292 TRANSACTIONS OFTHEROYALSOCIETY OF TROPICAL MEDICINE AND HYGIENE (1992) 86, 292-293
Detection
of circulating
antigens
in human
trichinellosis
Toshimasa Nishiyama’, Tsuneji Arakil, Naoto Mizuno l, Teruo Wadal, Takeshi Ide2 and Tomio Yamaguchi3 ‘Department of Parasitology and ‘Department of Chemistry, Nara Medical University, Kashihara, Nara, 634, Japan; 3Department of Parasitology, School of Medicine, Hirosaki University, Hirosaki, Aomori, 036, Japan Abstract A ‘sandwich’ enzyme-linked immunosorbent assay was established to detect circulating antigens of Trichinella spiralis in human sera, its sensitivity and specificity was evaluated using 4 antigens (Trichinella spiralis, Trichuris trichiura, Dirofilaria immitis and Ascaris suum), and it was found to be sensitive and specific for T. spiralis antigen. Samples of 347 individuals with suspected trichinellosis, who had eaten incompletely cooked bear meat containing larvae of T. spirahs, were examined. Among individuals showing clinical symptoms, circulating antigens were detected in 29.9%, and the prevalence of antibodies was 189%. Among individuals lacking clinical symptoms, antigens were detected in 21.4% and antibodies in 5.0%. It was concluded that detection of circulating antigens was more useful for making diagnoses than measurement of specific antibodies. Introduction TrichineZZa spiraZis is a nematode worm which encysts in skeletal muscles. The clinical symptoms of T. spiralis infection are varied (abdominal pain, diarrhoea, fever, myalgia, malaise, periorbital oedema, etc.), and the parasites often cannot be detected by direct microscopical examination. Serodiagnosis is therefore very important for identifying infected individuals. Several serodiagnostic methods have been developed to indicate the presence of specific antibodies during the course of this disease. Soluble antigen from larvae in muscle has been used in tests such as passive haemagglutination (PRICE & WEINER, 1956), indirect immunofluoresence (JACKSON, 1959) counter immunoelectrophoresis (DESPOMMIER et al., 1974), enzyme-linked immunosorbent assay (ELBA; ENGVALL & LJUNGSTR~M, 1975) and so on. However, cross-reactions with other parasitic diseases are common. In the present study, we used a ‘sandwich’ ELISA to demonstrate the presence of circulating antigens both in the sera of trichinellosis patients and in human sera from suspected cases collected during an outbreak in Yokkaichi city, Japan. The ‘sandwich’ ELISA was evaluated for its specificity and sensitivity. Materials and Methods Human sera Serum samples from 347 individuals were collected from people who had eaten incompletely cooked bear meat containing T. spiralis larvae at Yokkaichi city, Mie, Japan in 1982. Twenty-four individuals of this group gave the clinical symptoms of trichinellosis and a positive antibody response, using the latex agglutination test, circum-larval precipitation test and counter-current immunoelectrophoresis method (YAMAGUCHI et al., 1982; Table 1). Table 1. Distribution of the 347 persons studied according to results of serological investigations and clinical symptoms of trichinellosis Specific antibody
Clinical Positive
Positive Negative Total
1;: 127
symptoms Negative 2;; 220
Total 3:: 347
Sixty-four control sera were collected from healthy students and instructors at Nara Medical University who had no history of consuming raw bear meat. Crude antigens and antiserum Larvae of T. spiralis (Polish strain) in the muscle of infected mice were collected by pepsin-HCl digestion and lyophilized and stored until use. The lyophilized larvae were sonicated in nhosnhate-buffered saline (PBS,
pH 7.2) for 20 min and extracted with PBS for 24 h at 4°C. After centrifugation at 10 000 g for 30 min, the supernatant was lyophilized again and used as a crude antigen. Antigens of Trichuris trichiura, Dirofilaria immitis and Ascaris suum were prepared by the method of TSUJI (1974). The protein concentration of all antigens was measured by the method of LOWRY et al. (195 1). A Japanese white rabbit was immunized intracutaneously several times with T. spiralis antigen plus Freunds’ complete adjuvant. The antibody titre was checked by Ochterlony’s method. Purification of immunoglobuZin G and conjugation with horseradishperoxidase The immunoglobulin (Ig) G fraction of the antiserum against T. spiralis was obtained by ammonium sulphate precipitation, purified through a DE23 (Whatman, USA) column and conjugated with horseradish peroxidase (type VI, Sigma, USA) according to the method of NAKANE & KAWAOI (1974).
