Camargo, Mario E.

Hoshino-Shimizu, Sumie

Nagasse, Tereza. X.

English

Cadernos Espinosanos (E-Journal)

Rev.- lnst. Med. trop. São Paulo 20(4) :208-212, julho-agosto, 1978 UDC 593 .161 .13 616.937.3-092.9 A STABLE POLYSACCHARIDE-HEMAGGLUTINATION REAGENT FOR THE DIAGNOSIS OF ACUTE OR RECENT TRYPANOSOMA CRUZ! INFECTIONS ("J Sumi.e HOSHINO-SHil\llZU, l\fa.i.-io E. CAl\-IAJtGO and Tereza. X. NAGASSE SUIMIMARY A stable hemagglutination reagent was developed for antibodies against Trypanoso-ma cruzi polysaccharide components. Conditions for sensitizing aldeh,yde-fixed human erythrocyt·es with parasi.te polysaccharides are described, as. well as for obtaining a lyo-philized reagent. Hemagglutination of polysaccharide-sensitized cells was shown to be due to IgM antibodies. Thes,e were observed in every case studied of acute Chagas'disease, however they were absent or in very low titers in chronic cases. Possible seroepidemiologic applications of the test are considered. INTRODUCTION Sera from patients with acute or recent Chagas'disease were shown to react with po-lysaccharide components of Trypanosoma cruzi both in precipitin and complement fixa-, tion tests B,9. Preliminary evaluatioh of such antigens in the precipitin tests presented 97.9% to 100% positives in acute cases and 17% to 18% in chronic infections 9,12. Also in the complement fixation test, polysaccharide antigens furnished higher titers for acute than fbr chronic cases, only a fe'W of which showed positive results 9,11. The hemagglutination test with stable, preserved reagents, is a vecy practical proce-dure for Chagas'disease routine serologioal diagnosis, both for clinical and seroepidemio-logical purposes. However, the usual test with a reagient prepared by coating with T. cruzi extracts aldehyde-fixed cells treated by tannic acid or chromium chloride, although showing à high sensitivity for chroni-c c,a.seS s, is of li-mited diagnostic value for recent infections, wher,e low titers ar negative results are com-mon. Recently ANTUNES et ai. 1 have describ-ed an hemagglutination test. for muco-cuta-neous leishmaniasis, with a Leishmania bra-siliensis polysaccharide ,an tigen fixe d on fresh red cells. Positive results were observed for 91 % of the tested cases and only low-titered cross-reactions were seen with sera from Chagas'disea.se patients. However, whien trying to sensitize erythrocytes with T. cruzi poly-saccharide components, the techniques des-cribed for coating both fresh 1 and aldehyde-treated 5 -cells with polysaccharide antigens were not successful. This paper presents the standardization of a polysaccharide-hemagglu-tination test for Chagas'disease and results obtained with a few sera from acute or chro-nic Chagas'infection. MATERIAL ANO METHODS T. cruzi antigens a) Soluble saline extract - A suspension of 100 mg Iyophilized T. cruzi epimastigotes in 30 mi saline solution was desintegrated by ultrasonic oscillation for 2 minutes, in an ice-bath, and kept overnight at 4°C in a ro-tator. After centrifuging at 10,000 x g for 30 minutes at 4°C, the supernatant was stored at -70°C until used. Instituto de Medicina Tropical de São Paulo, São Paulo, Brasil (*) Supported in part by Brazilian «Conselho Nacional de Desenvolvimento Científico e Tecnológico» (CNPq), grant ll\> 2392 (SIP/08-061) 208 HOSHINO-SHIMIZU, S.; CAMARGO, M. E. & NAGASSE, T. K. - A stable polysaccharide-hemagglutination reagent for the diagnosis of acute or recent Trypanosoma cruzi infections. Rev. Inst. 1\Icd. trop. São Paulo 20:208-212, 1978. b) Saline-insoluble fraction - The pellet was resuspended in a large volume of saline solution