Short report: Diagnosis of human fascioliasis: detection of anti-cathepsin L antibodies in blood samples collected on filter paper.

We have developed an ELISA for the diagnosis of human fascioliasis based on the detection of IgG4 antibodies to Fasciola hepatica cathepsin LI cysteine protease. Use of this assay in the Bolivian Altiplano, a region with a high prevalence of the disease, was hampered by the reluctance of the indigenous population to provide blood. To overcome this problem, we have investigated the method of collecting small quantities of blood from the finger onto filter paper, followed by the elution of antibodies for use in the diagnostic assay. Serum samples and blood samples collected onto filter paper were obtained from 57 individuals living in the village of Cutusuma in 1987 and from 11 individuals in Chijipata in 1996. Analysis of the IgG4-ELISA results revealed that there is highly significant linear relationship (P < 0.001) between the two methods of sampling. Most importantly, a reliable diagnosis was made with the blood-filter samples from Cutusuma, which had been stored for 10 years at 40 degrees C. While some deterioration of the blood-filter samples from Cutusuma had occurred over the 10-year storage period, no deterioration occurred with the Chijipata samples, which were stored for one year. Therefore, the method of collecting blood onto filter paper should prove useful for large-scale epidemiologic studies on human fascioliasis in the Bolivian Altiplano and in other regions where this disease is prevalent.     

Evaluation of Fas2-ELISA for the serological detection of Fasciola hepatica infection in humans

The performance of Fas2-ELISA for the diagnosis of Fasciola hepatica infection in children living in areas of high endemicity for fascioliasis in the Peruvian Andes is analyzed. Fas2-ELISA is based on the detection of circulating IgG antibodies elicited in infected individuals against a F. hepatica antigen termed Fas2. The study was conducted in three Andean localities, Huertas-Julcan in Junin, Asillo in Puno, and Cajamarca, with a total population of 634 children in an age range 1 to 16 years old. Child fascioliasis prevalence was 21.1% in Huertas-Julcan, 25.4% in Asillo, and 24% in Cajamarca, estimated by coprological inspection. The seroprevalence of F. hepatica infection, determined by Fas2-ELISA, was 27.8% in Huertas-Julcan, 44.6% in Asillo, and 29.1% in Cajamarca. The overall sensitivity of Fas2-ELISA was 92.4%, the specificity 83.6%, and the negative predictive value 97.2%. No association between OD(450) Fas2-ELISA and infection intensity measured by egg counting was observed. Results show that Fas2-ELISA is a highly sensitive immunodiagnostic test for the detection of F. hepatica infection in children living in human fascioliasis endemic areas.     

Relationships between eosinophilia, anti-Fasciola IgG, and IgM rheumatoid factors, in urban and rural areas of north-western Spain

A study was carried out, among adult patients attending a hospital in Lugo, in north-western Spain, to investigate possible relationships between eosinophilia, IgG antibodies against the parasitic helminth Fasciola hepatica, and IgM rheumatoid factors (IgM-RF). Blood samples were collected from 1264 individuals and divided into three groups according to eosinophil count: normal (<0.5 x 10(9) eosinophils/litre), eosinophilic (0.5 x 10(9)-3 x 10(9)/litre) or hyper-eosinophilic (>3 x 10(9)/litre). Each sample was checked for IgG against F. hepatica, in an ELISA in which the excretory/secretory products of the trematode were used as the antigens, and for IgM-RF, in a latex agglutination test. Overall, 21% of the cases were found seropositive for fascioliasis and 15% were found to have IgM-RF. Women from rural areas not only showed the highest seroprevalence of fascioliasis but also the highest frequency of IgM-RF carriage. Men from rural areas were more likely to be eosinophilic than the other patients. Curiously, the patients found seronegative for fascioliasis were more likely (but not significantly more likely) to be eosinophilic than their seropositive counterparts, and the eosinophilic patients were significantly more likely (P=0.001) to be carrying IgM-RF auto-antibodies than the non-eosinophilic. It appears that, in the study area, those living in rural regions have a higher risk of F. hepatica infection (as indicated by IgG antibodies against the parasite) than those living in urban settings. The risk of developing eosinophilia appears to be positively associated with the presence of IgM-RF auto-antibodies.     

