Immunodiagnosis in oral candidiasis

Immunodiagnosis in oral candidiasis A review S. Jeganathan, NATIONAL

BDS, MSc,” and Yow Cheong Chan, PhD,b Singapore

UNIVERSITY

HOSPITAL

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OF SINGAPORE

Detection of anti-&&i& antibodies in sera and saliva of patients with oral candidiasis has been regarded as a valuable laboratory technique in the diagnosis of the lesion. However, despite considerable research, the value of candidal immunodiagnosis remains controversial. Conflicting conclusions about the sensitivities and specificities of these techniques as applied to human sera and saliva have appeared. These controversies have arisen because of the use of different antigen preparations and immunologic techniques. For the present, the use of purified cytoplasmic protein antigen of Ca~Mda albicam and the ELISA technique seems to be the most reliable laboratory method. (ORAL

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andidiasis varies in severity from a superficial localized oral infection, such as denture-induced stomatitis, to a severe systemic disease.Oral candidiasis is a common problem, which frequently appears as a chronic infection Localized chronic infections do not lead to serioussystemic involvement unlessthe person is otherwise compromised. Although antifungal agents are effective, recurrences are common in some patients after the cessation of medication. It is not understood why somepatients resist antifungal therapy, but we are tempted to suggestthat thesepatients may have an undetectable underlying immune defect. Diagnosis of candidiasis depends on the clinical signs, a demonstration of the organism in a direct smear, a culture of organisms in saliva, and the detection of specific antibodies to Candida antigen. The unequivocal diagnosisof candidiasis is difficult to establish. Clinically similar diseaseprocessesare produced by different mycotic agents as well as by certain bacterial and viral pathogens. In clinical mycology, the burden of diagnosisrests on direct detection, isolation, and identification of the fungus. In many instances these time-honored procedures cannot be fully used. For example, isolation and cultural identification may not always be successfulif the etiologic aDepartment of Restorative Dentistry, Faculty of Dentistry, National University Hospital. bDepartment of Microbiology, Faculty of Medicine, National University of Singapore. 7/13/36737

agent is sparse, nonviable, or overgrown with contaminants. Also, with the fungus, when cultural identification is possible, it may take a long time to accomplish and treatment may be delayed unnecessarily. In such situations, the clinician would welcome a rapid presumptive diagnosis basedon direct examination and histologic or immunologic evidence. Direct examination and histologic study, although useful in diagnosis,depend largely on morphologic characteristics. However, since a number of mycotic agents present similar morphologic forms in tissues, such examinations often do not provide a definitive diagnosis. Hence, immunodiagnosis may yet provide a useful adjunct for a definite diagnosis of oral candidiasis. The detection of candidal antibodies in the sera of patients has long been regarded as a rapid and valuable method in the diagnosisof candidiasis. However, despite considerable research effort by many laboratories, serodiagnosisof Candida remains a controversial subject. Clinical opinions on the value of Candida serodiagnosis vary substantially. Taschdjian et al.” concluded that “until a standardized antigen becomes available and standard testing methods have been agreed upon, the serological diagnosisof Candida infections will remain in status quo-promosing but uncertain.” Stone et a1.,2however, stated that “serological testing as a meansof establishing the diagnosis of an invasive yeast infection has not been reliable.” 451

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The principal reasons for the divergent opinions appear to be as follows: 1. The Candida antigens used in immunodiagnosis are usually prepared from a variety of yeast strains and by a variety of methods. Each laboratory, therefore, usually uses a unique, uncontrolled reagent in its tests. 2. Neither the types of tests suitable for the detection of Candida antibodies nor the details of their performance have been widely agreed upon or standardized, so that results of tests for Candida antibodies from different laboratories are often difficult to compare. 3. Because Candida is a ubiquitous component of the human microflora, antibodies to Candida may often be detected in the sera of persons without clinical candidiasis. These problems of test reproducibility and specificity are encountered throughout the literature on Candida immunodiagnosis.2 ANTIGENIC

EXTRACTS

OF CAND/~A

In immunologic tests, the Candida antigens used have been of three principal types: (1) cell wall antigens; (2) culture filtrate antigens; (3) cytoplasmic antigens. A fourth type, which is seldom used, is the nonviable yeast cell. Cell wall antigens The polysaccharides of a Candida cell wall are well known as determinants of the antigenic structures of different species. Mannans, glucans, mannoproteins, glucoproteins, and glucomannoproteins have all been extracted or labeled and shown to react with antibodies in sera raised against whole cells of Candida. Cell wall antigenic extracts, like culture filtrates, are usually crude preparations that contain a mixture of antigenically reactive components. Even preparation of mannans that appear to be reasonably pure in chemical and analytic terms have been found to produce as many as three precipitin arcs in immunodiffusion tests with human sera.3 Culture filtrate antigens Tran Van Ky et a1.4 showed that the antigenic components released by C. albicans into growth media were principally glycoproteins. They were able to detect seven individual antigens in unrefined culture filtrates by immunodiffusion test against rabbit antisera. However, culture filtrate antigens studied by Tran Van Ky et al. may have originated in the cell wall of C. albicans.

