Molecular Analysis of Anisakis Type I Larvae in Marine Fish from Three Different Sea Areas in Korea

Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA cox1 revealed that the larvae showed interesting distribution patterns depending on fish species and geographical locations. Anisakis pegreffii was predominant in fish from the Yellow Sea and the South Sea. Meanwhile, both A. pegreffii and A. simplex sensu stricto (A. simplex s.str.) larvae were identified in fish from the East Sea, depending on fish species infected. These results suggested that A. pegreffii was primarily distributed in a diverse species of fish in 3 sea areas around Korea, but A. simplex s.str. was dominantly identified in Oncorhynchus spp. in the East Sea.     

In vitro and in vivo activity of three sesquiterpenes against L(3) larvae of Anisakis type I

In order to investigate the possible use of terpenic derivatives to treat anisakiasis caused by L₃ larvae of Anisakis, we studied the in vitro and in vivo larvicidal activity of three sesquiterpenes (nerolidol, farnesol and elemol). In vitro experiments included the histological study of larval damage and in vivo studies the measurement of myeloperoxidase activity in rat gastrointestinal tract after administration of the sesquiterpenes. In the in vitro assays, the most active compound against the L₃ larvae was nerolidol, followed by farnesol; both caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture. In the in vivo assays, only 20% of infected rats treated with nerolidol or farnesol showed gastric wall lesions in comparison to 86.6% of control animals. According to these results, nerolidol and farnesol are good candidates for further research as biocidal agents against L₃ larvae of Anisakis type I.     

A Case of Anisakidosis Caused by Pseudoterranova decipiens Larva

Pseudoterranova decipiens larva is a rare cause of anisakiasis. Indeed, prior to the present study, there had been only 12 reports of larval P. decipiens infection in the Republic of Korea. In June 2011, an anisakid larva, 32.1 mm in length and 0.88 mm in width, and finally identified as the third stage larva of P. decipiens owing to the presence of an intestinal cecum but lacking ventricular appendage, was discovered in a 61-year-old woman during the course of endoscopy executed as a part of routine physical examinations. The patient had eaten raw a rockfish 13 hr prior to the endoscopy, but showed no symptoms of anisakiasis. This paper is the 13th report of P. decipiens infection in Korea.     

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.     

Molecular Identification of Diphyllobothrium nihonkaiense from 3 Human Cases in Heilongjiang Province with a Brief Literature Review in China

Human diphyllobothriasis is a widespread fish-borne zoonosis caused by the infection with broad tapeworms belonging to the genus Diphyllobothrium. In mainland China, so far 20 human cases of Diphyllobothrium infections have been reported, and the etiologic species were identified as D. latum and D. nihonkaiense based on morphological characteristics or molecular analysis. In the present study, proglottids of diphyllobothriid tapeworms from 3 human cases that occurred in Heilongjiang Province, China were identified as D. nihonkaiense by sequencing mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 5 (nad5) genes. Two different cox1 gene sequences were obtained. One sequence showed 100% homology with those from humans in Japan. The remaining cox1 gene sequence and 2 different nad5 gene sequences obtained were not described previously, and might reflect endemic genetic characterizations. D. nihonkaiense might also be a major causative species of human diphyllobothriasis in China. Meanwhile, the finding of the first pediatric case of D. nihonkaiense infection in China suggests that infants infected with D. nihonkaiense should not be ignored.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Fatal Balamuthia Amebic Encephalitis in a Healthy Child: A Case Report with Review of Survival Cases

Balamuthia mandrillaris is one of the 4 amebas in fresh water and soil that cause diseases in humans. Granulomatous amebic encephalitis (GAE), caused by B. mandrillaris, is a rare but life-threatening condition. A 4-year-old, previously healthy, Thai girl presented with progressive headache and ataxia for over a month. Neuroimaging studies showed an infiltrative mass at the right cerebellar hemisphere mimicking a malignant cerebellar tumor. The pathological finding after total mass removal revealed severe necrotizing inflammation, with presence of scattered amebic trophozoites. Cerebrospinal fluid (CSF) obtained from lumbar puncture showed evidence of non-specific inflammation without identifiable organisms. A combination of pentamidine, sulfasalazine, fluconazole, and clarithromycin had been initiated promptly before PCR confirmed the diagnosis of Balamuthia amebic encephalitis (BAE). The patient showed initial improvement after the surgery and combined medical treatment, but gradually deteriorated and died of multiple organ failure within 46 days upon admission despite early diagnosis and treatment. In addition to the case, 10 survivors of BAE reported in the PubMed database were briefly reviewed in an attempt to identify the possible factors leading to survival of the patients diagnosed with this rare disease.     

