Sung-Tae Hong

Epidemiology of cholangiocarcinoma: an update focusing on risk factors.

(Cancer Sci 2010; 101: 579–585)

Clonorchiasis and cholangiocarcinoma: Etiologic relationship and imaging diagnosis

SUMMARY Despite a gradual decrease in prevalence, clonorchiasis is still prevalent in East Asia. A large and compelling body of evidence links clonorchiasis and cholangiocarcinoma, although the mechanisms involved are not completely understood. Clonorchiasis induces biliary epithelial hyperplasia and metaplasia, and this could facilitate at least one stage of the carcinogenesis, which is promoting effect. In areas of endemic infection, more clonorchiasis cases are now diagnosed incidentally during radiological examinations such as cholangiography, ultrasonography, and computed tomography. Radiological findings are regarded as pathognomonic for clonorchiasis since they reflect the unique pathological changes of this disorder. These radiological examinations currently play important roles in the diagnosis, staging, and decision-making process involved in the treatment of cholangiocarcinoma. The morphological features and radiological findings of clonorchiasis-associated cholangiocarcinoma are essentially combinations of the findings for the two diseases. The morphological features of clonorchiasis- associated cholangiocarcinoma, observed in radiological examinations, do not differ from those of the usual cholangiocarcinoma. In patients diagnosed with or suspected to have clonorchiasis, radiological findings should be carefully scrutinized for occult cholangiocarcinoma.

Clonorchis sinensis and clonorchiasis, an update.

Clonorchis sinensis is the most common human liver fluke in East Asia. Several studies proved its carcinogenesis in humans and it was reclassified as a group 1 biological carcinogen in 2009. It is still actively transmitted in endemic areas of Korea, China, Russia, and Vietnam. Currently it is estimated that more than 200 million people are at risk of infection, 15-20 million people are infected and 1.5-2 million show symptoms or complications. Several molecules and genes of the fluke have been identified and characterized. Studies on its oncogenesis and omics-based findings have been especially encouraging. Diagnosis of its infection depends mainly on detection of eggs in feces but other methods have been developed. ELISA using crude extract antigen is now popular for its diagnosis. Diagnosis by detecting DNAs from eggs in feces has been developed using PCR, real-time PCR, and LAMP, which have been found sensitive and specific. Imaging diagnosis has been studied in depth and is widely used. Any evidence of clonorchiasis, such as eggs, DNAs, or images, may lead to recommendations of chemotherapy in endemic areas. Praziquantel is the major chemotherapeutic agent for clonorchiasis and recently tribendimidine was found effective and is now under investigation as a promising chemotherapeutic alternative. Sustainable control programs which include mass chemotherapy with praziquantel and education for prevention of re-infection may reduce its morbidity and eliminate its infections in endemic areas.

Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skin

Anthocyanin is the major color pigment in plants. In order to understand anthocyanin biosynthesis mechanism in apple, the cDNAs encoding flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were isolated from cDNA libraries prepared from the skin tissues of apple (Malus domestica Borkh. cv. Fuji). Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Each gene is a member of a multigene family. The mRNAs of anthocyanin biosynthetic genes were detected preferentially in the skin tissue and expression of the genes was coordinately induced by light. The transcripts were detected abundantly in the skins of cultivars with red skin, but rarely in that of a cultivar carrying non-red fruit, suggesting that these genes have major roles in determination of apple skin color.

Gymnophalloides seoi n. sp. (Digenea: Gymnophallidae), the first report of human infection by a gymnophallid.

Gymnophalloides seoi n. sp. (Digenea: Gymnophallidae) is described from worms expelled from a naturally infected women in Korea. The patient, a 66-yr-old housewife, experienced severe epigastric discomfort, and fecal examination revealed numerous parasite eggs of 0.019-0.021 x 0.014-0.016 mm. After anthelmintic treatment and purgation, 952 digeneans consisting of 910 adult gymnophallids and 42 heterophyids (2 species) were recovered. The gymnophallid flukes closely resemble Gymnophalloides tokiensis Fujita, 1925 (known only as metacercariae from oysters), type and only previously known species of the genus, except for a difference in the position and orientation of the seminal vesicle. Without experimental proof of identity, the adult gymnophallids have been described as a new species, G. seoi, the only gymnophallid known to infect humans.

The Kato-Katz method is reliable for diagnosis of Clonorchis sinensis infection.

We evaluated the sensitivity of three fecal examination methods, the Kato-Katz (KK) method, formalin-ether technique (FE) and direct smear, for the diagnosis of clonorchiasis. The KK method proved to be most sensitive in six groups with different egg counts. In cases of extremely light infection FE was found to be more sensitive than KK.

