- Email: [email protected]
Comparison of adult somatic and cysteine proteinase antigens of Fasciola gigantica in enzyme linked immunosorbent assay for serodiagnosis of human fasciolosis
Acta Tropica 88 (2003) 69 /75 www.elsevier.com/locate/actatropica
Comparison of adult somatic and cysteine proteinase antigens of Fasciola gigantica in enzyme linked immunosorbent assay for serodiagnosis of human fasciolosis M.B. Rokni a,*, J. Massoud a, A. Hanilo b a
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, P.O. Box 141556446, Tehran, Iran b Department Medical Parasitology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Received 29 January 2003; received in revised form 22 June 2003; accepted 22 June 2003
Abstract Fasciolosis caused by Fasciola hepatica and Fasciola gigantica is one of the major public health problems in the world and in Iran. Considering that stool examination for Fasciola eggs is not a sensitive method and immunodiagnosis methods are more applicable for this purpose, so the present study was conducted to compare the somatic (S) and cysteine proteinase (CP) antigens of F. gigantica in IgG-ELISA to diagnose human fasciolosis. Serum samples obtained from 100 individuals collected during the fasciolosis outbreak in 1999 in the Gilan province of Northern Iran that were coprologically positive for fasciolosis were analyzed by IgG-ELISA. Sera from healthy control individuals, not infected with any parasitic diseases (n/50) and from others with different parasitic infections including hydatidosis (n /40), toxocariosis (n /20), amoebiosis (n /10), and malaria (n /5) were examined as well. The cut-off point for (S) and CP was 0.40 and 0.35, respectively. All 100 individuals that showed clinical manifestations of fasciolosis, were also seropositive using both antigens, whereas all 50 non-infected controls were seronegative, therefore the sensitivity of the test was 100% for both antigens. The specificity of (S) and CP antigens were calculated as 96.9 and 98.4%, respectively. The positive and negative predictive values of the test regarding (S) antigen were 96 and 100%, whereas these values as for CP antigen were 98 and 100% correspondingly. Two individuals with hydatidosis and two with toxocariasis had antibodies that were reactive against (S) antigen, whereas concerning CP antigen, one individual with hydatidosis and another with toxocariasis showed cross-reactivity against it. We have demonstrated that altogether CP antigen provides a more conclusive diagnosis as possessing lower cut-off and enabling better to discriminate between seronegative and seropositive subpopulations. # 2003 Elsevier B.V. All rights reserved. Keywords: Fasciolosis; IgG-ELISA; Fasciola gigantica
1. Introduction * Corresponding author. Tel.: /98-21-406-6898; fax: /9821-646-2267. E-mail address: [email protected] (M.B. Rokni).
Fasciolosis caused by Fasciola hepatica and Fasciola gigantica is one of the major public health
0001-706X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0001-706X(03)00175-X
70
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
problems in the world and in Iran (WHO, 1995; Massoud, 1993). Epidemics of fasciolosis has caused extensive suffering and economic losses in some regions (Rokni et al., 2002). Humans are infected by ingestion of aquatic plants that contain the infected metacercariae (Markell and Voge, 1999). Diagnosis of fasciolosis is based on clinical findings and laboratory tests. The most reliable means is the finding of eggs in stool of infected individual despite the overwhelming consensus that this method is not wholly reliable (Hillyer, 1998). Using this method eggs are not detected until the latent period of infection when much of the liver damage has already occurred. In addition, eggs are released sporadically from the bile ducts and hence stool samples of infected patients can contain no eggs (Mas-Coma et al., 1998). Serological diagnosis is preferred particularly since antifasciola antibodies can be detected as early as 2 weeks post infection and can thus facilitate early chemotherapeutic intervention (Hillyer, 1998). Despite of the recent studies involving the diagnosis of fasciolosis using F. hepatica antigens (Cordova et al., 1999; O’Neill et al., 1998; Rokni et al., 2002), the effectiveness of F. gigantica somatic (S) and cysteine proteinase (CP) as antigens in ELISA have not been studied sufficiently and needs more investigations. Adult somatic extracts and excretory/secretory (ES) products of F. gigantica have been used in ELISA (Maleewong et al., 1996, 1999; Youssef and Mansour, 1991) and in Western Blott (Intapan et al., 1998) for serodiagnosis of human fasciolosis gigantica. In Iran this is the first report in this case. The purpose of this investigation was to compare aforementioned F. gigantica antigens in ELISA test for serodiagnosis of human fasciolosis.