Detection of Gnathostoma spinigerum Third-Stage Larvae in Snakeheads Purchased from a Central Part of Myanmar

To examine the infection status of freshwater fish with Gnathostoma spp. larvae in Myanmar, we purchased 15 snakeheads, Channa striatus, from a local market in a suburban area of Naypyidaw, the new capital city. Two larval gnathostomes were collected using an artificial digestion technique, and observed by a light microscope and a scanning electron microscope. The size of an intact larva was 2.65 mm long and 0.32 mm wide. The characteristic morphology of the larvae included the presence of a long esophagus (0.80 mm long), 2 pairs of cervical sacs (0.43 mm long), and a characteristic head bulb with 4 rows of hooklets. The number of hooklets in the 1st, 2nd, 3rd, and 4th row was 45, 48, 50, and 52, respectively. Based on these morphological characters, the larvae were identified as the advanced 3rd-stage larvae of Gnathostoma spinigerum. This is the first report of detection of G. spinigerum 3rd-stage larvae in the central part of Myanmar. Our study suggests that intake of raw meat of snakehead fish in Myanmar may result in human gnathostomiasis.     

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.     

Comorbid Gastric Adenocarcinoma and Gastric and Duodenal Strongyloides stercoralis Infection: A Case Report

Strongyloides stercoralis can cause systemic infection, termed strongyloidiasis, and gastrointestinal ulcer disease in immunocompromised patients. However, to our knowledge, there are no reported cases of comorbid gastric adenocarcinoma and S. stercoralis infection. Here, we report a case of an 81-year-old Korean man who presented with S. stercoralis infection coexisting with early gastric adenocarcinoma (T1aN0M0). S. stercoralis eggs, rhabditiform larvae, and adult females were observed in normal gastric and duodenal crypts. They were also observed in atypical glands representative of adenocarcinoma and adenoma. Preliminary laboratory tests revealed mild neutrophilic and eosinophilic leukocytosis. A routine stool test failed to detect rhabditiform larvae in the patient’s fecal sample; however, S. stercoralis was identified by PCR amplification and 18S rRNA sequencing using genomic DNA extracted from formalin-fixed paraffin-embedded tissues. Postoperatively, the patient had a persistent fever and was treated with albendazole for 7 days, which alleviated the fever. The patient was followed-up by monitoring and laboratory testing for 4 months postoperatively, and no abnormalities were observed thus far. The fact that S. stercoralis infection may be fatal in immunocompromised patients should be kept in mind when assessing high-risk patients.     

Trichuris trichiura Infection Diagnosed by Colonoscopy: Case Reports and Review of Literature

Trichuris trichiura, commonly referred to as a whipworm, has a worldwide distribution, particularly among countries with warm, humid climates. In Korea, trichuriasis was a highly prevalent soil-transmitted helminthiasis until the 1970s. However, the nationwide prevalence decreased to 0.02% in 2004 as a result of national control activities and improvement in the socioeconomic status of Koreans. Most infected individuals have no distinct symptoms, if lightly infected. The diagnosis is typically confirmed by detection of T. trichiura eggs on examination of a stool sample; few reports have described detection of the parasite during colonoscopy. Recently, we managed 4 patients with trichuriasis who were diagnosed by detection of the parasite on colonoscopy, and we reviewed the literature on the colonoscopic diagnosis of T. trichiura in Korea. We suggest that colonoscopy might be a useful diagnostic tool, especially when infected by only a few male worms with no eggs in the stool.     

First Report of Feline Intestinal Trichomoniasis Caused by Tritrichomonas foetus in Korea

Feline intestinal tritrichomoniasis by Tritrichomonas foetus was first recognized in USA in 1999 and has so far been reported from UK, Norway, Switzerland, and Australia, but not from the Far East Asian countries. In November 2008, 2 female and male littermate Siamese cats, 6-month old, raised in a household in Korea were referred from a local veterinary clinic with a history of chronic persistent diarrhea. A direct smear examination of fecal specimens revealed numerous trichomonad trophozoites which were isolated by the fecal culture in InPouch™ TF-Feline medium. A PCR testing of the isolate based on the amplification of a conserved portion of the T. foetus internal transcribed spacer (ITS) regions (ITS1 and ITS2) and the 5.8S rRNA gene, and the molecular sequencing of the PCR amplicons confirmed infection with T. foetus. This is the first clinical case of feline intestinal trichomoniasis caused by T. foetus in Korea.     

Pathogenicity of Korean isolates of Acanthamoeba by observing the experimental infection and zymodemes of five isoenzymes.

