article: Cystic echinococcosis (CE) is a globally distributed cestode zoonosis that causes hepatic cysts. Although Echinococcus granulosus sensu stricto (s. s.) is the major causative agent of CE worldwide, recent molecular epidemiological studies have revealed that E. canadensis is common in countries where camels are present. One such country is Mongolia. Forty-three human hepatic CE cases that were confirmed histopathologically at the National Center of Pathology (NCP) in Ulaanbaatar (UB) were identified by analysis of mitochondrial cox 1 gene as being caused by either E. canadensis (n = 31,72. 1%) or E. granulosus s. s. (n = 12,27. 9%). The majority of the E. canadensis cases were strain G6/7 (29/31,93. 5%). Twenty three haplotypes were identified. Sixteen of 39 CE cases with data on age, sex and province of residence were citizens of UB (41. 0%), with 13 of the 16 cases from UB caused by E. canadensis (G6/7) (81. 3%). Among these 13 cases, nine were children (69. 2%). All pediatric cases (n = 18) were due to E. canadensis with 17 of the 18 cases (94. 4%) due to strain G6/7. Serum samples were available for 31 of the 43 CE cases, with 22 (71. 0%) samples positive by ELISA to recombinant Antigen B8/1 (rAgB). Nine of 10 CE cases caused by E. granulosus s. s. (90. 0%) and 13 of 20 CE cases by E. canadensis (G6/7) (65. 0%) were seropositive. The one CE case caused by E. canadensis (G10) was seronegative. CE cases caused by E. granulosus s. s. showed higher absorbance values (median value 1. 131) than those caused by E. canadensis (G6/7) (median value 0. 106) (p = 0. 0137). The main species/strains in the study population were E. canadenis and E. granulossus s. s. with E. canadensis the predominant species identified in children. The reason why E. canadensis appears to be so common in children is unknown.
  Cystic echinococcosis (CE) is a globally distributed parasitic zoonosis caused by ingestion of the eggs of Echinococcus granulosus sensu lato (s. l.) [1]–[5]. Recent molecular re-evaluation of E. granulosus s. l. has revealed that it consists of 5 independent species, E. granulosus sensu stricto (s. s.) (G1–G3), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6–G10) and E. felidis [4]–[6]. Although E. granulosus s. s. (G1) is the major causative agent of human CE where sheep are grazed with dogs [6]–[11], recent molecular studies of human CE specimens have revealed that CE cases caused by E. canadensis (G6–G10) are common in some areas where camels and other livestock including cattle, pigs, and goats are distributed [4]–[6], [10]–[22]. Therefore, it is important to include molecular identification of human CE cases in epidemiological studies. In Mongolia, more than 50% of the population lives in the capital city of Ulaanbaatar (UB), with the remainder largely following the traditional nomadic lifestyle [23]–[28]. For many years, CE has been recognized as a common disease in Mongolia even though there is very little published data. Since the collapse of the Soviet Union in 1991, two meetings have been held in UB on the topic of CE. The first meeting was held at the National Center of Communicable Diseases (NCCD) in May 1995 and Tsoodol, Narantuya and Goosh from Mongolia provided overviews of human cases to date [29]. During this meeting, it was reported that in 1950,7. 8% of all surgical patients were diagnosed CE, whereas this value was only 1. 9% in 1990. CE was determined to be the cause of 18% of the surgical cases seen at State Central First Hospital (SCFH) in 1993 [25]–[28]. The second meeting was held at the Health Science University of Mongolia (HSUM) in June 2009 for the purpose of establishing a network of CE experts [27]. During this meeting, Ayushkhuu from the National Center for Maternal and Child Health (NCMCH) summarized 25 pediatric CE cases from 2008 and 2009 (19 cases in 2008 and 6 cases from Jan to May 2009). These cases consisted of 15 boys and 10 girls, including 4 children under the age of 5-years [27]. During this same meeting, Bazarragchaa from the SCFH summarized a total of 144 (63 males and 81 females) CE cases (1989–2009 June) [27]. None of the reported cases differentiate between infection with E. granulosus s. s. and E. canadensis. There have been several sero-epidemiology studies of CE in Mongolia [30]–[33]. These studies all have used hydatid cyst fluid (HCF) for screening with an ELISA. However, none of the studies used a confirmatory test to verify their findings. Most surgical cases in Mongolia are diagnosed by histopathological identification of resected lesions at the National Center of Pathology (NCP) in UB [27], [28]. The present study is the first to identify the primary species/strains of CE present in Mongolian patients that had been histopathologically confirmed as having CE as well as evaluate antibody responses to the recombinant Antigen B8/1 (rAgB) by ELISA [34]–[43].
