Four Human Cases of Diphyllobothrium nihonkaiense (Eucestoda: Diphyllobothriidae) in China with a Brief Review of Chinese Cases.

We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62-67×42-45 µm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.

[Analysis on parasitic infection of clinical samples from hospitals in Shanghai during 2011-2013].

OBJECTIVE: To analyze the results of parasitic pathogen detection on clinical samples from Shanghai hospitals during 2011-2013. METHODS: Samples of serum, stool, sputum, body fluid and biopsy were collected from hospitals. The etiological, serological and molecular biology methods were used to detect parasitic infection cases. RESULTS: During 2011-2013, a total of 16,151 clinical samples were collected. 855 parasitic infection were found from 5939 samples by pathogen detection, belonging to 32 species, with a detection rate of 14.4%. The positive rate of Blastocystis hominis and Entamoeba histolytica was 8.3% (494/5939) and 3.1% (186/5939), respectively. The rate of intestinal protozoa infection in under 20-year-old age group was higher than other age groups (P<0.05). No significant difference was found between males and females (P>0.05). Totally 10,212 serum samples were examined, the total antibody-positive rate was 7.1% (730/10,212). In the 730 positive samples, 173 (23.7%), 143 (19.6%), 139 (19.0%), 132 (18.1%), and 128 (17.5%) showed positive for the antibodies against Cysticercus cellulosae, Schistosoma japonicum, Paragonimus westermani, Toxoplasma gondii and Sparganum mansoni, respectively. The main source regions of protozoal infection were Shanghai (269 cases), Jiangsu (142 cases), Anhui (106 cases) and Zhejiang (82 cases). 89 cases were worm infection, the main source were Zhejiang (24 cases), Shanghai (18 cases), Jiangxi (11 cases). CONCLUSION: Among the samples from hospitals, the major intestinal protozoans are Blastocystis hominis and Entamoeba histolytica, and the sero-positive cases are mainly Cysticercus cellulosae and Schistosoma japonicum infection.

A protein microarray for the rapid screening of patients suspected of infection with various food-borne helminthiases.

BACKGROUND: Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3-98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2-99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1-96.3%) to 92.1% (95% CI: 83.5-100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4-92.6%) to 92.1% (95% CI: 83.5-100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. CONCLUSIONS/SIGNIFICANCE: The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

[In vitro effect of seven anthelmintic agents against adult Clonorchis sinensis].

OBJECTIVE: To observe the in vitro effect of praziquantel, tribendimidine, levamisole, artemether, artesunate, albendazole and mebendazole against adult Clonorchis sinensis. METHODS: Seventy rats infected with 50-100 C. sinensis metacercariae for 5-7 weeks were euthanized, and adult C. sinensis were collected from the common bile duct Three to four worms were placed in each well of a 24-well falcon plate, and treated by Hanks' balanced salt solution-20% calf serum containing aforementioned drugs at various concentrations. The motor activity and morphology change of the worms were observed under an inverted microscope at 4, 24, 48 and 72 h post treatment. RESULTS: Praziquantel could reduce the motor activity of the worms rapidly which resulted in detachment of oral sucker from the well wall, curl of the worm body and emergence of vacuoles from the tegument. The minimal concentration of praziquantel to kill adult C. sinensis was 0.1 g/ml. After adult C. sinensis exposed to tribendimidine at concentrations of 0.5, 1 and 10 g/ml, they revealed in paralysis, looseness and stretch of the worm body rapidly or immediately. The minimal concentration of tribendimidine to kill adult worms was 0.05 g/ml. When worms exposed to levamisole at 10 and 20 g/ml, there was a gradual decrease in the worm's motor activity accompanied by looseness of the worm body. But 48 h post exposure, most worms showed apparently recovery of motor activity. In a higher levamisole concentration of 50 g/ml, all worms revealed in stretch and paralysis which was similar to that induced by tribendimidine. When adult C. sinensis were exposed to artemether or artesunate 10 and 50 g/ml, the motor activity of worm body and oral sucker reduced which accompanied by worm contraction, then followed by looseness of the worm body and emergence of vacuoles along the tegument. At 72h post exposure, the worm mortalities induced by the two concentrations of the two drugs were about half, respectively. In adult C. sinensis exposed to albendazole and mebendazole at concentrations of 10 and 50 g/ml, only stimulation of motor activity of oral sucker was seen which revealed in vigorous contraction within 24 h post exposure. During 72 h observation period, no any other changes in worm activity and morphology were seen. CONCLUSION: Praziquantel and tribendimidine exhibit strong in vitro killing effect on adult C. sinensis. The minimal concentration of levamisole used to kill adult worm is 50 times higher than that of tribendimidine. The higher concentrations of artemether and artesunate show slower action to reduce the worm activity and kill part of the worms. Higher concentrations of albendazole and mebendazole exhibit no killing effect on C. sinensis, besides stimulating the motor activity of worm oral sucker.

