Antigenemia in paracoccidioidomycosis: probable demonstration of circulating antigen by counterimmunoelectrophoresis test: preliminary report

Antigenemia in paracoccidioidomycosis. probable demonstration of circulating antigen by counterimmunoelectrophoresis test. Preliminary report(¹)

Maria Conceição Rodrigues; Cintia Marzagão Cassaguerra; Carlos da Silva Lacaz

Instituto de Medicina Tropical de São Paulo and LIM/53 (Medical Mycology), HC-FMUSP. São Paulo — Brazil

Paracoccidioides brasiliensis presents as other dimorphic fungi a very complex antigenic structure. In vitro studies of STANDARD & KAUFMAN ¹⁷, and KAUFMAN et al.« on mycelial form cultures of that fungus demonstrate the production of three specific exoantigens designated 1, 2 and 3, able to react with preci-pitin-positive sera of patients with paracoccidioidomycosis. Peptide-polysaccharides containing mainly galactomannan were isolated from culture medium of the yeast form of P. brasiliensis (RODRIGUEZ V. et al.^IS). By the application of immunoenzymatic techniques and electron microscopy study, CAMPO-AASEN et al.² have shown the production and localization of the antigen Ej in yeast cells of P. brasiliensis. This antigen seems to be an important factor in the modulation of the host immune response. Circulating antigens have been detected in several experimental and human mycoses, as in cases of disseminated candidiasis (MILLER et al.9, WEINER & YOUNT²⁰, WARREN et al.^ls, RIPPON & ANDERSON^w, SEGAL et al.¹⁶, MECKSTROTH et al.⁸, NEGRONI et al.¹⁰), and in invasive aspergillosis (LEHMAN & REISS⁷, REISS & LEHMAN", RICHARDSON et al.« WEINER¹⁹). In the diagnosis of cryptococcosis, the detection of C. neoformans capsular polysaccharide antigen in body fluids is considered a useful routine test (KAUFMAN & BLU-MER 5, KAUFMAN *). It is valuable for starting specific therapy, when positive, even before the fungus is detected. Circulating immune complexes were detected and characterized in coccidioidomycosis (YOSHINOYA et al.²¹), and in cases of paracoccidioidomycosis (RAMOS e SILVA et al.¹¹, ARANGO et al.¹, CARVALHO et al.³). Indirectly, the presence of these immune complexes demonstrates the occurrence of circulating antigens in those mycoses.

In the evaluation of results of immunodiffusion test for demonstration of antibodies in sera from suspect cases of paracoccidioidomycosis we have verified the presence of precipitin lines at different positions as compared with those usually seen 'with precipitin-positive human sera. Some sera placed in the peripheral wells of the agar gel immunodiffusion slides with 2 seven-well patterns produced precipitin lines with the adjacent peripheral well. Apparently inespecific, these lines were not changed by wash in sodium citrate. This fact, with the additional presence of identity lines with the P. brasiliensis yeast form culture filtrate antigen placed in the center well suggested the presence of probable antigen in some of the studied sera.

In the last few years, counterimmunoelec-trophoresis test has been widely used in the se-rodiagnosis of systemic mycoses. Once standardized in our Laboratory, it has been a routine test in mycoserology. Due to its sensitivity, specificity and prognostic value, it was chosen as a reliable method for the present study. Proper 6 three-well patterns were used for the arrangement of the sera, in a trial to study the simultaneous presence of antibodies and antigens. Control antisera yet in preparation were replaced by precipitin-positive sera from patients with paracoccidioidomycosis, as mentioned in studies on other mycoses (WEINER & YOUNT²⁰, STANDARD & KAUFMAN ", KAUFMAN et al.⁶). The results of the counterimmunoelectrophore-sis correlated with those initially mentioned in the immunodiffusion test. The presence of precipitin lines has shown the occurrence of ananodic migration from the sera in study. This fact emphasizes the probable presence of antigen in the sera, as also suggested by the identity lines we have mentioned concerning the initial results of the immunodiffusion test (Pig. 1).

At the present we have studied ten samples of sera showing probable presence of antigen, one of which seemed to present antigen and antibody as reacting with other sera.

Antibody response do not always reflect active disease; they are variable and may be absent in certain compromised patients (KAUFMAN '). Although our study is yet in an early stage, next step :s the examination of negative sera from patients with clinically suspect relapse or active disease. High titer precipitin-positive sera were used as control antisera. Prom six negative sera studied, one has reacted as antigen with five of nine positive sera.

We did not establish yet the correlation of the probable presence of circulating antigen and the patients clinical data, forms of disease and therapy. Further studies with the addition oi polyetilenogbycol 6000 (PEG) to the agar or agarose gel in order to improve the sensitivity of precipitin tests (ZAROR et al.-) will be performed. The immunochemical characterization of the supposed antigen also must be accomplished by proper methods. "We consider coun-terimmunoelectrophoresis test to be a reliable and useful method also for the detection of circulating antigen in paracoccidioidomycosis.

Recebido para publicação em 4/4/1984.

