Pathological Lesions and Inducible Nitric Oxide Synthase Expressions in the Liver of Mice Experimentally Infected with Clonorchis sinensis

The nitric oxide (NO) formation and intrinsic nitrosation may be involved in the possible mechanisms of liver fluke-associated carcinogenesis. We still do not know much about the responses of inducible NO synthase (iNOS) induced by Clonorchis sinensis infection. This study was conducted to explore the pathological lesions and iNOS expressions in the liver of mice with different infection intensity levels of C. sinensis. Extensive periductal inflammatory cell infiltration, bile duct hyperplasia, and fibrosis were commonly observed during the infection. The different pathological responses in liver tissues strongly correlated with the infection intensity of C. sinensis. Massive acute spotty necrosis occurred in the liver parenchyma after a severe infection. The iNOS activity in liver tissues increased, and iNOS-expressing cells with morphological differences were observed after a moderate or severe infection. The iNOS-expressing cells in liver tissues had multiple origins.     

Human Infections with Liver and Minute Intestinal Flukes in Guangxi, China: Analysis by DNA Sequencing, Ultrasonography, and Immunoaffinity Chromatography

The prevalence of liver and intestinal fluke infections was determined by surveying inhabitants of Hengxuan, Fusui, and Shanglin villages which were known to be endemic for liver flukes in Guangxi, China in May 2010. A total of 718 people were examined for helminth eggs by the Kato-Katz thick smear technique, ultrasonography, immunoaffinity chromatography, and DNA sequencing. The overall egg positive rate was found to be 59.6% (28.0-70.6%) that included mixed infections with liver and intestinal flukes. Cases showing higher than 20,000 eggs per gram of feces (EPG) were detected between 1.3% and 16.2%. Ultrasonographic findings exhibited overall 28.2% (72 of 255 cases) dilatation rate of the intrahepatic bile duct. Clonorchis sinensis infection was detected serologically in 88.3% (38 of 43 cases) among C. sinensis egg positive subjects by the immunoaffinity chromatography using a specific antigen for C. sinensis. For differential diagnosis of the liver and intestinal flukes, more precise PCR and nucleotide sequencing for copro-DNA were performed for 46 egg positive cases. Mixed infections with C. sinensis and Metagonimus yokogawai were detected in 8 of 46 egg positive cases, whereas 29 specimens were positive for Haplorchis taichui. Ultrasonographic findings and immunoaffinity chromatography results showed usefulness, even in a limited way, in figuring out of the liver fluke endemicity.     

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.     

Painless Jaundice Caused by Clonorchis sinensis Infection: A Case Report

A man with only yellowing of the skin and eye sclera was diagnosed with clonorchiasis, which rarely manifested jaundice as the initial symptom. However, because of a lack of evidence for a diagnostic gold standard, the time until definitive diagnosis was more than a week. The diagnostic process relied on inquiring about the patient’s history, including the place of residence, dietary habits, and symptoms, as well as on serological findings, an imaging examination, and pathological findings. MRCP and CT results showed mild dilatation of intrahepatic ducts and increased periductal echogenicity. The eggs were ultimately found in stool by water sedimentation method after the negative report through direct smear. DNA sequencing of PCR production of the eggs demonstrated 98-100% homology with ITS2 of Clonorchis sinensis. After anti-parasite medical treatment, the patient’s symptoms were gradually relieved. Throughout the diagnostic procedure, besides routine examinations, the sedimentation method or concentration method could be used as a sensitive way for both light and heavy C. sinensis infection in the definite diagnosis.     

C3H/He Mice as an Incompatible Cholangiocarcinoma Model by Clonorchis sinensis,Dicyclanil and N-Nitrosodimethylamine

Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm³) from the Cs group was a hepatocellular adenoma and the other (280.6 mm³) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.     

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.     

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn²⁺ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.     

Cyst formation, increased anti-inflammatory cytokines and expression of chemokines support for Clonorchis sinensis infection in FVB mice

To verify the hypothesis that different pathology of Clonorchis sinensis infection by mouse strains is determined by different responses of cytokines and chemokines, we compared those responses of FVB with those of BALB/c mice. All of FVB mice infected with 30 metacercariae of C. sinensis showed cystic dilatation in the liver, whereas infected BALB/c mice did not. Mature worms were recovered from 19 of 20 liver sections of FVB mice while only one of 20 sections of BALB/c mice revealed a mature worm. In both strains the proportion of CD4⁺ T cells was lower in C. sinensis-infected than in the uninfected group. However, the proportion of CD8⁺ T cells was elevated in C. sinensis-infected from both strains compared to uninfected mice. The Th2-associated anti-inflammatory cytokines such as IL-4, IL-5 and IL-13, IL-10 and TGF-β, were significantly more produced by the lymphocytes of FVB than by those of BALB/c mice. Especially, the 2 anti-inflammatory cytokines, IL-10 and TGF-β, were presumably related with susceptibility and the development of worms in the liver. C. sinensis infected FVB mice also produced more chemokines such as RANTES and MIP-1α in the liver lymphocytes than BALB/c mice. In conclusion, the FVB mice provide the favorable niche for C. sinensis by cyst formation in the bile duct, increased production of Th2-associated anti-inflammatory cytokines and upregulation of chemokines.     