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults.

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.

[Detection and identification for Gnathostoma sp. in imported Monopterus albus].

OBJECTIVE: To inspect the third stage larvae of Gnathostoma in imported Monopterus albus, and identify its species. METHODS: Ten batches of M. albus imported to Shanghai were detected for nematode Gnathostoma from January 2010 to March 2011. Fifty-two M. albus imported from the Philippines (25), Indonesia (24) and Bangladesh (3) were sampled (3-10/batch), which were dissected, minced, and digested. The suspension was filtered with 10 mesh screen to take the deposit. The complete parasites were picked out under stereoscope followed by morphological identification. The rate and intensity of infection were calculated. Genomic DNA of Gnathostoma was extracted to amplify internal transcribed spacer region 2 (ITS-2) and cytochrome C oxidase subunit 1 (cox1) by PCR, the product of which was analyzed by electrophoresis and sequencing. The sequences were aligned with corresponding sequences in GenBank. RESULTS: The third stage larvae of Gnathostoma were detected in M. albus from Indonesia and Philippines with infection rate of 36.0% (9/25) and 50.0% (12/24) and average infectiosity of 7.8 (70/9) and 2.8 (34/12), respectively. No Gnathostoma was found in M. albus imported from Bangladesh. Under microscope, the larvae showed one cephalic bulb with 4 rings of hooklets on it, cross striations and small spines on the body surface. The front body spines were bigger and denser, while the rear spines were smaller and sparser. It had 1 cervical papilla and 4 cervical capsules. Morphological characteristics were similar to the third stage larvae of G. spinigerum. PCR results showed that the length of the ITS-2 and cox1 PCR products was 647 bp and 441 bp, respectively. Sequence alignment analysis showed that the two PCR products had 99%-100% consistency with G. spinigerum ITS-2 (GenBank Accession No. AB181155 and Z97175) and cox1 (GenBank Accession No. AY501388, AB180099, and AB551552). CONCLUSION: All the larvae detected in M. albus imported from the Philippines and Indonesia have been identified as G. spinigerum.

Comparative effect of mebendazole, albendazole, tribendimidine, and praziquantel in treatment of rats infected with Clonorchis sinensis.

The aim of the study is to understand the anti-Clonorchis sinensis properties of mebendazole and albendazole, and compare to praziquantel and tribendimidine. Two hundred and thirty rats were divided into five batches for experimental treatment. In four batches, each rat was infected orally with 50 or 100 C. sinensis metacercariae. Twenty-eight to 46 days post-infection, groups of rats were treated orally with single doses of mebendazole, albendazole, praziquantel, tribendimidine, or multiple daily doses of albendazole. While in the remaining batch, mebendazole or praziquantel was administered to groups of rats infected each with 50 metacercariae for 7 or 14 days. In each batch of test, untreated but infected rats served as control. All rats were euthanized 2-4 weeks post-drug administration for assessment of efficacy. In the first batch of test, rats treated with mebendazole or tribendimidine at single doses of 150, 75, and 37.5 mg/kg resulted in worm burden reductions of 99.0%, 94.0%, and 73.1%, or 98.0%, 80.6%, and 60.4%, respectively. When rats were treated with albendazole at the same dose levels, no or poor effect was seen. In the second batch of test, promising effect against adult C. sinensis in rats treated with mebendazole or tribendimidine at single doses of 100 and 50 mg/kg were also observed, but under the single dose of 25 mg/kg, only tribendimidine still remained the effect. In the third batch of test, the aforementioned three single dose levels of mebendazole, albendazole and praziquantel were applied. Again, mebendazole exhibited higher effect and albendazole exhibited no or poor effect. While praziquantel, administered at a higher dose of 300 mg/kg, also showed promising effect. In the fourth batch of test, oral administration of albendazole at a daily dose of 150 or 100 mg/kg for 2 or 3 days resulted in moderate or higher efficacy with worm burden reductions of 79.2% and 91.9%, respectively. In the fifth batch of test, single mebendazole doses of 150 or 75 mg/kg exhibited promising effect against 14-day-old C. sinensis in rats with worm burden reductions of 95.3% and 86.4%, respectively, but mebendazole was short of the effect against 7-day-old worms. Under the same dose level, praziquantel possessed an effect against both 7- and 14-day-old juvenile C. sinensis. The results confirm that in infected rats, mebendazole administered orally at a single dose of 150 mg/kg exhibits potential effect against juvenile (14-day-old) and adult C. sinensis. No or less effect is obtained from albendazole under the same dose levels, but extension of treatment course may enhance the effect of albendazole against this species of fluke. The single effective dose ranges of mebendazole and tribendimidine against C. sinensis in rats are similar with a broad window, while the window for praziquantel is narrow.

