Clonorchis Sinensis Eggs Research Articles

Ancient DNA analysis on Clonorchis sinensis eggs remained in samples from medieval Korean mummy

In the present study, Clonorchis sinensis (C. sinensis) ancient DNA (aDNA) was successfully extracted from human remains discovered in a tomb dating to the medieval Joseon dynasty of Korea. The presence of C. sinensis eggs was confirmed by microscopic observation, after which a PCR-based aDNA analysis was performed using primer sets designed for the amplification of nuclear ribosomal internal transcribed spacer 1 (ITS1), 2 (ITS2) and mitochondrial cytochrome c oxidase 1 (CO1) genes. The sequences obtained were 100% homologous to some contemporary C. sinensis gene sequences reported from Korea and other East Asian countries. We believe that the results of our analysis expand the temporal and geographical scopes of research on the history of C. sinensis infection in different human populations.

Cholecystolithiasis is associated with Clonorchis sinensis infection.

BackgroundThe objective of this study was to analyze gallbladder stones for direct evidence of a relationship between Clonorchis sinensis infection and gallbladder stones formation.MethodologyWe investigated one hundred eighty-three gallbladder stones for the presence of Clonorchis sinensis eggs using microscopy, and analyzed their composition using Fourier transform infrared spectroscopy. We confirmed the presence of Clonorchis sinensis eggs in the gallbladder stones using real-time fluorescent PCR and scanning electron microscopy.Principal Findings Clonorchis sinensis eggs were detected in 122 of 183 gallbladder stones based on morphologic characteristics and results from real-time fluorescent PCR. The proportion of pigment stones, cholesterol stones and mixed gallstones in the egg-positive stones was 79.5% (97/122), 3.3% (4/122) and 17.2% (21/122), respectively, while 29.5% (18/61), 31.1% (19/61) and 39.3% (24/61) in the egg-negative stones. The proportion of pigment stone in the Clonorchis sinensis egg-positive stones was higher than in egg-negative stones (P<0.0001). In the 30 egg-positive stones examined by scanning electron microscopy, dozens or even hundreds of Clonorchis sinensis eggs were visible (×400) showing a distinct morphology. Many eggs were wrapped with surrounding particles, and in some, muskmelon wrinkles was seen on the surface of the eggs. Also visible were pieces of texture shed from some of the eggs. Some eggs were depressed or without operculum while most eggs were adhered to or wrapped with amorphous particles or mucoid matter (×3000).Conclusion Clonorchis sinensis eggs were detected in the gallbladder stones which suggests an association between Clonorchis sinensis infection and gallbladder stones formation, especially pigment stones.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

Research report on intramural gallstones

Objective To investigate the relationship between intramural gallstones and intraluminal gallstones. Methods In this present study, the control study was applied to analysis on the gallstones from 116 cholecystolithiasis patients with intramural gallstones as well as intraluminal gallstones. All the patients were from the general surgery department and accepted gallbladder-preserving cholecystolithotomy during 2009.02-2009.08. Meanwhile, 171 patients with intraluminal gallstones were investigated the clinical traits; and gallstones from them were analyzed with FT-IR and microsopic examination. Results The results showed the coincidence rate of stone type was 97.4％, and it was mainly pigment gallstones and mixed gallstones in 116 cases; the coincident detection rate of clonorchis sinensis eggs was 98.3 ％, and it was much higher in the pigment gallstones and mixed gallstones than that in the cholesterol gallstones. The ratio of pigment gallstones from the 116 patients group was higher than the 171 patients group, and the detection rate of C. sinensis eggs of the two groups had no statistical difference (P＞0.05). Conclusions The stone type of intramural gallstones was mainly pigment gallstones and mixed gallstones. Intramural gallstones and intraluminal gallstones were homologous. Key words: Intramural Gallstones; Intraluminal Gallstones; Pigment Gallstones; Cholesterol Gallstones; Mixed Gallstones; Clonorchis sinensis eggs

Paleo-parasitological study on the soils collected from archaeological sites in old district of Seoul City

A paleo-parasitological study was performed on soil samples from archaeological sites in the old district of Seoul Metropolitan City (Old Seoul City) where the capital of the Joseon Kingdom was constructed about 600 years ago. The sampling sites were the original locations of the Royal Palace Wall, Yukjo Street and the Royal Arsenal. Microscopic examinations showed that parasite eggs of Clonorchis sinensis, Diphyllobothrium latum, Ascaris sp. and Trichuris sp. remained in the soil strata, which were estimated to represent the 14th-19th centuries. We suspect that the medieval citizens of Old Seoul City, numbering around 200,000, might have been made vulnerable to parasite infection by a combination of bad hygiene, inadequate sewer system and seasonal flooding.

