Establishment of an animal model of Sparganum mansoni infection and study on therapeutic methods II Establishment of a mouse model of sparganosis mansoni via oral administration of procercoids / 中国血吸虫病防治杂志

Objective To establish an animal model of sparganosis mansoni through oral administration of Cyclops infected with procercoids. Methods Domestic cats were infected with Sparganum mansoni under laboratory conditions, and fresh cat stool samples were collected, washed in dechlorinated water, and filtered. Spirometra mansoni eggs were collected and prepared into suspensions. Twenty C57BL/6j mice were randomly divided into the experimental group (n = 15) and the control group (n = 5). Wild Cyclops were infected with Spirometra mansoni coracidia to allow 3 to 5 procercoids in each Cyclop. Then, each mouse in the experimental group was given 15 Cyclops infected with procercoids by gavage, while mice in the control group were orally administered with the same volume of dechlorinated water. All mice were sacrificed after 5 months, and dissected, and suspicious Sparganum mansoni worms were collected. The serum specific IgG antibody against Sparganum mansoni was measured in mice using enzyme-linked immunosorbent assay (ELISA). Genomic DNA was isolated from suspicious Sparganum mansoni worms, and the specific Sparganum mansoni cytochrome oxidase I (COI) gene was amplified using PCR assay. Results Among the 15 mice in the experimental group, six were positive for the serum specific IgG antibody against Sparganum mansoni, and milky white worms were found and collected from the subcutaneous regions of 4 out of 6 mice. Only one worm was detected in each mouse, and the worm morphology was similar to Sparganum mansoni. Capillary electrophoresis of the PCR amplification products of COI gene presented a specific band with 151 bp in size, and sequencing analysis revealed 100% homology with Sparganum mansoni. Conclusions A mouse model of sparganosis mansoni is successfully created through oral administration of Cyclops infected with Spirometra mansoni procercoids.

Construction of a cDNA library for Sparganum mansoni and screening of diagnostic antigen cadidates / 中国血吸虫病防治杂志

Objective To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. Methods Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. Results The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. Conclusions A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

Breast Sparganosis Presenting with a Painless Breast Lump: Report of Two Cases

Sparganosis is a parasitic infestation caused by sparganum, a plerocercoid tapeworm larva of the genus Spirometra. Since the first case of human sparganosis reported in 1908, sparganosis has been a global disease, and is common in China, Japan, and Southeast Asian countries. Consumption of raw snakes, frogs, fish, or drinking contaminated beverages are sources of human infections. Human sparganosis usually manifests in subcutaneous fat in areas such as the abdomen, genitourinary tract, and limbs. Breast sparganosis cases are rare, representing less than 2% of total cases of human infections. Complete surgical extraction of the sparganum is the treatment of choice. Because of the rarity of the disease, clinical suspicion is vital to reach the diagnosis of breast sparganosis. Here we report 2 rare cases of breast sparganosis presenting with a painless breast lump, both treated with surgical excision and sparganum extraction.

Human Sparganosis in Korea

BACKGROUND: Sparganosis is a larval cestodiasis caused by the plerocercoid of Spirometra spp. Since the first description of human sparganosis in 1924, several hundred cases have been reported in Korea. However, systematic approaches for literature surveys of Korean sparganosis have seldom appeared. METHODS: We searched publicly available databases such as PubMed, Research Information Sharing Service, and Korea Medical Citation Index with relevant Medical Subject Headings. RESULTS: At least 438 Korean sparganosis cases have been described from 1924 to 2015. Preoperative diagnosis has been significantly increased since the 1980s due to popularization of serological and imaging diagnostics. Cases were largely detected from fifth decades in general, but cerebral sparganosis was detected in relatively young age groups (third and fourth decades). Sparganosis was prevalent in men (75.9%). Consumption of frog/snake and drinking unfiltered water were found in 63.4% and 16.9% of patients, respectively. Most frequently affected sites were subcutaneous tissues (49.9%), followed by the central nervous system (36.2%). Involvements of visceral organs (7.6%), ocular regions (3.6%), and muscles (2.7%) were noticed. In women, breast sparganosis constituted a large proportion (34.2%). Sparganosis associated with immunocompromised patients has recently been reported. CONCLUSION: Sparganosis has been continuously reported in Korea during the past 90 years, although its incidence has decreased during the last 20 years. The disease is mostly characterized by subcutaneous nodule, but infection of the worm in vital organs often results in serious illness. Continuous awareness is warranted to monitor sparganosis occurrence and associated clinical consequences.

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei (KJ599680) or S. decipiens (KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).

