Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.

Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.

Clonorchis sinensis and Opisthorchis spp. in Vietnam: current status and prospects.

Clonorchis sinensis and Opisthorchis viverrini are clinically important small liver flukes because of their known association with development of cholangiocarcinoma. In Vietnam, high prevalence of C. sinensis infection in humans was previously reported in northern provinces, and O. viverrini infection has been detected in several central provinces. However, diagnosis of C. sinensis and O. viverrini infections in the past was merely based on faecal egg examination. This method alone can lead to misidentification at the species level because of morphological similarity between the eggs of these liver flukes and minute intestinal trematodes of the family Heterophyidae. In fact, recent surveys in Vietnam revealed that infection with several minute intestinal flukes, such as Haplorchis pumilio and H. taichui, are much more common than infection with C. sinensis or O. viverrini, and they often co-infect humans. Thus, previously reported prevalence of small liver fluke infection in Vietnam was likely over-estimated due to mis identification of parasites in copro-parasitological examinations. In addition, there is some confusion about identification of cercariae, metacercariae and also adults of C. sinensis and O. viverrini in intermediate and definitive hosts. The aim of this review is, therefore, to draw realistic pictures of the past and present scientific reports on the epidemiology and biology of C. sinensis and Opisthorchis spp. infection in Vietnam.

Bile canalicular changes and defective bile secretion in Opisthorchis viverrini-infected hamsters.

Infection with the liver fluke Opisthorchis viverrini (Digenea) (Poirier, 1886) causes bile duct injury and periductal fibrosis by chronic overproduction of inflammatory-mediators and eventually results in cholangiocarcinoma development. While extensive research works have been done on O. viverrini infection-associated changes of bile ducts and periductal fibrosis, little attention was paid on morphological and biochemical changes of the bile canaliculi (BC), the origin of bile flow. We aimed to investigate the morphological and functional alterations of BC in the liver of hamsters infected with O. viverrini at one and three months post-infection. Ultrastructural changes of BC showed dilatation of BC and significant reduction of the density of microvilli as early as at one month post-infection. Immunohistochemistry revealed that CD10, a BC marker, expression was reduced early as one month post-infection. The mRNA expression of the genes encoding molecules related to bile secretion including bile acid uptake transporters (slc10a1 and slco1a1), bile acid dependent (abcb11) and independent (abcc2) bile flow and bile acid biosynthesis (cyp7a1 and cyp27a1) were significantly decreased at one month post-infection in association with the reduction of bile volume. In contrast, the expression of the mRNA of bile acid regulatory genes (fxr and shp-1) was significantly increased. These changes essentially persisted up to three months post-infection. In conclusion, O. viverrini infection induces morphological and functional changes of BC in association with the decrease of bile volume.

Fatty acid compositions of Taenia solium metacestode and its surrounding tissues.

Fatty acids (FAs) are the main energy sources of living organisms and are the major components of cellular and organelle membranes. Their compositions also affect the flexibility/rigidity of cells and cell vitality. The Taenia solium metacestode (TsM) causes neurocysticercosis (NC), which is one of the most common helminthic infections of the central nerve system. We investigated the FA composition of the cyst fluid (CF) and parenchyma of the TsM, together with those of the granuloma and swine tissue surrounding the granuloma. The FA fractions of the TsM CF and swine tissue showed a composition and proportional contents comparable to each other, in which C18:0 (stearic acid), C18:1n9c (oleic acid), C20:4 (arachidonic acid) and C16:0 (palmitic acid) constituted the major fractions. However, the relative amount of individual FAs of the TsM parenchyma and granuloma differed from those of TsM CF and swine tissue, which contained enriched C16:0 and a lower amount of C20:4. Saturated FAs were the major constituents in parenchyma and granuloma, 50.4% and 46.1%, respectively. Conversely, monounsaturated FAs were the major constituents of CF and swine tissue, 38.7% and 40.3%, respectively. Our results strongly suggest that host-derived FAs might translocate across the parasite syncytial membrane and be stored in the CF.

Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

BACKGROUND: Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic ß-barrel composed of 10 anti-parallel ß-strands and two α-helices. TsMFABP2 harbored two additional loops between ß-strands two and three, and ß-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol. CONCLUSIONS/SIGNIFICANCE: The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.

The current status of opisthorchiasis and clonorchiasis in the Mekong Basin.

This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the "96 Years of Opisthorchiasis. International Congress of Liver Flukes". It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor.

Molecular identification of a causative parasite species using formalin-fixed paraffin embedded (FFPE) tissues of a complicated human pulmonary sparganosis case without decisive clinical diagnosis.

PCR-based molecular diagnosis was made for the identification of causative agents of the clinically suspected pulmonary proliferative sparganosis case found in Thailand using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. As a reference, FFPE biopsy specimen from a typical cutaneous sparganosis case was examined together. DNA samples were extracted from tissues and two partial fragments of cytochrome c oxidase subunit 1 (cox1) gene were amplified for the detection of Spirometra DNA. Two cox1 fragments were amplified successfully for both specimens. After alignment of nucleotide sequences of the PCR-amplicons, the causative agents of both cases were identified as Spirometra erinaceieuropaei.

A novel sigma-like glutathione transferase of Taenia solium metacestode.

GSTs are a group of multifunctional enzymes, whose major functions involve catalysis of conjugation of glutathione thiolate anion with a multitude of bi-substrates or transportation of a range of hydrophobic ligands. Helminth GSTs are intimately involved in the scavenging of endogenously/exogenously-derived toxic compounds and xenobiotics. In this study, we identified a novel GST gene of Taenia solium metacestodes (TsMs), which is a causative agent of neurocysticercosis. The 804 bp-long cDNA encoded a 639 bp open reading frame (212 amino acid polypeptide), which exhibited the structural motif and domain organisation characteristic of GST. It formed a strong clade with trematode and insect sigmaGSTs. We designated this cDNA as TsM sigma-like GST (TsMsigmaGST). Native TsMsigmaGST identified through gel filtration combined with compatible immunoproteomics consisted of four isoforms at approximately 25 kDa with different pIs between 8.2 and 8.7. TsMsigmaGST showed an enzyme activity as a homodimer and was specifically expressed in the scolex cytosol. The recombinant TsMsigmaGST expressed in Escherichia coli showed sigma-like activity with 1-chloro-2,4-dinitrobenzene (CDNB). The Vmax and Km for CDNB and glutathione (GSH) were 1.08 and 0.78 micromol/min/mg, and 0.16 and 0.17 mM, respectively. Its optimal activity was observed at pH 8.0 and at 40 degrees C. The enzyme activity was potently inhibited by bromosulfophthalein, and to a lesser extent by rose bengal and triphenyltin chloride. Albendazole and praziquantel non-competitively inhibited both G- and H-sites of the enzyme. To our knowledge this is the first description of the sigma-class GST in cestode parasites. The enzyme might be involved in scavenging of intracellularly generated xenobiotics during homeostatic processes and anthelminthic metabolisms. Revelation of biochemical and biological properties of TsMsigmaGST might allow us to understand pathobiological events inherent to this long-standing parasitic disease, and thus to target therapeutic intervention.

Fulminant eosinophilic myocarditis associated with visceral larva migrans caused by Toxocara canis infection.

A 19-year-old man was transferred to hospital because of myocarditis with cardiogenic shock. Echocardiography showed a left ventricular ejection fraction of 23.8% and an intermediate amount of pericardial effusion. The patient immediately received an intra-aortic balloon pump and percutaneous cardiopulmonary support. Right ventricular endomyocardial biopsy was performed in the acute phase and showed extensive eosinophilic inflammatory cell infiltration, severe interstitial edema and moderate myocardial necrosis. High-dose corticosteroids were administered. Because the patient's antibody titer against Toxocara canis was high and his symptoms had appeared after eating raw deer meat, the diagnosis was fulminant eosinophilic myocarditis caused by a hypersensitivity reaction to visceral larval migrans. After starting high-dose corticosteroids, the ejection fraction dramatically improved, the eosinophilia decreased and the patient made a full recovery.