Hypersensitive Reaction to Praziquantel in a Clonorchiasis Patient

Praziquantel is the drug of choice for clonorchiasis. Since clonorchiasis is endemic in most river basins, praziquantel has been widely used for 30 years in Korea. A 54-year-old Korean woman suffered from hypersensitive reactions, such as nausea, dyspnea, rash, and urticaria after taking the first dose of praziquantel to treat clonorchiasis. She ingested one dose again and the same symptoms appeared, and she was treated at a clinic with anti-histamines. She tried one more dose with anti-histamines but found the same symptoms. Later, she was found to pass eggs of Clonorchis sinensis and medicated with flubendazole. The hypersensitive reaction to praziquantel is rare but occurs. This is the 5th case report in the world.

Epidemiological Survey on the Infection of Intestinal Flukes in Residents of Muan-gun, Jeollanam-do, the Republic of Korea

Infection status of intestinal flukes was investigated in residents of Muan-gun, Jeollanam-do, the Republic of Korea. Total 1,257 fecal samples of residents were examined by formalin-ether sedimentation technique and Kato-Katz thick smear method. Helminth eggs were detected from 95 (7.6%) residents, and eggs of heterophyid flukes and Clonorchis sinensis were found from 62 (4.9%) and 40 (3.2%) cases, respectively. The larger heterophyid eggs, somewhat dark-brown in color and 37.7 x 21.5 microm in average size, and found in 32 (2.6%) out of 62 egg positive cases of heterophyid flukes. To confirm the adult flukes, we performed worm recovery from 12 cases after praziquantel treatment and purgation with MgSO(4). A total of 1,281 adult flukes, assigned to 7 species, were recovered from 9 cooperative cases. Heterophyes nocens (total 981 specimens) was collected from 9 cases, Stictodora fuscata (80) from 7, Gymnophalloides seoi (75) from 5, Pygidiopsis summa (140) from 3, Stellantchasmus falcatus (3) from 2, and Stictodora lari and Acanthotrema felis (each 1 worm) from 1 case each. The intrauterine eggs of S. fuscata collected from the recovered worm were identical with the larger heterophyid eggs detected in the stool examination. By the present study, it was confirmed that A. felis is a new intestinal fluke infecting humans, and residents in Muan-gun, Jeollanam-do are infected with variable species of intestinal trematodes.

Changes in Sonographic Findings after Treatment of Patients with Clonorchiasis in a Heavy Endemic Area

We measured changes in sonographic findings of patients with clonorchiasis after a treatment in a highly endemic area. A total of 347 residents showed positive stool results for Clonorchis sinensis eggs in a village in northeastern China, and were treated with praziquantel. Of them, 132 patients underwent abdominal sonography both before and 1 year after treatment, and the changes in sonographic findings of 83 cured subjects were compared. Diffuse dilatation of intrahepatic bile ducts (DDIHD) was found in 82 patients (98.2%) before and 80 (96.4%) after treatment, which was improved in 3, aggravated in 1, and unchanged in 79 patients. Increased periductal echogenicity (IPDE) was observed in 42 patients (50.6%) before and 45 (54.2%) after treatment, which was improved in 5, aggravated in 8, and unchanged in 70 patients. Floating echogenic foci in the gallbladder (FEFGB) was detected in 32 patients (38.6%) before and 17 (20.5%) after treatment, which was improved in 20, aggravated in 5, and unchanged in 58 patients. Improvement of FEFGB only was statistically significantly (P = 0.004). The present results confirm that DDIHD and IPDE persist but FEFGB decreases significantly at 1 year after treatment. In a heavy endemic area, the sonographic finding of FEFGB may suggest active clonorchiasis 1 year after treatment.