Molecular Detection of Spirometra decipiens in the United States

The genus Spirometra belongs to the family Diphyllobothriidae and order Pseudophyllidea, and includes intestinal parasites of cats and dogs. In this study, a plerocercoid labeled as Spirometra mansonoides from the USA was examined for species identification and phylogenetic analysis using 2 complete mitochondrial genes, cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit 3 (nad3). The cox1 sequences (1,566 bp) of the plerocercoid specimen (USA) showed 99.2% similarity to the reference sequences of the plerocercoid of Korean Spirometra decipiens (GenBank no. KJ599679), and 99.1% similarity in regard to nad3 (346 bp). Phylogenetic tree topologies generated using 4 analytical methods were identical and showed high confidence levels with bootstrap values of 1.00, 100%, 100%, and 100% for Bayesian inference (BI), maximum-likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methods, respectively. Representatives of Diphyllobothrium and Spirometra species formed a monophyletic group, and the sister-genera status between these species was well supported. Trapezoic proglottids in the posterior 1/5 region of an adult worm obtained from an experimentally infected cat were morphologically examined. The outer uterine loop of the uterus coiling characteristically consisted of 2 complete turns. The results clearly indicated that the examined Spirometra specimen from the USA matched to S. decipiens very well, and indicated possible presence of the life cycle of this species in this region.

Infestación ocular por plerocercoide de Spirometra mansonoides: primer reporte en el Perú / Ocular infestation by plerocercoid of Spirometra mansonoides: first case report in Perú

La plerocercoidiosis es una zoonosis parasitaria producida por la larva plerocercoide de Spirometra. Esta larva migra por el tejido de la pared intestinal, mediante la ruta subcutánea y puede llegar a diferentes áreas del cuerpo humano como la cabeza, el cerebro y la órbita ocular. Se reporta el caso de un varón de 45 años procedente de la Amazonía peruana, quien presentó ardor asociado con edema y hemorragia conjuntival en el borde ocular externo del ojo derecho, durante once meses. Se observó un helminto localizado en la cavidad orbitaria derecha, el cual se extrajo y por estudios morfológicos e histopatológicos se identificó como Spirometra mansonoides de localización ocular el que se reporta por primera vez en el Perú.

Plerocercoidosis is a parasitic zoonosis caused by plerocercoid larvae of the genus Spirometra. The larvae migrate through the intestinal wall tissue, by subcutaneous route and can reach different areas of the body like the head, the brain and the eye socket. A case is reported of a 45 year-old man from the Peruvian Amazon with burning sensation associated with conjunctival edema and hemorrhage in the outer eye border of the right eye for eleven months. A localized worm in the right orbital cavity was observed, which was extracted. Morphological and histopathological studies identified it as Spirometra mansonoides localized in the eye, which is the first case reported in Perú.

Diagnostic Efficacy of a Recombinant Cysteine Protease of Spirometra erinacei Larvae for Serodiagnosis of Sparganosis

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient's sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.

Recurred Sparganosis 1 Year after Surgical Removal of a Sparganum in a Korean Woman

Sparganosis, an infection due to the plerocercoid of Spirometra erinacei, are found worldwide but the majority of cases occur in East Asia including Korea. This report is on a recurred case of sparganosis in the subcutaneous tissue of the right lower leg 1 year after a surgical removal of a worm from a similar region. At admission, ultrasonography (USG) of the lesion strongly suggested sparganosis, and a worm was successfully removed which turned out to be a sparganum with scolex. Since sparganum has a variable life span, and may develop into a life-threatening severe case, a patient once diagnosed as sparganosis should be properly followed-up for a certain period of time. Although imaging modalities were useful for the diagnosis of sparganosis as seen in this case, serological test such as ELISA should also be accompanied so as to support the preoperative diagnosis.

Scrotal Sparganosis Mimicking Scrotal Teratoma in an Infant: A Case Report and Literature Review

Sparganosis is an infection with a parasitic tapeworm larva that occurs by eating infected foods or drinking contaminated water. The larvae can migrate to a tissue or muscle in the chest, abdominal wall, extremities, eyes, brain, urinary tract, pleura, pericardium, spinal canal, or scrotum. Herein, we report a 5-month old infant with scrotal sparganosis who was initially suspected to have a scrotal inflammatory mass with a history of applying raw frog meat into the umbilicus. Preoperative ultrasound examinations and computed tomography (CT) scanning misdiagnosed the mass as a scrotal teratoma. The scrotal mass was surgically removed, and the histopathology proved it to be scrotal sparganosis. This case displays the youngest patient ever reported with scrotal sparganosis, and the first description of CT characteristics of scrotal sparganosis. A detailed medical history is necessary for patients with scrotal masses suspected of sparganosis. In addition, ultrasound and CT examinations are helpful to rule out other causes of a scrotal mass.

