Hoshino-Shimizu, Sumie

Kanamura, Herminia Y.

Silva, L. C. da

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. São Paulo 20(2) :76-81, março-abril, 1978 UDC 616.995.122.22 PATTERN OF CLASS-SPECIFIC FLUORESCENT ANTIBODIES ACCORDING TO THE STAGE OF THE INFECTION IN HUMAN SCHISTOSOMIASIS MANSONI Herminia Y. KANAMURA, Sumie HOSHINO-SHIMIZU and L. C. da SILVA SUMMARY The possible association of class specific antibodies to S. mansoni and stage of in-fection was studied by indirect fluorescent antibody test (IFT) in twenty-five selected sera from patients with acute and chronic schistosomiasis mansoni. IgA antibodies were detected only in sera from acute cases, but IgG, IgM and lgE antibodies were found in acute and chronic cases. Rheumatoid factor eventually present in the sera was removed by adsorbing the sera with immunoadsorbent of IgG, before the IFT was performed. In the acute stage the hemagglutination test (HAT) showed to be less sensitive than IFT. INTRODUCTION Immunofluorescence, hemagglutination, complement fixation and immunoprecipitation tests can detect anti-S. mansoni antibodies and are a very useful toai for the diagnosis of the disease. However, the correlation betlween serological findings and the stage of the in-foction has not definitely been .stablished. The leveis of different imn1unoglobulin classes in the sera of patients with schistoso-rniash; mansoni were studied by single radial immunodiffusion test 1,3,5,7,10,1s_ However, as far as the clinicai forms of the disease are concerned the results obtained by those Au-thors do not agree. Therefore, it might be more useful to determine the class-specific antibodies to S. mansoni instead of studying the total amount of immunoglobulin classes by radial immunodiffusion. Recently, the immunofluorescence test carried out with class-specific conjugates to immunoglobulins detected IgG, IgM, IgA and IgE antibodies to S. mansoni in sera of infect-ed patients 9,15,16, but thes.e findings were not related to the different stages of the disease. This report shows the results of a study based on class-specific fluorescent anti-S. mansoni antibodies in patients with different stages of the infection, clinically characteriz-ed as acute and chronic (intestinal, hepatoin-testinal and hepatosplenic) forros. Indirect hemagglutination test was also performed. M/ATERIAL AND IMIETHODS Sera ~ They were collected from 25 schis-tosomotic patients, with the following clinica! diagnosis rn: Acute form: 5 patients Chronic form: 20 patients (intestinal: 6, hepatointestinal: 5, hepatosplenic: 9 paticnts). Conjugates Commercially available conjugates to human IgG, IgM, IgA and IgE (H]yland Div. Travenol Lab. U.S.A.) were used after testing their specifici~y by immuno-electrophoresis against selected sera with high levei of immunoglobulins as determined by ra-dial immunodiffu.sion test. In arder to test the specificity of anti-IgE conjugate, one serum was lyophilized and concentrated 3 times. Indirect fluorescent antibody test (IFT) - Two S. mansoni antigens were prepared for cryostat sectioning as follows: 1) adult worms were embedded in Tissue-Tek O.C.T. medium (*) and frozen in liquid nitrogen according to WILSON et ai. 21. (") Ames Co., Miles Lab., U.S.A. ln'Stituto de Medicina Tropical de São Paulo, São Paulo, Brasil. Supported by a Grant from «Conselho Nacional dé Desenvolvimento Cientifico e Tecnológico (CNPq)» (SIP/08-110) 76 KANAMURA, H. Y.; HOSHINO-SHIMIZU, S. & SILVA, L. C. da ~ Pattern of class-specific fluorescent anti-bodies accordíng to the stage of the infection in human schistosomiasis mansoni. Rev, Jnst. Med. trop. São Paulo 20 :76-81, 1978. 