Infection of a group of boar-hunting dogs with Paragonimus westermani in Miyazaki Prefecture, Japan.

A 5-year-old male mixed-bred boar-hunting dog with a Plott hound background weighing 23 kg was brought to the Veterinary Teaching Hospital, University of Miyazaki, in October 2002. The dog was diagnosed with active infection with the lung fluke Paragonimus westermani by serological testing and also by detection of parasite eggs in his feces. Subsequent examination of four other dogs working with this dog as a boar-hunting team revealed that all five dogs were infected with P. westermani.

Description of a new lung fluke species, Paragonimus vietnamensis sp. nov. (Trematoda, Paragonimidae), found in northern Vietnam.

Based on morphology of metacercariae and adult worms together with molecular data from our previous study, we describe herein a new lung fluke species named Paragonimus vietnamensis sp. nov. Metacercariae of this new species is round and extremely large in size (nearly 800 mum in diameter) having a fragile outer cyst of variable thickness and a thin fragile inner cyst. There are little or no space between metacercaria and the inner cyst wall. These and other morphological features of metacercariae of P. vietnamensis sp. nov. are not completely identical with but have some similarities with those of P. microrchis, P. proliferus, or P. menglaensis. On the other hand, adult worms obtained by experimental infection of these large metacercariae are oval in shape, having a ventral sucker slightly larger than the oral one, and having singly arranged relatively short cuticular spines. These morphological features of adult worms are partly similar to but not identical with those of P. microrchis, P. skrjabini, P. yunnanenis, P. xiangshanensis, or P. harinasutai. Taken these morphological data and our previous molecular phylogenetic analyses of ITS2 and CO1 sequences of metacercariae and adult worms together, P. vietnamensis sp. nov. is different from any other known Paragonimus spp.

Intestinal intraepithelial lymphocytes sustain the epithelial barrier function against Eimeria vermiformis infection.

Eimeria spp. are intracellular protozoa that infect intestinal epithelia of most vertebrates, causing coccidiosis. Intestinal intraepithelial lymphocytes (IEL) that reside at the basolateral site of epithelial cells (EC) have immunoregulatory and immunoprotective roles against Eimeria spp. infection. However, it remains unknown how IEL are involved in the regulation of epithelial barrier during Eimeria sp. infection. Here, we demonstrated two distinct roles of IEL against infection with Eimeria vermiformis, a murine pathogen: production of cytokines to induce protective immunity and expression of junctional molecules to preserve epithelial barrier. The number of IEL markedly increased when oocyst production reached a peak. During infection, IEL increased production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and decreased transforming growth factor beta (TGF-beta) production. Addition of IFN-gamma and TNF-alpha or supernatants obtained from cultured IEL from E. vermiformis-infected mice reduced transepithelial electrical resistance (TER) in a confluent CMT93 cell monolayer, a murine intestine-derived epithelial line, but antibodies against these cytokines suppressed the decline of TER. Moreover, TGF-beta attenuated the damage of epithelial monolayer and changes in TER caused by IFN-gamma and TNF-alpha. The expression of junctional molecules by EC was decreased when IEL produced a high level of IFN-gamma and TNF-alpha and a low level of TGF-beta in E. vermiformis-infected mice. Interestingly, IEL constantly expressed junctional molecules and a coculture of EC with IEL increased TER. These results suggest that IEL play important multifunctional roles not only in protection of the epithelium against E. vermiformis-induced change by cytokine production but also in direct interaction with the epithelial barrier when intra-EC junctions are down-regulated.

Mucosal mast cell-derived chondroitin sulphate levels in and worm expulsion from FcRgamma-knockout mice following oral challenge with Strongyloides venezuelensis.