2. Materials and methods 2.1. Sera All samples from Fasciola-infected individuals were collected during the fasciolosis outbreak in 1999 (Bahonar and Poya, 2000; Rokni et al., 2002) in the Central Health Clinic of Rasht, Gilan
province of Northern Iran. Coprological analysis for Fasciola eggs was performed on faecal samples obtained from all individuals as previously described (Katz et al., 1972). Only individuals that were coprologically positive (n/100) and showed a history of the disease were included in the present study. Seventy five sera from patients with other parasitic diseases were also collected from the different laboratories in School of Public Health, Tehran University of Medical sciences: hydatidosis (n /40), toxocariosis (n/20), amoebiosis (n/10), and malaria (n /5). Negative control sera (n/50) belonged to cases with no clinical symptoms of fasciolosis and negative as for parasitic diagnostic tests were obtained from a non-endemic area. 2.2. Preparation of antigens Adult F. gigantica were obtained from infected bovine livers collected from local abattoirs. The worms were identified as F. gigantica based on criteria described by Sahba et al., 1972. F. gigantica (S) antigen was prepared by homogenizing adult worms in 0.045 M PBS/pH 7.2 using electrical homogenizator (Edmund Buhler Co., model Homo 4/A mit uhr) followed by sonication (Tomy Seiko model UP-200P, Tokyo), and then centrifugation at 15 000 /g at 4 8C for 30 min. Afterwards the supernatant (S antigen) was subjected to a 134 ml Sephacryle S200 HR gelfiltration column, equilibrated in 0.1 M Tris / HCl, pH 7.2. One-milliliter fractions were collected and monitored by absorbance at 280 nm for protein concentration. Each fraction was also assayed for CP activity using the flurogenic substrate Z -Phe /Arg /AMC (7-amino-4-methylcomarin). The release of the fluorescent leaving group, AMC was monitored in a Perkin/Elmer Luminescence Spectrometer model LS 50, at exciter and analyzer wavelengths of 370 and 440 nm, respectively (Smith et al., 1993). Fractions containing proteolytic activity were pooled, and then were subjected to ion exchange chromatography on QAE-Sephadex A50 equilibrated in 0.1 M Tris /HCl, pH 7.2. The first obtained fraction, which had the highest proteolytic activity, concentrated and used as CP antigen.
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
Both prepared antigens were analyzed by 15% reducing SDS-PAGE (Laemmeli, 1970). The concentration of each antigen preparation was measured using Bradford method (Bradford, 1976). In order to verify the class of these antigens specific inhibitors at final concentration of phenylmethylsulfonyl fluoride 10 mM; EDTA 10 mM; E-64 5 mg/ml; leupeptin 5 mg/ml and pepstatin 1 mM were used (Dalton and Heffernan, 1989). 2.3. ELISA test The immunodiagnostic assay was performed as previously described by O’Neill et al. (1999). Briefly, after the test was optimyzed, 100 ml (S) and CP antigens (30 and 5 mg/ml, respectively) was dispensed into the wells of microtiter plates (Nuclon, Kamstrup, Roskilde, Denmark) and then incubated overnight at 37 8C. Excess binding sites were blocked with 200 ml of bovine serum albumin (2% diluted in PBS/0.1% Tween 20) and incubated for 30 min at 37 8C. After the wells were washed three times with PBS /Tween 20, 100 ml of a serum sample (diluted 1:1250) was added to each plate and incubated for 60 min at 37 8C. Following another washing step, 100 ml of peroxidase-conjugated goat anti-human IgG (diluted 1:6000) was added to each well and the plates incubated for a further 60 min at 37 8C. Following a final washing step 100 ml of O -phenylendiamine dihydrochloride substrate (all from Sigma Chemical Co., Poole, Dorset, UK) was added to each well and the reaction stopped after 5 min by adding 50 ml of 12.5% H2SO4. The optical density (OD) of the samples was measured at 492 nm using a Titerteck (Helsinki, Finland) multiscan ELISA plate reader).
71
3. Results Following analysing the first peak gained from ion-exchange chromatography, i.e. CP antigen, by SDS-PAGE, a protein of about 27/28 kDa was identified (Fig. 1). Using aforementioned specific inhibitors, the class of this antigen was detected as CP. Serum samples obtained from 100 individuals that were coprologically positive for fasciolosis and 125 cases from non-fasciola infected and control group were analyzed by ELISA for total antibody responses against F. gigantica (S) and CP antigens. The absorbance readings obtained for these antigens were plotted against each other on scattergrams, which revealed two compact clusters of individuals with low and high absorbance readings (Fig. 2). An upper limit for ELISA absorbances, which defines the seronegative and putatively uninfected individuals was determined at 3.09 S.D. from the mean of this seronegative population (0.4 and 0.35 for S and CP antigens, respectively). This cuts off 99.9% of the seronegative peak below the cut-off point. With this cut-off point only one individual in 1000 seronegative individuals will be misdiagnosed as seropositive. Therefore, absorbance readings greater than the cut-off value were considered to be seropositive for fasciolosis. Accordingly, all 100 individuals that showed clinical manifestations of fasciolosis, were also seropositive using both (S) and CP antigens
2.4. Statistical analysis We used the mean plus 3.09 S.D. OD value of the healthy group sera as the lower limit of positivity. The sensitivity, specificity, and the predictive values were calculated as previously described (Galen, 1980). All assays were tested in triplicate and repeated twice. Statistical analysis was carried out using SPSS for Windows, version 10.
Fig. 1. SDS-PAGE of purified F. gigantica CP in a 15% polyacrylamide gel stained with coomassie blue. On the left shows the protein marker.