‘Sandwich’ ELBA The IgG fractions of rabbit antiserum against T. spiralis were diluted to 40 uggiml with 0.05 M carbonate buffer (pH 9.6). Sufficient amounts of IgG (100 pl) were used to coat each well of 96-well polystyrene ELISA plates (Ml29 A, Dynatech, West Germany). The plates were incubated for 3 h at 37°C and washed with PBS containing 0.02% Tween 20@. They were then incubated at 37°C for one hour with carbonate buffer containing 1% bovine serum albumin (BSA) to reduce non-specific binding of reagents to the plates (blocking). After washing, 100 ~1 of undiluted test serum or diluted antigens were added to the wells and further incubated at 37°C for one hour. The plates were then washed and 100 ~1 of peroxidase-labelled anti-Trichinella IgG diluted 1: 150 with PBS containing 1% BSA were added to each well. After one hour’s incubation at 37°C the plates were washed and 100 ~1 of freshly prepared substrate, o-phenylenediamine, were added. The reaction was stopped after 30 min by the addition of 100 ~1 of 3N sulphuric acid. The absorbence was read at 500 mn by an MTP 12A reader (Corona, Japan). Results Specificity and sensitivity of the ‘sandwich’ ELBA This method test was highly specific for detecting circulating antigens derived from T. spiralis (Figure). Detection of circulating antigen by the ‘sandwich’ ELISA Four hundred and eleven human sera were tested, and the protein concentration of circulating antigens was estimated (Figure). These results are summarized in Table 2. The antigen concentrations of normal control sera had a normal distribution. The threshold of positivity was calculated as the mean ulus 3 standard deviations (SD) of
293
b-.10.27 ngiml would be scored as positive. Among individuals showing clinical symptoms, 381127(29.9%) were antigen positive, and the prevalence of antibodies in their serawas 241127(18.9%). Among individuals lacking clinical symptoms, 471220 (21.4%) were scored as antigen positive, and the prevalence of antibodies was 111220(5.0%). About 30% of Table 2. The results of the ‘sandwich’
circulating
Circulating gfl$
antigens
of Trichinella
ELISA for detecting
spiralis
Clinical sym toms Positive Gegative Ab+” Ab-” Ab+ Ab-” 6 3
Normal controls 8
24 103 11 209 64 Total “Ab+ indicates presence of anti-T. spiralis antibody, detected by latex ag lutination and arcurn-larval recipitation tests an8 counter-current immunoef ectrophoresis; Ab- indicates absence of such antibody. individuals showing clinical symptoms of trichinellosis had detectable circulating antigens. Detection of circulating antigens was correlated more closely with clinical symptoms than was identification of specific antibodies. On the other hand, of the individuals showing clinical symptoms but not having detectable specific antibodies, 26/103 (14.6%) were antigen positive. However, false positives (2164) were observed with sera from healthy controls. Discussion
Detection of parasite antigens in the infected human fluids has recently been described in a number of parasitic diseases. The detection of circulating antigens in rats infected with T. spiralis was reported by GARCIA et al. (1979); LEIKINA et al. (1982) and SMITH & KENNEDY (1984) identified circulating antigens in experimentally infected guinea-pigs and mice. It is not yet known, however, whether detection of circulating antigens is a useful meansof serodiagnosis. This study shows that a ‘sandwich’ ELBA can be used to measure circulating antigens in human trichinellosis. The ‘sandwich’ ELISA was performed on 347 caseswith
