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Assessment of a 27-kDa antigen in Enzyme-Linked Immunosorbent Assay for the diagnosis of fasciolosis in Vietnamese patients

2010; Wiley; Linguagem: Inglês

10.1111/j.1365-3156.2010.02468.x

1365-3156

Thanh Giang Thi Nguyen, Thanh Hoa Le, Nguyen Van De, Ha Thi-Ngoc Doan, Thi Ha Thanh Dao, Jozef Vercruysse, Pierre Dorny,

Parasite Biology and Host Interactions

Fasciolosis has emerged as an important zoonotic disease in many parts of the world. In recent years, an increasing number of human cases were reported in Vietnam. In this study, the 27-kDa component protein from the excretory/secretory production of adult Fasciola gigantica, purified by high performance liquid chromatography, was assessed in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola spp. for diagnosis of human fasciolosis. The ELISA showed a high sensitivity (100%) and specificity (97.67%) when tested on patients with fasciolosis, other parasitic infections, cholangiocarcinoma and on healthy controls. The assay was applied for diagnosis on 143 patients in the Viet Duc-Hanoi hospital who presented with clinical signs of liver disease and lesions in their livers as shown by imaging techniques. Antibodies were found in 37 (25.9%) of these patients, of whom only 3 shed Fasciola eggs in their stools (2.1%). The excellent response to triclabendazole treatment of 37 sero-positive patients confirmed the diagnosis of fasciolosis. This study demonstrated the diagnostic potential for human fasciolosis of the 27-kDa antigen ELISA. Fasciolosis should be considered in the differential diagnosis of hepatic disease in Vietnam. Évaluation d'un antigène de 27 kDa dans un test ELISA pour le diagnostic de la fasciolose chez des patients vietnamiens La fasciolose a émergé comme une zoonose importante dans de nombreuses régions du monde. Ces dernières années, un nombre croissant de cas humains ont été signalés au Vietnam. Dans cette étude, la composante protéine de 27 kDa du système de production excrétion/sécrétion des adultes de Fasciola gigantica, purifiée par chromatographie liquide haute performance, a étéévaluée dans un test ELISA afin de détecter les anticorps contre Fasciola spp. dans le diagnostic de la fasciolose humaine. Le test ELISA a montré une sensibilité (100%) et spécificité (97.67%) élevées lorsqu'il est testé sur des patients présentant la fasciolose, d'autres infections parasitaires, le cholangiocarcinome et sur des contrôles sains. Le test a été appliqué pour le diagnostic de 143 patients à l'hôpital de Viet Duc-Hanoi qui présentait des signes cliniques de maladie du foie et des lésions dans le foie tel que le montraient les techniques d'imagerie. Des anticorps ont été retrouvés chez 37 (25,9%) de ces patients, dont seuls 3 libéraient des oeufs de Fasciola dans leurs selles (2,1%). L'excellente réponse au traitement avec le triclabendazole de 37 patients séro-positifs a confirmé le diagnostic de la fasciolose. Cette étude a démontré le potentiel de diagnostic de la fasciolose humaine par ELISA sur l'antigène de 27 kDa. La fasciolose devrait être prise en compte dans le diagnostic différentiel de la maladie hépatique au Vietnam. Evaluación de un antígeno de 27 kDa en un ensayo de ELISA para el diagnóstico de la fasciolosis en pacientes vietnamitas. La fasciolosis ha emergido como una importante enfermedad zoonótica en muchos lugares del mundo. En los últimos años ha aumentado el número de casos humanos reportados en Vietnam. En este estudio, la proteína de 27 kDa de la producción excretora/secretora de la Fasciola gigantita adulta, purificada por Cromatografía de Alto Rendimiento (HPLC) fue evaluada mediante inmunoensayo enzimático (ELISA) para detectar anticuerpos contra Fasciola spp. para el diagnóstico de fasciolosis humana. El ELISA demostró una alta sensibilidad (100%) y especificidad (97.67%) cuando se probó con muestras de pacientes con fasciolosis, otras infecciones parasitarias, colangiocarcinoma y sobre controles sanos. El ensayo se aplicó para el diagnóstico de 143 pacientes en el hospital Viet Duc-Hanoi que se presentaban con signos clínicos de enfermedad hepática y lesiones en sus hígados, visibles mediante técnicas de imagen. Se encontraron anticuerpos en 37 (25.9%) de estos pacientes, de los cuales solo 3 excretaron huevos de Fasciola en sus heces (2.1%). La excelente respuesta al tratamiento con triclabendazol en 37 pacientes seropositivos