Mucosal mast cell-derived chondroitin sulphates (sulphated proteoglycans) were assayed in gut washings and homogenate of FcRgamma-knockout (KO) and wild-type (WT) C57BL/6 mice challenged with Strongyloides venezuelensis in order to assess their possible role in secondary immunity against enteric nematodes. Groups of immune KO and WT mice were challenged by oral gavage with 300 infective larvae (L3). Establishment of infection was assessed by daily faecal analysis to determine the number of eggs per gram of faeces (EPG) and by adult worm recovery on days 5 and 13 post challenge. Mucosal mast cell (MMC) counts were done on days 5 and 13 post challenge while MMC-derived chondroitin sulphates in gut washings (days 1 and 5) and homogenate (day 8) were assayed by high performance liquid chromatography (HPLC). Results showed that patent infection occurred in challenged KO but not WT mice despite significantly higher mastocytosis in jejunal sections of KO than WT mice (p<0.001). Similarly but against prediction, significantly higher concentration of MMC-derived chondroitin sulphates was observed in gut homogenate of KO than WT mice (p<0.05). In contrast, significantly higher concentration of chondroitin sulphates was observed in gut washings of WT than KO mice (p<0.05). These results suggest that MMC in KO mice failed to release sufficient amount of sulphated proteoglycans into the gut lumen as did the WT mice, which may have been part of the hostile environment that prevented the establishment in and eventual expulsion of adult S. venezuelensis from the gut of WT mice following challenge.

Paragonimiasis cases recently found among immigrants in Japan.

OBJECTIVE: Paragonimiasis is a typical food-borne parasitic disease mainly endemic in Southeast Asia. In Japan, the disease has been re-emerging since the 1980s. In addition, recently we encountered an increasing number of immigrants with paragonimiasis in Japan. In this study we summarized the clinical features of immigrants. PATIENTS AND METHODS: Among a total of 152 paragonimiasis cases referred to and diagnosed in our laboratory during 1998 to 2002, 18 were immigrants. Their clinical features including laboratory data such as eosinophilia and total IgE level were gathered from the consultation sheets from attending physicians. RESULTS: Among a total of 18 immigrant cases, 16 were from China and 2 from Thailand. A majority of immigrants had eaten freshwater crabs. Most of the Chinese patients were infected as small groups of family and/or compatriots. Chest radiographic findings were variable and multiple lung lesions were seen in about one-half of the patients. About 80% of patients had peripheral blood eosinophilia and 65% had elevated serum IgE level. CONCLUSION: The clinical features of paragonimiasis in immigrants in Japan were much more severe compared to those of Japanese patients.

A case of cutaneous paragonimiasis with pleural effusion.

BACKGROUND: Paragonimiasis is an infectious disease caused by Paragonimus, which persists in the lung of mammals. Infection in the skin is very rare. RESULTS: A subcutaneous tumor with itching developed on the right lateroabdominal region of a 55-year-old man 10 days after eating fresh-water crab. It was surgically excised 2 months later. Examination of a specimen of the surgically excised tumor revealed an abscess containing many eosinophils. Parasites or other infectious organisms were not found in sections. The blood eosinophil count of the patient was elevated, and P. westermani-specific immunoglobulin G (IgG) antibody titer was strongly detected in the serum. The tumor was diagnosed as cutaneous paragonimiasis caused by P. westermani. Three months after eating the crab, a chest X-ray film showed a pleural effusion. P. westermani-specific IgG antibody was also detected in the pleural fluid. The patient was given 75 mg/kg/day of praziquantel for 3 days. The pleural effusion gradually disappeared after the medication. CONCLUSIONS: This patient had cutaneous paragonimiasis with a skin tumor that was diagnosed prior to the expression of a pleural effusion.

The current status of parasitic diseases in Japan.