Prevalence of Clonorchiasis in Southern Endemic Areas of Korea in 2006

This study was performed to investigate prevalence of clonorchiasis among the inhabitants living in villages along the 4 major rivers, Nakdong-gang (=river), Seomjin-gang, Youngsan-gang, and Guem-gang in southern Korea. From January to December 2006, a total of 24,075 stool samples (1 sample per an inhabitant) were collected in 23 localities and examined by the formalin-ether sedimentation technique. Of the inhabitants examined, 3,441 (14.3%) were found to harbor various types of intestinal parasite eggs, cysts or larvae. Numbers of infected people were 2,661 (11.1%) for Clonorchis sinensis, 431 (1.8%) for heterophyids, 226 (0.9%) for Entamoeba spp., 57 (0.2%) for Giardia lamblia, 30 (0.1%) for Trichuris trichiura, and 18 (0.07%) for echinostomes. Prevalence rates of clonorchiasis according to the river basin were 17.1% in Nakdong-gang, 11.2% in Seomjin-gang, 5.5% in Youngsan-gang and 4.6% in Guem-gang. Of the 2,661 C. sinensis egg-positive cases, 57.7% was male. The present findings suggest that clonorchiasis is still highly prevalent among inhabitants in the riverside areas of southern Korea, and it is necessary to implement a systematic control program in the endemic areas.

Detection of Clonorchis sinensis in stool samples using real-time PCR

Abstract Human clonorchiasis, caused by infection with the trematode Clonorchis sinensis, is a common health problem in East Asia. In an attempt to develop a new, sensitive method for the diagnosis of the disease, the use of a real-time PCR (targeting the internal-transcribed-spacer-2 sequence of the parasite) to detect C. sinensis-specific DNA in faecal samples has recently been evaluated. The PCR-based assay, which included an internal control to detect any inhibition of the amplification by faecal constituents in the sample, was performed on stool samples and on DNA controls representing a wide range of intestinal microorganisms. The assay appeared very specific, only showing positivity with C. sinensis and Opisthorchis felineus. The sensitivity of the assay was explored by testing 170 preselected samples of human faeces, from an endemic area of South Korea, which had known (microscopically-determined) densities of C. sinensis eggs. The sensitivity of the assay was 100% for the 74 samples that each had > 100 eggs/g and 91.4% for the other 70 samples found egg-positive by microcopy (i.e. those that had ≤ 100 eggs/g). Three of the 26 samples that appeared egg-negative by microscopy were found PCR-positive. Encouragingly, the PCR cycle-threshold values, which reflect parasite-specific DNA loads, showed significant correlation with the egg counts. The real-time PCR used in this study therefore appears to be a powerful tool for both the detection and quantification of C. sinensis infections.

Proliferative effects of excretory/secretory products from Clonorchis sinensis on the human epithelial cell line HEK293 via regulation of the transcription factor E2F1

Clonorchis sinensis is one of the most prevalent parasitic helminths of humans in East Asia. Although several complications in bile duct epithelial cells are caused by C. sinensis infection, the mechanism is not clearly understood. To clarify the effects of C. sinensis excretory–secretory products (ES products) on bile duct epithelial cells, we investigated their effects on the human embryonic kidney epithelial cell line HEK293 in vitro. Our results show that ES products alter the proportion of cells in each stage of the cell cycle and induce HEK293 cell proliferation. Among cell cycle-related proteins, the expression of cyclin E increased markedly after treatment with ES products, indicating that the G1/S transition occurred. In addition, the expression of the transcription factor E2F1 was up-regulated by the addition of ES products. Small interfering RNA (siRNA) was used to demonstrate that the transcription factor E2F1 is a key factor in the control of cell proliferation in HEK293 cells. The present results demonstrate that ES products from C. sinensis stimulate cell proliferation by inducing E2F1 expression. We suggest that the ES products released from C. sinensis during infection may play an important role in the development of cholangiocarcinoma via the overgrowth of the bile duct epithelium.

Effect of Control Strategies on Prevalence, Incidence and Re-infection of Clonorchiasis in Endemic Areas of China

Background A pilot clonorchiasis control project was implemented to evaluate the efficacies of various chemotherapy strategies on prevalence, incidence and re-infection in Heilongjiang Province, China. Methods and Findings Seven intervention groups (14,139 residents, about 2000 in each group) in heavily or moderately endemic areas were subjected to repeated praziquantel administration from 2001 to 2004. In the selective chemotherapy groups, residents were examined for fecal eggs, and those who tested positive were treated with three doses of 25 mg/kg praziquantel at 5-hour-intervals in one day. However, all residents were treated in the mass chemotherapy groups. In heavily endemic areas, two mass treatments of all residents in 2001 and 2003 reduced the prevalence from 69.5% to 18.8%, while four annual mass treatments reduced the prevalence from 48.0% in 2001 to 8.4% in 2004. Selective annual treatments for egg-positive subjects reduced the egg-positive rates from 54.9% in 2001 to 15.0% in 2004 or from 73.2% in 2001 to 12.3% in 2004. Selective treatments every 6 months significantly reduced the prevalence from 59.5% in 2001 to 7.5% in 2004. All of the repeated treatments reduced EPG (eggs per gram of feces) significantly. The annual mass treatment and selective treatment every 6 months produced lower prevalence and re-infection rates and higher egg reduction rate than annual selective treatments did. In the moderate endemic areas, egg positive rates were 24.8% and 29.7% in 2001 but were 1.9% and 1.3% after 2 or 3 selective treatments. The prevalence, incidence, re-infection rates in a moderately endemic area were significantly lower than those of heavy endemic areas. Conclusions Repeated mass treatment or selective treatment with praziquantel every 6 to 12 months is highly effective for clonorchiasis control in heavily endemic areas. In contrast, one or two selective treatments with health education is effective in moderately endemic areas.