Establishment and preliminary application of a recombinase-aided isothermal amplification assay-based multiplex nucleic acid assay for detection of three Echinococcus species / 中国血吸虫病防治杂志

Objective To establish a multiplex nucleic acid assay for rapid detection of Echinococcus multilocularis, E. granulosus and E. shiquicus based on the recombinase-aided isothermal amplification assay (RAA) and to preliminarily assess its diagnostic efficiency. Methods The mitochondrial genomic sequences of E. multilocularis (GenBank accession number: NC_000928), E. granulosus (GenBank accession number: NC_044548) and E. shiquicus (GenBank accession number: NC_009460) were used as target sequences, and three pairs of primers were designed based on the RAA primer design principle and synthesized for the subsequent multiple RAA amplification. The genomic DNA of E. multilocularis, E. granulosus and E. shiquicus at different concentrations and the recombinant plasmids containing the target gene at various concentrations were amplified to evaluate the diagnostic sensitivity of the multiplex RAA assay, and the genomic DNA of E. multilocularis, E. granulosus, E. shiquicus, Taenia multiceps, T. saginata, T. asiatica, Dipylidium caninum, T. hydatigena, Toxocara canis, Fasciola hepatica, T. pisiformis, Mesocestoides lineatus and Cryptosporidiumn canis was detected using the multiplex RAA assay to evaluate its specificity. In addition, the reaction condition of the multiplex RAA assay was optimized, and was then employed to detect the tissues with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples to examine its application values. Results The multiplex RAA assay was effective to specifically amplify the mitochondrial gene fragments of E. multilocularis, E. granulosus and E. shiquicus within 40 min at 39 °C, with sequence lengths of 540, 430 bp and 200 bp, respectively. This multiplex RAA assay showed the minimum detection limits of 2.0, 2.5 pg/μL and 3.1 pg/μL for detection of the genomic DNA of E. multilocularis, E. granulosus and E. shiquicus, and presented the minimum detection limit of 200 copies/μL for detection of the recombinant plasmids containing E. multilocularis, E. granulosus and E. shiquicus target genes. This multiplex RAA assay was effective to simultaneously detect single and multiple infections with E. multilocularis, E. granulosus and E. shiquicus, but failed to amplify the genomic DNA of T. multiceps, T. saginata, T. asiatica, D. caninum, T. hydatigena, T. canis, F. hepatica, T. pisiformis, M. lineatus and C. canis. In addition, the optimized multiplex RAA assay was effective to detect all positive samples from the tissue samples with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples, which was fully consistent with the detection of the single PCR assay. Conclusion A sensitive and specific multiplex nucleic acid assay for rapid detection of E. multilocularis, E. granulosus and E. shiquicus has been successfully established.

Taenia saginata Infection Misdiagnosed as Acute Cholecystitis in a Tibetan Patient, in China

The present study reports a rare case of Taenia saginata infection, which was initially diagnosed as acute cholecystitis in a Tibetan patient at the Qinghai-Tibetan Plateau pastoral area, China. A 45-year-old female was initially diagnosed with acute cholecystitis at a hospital in China. She had a slight fever, weight loss and constipation and complained of pain in the upper abdomen and left back areas. Increase of monocyte, eosinophil and basophil levels were shown. Taenia sp. eggs were detected in a fecal examination. An adult tapeworm approximately 146 cm in length, whitish-yellow color, was collected from the patient after treatment with traditional Chinese medicine. The adult tapeworm had a scolex and proglottids with genital pores. The scolex was rectangular shape with 4 suckers and rostellum without hooklet. The cox1 gene sequence shared 99.5-99.8% homology with that of T. saginata from other regions in China. The patient was diagnosed finally infected with T. saginata by morphological and molecular charateristics.