Spargana in a Weasel, Mustela sibirica manchurica, and a Wild Boar, Sus scrofa, from Gangwon-do, Korea

To know the status of sparganum (plerocercoid of Spirometra erinacei) infection in the Korean wild life, several species of wild animals were captured in Gangwon-do and examined for their status of infection with spargana. From February to December 2011, a total of 62 wild boars, 5 badgers, 1 weasel, 1 Siberian chipmunk, and 53 wild rodents were captured, and their whole muscles were examined with naked eyes for the presence of spargana worms. From the weasel and 1 wild boar, a total of 5 spargana specimens were extracted. The weasel was for the first time recorded as an intermediate or paratenic/transport host of S. erinacei in Korea, and both the weasel (Mustela sibirica manchurica) and wild boar (Sus scrofa) were added to the list of wild animals carrying spargana.

Massive Scrotal Hematoma with Active Bleeding Caused by Sparganum

Development of swelling and pain without trauma in a scrotal hematoma is very rare. We report on a case of scrotal hematoma with active bleeding caused by sparganosis. A 75-year-old male patient who presented with left scrotal swelling and moderate pain that started one day ago was admitted to the emergency department. On the computed tomography (CT) scan, a hematoma of greater than 10 cm was observed in the left scrotum and contrast extravasation was observed on the post-enhanced CT scan. Therefore, we concluded massive left scrotal hematoma with active bleeding. The patient underwent immediate surgery, and approximately 200 cc of hematoma was evacuated, and a movable whitish colored sparganum was found and removed. In cases involving development of scrotal hematoma without trauma, confirmed by surgery, sparganosis should be suspected, and should be removed.

A Case of Inguinal Sparganosis Mimicking Myeloid Sarcoma

We report here a case of inguinal sparganosis, initially regarded as myeloid sarcoma, diagnosed in a patient undergone allogeneic hematopoietic transplantation (HSCT). A 56-year-old male patient having myelodysplastic syndrome was treated with allogeneic HSCT after myeloablative conditioning regimen. At day 5 post-HSCT, the patient complained of a painless palpable mass on the left scrotum and inguinal area. Pelvic magnetic resonance imaging and computed tomography revealed suspected myeloid sarcoma. Gun-biopsy was performed, and the result revealed eosinophilic infiltrations without malignancy. Subsequent serologic IgG antibody test was positive for sparganum. Excisional biopsy as a therapeutic diagnosis was done, and the diagnosis of sparganosis was confirmed eventually. This is the first report of sparganosis after allogeneic HSCT mimicking myeloid sarcoma, giving a lesson that the physicians have to consider the possibility of sparganosis in this clinical situation and perform adequate diagnostic and therapeutic approaches.

Electron Microscopy of the Separated Outer Tegument of the Sparganum and Its Antigenicity

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.

Case of sparganosis: A diagnostic dilemma.

Sparganosis, also known as larval diphyllobothriasis, is a rare disease of humans as man is not a natural host in the life cycle of Spirometra spp. Diagnosis of the latter is difficult as it mimics other conditions that commonly cause subcutaneous or visceral fluid collection. Clinical diagnosis of this particular case was also erroneously labelled as tuberculosis but later labelled as a case of sparganosis. To the best of our knowledge, this is the first case from India where a sparganum-like parasite was isolated in drain fluid from the perinephric area.

Human ocular sparganosis in southern Brazil / Esparganose ocular humana no sul do Brasil

We report the first case of human ocular sparganosis in the state of Santa Catarina, southern Brazil. A young female patient presented with three periocular moveable inflammatory masses in her right eye, during two years. By surgical excisional biopsy, a helminth larval stage was removed and identified as sparganum. Clinical, laboratory and epidemiological data on this parasite are presented.

Registra-se o primeiro caso de esparganose ocular humana no estado de Santa Catarina, sul do Brasil a partir de paciente adulta que apresentou três massas inflamatórias móveis perioculares, localizadas no olho direito, durante dois anos. Com a excisão cirúrgica o material foi para a biópsia e um estágio larval de helminto foi identificado como espargano. Dados clínicos, laboratoriais e epidemioógicos são apresentados neste trabalho.

