Molecular determination of suspected alveolar echinococcosis requiring surgical treatment in human cases from Poland

In this study five cases of suspected alveolar echinococcosis from Poland in which surgical treatment was needed, previously diagnosed by means of imaging and serological techniques, were analyzed in terms to identify the causative agent. Samples of the parasite tissues taken perioperatively from the liver lesions were used for the histopathological and molecular examinations. The sequences of all isolates were identical to Echinococcus multilocularis; all nad1 sequences have been deposited in GenBank The histopathological examination revealed Passpositive fragments of laminated layers typical for E. multilocularis metacestode. Obtained results confirm that the use of imaging techniques only may be insufficient to diagnose alveococcosis thus the recognition of the zoonosis should base on several procedures; especially valuable are highly sensitive and specific molecular methods.

Development of a movement-based in vitro screening assay for the identification of new anti-cestodal compounds.

Intestinal cestodes are infecting millions of people and livestock worldwide, but treatment is mainly based on one drug: praziquantel. The identification of new anti-cestodal compounds is hampered by the lack of suitable screening assays. It is difficult, or even impossible, to evaluate drugs against adult cestodes in vitro due to the fact that these parasites cannot be cultured in microwell plates, and adult and larval stages in most cases represent different organisms in terms of size, morphology, and metabolic requirements. We here present an in vitro-drug screening assay based on Echinococcus multilocularis protoscoleces, which represent precursors of the scolex (hence the anterior part) of the adult tapeworm. This movement-based assay can serve as a model for an adult cestode screen. Protoscoleces are produced in large numbers in Mongolian gerbils and mice, their movement is measured and quantified by image analysis, and active compounds are directly assessed in terms of morphological effects. The use of the 384-well format minimizes the amount of parasites and compounds needed and allows rapid screening of a large number of chemicals. Standard drugs showed the expected dose-dependent effect on movement and morphology of the protoscoleces. Interestingly, praziquantel inhibited movement only partially within 12 h of treatment (at concentrations as high as 100 ppm) and did thus not act parasiticidal, which was also confirmed by trypan blue staining. Enantiomers of praziquantel showed a clear difference in their minimal inhibitory concentration in the motility assay and (R)-(-)-praziquantel was 185 times more active than (S)-(-)-praziquantel. One compound named MMV665807, which was obtained from the open access MMV (Medicines for Malaria Venture) Malaria box, strongly impaired motility and viability of protoscoleces. Corresponding morphological alterations were visualized by scanning electron microscopy, and demonstrated that this compound exhibits a mode of action clearly distinct from praziquantel. Thus, MMV665807 represents an interesting lead for further evaluation.

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 µM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 µM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 µM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

[Mixed infection of Echinococcus granulosus and Echinococcus multilocularis in dog].

OBJECTIVE: To identify mixed infection of Echinococcus granulosus and E. multilocularis in a dog from Xinjiang. METHODS: Thirty dogs from the pasture area were dissected and over 10,000 Echinococcus adult worms were found from one dog. Morphological observation revealed possible mixed infection of the two Echinococcus species. Further identification was made by amplification of the target gene DNA fragment (mitochondrial 12S rRNA gene). RESULTS: The adult worms of E. granulosus showed a relatively longer and larger gravid proglottid, its genital pore situated near or below the middle-side of the segment. The uterus was in a sacculate shape with irregular branches and approximately over 200 - 800 eggs in it. Morphology of the adult worms of E. multilocularis was similar to E. granulosus, slightly smaller, consisting of 4 to 5 proglottids. The uterus was not sacculate and with no branch. Its lateral genital pore often situated in the anterior part of the segment. Sequence analysis of mitochondrial 12S rRNA gene showed that amplification with the Eg1f/r primers shared complete identity with E. granulosus G1 genotype (GenBank accession no. AY462129), while that witht the EmH15/17 primers shared complete identity with E. multilocularis (GenBank accession no. AB031351). The presence of both E. granulosus and E. multilocularis was confirmed by microscopy and gene identification. CONCLUSION: Mixed infection of the two species of Echinococcus has been confirmed in the dog by morphological observation and PCR technique.

Morphological and molecular characteristics of Echinococcus multilocularis and Echinococcus granulosus mixed infection in a dog from Xinjiang, China.

The Xinjiang plateau of western China has been shown to have a high prevalence for human cystic echinococcosis (CE) caused by Echinococcus granulosus, and human alveolar echinococcosis (AE) caused by Echinococcus multilocularis. The domestic dog is suspected to be the primary definitive host for the transmission of both E. granulosus and E. multilocularis to humans in this locality. Seventeen of 30 stray dogs from Hejing County of Xinjiang were found positive for E. granulosus post mortem, and one double infection was suspected. Worm samples were collected, dyed by carmine, and observed microscopically. Carmine staining examination clearly revealed the differences in number of proglottids and appearance of uterine branches and lateral genital pore for those two species of Echinococcus. Furthermore, gene target DNA fragments were amplified for formal identification of the two parasite species, based on 12s rRNA mitochondrial gene. The PCR products were purified and sequenced. Compared with NCBI GenBank, the DNA sequences demonstrated 100% identity with E. granulosus (sheep strain, G1 genotype) and E. multilocularis.