[Effect of six anthelmintics in oral treatment of mice infected with Armillifer agkistrodontis nymphs].

OBJECTIVE: To observe the effect of praziquantel, mebendazole, tribendimidine, ivermectin, artemether and dihydroartemisinin against Armillifer agkistrodontis nymphs harbored in mice. METHOD: Thirty-five mice infected each with 40 eggs of Armillifer agkistrodontis for 25-37 weeks were divided into 10 groups (2-8 mice per group). Among them, nine groups were treated orally with praziquantel 500 mg/(kg x d) x 5 d or 500 my/(kg x d) x 14 d, mebendazole 300 mg/(kg x d)x 5 d, tribendimidine 300 mg/(kg x d) x 5 d, ivermectin 8-10 mg/(kg x d) x 3 d or 15 mg/(kg x d) x l4 d, artemether 400 mg/(kg x d) x 5 d and dihydroartemisinin 200 mg/(kg x d) x 5 d, respectively. The remaining untreated group served as control. All mice were sacrificed 1-3 weeks post-treatment for collection and separation of Armillifer agkistrodontis nymph cysts from abdominal wall, lipid tissue and viscera including liver, spleen and outer wall of intestine. The nymphs were released after tearing up the cyst with forceps, placed in the normal saline to observe their motor activity and counted. RESULTS: The difference of mean nymph burden between the drug treated groups and control group was not statistically significant (P > 0.05) with mean nymph reductions of 8.3%-35.0%. Meanwhile, the appearance of the cyst, the size, colour, morphology and motor activity of nymphs were also similar to that of the control. CONCLUSION: Praziquantel, mebendazole, tribendimidine, ivermectin, artemether and dihydroartemisinin exhibit no effect against Armillifer agkistrodontis harbored in mice under the conditions in the experiment.

[Treatment of rats infected with Clonorchis sinensis using clinical administration regimens of tribendimidine, praziquantel and artesunate].