72
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
Fig. 2. Scatter graph of the combined data for 100 Fasciolainfected and 150 non-infected cases obtained in the total IgGELISA using somatic (S) and CP antigens of F. gigantica antigens. The K-means cluster analysis using the combined data obtained for (S) and CP in the total IgG-ELISA separated the population into seronegative (indicated by closed squares) and seropositive (indicated by open squares) subpopulations. The vertical and horizontal lines in the Figure indicate the calculated cut-off points for each antigen in the total IgG-ELISA.
(Fig. 2, indicated by open squares), whereas 121 and 123 cases (regarding somatic and CP antigens, respectively) from non-fasciola group clustered together as seronegative (Fig. 2, indicated by closed squares). Therefore, the sensitivity of the test was 100% for both antigens. There was no significant difference between the different non-fasciolosis groups in the ELISA readings recorded, instead, the absorbance readings from all samples from the Fasciola seropositive individuals were significantly higher than those obtained from patients that were seronegative or were infected with other parasites (P B/ 0.001). Four individuals from the non-fasciolosis group (two with hydatidosis and two with toxocariosis) were seropositive concerning (S) antigen, while two cases (one with hydatidosis and another with toxocariasis) showed seropositivity for CP antigen (Fig. 3). Consequently the specificity of (S) and CP antigens were calculated as 96.9 and 98.4%, respectively. The positive and negative
Fig. 3. Analysis of sera from patients with various single infections by IgG-ELISA using F. gigantica somatic (S) and CP as antigens. Serum samples obtained from patients with fasciolosis (100, lanes 1), hydatidosis (40, lanes 2), toxocariasis (20, lanes 3), amoebiasis (10, lanes 4), malaria (5, lanes 5), and control human sera (50, lanes 7).
predictive values of the test regarding (S) antigen were 96 and 100%, respectively, whereas these values as for CP antigen were 98 and 100%, correspondingly. Fig. 4 shows the boxplots of obtained ELISA absorbances for two antigens from infected and uninfected groups. Boxes belong to CP antigen showed smaller measures than (S) one.
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
73
Fig. 4. Boxplots of obtained IgG-ELISA absorbances from F. gigantica (S) and CP antigens for Fasciola-infected and uninfected groups.
4. Discussion Stool examination for Fasciola eggs is a reliable method, but the sensitivity is too low (Hillyer, 1998). The infected individuals during prepatent period failed to release the eggs in the stool as well, so immunodiagnosis methods are more applicable for this purpose (Hillyer, 1998; O’Neill et al., 1999). Recently more emphasis is on the antigenic product of CP from F. hepatica (Cordova et al., 1999; O’Neill et al., 1998). Fagbemi and Hillyer (1992) have reported the purification of a CP from F. gigantica adult worms. CP is immunogenic at all stages of liver fluke development in the definitive host and can be used to diagnose both the acute and chronic stages of infections. In some previous studies the sensitivity and specificity of F. gigantica somatic antigen has been reported as 100 and 98% (Maleewong et al., 1996), 100 and 82.5% (Youssef and Mansour, 1991), respectively. Using immunoblotting technique one antigenic component of F. gigantica somatic extract, i.e. 38 kDa was found to give a consistent reaction with sera of patients with fasciolosis as 100% sensitivity and 96.7% specificity (Maleewong et al., 1997).
As is shown in Fig. 3, antibodies in the sera of four patients infected with hydatidosis (two cases) and toxocariasis (two cases) were reactive with F. gigantica (S) antigen, while pertaining to CP antigen the number of cross-reaction cases decreased to two sera infected with hydatidosis (one case) and toxocariasis (one case). This finding suggests that using of more purified antigen than (S) improves the output. There is possibility that an antigen other than the protease in the F. gigantica (S) and CP shares epitope(s) with a hydatid or toxocara immunogen. More to the point, subclinical Fasciola infection may be involved. The present report is the first to demonstrate the use of F. gigantica (S) and CP antigens for the diagnosis of fasciolosis in Iran. Up-to-date, there is no data which species of Fasciola is responsible to fasciolosis in Iran (Massoud, 1989, 1997). It is demonstrated that using of F. gigantica purified antigens could be used as antigen in diagnose of infections with F. hepatica (Weiyi et al., 1997). Our study shows higher specificity of CP than somatic antigens. We have demonstrated that altogether CP antigen provide a more conclusive
74
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
diagnosis as possessing lower cut-off and enabling better to discriminate between seronegative and seropositive subpopulations. Fig. 4 shows the boxplot diagram of obtained OD absorbances relevant to CP and (S) antigens. The smaller box encompasses the less variability around median, hence it is concluded that CP antigen demonstrates better quality to discriminate between seropositive and seronegative populations than (S) one. Based on coprological tests, acute and chronic fasciolosis have the same diagnostic dilemma for the physician since in acute phase there is no eggs in stool and in chronic phase the sensitivity is too low. So it is essential to establish a reliable test like ELISA aiming at diagnosis and seroepidemiology of the disease. This study may be functional to implement control measures against the spread of the infection and to establish a reliable diagnostic tool.