2) liver specimen with eggs and granulo-mata obtained from infected hamster was also frozen as described by COUDER~ et al. 6. Sections were cut at 4 µ thickness, fixed on microscope slides and stored at -20ºC until used. The slides were then fixed in cold ace-tone for 5 minutes before the immunofluo-rescent technique. Serum dilutions from 1/10 to 1/5,120 were added on tissue sections and, after incubating for 30 minutes at 37°C, these were washed for 20 minutes with two changes of PBS (0.15M NaCI; 0.01M phosphates; pH 7 .2). An appropriate dilution of conjugate was placed on the specimens, and the slides were again incubated for 30 minutes at 37ºC, twice washed in PBS and mounLed wiLh glice-rol (pH 8.5) and a coverslip. All immunofluo-rescence tests included both positive and. ne-gative controls. The slides were examined under a Zeiss fluorescence-microscope provided with a HBO-200 bulb, KP-500 exciter filter, Zeiss 50 barrier filter and dark field condenser. Passive hemagglutination test - For the EB] Acute wm1 1nte6tinol hemagglutination testa preserved reagent was used according to HOSHIINO et ai. 13. Thi.s was prepared by Lyophilizing formalin treated cells which had been preserved by aldehyde fixation after tannic acid treatment and sen-sitization with S. mansoni extracts. Sera were serially diluted from 1/20 to 1/5,120 in O. 85% NaCI and plastic microtitration plates used for thi.s test. Rheumatoid factor - A search for rheu-matoid factor wa.s performed in ali sera by latex test (Behringwerke AG, Germany). Ten selected sera, in which IgM was de-tected, were also adsorbed with immunoadsor-bent of human IgG2 and retested by immuno-fluorescence technique, in arder to identify the immunological specificity of IgM. RESULTS Monospecific anti-human globulin.s immu-nofluorescence tests and hemagglutination test titers obtained in 25 sera from patients with acute and chronic schis.tosomiasis are presented in Table I. Figure 1 shows the fin-____ E]----1::l_fil:!o to i n testlnol ~ Hepatosplenlc [3 Meoh titers {log.) ► ~ > . .: 00 o ~ " 3. 5 'ºº + + + 3.0 + + 80 + + + 2. 5 + + + + 60 + 2.0 + + + + + + r. 5 40 + + + ,. o + + 20 + + + + o. 5 + + + o + o lgM !gE '-----------~ANTIBODY-----------lgG lgA 1 Fig. 1 - Oislribulion ond meon lilers of closs specific ontibodies found in sera from schístosomolic polients occording to lhe cllnlcol form of lhe diseose. .; o " m "' w~ .: 77 KANAMURA, H. Y.; HOSHINO-SHIMIZU, S. & SILVA, L. C. da - Pattern of class-specific fluorescent anti-bodies according to the stage of the infection in human schistosomiasis mansoni. Rev. Inst. Med. trop. São Paulo 20:76-81, 1978. TABLE I Titers of 25 sera from patíents with acute and chronic schistosomiasis mansoni obtained by immunofluorescence test (IFT), using monospecific conjugates, and by passive hemagglutínation test (HAT) I F T " " Patient o E IgG Ab IgM .Ab IgE Ab IgA Ab HAT ·a :... § .8 Liver Worm Liver ( * *) 1. css 320 320 80 2. IGO ., 320 80 40 E< 3. JAL :o 640 320 320 4. JRA <.) 640 640 320 < 5. wss 640 320 160 6. AFN :320 320 160 7. ARL 40 80 20 8. ATS 20 40 10 9. ESN 640 640 40 10. FAM 640 320 40 11. VAS 80 80 -12. CRX 640 320 20 13. DDS ' 640 320 40 14. DMJ 320 320 40 15. MCN <.) 2560 320 80 H 16. ZLS z 640 80 -o " 17. AMD iI1 640 640 20 18. ASS <.) 320 160 40 19. EAV 1280 320 20 20. HVS 2560 1280 80 21. JBA 2560 640 20 22. JJS 640 640 80 23. LVS 1280 820 80 24. MAS 640 320 20 25. MDX 640 640 40 (*) Chronic forms: intestinal (No. 6 to 11) hepatointestinal (No. 12 to 16) hepatosplenic (No. 17 to 25) Worm Liver Worm Liver Worm 160 20 20 40 20 40 80 40 ('...'._'.) 