A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers for Echinococcus multilocularis (amplicon size 395 bp) were species-specific as assessed by in silico analysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA from E. granulosus or Taenia spp. was not possible. The primers designed for E. granulosus also amplified DNA (117 bp) from E. vogeli, and those designed for Taenia spp. amplified products (267 bp) from species of Mesocestoides, Dipylidium and Diphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the described E. granulosus genotypes. Taenia spp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.

Echinococcosis is a major emerging zoonosis in central Asia. A cross-sectional study of dogs in four villages in rural Kyrgyzstan was undertaken to investigate the epidemiology and transmission of Echinococcus spp. A total of 466 dogs were examined by arecoline purgation for the presence of Echinococcus granulosus and Echinococcus multilocularis. In addition, a faecal sample from each dog was examined for taeniid eggs. Any taeniid eggs found were investigated using PCR techniques (multiplex and single target PCR) to improve the diagnostic sensitivity by confirming the presence of Echinococcus spp. and to identify E. granulosus strains. A total of 83 (18%) dogs had either E. granulosus adults in purge material and/or E. granulosus eggs in their faeces as confirmed by PCR. Three genotypes of E. granulosus: G1, G4 and the G6/7 complex were shown to be present in these dogs through subsequent sequence analysis. Purge analysis combined with PCR identified 50 dogs that were infected with adult E. multilocularis and/or had E. multilocularis eggs in their faeces (11%). Bayesian techniques were employed to estimate the true prevalence, the diagnostic sensitivity and specificity of the procedures used and the transmission parameters. The sensitivity of arecoline purgation for the detection of echinococcosis in dogs was rather low, with a value of 38% (Credible intervals (CIs) 27-50%) for E. granulosus and 21% (CIs 11-34%) for E. multilocularis. The specificity of arecoline purgation was assumed to be 100%. The sensitivity of coproscopy followed by PCR of the isolated eggs was calculated as 78% (CIs 57-87%) for E. granulosus and 50% (CIs 29-72%) for E. multilocularis with specificity of 93% (CIs 88-96%) and 100% (CIs 97-100), respectively. The 93% specificity of the coprological-PCR for E. granulosus could suggest coprophagia rather than true infections. After adjusting for the sensitivity of the diagnostic procedures, the estimated true prevalence of infection of E. granulosus was 19% (CIs 15-25%) and the infection pressure in the dog population was estimated to be 0.29 infections per year (CIs 0.014-0.75). Logistic regression analysis failed to identify any significant risk factors for infections for E. granulosus. After adjusting for the sensitivity of the test procedures, the estimated true prevalence was 18% (CIs 12-30%). Dogs that were restrained had a significantly lower prevalence of E. multilocularis of 11% (CIs 6-29%) compared with 26% in free-roaming dogs (CIs 17-44%) and