suspected T. spiralis infection; of 85 sera containing circulating antigens, 17 were also positive for specific antibodies and 68 were negative. It is interesting that the 10 individuals with the hiihest concentration 01 circulating antigens were negative for specific antibodies. This may be becauseof a low immunological responseto infection with T. spiralis or becausethey were at an early stageof infection when specific antibody titres had not yet increased. Among caseswith clinical symptoms, the prevalence of circulating antigen (29.9%) was higher than that of specific antibody (18.9%). These results show that detection of the circulating antigens by the ‘sandwich’ ELISA was more sensitive than the measurement of specific antibodies by the other serological tests used. The ‘sandwich’ ELISA can be considered to be specific for T. spiralis antigens, but sera from 2 healthy control individuals were scored as positive. These 2 individuals had travelled in the epidemic area of trichinellosis and had eaten porcine meat during their travels; it is possible that they may have been infected with T. spiralis. Serodiagnosisis a useful diagnostic tool in human trichinellosis, but it should be confirmed by several different serodiagnostic methods. The ‘sandwich’ ELBA based on detection of circulating antigens may be an important new addition to currently available tests for the serodiagnosis of human trichinellosis. Acknowledgements We are greatly indebted to Drs Shiro Tabata, Isamu Sugawara, Sohei Shinka and Masamichi Aikawa for their advice and for reviewingthe manuscript.This study wassupportedin part by a grant from the Ministry of Education of Japan (nos 02304036 and 02454173). References Despommier, D. D., Muller, M., Jenks, B. & Fruitstone, M. (1974). Immunodiagnosis of human trichinosis using counterelectrophoresis and agar gel diffusion. AmericanJournal of Tropical Medicine and Hygiene, 23,41-%4. Engvall, E. & Ljungstram, I. (1975). Detection of human antibodies to Trichinella spiralis by enzyme-linked irnrnunosorbActa Pathologica et Microbiologica ent assay, ELBA. Scandinavica, section C, 83,231-237. Garcia, G. V., Osorio, R. M. & Castro, G. J. (1979). Application of the micro-ELBA technique for double antibodies to investigations of antigens in experimental trichinellosis of rats. Revista Iberica de Parasitologia, 39,55-63. Jackson, G. J. (1959). Fluorescent antibody studies of Trichinella spiralis infections.gournal of Infectious Diseases, 107,97117. Leikina, E. S., Kovrova, E. A. & Krusovskaya, N. N. (1982). Detection of circulating antigens in the sera of patients with unilocular or multilocular hydatidosis or with trichinelliasis. Meditsinskaya Parazitologiya i Parazitarnye Bole+ 51,7-15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193,265-275. Nakane, P. K. & Kawaoi, A. (1974). Peroxidase labelled antibody, a new method of conjugation. Journal of Histochemisty and Cytochemisty, 22, 1084-1091. Price, S. G. & Weiner, L. M. (1956). Use of haemagglutination in the diagnosis of trichinosis. American Journal of Clinical Pathology, 26, 1261-1269. Smith, H. V. & Kennedy, M. W. (1984). Soluble antigens and antibodies in the sera of mice infected with Trichinella spiral@, detected by modified double counter imtnunoelectrophoresls technique. Journal of Helminthologv, 58,7 l-78. Tsuji, M. (1974). On the immunoelectrophoresis for hehninthological researches. Japanese Journal of Parasitolog;v, 23, 335-
345.
