Oxamniquine

SUMMARY

The circumoval precipitin test (COPT), enzyme-linked immunosorbent assay (ELISA) and the immunoblotting anti-adult worm antigen (AWA) and soluble egg antigen (SEA) tests were applied to 17 chronically schistosome-infected patients for the detection of anti-Schistosoma mansoni antibodies before and on four occasions after oxamniquine administration over a period of six months. Compared to a control group, schistosomiasis patients showed high levels of IgG antibodies in AWA and SEA-ELISA. A decrease in IgG levels was observed six months after treatment, although negative reactions were not obtained. Significant decreases in IgG,, IgG 3 and, mainly, IgG, but not anti-SEA IgG 2 levels were observed six months after treatment, again without negativity. Analysis of anti-AWA IgG antibodies by immunoblotting before treatment showed a 31 kDa strand in 14 patients (82%) which disappeared in three cases up to six months after treatment; furthermore, anti-SEA IgG antibodies showed the same band in nine patients (53%) before treatment, which disappeared in only four cases up to six months after treatment.

KEYWORDS: Schistosomiasis diagnosis; Schistosomiasis cure evaluation; Immunenzymatic tests; Immunoblotting.

INTRODUCTION

In Brazil there are at least 2,500,000 individuals infected with Schistosoma mansoni and about 25 millions live in areas where the transmission of this helminthiasis may be possible32. The laboratory diagnosis of active schistosomiasis mansoni is made principally by the finding of S. mansoni eggs in the stool9. However, fecal parasitologic tests are not considered to have high sensitivity because of factors such as irregularity in Schistosoma egg shedding3,8 absence of egg laying just after anti-schistosome treatment even if not successful and tissue egg retaining due to intestinal fibrosis29. Thus, several immunological tests using crude or purified egg and adult worm antigens have been developed in the last decades to detect anti-S. mansoni antibodies and some of them have been proposed for treatment evaluation.

The circumoval precipitin test (COPT) is one of the exams used for the diagnosis of schistosomiasis and for the evaluation of antischistosome treatment" and consists of immune complex formation around schistosome eggs incubated with serum from schistosomeinfected individuals. It is considered as a highly sensitive and specific test and usually becomes non-reactive from 6 to 12 months after antischistosome treatment14,33. However, in some instances, patients continue to have positive COPT results after specific anti-schistosome treatment or test negativity occurs only a long time after treatment 16.

Immunoenzymatic tests for anti-S. mansoni antibodies utilize antigens obtained from either adult worms or eggs. Some of these tests show a decrease in anti-schistosome antibody levels soon after specific treatment', while others show the persistence of high antibody levels up to 18 months". When IgG subclasses are analyzed there is a decrease in all subclasses, with special relevance in the case of IgG 42.

Immunoblotting tests have been used to detect AWA antibodies since an immunogenic fraction with a molecular weight of 31-32 kDa (Sm 31/32) was considered to be the most frequently recognized fraction and could therefore be used as a serologic marker35,36,43. Furthermore this fraction became negative or decreased in the serum of treated patients exposed to a low infection risk35,36 On the other hand, KIMURA22 did not obtain the same results.

Using the immunoblotting test for SEA, NOYA et al.30 studied a group of patients after therapy and identified a special fraction of about 31 kDa which decreased after specific treatment. This fraction, corresponding to the Omega 1 antigen described by DUNNE et al.7, was present in a special schistosome egg antigen known as cation exchanging fraction-6 (CEF-6) and has been considered an important diagnostic marker for cure control. Moreover, this antigen is one subcomponent of COPT and has been considered as a very sensitive and specific antigen5,27,28 DOENHOFF et al.4, employing this antigen as a serologic marker, observed 50% and 80% negative reactions in schistosomiasis patients from Santa Lucia, West Indies, six and 12 months after treatment, respectively. However, a similar evaluation of patients from Puerto Rico did not show the same results6,15.

The occurrence of controversial results when the same immunological technique is used in different populations justifies the evaluation of tests usually employed in the diagnosis and cure control of schistosomiasis under our operational conditions in order to standardize immunological evaluation of schistosomiasis treatment. Furthermore, the decrease of the rate of infection with S. mansoni in Brazil, particularly in Sao Paulo State, strengthens the importance of using an immunological technique of diagnosis rather than parasitological tests in epidemiological surveys.