Detection of Fasciola hepatica infection in a community located in the Ecuadorian Andes

The presence of fascioliasis was assessed in four Andean communities using an enzyme-linked immunosorbent assay (ELISA) test to measure antibodies against Fasciola hepatica excretion-secretion antigens. Six percent (9 out of 150) of the individuals in one community were ELISA-positive for these antibodies. Fecal samples from two of the ELISA-positive individuals contained F. hepatica ova. All of the ELISA-positive cases, except for one, were children within the ages of 9 to 12 years.     

Use of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano.

A collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotransfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

肝片形吸虫感染宿主细胞免疫的研究进展

肝片形吸虫病是一种危害严重的人兽共患病,肝片形吸虫主要感染反刍动物及包括人类在内的哺乳动物,是一个严重威胁健康、影响经济发展的公共卫生问题。目前治疗肝片形吸虫病的药物少、疗效有限。大量研究表明,肝片形吸虫受宿主免疫应答的调控,本文对肝片形吸虫病的发病机制、肝片形吸虫主要参与诱导的几种机体细胞免疫反应及相关的研究进展进行综述.以期为肝片形吸虫病的临床治疗提供资料。     

Parasite-specific antibody profile in human fascioliasis: application for immunodiagnosis of infection

The antibody isotype response to an adult Fasciola worm antigen preparation (FWAP) was examined in sera from 60 Egyptians with parasitologically confirmed fascioliasis by an ELISA. The FWAP-specific IgG1 and IgG4 antibodies were found in 97-100% of the patients. The ratio of the mean absorbance values between infected patients and healthy controls was 9.7 and 29.7 for IgG1 and IgG4 antibodies, respectively. The IgM, IgA, IgG2, and IgG3 antibodies were less dominant. In contrast to IgG1 antibodies, which were often detected in sera from patients infected with Schistosoma, Echinococcus granulosus, Ascaris lumbricoides, Ancylostoma duodenale, or Hymenolepis nana, FWAP-specific IgG4 antibodies were detected exclusively in the sera of patients with fascioliasis. The data thus support the conclusion that an IgG4/ELISA with crude FWAP as antigen may be used for sensitive and accurate immunodiagnosis of human fascioliasis.     

High fascioliasis infection in children linked to a man-made irrigation zone in Peru

We detected 10 protozoan and nine helminth species in surveys of 338 5-15 year-old Quechua schoolchildren in three communities of the Asillo zone of the Puno region, located at a very high altitude of 3910 m in the Peruvian Altiplano. The area proved to be hyperendemic for human fascioliasis with a 24.3% overall mean prevalence of Fasciola hepatica, local prevalences ranging between 18.8 and 31.3%, and infection intensities of up to 2496 eggs per gram of faeces (epg), with 196-350 epg (mean: 279 epg) and 96-152 epg (123 epg) as arithmetic and geometric means, respectively. Prevalences did not significantly vary between schools and in relation to sex. No statistical differences were found in intensities between schools, nor according to sex or age groups between and within schools, although the highest overall egg counts were detected in girls and in the youngest age group. Asillo zone is a man-made irrigation area built only recently to which both liver fluke and lymnaeid snails have quickly adapted. The region appears to be isolated from the Northern Bolivian Altiplano natural endemic area. Such man-made water resources in high altitude areas of Andean countries pose a high fascioliasis risk. Significant positive association of F. hepatica with protozooses following a one host life cycle, such as Giardia intestinalis, suggests that human infection mainly occurs through drinking water. This is supported by additional evidence such as the absence of typical aquatic vegetation in the drainage channels inhabited by lymnaeid snails, the absence of aquatic vegetables in the traditional nutrition habits of the Quechua inhabitants, and the lack of potable water systems inside dwellings, which requires inhabitants to obtain water from irrigation canals and drainage channels.     

Analysis of climatic data and forecast indices for human fascioliasis at very high altitude