In Japan parasitic diseases have been considered to be successfully controlled in the last 30 years. However, some parasitic diseases, such as food-borne zoonoses and/or larva migrans, are emerging and/or re-emerging in Japan. Furthermore, imported parasitic diseases like malaria are also gradually increasing. Unfortunately accurate numbers of parasitic diseases other than echinococcosis, malaria, amebiasis, giardiasis, or cryptosporidiosis are obscure in Japan because of the lack of a legal registration system. Since symptoms and diagnostic imaging patterns of parasitic diseases are non-specific and have similarities with other infectious diseases or cancer, parasitic diseases are sometimes overlooked or left misdiagnosed. In this review, the current status of parasitic diseases in Japan is briefly summarized based on the analysis of the accumulated cases seen in our department. We also outline the clinical features, differential diagnosis and treatment of representative parasitic diseases for the better understanding and management of the parasitic diseases in Japan.

A clinical study of eosinophilic meningoencephalitis caused by angiostrongyliasis.

OBJECTIVE: To improve the clinician's awareness of angiostrongyliasis. METHODS: The clinical and laboratory data as well as the epidemiological information concerning 18 patients with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis were analyzed. RESULTS: All patients had a history of eating raw fresh water snail (Ampularium canaliculatus) before the onset of the disease. Incubation period ranged from 1 to 25 days. The major symptoms of the patients had severe headache and pain in the trunk and limbs. Increased eosinophlic count in peripheral blood and cerebrospinal fluid was noted. Tested by enzyme-linked immunoadsorbent assay (ELISA), sera were specifically IgG-antibody positive against Angiostrougylus cantonensis antigen, but were negative against other parasitic antigens such as Paragonimus westermani, Cysticerus, Cellulosae hominis, Echinococcus granulosus and Trichinella spiralis. Abnormal spotty signals were found in 2 cases with brain magnetic resonance imaging. Electroencephalogram (EEG) showed slow alpha rhythm. All the patients were effectively treated with combined administration of albendazole and dexamethazone. CONCLUSIONS: Angiostrongyliasis is one of the common causes leading to eosinophilic meningoencephalitis. To our knowledge, Wenzhou is the first small outbreak site of angiostrongyliasis discovered in Chinese mainland.

[A case of neurocysticercosis suggestive of a reinfection, 20 years after the initial onset].

A 70-year-old man, who had frequently visited the Southeast Asian countries as a tour conductor, presented convulsion as an initial symptom twenty years ago. A diagnosis of neurocycticercosis was made by a stereotactic brain biopsy. Thereafter, anticonvulsant therapy was initiated, and he continued his travelling without any troubles. After travelling to Philippines, he suffered high-grade fever and consciousness disturbance, and his general status deteriorated. His consciousness level was evaluated as Japan Coma Scale 3 and his status was bedridden on admission to our hospital in December 1999. Anti-cysticercus cellulosae antibody was clearly detected both in his serum and cerebrospinal fluid (CSF). An MRI brain scan revealed disseminating lesions showing ring enhancement after administration of gadolinium-DTPA both intra and extra brain parenchyma. Steroid therapy and a cysticidal therapy relieved his symptoms, and CSF and MRI findings. Because it may be impossible for the cysticercosis cellulosae to be latent in the central nervous system as long as 20 years and to be reactivated, the reinfection of cysticercus cellulosae was supposed to be the cause of his symptoms.

Active hepatic capsulitis caused by Paragonimus westermani infection.

Paragonimiasis is an important re-emerging parasitosis in Japan. Although the lungs and pleural cavity are the principal sites affected with the parasite, ectopic infection can occur in unexpected sites such as skin and brain. This case report describes a patient with active hepatic capsulitis due to Paragonimus westermani infection. The patient was successfully treated with praziquantel at the dose of 75 mg/kg/day for 3 days.

A praziquantel-ineffective fascioliasis case successfully treated with triclabendazole.

Human fascioliasis cases in Japan have been reported infrequently and the most appropriate therapy for this disease remains to be determined. This case report describes a patient with the infection unsuccessfully treated with multiple high doses of praziquantel for over 1 year and eventually cured by the administration of triclabendazole in two single doses of 10 mg/kg and 12.5 mg/kg 5 months apart.