confirmó el diagnóstico de fasciolosis. Este estudio demostró el potencial del ELISA con el antígeno de 27 kDa para realizar el diagnóstico de la fasciolosis humana. La fasciolosis debería ser considerada dentro del diagnóstico diferencial de enfermedades hepáticas en Vietnam. Fasciolosis is a parasitic infection of great concern, not only because of its high prevalence and economic significance to the animal stock (Schweizer et al. 2005; Mungube et al. 2006), but also because it appears to be an emerging public health problem in many regions of the world (Mas-Coma et al. 1999a). It is estimated that 2.4 million people are infected worldwide and that the number of people at risk is more than 180 million (WHO 1995). Humans get infected by eating aquatic vegetables contaminated with metacercariae. Fasciolosis is an example of an emerging/re-emerging parasitic disease in many countries as a consequence of environmental changes as well as man-made modifications (Mas-Coma et al. 2005). Fasciolosis has a worldwide distribution, but while Fasciola hepatica is predominating in temperate zones, Fasciola gigantica is found mainly in tropical and subtropical regions of Asia and Africa (Andrews 1999). In eastern Asia, human cases have been reported rather sporadically in Korea (Lee & Kim 2006), Japan (Adachi et al. 2005) and China (Ying et al. 2007) and more commonly in the northern part of Thailand (Tesana et al. 1989; Wongkham et al. 2005). In Vietnam, few human cases were reported before the 1990s; however, in the last decade, a marked increase was observed, with over 500 cases during 1997–2000 (De et al. 2003) and 4585 patients in the period 2000–2006 (De et al. 2006). These figures are probably an underestimation because many cases are not registered, diagnostic methods are not available and doctors in the local hospitals have little experience with diagnosis of this parasitic infection. Almost all Vietnamese cases have been ascribed to F. gigantica (De et al. 2003). In endemic areas, clinical examination and imaging techniques are used for diagnosis of fasciolosis, but these methods cannot always differentiate between fasciolosis and other causes of hepatitis (Marcos et al. 2008). Therefore, coprological and immunological methods have to be used to confirm suspected cases of fasciolosis. However, diagnosis by coprological examination is complicated by the long prepatent period (3–4 months) of Fasciola spp., during which no eggs are shed, and by the fact that in humans not all flukes develop to adult egg-laying worms or flukes migrate to non-hepatic areas (Mas-Coma et al. 1999b). Indeed, in only 42 of 249 Vietnamese fasciolosis patients (16.9%) eggs were found in the stools (De et al. 2005). Immunological techniques offer an interesting option for diagnosis (Espino et al. 1998; Almazán et al. 2001; Dixit et al. 2002). Among other tests, enzyme-linked immunosorbent assays (ELISA) have been developed using excretory-secretory (E/S) antigens from adult flukes, purified antigens or recombinant antigens (Maleewong et al. 1996; Dalimi et al. 2004; Silva et al. 2004). An ELISA using the 27-kDa native protein isolated from E/S products of Fasciola spp. has shown high sensitivity and specificity in the diagnosis of human fasciolosis (Maleewong et al. 1996, 1999). The objectives of the present study were, first, to confirm the performance of the 27-kDa ELISA in Vietnamese patients presenting with fasciolosis and other parasitic and hepatic diseases, and second, to assess the potential of this assay as a practical tool for diagnosis of fasciolosis in Vietnamese patients presenting with hepatic disease. Excretory-secretory antigens (E/S) were produced from F. gigantica adult parasites collected from bovine livers at the slaughterhouse of Hanoi, following the protocol described by Mezo et al. (2003). In brief, after washing the adult flukes in sterile saline and in enriched Roswell Park Memorial Institute 1640 (RPMI–1640) cell culture medium, containing penicillin and streptomycin, 50 worms were incubated at 38 °C under 5% CO2 for 12 h in 150 ml RPMI 1640 medium. Next, a cocktail of protease inhibitors (1 mm n-ethylmaleimide, 1 mm phenylmethylsulfonylfluoride, 1 mm Ethylenediaminetetraacetic acid, 0.001 mm pepstatin A) (Sigma) was added to the medium containing the E/S antigens, and this solution was centrifuged at 10 000 g for 20 min at 4 °C. The supernatant was collected and dialyzed