• 1. ARANGO. M.; OROPEZA, F.; ANDERSON, O.; CON-TRERAS. ft; BIANCO. N. if YARZABAL. L, Circulating immune complexes and in vitro cell reactivity in paracoccidioidomycosis. Mycopathologia 79: 153-158, 1982.
• 2. CAMPO-AASEN, I.; CABRAL. N. A. & YARZABAL. L. Sub-cellular locoliralion of antigen E. of Paracoccidioides brasiliensis. An lmmunoeraymatlc electron microscopy study. Sabouraudia 18: 167171. 1380.
• 3. CARVALHO, I. P. de: COSTA. J. ft; ROSSI. B. C. & MARTINEZ. R. Imuno-complexos circulantes em pacientes com paracoccidioidomicose. Anais do II Encontro sobre paracoccidioidomicose. Campus de Botucatu, UNESP. São Paulo. Brasil, 1983.
• 4 KAUFMAN. L. Current methods for serodiagnosing systemic fungus infections and identifying their etiologie agents. L' Igiene Moderna 26: 427-442, 1981.
• 5. KAUFMAN. L. A BLUMER. S. Cryptococcosis: lhe awakening giant. In Proceedings of the Fourth International Symposium on Mycoses. Scientific Publ. N.ş 356. Pan American Health Organization. Washington. D.C. p. 176-184, 1978.
• 6. KAUFMAN, L.; STANDARD, P. & PADHYE, A, A. Exoantigen tests for the immunoidentification of fungal cultures. Mycopathologia (Den Haag) 82: 3-12, 1983.
• 7. LEHMAN. P. P. & REISS, E. Invasive aspergillosis: antiserum for circulating antigen produced after immunization with serum from infected rabbits. Infect. Immun. 20: 570-572. 1978.
• 8 MECKSTROTH. K. L.: REISS, E.; KELLER, J. W. & KAUFMAN. L. Detection of antibodies and anti- genemia in leukemic patients with candidiasis by enzyme-linked immunosorbent assay. J. Infect. Dis. 144: 24-32, 1981.
• 9. MILLER, G. G.; WITWER, M. W.; BRAUDE, A. I. & DAVID, C. E. Rapid identification of Candida albicans septicemia in man by gas-liquid chromatography. J. Clin. Invest. 54: 1235-1240, 1974.
• 10. NEGRONI, R.; ELIAS COSTA, M. R. I. de & GIMANO, S. Determinación de antigenemia en candidiasis disseminada experimental y humana. Rev. Arg. Micologia 6: 7-9, 1983.
• 11. RAMOS E SILVA, M.; MARQUES, A. S.; CAMPOS, D. S.; TABOADA, D. C.; SOARES, G. H.; BRAS-CHER, H. M.; VARGENS NETO, J. R.; CRUZ, M. Q. da; LABARTHE, N. V.; ROCHA, G. L. & LIMA, A. O. Imunologia da paracoccidioidomicose. An. bras. Dermatol. 56: 227-234, 1981.
• 12. REISS, E. & LEHMAN, P. P. Galactomannan antigenemia in invasive aspergillosis. Infect. Immun. 25: 357-365, 1979.
• 13. RICHARDSON, M. D.; WHITE, L. O. & WARREN, R. C. Detection of circulating antigen of Aspergillus fumigatus in sera of mice and rabbits by enzyme linked immunosorbent assay. Mycopathologia (Den Haag) 67: 83-88, 1979.
• 14. RIPPON, J. W. & ANDERSON, D. N. Detection of antigen by passive hemagglutination in experimental chronic candidiasis of rabbits treated with cyclophosphamide. Proc. IV International Conf. on the Mycoses. Pan American Health Organization, Washington, D. C, p. 288-294, 1978.
• 15. RODRIGUEZ V., V. J.; BIÈVRE, C. de & MARIAT, F. Étude des peptidopolyosides extraits du millieu de culture de la phase levure du champignon patho-gène de l'homme Paracoccidioides brasiliensis. Note. C. R. Acad. Sc. (Paris) 290: 1177-1179, 1980.
• 16. SEGAL, E.; BERG, R. A.; PIZZO, P. A. & BENNETT, J. E. Detection of Candida antigen in sera of patients with candidiasis by an enzyme-linked immunosorbent assay-inhibition technique. J. Clin. Microbiol. 10: 116-118, 1979.
• 17. STANDARD, P. G. & KAUFMAN, L. A rapid and specific method for the immunological identification of mycelial form cultures of Paracoccidioides brasiliensis Current Microbiology 4: 297-300, 1980.
• 18. WARREN, R. C; BARTLETT, A.; BIDWELL, D. E.: RICHARDSON, M. D.; VOLLER, A. & WHITE, L. Diagnosis of invasive candidosis by enzyme immunoassay of serum antigen. Brit. Med. J. 1: 1183-1185, 1977.
• 19. WEINER, M. H. Antigenemia detected by radioimmunoassay in systemic aspergillosis. Ann. Intern. Med. 92 : 793-796, 1980.
• 20. WEINER, M. H. & YOUNT, W. J. Mannan antigenemia in the diasnosis of invasive Candida infections. J. Clin. Invest. 58: 1045-1053, 1976.
• 21. YOSHINOYA, S.; COX, R. A. & POPE, R. M. Circulating immune complexes in coccidioidomycosis. Detection and characterization. J. Clin. Invest. 66: 655-663, 1980.
• 22. ZAROR, L.; ROBLES, A. M. & NEGRONI, R. Pruebas de inmunodifusion en medios gelosados con el agregado de polietilenglicol 6000 para el serodiagnostico de las micosis. Rev. Asoc. Arg. Microbiol. 10: 61-64, 1978.

(1

) Report presented at the 219th Seminary of the Instituto de Medicina Tropical de São Paulo in September 29, 1983

Publication Dates

• Publication in this collection
  08 Apr 2013
• Date of issue
  Oct 1984