Acknowledgements We show great appreciation to Prof. John P. Dalton and Sandra O’Neill from Dublin City University, Dublin, Ireland and Mr Ahmad Siavash poori from the Tehran University of Medical Sciences for their sincer cooperation. This study was supported by research assistance of Tehran University of Medical Sciences Grant No. 712 to Prof. Massoud and Dr Rokni.
References Bahonar, A., Poya, B., 2000. A descriptive epidemiology of patients infected with fasciolosis in Rasht and Bandar Anzali in 1999. Third National Congress of Medical Parasitology, Sari, Iran, April 9 /11, p. 67. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantitive protein utilizing the principle of protein /dye binding. Anal. Biochem. 72, 248 / 254. Cordova, M., Reategui, L., Espinoza, R., 1999. Immunodiagnosis of human fasciolosis with Fasciola hepatica cysteine proteinase. Trans. R Soc. Trop. Med. Hyg. 93, 54 /57. Dalton, J.P., Heffernan, M., 1989. Thiol protease released in vitro by Fasciola hepatica . Mol. Biochem. Parasitol. 35, 161 /166.
Fagbemi, B.O., Hillyer, G.V., 1992. The purification and characterization of a cysteine proteinase of F. gigantica adult worms. Vet. Parasitol. 43, 223 /232. Galen, R.S., 1980. Predictive values and efficiency of laboratory testing. Pediatr. Clin. North Am. 27, 861 /869. Hillyer, G.V., 1998. Immunodiagnosis of human and animal fasciolosis. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Oxon, Wallingford, UK, pp. 435 /448. Intapan, P.M., Maleewong, W., Wongkham, C., Tomanakarn, K., Ieamviteevanich, K., Piptigool, V., Sukolapong, V., 1998. Excretory-secretory antigenic components of adult Fasciola gigantica recognized by infected human sera. Southeast Asian J. Trop. Med. Publ. Health 29, 579 /583. Katz, N., Chaves, A., Pellegrin, J., 1972. A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Rev. Inst. Med. Trop. Sao Paulo 14, 397 /402. Laemmeli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 /685. Maleewong, W., Intapan, P.M., Wongkham, C., Tomanakan, K., 1996. Comparison of adult somatic and ES antigen in ELISA for the serodiagnosis of human infection with F. gigantica . Southeast Asian J. Trop. Med. Publ. Health 27, 566 /569. Maleewong, W., Intapan, P.M., Tomanakarn, B., Wongkham, C., 1997. Antigenic components of somatic extract from adult Fasciola gigantica recognized by infected human sera. Asian Pac. J. Allergy Immunol. 15, 213 /218. Maleewong, M., Wongkham, C., Intapan, P.M., Pipitgool, V., 1999. Fasciola gigantica */specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fasciolosis. Am. J. Trop. Med. Hyg. 61, 648 /651. Markell, E.K., Voge, M., 1999. Medical Parasitology, eigth ed.. Saunders Company Publication, pp. 185 /188. Mas-Coma, S., Bargues, M.D., Esteban, J.G., 1998. Human fasciolosis. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Oxon, Wallingford, UK, pp. 411 /434. Massoud, J., 1989. Fasciolosis outbreak in man and drug test (Triclabandazole) in Caspian littoral, northern part of Iran. Bull. Soc. France Parasitol. 8, 438. Massoud, J., 1993. Present status of fasciolosis in Iran. WHO Mimeogr. Rep. SCH/SG/93/WP 19. Massoud, J., 1997. The importance of helminthic diseases in Iran. Second National Congress of Parasitic Diseases in Iran. Tehran, Iran, October 19 /22. p. 74. O’Neill, S.M., Parkinson, M., Strauss, W., Angles, R., Dalton, J.P., 1998. Immunodiagnosis of Fasciola hepatica infection (fasciolosis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am. J. Trop. Med. Hyg. 58, 417 /423. O’Neill, S.M., Parkinson, M., Dowd, A.J., Strauss, W., Angles, R., Dalton, J.P., 1999. Immunodiagnosis of human fasciolosis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am. J. Trop. Med. Hyg. 60, 749 /751. Rokni, M.B., Massod, J., Parkinson, M., O’Neill, S., Dalton, J.P., 2002. Diagnosis of human fasciolosis in the Gilan
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75 province of Northern Iran: application of cathepsin LELISA. Diagn. Microbiol. Infect. Dis. 44, 175 /179. Sahba, G.H., Arfaa, F., Farahmandian, I., Jalali, H., 1972. Animal fasciolosis in Khuzestan, southwestern Iran. J. Parasitol. 58, 712 /716. Smith, A.M., Dowd, A., McGonigle, S.M., Keegan, P., Brennan, G., Trudget, A., Dalton, J.P., 1993. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica . Mol. Biochem. Parasitol. 62, 1 /8.
75
Weiyi, H., Zhang, W., Tian, E., 1997. Analysis of F. gigantica ES antigens, vol. 16. J. Guanxi Agricultural University, pp. 115 /124. World Health Organization (WHO), 1995. Control of foodborne trematode infection. WHO Technical Report Series, WHO, Geneva, 845, pp. 1 /157. Youssef, F.G., Mansour, N.S., 1991. A purified Fasciola gigantica worm antigen for the serodiagnosis of human fasciolosis. Trans. R Soc.Trop. Med. Hyg. 85, 535 /537.