Strongyloidiasis in a Diabetic Patient Accompanied by Gastrointestinal Stromal Tumor: Cause of Eosinophilia Unresponsive to Steroid Therapy

We report here a case of strongyloidiasis in a 72-year-old diabetic patient (woman) accompanied by gastrointestinal stromal tumor receiving imatinib therapy, first diagnosed as hypereosinophilic syndrome and treated with steroids for uncontrolled eosinophilia. She suffered from lower back pain and intermittent abdominal discomfort with nausea and diagnosed with gastrointestinal stromal tumor. After post-operative imatinib treatment eosinophilia persisted, so that steroid therapy was started under an impression of hypereosinophilic syndrome. In spite of 6 months steroid therapy, eosinophilia persisted. Stool examination was performed to rule out intestinal helminth infections. Rhabditoid larvae of Strongyloides stercoralis were detected and the patient was diagnosed as strongyloidiasis. This diagnosis was confirmed again by PCR. The patient was treated with albendazole for 14 days and her abdominal pain and diarrhea improved. This case highlights the need for thorough investigation, including molecular approaches, to test for strongyloidiasis before and during steroid therapies.     

Human Neurocysticercosis Case and an Endemic Focus of Taenia solium in Lao PDR

A male patient with neurocysticercosis was identified in Montai Village, Xay District, Oudomxay Province, Lao PDR in February 2004. He had a history of diagnosis for neurocysticercosis by a CT scan in Thailand after an onset of epileptic seizure in 1993. A pig in the same district was found to contain Taenia solium metacestodes (=cysticerci); the slaughtered pig body contained more than 2,000 cysticerci. In addition to morphological identification, molecular identification was also performed on the cysticerci by DNA sequencing analysis of the mitochondrial cox1 gene; they were confirmed as T. solium metacestodes. The patient is regarded as an indigenous case of neurocysticercosis infected in an endemic focus of T. solium taeniasis/cysticercosis in Oudomxay Province, Lao PDR.     

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

Cutaneous Gnathostomiasis with Recurrent Migratory Nodule and Persistent Eosinophilia: a Case Report from China

The present study reports a human case of cutaneous gnathostomiasis with recurrent migratory nodule and persistent eosinophilia in China. A 52-year-old woman from Henan Province, central China, presented with recurrent migratory reddish swelling and subcutaneous nodule in the left upper arm and on the back for 3 months. Blood examination showed eosinophila (21.2%), and anti-sparganum antibodies were positive. Skin biopsy of the lesion and histopathological examinations revealed dermal infiltrates of eosinophils but did not show any parasites. Thus, the patient was first diagnosed as sparganosis; however, new migratory swellings occurred after treatment with praziquantel for 3 days. On further inquiring, she recalled having eaten undercooked eels and specific antibodies to the larvae of Gnathostoma spinigerum were detected. The patient was definitely diagnosed as cutaneous gnathostomiasis caused by Gnathostoma sp. and treated with albendazole (1,000 mg/day) for 15 days, and the subsequent papule and blister developed after the treatment. After 1 month, laboratory findings indicated a reduced eosinophil count (3.3%). At her final follow-up 18 months later, the patient had no further symptoms and anti-Gnathostoma antibodies became negative. Conclusively, the present study is the first report on a human case of cutaneous gnathostomiasis in Henan Province, China, based on the past history (eating undercooked eels), clinical manifestations (migratory subcutaneous nodule and persistent eosinophilia), and a serological finding (positive for specific anti-Gnathostoma antibodies).     

Immunodiagnosis of clonorchiasis using a recombinant antigen

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a β-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients'' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.     