20 - 80 320 80 80 40 20 80 640 80 80 40 80 160 160 80 40 80 40 80 80 40 10 - - 640 20 - 10 - - 320 10 - - - - 80 40 80 40 - - 40 80 20 40 - - 320 - - - - - 40 40 40 40 - - 320 40 40 40 - - 320 40 20 20 - - 320 160 320 80 -- - 640 - 40 40 - - 320 40 80 40 - - 160 20 20 - - - 320 80 80 40 - - 320 160 :~20 80 -· - 2560 80 160 160 - - 640 160 80 80 320 40 40 40 610 - 40 20 - - 1280 80 20 10 - - 160 (**) Liver = Jiver sections with egg and granulomata worm = adult worm sections (::_:) = negative rcsult dings of IgG, IgM, IgA and IgE antibodies to S. mansoni, according to clinical form of dis-ease and estimated in percentages and mean titers. Fluorescent antibody to S. mansoni IgG - All sera gave a positive reaction to adult worm and to egg Oiver section) an-tigens, respectivelly with mean titers (*) 280 and 480 in acu te cases, and 290 and 500 in chronic cases. (*) mean titers geometric mean titcrs 78 lgM - 17 Out of 20 sera from chronic ca-ses gave a positive reaction to ~ult worm and egg antigens, and both mean titers were 30. Two sera gave negative results to worm and egg antigens and one, showed positive re-sul t onjy to egg an tigen. All 5 sera from acute cases were positive to IgM antibodies, and the titers tended to be higher than in chronic cases. The mean titers 210 and 140 were respectively obtained with wo-rm and egg antigens. lgE - Most sera (23) gave a po.sitive anti-IgE flu,orescent reaction. fÍOWever, in 3 sera, KANAMURA, H. Y.; HOSHINO-SHIMIZU, S. & SILVA, L. C. da - Pattern ~f cla.ss-specific fluoroocent anti-bodies according to tho stage of the infection in human schistosomia.sis ma.nsoni. Rev. Inst. l\:led. trop. São Paulo 20 :76-81, 1978. 2 from chronic and one from acute schistoso-miasis, positive reactions were observed with one antigen, either worm or egg. Moreover, there were 2 chronic cases in which IgE anti-bodies could not be detected. In acute cases, mean titers were 20 for worm and 50 for egg antigen. In chronic cases, mean titers were respectively 20 and 30. Negative and lower titers were mostly found in intestinal forros. lgA - IgA antibodies were detected only in the acute forro of the disea.se, with worm and egg antigens (mean titers 20 and 40, res-pectively). Hemagglutination test (HAT) - Mean. ti-ters were lower in acute (80) than in chronic schistosomiasis (320). Rheumatoid factor .:........ The latex test re-vealed antibodies to IgG in two sera (AFN e LVS). The adsorption of 10 selected sera (IGO, JAL, AFN, Fil:M, MCN, ZLS, HVS, JJS, LVS and MAS) -with immunoadsorbent of IgG in-dicated that IgM antibodies first detected by immunofluorescent test were specific to S. mansoni. The rheumaitoid factor detected in those two sera did not interfere with the ti-tration of specific antibody. DISCUSSION Although some Author.s 8,9,15,16 have alrea-dy studied immunoglobulin classes of anti-8. mansoni antibodies in infected patients and animals, to our knowledge there is. no investi-g-ation about the significance of these anti-bodies in different stages of the disease in humans. The fluorescent antibody test showed IgG, IgM and IgE anti-S. mansoni antibodies in sera from acute and chronic cases. The finding of IgA antibodies only in acute cases differed strikingly from the chronic cases in which these antibodies were absent. The presence of IgA antibodies in schis-tosomiasis seems to be significan t since in other parasitic infections such as toxoplasmo-sis, trichinosis and hydatidosis IgA antibodies were found to be an indication of recent anti-genic stimuli 15,17. In $. mansoni infection, HULDT et al. 15 have found IgA antibodies in 6 out of 103 pa-tients and DEELDER et al.9 in every case from 19 selected patients. Also in S. haematobium infection IgA antibodies were detected in all 24 patients studied by KANE et ai. 16. How-ever, the stage of the .infection was not mentioned by those Authors. Unlike virus or protozoan infection, IgM response in schistosomiasis does not necessari-ly reflect a recent infection. However, it was