Comparison of adult somatic and cysteine proteinase antigens of Fasciola gigantica in enzyme linked immunosorbent assay for serodiagnosis of human fasciolosis M.B. Rokni a,*, J. Massoud a, A. Hanilo b a
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, P.O. Box 141556446, Tehran, Iran b Department Medical Parasitology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Received 29 January 2003; received in revised form 22 June 2003; accepted 22 June 2003
Abstract Fasciolosis caused by Fasciola hepatica and Fasciola gigantica is one of the major public health problems in the world and in Iran. Considering that stool examination for Fasciola eggs is not a sensitive method and immunodiagnosis methods are more applicable for this purpose, so the present study was conducted to compare the somatic (S) and cysteine proteinase (CP) antigens of F. gigantica in IgG-ELISA to diagnose human fasciolosis. Serum samples obtained from 100 individuals collected during the fasciolosis outbreak in 1999 in the Gilan province of Northern Iran that were coprologically positive for fasciolosis were analyzed by IgG-ELISA. Sera from healthy control individuals, not infected with any parasitic diseases (n/50) and from others with different parasitic infections including hydatidosis (n /40), toxocariosis (n /20), amoebiosis (n /10), and malaria (n /5) were examined as well. The cut-off point for (S) and CP was 0.40 and 0.35, respectively. All 100 individuals that showed clinical manifestations of fasciolosis, were also seropositive using both antigens, whereas all 50 non-infected controls were seronegative, therefore the sensitivity of the test was 100% for both antigens. The specificity of (S) and CP antigens were calculated as 96.9 and 98.4%, respectively. The positive and negative predictive values of the test regarding (S) antigen were 96 and 100%, whereas these values as for CP antigen were 98 and 100% correspondingly. Two individuals with hydatidosis and two with toxocariasis had antibodies that were reactive against (S) antigen, whereas concerning CP antigen, one individual with hydatidosis and another with toxocariasis showed cross-reactivity against it. We have demonstrated that altogether CP antigen provides a more conclusive diagnosis as possessing lower cut-off and enabling better to discriminate between seronegative and seropositive subpopulations. # 2003 Elsevier B.V. All rights reserved. Keywords: Fasciolosis; IgG-ELISA; Fasciola gigantica
1. Introduction * Corresponding author. Tel.: /98-21-406-6898; fax: /9821-646-2267. E-mail address: [email protected] (M.B. Rokni).
Fasciolosis caused by Fasciola hepatica and Fasciola gigantica is one of the major public health
0001-706X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0001-706X(03)00175-X
70
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
problems in the world and in Iran (WHO, 1995; Massoud, 1993). Epidemics of fasciolosis has caused extensive suffering and economic losses in some regions (Rokni et al., 2002). Humans are infected by ingestion of aquatic plants that contain the infected metacercariae (Markell and Voge, 1999). Diagnosis of fasciolosis is based on clinical findings and laboratory tests. The most reliable means is the finding of eggs in stool of infected individual despite the overwhelming consensus that this method is not wholly reliable (Hillyer, 1998). Using this method eggs are not detected until the latent period of infection when much of the liver damage has already occurred. In addition, eggs are released sporadically from the bile ducts and hence stool samples of infected patients can contain no eggs (Mas-Coma et al., 1998). Serological diagnosis is preferred particularly since antifasciola antibodies can be detected as early as 2 weeks post infection and can thus facilitate early chemotherapeutic intervention (Hillyer, 1998). Despite of the recent studies involving the diagnosis of fasciolosis using F. hepatica antigens (Cordova et al., 1999; O’Neill et al., 1998; Rokni et al., 2002), the effectiveness of F. gigantica somatic (S) and cysteine proteinase (CP) as antigens in ELISA have not been studied sufficiently and needs more investigations. Adult somatic extracts and excretory/secretory (ES) products of F. gigantica have been used in ELISA (Maleewong et al., 1996, 1999; Youssef and Mansour, 1991) and in Western Blott (Intapan et al., 1998) for serodiagnosis of human fasciolosis gigantica. In Iran this is the first report in this case. The purpose of this investigation was to compare aforementioned F. gigantica antigens in ELISA test for serodiagnosis of human fasciolosis.