Yamaguchi, T., Yagisawa, M., Inaba, T., Huang, W. H. & Ishida, K. (1982). Epidemiological aspects of trichinellosis in Japan. Summary of Sino-Japanese Seminar on Parasitic Zoonoses 1982, pp. 117-124. Received 30 May 1991; revised for publication 28 August 1991
28 August 1991; accepted
Detection
of circulating
antigens
in human
trichinellosis
Toshimasa Nishiyama’, Tsuneji Arakil, Naoto Mizuno l, Teruo Wadal, Takeshi Ide2 and Tomio Yamaguchi3 ‘Department of Parasitology and ‘Department of Chemistry, Nara Medical University, Kashihara, Nara, 634, Japan; 3Department of Parasitology, School of Medicine, Hirosaki University, Hirosaki, Aomori, 036, Japan Abstract A ‘sandwich’ enzyme-linked immunosorbent assay was established to detect circulating antigens of Trichinella spiralis in human sera, its sensitivity and specificity was evaluated using 4 antigens (Trichinella spiralis, Trichuris trichiura, Dirofilaria immitis and Ascaris suum), and it was found to be sensitive and specific for T. spiralis antigen. Samples of 347 individuals with suspected trichinellosis, who had eaten incompletely cooked bear meat containing larvae of T. spirahs, were examined. Among individuals showing clinical symptoms, circulating antigens were detected in 29.9%, and the prevalence of antibodies was 189%. Among individuals lacking clinical symptoms, antigens were detected in 21.4% and antibodies in 5.0%. It was concluded that detection of circulating antigens was more useful for making diagnoses than measurement of specific antibodies. Introduction TrichineZZa spiraZis is a nematode worm which encysts in skeletal muscles. The clinical symptoms of T. spiralis infection are varied (abdominal pain, diarrhoea, fever, myalgia, malaise, periorbital oedema, etc.), and the parasites often cannot be detected by direct microscopical examination. Serodiagnosis is therefore very important for identifying infected individuals. Several serodiagnostic methods have been developed to indicate the presence of specific antibodies during the course of this disease. Soluble antigen from larvae in muscle has been used in tests such as passive haemagglutination (PRICE & WEINER, 1956), indirect immunofluoresence (JACKSON, 1959) counter immunoelectrophoresis (DESPOMMIER et al., 1974), enzyme-linked immunosorbent assay (ELBA; ENGVALL & LJUNGSTR~M, 1975) and so on. However, cross-reactions with other parasitic diseases are common. In the present study, we used a ‘sandwich’ ELISA to demonstrate the presence of circulating antigens both in the sera of trichinellosis patients and in human sera from suspected cases collected during an outbreak in Yokkaichi city, Japan. The ‘sandwich’ ELISA was evaluated for its specificity and sensitivity. Materials and Methods Human sera Serum samples from 347 individuals were collected from people who had eaten incompletely cooked bear meat containing T. spiralis larvae at Yokkaichi city, Mie, Japan in 1982. Twenty-four individuals of this group gave the clinical symptoms of trichinellosis and a positive antibody response, using the latex agglutination test, circum-larval precipitation test and counter-current immunoelectrophoresis method (YAMAGUCHI et al., 1982; Table 1). Table 1. Distribution of the 347 persons studied according to results of serological investigations and clinical symptoms of trichinellosis Specific antibody
Clinical Positive
Positive Negative Total
1;: 127
symptoms Negative 2;; 220
Total 3:: 347
Sixty-four control sera were collected from healthy students and instructors at Nara Medical University who had no history of consuming raw bear meat. Crude antigens and antiserum Larvae of T. spiralis (Polish strain) in the muscle of infected mice were collected by pepsin-HCl digestion and lyophilized and stored until use. The lyophilized larvae were sonicated in nhosnhate-buffered saline (PBS,
pH 7.2) for 20 min and extracted with PBS for 24 h at 4°C. After centrifugation at 10 000 g for 30 min, the supernatant was lyophilized again and used as a crude antigen. Antigens of Trichuris trichiura, Dirofilaria immitis and Ascaris suum were prepared by the method of TSUJI (1974). The protein concentration of all antigens was measured by the method of LOWRY et al. (195 1). A Japanese white rabbit was immunized intracutaneously several times with T. spiralis antigen plus Freunds’ complete adjuvant. The antibody titre was checked by Ochterlony’s method. Purification of immunoglobuZin G and conjugation with horseradishperoxidase The immunoglobulin (Ig) G fraction of the antiserum against T. spiralis was obtained by ammonium sulphate precipitation, purified through a DE23 (Whatman, USA) column and conjugated with horseradish peroxidase (type VI, Sigma, USA) according to the method of NAKANE & KAWAOI (1974).