OBJECTIVE: To evaluate the efficacy in treatment of Clonorchis sinensis-infected rats using the administration regimens of tribendimidine, artesunate and praziquantel applied in clinical treatment of clonorchiasis. METHODS: The doses of tribendimidine, artesunate and praziquantel used in clinical treatment of clonorchiasis were converted to the doses used in rats by the method of equal effective dose conversion among different animals, while the administration regimens of the drugs were designed basing on the regimens used in clinical trials. Thus, the following dose schedules were set up, i.e., tribendimidine 16 or 32 mg/(kg x d) x 1, 2 or 3 d (bid), 8 or 16 mg/(kg x d) x 3 d; artesunate 12 mg/(kg x d) x 3 d (tid) and 16 mg/(kg x d) x 3 d (bid); praziquantel 143 mg/(kg x d) x 2 or 3 d (tid), 143 mg/(kg x d) x 2 or 3 d (bid), 47.7 or 71.5 mg/(kg x d) x 3 d. 151 rats were divided into 2 batches and each rat was infected orally with 50 metacercariae of C. sinensis. In the first batch of test, 79 rats were divided into 13 groups of 5-6 rats 5 weeks post-infection. Among them 6 groups were treated orally only with tribendimidine, artesunate or praziquantel, while other 7 groups were treated with tribendimidine combined with artesunate or praziquantel, or praziquantel combined with artesunate. The remaining 8 untreated rats served as control. In the second batch of test, 72 rats were divided into 13 groups of 5 rats. Among them, 7 and 6 groups were treated with tribendimidine and praziquantel, respectively, 6 weeks post-infection. The remaining 8 untreated rats served as control. Rats were sacrificed 14 days post-treatment, worms were recovered from the bile duct and the liver tissue. The mean worm reduction rate was calculated and compared among the groups by non-parametric method (Mann-Whitney test). RESULTS: In the first batch of test, the mean worm burdens in rats infected with C. sinensis and treated orally with tribendimidine 16 or 32 mg/(kg x d) x 3 d (bid), praziquantel 143 mg/(kg x d) x 3 d (tid), or 143 mg/(kg x d) x 3 d (bid) were significantly lower than that of the control (P < 0.01) with mean worm burden reductions of 94.2%-96.0%. No efficacy was seen when infected rats were treated orally with artesunate 12 mg/(kg x d) x 3 d (tid). But in those treated with artesunate 16 mg/(kg x d) x 3 d (bid), the mean worm burden was significantly lower than that of the control (P < 0.05) with a mean worm reduction of 57.2%. In combined treatment, the infected rats treated with tribendimidine 16 or 32 mg/(kg x d) x 3 d (bid) in combination with praziquantel 143 mg/(kg x d) x 3 d(bid) or artesunate 16 mg/(kg x d) x 3 d (bid), the difference of mean worm burden between each combined treatment group and control group was statistically significant (P < 0.01) with mean worm reductions of 94.2% -99.4% which revealed that the worm reduction rate in combined treatment group was similar to the corresponding group treated with tribendimidine or praziquantel alone, but significantly higher than that of the group treated with artesunate alone. In infected rats treated with praziquantel 143 mg/(kg x d) x 3 d (tid) plus artesunate 12 mg/(kg x d) x 3 d (tid) or praziquantel 143 mg/(kg x d) x 3 d (bid) plus artesunate 16 mg/(kg x d) x 3 d (bid), the mean worm burden reductions were 93.6% -100%. In the second batch of test, the efficacy of tribendimidine obtained from infected rats treated with the drug 16 or 32 mg/(kg x d) x 2 d (bid) and 3 d (bid), the difference of mean worm burdens between them was not statistically significant with mean worm reductions of 86.5%-95.1%. When rats were treated with tribendimidine 32 mg/(kg x d) x 1 d (bid), the mean worm reduction was 73.0%, while the dose of the drug was given to the rats at 8 or 16 mg/kg daily for 3 days the mean worm reduction rates were 88.3%-92.6%. Treatment of praziquantel 143 mg/(kg x d) x 3 d (tid) resulted in a worm reduction of 96.9%, if the treatment course reduced to 2 d, the rate was 63.2%. Similar results were obtained in rats treated with praziquantel 143 mg/(kg x d) x 2 d (bid) and 3 d (bid). Finally, administration of praziquantel at a daily dose of 47.7 or 71.5 mg/kg for 3 d exhibited no effect against C. sinensis. CONCLUSION: When the dose schedules of tribendimidine, artesunate and praziquantel used in humans are converted to the doses for use in rats, tribendimidine and praziquantel exhibit satisfactory effect against C. sinensis, but artesunate shows no or less effect; the treatment course of tribendimidine can be reduced from 3 d to 2 d. Since tribendimidine and praziquantel used alone have endorsed high efficacy against C. sinensis in rats, combinations among the 3 drugs do not show better effect.

Effectiveness of mefloquine against Clonorchis sinensis in rats and Paragonimus westermani in dogs.