observed that in acute cases IgM levels were slightly higher than in chronic cases. A continuous stimulation with different antigenic determinants might elicit IgM anti-body response in the host even at a late stage of the disease. IgM antibodies to S. mansoni in chronic cases of human schistosomiasis were aLso described by SILVA & FERRI 20 and HILLYER 12. Since rheumatoid factors are referred as usually occurring in many parasitic dis-eases 4,II.14, we considered the possibiliJty of IgM reactions seen in the IFT being due to this factor. However, the tests showed that IgM detected in the sera of acute and chronic cases werc specific to S. mansoni. For Lhe Iatex test, on\y two revealed the pre-sence of rheumatoid factor. Besides., after complete remova! of rheumatoid factor from the,-;e two sera, both from pat,ients with chro-nic infection, IgM antibodies to S. mansoni could still be· detected. The other sera also showed no change in Ig·M antibody titers after treatment with insoluble IgG. IgE antibodies to S. mansoni were de-monstrated in 23 out of 25 sera. Some Authors 9,10 have found IgE in about 50% to 70% o! infected patients .. In our hands IgE as well as IgM and IgG antibodies did not differentiate acute from chronic infections. Our results suggest distribution of class specific antibodies to vary in the course of the infection. The observed modifications per-mit,. thus, to select more appropriate tests to be applied either for diagnostic purposes or to follow the course of the infection. More de-tailed studies are under wa:y in a Iarger group of patients from an endemic area. 79 KANAMURA, H. Y.; HOSHINO-SHIMIZU, S. & SILVA, L. C. da - Pattern of class-.specific fluorescent anti-bodies according to the stage of the infection in human schistosomiasis mansoni. Rev. Jnst. 1\-led. trop. São Paulo 20 :76-81, 1978. As far as HAT is concerned, it showed to be a less sensitive test than IFT in the acute stage of the disease. RESUMO Comportamento das imuneglobulinas específi-cas nas diferentes formas clínicas da esquistossomose mansônica A possível associação de anticorpos anti-S. mansoni de diferentes classes de imuneglo-bulinas com o estágio de infecção foi estudada por técnica de imunofluorescência (IF), em 25 soros selecionados de pacientes com esquistos-somose aguda e crônica. Anticorpos IgA fo-ram detectados somente em soro de casos agu-dos, tendo-se encontrado anticorpos IgG, IgM e IgE, tanto em casos agudos como crônicos. O fator reumatóide, eventualmente presente nos soros, foi removido por adsorção com imu-noadsorvente de IgG, antes de se efetuar o teste de IF. A reação de hemaglutinação pas-siva mostrou-se menos sensível do que a IF para detecção de anticorpos em casos agudos da doença. ACKNOWLEDGEMENT We wish to express our gratitude to Maria do Carmo Berthe Rosa for the elaboration of the bibliographieal references. REFERENCES 1. ANTUNES, L. J.; REIS, A. P.; PELLEGRINO, J.: TAVARES, C. A. & KATZ, N. - Immunoglo-bulins in human ·schistosomiasis mansoni. J, Pa-rasit. 57 :539-542, 1971, 2. A VRAMEAS, A. - Coupling of encymes of pro-teim::; with glutaraldehyde. Use of conjugate for the detection of antigens and antibodies. Immn-uochemistry 6 :43-55, 1969. 3. BASSILY, S.; HIGASHI G. I.; F,ARID, Z. & WILLIAM:S, R. E. - Se~um immunoglobulins in sC:histosomia.sis mansoni. J, Trop. l\led. Hyg. 75: 73-75, 1972. 4. CAMARGO, M. E.; LESER, P. G. & ROCCA, A. - Rhoumatoid factors as a c;mse for false po-sitive IgM anti-toxoplasma fluorescent · tests. A technique for specific results. Rev, Inst. Med. trop, São Paulo 14':310-313, 1972. 5. COOK, J. A.; WOODSTOCK, L. & JORDAN, P. - Immunological studies in Schistosoma mansoni 80 infection in St. Lucia. Ann, Trop. l\'led. Parasit. 66 :369-373, 1972. 