Dirofilaria repens in Vietnam: detection of 10 eye and subcutaneous tissue infection cases identified by morphology and molecular methods

From 2006 to 2010, hospitals in Hanoi treated 10 human patients for dirofilariasis. The worms were collected from parasitic places, and identification of the species was completed by morphology and molecular methods. Ten parasites were recovered either from the conjunctiva (n=9) or subcutaneous tissue (n=1). The parasites were 4.0-12.5 cm in length and 0.5-0.6 mm in width. Morphological observations suggested all parasites as Dirofilaria repens. Three of the 10 parasites (1 from subcutaneous tissue and 2 from eyes) were used for molecular confirmation of the species identification. A portion of the mitochondrial cox1 (461 bp) was amplified and sequenced. Nucleotide and amino acid homologies were 95% and 99-100%, respectively, when compared with D. repens (Italian origin, GenBank AJ271614; DQ358814). This is the first report of eye dirofilariasis and the second report of subcutaneous tissue dirofilariasis due to D. repens in Vietnam.     

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.     

Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

A Case of Ocular Angiostrongyliasis with Molecular Identification of the Species in Vietnam

A 23-year-old female residing in a village of Cao Bang Province, North Vietnam, visited the Hospital of Hanoi Medical University in July 2013. She felt dim eyes and a bulge-sticking pain in her left eye for some days before visiting the hospital. In the hospital, a clinical examination, an eye endoscopy, and an operation were carried out. A nematode specimen was collected from the eye of this patient. The body of this worm was thin and long and measured 22.0×0.3 mm. It was morphologically suggested as an immature female worm of Angiostrongylus cantonensis. By a molecular method using 18S rRNA gene, this nematode was confirmed as A. cantonensis. This is the first molecular study for identification of A. cantonensis in Vietnam.     

Orbital Dirofilariasis in Iran: A Case Report

Dirofilariasis is a common parasitic disease in both domestic and wild animals around the world, with canines as the principal reservoir host and mosquitoes as the vector. Human ophthalmic dirofilariasis is an uncommon condition, but there have been reports from many parts of the world, including Africa, Australia, the Americas, Europe, and Asia. Ophthalmic involvement with Dirofilaria may present itself as periorbital, subconjunctival, orbital, or intraocular infections. In this report, we present a case of orbital dirofilariasis with lateral rectus muscle involvement. To our knowledge, this is the first orbital dirofilariasis case reported in Iran. Although debulking of the tumor usually leads to resolution, our patient showed an episode of recurrence after biopsy. Complete recovery was achieved after a spontaneous discharge, without the need for systemic medication.     

A Human Case of Clinostomum complanatum Infection in Korea

A 33-year-old Korean man visited a medical clinic with complaints of throat discomfort and pain for one week. The symptoms occurred one day after eating raw brackish water fish, perch. Endoscopy revealed a fluke, about 5 mm in length, attaching to and peristaltically moving on the surface of the mucosa at the arytenoid region of the larynx. Microscopically, the testes were triangular, tandem, and separated by the uterus. The ovary and cirrus pouch were placed apart from median line between testes. Numerous blood cells were observed in the ceca. The worm was identified to be Clinostomum complanatum. This is the second human case of clinostomiasis in Korea.     

Sparganosis Presenting as Cauda Equina Syndrome with Molecular Identification of the Parasite in Tissue Sections

A 52-year-old woman presented with lower back pain, progressive symmetrical paraparesis with sensory impairment, and sphincter disturbance. Magnetic resonance imaging (MRI) of the whole spine revealed multiple intradural extramedullary serpiginous-mass lesions in the subarachnoid space continuously from the prepontine to the anterior part of the medulla oblongata levels, C7, T2-T8, and T12 vertebral levels distally until the end of the theca sac and filling-in the right S1 neural foramen. Sparganosis was diagnosed by demonstration of the sparganum in histopathological sections of surgically resected tissues and also by the presence of serum IgG antibodies by ELISA. DNA was extracted from unstained tissue sections, and a partial fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using a primer set specific for Spirometra spp. cox1. After sequencing of the PCR-amplicon and alignment of the nucleotide sequence data, the causative agent was identified as the larva of Spirometra erinaceieuropaei.     