and kept in a rotator at 4ºG for 24 hours, with 3 changes of saline solution in arder to remove all soluble components. To the washed pellet, 15 mi Df 0.15 M NaOH solution Were added and the mixture kept at 4°0 for 18 hours. After neutralizing with an equal vo-lum1e of O .15 M HCl solution, eventual insolu-ble particles were removed by centrifugation of the mixture and the supernatant was kept at -70°0 until used. e) 'Whole crude antigen - One hundved milligranis lyophilized T. cruzi epimastigotes were mixed with 15 mi O .15M NaOH, in a tis-sue homogenizer, and kept overnight at 4°C. After neutralizing with HCl solution, any in-solubl1e residue w,as removed by centrifugation. d) Po~ysaccharide antigens - Parasite polysaccharide fractions w-ere prepared ac-cording tD the techniques described by MU-NIZ & FREITAS 9, GONZALEZ ,& YAMAHA 4 and NASH et al. 10. Protein and polysaccharide determinations Proteins were determined according to LOWRY et ai. 6 and pD\ysaccharides according to MARTIRANI et ai. 7, in the different anti-gens. Formalin-fixed erythrocytes Freshly collected human group o, Rh-ne-gative erythrocytes were washed 4 times with a O .15M NaOI solutiDn and suspended at 10% in PBS (0.15 M NaCI, O.O! M phosphates, pH 7 .2). An equal volume of 4% formaMehyde sol u tion in PBS was added and the mixture kept at 37°0 for 18 hours, With occasional stirr_ ing. Oells were washed 3 times in disti!led water or saline solutión and suspended at 20% in 0.15 NaOI solutiDn with 0.8% fDrmaldehy-d,e. Such fDrmalin-fixed cells could be kept at 4ºC for at least 2 years with no apparent de-teriora tion. Sensitization o.f formalin-fixed erythrocytes with T. cruzi antigens a) Polysaccharid.e antigens - Oells were incubated for varying periods with po\ysaccha-ride antigens diluted in different solutions at different te-mperatures and pH values. To the suspension was then added an equal vo-lume of 0.1 % glutaraldehyde saline solution, the mixture incubated for 30 minutes at 37°0. After wa.shing twice with saline solution, the cells were suspended for immediate use at 2% in PBS pH 6.4 containing 0.9% dried skim milk. b) Protein antigens - The procedure described by OAMARGO et ai. 2 was follDwed, with minar modifications. In brief, sarne vo--Iumes of a 2% cells suspension and a 1: 15,000 tannic acid dilution, both in saline solution, were mixed and incubated at 56°C for 15 mi-nutes. Oells were washed twice in- saline solu-tion 1and res.us,pended at 2% in an'Gigen dilut-ed in PBS pH 6.4. After incubating at 37°0 for 50 minutes, an equal volume of O .1 % glu-taraldeb,yde in PBS pH 6. 4 was added to the suspension and the mixture maintained at 37°0 for 30 more minutes. After washing for three times in saline solution, cells were sus-pended a t 2 % in PBS 6 . 4 contalning O. 9 % dried skim mi1k, for immediate use. Preservation and storage of hemagglutination reagents Sensitized erythrocytes were suspended at 6% in a presierving solution (PBS. pH 6 .4 con-taining 4% glycine, 2. 7% dried skim milk, 2% sodium glutamate, 0.1% sodium thioglycolate and O .1 % merthiDlate) and lyophilized in 1.0 mi aliquots. For use -each aliquot was recons-tituted with 3. O mi distilled water. Reconsti-tuted reagent CDuld be kept at 4º0 for at least two months with no Ioss of activity. Serologic tests Passive hemaggluti~ation, complement fi-xation, anti-IgG immunofluorescence and an-ti-IgM immunofluorescence tests were per-formed as already described 2. Serum samples As soon as parasitemia was detected in patients with acute Chagas'disease, serum samples were collected at different succe.ssive intervals. These were mostly post-transfusio-nal pa tien ts. 209 HOSHINO-SHIMIZU, S.; CAMARGO, M. E. & NAGASS.E, T. K. - A stable polysaccharide-hemagglutination reagent for the diagnosis of acute or recent Trypanosoma Cl'nzi infections. Rev. Inst. Med. trop. São Paulo 20 :208-212, 1978. Sera were also obtained from chronic ca-ses of Chagas'disease and from non-infected individuals. Trea.tment of sera 'with Z.