6. COUDERT, J.; GARIN, J. P.; AM:BRIOSE-THOMAS, P. & POTHIER, M. A. - Premiers résultats à propos du diagno,stic séroJogique de la bilharziose par immunofluorescence sur coupes à Ia congelation de Scllistosoma mansoni. Ann. Parasit. Hum. Comp. 42:483-492, 1967. 7. CURIEL, M.; CHAVES, J. & TORREALBA, J. W. - Immunoglobulin levels in schistosomiasis. Rev, Inst. Med. trop. São Paulo 14:384-387, 1972. 8. DEELDER, A. M. - Immunology of experimental infcction-s with Schistosoma mansoni in the swiss mouse and Faséiola hepatica in the rabbit. Acta Lcidensia 39:1-107, 1973. 9. DEELDER, A. M,; SNOIJINK, J. J. & PLOEM, J. S. - Immunoprecipitation and class-specific immunofluorescence titration of human serum anti-bodies to Schistosoma mansoni antigen.s. Z. Pa-rasitenk. 46 :195-201, 19-75. 10. DESSAINT, J. P.; CAPRON, M.; BOUT, D. & CAPRON, A. - Quantitative determination of specific IgE antibodies to schistosome antigens and serum IgE leveis in patients with schistoso-miasis (S. mansoni or S. haematobium). Clin. Exp. Immunol. 20:427-436, 1975. 11. GREENWOOD, B. M.; MULLER, A. S, & VAL-KENBURG, H. A. - Rheumatoid factor in Nige-rian sera. CH.n, Exp, Immunol, 8:161-173, 1971, l:l. Hl 1,1, V l<],H., G-. V. - 1 mmlHJOJ)l'(·l(lipit.jn,; ln Schis-tosoma mansoni mfections. lV - Human infec-tions, Exp. Parasit, 25:376-381, 1969. 13. HOSHTNO, R.; CAMARfl.O, M. E. & RTT.,VA, L. C. da - Standardization of a hemagglutination test for ,schistosomiasi.s with formalin-tr-eated human erythrocyte.s. Amer, J. Trop. l\Ied. Hyg, 19 :463-470, 1970. 14. ROUBA, V. & ALLISON, A. C. - M-antiglobulins (rhe-umatoid-factor-like globulins) and other gam-ma-globulins in relation to tropical parasitic in-fections. Lancet 1 :'843-852, 1966. 15. HULDT, G.; LJUNGSTROM, I. & AUST-KE-TTIS, A. - Detection by immunofluorescence of anti-bodies to para.sitie agents. Use of cla.ss-specific conjugates. Ann. N.Y. Acad. Sei. 254:304-313, 1975. 16. KANE, G. J; MATOSSIAN, R. & BATTY, I. -Fluorochrome-labelled anti-immunoglobulin frac-tions used with stabilized antigen preparations for the assessment of para.sitie diseases. Ann. N.Y. Acad. Sei. 177:134-145, 1971. 17. MATOSSIAN, R. M,; KANE, G. J".; CHANTLER, S, M.; BATTY, I. & SARHADIAN, H. - The spe-cific immunoglobulin in hydatic disease. lmmu-nology 22 :423-430, 1972. KANAMURA, H. Y.; HOSHINO-SHIMIZU, S. & SILVA, L. C. da - Pattern of class-specific fluorescent anti-bodies according to the stage of the infection in lmman schistosomiasis mansoni. Rcv. Inst, 1\-Ie,d. trop. SiiQ Paulo 20 :76-81, 1978. 18. MORIEARTY, P. L. & LEWERT R. M. - De-Jayed hypersentivity in Ugandan schistosomiasis mansoni. III - Examination of serological res-ponses and clinica! states. Amer, J. T1•op, 1\-Icd. H~·g. 23:190-196, 1974. 19. SILVA, L. C. da - Anticorpos e eosinõfilos cir-culantes na csquistossomose mansônica-. Contribui-!.liio ao estudo de efeitos da <Iuimiotcrapia.. [Tese de livre-docência]. São Paulo, Faculdade de Me-dicina da Universidade de São Paulo, 1974. 20. SILVA, L. C. da & FERRI, R. G. - Immuno-diffusion studies in human schistosomiasis man-soni. II - Localization of antibodies by immu-noelectrophoresis. Rev. Inst. Med. tro1>. São Pau-lo 7 :7-10, 1965. 21. WILSON, M.: SULZER A. J. & WALLS, K. W. - Modified antigens in the indirect immunofluo-rescence test for schistosomiasis. Amer, J, Trop. Med. Hy.g, 23:1072-1076, 1974. Recebido para publicação em 30/5/1977. 81 

Pattern of class-specific fluorescent antibodies according to the stage of the infection in human schistosomiasis mansoni

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Pattern of class-specific fluorescent antibodies according to the stage of the infection in human schistosomiasis mansoni

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