To determine the pathogenicity of Acanthamoeba spp. isolated in Korea and to develop a isoenzymatic maker, the mortality rate of infected mice, in vitro cytotoxicity against target cells and isoenzyme band patterns were observed. Five isolates of Acanthamoeba spp. (YM-2, YM-3, YM-4, YM-5, and YM-7) were used in this study as well as three reference Acanthamoeba spp. (A. culbertsoni, A. hatchetti, and A. royreba). According to the mortality rate of infected mice, Korean isolates could be categorized into three groups high virulent (YM-4), low virulent (YM-2, YM-5, YM-7) and the nonpathogenic group (YM-3). In addition, the virulence of Acanthamoeba spp. was enhanced by brain passage in mice. In the cytotoxicity assay against chinese hamster ovary cells, especially, the cytotoxicity of brain-passaged amoebae was relatively higher than the long-term cultivated ones. The zymodeme patterns of glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), hexokinase (HK), glutamate oxaloacetate transaminase (GOT) and malic enzyme (ME) of Acanthamoeba spp. were different among each isolate, and also between long-term cultured amoebae and brain passaged ones. In spite of the polymorphic zymodemes, a slow band of G6PD and HK, and an intermediate band of MDH were only observed in pathogenic Acanthamoeba spp., which should be used as isoenzymatic makers.     

First Report of Myiasis Caused by Cochliomyia hominivorax (Diptera: Calliphoridae) in a Diabetic Foot Ulcer Patient in Argentina

Myiasis is usually caused by flies of the Calliphoridae family, and Cochliomyia hominivorax is the etiological agent most frequently found in myiasis. The first case of myiasis in a diabetic foot of a 54-year-old male patient in Argentina is reported. The patient attended the hospital of the capital city of Tucumán Province for a consultation concerning an ulcer in his right foot, where the larval specimens were found. The identification of the immature larvae was based on their morphological characters, such as the cylindrical, segmented, white yellow-coloured body and tracheas with strong pigmentation. The larvae were removed, and the patient was treated with antibiotics. The larvae were reared until the adults were obtained. The adults were identified by the setose basal vein in the upper surface of the wing, denuded lower surface of the wing, short and reduced palps, and parafrontalia with black hairs outside the front row of setae. The main factor that favoured the development of myiasis is due to diabetes, which caused a loss of sensibility in the limb that resulted in late consultation. Moreover, the poor personal hygiene attracted the flies, and the foul-smelling discharge from the wound favoured the female''s oviposition. There is a need to implement a program for prevention of myiasis, in which the population is made aware not only of the importance of good personal hygiene and home sanitation but also of the degree of implication of flies in the occurrence and development of this disease.     

Two cases of human thelaziasis and brief review of Korean cases

The present study was performed to describe 2 cases of human thelaziasis (HT) which occurred in Gyeongsangnam- do and to briefly review the previously reported Korean cases. A 58-year old woman, residing in Hadong-gun, Gyeongsangnam-do, came to Gyeongsang National University Hospital (GNUH) complaining of foreign body sensation and itching of the right eye in March 2000. Total 6 adult nematodes of Thelazia callipaeda (2 males and 4 females) were detected in her right eye. A 80-year old man, residing in Jinju-si, Gyeongsangnam-do, came to GNUH complaining of foreign body sensation, itching, and pain of the right eye in December 2007. A total of 5 worms (4 females and 1 degenerated) were removed from his right eye. We analyzed characteristics of the total 39 Korean HT cases reported to date, including the present 2 cases. Most of the cases (71.8%) occurred in Seoul and Gyeonggi-do before 2000, and 21 cases (53.8%) were males and 18 (46.2%) were females. The prevalence was higher in younger ages below 30 years (48.7%) than 31-60 years (41.0%) and over 61 years (10.3%). The seasonal prevalence showed a higher incidence in autumn (43.6%) than in other seasons. Most of the cases (94.9%) were conjunctival sac infections and only 2 (5.1%) were intraocular cases. The present 2 HT cases are the first reported cases in Gyeongsangnam-do. Some characteristics of Korean HT cases were analyzed.     

Identification and Clinical Implications of Novel MYO15A Mutations in a Non-consanguineous Korean Family by Targeted Exome Sequencing

Mutations of MYO15A are generally known to cause severe to profound hearing loss throughout all frequencies. Here, we found two novel MYO15A mutations, c.3871C>T (p.L1291F) and c.5835T>G (p.Y1945X) in an affected individual carrying congenital profound sensorineural hearing loss (SNHL) through targeted resequencing of 134 known deafness genes. The variant, p.L1291F and p.Y1945X, resided in the myosin motor and IQ2 domains, respectively. The p.L1291F variant was predicted to affect the structure of the actin-binding site from three-dimensional protein modeling, thereby interfering with the correct interaction between actin and myosin. From the literature analysis, mutations in the N-terminal domain were more frequently associated with residual hearing at low frequencies than mutations in the other regions of this gene. Therefore we suggest a hypothetical genotype-phenotype correlation whereby MYO15A mutations that affect domains other than the N-terminal domain, lead to profound SNHL throughout all frequencies and mutations that affect the N-terminal domain, result in residual hearing at low frequencies. This genotype-phenotype correlation suggests that preservation of residual hearing during auditory rehabilitation like cochlear implantation should be intended for those who carry mutations in the N-terminal domain and that individuals with mutations elsewhere in MYO15A require early cochlear implantation to timely initiate speech development.     

