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Serology of human sarcosporidiosis
CORRESPONDENCE
415
others the reactions disappeared after a few hours. All 7 patients had a positive complement fixation test for Chagas's disease. T h e insoluble antigen did not give an immediate reaction; a weakly positive reaction (small indurated nodule) was observed in 5 of the patients after 48-72 hours, particularly to the 20 and 50 gamma doses. Only one of them gave a similar reaction to a regular Montenegro antigen (4 gamma N/ml.). I n the control group, 7 patients were negative to both types of antigens. Two patients had a positive reaction only to a 300 gamma dose of the soluble antigen (considered as false positives) and 2 gave a clear positive immediate reaction to 100 and 300 gamma dose, one of them also presenting a weakly positive delayed reaction. A subsequent complement fixation test confirmed that these 2 patients had a previously undetected Chagas's infection. All control patients had a negative Montenegro test. 4 of the 6 proved cases of cutaneous leishrnaniasis presented a clear positive immediate reaction to the soluble antigen (100 gamma). All had a positive Montenegro test which was also elicited by the T. cruzi insoluble antigen. I n conclusion, it is possible to obtain a clear immediate skin reaction in chronic Chagas's disease by using soluble protein antigen from parasites grown in culture. T h e optimum amount of nitrogen in the antigen is around 100 gamma per ml. T h e reaction can be positive in cutaneous leishmanlasis but the Montenegro test will differentiate it. T h e delayed skin test in Chagas's disease is not predictable and when present tends to be rather weak. We are, etc., RODRIGO ZELEDON, Departmento de Parasitologia, Universidad de Costa Rica and L . S . U . - - I . C . M . R . T . , Apartado 10155 San Jos6, Costa Rica. CARLOS PONCE, Departamento de Microbiologia, Universidad de Honduras and L . S . U . - - I . C . M . R . T . , Costa Rica. 1 ffuly, 1974. REFERENCES FREITAS, J. L. P. de, (1947). Contribuicao para o estudo do diagn6stico da Molestia de Chagas por processos de laboratorio. Tese de Doutoramento, Sao Paulo, 160 pp. AMATO NETO, V., MAGALDI, C., & PESSOA, S. (1964). Revta. Goiana Med., 10~ 121. TOBIE, E. J., & REES, C. (1948).ff. Parasit., :141, 162. LANG, C. A. (1958). Anal. Chem., 30, 1962.
SEROLOGYOF HUMANSARCOSPORIDIOSIS SIR,--In his review of sarcosporidiosis in man, JEFFREY (1974) suggested that the complement-fixation test would be of value in distinguishing between toxoplasmosis and sarcosporidiosis in cases where morphological study of small cysts in muscle gives an equivocal diagnosis. While much information concerning the serodiagnosis of toxoplasmosis is available, this is not true of sarcosporidiosis. ROMMEL and HEYOORN (1972) found that after eating raw beef or pork containing Sarcocystis, human volunteers shed Isospora hominis in their faeces. Thus, Sarcocystis and I. hominis are different stages of the same parasite. Antibodies to Sarcocystis detectable by the indirect fluorescent antibody test were not shown in the serum of a cat with an intestinal infection (analogous to I. hominis in man) which followed the ingestion of tissue cysts of Sarcocystis in raw cattle diaphragm (MARKUS et al., 1974; MARKUSet al., in press). T h e reason may have been that extra-intestinal infection is sometimes or always necessary for antibody production. Unlike Toxoplasma, Sarcocystis in carnivores does not normally develop extra-intestinal stages (see MARKUS et al., in press), which play a r61e in stimulating the production of antibody. However, circulating antibody has apparently been demonstrated in both known I. hominis carriers and non-carriers (including vegetarians), with use of bovine Sarcocystis antigen and I. hominis sporozoites in the indirect fluorescent antibody test, but was only occasionally observed in young babies (LAARMANet al., 1974; TADROS et al., 1974). Whether this test can give false positives as it occasionally does with Toxoplasma antigen, is an aspect requiring investigation (see ARAU]O et al., 1971; M.ARKUS, 1973; REMINGTON and DESMONTS, 1973). Furthermore, it should be noted that there is a serum factor which causes polar fluorescence of Toxoplasma in approximately 30% of seronegative individuals. This factor is heat stable, can be titrated, and does not usually occur in the blood of infants of less than 6 months of age (SULZERand WILSON,1971 ; KAUFMANet al., 1973). Subclinical coccidiosis caused by L hominis is common (see TADROS et al., 1974). I f antibody is indeed formed, it follows that tests using Sarcocystis antigen on sera of persons having unidentified muscle cysts of Toxoplasma could be positive for Sarcocystis because of a past or concomitant intestinal L hominis infection. On the basis of present knowledge, therefore, a positive result from a serological test for sarcosporidiosis in a person with unidentified muscle cysts might be difficult to interpret, particularly if tests for Toxoplasma are also positive. T h e chances that a test for toxoplasmosis will be positive are good, since it is now a well-established fact that antibodies to Toxoplasma are found in a large number of healthy persons. Only a negative test for toxoplasmosis would help to exclude this protozoon in a patient having unidentified cysts in muscle. However, the absence of demonstrable Toxoplasma antibody cannot be taken as absolute proof that the parasite is not present in the tissues (REMINGTON and ARAUJO, 1974).