The aim of the study is to explore the effect of mefloquine against Clonorchis sinensis and Paragonimus westermani. For anti-C. sinensis study, a total of 71 rats were divided into four batches for oral infection of each rat with 50 C. sinensis metacercariae. Five to 7 weeks post-infection, groups of rats were treated orally with mefloquine at single doses or multiple daily doses while infected, but untreated rats served as control. All treated rats were euthanized 2 weeks post-treatment for assessment of efficacy. For anti-P. westermani study, two batches of eight and ten dogs were each infected intraperitoneally with 100 P. westermani metacercariae. Eighty-five to 96 days post-infection, groups of two or three dogs were treated orally with mefloquine and groups of two dogs were treated with praziquantel at a single dose or multiple doses. In each batch of test, three untreated but infected dogs served as control. All treated dogs were euthanized 26-30 days post-treatment for evaluation of efficacy. In rats infected with C. sinensis and treated orally with mefloquine at a single dose of 75 and 150 mg/kg, no effect against C. sinensis was observed. When the dose of mefloquine was increased to 250 mg/kg, one third (five out of 15) rats died 3-5 days post-treatment. Although the mean worm burden was lower than that of the control, the difference between the treated and control groups was not statistically significant (P>0.05) with worm burden reduction of 22.4%. Whereas, the group of infected rats received mefloquine at a daily dose of 100 mg/kg for 3 days, one out of five rats died after the last administration. The mean worm burden was significantly lower than that of the control with worm burden reduction of 67.6% (P<0.01). In the first test of mefloquine against P. westermani, three infected dogs received two oral doses of the drug, 50 mg/kg, given at a 4-h interval, the mean worm burden were similar to that of the control. While other two dogs were treated with praziquantel at the same dose schedule, the worm burden reduction of 78% was observed. In the second test, three and two dogs were treated with mefloquine 50 mg/kg daily for 5 days or 100 mg/kg daily for 2 days; the mean worm burdens of the two groups were lower than that of the control with worm burden reduction of 65.6% and 51.9%, respectively. However, only the difference of mean worm burdens between mefloquine 50 mg/kg given daily for 5 days and the control was statistically significant (P<0.05). Other two dogs treated with praziquantel at a single dose of 100 mg/kg were cured. The results indicate that under the appropriate dose schedule mefloquine exhibits less effect against C. sinensis in rats and P. westermani in dogs.

[Efficacy of tribendimidine and albendazole in treating mice infected with Trichinella spiralis].

OBJECTIVE: To observe the efficacy of tribendimidine and albendazole against Trichinella spiralis in mice. METHODS: A total of 85 Kunming strain mice, infected orally with 100 T. spiralis larvae, was divided into 3 groups: group A (adult stage, 7 d after infection), group B (migrating larva stage, 15 d after infection), and group C (encapsulated larva stage, 35 d after infection). Group A (35 mice) was equally divided into 7 sub-groups, tribendimidine and albendazole were each orally administered to 3 sub-groups both with doses of 6.25, 12.5, and 25 mg/kg respectively, the untreated sub-group served as control. Groups B and C (25 mice each) were both divided equally into 5 sub-groups. Mice in 2 sub-groups were treated respectively with the 2 drugs in a dose of 100 or 200 mg/kg, the untreated sub-group served as control. Mice in group A were sacrificed 2 d post-treatment and adult worms recovered from the small intestine were counted. Those in groups B and C were sacrificed 15 d post-treatment and intact diaphragm was then removed from each mouse. The muscle of diaphragm was digested by digestive solution and the larvae were counted by stereomicroscope. Mean worm burden and mean worm reduction of each treated group were calculated and statistically compared with the control. RESULTS: The mean worm burden in sub-groups of group A treated with tribendimidine was significantly lower than that of the control (P<0.01) with a mean worm reduction of 63.3%, 86.2%, and 98.5%, respectively. In the same batch of mice treated with albendazole at a single dose of 6.25 and 12.5 mg/kg resulted in similar mean worm burden compared to the control (P<0.05). While in the sub-group received albendazole at a higher dose of 25 mg/kg, the mean worm burden was significantly lower than that of the control (P<0.05), with a mean worm reduction of 41.2%. The mean worm burden in group B was significantly lower than that of the control (P<.01). The mean worm reduction in the 2 sub-groups treated with tribendimidine or albendazole was 64.4% and 89.6%, or 56.7% and 78.4%, respectively. In group C, significantly lower mean worm burden was only found in the subgroup treated with albendazole at a higher dose of 200 mg/kg than the control (P<0.01) with a mean worm reduction of 71.8%. No effect was seen in the other 3 groups. CONCLUSION: Tribendimidine exhibits potential effect against adult and migrating larva stage of T. spiralis in mice, but lacks effect against encapsulated larva stage of the parasite. Albendazole administered at a larger or multiple doses to mice endorses effect against its adult, migrating larva and encapsulated larva stages.

Multi-host model-based identification of Armillifer agkistrodontis (Pentastomida), a new zoonotic parasite from China.