Bronchopulmonary Infection of Lophomonas blattarum: A Case and Literature Review

Human infections with Lophomonas blattarum are rare. However, the majority of the infections occurred in China, 94.4% (136 cases) of all cases in the world. This infection is difficult to differentiate from other pulmonary infections with similar symptoms. Here we reported a case of L. blattarum infection confirmed by bronchoalveolar lavage fluid smear on the microscopic observations. The patient was a 21-year-old female college student. The previous case which occurred in Chongqing was 20 years ago. We briefly reviewed on this infection reported in the world during the recent 20 years. The epidemiological characteristics, possible diagnostic basis, and treatment of this disease is discussed in order to provide a better understanding of recognition, diagnosis, and treatment of L. blattarum infection.     

Paragonimiasis in the Abdominal Cavity and Subcutaneous Tissue: Report of 3 Cases

Paragonimiasis is a parasitic disease caused by the lung fluke, Paragonimus spp. Lung flukes may be found in various organs, such as the brain, peritoneum, subcutaneous tissues, and retroperitoneum, other than the lungs. Abdominal paragonimiasis raises a considerable diagnostic challenge to clinicians, because it is uncommon and may be confused with other abdominopelvic inflammatory diseases, particularly peritoneal tuberculosis, and peritoneal carcinomatosis. Also, subcutaneous paragonimiasis does not easily bring up clinical suspicion, due to its rarity. We herein report 2 cases of abdominal paragonimiasis and 1 case of subcutaneous paragonimiasis in Korea.     

Anisakis simplex larvae: infection status in marine fish and cephalopods purchased from the Cooperative Fish Market in Busan, Korea

The infection status of marine fish and cephalopods with Anisakis simplex third stage larva (L3) was studied over a period of 1 year. A total of 2,537 specimens, which consisted of 40 species of fish and 3 species of cephalopods, were purchased from the Cooperative Fish Market in Busan, Korea, from August 2006 to July 2007. They were examined for A. simplex L3 from the whole body cavity, viscera, and muscles. A. simplex L3 were confirmed by light microscopy. The overall infection rate reached 34.3%, and average 17.1 larvae were parasitized per infected fish. Fish that recorded the highest infection rate was Lophiomus setigerus (100%), followed by Liparis tessellates (90%), Pleurogrammus azonus (90%), and Scomber japonicus (88.7%). The intensity of infection was the highest in Gadus macrocephalus (117.7 larvae per fish), followed by S. japonicus (103.9 larvae) and L. setigerus (54.2 larvae). Although abundance of A. simplex L3 was not seasonal in most of the fish species, 10 of the 16 selected species showed the highest abundance in February and April. A positive correlation between the intensity of L3 infection and the fish length was obvious in S. japonicus and G. macrocephalus. It was likely that A. simplex L3 are more frequently infected during the spring season in some species of fish. Our study revealed that eating raw or undercooked fish or cephalopods could still be a source of human infection with A. simplex L3 in Korea.     

The I2 resistance gene homologues in Solanum have complex evolutionary patterns and are targeted by miRNAs

Background

Several resistance traits, including the I2 resistance against tomato fusarium wilt, were mapped to the long arm of chromosome 11 of Solanum. However, the structure and evolution of this locus remain poorly understood.

Results

Comparative analysis showed that the structure and evolutionary patterns of the I2 locus vary considerably between potato and tomato. The I2 homologues from different Solanaceae species usually do not have orthologous relationship, due to duplication, deletion and frequent sequence exchanges. At least 154 sequence exchanges were detected among 76 tomato I2 homologues, but sequence exchanges between I2 homologues in potato is less frequent. Previous study showed that I2 homologues in potato were targeted by miR482. However, our data showed that I2 homologues in tomato were targeted by miR6024 rather than miR482. Furthermore, miR6024 triggers phasiRNAs from I2 homologues in tomato. Sequence analysis showed that miR6024 was originated after the divergence of Solanaceae. We hypothesized that miR6024 and miR482 might have facilitated the expansion of the I2 family in Solanaceae species, since they can minimize their potential toxic effects by down-regulating their expression.

Conclusions

The I2 locus represents a most divergent resistance gene cluster in Solanum. Its high divergence was partly due to frequent sequence exchanges between homologues. We propose that the successful expansion of I2 homologues in Solanum was at least partially attributed to miRNA mediated regulation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-743) contains supplementary material, which is available to authorized users.