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When yeasts are disrupted mechamcally and the insoluble debris, such as cell wall, nuclei, and mitochondria, is removed by centrifugation, the supernatant is known as cytoplasmic extract. This often contains components that are antigenically indistinguishable from the glycoproteins present in Candida culture filtrates or cell wall extracts. It is likely that these components appear in the cytoplasmic extract as contaminants leached from the cell wall as a result of the strong forces used to disintegrate yeast cells. Cytoplasmic extracts appear to contain large numbers of immunologically reactive components. As many as 78 antigenic protein components in Candida cytoplasmic materials have been detected by two-dimensional antigen-antibody-crossed immunoelectrophoresis5 Greenfield and Jones6 have purified and characterized a major cytoplasmic antigen from C. albicans. This antigen is a single polypeptide chain that contains about 435 amino acids with a molecular weight of 54 kDa. Patients with disseminated candidiasis have been found to have significantly higher levels of antibody directed against this protein antigen than patients with noninvasive forms of candidiasis, patients with other fungal infections, or healthy persons.7 Recently, immunoblot analysis of serum antibody specificities against antigens contained in a soluble extract of C. albicans showed that a significantly higher proportion of sera from patients with oral and vulvovaginal candidiasis reacted with a 65 kDa antigen band than did sera from controls with clinically healthy oral mucosa. In addition, sera of patients with superficial candidiasis reacted more often than control sera to 38 kDa, 32 kDa, and 29 kDa antigens of C. albicans.8 LABORATORY ANTIBODIES

TESTS FOR CANDKIA

The increase in the number of tests available for the detection of Candida antibodies has paralleled the advances in immunology. Antibodies that agglutinate killed cells of Candida have been recognized and assayed since the beginning of this century.9 Agglutinins have been found in both healthy persons and those with disease. And the problem of cross-reactivity casts further doubts on the diagnostic value of agglutinins in candidiasis.1° Complement-fixing antibodies to Candida of similar titers have been detected in the sera of patients with and without candidiasis, so that the complement fixation test has been virtually abandoned in the serodiagnosis of candidiasis.2

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Precipitating antibodies to Candida have been detected by several methods that include Preer tube precipitation, immunodiffusion, and several variants of immunoelectrophoresis.” As with agglutinins and complement-fixing antibodies, precipitins were also demonstrated in sera from healthy volunteers and from patients with candidiasis.’ I All these conventional serologic tests for the assessment of serum antibody against Candida antigens in healthy and diseased subjects’ gave controversial results.t2 The reasons for this are poor standardization of methods and the nature of antigenic extracts used. Thus, without precise quantitation of antibody levels and adequate standardization, detection of antibody to C. albicans is of little value. A highly sensitive solid-phase radioimmunoassay has been used to detect serum levels of antibody to C. albicans in patients with superficial or deep-seated candidiasis over an extended period.13 The Candida solid-phase radioimmunoassay has been shown to be a convenient method for the detection of rapidly changing antibody levels and thus appears to be of diagnostic value. Sassi et a1.14 used a carefully standardized, sensitive, and quantitative: ELISA to measure antibody to purified cytoplasmic protein antigen from C. albicans. They found that healthy subjects had low antibody titers against this antigen whereas patients with denture-induced stomatitis had increased antibody titers. With the same technique, Ravindran et all5 reported that there was a significant decrease in antibody levels after antifungal treatment. It may be argued that a serologic test is an invasive procedure and its practicality in clinical practice may be questioned. Proponents of serodiagnosis argue that patients with candidal stomatitis often may have to undergo hematologic and biochemical investigations to exclude blood dyscrasias, hematinic deficiencies, and other conditions that may predispose to oral candidiasis. However, hematologic investigations may be required for only a small proportion of these patients. Furthermore, the need to obtain the patient’s consent and cooperation to c8011ectblood from a vein and to separate serum before tests often makes serodiagnosis difficult to carry out. Alternative fluids that do not require an invasive procedure for collection would be preferred for antibody tests. Saliva, is an ideal alternative specimen for antibody tests since it contains immunoglobulins, especially IgA. Although there is a considerable volume of literature on candidal antibodies in serum, salivary antibodies have been a subject of only a few immunologic