416
CORRESPONDENCE
An obvious question which springs to mind is whether there is cross-reaction between Sarcocystis and Toxoplasma. I t would appear that they are immunologically distinct. Cats which are shedding I. cati (one of the coccidia that has in the past been called I. bigemina), now known to be a resistant stage of Sarcocystis (HEYDORN and ROMMEL, 1972), can be superinfected with Toxoplasma, resulting in production of o6cysts of the latter parasite as well (see FRENKEL, 1973). However, although several workers have shown that immunity usually develops in cats in which Toxoplasma has undergone gametogony, the degree to which this happens following sexual reproduction of Sarcocystis has yet to be determined. T h e indications are that cats can be re-infected with Sarcocystis relatively easily (e.g. MARKIJS et al., in press). Additional evidence suggesting an absence of any marked common antigenicity between Sarcocystis and Toxoplasma is provided by serological studies by MANDOUR (1965), MGCONNELL et al. (1973) and TADROS et al. (1974). In a summary of other work on this subject, the conclusion was reached (MARKIJS, 1973) that there are as yet no grounds for questioning the reliability of routine serological tests for human toxoplasmosis. T h e opinion of DE OLIVEIRAet al. (1973) that there is "group" cross-reaction between Toxoplasma on the one hand and I. hominis and I. belli on the other is open to question and needs to be confirmed. While a negative serological test for toxoplasmosis in a patient having unidentified cysts in muscle would be useful information, more experimental work on antibody production in Sarcocystis infections in relation to the use of tests for sarcosporidiosis is indicated. I am, etc., .MILES B. MARKUS, Department of Zoology and Applied Entomology, Imperial College, University of London, London S.W.7. 8 Ju/y, 1974 REFERENGES ARAUJO, F. G., BARNETT, E. V., GENTRY, L. O. & REMINGTON, J. S. (1971). Appl. Microbiol., 22, 270. DE OLIVEIRA, G. S. C., BARBOSA,W. & DA SILVA, A. L. (1973). Revta Patol. trop., 2, 387. FRENKEL, J. K. (1973). In: HAMMOND, D. M. & LONG, P. L. (eds), The coccidia, p. 343. Baltimore: University Park Press and London: Butterworths. HEYDORN, A.-O. & ROMMEL, M. (1972). Berl. Munch, tier~irztl. Wschr., 85, 121. JEFFREY, H. C. (1974). Trans. R. Soc. trop. Med. Hyg., 68, 17. KAUFMAN, G. 1. REMINGTON,J. S. & WATERS, H. C. (1973). Appl. Microbiol., 2,5, 724. LAARMAN,J. J., TADROS, W. & VAN DEN EIJI(, A. A. (1974). Unpublished paper read at 3rd Int. Congr. Parasit. (Munich). McCoNNELL, E. E., BASSON,P. A., WOLSTENHOLME,B., DE Vos, V. & MALHERBE, H. H. (1973). Trans. R. Soc. trop. Med. Hyg., 87, 851. MANDOtm, A. M. (1965). Ph.D. thesis, University of London. MARKUS, M. B. (1973). New Engl. J. Med., 289, 980. , DRAPER, C. C., HUTCHISON, W. M., KILLICK- KENDRICK, R. & GARNHAM,P. C. C. (1974). Trans. R. Soc. trop. Med. Hyg., 88, 3. , KILLICK-KENDRICK, R. & GARNHAM, P. C. C. 97. trop. Med. Hyg. (In press). REMINGTON, J. S. & ARAUJO, F. G. (1974). Proc. 3rd Int. Congr. Parasit. (Munich), 1, 294. & DESMONTS, G. (1973). J. Pediat., 83, 27. ROMMEL, M. & HEYDORN, A.