MATERIAL AND METHODS

The reaction was stopped by washing the strips in distilled water. The strips showing protein recognition by the schistosomiasis patient sera were scanned using an optical densitometer and measured by analysis of one-dimensional separation and dot blots using an Image Master Program 1.20 produced by Pharmacia Biotech AB.

Statistical analysis: The results are expressed as mean and standard deviation. The ANOVA test with the Student Newman-Keuls contrast post-test was used for statistical analysis of the various groups. The paired and impaired Student test and Pearson correlation were used for statistical comparison of two groups. A level of confidence of 95% (p

RESULTS

This study was carried out on 17 patients with a parasitological diagnosis of schistosomiasis mansoni made by the Kato-Katz method. Ninety days after treatment with oxamniquine (a single oral dose of 15 mg/kg weight) all patients yielded negative results in fecal exams; the COPT, on the other hand, remained positive in one case (Table 1).

A heterogenous pattern of reactivity was observed when the immunoblotting for anti-AWA and SEA IgG antibodies of all 17 patients were analyzed before and after 180 days after treatment (Tables 2 and 3). Table 4 shows the percentage of positive bands found in all patients before and 180 days after treatment with oxamniquine.

DISCUSSION

In the present study, 17 schistosomiasis patients, 14 of them harboring moderate or low parasite burdens, living for several years in a nonendemic area for S. mansoni infection were examined before and after treatment with oxamniquine. All of them had been considered cured by fecal tests and all but one by COPT in the evaluation carried out six months after treatment (Table 1). In contrast to reports by others 14,31, 33, under our operational conditions COPT showed a relatively low sensitivity before treatment. Of 16 patients submitted to this diagnostic procedure only 11 (68.7%) yielded positive results.

The results of immunological tests (anti-AWA and anti-SEA IgGELISA) performed before specific treatment were similar to those obtained by others13,26,28,40 and support the usefulness of these techniques for schistosomiasis diagnosis, mainly in areas where the parasite burden is low and consequently fecal tests are less sensitive20.

Analysis of IgG anti-AWA and anti-SEA immunoblottings showed a heterogeneous pattern before and after specific treatment in the 17 patients studied. The bands of 31-32 kDa, considered to be of high diagnostic value17,35.36,37, were found in 82%. and 53% of cases, respectively, before treatment when AWA and SEA were analyzed (Tables 2 and 3). This kind of diversity was, in fact, previously pointed out by others in Brazilian schistosomiasis patients: VALLI et al.43 found 98% positivity for the 31 kDa band and KIMURA 12 found only 12.5% positivity for the same strand.

Although the 31-32 kDa bands have been considered as good markers for schistosomiasis cure after specific treatment by some researcherS13,11, in the evaluation performed 180 days after oxamniquine administration these bands vanished in only three cases (Tables 2 and 3), persisting in 65% and 29% of the patients studied when AWA and SEA antigens were used, respectively (Table 4).

In summary, the use of COPT, immunoenzymatic tests as well as the immunoblotting techniques did not permit a safe and definitive early evaluation of schistosomiasis treatment in the 17 patients studied. Thus, the ideal diagnostic method for schistosomiasis cure control seems to be still far from available, but IgG, subclass levels showed a significant decrease up to 180 days after specific treatment. Long term post treatment follow-up would be relevant and other studies are necessary to define in further detail the role of IgG4 and its promising usefulness as a possible cure marker.

RESUMO

Avaliacao da presenqa de anticorpos IgG anti-Schistosoma mansoni no soro de pacientes com esquistossomose mansonica cronica, antes e apos tratamento especifico

Em 17 pacientes com infeccao cronica por Schistosoma mansoni utilizaram-se os testes de reacao periovular, imunoenzimatico (ELISA) e imunoblotting, empregando-se antigenos obtidos a partir de vermes adultos (AWA) ou de ovos de S. mansoni (SEA), para deteccao de anticorpos antiS. mansoni, antes e em quatro ocasibes apos tratamento com oxamniquine. Quando cotejados a grupo controle os pacientes esquistossomoticos revelaram altos niveis sericos de anticorpos IgG nos testes ELISA (antiAWA e anti-SEA), nao se observando, porem, negativacao ate seis meses apos tratamento especifico. Encontrou-se, entretanto, decrescimo significativo, sem negativaqao, dos niveis de IgG1, IgG3 e, principalmente, IgG4, quando se utilizou antigeno soluvel obtido a partir de ovos de S. mansoni (SEA), seis meses apos administraqao de oxamniquine. O mesmo nao foi observado no caso de anticorpos da subclasse IgG2.