Human infection with Fasciola hepatica has recently been recognized as an important health problem worldwide, and particularly at very high altitudes in South America. The highest prevalences and intensities of human fascioliasis known are those of the northern Bolivian Altiplano, where infected Lymnaea truncatula occur at altitudes of 3800-4100 m. In the present study, the climatic data for this area of the Altiplano, which differ markedly from those of endemic areas in the lowlands, were analysed. There is no marked seasonality in temperature but there are large variations in temperature within a daily, 24-h period. Rainfall is seasonal, with a long dry season, coinciding with the lowest minimum temperatures, and a long wet season. The rate of evapotranspiration is very high, and temporary water bodies dry out very quickly. Solar radiation at ground level is intense, not only because of the altitude but also because of the lack of trees and shrubs. Two climatic indices for forecasting fascioliasis, Mt and Wb-bs, were calculated. Modifications in these forecast indices are proposed, to reflect the environment at high altitude and low latitude. Estimates, based on climadiagrammes, of the durations of the wet and dry seasons were greatly effected by the inclusion of an aridity-index modification. The usefulness of the modified indices was examined using prevalence data for human and cattle fascioliasis collected in the neighbourhoods of the stations providing the meteorological data. Values for both indices indicated that conditions were optimum for transmission between December and March. The results were statistically significant for the modified Wb-bs index when the data for a meteorological station in which no lymnaeids were found were excluded. The modified Mt index did not appear sufficiently accurate to be useful. The values for the modified Wb-bs index permitted the study areas to be designated low-, moderate- or high-risk areas for the transmission of fascioliasis to man and domestic animals.     

Comparison of adult somatic and cysteine proteinase antigens of Fasciola gigantica in enzyme linked immunosorbent assay for serodiagnosis of human fasciolosis

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.     

Fascioliasis imported to Norway

We report 3 cases of fascioliasis imported to Norway: a 24-y-old male Vietnamese immigrant and a 51-y-old female Vietnamese immigrant with acute fascioliasis, and a 2-y-old male Ethiopian adoptee with chronic fascioliasis. Diagnosis was confirmed by detection of specific antibodies to Fasciola hepatica in the 2 first cases and by demonstration of Fasciola hepatica eggs in stool samples in the latter case. Treatment with oral triclabendazole led to prompt cure in all 3 patients. Imported fascioliasis is rarely reported but should be suspected in patients with a travel history to endemic areas, abdominal symptoms and hypereosinophilia of the blood.     

Serum antiimmunoglobulins reactive with human and rabbit IgG in rheumatoid arthritis and other conditions

IgG, IgA, and IgM antiimmunoglobulins reactive with human and rabbit IgG were measured in patients with rheumatoid arthritis (RA), patients with rheumatic fever or osteoarthritis, and normal individuals. Values of all antiimmunoglobulins were significantly evaluated in RA patients as compared with other groups and depended upon activity and stage of the disease. IgM antibodies with specificity for human IgG predominated quantitatively over others in sera of RA patients with high titers of RF, whereas most of those reactive with rabbit IgG in latex negative or positive RA patients belonged to IgG class. The reaction with human IgG included thermostable and thermolabile IgM antiimmunoglobulins but in that with rabbit IgG only thermostable antibodies were active.     

Increased anti-Echinococcus granulosus antibody positivity in Fasciola hepatica infection

Background/aims: Parasitic helminths express some antigen, which often accounts for serological cross-reactions. The aim of this study was to determine the prevalence of antiEchinococcus granulosus antibody in patients with Fasciola hepatica infection using indirect immunofluorescence assay. Materials and Methods: The study population consisted of the following groups: Fasciola hepatica group (n=22), hydatid disease group (n=22) and healthy control group (n=24). Indirect immunofluorescence assay for Echinococcus granulosus was performed in all groups. Results: Indirect immunofluorescence assay was positive in all patients with hydatid disease, in 13 of 22 (59%) patients with fascioliasis and in 2 of 24 (8%) healthy subjects. The positivity rate of indirect immunofluorescence assay was significantly higher in the hydatid disease group compared to the fascioliasis group (p<0.001) and compared to the control group (p<0.001), and it was significantly higher in the fascioliasis group compared to the control group (p=0.001). Antibody titer was 1/100 in 7 patients, 1/320 in 12 patients and 1/1000 in 3 patients with hydatid disease. Indirect immunofluorescence assay was positive in 10 of 15 patients with hepatic phase and in 3 of 7 patients with biliary phase of fascioliasis. The antibody titer was 1/100 in 6 and 1/320 in 7 patients with fascioliasis. The antibody titer was 1/100 in both healthy subjects. Conclusions: Indirect immunofluorescence assay for Echinococcus granulosus may be positive in a majority of patients with Fasciola hepatica infection and in some healthy subjects.     