-O. (1972). Berl. Miinch. tiertirzd. Wschr., 85, 143. SULZER, A. J. & WILSON, M. (1971). Expl. Parasit., 20, 197. TADROS, W., LAARMAN,J. J. & VAN DEN EIIK, A. A. (1974). Z. Parasit Kde, 43, 221. SYNGYTIAL MASSES IN PANSTRONGYLUS MEGISTUS NATURALLY INFECTED WITH TRFPANOSOMA CRUZI SIR,--In smears from intestinal contents of P. megistus naturally infected with T. crugi we detected a certain number of syncytial masses indicating a previously undescribed process of T. cruzi life-cycle in this invertebrate host. T h e multinucleated syncytial masses show indefinite cytoplasm with a large number of nuclei and kinetoplasts, indicating fusion of the unicellular parasites. T h e liberation of flagellated forms from the syncytial masses were observed. This sequence suggests that this process in the T. cruzi life-cycle has not been studied in the invertebrate host and that it may involve genetic exchange as has been reported in Triatoma infestans by BRENER and in cultures of T. cruzi by ADLER. I am, etc., R. P. BRAZIL, Lab. Leishmanoses, C.P.P.N., I.C.B., F u n d i o , Guanabara, Brasil. 16 July, 1974. REFERENCES BRENER, Z. (1972). 97. Protozool., 19, (1), 23. ADLER, S. (1958). Ann. trop. Med. Parasit., 52, 282.
415
others the reactions disappeared after a few hours. All 7 patients had a positive complement fixation test for Chagas's disease. T h e insoluble antigen did not give an immediate reaction; a weakly positive reaction (small indurated nodule) was observed in 5 of the patients after 48-72 hours, particularly to the 20 and 50 gamma doses. Only one of them gave a similar reaction to a regular Montenegro antigen (4 gamma N/ml.). I n the control group, 7 patients were negative to both types of antigens. Two patients had a positive reaction only to a 300 gamma dose of the soluble antigen (considered as false positives) and 2 gave a clear positive immediate reaction to 100 and 300 gamma dose, one of them also presenting a weakly positive delayed reaction. A subsequent complement fixation test confirmed that these 2 patients had a previously undetected Chagas's infection. All control patients had a negative Montenegro test. 4 of the 6 proved cases of cutaneous leishrnaniasis presented a clear positive immediate reaction to the soluble antigen (100 gamma). All had a positive Montenegro test which was also elicited by the T. cruzi insoluble antigen. I n conclusion, it is possible to obtain a clear immediate skin reaction in chronic Chagas's disease by using soluble protein antigen from parasites grown in culture. T h e optimum amount of nitrogen in the antigen is around 100 gamma per ml. T h e reaction can be positive in cutaneous leishmanlasis but the Montenegro test will differentiate it. T h e delayed skin test in Chagas's disease is not predictable and when present tends to be rather weak. We are, etc., RODRIGO ZELEDON, Departmento de Parasitologia, Universidad de Costa Rica and L . S . U . - - I . C . M . R . T . , Apartado 10155 San Jos6, Costa Rica. CARLOS PONCE, Departamento de Microbiologia, Universidad de Honduras and L . S . U . - - I . C . M . R . T . , Costa Rica. 1 ffuly, 1974. REFERENCES FREITAS, J. L. P. de, (1947). Contribuicao para o estudo do diagn6stico da Molestia de Chagas por processos de laboratorio. Tese de Doutoramento, Sao Paulo, 160 pp. AMATO NETO, V., MAGALDI, C., & PESSOA, S. (1964). Revta. Goiana Med., 10~ 121. TOBIE, E. J., & REES, C. (1948).ff. Parasit., :141, 162. LANG, C. A. (1958). Anal. Chem., 30, 1962.