-mercaptoethanol The technique of DEUTSCH & NORTON 3 w.a.s followed for treating sera with 2-ME. RESULTS Standardization of fixed-erythrocytes polysar ccharide sensitization When incubating aldehyde-fixed cells un-der variable conditions of temperature and time with T. cruzi polysaccharide antigens di-luted in a serles of different solutions and buffers with a wide pH range, it w.a.s. observed that only alkaline NaOH antigenic solutions were able to sensitize the cells. Optimal conditions wer•e investigated first with extracts prepared according to NASH et ai. 10, but similar results were later found for parasite fractions obta.ined as described by MUNIZ & FREITM 9 and by GONÇALVES & YAMAHA 4_ strikingly, it became patent that previous isolation of such components was not necessary, since at optimal conditions sensiti-zation results were the sarne with either pu-rified polysaccharides or T. cruzi total ex-tracts. In Table I results of siensiti~a~ions both with purified components or total extract, for different NaOH concentrations, are expressed by test titers of a serum sample from a-n acute case of Chagas'disease. Results were negative for sera from chronic cases and from non-in-fected individuais. TABLE I Serum titers for antig·ens diluted in different NaOH solutions (sensitization at 56QC for 15 minutes) Antigens NaOH concentration: 2M lM 0.5M 0.2M 0.1M 0.05M 0.02M 0.01M Polysaccharide - (10 µg/ml) 640 1,280 2,560 1,280 1,280 640 Total extract (Polysaccharide - 10 µg/ml 640 2,560 1,280 2,560 1,280 1,280 640 Protein - 170 µg/ml) It was obs.erved that cell coating with po-lysaccharide antigens could be obtained in a temperature range of 4ºC to 56°C, as long as suitable incubation was allowed. Longier pe-riods were required at lower temperatures but, at 56ºC, 5 minutes were enough for maximal sensitiZation. No increase in titers were observ-ed when incubation was. lengthened to as much a.s 60 minutes. The study of antigen concentration show-ed that maximum sensitization could be obtained with as Iow as 3 µg/ml polysaccharide (Table II). TABLE II Serum titers for different polysaccharide antigen concentrations (sensitization in NaOH 0.5 M, for 10 minutes at 56QC) Antigens Polysaccharide concentration (sugar contents µg/ml) 25.0 12.5 6.0 3.0 1. 6 0.8 0.4 Purified fraction 1,280 2,560 1,280 1,280 640 640 160 Total extract 2,560 2,560 1,280 1,280 1,280 640 320 210 HOSHINO-SHIMIZU, S.; CAMARGO, M. E. & NAGASSE, T. K. - A stable polysaccharide~hemagglutination roagent for the diagnosis of acute or rccent T1·ypa-nosuma cl'uzi infections. Rev. Inst. l\Ied. trop, São :Paulo 20:208-212, 1978. Yield and antigenic activity of polysaccharide components from T. cruzi extracts or fractions For 100 mg lyophilized trypanosomes, a to-tal 2 .14 mg polysaccharides could be obtained from the crude T. cruzi extract by the techni-que of NASH et al. 10. Such amounts. were res-pectively O. 68 mg .and 1. 