Expression of TNF-alpha and IL-1beta in Splenic Dendritic Cells and Their Serum Levels in Mouse Sparganosis

Sparganosis is a tissue invading helminthiasis infecting intermediate hosts, including humans. Strong immune responses are expected to occur in early phases of infection. Thus, we investigated cytokine expressions in splenic dendritic cells and in sera after experimental infection of mice. In splenic dendritic cells, TNF-alpha and IL-1beta expression peaked at week 1 and week 3 post-infection (PI), respectively, and also early phase (week 2 PI) depressed cytokine expression was noticed. Serum IL-1beta concentration increased significantly at week 2 PI and peaked at week 6 PI, and that of TNF-alpha peaked at week 6 PI. These results showed that pro-inflammatory cytokines, TNF-alpha and IL-1beta, are chronologically regulated in mouse sparganosis.

Therapeutic effect of different dosages of praziquantel on mice infected with Sparganum mansoni / 中国血吸虫病防治杂志

Objective To observethe efficacy of mice infected with Sparganum mansoni by using different dosages of praziquantel.Methods A total of 156 Kunming mice were divided into 2 batches.each of them wag orally infted with 5 spargana.Thirty-six mice in the first batch were equally divided into 6 groups.the mice in group 1-5 were inoculated with spargana cultured in different concentrations of praziquantel for 3 days,the group 6 served as a control.One hundred and twenty mice in the second batch were equally divided into 12 groups,each mouse was inoculated with spargana obtained from frogs or tadpoles,group 1-9 were treated by different desages of praziquantel 1 or5 weeks post infection.group 10-12 served as controls.All of the mice wore sacriftced and dissectedl or 2 weeks after the treatment.the mean number of worms recovered was cmculated and worm reduction rates were determined.Results The number of worm recovered from mice infected with spargana cultured in 10-40 μg/ml of praziquantel had no significant difference with that of the control(P>0.05).The worm reduction rate wag 16.60%while the spargana beins cultured in 50 μg/ml of praziquantel.The worm reduction rates of the mice that sacrificed 1 week or 2 weeks after being treated by the same dosage of praziquantel had no significant difference(P>0.05).When being treated with 200.400 or 800 mg/kg of praziquantel 1 week post infection,the number of worm recovered from mice infected with spargana from frogs had no significant difference with those of the control 1 and 2 weeks after the treatment(P>0.05).The worm reduction rates between the groups with the same dosage 1 week and 2 weeks post treatment had no significant difference(P>0.05).When being treated with 200 or 400 mg/kg of praziquantel 1 week post infection,the number of worm recovered from mice infected with spargana from tadpoles had no statistically difference with that of the control 1 week after treatment (P>0. 05). The worm reduction rate of mice was only 17.02% while being treated with 800 mg/kg of praziquantel. The worm reduction rates among groups with different dosages had no significant difference (P>0.05). Compared with the mice infected with spargana from frogs treated with 1 200 or 1 800 mg/kg of praziquantel 5 weeks post infection, the difference between the numbers of worm recovered from mice 1 week and 2 weeks after treatment had no statistically significance (P > 0.05), but they were significantly higher than those of the controls (P0.05). ConclusionsPraziquantel (10-50 μg/ml) has no evident killing effect on spargnna in vitro, but when the dosage is higher(1 800 mg/kg), it has certain efficacy for treating the mice infected with spargana by oral inoculation.

A Case of Sparganosis in the Leg

The life-span of the sparganum in humans is not exactly known, but it may survive longer than 5 years in some patients. We experienced a case infected with a sparganum that is presumed to have lived for 20 years in a patient's leg. The patient was a 60-year-old woman, and she was admitted to a hospital due to ankle pain that was aggravated on dorsiflexion. She had noticed a mass on her knee some 20 years ago, but she received no medical management for it. The mass moved into the ankle joint 3 months before the current admission, and then the aforementioned symptoms appeared. A living sparganum was recovered by surgery, and the calcified tract near the knee was proved to be the pathway along which the larva had passed.

Production of Polyclonal Antibodies against the Tegument of Sparganum (Plerocercoid of Spirometra mansoni) and Its Immunolocalization

In a previous study, the author developed a method for separation of the tegument of spargana (plerocercoids of Spirometra mansoni) from the parenchyme using urea. The present study, as a next step, was performed to evaluate which molecules are present in the outer tegument. Two major proteins, 180 and 200 kDa, are present in the tegument and we could make polyclonal antibodies against these molecules. Their immunolocalization was processed and the outermost layer of the spargana showed strong positive staining. Conclusively, we could confirm that the 180 and 200 kDa molecules might be tightly bound membrane proteins in the tegument of spargana.