tissue. Amer. J. Path. 33: , G e r e n c s e b, M. A. a n d Sl a c k, J. M.: Isolation and characterization of Actinomyces propionicus. J. Bact. 94: , G o b d o n, M. A., E l l i o t t, J. D., a n d H a w k in s, T. W.: Identification of Candida albicans, other Candida species and Torulopsis glabrata by means of immunofluorescence. Sabouraudia 5: , H o t c h i, M.: The application of fluorescent antibody technique to the identification of pathogenic fungi in tissue specimens. Med. J. Shunshu Univ. 12: , H o t c h i, M., S c h w a r z, J., a n d K a p l a n, W.: Limitations of fluorescent antibody staining of Histoplasma capsulatum in tissue sections. Sabouraudia 10: , K a p l a n, W. a n d C l i f f o r d, M. K.: Production of fluorescent antibody reagents specific for the tissue form of Coccidioides immitis. Amer. Rev. Resp. Dis. 59: , K a p l a n, W. a n d G o n z a l e z -O c h o a, A.: Application of the fluorescent antibody technique to the rapid diagnosis of sporotrichosis. J. Lab. Clin. Med. 62: , K a p l a n, W. a n d I v e n s, M. S.: Fluorescent antibody staining of Sporotrichum schenckii in cultures and clinical materials. J. Invest. Dermat. 35: , K a p l a n, W. a n d K a u f m a n, L.: Specific fluorescent antiglobulins for the detection and identification of Blastomyces dermatitidis yeastphase cells. Myocpathologia 19: , K a p l a n, W. a n d K r a f t, D. E.: Demonstration of pathogenic fungi in formalin-fixed tissues by immunofluorescence. Amer. J. Clin. Path. 52: , K a u f m a n, L. a n d B l u m e b, S.: Occurrence of serotypes among Histoplasma capsulatum strains. J. Bact. 19: , K a u f m a n, L. a n d B l u m e r, S.: Development and use of a polyvalent conjugate to differentiate Histoplasma capsulatum and Histoplasma duboisii from other pathogens. J. Bact. 95: , K a u f m a n, L. a n d K a p l a n, W.: Preparation of a fluorescent antibody specific for the yeast phase of Histoplasma capsulatum. J. Bact. 82: , L a m b e r t, F. W., B r o w n, J. M., a n d G e o r g, L. K.: Identification of Actinomyces israelii and Actinomyces naeslundii by fluorescent antibody and agar gel diffusion techniques. J. Bact. 94: , M a r s h a l l, J. D., I v e n s o n, L., E v e l a n d, W. C., a n d K a s e, A.: Application and limitations of the fluorescent antibody stain in the specific diagnosis of cryptococcosis. Lab. Invest. 10: , M i y a k e, M., O k u d a ir a, M., N a s u, T., H o t c h i, M., U y e t s u k a, A., M i n e, Y., N a n t a, M., a n d H a m a s h im a, K.: Identification of pathogenic fungi in paraffin embedded tissue sections by means of fluorescent antibody technic. Jap. J. Exp. M ed. 38:95-104, P id c o e, V. a n d K a u f m a n, L.: Fluorescentantibody reagent for the identification of Cryptococcus neoformans. Appl. Microb. 16: , P i n e, L., K a u f m a n, L., a n d B o o n e, C. F.: Comparative fluorescent-antibody staining of Histoplasma capsulatum and Histoplasma duboisii with a specific anti-yeast phase H. capsulatum conjugate. Mycopathologia 22: , W i l l i a m s, E. R. a n d K a p l a n, W.: Application of the fluorescent antibody technique to the differentiation of aspergilli, Candida species and zygomycetes in paraifin sections of formalin-fixed tissues. (Manuscript in preparation.)