‘Sandwich’ ELBA The IgG fractions of rabbit antiserum against T. spiralis were diluted to 40 uggiml with 0.05 M carbonate buffer (pH 9.6). Sufficient amounts of IgG (100 pl) were used to coat each well of 96-well polystyrene ELISA plates (Ml29 A, Dynatech, West Germany). The plates were incubated for 3 h at 37°C and washed with PBS containing 0.02% Tween 20@. They were then incubated at 37°C for one hour with carbonate buffer containing 1% bovine serum albumin (BSA) to reduce non-specific binding of reagents to the plates (blocking). After washing, 100 ~1 of undiluted test serum or diluted antigens were added to the wells and further incubated at 37°C for one hour. The plates were then washed and 100 ~1 of peroxidase-labelled anti-Trichinella IgG diluted 1: 150 with PBS containing 1% BSA were added to each well. After one hour’s incubation at 37°C the plates were washed and 100 ~1 of freshly prepared substrate, o-phenylenediamine, were added. The reaction was stopped after 30 min by the addition of 100 ~1 of 3N sulphuric acid. The absorbence was read at 500 mn by an MTP 12A reader (Corona, Japan). Results Specificity and sensitivity of the ‘sandwich’ ELBA This method test was highly specific for detecting circulating antigens derived from T. spiralis (Figure). Detection of circulating antigen by the ‘sandwich’ ELISA Four hundred and eleven human sera were tested, and the protein concentration of circulating antigens was estimated (Figure). These results are summarized in Table 2. The antigen concentrations of normal control sera had a normal distribution. The threshold of positivity was calculated as the mean ulus 3 standard deviations (SD) of
293
b-.10.27 ngiml would be scored as positive. Among individuals showing clinical symptoms, 381127(29.9%) were antigen positive, and the prevalence of antibodies in their serawas 241127(18.9%). Among individuals lacking clinical symptoms, 471220 (21.4%) were scored as antigen positive, and the prevalence of antibodies was 111220(5.0%). About 30% of Table 2. The results of the ‘sandwich’
circulating
Circulating gfl$
antigens
of Trichinella
ELISA for detecting
spiralis
Clinical sym toms Positive Gegative Ab+” Ab-” Ab+ Ab-” 6 3
Normal controls 8
24 103 11 209 64 Total “Ab+ indicates presence of anti-T. spiralis antibody, detected by latex ag lutination and arcurn-larval recipitation tests an8 counter-current immunoef ectrophoresis; Ab- indicates absence of such antibody. individuals showing clinical symptoms of trichinellosis had detectable circulating antigens. Detection of circulating antigens was correlated more closely with clinical symptoms than was identification of specific antibodies. On the other hand, of the individuals showing clinical symptoms but not having detectable specific antibodies, 26/103 (14.6%) were antigen positive. However, false positives (2164) were observed with sera from healthy controls. Discussion
Detection of parasite antigens in the infected human fluids has recently been described in a number of parasitic diseases. The detection of circulating antigens in rats infected with T. spiralis was reported by GARCIA et al. (1979); LEIKINA et al. (1982) and SMITH & KENNEDY (1984) identified circulating antigens in experimentally infected guinea-pigs and mice. It is not yet known, however, whether detection of circulating antigens is a useful meansof serodiagnosis. This study shows that a ‘sandwich’ ELBA can be used to measure circulating antigens in human trichinellosis. The ‘sandwich’ ELISA was performed on 347 caseswith