in phosphate-buffered saline (PBS) for 24 h and sterilized by filtration using a 0.45-μm-pore filter disc. Crude E/S antigen was purified by high performance liquid chromatography (HPLC): 0.5 ml E/S antigen (protein concentration 3.5 mg/ml) were loaded onto a Superdex 75 HR 10/30 column (Amersham Biosciences), equilibrated with PBS solution as elution buffer at a flow rate of 0.3 ml/min. The effluent was monitored for protein concentration at 280 nm and peak fractions were collected and concentrated by using an Amicon 8050 ultrafiltration cell (Amicon, Inc., Massachusetts, USA), equipped with a YM10 membrane (10 000 kDa cut-off). Protein concentration was determined by the method of Bradford (1976). Purification of the E/S fractions was confirmed by polyacrylamide gel electrophoresis (SDS–PAGE). The ELISA was performed on microtiter plates (Nunc Immuno plate Maxisorp surface, NUNC, Roskilde, Denmark) with purified E/S antigen of F. giantica at a concentration of 0.5 μg/ml in a carbonate coating buffer (0.05 m, pH 9.6) and incubated at 4 °C overnight. Next, plates were washed three times in PBS with 0.05% Tween 20 (PBS-T) and non-specific binding sites were blocked by addition of 200 μl of 3% skimmed milk in PBS-T for 1 h at 37 °C. After washing three times with PBS-T, 100 μl serum at a dilution of 1:400 in PBS-T were added to each well (duplicate wells/serum dilution). After incubation for 1 h at 37 °C, the plates were washed four times in PBS-T. Then, 100 μl of anti human IgG conjugate HRPO (Sigma), diluted 1:80 000 in PBS were added to each well, and then plates were incubated for 1 h at 37 °C. After washing 5 times in PBS-T, 100 μl of the substrate solution TMB (3.3′, 5.5′-tetramethylbenzidine) and H2O2 30% were added to each well and incubated for 10 min at room temperature, which was stopped by adding 50 μl of 2 m H2SO4. The optical density (OD) was measured at 450 nm in a microplate reader (Bio-Rad). To evaluate the sensitivity and potential cross-reactivity of the ELISA, serum samples were collected at the clinic of the National Institute of Malariology, Parasitology and Entomology (NIMPE) from patients who underwent clinical, coprological and imaging (ultrasound and/or CT scans) examinations. Four groups of patients were available: Group 1 (n = 50) were individuals with clinical fasciolosis; Group 2 (n = 103) were patients with other parasitic infections i.e. paragonimosis (n = 5), clonorchiosis (n = 30), taeniosis/cysticercosis (n = 16), intestinal nematode infections, including ascariosis, trichuriosis and hookworm infections (n = 52); Group 3 were patients with cholangiocarcinoma by clinical signs and imaging (CT scan and magnetic resonant cholangiopancreatography (MRCP)) (n = 34); and Group 4 were healthy adults without parasitic infection by stool examination and clinical signs (n = 51). The 27-kDa ELISA was applied on all serum groups. The mean optical density (OD) of the negative controls (Group 4) plus three standard deviations was taken as the cut-off value. Based on the OD from serum groups, the diagnostic ability of the ELISA test was determined by using receiver operating characteristic (ROC) analysis using the GraghPad Prism 5.0 programme. Patients from different localities in Northern Vietnam, who registered for examination at the 'Liver and gall bladder surgical department' of the Viet Duc hospital in Hanoi, North Vietnam between 2006–07, with clinical symptoms such as, fever, pain in the right upper quadrant of the abdomen, indigestion, weight loss and pruritis, and on which lesions or tumours were detected in the hepatic area by non-invasive imaging examination, ultrasound, computer tomography (CT) or magnetic resonant cholangiopancreatography (MRCP) were included in this study. Most of these patients were referred to the Viet Duc hospital by local hospitals. Following the advice of the physician to eliminate parasitic infection as the cause of disease, the patients agreed to send their serum and stool samples to the Laboratory of Parasitology at the NIVR for serological and coprological diagnosis of fasciolosis. The Kato–Katz technique (Katz et al. 1972) was applied for detection of Fasciola eggs in the stool samples and antibodies to Fasciola were detected by ELISA using the 27-kDa antigen. A short questionnaire was given to each patient for obtaining personal information, the history of hepatic disease, the habit of eating fresh aquatic vegetables