2. Materials and methods 2.1. Sera All samples from Fasciola-infected individuals were collected during the fasciolosis outbreak in 1999 (Bahonar and Poya, 2000; Rokni et al., 2002) in the Central Health Clinic of Rasht, Gilan
province of Northern Iran. Coprological analysis for Fasciola eggs was performed on faecal samples obtained from all individuals as previously described (Katz et al., 1972). Only individuals that were coprologically positive (n/100) and showed a history of the disease were included in the present study. Seventy five sera from patients with other parasitic diseases were also collected from the different laboratories in School of Public Health, Tehran University of Medical sciences: hydatidosis (n /40), toxocariosis (n/20), amoebiosis (n/10), and malaria (n /5). Negative control sera (n/50) belonged to cases with no clinical symptoms of fasciolosis and negative as for parasitic diagnostic tests were obtained from a non-endemic area. 2.2. Preparation of antigens Adult F. gigantica were obtained from infected bovine livers collected from local abattoirs. The worms were identified as F. gigantica based on criteria described by Sahba et al., 1972. F. gigantica (S) antigen was prepared by homogenizing adult worms in 0.045 M PBS/pH 7.2 using electrical homogenizator (Edmund Buhler Co., model Homo 4/A mit uhr) followed by sonication (Tomy Seiko model UP-200P, Tokyo), and then centrifugation at 15 000 /g at 4 8C for 30 min. Afterwards the supernatant (S antigen) was subjected to a 134 ml Sephacryle S200 HR gelfiltration column, equilibrated in 0.1 M Tris / HCl, pH 7.2. One-milliliter fractions were collected and monitored by absorbance at 280 nm for protein concentration. Each fraction was also assayed for CP activity using the flurogenic substrate Z -Phe /Arg /AMC (7-amino-4-methylcomarin). The release of the fluorescent leaving group, AMC was monitored in a Perkin/Elmer Luminescence Spectrometer model LS 50, at exciter and analyzer wavelengths of 370 and 440 nm, respectively (Smith et al., 1993). Fractions containing proteolytic activity were pooled, and then were subjected to ion exchange chromatography on QAE-Sephadex A50 equilibrated in 0.1 M Tris /HCl, pH 7.2. The first obtained fraction, which had the highest proteolytic activity, concentrated and used as CP antigen.
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
Both prepared antigens were analyzed by 15% reducing SDS-PAGE (Laemmeli, 1970). The concentration of each antigen preparation was measured using Bradford method (Bradford, 1976). In order to verify the class of these antigens specific inhibitors at final concentration of phenylmethylsulfonyl fluoride 10 mM; EDTA 10 mM; E-64 5 mg/ml; leupeptin 5 mg/ml and pepstatin 1 mM were used (Dalton and Heffernan, 1989). 2.3. ELISA test The immunodiagnostic assay was performed as previously described by O’Neill et al. (1999). Briefly, after the test was optimyzed, 100 ml (S) and CP antigens (30 and 5 mg/ml, respectively) was dispensed into the wells of microtiter plates (Nuclon, Kamstrup, Roskilde, Denmark) and then incubated overnight at 37 8C. Excess binding sites were blocked with 200 ml of bovine serum albumin (2% diluted in PBS/0.1% Tween 20) and incubated for 30 min at 37 8C. After the wells were washed three times with PBS /Tween 20, 100 ml of a serum sample (diluted 1:1250) was added to each plate and incubated for 60 min at 37 8C. Following another washing step, 100 ml of peroxidase-conjugated goat anti-human IgG (diluted 1:6000) was added to each well and the plates incubated for a further 60 min at 37 8C. Following a final washing step 100 ml of O -phenylendiamine dihydrochloride substrate (all from Sigma Chemical Co., Poole, Dorset, UK) was added to each well and the reaction stopped after 5 min by adding 50 ml of 12.5% H2SO4. The optical density (OD) of the samples was measured at 492 nm using a Titerteck (Helsinki, Finland) multiscan ELISA plate reader).
71
3. Results Following analysing the first peak gained from ion-exchange chromatography, i.e. CP antigen, by SDS-PAGE, a protein of about 27/28 kDa was identified (Fig. 1). Using aforementioned specific inhibitors, the class of this antigen was detected as CP. Serum samples obtained from 100 individuals that were coprologically positive for fasciolosis and 125 cases from non-fasciola infected and control group were analyzed by ELISA for total antibody responses against F. gigantica (S) and CP antigens. The absorbance readings obtained for these antigens were plotted against each other on scattergrams, which revealed two compact clusters of individuals with low and high absorbance readings (Fig. 2). An upper limit for ELISA absorbances, which defines the seronegative and putatively uninfected individuals was determined at 3.09 S.D. from the mean of this seronegative population (0.4 and 0.35 for S and CP antigens, respectively). This cuts off 99.9% of the seronegative peak below the cut-off point. With this cut-off point only one individual in 1000 seronegative individuals will be misdiagnosed as seropositive. Therefore, absorbance readings greater than the cut-off value were considered to be seropositive for fasciolosis. Accordingly, all 100 individuals that showed clinical manifestations of fasciolosis, were also seropositive using both (S) and CP antigens
2.4. Statistical analysis We used the mean plus 3.09 S.D. OD value of the healthy group sera as the lower limit of positivity. The sensitivity, specificity, and the predictive values were calculated as previously described (Galen, 1980). All assays were tested in triplicate and repeated twice. Statistical analysis was carried out using SPSS for Windows, version 10.
Fig. 1. SDS-PAGE of purified F. gigantica CP in a 15% polyacrylamide gel stained with coomassie blue. On the left shows the protein marker.
72
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
Fig. 2. Scatter graph of the combined data for 100 Fasciolainfected and 150 non-infected cases obtained in the total IgGELISA using somatic (S) and CP antigens of F. gigantica antigens. The K-means cluster analysis using the combined data obtained for (S) and CP in the total IgG-ELISA separated the population into seronegative (indicated by closed squares) and seropositive (indicated by open squares) subpopulations. The vertical and horizontal lines in the Figure indicate the calculated cut-off points for each antigen in the total IgG-ELISA.