BACKGROUND: Pentastomiasis is a rare parasitic infection of humans. Pentastomids are dioecious obligate parasites requiring multiple hosts to complete their lifecycle. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, their systematic position is not uncontested and historically, they have been considered as a separate phylum. METHODOLOGY/PRINCIPAL FINDINGS: An appraisal of Armillifer agkistrodontis was performed in terms of morphology and genetic identification after its lifecycle had been established in a multi-host model, i.e., mice and rats as intermediate hosts, and snakes (Agkistrodon acutus and Python molurus) as definitive hosts. Different stages of the parasite, including eggs, larvae and adults, were isolated and examined morphologically using light and electron microscopes. Phylogenetic and cluster analysis were also undertaken, focusing on the 18S rRNA and the Cox1 gene. The time for lifecycle completion was about 14 months, including 4 months for the development of eggs to infectious larvae in the intermediate host and 10 months for infectious larvae to mature in the final host. The main morphological difference between A. armillatus and Linguatula serrata is the number of abdominal annuli. Based on the 18S rRNA sequence, the shortest hereditary distance was found between A. agkistrodontis and Raillietiella spp. The highest degree of homology in the Cox 1 nucleic acid sequences and predicted amino acid sequences was found between A. agkistrodontis and A. armillatus. CONCLUSION: This is the first time that a multi-host model of the entire lifecycle of A. agkistrodontis has been established. Morphologic and genetic analyses supported the notion that pentastomids should be placed into the phylum Arthropoda.

[Therapeutic effect of tribendimidine, artesunate and praziquantel administered to hamsters infected with Clonorchis sinensis].

OBJECTIVE: To observe the effect of tribendimidine, artesunate and praziquantel in treatment of hamsters (Mesocricetus auratus) infected with Clonorchis sinensis. METHODS: A total of 93 hamsters, each infected with 30 C. sinensis metacercariae, were treated intragastrically with above-mentioned drugs at a single dose. (1) In order to observe the effect of the drugs against juvenile C. sinensis, 20 out of 31 infected hamsters were randomly divided into 4 groups (5 hamsters per group) 14 d post-infection: artesunate 300 mg/kg, tribendimidine 100 mg/kg or 200 mg/kg, and praziquantel 200 mg/kg. Other 6 hamsters were divided equally into 2 groups 24 d post-infection and treated with tribendimidine 200 mg/kg and artesunate 300 mg/kg, respectively. The remained 5 untreated hamsters served as control. (2) Twenty-two hamsters were randomly divided into 5 groups (4-5 hamsters per group) 28 d post-infection and treated with tribendimidine 25 mg/kg or 50 mg/kg, artesunate 25 mg/kg and praziquantel 50 mg/kg, respectively. Other untreated hamsters served as control. (3) Forty hamsters 28 d after infection were randomly divided into 8 groups (4-6 hamsters per group) and treated with tribendimidine 50 mg/kg, 100 mg/kg or 200 mg/kg, artesunate 100 mg/kg or 200 mg/kg, praziquantel 100mg/kg or 200mg/kg, respectively. The remained hamsters served as control. All hamsters were sacrificed 14 d post-treatment and worms were recovered from the bile duct and liver tissue. The mean worm burden and its reduction were calculated. The differences of mean worm burden between each treated group and the corresponding control were analyzed statistically. RESULTS: In hamsters infected with 14-d-old C. sinensis and treated orally with tribendimidine at a single dose of 100 or 200 mg/kg, the mean worm burdens were significantly lower than that of the control (P<0.01) with a worm reduction of 90.6% and 85.9% respectively. The mean worm burden obtained from the infected hamsters treated with praziquantel at a single dose of 200 mg/kg was also significantly lower than that of the control (P<0.05) with a worm reduction of 71.9%. However, the difference of mean worm burden between artesunate and control groups was not statistically significant. The juvenile parasites developed into adult worms 24 d after infection. By administering tribendimidine 200 mg/kg to the adult C. sinensis-infected hamsters, the mean worm burden was significantly lower than that of the control (P<0.01) with a worm reduction of 89.8%. Whilst the administration of artesunate at a higher dose of 300 mg/kg, all hamsters were cured. Further tests indicated that tribendimidine in a lower dose of 25 mg/kg to the hamsters 28 d after infection resulted in a significantly lower mean worm burden compared to the control (P<0.05) with a worm reduction of 71.8%. With an increased dose of tribendimidine 100 mg/kg, all hamsters were cured. The worm reduction was only 20.0% and 56.4% when 25 mg/kg and 100 mg/kg of artesunate were administered. With 200 mg/kg artesunate, the worm reduction reached as high as 98.5% and the mean worm burden was significantly lower than that of the control (P<0.01). Furthermore, administration of praziquantel at a dose of 100 mg/kg or 200 mg/kg at 28 d post-infection resulted in a significantly lower mean worm burden than that of the control (P<0.05) with a worm reduction of 78.9% and 83.5% respectively. CONCLUSION: In hamster model, tribendimidine and praziquantel exhibit promising effect against both juvenile and adult C. sinensis, while artesunate is only efficacious against adult worms.