PALEOPARASITOLOGICAL REPORT ON THE STOOL FROM A MEDIEVAL CHILD MUMMY IN YANGJU, KOREA

Previous studies have successfully shown evidence for parasitic infections in human remains from various archaeological sites. However, in the case of Korea, since there have been very few paleoparasitological reports published, pre-20th century parasitic infection patterns remain obscure. Therefore, in order to partly fill this gap, we are reporting on a case of paleoparasitic infection from the feces of a 15th century child mummy from Yangju, Korea. In the course of the present study, we found the eggs of Clonorchis sinensis, Ascaris lumbricoides, and Trichuris trichiura in the feces of the mummy. Trichuris trichiura eggs were found in far greater numbers than other parasite eggs; in fact, intact bipolar plugs were clearly observed and even the larvae were still visible in some eggs. The eggs of C. sinensis and A. lumbricoides were also well preserved, though not in as great a number. Since we could find a number of well-preserved larvae-containing eggs, we are encouraged that successful extraction, amplification, and sequence determination of ancient DNA from the paleoparasite eggs might be possible in future studies. With additional paleoparasitological investigation using feces from Korean mummies, we hope that a history of parasite infection in Korea will be reconstructed.

Comparison of ancient and modern Clonorchis sinensis based on ITS1 and ITS2 sequences

In 1975, an ancient corpse buried in 167 BC was found at Jiangling County, Hubei Province of China. The eggs of Clonorchis sinensis found in the gall bladder of the corpse were preserved well. In the present paper, we extracted the genomic DNA from the ancient eggs and modern eggs, respectively, and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) at ribosomal RNA genes were studied. The results show that ITS2 sequences from the ancient sample were identical with those from modern samples, but in ITS1 differences in 15 nucleotide positions were found between the ancient and modern samples. The results demonstrated that it is possible to extract and sequence DNA from ancient parasite eggs. The ITS1 sequence obtained differed from all modern ones available to date. This might indicate sequence divergence through time, or might reflect a sequence polymorphism that may eventually be found also in modern samples.

Collection of Clonorchis sinensis adult worms from infected humans after praziquantel treatment

A cohort was established for evaluation of cancer risk factors in Sancheong-gun, Gyeongsangnam-do, Korea. As one of the cohort studies, stools of 947 residents (403 males and 544 females, age range: 29-86 years) were screened for Clonorchis sinensis eggs using both Kato-Katz method and formalin-ether sedimentation technique. The overall egg positive rate of C. sinensis was 37.7% and individual EPG (eggs per gram of feces) counts ranged from 24 to 28,800. Eight egg positive residents voluntarily joined a process of collection of the passed worms after praziquantel treatment. A total of 158 worms were recovered from 5 of the 8 treated persons, ranged from 3 to 108 in each individual. The worms were 15-20 mm x 2-3 mm in size, and showed brown-pigmented, red, or white body colors. This is the first collection record of C. sinensis adult worms from humans through anthelmintic treatment and purgation. The adult worms of C. sinensis may be paralyzed by praziquantel and then discharged passively through bile flow in the bile duct and by peristaltic movement of the bowel.

A Bile Based Study of Clonorchis sinensis Infections in Patients with Biliary Tract Diseases in Ulsan, Korea

Stool examination is believed to be the most reliable method for detecting Clonorchis sinensis (CS) eggs. However, it has limited value for diagnosing clonorchiasis when the biliary tract is obstructed or when there is a light infection. We evaluated the infection states of CS in patients with biliary tract diseases using a bile sample. From January 2001 to August 2003, 238 patients who had undergone endoscopic biliary drainage were prospectively included in the study. The patients' bile samples were obtained directly from the nasobiliary drainage tube and then analyzed to detect CS eggs. The overall CS egg positive rate was 28.2% (35.4% in males, 19.4% in females). The egg positive rate was similar in all age groups examined: 26.7% in 30-39 years, 25.0% in 40-49 years, 24.4% in 50-59 years, 30.2% in 60-69 years, 35.3% in 70-79 years, and 25.0% in 80 years of age and over. There were no significant differences in the egg positive rate between the disease groups: 32.6% in bile duct cancer, 38.5% in gallbladder cancer, and 26.4% in gallstone diseases. Our results show that the CS infection rate was very high, regardless of the age, gender, and type of diseases of the patients. Although the study population was limited to patients with biliary tract diseases, it is assumed that clonorchiasis is still an endemic disease in Ulsan, Korea.