(Fig. 2, indicated by open squares), whereas 121 and 123 cases (regarding somatic and CP antigens, respectively) from non-fasciola group clustered together as seronegative (Fig. 2, indicated by closed squares). Therefore, the sensitivity of the test was 100% for both antigens. There was no significant difference between the different non-fasciolosis groups in the ELISA readings recorded, instead, the absorbance readings from all samples from the Fasciola seropositive individuals were significantly higher than those obtained from patients that were seronegative or were infected with other parasites (P B/ 0.001). Four individuals from the non-fasciolosis group (two with hydatidosis and two with toxocariosis) were seropositive concerning (S) antigen, while two cases (one with hydatidosis and another with toxocariasis) showed seropositivity for CP antigen (Fig. 3). Consequently the specificity of (S) and CP antigens were calculated as 96.9 and 98.4%, respectively. The positive and negative
Fig. 3. Analysis of sera from patients with various single infections by IgG-ELISA using F. gigantica somatic (S) and CP as antigens. Serum samples obtained from patients with fasciolosis (100, lanes 1), hydatidosis (40, lanes 2), toxocariasis (20, lanes 3), amoebiasis (10, lanes 4), malaria (5, lanes 5), and control human sera (50, lanes 7).
predictive values of the test regarding (S) antigen were 96 and 100%, respectively, whereas these values as for CP antigen were 98 and 100%, correspondingly. Fig. 4 shows the boxplots of obtained ELISA absorbances for two antigens from infected and uninfected groups. Boxes belong to CP antigen showed smaller measures than (S) one.
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
73
Fig. 4. Boxplots of obtained IgG-ELISA absorbances from F. gigantica (S) and CP antigens for Fasciola-infected and uninfected groups.
4. Discussion Stool examination for Fasciola eggs is a reliable method, but the sensitivity is too low (Hillyer, 1998). The infected individuals during prepatent period failed to release the eggs in the stool as well, so immunodiagnosis methods are more applicable for this purpose (Hillyer, 1998; O’Neill et al., 1999). Recently more emphasis is on the antigenic product of CP from F. hepatica (Cordova et al., 1999; O’Neill et al., 1998). Fagbemi and Hillyer (1992) have reported the purification of a CP from F. gigantica adult worms. CP is immunogenic at all stages of liver fluke development in the definitive host and can be used to diagnose both the acute and chronic stages of infections. In some previous studies the sensitivity and specificity of F. gigantica somatic antigen has been reported as 100 and 98% (Maleewong et al., 1996), 100 and 82.5% (Youssef and Mansour, 1991), respectively. Using immunoblotting technique one antigenic component of F. gigantica somatic extract, i.e. 38 kDa was found to give a consistent reaction with sera of patients with fasciolosis as 100% sensitivity and 96.7% specificity (Maleewong et al., 1997).
As is shown in Fig. 3, antibodies in the sera of four patients infected with hydatidosis (two cases) and toxocariasis (two cases) were reactive with F. gigantica (S) antigen, while pertaining to CP antigen the number of cross-reaction cases decreased to two sera infected with hydatidosis (one case) and toxocariasis (one case). This finding suggests that using of more purified antigen than (S) improves the output. There is possibility that an antigen other than the protease in the F. gigantica (S) and CP shares epitope(s) with a hydatid or toxocara immunogen. More to the point, subclinical Fasciola infection may be involved. The present report is the first to demonstrate the use of F. gigantica (S) and CP antigens for the diagnosis of fasciolosis in Iran. Up-to-date, there is no data which species of Fasciola is responsible to fasciolosis in Iran (Massoud, 1989, 1997). It is demonstrated that using of F. gigantica purified antigens could be used as antigen in diagnose of infections with F. hepatica (Weiyi et al., 1997). Our study shows higher specificity of CP than somatic antigens. We have demonstrated that altogether CP antigen provide a more conclusive
74
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75
diagnosis as possessing lower cut-off and enabling better to discriminate between seronegative and seropositive subpopulations. Fig. 4 shows the boxplot diagram of obtained OD absorbances relevant to CP and (S) antigens. The smaller box encompasses the less variability around median, hence it is concluded that CP antigen demonstrates better quality to discriminate between seropositive and seronegative populations than (S) one. Based on coprological tests, acute and chronic fasciolosis have the same diagnostic dilemma for the physician since in acute phase there is no eggs in stool and in chronic phase the sensitivity is too low. So it is essential to establish a reliable test like ELISA aiming at diagnosis and seroepidemiology of the disease. This study may be functional to implement control measures against the spread of the infection and to establish a reliable diagnostic tool.
Acknowledgements We show great appreciation to Prof. John P. Dalton and Sandra O’Neill from Dublin City University, Dublin, Ireland and Mr Ahmad Siavash poori from the Tehran University of Medical Sciences for their sincer cooperation. This study was supported by research assistance of Tehran University of Medical Sciences Grant No. 712 to Prof. Massoud and Dr Rokni.