Nos imunoblottings efetuados com o emprego de antigeno de verme adulto (AWA), antes do tratamento com oxamniquine, evidenciou-se a presenga de banda com 31 kDa em 14 (82%) dos 17 pacientes estudados, observando-se seu desaparecimento em tres pacientes examinados seis meses apos tratamento especifico. Quando se utilizou antigeno obtido a partir de ovos de S. mansoni (SEA) a mesma banda foi evidenciada em nove pacientes, desaparecendo em quatro casos, apos o tratamento.

REFERENCES

1. BOROS, D.L. &WARREN, K.S. - Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. exp. Med., 132: 488-507, 1970.

2. BOCTOR, F.N. & PETER, LB. - IgG subclasses in human chronic schistosomiasis: over-production of schistosome-specific and non-specific IgG4. Clin. exp. Immunol., 82: 574-578, 1990.

3. CHIEFFI, P.P.; MARQUES, R.M. & SIQUETRA, J.G.V. - Avaliacao da eficacia do m6todo de Kato-Katz no diagnostico parasitologico da esquistossomose mansonica. Rev. Inst. Adolfo Lutz, 41: 23-30, 1981.

4. DOENHOFF, M.J.; DUNNE, D.W. & LILLYWHITE, J.E. - Serology of Schistosoma mansoni infection after chemoterapy. Trans. roy. Soc. trop. Med. Hyg., 83: 237238, 1989.

5. DUNNE, D.W.; LUCAS, S.; BICKLE, Q. et al. - Identification and partial purification of an antigen (co,) from Schistosoma mansoni eggs which is putatively hepatotoxic in T cell deprived mice. Trans. roy. Soc. trop. Med. Hyg., 75: 54-71, 1981.

6. DUNNE, D.W.; HILLYER, G.V. & VAZQUEZ, G. - Schistosoma mansoni cationic egg antigens (CEF-6): immunoscrology with oxamniquine-treated patients and involvement of CEF-6 in the circumoval precipitin reaction. Amer. J. trop. Med. Hyg., 38: 508-514, 1998.

7. DUNNE, D.W.; JONES, F.M. & DOENHOFF, M.J. - The purification, characterization, serological activity and hepatotoxic properties of two cationic glycoproteins (a, and io),) from Schistosoma mansoni eggs. Parasitology, 103: 225-236, 1991.

8. ENGELS, D; SINZINKAYO, E. & GRYSEELS, B. - Day-to-day egg count fluctuation in Schistosoma mansoni infection and its operational implications. Amer. J. trop. Med. Hyg., 54: 319-324,1996.

9. FELDMEIER, H. & POGGENSEE, G. - Diagnostic techniques in schistosomiasis control. A review. Acta trop. (Basel), 52: 205-220, 1993.

10. GAZZINELLI, G.; LAMBERTUCCI, J.R.; KATZ, N. et aL - Immune responses during human schistosomiasis mansoni. XI. Immunologic status of patients with acute infections and after treatment. J. Immunol., 135: 2121-2127, 1985.

11. GROGAN, J.L.; KREMSNER, P.G.; VAN DAM, G.J. et al. - Antischistosome IgG4 and IgE responses are affected differentially by chemotherapy in children versus adults. J. infect. Dis., 173: 1242-1247, 1996.

12. HASSAN, M.M.; BADAWI, M.A. & STRAND, M. - Circulating schistosomal antigen in diagnosis and assessment of cure in individuals infected with Schistosoma manson. Amer. J. trop. Med. Hyg., 46: 737-744, 1992.

13. HILLYER, G.V. & GOMEZ DE RIOS, 1. - The enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of schistosomiasis. Amer. J. trop. Med. Hyg., 28: 237-241, 1979.

14. HILLYER, G.V; RUIZ-TIBEN, E.; KNIGHT, W.B.; GOMEZ de RIOS, 1. & PELLEY, R.P. - Immunodiagnosis of infection with Schistosoma mansoni: comparison of ELISA, radioimmunoassay, and precipitation tests performed with antigens from eggs. Amer. J. trop. Med. Hyg., 28: 661-669, 1979.

15. HILLYER, G.V; NIEVES-FRAU, L.F. & VAZQUEZ, G. - Identification of a genusspecific Schistosoma man.soni soluble egg antigen reactive with the serum of infected patients. Amer. J. trop. Med. Hyg., 35: 1198-1204, 1986.