Sonographic findings of active Clonorchis sinensis infection.

The aim of this study was to document the characteristic sonographic findings of clonorchiasis for the diagnosis of active infection in an endemic area. In a village in northeastern China, residents underwent fecal examinations for detection of Clonorchis sinensis eggs. Shortly thereafter, residents were examined with abdominal sonography. An experienced radiologist performed the sonographic examinations and analyzed the findings. Subjects whose fecal examinations were positive for eggs were considered to have active clonorchiasis; those whose examinations were negative for eggs were used as control subjects. The distinguishing sonographic features of active clonorchiasis were identified by stepwise logistic regression analysis. The study population comprised 457 subjects; fecal examinations revealed C. sinensis eggs in 316 and no eggs in 141. Four sonographic findings distinguished subjects with active clonorchiasis from control subjects: increased periductal echogenicity (p < 0.001; R = 0.11; sensitivity, 35%; specificity, 91%), floating echogenic foci in the gallbladder (p < 0.001; R = 0.09; sensitivity, 28%; specificity, 94%), diffuse dilatation of the intrahepatic bile ducts (p < 0.01; R = 0.03; sensitivity, 67%; specificity, 48%), and gallbladder distention (p < 0.05; R = 0.02; sensitivity, 3%; specificity, 100%), in decreasing order of significance. Among these 4 sonographic findings, increased periductal echogenicity and floating echogenic foci in the gallbladder were more significantly associated with active infection than were the other 2. Increased periductal echogenicity and floating echogenic foci in the gallbladder were identified as the 2 most significant findings for the sonographic diagnosis of active C. sinensis infection.

Detection of parasite eggs from archaeological excavations in the Republic of Korea.

Excavations at two sites dating from 2000 BC-1900 AD in southeastern areas of the Republic of Korea, revealed the remains of several structures. Examination of the contents suspected privies revealed the presence of eggs from 5 kinds of parasite: Ascaris, Trichuris, Clonorchis, and two species of unknown trematodes. Clonorchis sinensis eggs were found in a soil dating from around AD 668-935. This is the first record of C. sinensis eggs in archaeological materials in the Republic of Korea.

Study of an ancient corpse of the Warring States period unearthed from Tomb No. 1 at Guo-Jia Gang in Jingmen City (A comprehensive study)

An ancient female corpse excavated from the robbed tomb No. 1 at Guo-Jia-Gang in Jingmen city of Hubei Province was studied. It was confirmed that the corpse was buried more than 2,300 years ago, namely at the middle stage of the Warring States period and is the earliest one of the ancient coffin corpses unearthed in China so far. It is of the same type as the ancient corpses of Weatern Han Dynasty excavated from tomb No. 1 at Ma-Wang-Dui in Changsha City and from the tomb No. 168 at Phoenix Hill in Jinagling City respectively. The collagenous fibers of connective tissue from all parts in the corpse were well preserved, showing fine ultrastructure with clear periodical cross banding, and a lot of bacterial spores were found between them. In addition, a large number of eggs of clonorchis sinensis and trichuris trichiura were found in the content of the bowel. The level and the cause of good preservation were discussed and it was believed that the good preservation may result from the combined effect of the internal and external environmental factors around the corpse.

Intestinal parasitic infections of Korean Army soldiers in Whachon-gun, Korea.

We examined stools of 113 soldiers of Korean Army in Whachon-gun, Korea in 1992. The eggs of Clonorchis sinensis (7/113) and Metagonimus sp. (3/113), and cysts of Giardia lamblia (4/113) were detected. No eggs of soil-transmitted helminths were found. Most of the Clonorchis-infected soldiers (6/7) were recruited from Kyongsangnam-do.

Intestinal parasites of cats purchased in Seoul.

