Environmental distribution of Echinococcus- and Taenia spp.-contaminated dog feces in Kyrgyzstan.

Recently, there have been epidemics of human cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Kyrgyzstan. This study investigated 2 districts for the presence of Echinococcus granulosus s.l. and Echinococcus multilocularis eggs; species identity was confirmed by polymerase chain reaction in dog feces and the level of environmental contamination with parasite eggs in 2017­2018 was also investigated. In the Alay district 5 villages with a high reported annual incidence of AE of 162 cases per 100 000 and 5 villages in the Kochkor district which had a much lower incidence of 21 cases per 100 000 were investigated. However, the proportion of dog feces containing E. granulosus s.l. eggs was ~4.2 and ~3.5% in Alay and Kochkor respectively. For E. multilocularis, the corresponding proportions were 2.8 and 3.2%. Environmental contamination of Echinococcus spp. eggs was estimated using the McMaster technique for fecal egg counts, weight and density of canine feces. The level of environmental contamination with E. multilocularis eggs was similar at 4.4 and 5.0 eggs per m2 in Alay and Kochkor respectively. The corresponding values for E. granulosus s.l. were 8.3 and 7.5 eggs per m2. There was no association between village or district level incidence of human AE or CE and the proportion of dog feces containing eggs of Echinococcus spp. or the level of environmental contamination. Increased contamination of taeniid eggs occured in the autumn, after the return of farmers with dogs from summer mountain pastures.

Serological Assays for Alveolar and Cystic Echinococcosis-A Comparative Multi-Test Study in Switzerland and Kyrgyzstan.

Both alveolar (AE) and cystic echinococcosis (CE) are lacking pathognomonic clinical signs; consequently imaging technologies and serology remain the main pillars for diagnosis. The present study included 100 confirmed treatment-naïve AE and 64 CE patients that were diagnosed in Switzerland or Kyrgyzstan. Overall, 10 native Echinococcus spp. antigens, 3 recombinant antigens, and 4 commercial assays were comparatively evaluated. All native E. multilocularis antigens were produced in duplicates with a European and a Kyrgyz isolate and showed identical test values for the diagnosis of AE and CE. Native antigens and three commercial tests showed high diagnostic sensitivities (Se: 86-96%) and specificities (Sp: 96-99%) for the diagnosis of AE and CE in Swiss patients. In Kyrgyz patients, values of sensitivities and specificities were 10-20% lower as compared to the Swiss patients' findings. For the sero-diagnosis of AE in Kyrgyzstan, a test-combination of an E. multilocularis protoscolex antigen and the recombinant antigen Em95 appears to be the most suitable test strategy (Se: 98%, Sp: 87%). For the diagnosis of CE in both countries, test performances were hampered by major cross-reactions with AE patients and other parasitic diseases as well as by limited diagnostic sensitivities (93% in Switzerland and 76% in Kyrgyzstan, respectively).

LAMP Assay for the Detection of Echinococcus multilocularis Eggs Isolated from Canine Faeces by a Cost-Effective NaOH-Based DNA Extraction Method.

The detection of Echinococcus multilocularis in infected canids and the environment is pivotal for a better understanding of the epidemiology of alveolar echinococcosis in endemic areas. Necropsy/sedimentation and counting technique remain the gold standard for the detection of canid infection. PCR-based detection methods have shown high sensitivity and specificity, but they have been hardly used in large scale prevalence studies. Loop-mediated isothermal amplification (LAMP) is a fast and simple method to detect DNA with a high sensitivity and specificity, having the potential for field-application. A specific LAMP assay for the detection of E. multilocularis was developed targeting the mitochondrial nad1 gene. A crucial step for amplification-based detection methods is DNA extraction, usually achieved utilising silica-gel membrane spin columns from commercial kits which are expensive. We propose two cost-effective and straightforward methods for DNA extraction, using NaOH (method 1A) and InstaGeneTM Matrix (method 1B), from isolated eggs circumventing the need for commercial kits. The sensitivity of both assays with fox samples was similar (72.7%) with multiplex-PCR using protocol 1A and LAMP using protocol 1B. Sensitivity increased up to 100% when testing faeces from 12 foxes infected with more than 100 intestinal stages of E. multilocularis. For dogs, sensitivity was similar (95.4%) for LAMP and multiplex-PCR using protocol 1B and for both methods when DNA was extracted using protocol 1A (90.9%). The DNA extraction methods used here are fast, cheap, and do not require a DNA purification step, making them suitable for field studies in low-income countries for the prevalence study of E. multilocularis.

Genetic diversity of Echinococcus multilocularis and Echinococcus granulosus sensu lato in Kyrgyzstan: The A2 haplotype of E. multilocularis is the predominant variant infecting humans.

Alveolar and cystic echinococcosis (AE, CE) caused by E. multilocularis and E. granulosus s.l., respectively, are considered emerging zoonotic diseases in Kyrgyzstan with some of the world highest regional incidences. Little is known regarding the molecular variability of both species in Kyrgyzstan. In this study we provide molecular data from a total of 72 parasite isolates derived from humans (52 AE and 20 CE patients) and 43 samples from dogs (23 infected with E. multilocularis and 20 with E. granulosus s.l.).Genetic variability in E. multilocularis was studied using the concatenated complete sequences of the cob, nad2 and cox1 mitochondrial genes adding a total of 3,558bp per isolate. The cob/nad2/cox1 A2 haplotype was identified in 63.4% of the human and in 65.2% of the dog samples. This haplotype was originally described in samples from Kazakhstan and St. Lawrence Island (Alaska, USA). We also describe here 16 non-previously defined variants of E. multilocularis (called A11-A26). All haplotypes cluster together within the Asian group in the haplotype network. Based on Fst values, low level of genetic differentiation was found between the populations of E. multilocularis isolated from different regions within the country. However, high degree of differentiation was found when all the concatenated sequences from Kyrgyzstan are considered as a single population and compared with the population of the parasite from the neighbouring country China. In the case of E. granulosus s.l. the analysis was based in 1,609bp of the cox1 gene. One isolate from a dog was identified as E. equinus, while all the other sequences were identified belonging to E. granulosus s.s. In total, 24 cox1 haplotypes of E. granulosus s.s. were identified including the already described variants: Eg01 (in 6 samples), Eg33 (in 4 samples), EgCl04 (in 2 samples), Eg03 (in 1 sample) and Eg32 (in 1 sample). From the twenty-five other isolates of E. granulosus s.s. a total of 19 non-previously described cox1 haplotypes were identified and named as EgKyr1 to EgKyr19. The most common haplotype infecting human is the EgKyr1 which was found in 5 isolates.The cob/nad2/cox1 A2 haplotype of E. multilocularis is responsible for the majority of human infections in Kyrgyzstan and is also found in the majority of dogs included in this study. Further similar studies in different parts of Asia could elucidate if it is also the most common variant infecting humans in other countries. It remains unknown if this particular haplotype presents differences in virulence which could have contributed to the emergency of alveolar echinococcosis in Kyrgyzstan. In the case of E. granulosus s.s. it seems that there is no dominant haplotype infecting humans in Kyrgzstan. Further characterization of biological or antigenic features of dominant mitochondrial haplotypes could help to elucidate if they present differences which could be relevant in the diagnostic, pathogenicity or in the host/parasite interaction when infecting humans.