16. HILLYER, G.V & GALANES, M.S. - Seroepidemiology of schistosomiasis in Puerto Rico: evidence for vanishing endemicity. Amer. J. trop. Med. Hyg., 60: 827-830, 1999.

17. IDRIS, M.A. & RUPPEL, A. - Diagnostic Mr 31/32,000 Schistosoma mansoni proteins (SM31132): reactions with sera from Sudanese patients infected with S. mansoni or S. haematobium. J. Helminth., 62: 95-101, 1988.

18. ISKANDER, R.; DAS, P.K. & AALBERSE, R.C. - IgG4 antibodies in Egyptian patients with schistosomiasis. InC. Arch. Allergy, 66: 200-207, 1981.

19. JASSIN, A.; HASSAN, K. & CATTY, D. - Antibody isotypes in human schistosomiasis mansoni: Paras. Immunol., 9: 627-650, 1987.

20. KANAMURA, H.Y.; DIAS, L.C.S.; DA SILVA, R.M. et al. - A comparative epidemiologic study of specific antibodies (IgM and IgA) and parasitological findings in an endemic area of low transmission of Schistosoma mansoni. Rev. Inst. Med. trop. S. Paulo, 40: 85-91, 1998.

21. KATZ, N.; CHAVES, A. & PELLEGRINO, J. - A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Rev. Inst. Med. trop. S. Paulo, 14: 397-400, 1972.

22. KIMURA, E.A.S. - S. mansoni: analise por "western-blot" dos antigenos envolvidos na resposta humoral de pacientes submetidos ao tratamento. Sao Paulo, 1986. [Master's Thesis - Instituto de Ciencias Biom6dicas da Universidade de SAo Paulo].

23. KLINKERT, M.Q.; BOMMERT, K.; MOSER, D. et al. - Immunological analysis of cloned Schistosoma mansoni antigens Sm31 and Sm32 with sera of schistosomiasis patients. Trop. Med. Parasit., 42: 319-324, 1991.

24. LAEMMLI, U.K. - Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.

25. LOWRY, O.11.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.

26. McLAREN, M.L.; LONG, E.G.; GOODGAME, R.W. & LILLYWHITE, J.E. Application of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Schistosoma mansoni infections in St. Lucia. Trans. roy. Soc. trop. Med. Hyg., 73: 636-639, 1979.

27. McLAREN, M.L.; LILLYWHITE, J.E.; DUNNE, D.W. & DOENHOFF, MJ. Serodiagnosis of human Schistosoma mansoni infections: enhanced sensitivity and specificity in ELISA using a fraction containing S. mansoni egg antigens co, and a,. Trans. roy. Soc. trop. Med. Hyg., 75: 72-79, 1981.

28. MOTT, K.E. & DIXON, H. - Collaborative study on antigens for immunodiagnosis of schistosomiasis. Bull. Wld. Hlth. Org., 60: 729-753, 1982.

29. NASH, TE. - Schistosomiasis and other trematode infections. In: ISSELBACKER, K.J. et aL, ed. Harrison's principles of internal Medicine. 13. ed. New York, Mc Graw Hill International, 1994. pt. 6, p. 924-931.

30. NOYA, O.; LOSADA, S.; ALARC6N DE NOYA, B. et al. - Effect of chemotherapy on immune response to egg antigens of Schistosoma mansoni in chronically infected children from areas of low transmission. Paras. Immunol., 17: 1111-117, 1995.

31. OLIVER-GONZALEZ, J. - Anti-egg precipitins in sera of humans infected with Schistosoma mansoni. J. infect. Dis., 95: 86-91, 1954.

32. PASSOS, A.D.C. & AMARAL, R.S. - Esquistossomose mans6nica: aspectos epidemiologicos e de controle. Rev. Soc. bras. Med. trop., 31(supl. 2): 61-74, 1998.

33. RAMIREZ, R.M.; CEBALLOS, E.; ALARCON DE NOYA, B.; NOYA, 0. & BIANCO, N. - The immunopathology of human schistosomiasis. 111. Immunoglobulin isotype profiles and response to praziquantel. Mem. Inst. Oswaldo Cruz,91: 593-599, 1996.