A Case of Diphyllobothrium nihonkaiense Infection as Confirmed by Mitochondrial COX1 Gene Sequence Analysis

Diphyllobothrium nihonkaiense has been reported in Korea as Diphyllobothrium latum because of their close morphologic resemblance. We have identified a human case of D. nihonkaiense infection using the mitochondrial cytochrome c oxidase subunit I (cox1) gene sequence analysis. On 18 February 2012, a patient who had consumed raw fish a month earlier visited our outpatient clinic with a long tapeworm parasite excreted in the feces. The body of the segmented worm was 2 m long and divided into the scolex (head) and proglottids. It was morphologically close to D. nihonkaiense and D. latum. The cox1 gene analysis showed 99.4% (340/342 bp) homology with D. nihonkaiense but only 91.8% (314/342 bp) homology with D. latum. The present study suggested that the Diphyllobothrium spp. infection in Korea should be analyzed with specific DNA sequence for an accurate species identification.     

Diagnostic Probes Targeting the Major Sperm Protein Gene That May Be Useful in the Molecular Identification of Nematodes

Discrimination of closely related nematode species is typically problematic when traditional identification characteristics are prone to intraspecific variation. In this study, a molecular approach that can distinguish Pratylenchus penetrans and P. scribneri is described. The approach uses universal primers in conjunction with polymerase chain reaction (PCR) to amplify equivalent fragments of the major sperm protein (msp) gene from any nematode. This gene fragment typically includes an intron of variable sequence. The presence of this highly variable segment in an otherwise conserved gene sequence allows P. penetrans and P. scribneri to be distinguished by either a species-specific amplification or by dot-blot hybridization. The approach is potentially of general utility in species-specific identification of nematodes.     

Human Infections with Spirometra decipiens Plerocercoids Identified by Morphologic and Genetic Analyses in Korea

Tapeworms of the genus Spirometra are pseudophyllidean cestodes endemic in Korea. At present, it is unclear which Spirometra species are responsible for causing human infections, and little information is available on the epidemiological profiles of Spirometra species infecting humans in Korea. Between 1979 and 2009, a total of 50 spargana from human patients and 2 adult specimens obtained from experimentally infected carnivorous animals were analyzed according to genetic and taxonomic criteria and classified as Spirometra erinaceieuropaei or Spirometra decipiens depending on the morphology. Morphologically, S. erinaceieuropaei and S. decipiens are different in that the spirally coiled uterus in S. erinaceieuropaei has 5-7 complete coils, while in S. decipiens it has only 4.5 coils. In addition, there is a 9.3% (146/1,566) sequence different between S. erinaceieuropaei and S. decipiens in the cox1 gene. Partial cox1 sequences (390 bp) from 35 Korean isolates showed 99.4% (388/390) similarity with the reference sequence of S. erinaceieuropaei from Korea (G1724; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) and an additional 15 Korean isolates revealed 99.2% (387/390) similarity with the reference sequences of S. decipiens from Korea (G1657; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679). Based on morphologic and molecular databases, the estimated population ratio of S. erinaceieuropaei to S. decipiens was 35: 15. Our results indicate that both S. erinaceieuropaei and S. decipiens found in Korea infect humans, with S. erinaceieuropaei being 2 times more prevalent than S. decipiens. This study is the first to report human sparganosis caused by S. decipiens in humans in Korea.     

In Vitro Culture of Subanguina picridis in Acroptilon repens Callus,Excised Roots,and Shoot Tissues

The knapweed nematode Subanguina picridis is a foliar parasite that is of interest as a biological weed control agent of Russian knapweed. Attempts were made to culture the nematode in callus, excised roots and in shoots derived from roots of Russian knapweed. In callus tissues, the nematode developed from second-stage juvenile to adult but failed to reproduce; it developed only to the fourth stage in excised roots. However, S. picridis was successfully cultured in vitro in shoots derived from roots. The nematode induced galls on the leaves, petioles, and shoot apices and developed and reproduced inside the galls. Gibberellic acid increased the development rate of the nematode and promoted the formation of males. This is the first gnotobiotic culture of a nematode used for biological weed control.     

Taxonomic and Molecular Identification of Mesocriconema and Criconemoides Species (Nematoda: Criconematidae)

Populations of Mesocriconema curvatum, M. kirjanovae, M. onoense, M. ornatum, M. sphaerocephala, M. surinamense, M. vadense, M. xenoplax, and Criconemoides informis from different geographical areas in the continental United States were characterized morphologically and molecularly. A new ring nematode from Washington County, Arkansas, is also described and named Mesocriconema ozarkiense n. sp., This new species is characterized by females with small flattened submedian lobes, lower than or at the same level as the labial disc, vagina straight, very well developed spermatheca without sperm, no more than one anastomoses, L=379-512 μm, V=89-93, stylet length = 49-61 μm, R=107-119, annuli with slightly crenate margins on tail portion and a simple anterior vulval lip. The molecular characterization of M. ozarkiense n. sp. using the ITS rRNA gene sequence and the phylogenesis relationship of this new species with the ring nematodes included in this study are provided.     