SEROLOGYOF HUMANSARCOSPORIDIOSIS SIR,--In his review of sarcosporidiosis in man, JEFFREY (1974) suggested that the complement-fixation test would be of value in distinguishing between toxoplasmosis and sarcosporidiosis in cases where morphological study of small cysts in muscle gives an equivocal diagnosis. While much information concerning the serodiagnosis of toxoplasmosis is available, this is not true of sarcosporidiosis. ROMMEL and HEYOORN (1972) found that after eating raw beef or pork containing Sarcocystis, human volunteers shed Isospora hominis in their faeces. Thus, Sarcocystis and I. hominis are different stages of the same parasite. Antibodies to Sarcocystis detectable by the indirect fluorescent antibody test were not shown in the serum of a cat with an intestinal infection (analogous to I. hominis in man) which followed the ingestion of tissue cysts of Sarcocystis in raw cattle diaphragm (MARKUS et al., 1974; MARKUSet al., in press). T h e reason may have been that extra-intestinal infection is sometimes or always necessary for antibody production. Unlike Toxoplasma, Sarcocystis in carnivores does not normally develop extra-intestinal stages (see MARKUS et al., in press), which play a r61e in stimulating the production of antibody. However, circulating antibody has apparently been demonstrated in both known I. hominis carriers and non-carriers (including vegetarians), with use of bovine Sarcocystis antigen and I. hominis sporozoites in the indirect fluorescent antibody test, but was only occasionally observed in young babies (LAARMANet al., 1974; TADROS et al., 1974). Whether this test can give false positives as it occasionally does with Toxoplasma antigen, is an aspect requiring investigation (see ARAU]O et al., 1971; M.ARKUS, 1973; REMINGTON and DESMONTS, 1973). Furthermore, it should be noted that there is a serum factor which causes polar fluorescence of Toxoplasma in approximately 30% of seronegative individuals. This factor is heat stable, can be titrated, and does not usually occur in the blood of infants of less than 6 months of age (SULZERand WILSON,1971 ; KAUFMANet al., 1973). Subclinical coccidiosis caused by L hominis is common (see TADROS et al., 1974). I f antibody is indeed formed, it follows that tests using Sarcocystis antigen on sera of persons having unidentified muscle cysts of Toxoplasma could be positive for Sarcocystis because of a past or concomitant intestinal L hominis infection. On the basis of present knowledge, therefore, a positive result from a serological test for sarcosporidiosis in a person with unidentified muscle cysts might be difficult to interpret, particularly if tests for Toxoplasma are also positive. T h e chances that a test for toxoplasmosis will be positive are good, since it is now a well-established fact that antibodies to Toxoplasma are found in a large number of healthy persons. Only a negative test for toxoplasmosis would help to exclude this protozoon in a patient having unidentified cysts in muscle. However, the absence of demonstrable Toxoplasma antibody cannot be taken as absolute proof that the parasite is not present in the tissues (REMINGTON and ARAUJO, 1974).