suspected T. spiralis infection; of 85 sera containing circulating antigens, 17 were also positive for specific antibodies and 68 were negative. It is interesting that the 10 individuals with the hiihest concentration 01 circulating antigens were negative for specific antibodies. This may be becauseof a low immunological responseto infection with T. spiralis or becausethey were at an early stageof infection when specific antibody titres had not yet increased. Among caseswith clinical symptoms, the prevalence of circulating antigen (29.9%) was higher than that of specific antibody (18.9%). These results show that detection of the circulating antigens by the ‘sandwich’ ELISA was more sensitive than the measurement of specific antibodies by the other serological tests used. The ‘sandwich’ ELISA can be considered to be specific for T. spiralis antigens, but sera from 2 healthy control individuals were scored as positive. These 2 individuals had travelled in the epidemic area of trichinellosis and had eaten porcine meat during their travels; it is possible that they may have been infected with T. spiralis. Serodiagnosisis a useful diagnostic tool in human trichinellosis, but it should be confirmed by several different serodiagnostic methods. The ‘sandwich’ ELBA based on detection of circulating antigens may be an important new addition to currently available tests for the serodiagnosis of human trichinellosis. Acknowledgements We are greatly indebted to Drs Shiro Tabata, Isamu Sugawara, Sohei Shinka and Masamichi Aikawa for their advice and for reviewingthe manuscript.This study wassupportedin part by a grant from the Ministry of Education of Japan (nos 02304036 and 02454173). References Despommier, D. D., Muller, M., Jenks, B. & Fruitstone, M. (1974). Immunodiagnosis of human trichinosis using counterelectrophoresis and agar gel diffusion. AmericanJournal of Tropical Medicine and Hygiene, 23,41-%4. Engvall, E. & Ljungstram, I. (1975). Detection of human antibodies to Trichinella spiralis by enzyme-linked irnrnunosorbActa Pathologica et Microbiologica ent assay, ELBA. Scandinavica, section C, 83,231-237. Garcia, G. V., Osorio, R. M. & Castro, G. J. (1979). Application of the micro-ELBA technique for double antibodies to investigations of antigens in experimental trichinellosis of rats. Revista Iberica de Parasitologia, 39,55-63. Jackson, G. J. (1959). Fluorescent antibody studies of Trichinella spiralis infections.gournal of Infectious Diseases, 107,97117. Leikina, E. S., Kovrova, E. A. & Krusovskaya, N. N. (1982). Detection of circulating antigens in the sera of patients with unilocular or multilocular hydatidosis or with trichinelliasis. Meditsinskaya Parazitologiya i Parazitarnye Bole+ 51,7-15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193,265-275. Nakane, P. K. & Kawaoi, A. (1974). Peroxidase labelled antibody, a new method of conjugation. Journal of Histochemisty and Cytochemisty, 22, 1084-1091. Price, S. G. & Weiner, L. M. (1956). Use of haemagglutination in the diagnosis of trichinosis. American Journal of Clinical Pathology, 26, 1261-1269. Smith, H. V. & Kennedy, M. W. (1984). Soluble antigens and antibodies in the sera of mice infected with Trichinella spiral@, detected by modified double counter imtnunoelectrophoresls technique. Journal of Helminthologv, 58,7 l-78. Tsuji, M. (1974). On the immunoelectrophoresis for hehninthological researches. Japanese Journal of Parasitolog;v, 23, 335-
345.
Yamaguchi, T., Yagisawa, M., Inaba, T., Huang, W. H. & Ishida, K. (1982). Epidemiological aspects of trichinellosis in Japan. Summary of Sino-Japanese Seminar on Parasitic Zoonoses 1982, pp. 117-124. Received 30 May 1991; revised for publication 28 August 1991
28 August 1991; accepted