Taenia saginata Infection Misdiagnosed as Acute Cholecystitis in a Tibetan Patient, in China

The present study reports a rare case of Taenia saginata infection, which was initially diagnosed as acute cholecystitis in a Tibetan patient at the Qinghai-Tibetan Plateau pastoral area, China. A 45-year-old female was initially diagnosed with acute cholecystitis at a hospital in China. She had a slight fever, weight loss and constipation and complained of pain in the upper abdomen and left back areas. Increase of monocyte, eosinophil and basophil levels were shown. Taenia sp. eggs were detected in a fecal examination. An adult tapeworm approximately 146 cm in length, whitish-yellow color, was collected from the patient after treatment with traditional Chinese medicine. The adult tapeworm had a scolex and proglottids with genital pores. The scolex was rectangular shape with 4 suckers and rostellum without hooklet. The cox1 gene sequence shared 99.5-99.8% homology with that of T. saginata from other regions in China. The patient was diagnosed finally infected with T. saginata by morphological and molecular charateristics.

Coenurosis of Yak, Bos grunniens, caused by Taenia multiceps: A Case Report with Molecular Identification in Qinghai Tibetan Plateau Area, China

Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.

A Case of Coenurosis in a Wild Rabbit (Lepus sinensis) Caused by Taenia serialis Metacestode in Qinghai Tibetan Plateau Area, China

Six cystic metacestodes were found in the abdominal muscles of a wild rabbit, Lepus sinensis, in China. The coenurus contained one or more scolices armed with hooklets. Mitochondrial cox1 (1,623 bp) confirmed 98% homology with cox1 of Taenia serialis. This is the first report of T. serialis infection in an intermediate host in the Qinghai Tibetan Plateau Area, China.

The First Case of Diarrhoea in Tibetan Sheep, Ovis aries, Caused by Balantidium coli in the Qinghai Tibetan Plateau Area, China

This study was carried out to determine the pathogen-causing diarrhoea in sheep Ovis aries in the Qinghai Tibetan Plateau Area, China. A trophozoite was identified as species of ciliate alveolates infecting the sheep based on morphological characteristics examined by microscope. It was mostly spherical, colourless and transparent, with many vesicles. Macronucleus and contractile vacuoles could not be distinguished. Size of the trophozoite was 80–180×70–150 μm and its surface was covered with cilia. Molecular analysis based on sequences of 18S rRNA and ITS genes confirmed the ciliate species as Balantidium coli. According to the literature, there have been many epidemiological investigations of B. coli infection in pigs, monkeys and humans. To our knowledge, this was the first report of B. coli infections in sheep in the Qinghai Tibetan Plateau Area of China, or eleswhere around the world. Importantly, the sheep case was rare but raised our concern that B. coli may spread across species and expand its host range.

Genotyping and gene nucleotide polymorphism of epidemic strain of Echino-coccus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province / 中国血吸虫病防治杂志

Objective To understand the genotypes and nucleotide polymorphisms of Echinococcus granulosus metacestode from humans and sheep in Tianjun region,Qinghai Province. Methods The specific primers were designed according to the cox1 and nad1 genes of E.granulosus mitochondrial genome sequences accessed by GenBank.The primers were used to detect the cyst samples from 16 sheep and 2 humans infected with E.granulosus in Tianjun region of Qinghai Province by PCR,then the PCR amplification products were sequenced,the genotypes and nucleotide polymorphisms of the cox1 and nad1 genes were analyzed.Results The 18 isolated samples all belonged to E.granulosus G1 genotype.Among all the isolates,9 haplotypes ex-isted in the cox1 gene with 16 nucleotide mutation sites,and there were 0 to 5 nucleotide differences with the highest variation rate of 0.31%,whereas 7 haplotypes occurred with 15 nucleotide mutation sites,and there were 1 to 8 nucleotide differences with the highest variation rate of 0.89% for the nad1 gene.Conclusions The epidemic genotype of E.granulosus is G1 in hu-mans and sheep in Tianjun region of Qinghai Province,and the nucleotide polymorphisms of the cox1 gene were more abundant than those of the nad1 gene,and the resolution of the nucleotide polymorphisms of cox1 gene is higher than that of the nad1 gene used in E.granulosus isolates.