416
CORRESPONDENCE
An obvious question which springs to mind is whether there is cross-reaction between Sarcocystis and Toxoplasma. I t would appear that they are immunologically distinct. Cats which are shedding I. cati (one of the coccidia that has in the past been called I. bigemina), now known to be a resistant stage of Sarcocystis (HEYDORN and ROMMEL, 1972), can be superinfected with Toxoplasma, resulting in production of o6cysts of the latter parasite as well (see FRENKEL, 1973). However, although several workers have shown that immunity usually develops in cats in which Toxoplasma has undergone gametogony, the degree to which this happens following sexual reproduction of Sarcocystis has yet to be determined. T h e indications are that cats can be re-infected with Sarcocystis relatively easily (e.g. MARKIJS et al., in press). Additional evidence suggesting an absence of any marked common antigenicity between Sarcocystis and Toxoplasma is provided by serological studies by MANDOUR (1965), MGCONNELL et al. (1973) and TADROS et al. (1974). In a summary of other work on this subject, the conclusion was reached (MARKIJS, 1973) that there are as yet no grounds for questioning the reliability of routine serological tests for human toxoplasmosis. T h e opinion of DE OLIVEIRAet al. (1973) that there is "group" cross-reaction between Toxoplasma on the one hand and I. hominis and I. belli on the other is open to question and needs to be confirmed. While a negative serological test for toxoplasmosis in a patient having unidentified cysts in muscle would be useful information, more experimental work on antibody production in Sarcocystis infections in relation to the use of tests for sarcosporidiosis is indicated. I am, etc., .MILES B. MARKUS, Department of Zoology and Applied Entomology, Imperial College, University of London, London S.W.7. 8 Ju/y, 1974 REFERENGES ARAUJO, F. G., BARNETT, E. V., GENTRY, L. O. & REMINGTON, J. S. (1971). Appl. Microbiol., 22, 270. DE OLIVEIRA, G. S. C., BARBOSA,W. & DA SILVA, A. L. (1973). Revta Patol. trop., 2, 387. FRENKEL, J. K. (1973). In: HAMMOND, D. M. & LONG, P. L. (eds), The coccidia, p. 343. Baltimore: University Park Press and London: Butterworths. HEYDORN, A.-O. & ROMMEL, M. (1972). Berl. Munch, tier~irztl. Wschr., 85, 121. JEFFREY, H. C. (1974). Trans. R. Soc. trop. Med. Hyg., 68, 17. KAUFMAN, G. 1. REMINGTON,J. S. & WATERS, H. C. (1973). Appl. Microbiol., 2,5, 724. LAARMAN,J. J., TADROS, W. & VAN DEN EIJI(, A. A. (1974). Unpublished paper read at 3rd Int. Congr. Parasit. (Munich). McCoNNELL, E. E., BASSON,P. A., WOLSTENHOLME,B., DE Vos, V. & MALHERBE, H. H. (1973). Trans. R. Soc. trop. Med. Hyg., 87, 851. MANDOtm, A. M. (1965). Ph.D. thesis, University of London. MARKUS, M. B. (1973). New Engl. J. Med., 289, 980. , DRAPER, C. C., HUTCHISON, W. M., KILLICK- KENDRICK, R. & GARNHAM,P. C. C. (1974). Trans. R. Soc. trop. Med. Hyg., 88, 3. , KILLICK-KENDRICK, R. & GARNHAM, P. C. C. 97. trop. Med. Hyg. (In press). REMINGTON, J. S. & ARAUJO, F. G. (1974). Proc. 3rd Int. Congr. Parasit. (Munich), 1, 294. & DESMONTS, G. (1973). J. Pediat., 83, 27. ROMMEL, M. & HEYDORN, A.-O. (1972). Berl. Miinch. tiertirzd. Wschr., 85, 143. SULZER, A. J. & WILSON, M. (1971). Expl. Parasit., 20, 197. TADROS, W., LAARMAN,J. J. & VAN DEN EIIK, A. A. (1974). Z. Parasit Kde, 43, 221. SYNGYTIAL MASSES IN PANSTRONGYLUS MEGISTUS NATURALLY INFECTED WITH TRFPANOSOMA CRUZI SIR,--In smears from intestinal contents of P. megistus naturally infected with T. crugi we detected a certain number of syncytial masses indicating a previously undescribed process of T. cruzi life-cycle in this invertebrate host. T h e multinucleated syncytial masses show indefinite cytoplasm with a large number of nuclei and kinetoplasts, indicating fusion of the unicellular parasites. T h e liberation of flagellated forms from the syncytial masses were observed. This sequence suggests that this process in the T. cruzi life-cycle has not been studied in the invertebrate host and that it may involve genetic exchange as has been reported in Triatoma infestans by BRENER and in cultures of T. cruzi by ADLER. I am, etc., R. P. BRAZIL, Lab. Leishmanoses, C.P.P.N., I.C.B., F u n d i o , Guanabara, Brasil. 16 July, 1974. REFERENCES BRENER, Z. (1972). 97. Protozool., 19, (1), 23. ADLER, S. (1958). Ann. trop. Med. Parasit., 52, 282.