The First Human Case of Thelazia callipaeda Infection in Vietnam

A 26-year-old man residing in a village of Thai Nguyen Province, North Vietnam, visited the Thai Nguyen Provincial Hospital in July 2008. He felt a bulge-sticking pain in his left eye and extracted 5 small nematode worms by himself half a day before visiting the hospital. Two more worms were extracted from his left eye by a medical doctor, and they were morphologically observed and genetically analyzed on the mitochondrial cytochrome c oxidase 1 gene. The worms were 1 male and 1 female, and genetically identical with those of Thelazia callipaeda. By the present study, the presence of human T. callipaeda infection is first reported in Vietnam.     

Sympatric Distribution of Three Human Taenia Tapeworms Collected between 1935 and 2005 in Korea

Taeniasis has been known as one of the prevalent parasitic infections in Korea. Until recently, Taenia saginata had long been considered a dominant, and widely distributed species but epidemiological profiles of human Taenia species in Korea still remain unclear. In order to better understand distribution patterns of human Taenia tapeworms in Korea, partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were determined, along with morphological examinations, on 68 Taenia specimens obtained from university museum collections deposited since 1935. Genomic DNA was extracted from formalin-preserved specimens. Phylogenetic relationships among the genotypes (cox1 haplotype) detected in this study were inferred using the neighbor-joining method as a tree building method. Morphological and genetic analyses identified 3 specimens as T. solium, 51 specimens as T. asiatica, and 14 specimens as T. saginata. Our results indicate that all 3 Taenia tapeworms are sympatrically distributed in Korea with T. asiatica dominating over T. saginata and T. solium.     

Serological and Molecular Characteristics of the First Korean Case of Echinococcus multilocularis

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient''s serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.     

Taxonomic and Molecular Identification of Bakernema,Criconema, Hemicriconemoides,Ogma and Xenocriconemella Species (Nematoda: Criconematidae)

Populations of Bakernema inaequale, C. petasum, C. sphagni, C. mutabile, Ogma octangulare, Xenocriconemella macrodora and Hemicriconemoides chitwoodi were identified and re-described from different geographical areas in the continental United States and molecularly characterized. Two new species of spine nematodes Criconema arkaense n. sp. from Washington County and Lee County, Arkansas and Criconema warrenense n. sp from Warren, Bradley County, Arkansas are also described and named. Criconema arkaense is characterize by having a conspicuous lip region offset from the body with two annuli, short rounded tail with a thin cuticular sheath and subterminal anus. Criconema warrenense n. sp. has two lip region annuli about the same width, first annulus directed posteriorly, separated by a narrow neck annulus and a short conoid tail, unilobed non-folded annulus. The molecular characterization of Criconema arkaense and Criconema warrenense using ITS1 rDNA gene sequence and the molecular phylogenetic relationships of these new species along with the known spines nematodes are provided.     

Infection Status of Estuarine Fish and Oysters with Intestinal Fluke Metacercariae in Muan-gun, Jeollanam-do, Korea

The source of human infection with intestinal flukes was surveyed in estuarine fishes, including the dotted gizzard shad, common sea bass, common blackish goby, redlip mullet, black sea bream, and oyster collected from Muan-gun, Jeollanam-do, Korea during August and September 2007. Collected fishes and oysters were artificially digested in pepsin-HCl solution and examined under a stereomicroscope. In 36 shads (Konosirus punctatus) and 20 basses (Lateolabrax japonicus) examined, Heterophyopsis continua metacercariae were found in 58.3% and 100%, and their average numbers were 12.0 and 6.3 per infected fish, respectively. In 34 gobies (Acanthogobius flavimanus) examined, metacercariae of H. continua were detected in 79.4%, Stictodora lari in 97.1%, and Acanthotrema felis in 92.1%, and their average numbers were 45.8, 189.3, and 235.3 per infected fish, respectively. In 37 redlip mullets (Chelon haematocheilus), Heterophyes nocens metacercariae were found in 56.8%, Pygidiopsis summa in 94.6%, and Stictodora fuscata in 45.9%, and the average metacercarial densities were 17.4, 31.3, and 35.1 per infected fish, respectively. In 30 black sea breams (Acanthopagrus schlegeli) and 45 oysters (Crassostrea gigas) examined, no metacercariae were detected. From the above results, it has been confirmed that the dotted gizzard shad, common sea bass, common blackish goby, and redlip mullet from Muan-gun, Jeollanam-do, Korea are infected with the metacercariae of heterophyid flukes.     

Rapid Detection and Identification of Wuchereria bancrofti,Brugia malayi,B. pahangi,and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.     

The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.