Immunodiagnosis in oral candidiasis 453

studies. The probable reason is that all classes of immunoglobulin are present in saliva in much lower concentrations than in blood.16 Earlier immunologic techniques, which were less sensitive and thus are dependent on the concentration of immunoglobulin, could not be used on saliva for detection of antibody. However, with the availability of more sensitive immunologic techniques, the use of saliva for antibody tests has become feasible. With the use of the immunofluorescence technique, various workers had reported raised salivary IgA anti-Candida antibodies in patients with oral candidiasis.17, t* With the same technique, Epstein et a1.18reported no change in specific anti-Candida antibodies in saliva after antifungal treatment. These studies showed that even in patients with candidiasis, anti-Candida antibodies were detectable only at very low dilutions of saliva. Indeed, Lehner17 stated that “a saliva. titer of 1:l or above indicates clinical infection.” Therefore, although immunofluorescence is useful in the detection of antibodies and thereby aids in the differential diagnosis of candidiasis, it may be less useful for the quantification of antibody levels. A more sensitive technique, such as the ELISA, is required to monitor antibody levels in saliva. Recently, Jeganathan et al., l9 with the use of the ELISA, reported significantly increased salivary IgA antiCandida antibodies in patients with oral candidiasis. A significant decrease in salivary IgA antibody levels was found in all patients after antifungal therapy. Hence, the determination of levels of serum and salivary antibody to cytoplasmic Candida antigen may be a useful laboratory method in the diagnosis of oral candidiasis. Monitoring of these antibody levels may be a useful tool in the assessment of the success of antifungal therapy. CONCLUSION

Numerous immunodiagnostic methods for oral candidiasis have been described in the literature. Because of the use of different techniques and antigenic preparations, controversial results have appeared. A standardized and sensitive technique, together with an antigenic preparation that would be specific to Candida infection, is an important requirement if laboratory methods are to be of any value as an adjunct to clinical diagnosis. One such test that satisfies these ideal requirements is the ELISA, which uses the purified cytoplasmic protein of C. albicans as antigen. The ELISA has been proven to be a sensitive, cheap, and quick method for the quantification of antibody titers and would undoubtedly be the laboratory method of choice.

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REFERENCES 1. Taschdjian CL, Seelig MS, Kozinn PJ. Serological diagnosis of Candidal infections. CRC Crit Rev Clin Lab Sci 1973:4: 1959. 2. Stone HH, Kolb LD, Currie CA, Geheber GE, Cuzzell JZ. Candida species: pathogenesis and principles of treatment. Ann Surg 1974;179:697-711. Leicester: Leicester Uni3. Odds FC. Candida and candidosis. versity Press 1979. 4. Tran Van Ky P, Biquet J, Andrieu S. Etude par l’electrophorese et la double diffusion en gelose des substances antigeniques excretes dans le milieu de culture de Candida albicans. Sabouradia 1963;2:164-70. 5. Axelsen NH. Antigen-antibody crossed electrophoresis (Laurell) applied to the study of the antigenic structure of C. albicans. Infect Immun 1971;4:525-7. 6. Greenfield RA, Jones JM. Purification and characterization of a major cytoplasmic antigen of Candida albicans. Infect Immun 1981;34:469-77. I. Strockbine NA, Larger MT, Zweibel SM, Buckley HR. Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera. Infect Immun 1984;43:715-2. 8. Weller BI, Simmons PD, Ivanyi L. Identification of immunodominant antigens of Candida albicans in patients with suoerficial candidosis. Clin Immunol Immunooathol 1990: 541347-53. 9. Winner HI, Hurley R. Candida albicans. London: Churchill Livingstone 1964. 10. Salvin SB. Ouantitative studies on the serologic relationshio of fungi. J Immunol 1950;65:617-20. w 11. Chew WH, Theus TL. Candida precipitins. J Immunol 1967; 98:220-4. 12. Rogers TJ, Balish E. Immunity to Candida albicans. Microbiol Rev 1980;44:660-82.

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13. Mauch II. Diagnostic value of monitoring kinetics of antibody responses in candidiasis by a solid-phase radioimmunoassay. Am J Clin Path01 1983;79:200-5. 14. Sassi KOH, Ivanyi L, Hobkirk JA. Comparison of serum antibody levels to Candida albicans in patients with denture induced stomatitis and in controls with clinically healthy oral mucosa. (Abstract #247). J Dent Res 1986;65:515. 15. Ravindran N, Ivanyi L, Zakrzawska J, Nally FF. Comparison of serum antibody levels to Candida a1bica.m in patients with chronic oral candidosis before and after antifungal therapy (Abstract #251). J Dent Res 1986;65:515. 16. Jenkins GN. The physiology and biochemistry of the mouth, 4th ed. Oxford: Blackwell Scientific Publications, 1978: 324-7. 17. Lehner T. Immunofluorescence investigation of Candida albicans antibodies in human saliva. Arch Oral Biol 1965;10:97580. 18. Epstein JB, Pearsall NN, Truelove EL. Oral candidiasis: effects of antifungal therapy upon clinical signs and symptoms, salivary antibody and mucosal adherence of Candida albicans. ORALSURGORALMEDORALPATHOL 1981;51:32-6. 19. Jeganathan S, Ufomata D, Hobkirk JA, Ivanyi L. Immunoglobulin Al and A2 subclass of salivary antibodies to Candida albicans in patients with oral candidosis. Clin Exp Immunol 1987;70:316-21. Reprint requests: S. Jeganathan, BDS, MSc Department of Restorative Dentistry Faculty of Dentistry National University Hospital Lower Kent Ridge Road Singapore 05 11