The in vitro and in vivo effect of tribendimidine and its metabolites against Clonorchis sinensis.

The purpose of the study was to understand the in vitro and in vivo effect of tribendimidine (TBD) and its metabolites of p-(1-dimethylamino ethylimino)aniline (aminoamidine, deacylated amidantel, BAY d 9216, dADT), acetylated dADT (AdADT), terephthalaldehyde (TPAL), and terephthalic acid (TPAC) against adult Clonorchis sinensis. In in vitro test, the adults of C. sinensis were placed to each of the 24 wells of a Falcon plate and maintained in Hanks' balanced salt solution-20% calf serum. Besides observation on the direct in vitro effect of TBD and its metabolites, the worms exposed to TBD and its metabolites for 1-24 h were transferred to the medium without drug and incubated continually for another 72 h. The reversible effect of TBD and its metabolites was assessed by the recovery of worm motor activity and parasite survival. In in vivo test, 235 rats were divided into five batches for oral infection of each rat with 50 C. sinensis metacercariae. Five to 6 weeks post-infection, groups of rats were treated orally or intramuscularly with a single dose of TBD or its metabolites, while untreated but infected rats served as control. All treated rats were killed 2 weeks post-treatment for assessment of efficacy. When adult C. sinensis were exposed to TBD or dADT 0.5 microg/mL, they were paralyzed rapidly accompanied by dilatation of the gut. The in vitro effect of AdADT decreased significantly, which was at least lower than 20- to 40-fold compared with TBD and dADT. TPAL and TPAC at a high concentration of 100 microg/mL exhibited no effect against adult C. sinensis. In the worms exposed to TBD or dADT 1 microg/mL for 1 h, well recovery of the worm motor activity from paralysis was seen in the medium without drug. If exposure time extended to 4-24 h before transferred to the medium without drug, few worms were dead and most worms showed very poor recovery of their activity. When the worms exposed to TBD or dADT 10 microg/mL for 1, 4, and 24 h were transferred to the drug-free medium, recovery of poor motor activity of worms or worm death was seen. In the worms exposed to AdADT 20 and 40 microg/mL for 1-24 h, more worms recovered poor motor activity in the medium without drug. In rats infected with C. sinensis and treated orally with TBD or dADT, the ED(50) and ED(95) were 20.318 and 195.358 mg/kg or 18.969 and 268.882 mg/kg. Under the equal dosages used in the treatment of rats infected with C sinensis, the effects between TBD and dADT or TBD and AdADT were similar. Intramuscular TBD or dADT at a single dose of 12.5-75 mg/kg showed effect against adult C. sinensis harbored in rats. TPAL and TPAC exhibit no effect against C sinensis harbored in rats treated orally with a higher dose of 1 g/kg. The results indicate that TBD and dADT exhibit a strong in vitro effect to paralyze the adult C. sinensis, but less in vitro effect was seen in AdADT. TBD, dADT, and AdADT exhibit similar therapeutic effect in oral treatment of rats infected with C. sinensis, and intramuscular TBD and dADT also show promising effect against C. sinensis in rats. TPAL and TPAC are ineffective metabolites of TBD.

Artemether and tribendimidine lack activity in experimental treatment of Paragonimus westermani in the dog.

Artemether and tribendimidine are active against several trematode species, but no data are available regarding the lung fluke Paragonimus westermani. We infected six dogs with 100 P. westermani metacercariae each. At day 103 post-infection, four dogs were treated orally for 3 days with either artemether (total dose, 66.7 and 75 mg/kg) or tribendimidine (total dose, 100 mg/kg). The remaining dogs were left untreated and served as control. Sixteen days after the final dosing, dogs were killed, and P. westermani flukes were recovered from the lungs and counted. Neither artemether nor tribendimidine showed activity against P. westermani at this dose regimen in dogs.