V-shaped Pits in Regions of Ancient Baekje Kingdom Paleoparasitologically Confirmed as Likely Human-Waste Reservoirs

In a paleo-parasitological analysis of soil samples obtained from V-shaped pits dating to the ancient Baekje period in Korean history, we discovered Ascaris lumbricoides, Trichuris trichiura, and Clonorchis sinensis eggs. In light of the samples'' seriously contaminated state, the V-shaped pits might have served as toilets, cesspits, or dung heaps. For a long period of time, researchers scouring archaeological sites in Korea have had difficulties locating such structures. In this context then, the present report is unique because similar kind of the ancient ruins must become an ideal resource for successful sampling in our forthcoming paleoparasitological studies.     

Infection Status of Freshwater Fish with Metacercariae of Clonorchis sinensis in Korea

This study investigated freshwater fish for their current infection status with metacercariae of Clonorchis sinensis in Korea. Twenty-one species of freshwater fish (n = 677) were collected from 34 regions nationwidely from February 2007 to June 2008. They were individually examined by digestion technique. Eight species of freshwater fish from 17 different regions were recognized positive for the metacercariae of C. sinensis. The positive rates (range of metacercariae number per fish) of fish by the species were as follows: 48% (1-1,142) in Pseudorasbora parva, 60% (1-412) in Pungtungia herzi, 15.7% (1-23) in Pseudogobio esocinus, 29% (1-7) in Acheilognathus intermedia, 21% (1-4) in Odontobutis interrupta, 33% (1-6) in Zacco temmincki, 3.6% (1-4) in Zacco platypus, and 26.3% (1) in Hemibarbus labeo. The two species, P. parva and P. herzi, are able to be the index fish for estimation of C. sinensis transmission in a certain locality. Still several species of freshwater fish are briskly transmitting C. sinensis infection in many riverside areas of southern Korea.     

MTHFR Gene variants C677T, A1298C and association with Down syndrome: A Case-control study from South India

BACKGROUND:

The 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphisms and low folate levels are associated with inhibition of DNA methyltransferase and consequently DNA hypomethylation. The expanding spectrum of common conditions linked with MTHFR polymorphisms includes certain adverse birth outcome, pregnancy complications, cancers, adult cardiovascular diseases and psychiatric disorders, with several of these associations remaining still controversial. Trisomy 21 or Down syndrome (DS) is the most common genetic cause of mental retardation. It stems predominantly from the failure of chromosome 21 to segregate normally during meiosis. Despite substantial research, the molecular mechanisms underlying non-disjunction leading to trisomy 21 are poorly understood.

MATERIALS AND METHODS:

Two common variants C677T and A1298C of the MTHFR gene were screened in 36 parents with DS children and 60 healthy couples from Tamil Nadu and Karnataka. The MTHFR genotypes were studied by RFLP analysis of PCR-amplified products and confirmed by sequencing.

RESULTS:

The CT genotype was seen in three each (8.3%) of case mothers and fathers. One case father showed TT genotype. All the control individuals exhibited the wild type CC genotype. A similar frequency for the uncommon allele C of the second polymorphism was recorded in case mothers (0.35) and fathers (0.37) in comparison with the control mothers (0.39) and fathers (0.37).

CONCLUSION:

This first report on MTHFR C677T and A1298C polymorphisms in trisomy 21 parents from south Indian population revealed that MTHFR 677CT polymorphism was associated with a risk for Down syndrome.     

rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia

Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones & Spatafora (syn. Cordyceps sinensis) one of the entomopathogenic fungi, is a rare Traditional Chinese Medicine (TCM) found in the Qinghai-Tibetan Plateau. Polymerase Chain Reaction (PCR) and Fluorescence in situ hybridization (FISH) methods are necessary to identify the mycelia or spores of O. sinensis from its habitat and to monitor its dispersal, colonization and infectivity. To develop both primers and probe specific to O. sinensis, ribosomal DNA (rDNA) amplified with universal primers from O. sinensis genomic DNA and seven closely related fungi were sequenced. According to these sequences, the upper and lower primers (OsT-F and OsT-R) were designed within internal transcribed spacer region 1 (ITS1) and ITS2 and flanked by universal primers ITS5 and ITS4, respectively. The designed primers were used for general PCR, touchdown PCR, or both together with the universal primers for nested-touchdown PCR. The results showed that only the extracted DNA of O. sinensis was specifically amplified. The sensitivity of nested-touchdown PCR with extracted DNA of O. sinensis is as low as 10⁻¹⁴ g (10 fg) and at least 1000 times higher than the other PCR methods. In addition, Cy5-labeled probe (OsLSU) for cytoplasmic LSU rRNA was hybridized with the ascospores of O. sinensis. It showed a strong red fluorescence throughout the whole cell but did not cross-react with other entomopathogenic fungi. Taken together, these methods were useful for studying the biology and ecology of O. sinensis.