Fecal samples of cats purchased in Seoul were examined for helminth ova or protozoan oocysts from December 1987 to March 1988. Out of the 41 samples, 31 (75.6%) were positive and 60 (146.3%) were cumulative positive for parasites. The followings were identified in the samples: Eggs of Toxocara cati, Clonorchis sinensis, Metagonimus sp., Pharyngostomum cordatum, Spirometra erinacei, Taenia taeniaeformis and oocysts of Isospora sp. From nine autopsied cats, larvae of Anisakis simplex, adults of C. sinensis, M. yokogawai, P. cordatum, S. erinacei and T. taeniaeformis were identified. This is the first report on the detection of Anisakis larvae from cats in Korea. The possible role of cats as a source of human infection with each parasite was discussed.

Comparative Morphology Of Eggs Of Heterophyids And Clonorchis Sinensis Causing Human Infections In Korea.

In order to provide some clues for differential diagnosis of trematode infections in fecal examination, the comparative morphology of eggs of 5 kinds of heterophyid flukes (Metagonimus yokogawai, Heterophyes heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus and Pygidiopsis summa) and Clonorchis sinensis was studied. The eggs were obtained from distal portion of uteri of worms which were recovered from men after treatment. The characteristic shape and apperance of each kind of eggs were observed in detail under light microscope, and their length and width measured and compared one another. The results are as follows: Eggs of C. sinensis are elongated ovoidal in shape with attenuated anterior end, 25.3-33.2 (28.3 in average) micrometer long and 14.2-17.4(15.9) micrometer wide with length /width ratio of 1.60-2.00 (1.78). They differ from all heterophyid eggs in that they have prominent wrinkling (muskmelon pattern) at their shell surface. P. summa eggs are ovoid to pyriform in shape and characterized by the smallest size of all kinds examined, 19.8-22.9(21.6) micrometer long and 11.1-13.4 (12.1) micrometer wide and the ratio 1.63-1.99 (1.78). Eggs of S. falcatus are elongated ovoidal and most slender form, 25.3-29.2 (27.2) micrometer long and 11.1-13.4 (12.5) micrometer wide with the ratio of 2.00-2.57 (2.17). Eggs of M. yokogawai are ellipsoid to elliptical in shape with round both ends, 26.9-31.6 (28.5) micrometer long and 14.2-18.2 (16.8) micrometer wide with the ratio of 1.48-2.11(1.70). H. continua eggs are oval in shape, sometimes similar to M. yokogawai or H. h. nocens eggs, however, the relative breadth is broadest among all kinds, with maximum width at posterior half portion. They are 23.7-27.7 (25.0) micrometer long, 15.8-18.9 (16.4) micrometer wide with the ratio of 1.33-1.75 (1.53). Eggs of H. h. nocens are ellipsoid to ovoid in shape but sometimes more slender than M. yokogawai and have slightly pointed both ends. They are 23.7-29.2 (25.7) micrometer long, 14.2-15.8 (15.4) micrometer wide, and the ratio 1.50-2.06 (1.67). From the results, it is concluded that eggs of 5 kinds of heterophyids and C. sinensis can be morphologically differentiated one another, however, careful observation and measurement on sufficient number of eggs are needed.

Studies On Intestinal Trematodes In Korea: VIII. A Human Case Of Echinostoma Hortense Infection.

Echinostomatid eggs were found from a 21-year old man, whole native village is Munkyong-gun, Kyongsangnam-do during fecal examination in October 1983. The eggs were detected again one week later concommitantly with the eggs of Clonorchis sinensis. He had no subjective symptoms related with these fluke infections. He was treated with praziquantel(Distocide(R)) 20 mg/kg in single dose and purgated with magnesium salt. One echinostomatid fluke and 4 adults of C. sinensis were collected from the diarrheal stools. After morphological observation, the echinostomatid fluke was identified as Echinostoma hortense Asada, 1926. This is the first record on human E. hortense infection in Korea.

Les œufs operculés de plathelminthes : Descriptions d’atypies et essais d’explication de leur genèse

The authors remind the formation of operculated eggs. Atypical eggs of Clonorchis sinensis, Fasciola hepatica and Diphyllobothrium latum are described: no operculum for the two first species and existence of non-described buttons for the two last species. These anomalies could be explained by the way of formation of these eggs.