22 mg for the saline-soluble and for the saline-insoluble fractions. E?ensitization patterns and antigenic ac-tivities were similar for polysaccharide com-ponent.s obtained from either T. cruzi crude extract or its fractions. Heat-stability of T. cruzi polysaccharide anti-gens No differences were observed for sensitiz-ing patterns or antigenic activity of T. cruzi polysaccharide components after boiling for 1 hour in a water-bath or autoclaving at 120°0 for 30 minutes. Treatment of sera with 2--mercaptoethanol Serum samplies from acute and chronic cases of Chaga.s'disease were submitted to different serologic tests, before and after treatment by 2-ME. The tests included anti-IgG and anti-IgM immunofluorescence, com-plement fixation, protein-hemagglutination and polysaccharide-hemagglutinátion tests. Results shown in Table III indica te polysa-ccharide-hemagglutination to d,epend on anti-bodies which are reduced by 2-ME. TABLE III Serum titers for cases of recent and chronic Chagas' infection before and after treating samples with 2-mer-captoethanol Test titers Before 2-ME trea,tment After 2-ME treatment PolyHA ProtHA IgM-IF IgG-IF (") Poly HA ProtHA IgM-IF IgG-IF Acute case-s Al 640 160 160 160 A2 320 80 160 160 A3 320 160 320 160 A4 640 o 160 160 A5 320 160 160 320 A6 160 80 160 320 Chronic cases Cl 40 1,280 o 640 C2 o 1,280 o 640 C3 o 640 o 320 e, .O 640 o 160 C5 20 2,560 o 160 C6 o 2,560 o 320 (*) PolyHA - polysaccharide hemagglutination test ProtHA - protein hemagglutination test o o o 160 o o o 160 o o o 80 o o o 320 o o o 320 o o o 160 o 1,280 o 640 o 1,280 o 320 o 1,280 o 320 o 640 o 160 o 2,560 o 160 o 1,280 o 160 IgM-IF - immunofluorescence te.st for IgM antibodies IgG-IF - immunofluorescence te.st for IgG antibodies Results of the polysaccharide hemagglutina,-tion tests in T. cruzi infected and non-infected patiénts Serum samples from 44 non-infected in-dividuals taken at random were tested, and 40 showed negative results while in the re'-maining 4 a 1 : 40 ti ~er was o bserved. Thirty nine chronic Chag,as'infection ca-ses were studi1ed. Positive tests were seen in 5 cases, 4 at 1:40 and 1 at 1:80, with 34 nega-211 HOSHINO-SHIMIZU, S.; CAMARGO, M, E. & NAGASSE, T, K. - A stable polysaccharide-hemagglutination reagent for the diagnosis of acute or recent 'l'•·ypanosorna c1·uzi infecti:ons. Rev. Inst. 1\fod. trop, São Paulo 20:208-212, 1978. tives, whe'rea.s much higher titers were detect-ed in ali 12 cases in the acute phase of the disease. Thus, titers: of 1:80 and 1:160 were observed only in two cases. The others showed· tibers of 1:320 (2 cases), 1:640 (1 case), 1:1,280 (4 cases) and 1:2,560 (3 cases). Periods ,after ·infection during which the polys.accharide test remains positive could not be determined, although this could be an im-portant information for seroepidemiological applications. Most of our acute cases have been submitted to chemotherapy and in two, negativation was observed for ali serologic tests performed, including the polysaccharide hemagglutination test. For both cases, this test became negative respectively 15 and 36 months after the infection initiated. For other 7 cases of acute infection, polysaccharide he-magglutination test, as well as the other sero-Iogic tests, are still positive after periods varying from 3 to 31 months. DISCUSSION In endemic areas of American trypanoso-miasis, recognizing recent T. cruzi infections presents a difficult problem, both on clinical and Iaboratory grounds. Antibodies against po-lysaccharide components of the parasitie are known to occur in acute Chagas'disease but only occasionally in chronic cases. However available tests are not satisfactory for sero-epidemiology purposes. Devielopment of stable, Iyophilized, human cells sensitlzed with T. cruzi polysaccharide components introduces new possibilities. Besides very practical for routine purpos1es, the hemagglutination test can be performed both for serum samples and for