34. ROBERTS, S.M.; WILSON, R.A.; OUMA, J.H. et aL - Immunity after treatment of human schistosomiasis mansoni: quantitative and qualitative antibody responses to tegumental membrane antigens prepared from adult worms. Trans roy. Soc. trop. Med. Hyg., 81: 786-793, 1987.

35. RUPPEL, A.; DIESFELD, H.J. & ROTHER, U. - Immunoblot analysis of Schistosoma mansoni antigens with sera of schistosomiasis patients: diagnostic potential of an adult schistosome polypeptide. Clin. exp. Immunol., 62: 499-506, 1985.

36. RUPPEL, A.; SHI, YE.; WEI, DX. & DIESFELD, H.J. - Sera of Schistosoma japonicuminfected patients cross-react with diagnostic 31/32 kD proteins of S. mansoni. Clin. exp. Immunol., 69: 291-298, 1987.

37. RUPPEL, A.; IDRIS, M.A.; SULAIMAN, S.M. & HILALI, A.M.H. - Schistosoma mansoni diagnostic antigens (Sm3l/32): a sero-epidemiological study in the Sudan. Trop. Med. Parasit., 41: 127-130, 1990.

38. RUPPEL,A.; XING, Y.; DELL, R.; NUMRICH, P. & SHI,YE. -Schistosoma mansoni and S.japonicum: decline of antibodies against diagnostic adult worm antigens (Sm31/32) following praziquantel treatment of mice. Trop. Med. Parasit, 42: 325-331, 1991.

39. SILVA, L.C. da: HOSHINO-SHIMIZU, S.; KANAMURA, H. et al - Serum antibody changes after chemotherapy of patients with schistosomiasis mansoni. A statistical analysis. Rev. Inst. Med. trop. S. Paulo, 17: 344-349, 1975.

40. SPENCER, L.; DE NOYA, B.A.; NOYA, 0. & MASROUA, G. - Analisis comparativo entre la prueba de precipitacion circumoval y ELISA con antigenos crudos para el diagnostico de la esquistosomiasis en Venezuela. G.E.N. (Caracas), 45:77-83, 1991.

41. SOBH, M.A.; MOUSTAFA, FE.; HAMED, S.M. & GHONEIM, M.A. - Infectious glomerulopathy induced by a defined agent (Schistosoma mansoni): progression despite early elimination of the causal agent. Exp. Nephrol., 1: 261-264, 1993.

42. TANABE, M.; OKAZAKI, M.; OKAZAKI, M. et at. - Serological studies on schistosomiasis mansoni in the Northeast Brazil (I). Rev. Inst. Med. Trop. S. Paulo, 32: 121-131, 1990.

43. VALLI, L.C.; KANAMURA, H.Y.; DA SILVA, R.M.; RIBEIRO-RODRIGUES, R. & DIETZE, R. - Schistosomiasis mansoni: immunoblot analysis to diagnose and differentiate recent and chronic infection. Amer. J. trop. Med. Hyg., 61: 302-307, 1999.

44. WORLD HEALTH ORGANIZATION - The control of schistosomiasis. WId. Hlth. Org. techn. Rep. Ser., (830), 1993.

Received: 22 December 2000 Accepted: 02 February 2001

Cilia Maria V. VENDRAME(I), Marcia Dias T. CARVALHO(2), Celia Regina F. YAMAMOTO(3), Maria Cristina NAKHLE(I), Silvino Alves CARVALHO(I) & Pedro Paulo CHIEFFI(1,4)

(1) Laboratorio de Imunopatologia da Esquistossomose (LIM/06) do Instituto de Medicina Tropical de Sao Paulo, Faculdade de Medicina da Universidade de Sao Paulo (FMUSP), Sao Paulo, Brasil.

(2) Laboratorio de Lipides (LIM/10) do Instituto de Medicina Tropical de Sbo Paulo, FMUSP, Sao Paulo, Brasil.

(3) Laboratorio de Imunologia (LIM/48), do Instituto de Medicina Tropical de Sao Paulo, FMUSP, Silo Paulo, Brasil.

(4) Faculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo, Brasil.

Correspondence to: Pedro Paulo Chieffi, Laboratorio de Imunopatologia da Esquistossomose (LIM/06), Instituto de Medicina Tropical de SIlo Paulo. Av. Dr. Eneas de Carvalho Aguiar 500, 2 andar, 05403-000 Sio Paulo, SP, Brasil. Fone/Fax: +55-11-3064-5132. E-mail: pchieffi @usp.br

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