Use of a partially purified Fasciola gigantica worm antigen in the serological diagnosis of human fascioliasis in Egypt

Crude extracts of Fasciola gigantica adult worms, when used as an antigen in indirect hemagglutination (IHA) and counterimmunoelectrophoresis (CIEP) tests, detected all independently diagnosed human F. gigantica and F. hepatica infections but cross-reacted with sera of patients with schistosomiasis and amebiasis. Fractionation of this crude worm extract using Sephadex G-200 chromatography demonstrated four major protein peaks. Antigen from the crest and descending portion of peak II (mol. wt. approximately 20 x 10(3)) and all of peak III (mol. wt. approximately 6 x 10(3)) were pooled and used as a source of partially purified antigen. This partially purified fraction, when used in the CIEP test, reacted with sera from patients with fascioliasis but not those from schistosomiasis or amebiasis patients, whether undiluted or concentrated fivefold, but failed to react by IHA with fascioliasis sera. It reacted with undiluted sera from all individuals passing F. gigantica eggs except one, a possibly spurious infection, and with eight of 20 sera from individuals passing F. hepatica eggs, while the remaining 12 sera became positive after fivefold concentration. It also reacted with two sera from individuals passing eggs of both Fasciola species and with five of 11 sera from individuals negative microscopically but positive serologically with the crude antigen.     

Assessing the validity of an ELISA test for the serological diagnosis of human fascioliasis in different epidemiological situations

Objectives To improve the diagnosis of human fascioliasis caused by Fasciola hepatica and Fasciola gigantica, we evaluated the diagnostic accuracy of an enzyme‐linked immunosorbent assay (ELISA), with Fasciola antigen from the adult liver fluke, for the detection of IgG against fascioliasis in human sera. Methods The sera of 54 fascioliasis cases, originating from three endemic areas, were used in this evaluation: (i) a hyperendemic F. hepatica area where humans usually shed a great number of parasite eggs in faeces (11 sera); (ii) an epidemic F. hepatica area where humans usually shed small amounts of parasite eggs (24 sera) and (iii) an overlap area of both Fasciola species and where human shedding of parasite eggs in faeces is usually scarce or non‐existent (19 sera). One hundred and sixty‐eight patients with other parasitic infections and 89 healthy controls were also analysed. Results The respective sensitivity and specificity of this assay were 95.3% (95% confidence intervals, 82.9–99.2%) and 95.7% (95% confidence intervals, 92.3–97.5%). No correlation between egg output and the OD450 values of the F. hepatica IgG ELISA test was observed. Conclusions This test could be used both as an individual serodiagnostic test for human fascioliasis when backed up by a compatible clinical history together with a second diagnostic technique for other cross‐reactive helminth infections, and in large‐scale epidemiological studies of human fascioliasis worldwide.     

Hyperendemic fascioliasis associated with schistosomiasis in villages in the Nile Delta of Egypt

Coprologic surveys were carried out in villages of the Behera Governorate in the Nile Delta region of Egypt to characterize the epidemiologic features of human fascioliasis caused by Fasciola hepatica and F. gigantica in this lowland endemic area by comparison with fascioliasis caused by only F. hepatica in areas hyperendemic for human disease in the Andean highlands of South America. The fascioliasis prevalences detected (range = 5.2-19.0%, mean = 12.8%) are the highest obtained in Egypt. The comparison with previous results suggests that in the Nile Delta, fascioliasis is spreading from an original situation of sporadic human cases in well-known endemic foci for animal disease to an endemic distribution in humans, which may be characterized as a mesoendemic region that includes several hyperendemic areas for human disease. As in Andean countries, a relationship with sex was found, although in Egypt prevalences, but not intensities, appeared to be significantly higher in females. All ages appear to be susceptible to liver fluke infection, with prevalences and intensities being lower before and after school age, a situation that is consistent with that detected in Andean countries, although the peak in the 9-11-year-old age group appears less pronounced in Egypt. The intensities were very high when compared with those found in subjects sporadically infected in areas endemic for animal disease, but relatively low for a hyperendemic situation, although the intensities may not be conclusive because of the transmission seasonality of fascioliasis in the Nile Delta. The marked similarities in the qualitative and quantitative spectrums of protozoans and helminths, multiparasitisms, and associations between liver flukes and other parasitic species suggest physiographic-hydrographic and behavioral-social characteristics similar in all areas hyperendemic for human fascioliasis, which are independent of other factors such as climate, altitude, and cultural or religious features. The significant positive association between liver fluke infection and schistosomiasis mansoni detected in one locality has never been described elsewhere, and must be considered relevant from clinical, pathologic, diagnostic, and therapeutic points of view. Interestingly, the relationships of schistosomiasis prevalences and intensities with sex and age follow patterns similar to those found in fascioliasis.     