References Bahonar, A., Poya, B., 2000. A descriptive epidemiology of patients infected with fasciolosis in Rasht and Bandar Anzali in 1999. Third National Congress of Medical Parasitology, Sari, Iran, April 9 /11, p. 67. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantitive protein utilizing the principle of protein /dye binding. Anal. Biochem. 72, 248 / 254. Cordova, M., Reategui, L., Espinoza, R., 1999. Immunodiagnosis of human fasciolosis with Fasciola hepatica cysteine proteinase. Trans. R Soc. Trop. Med. Hyg. 93, 54 /57. Dalton, J.P., Heffernan, M., 1989. Thiol protease released in vitro by Fasciola hepatica . Mol. Biochem. Parasitol. 35, 161 /166.
Fagbemi, B.O., Hillyer, G.V., 1992. The purification and characterization of a cysteine proteinase of F. gigantica adult worms. Vet. Parasitol. 43, 223 /232. Galen, R.S., 1980. Predictive values and efficiency of laboratory testing. Pediatr. Clin. North Am. 27, 861 /869. Hillyer, G.V., 1998. Immunodiagnosis of human and animal fasciolosis. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Oxon, Wallingford, UK, pp. 435 /448. Intapan, P.M., Maleewong, W., Wongkham, C., Tomanakarn, K., Ieamviteevanich, K., Piptigool, V., Sukolapong, V., 1998. Excretory-secretory antigenic components of adult Fasciola gigantica recognized by infected human sera. Southeast Asian J. Trop. Med. Publ. Health 29, 579 /583. Katz, N., Chaves, A., Pellegrin, J., 1972. A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Rev. Inst. Med. Trop. Sao Paulo 14, 397 /402. Laemmeli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 /685. Maleewong, W., Intapan, P.M., Wongkham, C., Tomanakan, K., 1996. Comparison of adult somatic and ES antigen in ELISA for the serodiagnosis of human infection with F. gigantica . Southeast Asian J. Trop. Med. Publ. Health 27, 566 /569. Maleewong, W., Intapan, P.M., Tomanakarn, B., Wongkham, C., 1997. Antigenic components of somatic extract from adult Fasciola gigantica recognized by infected human sera. Asian Pac. J. Allergy Immunol. 15, 213 /218. Maleewong, M., Wongkham, C., Intapan, P.M., Pipitgool, V., 1999. Fasciola gigantica */specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fasciolosis. Am. J. Trop. Med. Hyg. 61, 648 /651. Markell, E.K., Voge, M., 1999. Medical Parasitology, eigth ed.. Saunders Company Publication, pp. 185 /188. Mas-Coma, S., Bargues, M.D., Esteban, J.G., 1998. Human fasciolosis. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Oxon, Wallingford, UK, pp. 411 /434. Massoud, J., 1989. Fasciolosis outbreak in man and drug test (Triclabandazole) in Caspian littoral, northern part of Iran. Bull. Soc. France Parasitol. 8, 438. Massoud, J., 1993. Present status of fasciolosis in Iran. WHO Mimeogr. Rep. SCH/SG/93/WP 19. Massoud, J., 1997. The importance of helminthic diseases in Iran. Second National Congress of Parasitic Diseases in Iran. Tehran, Iran, October 19 /22. p. 74. O’Neill, S.M., Parkinson, M., Strauss, W., Angles, R., Dalton, J.P., 1998. Immunodiagnosis of Fasciola hepatica infection (fasciolosis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am. J. Trop. Med. Hyg. 58, 417 /423. O’Neill, S.M., Parkinson, M., Dowd, A.J., Strauss, W., Angles, R., Dalton, J.P., 1999. Immunodiagnosis of human fasciolosis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am. J. Trop. Med. Hyg. 60, 749 /751. Rokni, M.B., Massod, J., Parkinson, M., O’Neill, S., Dalton, J.P., 2002. Diagnosis of human fasciolosis in the Gilan
M.B. Rokni et al. / Acta Tropica 88 (2003) 69 /75 province of Northern Iran: application of cathepsin LELISA. Diagn. Microbiol. Infect. Dis. 44, 175 /179. Sahba, G.H., Arfaa, F., Farahmandian, I., Jalali, H., 1972. Animal fasciolosis in Khuzestan, southwestern Iran. J. Parasitol. 58, 712 /716. Smith, A.M., Dowd, A., McGonigle, S.M., Keegan, P., Brennan, G., Trudget, A., Dalton, J.P., 1993. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica . Mol. Biochem. Parasitol. 62, 1 /8.
75
Weiyi, H., Zhang, W., Tian, E., 1997. Analysis of F. gigantica ES antigens, vol. 16. J. Guanxi Agricultural University, pp. 115 /124. World Health Organization (WHO), 1995. Control of foodborne trematode infection. WHO Technical Report Series, WHO, Geneva, 845, pp. 1 /157. Youssef, F.G., Mansour, N.S., 1991. A purified Fasciola gigantica worm antigen for the serodiagnosis of human fasciolosis. Trans. R Soc.Trop. Med. Hyg. 85, 535 /537.