Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCRbased detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections.Taenia solium, Taenia saginata, and Taenia asiatica are known as causative agents of taeniasis in humans. T. solium also causes cysticercosis in humans. In particular, neurocysticercosis caused by larval T. solium cysticerci developed in the central nervous system is the most serious disease characterized by diverse neurologic symptoms, most commonly epileptic seizure (5,20). In contrast to cysticercosis, taeniasis is relatively innocuous, with the adult stages of these cestodes infecting the small intestine of humans and causing a few specific symptoms, such as abdominal pain and nausea (15). However, gravid proglottids filled with eggs expelled from tapeworm carriers serve as a new source of infection for intermediate hosts, particularly in developing countries where sanitary conditions are poor. Therefore, early detection and adequate treatment of taeniasis is important for the prevention of cysticercosis infections. Furthermore, reliable epidemiological information for use in the effective control of taeniasis or cysticercosis, including accurate tools for parasite identification, is needed. To date, proglottids and scolices expelled from tapeworm carriers or cysticerci collected from intermediate hosts have been identified morphologically. In Asian regions, however, T. saginata and T. asiatica are frequently confused due to their morphological similarities. Moreover, a recent study demonstrates that two distinct genotypes of T. solium exist, i.e., Asian and American/African genotypes (14). DNA differential diagnosis is considered very useful for the accurate identification of human taeniid...

The genetic polymorphisms of Echinococcus spp. in the eastern Tibetan Plateau and the Xinjiang Uyghur Autonomous Region were evaluated by DNA sequencing analyses of genes for mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear elongation factor-1 alpha (ef1a). We collected 68 isolates of Echinococcus granulosus sensu stricto (s.s.) from Xinjiang and 113 isolates of E. granulosus s. s., 49 isolates of Echinococcus multilocularis and 34 isolates of Echinococcus shiquicus from the Tibetan Plateau. The results of molecular identification by mitochondrial and nuclear markers were identical, suggesting the infrequency of introgressive hybridization. A considerable intraspecific variation was detected in mitochondrial cox1 sequences. The parsimonious network of cox1 haplotypes showed star-like features in E. granulosus s. s. and E. multilocularis, but a divergent feature in E. shiquicus. The cox1 neutrality indexes computed by Tajima's D and Fu's Fs tests showed high negative values in E. granulosus s. s. and E. multilocularis, indicating significant deviations from neutrality. In contrast, the low positive values of both tests were obtained in E. shiquicus. These results suggest the following hypotheses: (i) recent Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

This area has the highest prevalences of both forms of this disease in the world.

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/2, EgAgB8/3, and EgAgB8/4, respectively. The remaining clone shared 51-58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.

Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out "blindly" using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.Alveolar echinococcosis (AE), caused by the metacestode of the fox tapeworm, Echinococcus multilocularis, is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE), caused by the metacestodes of the dog tapeworm, E. granulosus, is also endemic. For example, both AE and CE are highly endemic in China (8,19) and serological detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems, since the pathogenicity of these two types of echinococcosis and the treatment of patients with these diseases are critically different (14,16).In Japan and France, immunoblotting (Western blotting [WB]) assay systems have been developed for differentiation of AE from other diseases (4-7). The Asahikawa Medical College (AMC) group in Japan has focused on the detection of antibody response to the Em18 antigen (approximately 18 kDa) extracted from protoscoleces of E. multilocularis (4, 9, 15) and has tried to purify Em18, which shows a single band in WB, and to make it available for enzyme-linked immunosorbent assay (ELISA), using preparative isoelectric focusing (PIEF). Since purification of Em18 by PIEF takes longer and the yield is not as great, we have shifted to purification of Em18 by affinity chromatography (AffEm18) and production of a recombinant Em18 (RecEm18) for WB and ELISA (7, 9, 10, 13, 18). Echinococcus WB immunoglobulin G (IgG) (EchWB IgG; LDBIO Diagnostics, Lyon, France), which has a high sensitivity for the detection of both AE and CE, is basically very similar to the AMC system since it also focuses on differentiation of AE and CE based on different banding patterns including antigen B (most predominant at 8 and 26 to 28 kDa), Em16, and Em18 in crude antigens. The merit of the latter system is that it detects both AE and CE on a single strip based on the difference in the banding...

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To further evaluate recombinant Em18 antigen (rEm18) for immunodiagnosis of human alveolar echinococcosis, 208 serum samples were examined by enzyme-linked immunosorbent assay (ELISA). To comparatively assess the results of rEm18-ELISA, ELISA and immunoblot analysis with two affinity-purified native antigens were also performed with 45 selected serum samples. The results indicate that rEm18 is highly useful for serodiagnosis.

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