and knowledge of hepatic disease caused by parasites. Based on the results of the different diagnostic techniques, the patients were given appropriate treatment as decided by the physician. The patients in whom diagnosis of fasciolosis was made were monitored in the hospital for 1 week following treatment with triclabendazole. This study was approved by the Hanoi Medical University Ethical Committee, Ministry of Health, Vietnam. One ml of crude E/S antigen at a 5 mg/ml concentration was loaded onto a Superdex 75 HR 10/30 column (Amersham Biosciences). Two major peaks were obtained in HPLC on crude F. gigantica E/S antigen. Peak 2 (P2), containing the major component proteins of approximately 27 kDa, as shown on a 12% acrylamide separating gel, using SDS–PAGE analysis and Coomassie brilliant blue staining, was collected and used as the antigen in the ELISA. The ELISA procedure was obtained after optimizing the dilutions of coating antigen, serum samples, and conjugate to 0.5 μg/ml, 1/400 and 1/80 000, respectively. The cut-off was set at OD 0.35, as determined from the mean OD value of 51 negative samples (Group 4) plus three standard deviations. On average, the mean OD of samples in Group 1 was twice that of samples in Groups 2, 3 and 4 (Figure 1). One serum sample of a patient with taenosis/cysticercosis and two serum samples from Clonorchis-infected patients had a OD higher than the calculated cut-off value. The diagnostic sensitivity and specificity of purified antigen were 100% and 97.7% (P = 0.05), respectively. The positive and negative predictive values were 94.3% and 100%, respectively. Evaluation of the cross-reactivity of the P2 fraction of Fasciola gigantica excretory/secretory antigen, collected by HPLC, in ELISA, on serum samples of humans infected with homologous and heterologous parasitic infections, cholangiocarcinoma patients and healthy individuals: Fas = Fasciolosis (n = 50); Cyst = Taeniosis and cysticercosis (n = 16); Clo = Clonorchiosis (n = 30) ; Par = Paragonimosis (n = 5); Nem = nematode infections* (n = 52); Cho = Cholangiocarcinoma (n = 34); Non = Healthy controls (n = 51). *Nematode infections are caused by Ascaris lumbricoides, Trichuris trichiura and hookworms. A total of 143 hepatic patients, from different provinces in Northern Vietnam, were included in the study. All these patients presented clinical symptoms of hepatic disease as well as lesions in the liver as shown by diagnostic imaging methods. The age of these patients ranged between 17 and 74 years, with a peak between 40 and 60 years. In 37 (25.9%) of these patients, serum antibodies against Fasciola spp., as shown by the 27-kDa ELISA, were observed. Among these patients were 22 women and 15 men; 17 patients were between 40 and 60 years old. In the 106 seronegative patients, other liver diseases, including, hepatic cancer, viral hepatitis, toxocarosis, pyogenic or amoebic liver abscesses, were diagnosed. Appropriate treatment was given to these patients upon the physician's decision. Coprological examination revealed the presence of Fasciola eggs in three stool samples (2.1%) from patients who were also positive in ELISA. Nematode eggs were detected in about one-third of all hepatic patients. Data obtained from the questionnaires showed that eating of fresh vegetables was very common in most of the patients. None of them had any knowledge on the risks associated with eating raw vegetables and on fasciolosis. Thirty-seven seropositive patients were given anthelmintic treatment (Egaten@– triclabendazole) at a single oral dose of 10 mg/kg. Upon the physician's decision, the same treatment was repeated 24 h later in 15 patients presenting with severe clinical signs and lesions in their livers. The clinical signs resolved within 1 week post treatment in all patients given triclabendazole treatment, confirming the diagnosis of fasciolosis. Human fasciolosis has emerged as an important zoonosis in Vietnam in the last decade. The availability of an effective immunological method for confirmation of clinical and imaging diagnosis would improve the diagnostic accuracy and help assessing the epidemiological situation in the country. In this study, the 27-kDa ELISA showed to be a promising test for diagnosis of fasciolosis in Vietnamese patients presenting with hepatic disease. When fixing the cut-off value in the ELISA at OD 0.35, all serum samples from Fasciola-infected patients were