eluates from blood samples dried on filter paper. Our preliminary results confirm the described occurrence of antibodies against T. cruzi polysaccharide antigens in recent Cha-gas'infections. However, investigations are ne-cessary to evaluate the new hemagglutination test for seroepidemiological applications. RESUMO Reagente estável para teste de hemaglutina .. ção com antígenos polissacarídicos do T. cruzi, para diagnóstico de infecção chagásica aguda Descreve-se reação de hemaglutinação pa-ra anticnrpos antipolissacárides do T. cruzi, definindo-se condições para preparo de rea-212 gente estável, liofilizado. Demonstrou-se que tais anticorpos pertencem à classe IgM. O tes-te foi positivo em todos os casos de infecção chagásica aguda es.tudados, mas negativo ou com títulos muito baixos nos casos crônicos. Estes resultados sug,erem a possibilidade de aplicação do teste para estudos soroepidemio-lógicos de incidência da infecção. REFERENCES 1. ANTUNES, L. J.; REIS, A. R.; TAVARES, C. A. P .. & PELLEGRINO, J. - Dosagem das imu-noglobulinas e reação de hemaglutinação passiva em pacientes com leishmaniose cutaneo-mucnsa. Rev. Inst. illed. trop. São Paulo• 14: 203-206, 1972. 2. CAMÁRGO, M, E.; HOSHINO-SHIMIZU, S:; MACEDO, V.; PERES, B. A. & CASTRO, C. -Diagnóstico sorológico da infocção pelo T. cruzi. Estudo comparativo de te'Stes de fixação do com-pl,emento, imunofluorescência, hemaglutinação e floculação em 3.624 soros. Rev. lnst. 1\Ied. trop. São Paulo 19: 254-260, 1977. 3. DEUTSCH, H. F. & MORTON, J. I. - Disso-ciation of human serum macroglobulins. Science 125: 600-601, 1957. 4, GONÇALVES, J. M, & YAMAHA, T, - Immu-nochemical polysaccharide from T, cruzi. J, Trop. l\fod. H,yg. 72: 39-44, 1969. 5. HIRATA, A. A. & BRANDRISS, M. W, - Pas-sive hemagglutination procedures for protein and polysaccharide antigen.s using erythrocytes stabi-lized by aldehydes. J. Immunoi. 100: 641-646, 1968. 6. LOWRY, O. H.; ROSENBROUGH, N; J,; FARR, A. L. & RANDALL, R. J. - Protein measure-ment with the folin phenol reagent. J, Biol. Chem, 193: 265-275, 1951. 7. MARTIRANI, I.; HOXTER, G.; WAYCHEN-BERG, B. L.; MARIANI, I. <& CINTRA, A. B. U. - Dctermination of polysaccharide he-xoses and hexosamines in normal hum.a,n sera. J. Lab. Clin. Med. 54: 773-778, 1959. 8. MUNIZ, J. - Do valor da reação de precipitina no diagnóstico das forma'S agudas e subagudas da doença de _Chagas (Trypanosomiasis america-na). Mem, lnst. Oswal!do Cl'uz 45: 53.7-549, 1947. 9, MUNIZ, J. & FREITAS, G. - Contribuição para o diagnóstico da doença de Chagas pelas reações de imunidade. II - Isolamento de polissacarídeos de Schizotripanum cruzi e de outros tripanosomi-deos, seu comportamento nas reações de precipi-taçij.o, de fixação do complemento e de hipersen-sibilidade. Re;, Brasil, Biol. 4: 421-438, 1944. 10. NASH, T. E.; PRESCOTT, B, & NE,VA, F .. A. - The caracteristios of a circulating antigen in schistosomiasis. J. Irnm11.Ilol. 112: 1500-1507, 1974. 11, PELLEGRINO, J, & BRENER, A. - A reação de fixação do complemento na doença de Chagas. Hospital (Rio) 42: 115-121, 1952. 12. PELLEGRINO, J.; BRENER, Z, & JACOMO, R. - A reação de precipitina na fase aguda da doen-ça d,e Chagas, Rev. Bmsil. Malariol. Doen!;as Trop. 8: 247-252, 1956. Recebido para publicação em 14/3/1978. 

Reagente estável para teste de hemaglutina ção com antígenos polissacarídicos do T. cruzi, para diagnóstico de infecção chagásica aguda

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A stable polysaccharide-hemagglutination reagent for the diagnosis of acute or recent trypanosoma cruz! Infections

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