Protection against Fasciola hepatica in the intestine is highly correlated with eosinophil and immunoglobulin G1 responses against newly excysted juveniles

Rats were infected with Fasciola hepatica and challenged at regular intervals up to 38 weeks using an ex vivo gut loop, a technique developed in our laboratory. The kinetics of the observed immune responses against F. hepatica in gut tissue and serum were investigated and correlated to protection. Immunohistochemical methods were used to measure the frequency of eosinophils, immunoglobulin (Ig)E-positive cells, and mucosal mast cells in the gut loop, and to determine whether the newly excysted juveniles were coated with IgG antibodies or surrounded by eosinophils, or both. Enzyme-linked immunosorbent assays and a radioimmuno assay were used to measure serum antibody reactive with newly excysted juveniles. Results showed that protection was highly correlated with the frequency of eosinophils and IgE-positive cells in the gut, but was only moderately correlated with the frequency of mucosal mast cells. Newly excysted juveniles taken from rats exhibiting high levels of protection were always coated with IgG antibodies and surrounded by eosinophils. Protection was highly correlated with titers of serum IgG1 antibodies directed against newly excysted juveniles, but was only weakly correlated with titers of serum IgA and IgE antibodies. Because protection was highly correlated with IgG1 in gut tissue and serum, and with eosinophils in gut tissue, we suggest that IgG1 and eosinophils are important in protecting rats against F. hepatica.     

Identification of Trichomonas vaginalis alpha-actinin as the most common immunogen recognized by sera of women exposed to the parasite.

A study on presence of antibodies to Trichomonis vaginalis in serum was done on a group of 500 pregnant, asymptomatic Angolan women. A serologic screening, done by ELISA, revealed that 41% of the women had IgG and IgM against the parasite. Analysis of sera by immunoblotting revealed that 94.4% of sera with anti-T. vaginalis IgG class antibodies were reactive against a common immunogenic protein of 115 kDa. The common immunogen was identified as the protozoan alpha-actinin. All sera recognizing the 115-kDa antigen were reactive against both native and recombinant T. vaginalis alpha-actinin and nonreactive against human alpha-actinin. The findings presented in this work offer a new tool for epidemiologic studies and open new perspectives for vaccination.     

Asymptomatic fascioliasis

A 72-year-old Japanese man displayed asymptomatic eosinophilia for 4 months. Computed tomography showed multiple space-occupying lesions in the liver. Zoonotic liver flukes were suspected based on occupational exposure to cattle, serological and radiological findings. Immunological examination was helpful in diagnosing the disease and laparoscopy was crucial in confirming Fasciola hepatica and excluding the possibility of malignant hepatic tumors. Human fascioliasis was finally diagnosed and praziquantel administered. Blood eosinophilia resolved within 4 months and liver tumors almost disappeared within 12 months. From our experience, laparoscopy with liver biopsy is very important for diagnosing human fascioliasis, particularly for asymptomatic fascioliasis.     

Fasciola hepatica infection: clinical and computerized tomographic findings of ten patients.

BACKGROUND/AIMS: Fasciola hepatica is the cause of liver infection, fascioliasis. Although rare, it is still a problem even in developed countries. In this study, the clinical and computerized tomographic findings of 10 patients diagnosed with fascioliasis are summarized. METHODS: The medical records of the patients with fascioliasis were retrospectively examined. Clinical, laboratory findings and computerized tomographic results were recorded. RESULTS: Abdominal pain, fever, eosinophilia and abnormal liver function tests were the most commonly encountered symptoms and signs. One patient was human immunodeficiency virus -positive with active tuberculosis. Serologic test for fasciola hepatica was positive in all patients. Nodular masses without prominent enhancement, and branching low-attenuated tubular lesions were the most commonly seen tomographic findings and were supportive for the diagnosis. All except the HIV-positive patient received bithionol therapy; six patients responded well, two lost contact with the clinic and one patient who was unresponsive to bithionol therapy received triclabendazole. During follow-up of the six patients who responded, all the clinical and radiological findings regressed. CONCLUSION: In any patient with peripheral eosinophilia, abdominal pain and elevated liver enzymes, especially when CT reveals tubular and nodular hypodense lesions particularly in subcapsular area, F. hepatica infection should be considered. Either triclabendazole or bithionol can be used effectively for the treatment.