positive, and only three serum samples from heterologous parasitic infections gave a false positive result. An apparent sensitivity and specificity of 100% and 97.6 7%, respectively, confirms the diagnostic potential of the assay, but these excellent performances should be confirmed by applying the test on a larger number of documented samples. Maleewong et al. (1999), in Thailand observed relatively high cross-reactivity with the 27-kDa protein ELISA in 7/27 (25.9%) serum samples from cholangiocarcinoma patients. According to those authors, those patients might have been infected with Opisthorchis viverrini, a liver fluke that has been associated with cholangiocarcinoma development (Sripa et al. 2007). Opisthorchis viverrini is closely related to Clonorchis sinensis, a liver fluke species that occurs in Northern Vietnam. In our study, 2 of the 3 serum samples that gave a false positive result were from C. sinensis infections, confirming that cross reactions with this parasite should be taken into account. However, in contrast to fasciolosis, clinical manifestations of clonorchiosis and opisthorchiosis occur mainly in the chronic phase of the infection and are usually associated with faecal egg excretion. On sonography, clonorchiosis and opisthorchiosis are characterized by enlargement of the gall bladder (Sripa et al. 2007), and diffuse, mild, uniform dilatation of the small intra-hepatic bile ducts with no or minimal dilatation of larger bile ducts and without an obstructing lesion (Choi et al. 2004). The hospital-based study in Viet Duc, Hanoi demonstrated the diagnostic potential of the 27-kDa ELISA in hepatic patients. On a total of 143 patients presenting with symptoms of hepatic disease and examined by clinical examination by experienced physicians, biochemical tests and imaging methods, Fasciola spp. antibodies were detected in 37 (25.9%) patients. Triclabendazole is the drug of choice for treatment of Fasciola spp., with high efficacy against both adult and immature flukes (De et al. 2005). It has no therapeutic effects against other hepatic diseases, including other trematode infections such as clonorchiosis and opisthorchiosis (Graham et al. 2001; Marcos et al. 2008). Therefore, the quick response to triclabendazole treatment of the seropositive patients can be considered as a confirmation of the diagnosis of fasciolosis. Eating fresh aquatic plants is a deeply rooted feature of Vietnamese cuisine, and it is considered an important risk factor for contracting fasciolosis. The patients presenting at the hepatic diseases unit in Viet Duc hospital originated from various provinces of North Vietnam. From the dataset it was not possible to classify the rate of human Fasciola spp. infection according to urban or rural origin. However, the data did show a concentration of cases in the 20- to 60-year age group (24/37). This finding agrees with a previous hospital-based study in IMPE Quy Nhon and NIMPE, where 197 out of 249 (79.1%) fasciolosis patients were between 20 and 60 years old (De et al. 2005). The results from 31 surveys performed in 24 localities in Bolivia, a country where zoonotic fasciolosis is very well established, show that the highest prevalence, estimated by coprological examination, was found in the age class <20 years (concentration at school age). The overall prevalence in that study was 15.4% (Esteban et al. 1999). Community-based studies should be initiated in endemic areas of Vietnam to investigate the effect of age on infection. Such studies will help understanding transmission dynamics of fasciolosis in this country. In conclusion, this study showed that fasciolosis must be considered as an important cause of hepatic disease in Vietnamese patients. The 27-kDa ELISA proved to be a sensitive and fairly specific tool for individual diagnosis of fasciolosis in patients presenting with hepatic disease. Studies on prevalence and risk factors should be undertaken in the country to document the extent of the problem and to develop an appropriate control programme. This study was financially supported by the Flemish Inter-University Council (VLIR) PhD programme, Belgium. The authors wish to thank the staff of the Parasitology Department of NIVR, the clinical Department of NIMPE, Viet Duc-Hanoi hospital, the Department of Animal Health of ITM and the Laboratory Department of Parasitology, Faculty of Veterinary Medicine, Ghent University, for technical help.