  Molecular analysis of human specimens and serological analysis of antibody responses, and junior researchers from several institutes in UB, Mongolia, to do laboratory analysis of these all specimens were approved by the Asahikawa Medical University (AMU) Institutional Review Board (AMU-IRB-1435). A total of 43 CE cases were evaluated, consisting of 18 pediatric cases from the NCMCH and 27 cases from the SCFH. In total, 43 hepatic CE cysts (1 cyst per patient) were obtained and confirmed to be CE histopathologically at the NCP. Pre-surgical serum samples were available for 31 of the 43 study patients (Table 1). De-identified data on patient age, sex, and province of residence were also obtained. Genomic DNA was extracted from 43 ethanol-fixed samples from hepatic CE patients (Table 1) using a DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer' s instructions. The extracted DNA was kept at −20°C until further analysis could be performed. DNA obtained was used as templates for polymerase chain reaction (PCR). The complete or partial mitochondrial cytochrome c oxidase subunit I (cox 1) gene was amplified by PCR as reported previously [44]–[46]. PCR products were treated with illustra ExoStar (GE Healthcare) to remove excess primers and dNTPs, and directly sequenced with a BigDye Terminator v3. 1 and a 3500 DNA sequencer (Life Technologies). Obtained sequences were edited using Geneious Pro version 7. 0. 4 (created by Biomatters, available from http: //www. geneious. com), and multiple alignments of each cox1 haplotype were made by the program MAFFT [47] with the homologous sequences of other Echinococcus species available in the GenBank database. A phylogenetic tree was constructed using the neighbour-joining method and Kimura' s two-parameter model [48] in Phylogenetic Analysis Using Parsimony (PAUP) version 4. 0b [49]. The robustness of the phylogenetic tree was tested by bootstrapping with 1000 replicates. For tree construction, Versteria mustelae was used as an outgroup because it is a sister to all members of the genus Echinococcus [50]. Recombinant Antigen B8/1 (rAgB) produced from E. multilocularis (rEmAgB) was applied for ELISA and immunoblot tests as reported previously [36]. Several specimens with optical density (OD) values around the cut-off were re-checked by immunoblot using the same antigen for confirmation (figure not shown) [36], [40], [41]. ELISA was carried out in flat-bottom 96-well microplates (Nunc, Maxisorp, Roskilde, Denmark) as previously described [36]. The microplates were coated with 100 ng/ml of rEmAgB diluted in phosphate-buffered saline (PBS) and incubated at 4 °C overnight. Excess antigen was removed by washing with PBS. Blocking was performed with blocking solution [1% casein in 20 mM Tris-HCl (pH 7. 6) containing 150 mM NaCl] and the plates were incubated at 37 °C for 1 hr. The plates were washed twice with PBS containing 0. 05% Tween 20 (PBST). 100 µl of diluted sera (1/100 dilution in blocking solution) was added and plates were incubated at 37 °C for 1. 5 hrs. After washing five times with PBST, 100 µl of protein G-peroxidase conjugate (1/4000 dilution in blocking solution) (Invitrogen, Camarillo, CA) was added to each well and the plates were incubated at 37 °C for 1. 5 hrs. Plates were washed six times with PBST and one time with PBS and incubated with 100 µl of substrate solution [0. 4 mM 2,2-azino-di (3-ethyl-benzthiazoline-6-sulfonate) (ABTS) in 0. 2 M citric acid buffer (pH 4. 7) ] at room temperature for 30 min. The color reaction was stopped by addition of 1% SDS. The optical density at 405 nm (OD405) was determined using an ELISA plate reader (Immuno Mini NJ2300, Tokyo, Japan). The cut-off point was set as the mean OD405 plus 3SD for 30 negative control samples. ELISA OD value results and positive ratios in CE cases caused by E. granulosus s. s. (G1) and E. canadensis (G6/7) were assessed by the Wilcoxon rank sum test and Fisher' s exact test, respectively. All analyses were two-tailed and a p-value < 0. 05 was considered statistically significant.
  Data on patient age, sex, and province of residence are shown in Table1. Nucleotide sequences of the cox1 gene (1608-1609 bp) were determined for 40 specimens, and consequently 23 haplotypes were obtained. Among these, 21 haplotypes (EgMGL1-10, EcMGL5-15) were newly identified in Mongolia. The nucleotide sequences of all haplotypes were deposited into DDBJ/EMBL/GenBank databases under the accession numbers AB893242-AB893264. Phylogenetic analysis clearly showed that 12 specimens (10 haplotype) were E. granulosus s. s. (G1) and 29 (12 haplotypes) and 2 (1 haplotype) specimens were E. canadensis (G6/7) and (G10), respectively (Table 1, Figure 1). For three specimens (Nos. 19,27,45), only a partial sequence (828 bp) was determined. BLAST search revealed that the sequence was 100% identical to the cox1 gene sequence of E. canadensis (G6/7) (Accession Number = AB813182). Among 43 specimens, 39 [11 E. granulosus s. s. (G1), 27 E. canadensis (G6/7), one E. canadensis (G10) ] had data available on patient age, sex and province of residence (Table 1, Figure 2). CE cases were found from 14 provinces, including UB (Figure 2). As shown in Table 1, all pediatric CE cases (n = 18) were caused by E. canadensis (100%). Of the 16 CE cases from UB (16/39,41. 0%), 13 cases were caused by E. canadensis (G6/7) (13/16,81. 3%). Among these 13 cases caused by E. canadensis (G6/7), nine were children (9/13,69. 2%). Among 31 serum samples examined, 22 samples (64. 7%) were antibody positive to rEmAgB: 9 of 10 E. granulosus s. s. (G1) (90%) cases and 13 of 20 E. canadensis (G6/7) (65%) cases. The one E. canadensis (G10) case was sero-negative (Table 1). Figure 3 illustrates antibody responses of these 30 serum samples to rEmAgB by ELISA. The median absorbance ratios were 1. 131 for E. granulosus (G1) and 0. 106 for E. canadensis (G6/7). The p-values for sero-positive ratio and absorbance value between the two species were 0. 2103 and 0. 0137, respectively. The average ages of all CE patients were 47. 6 years (range: 21 to 72 years) and 22. 5 years (range: 4 to 79 years) for patients infected with E. granulosus s. s. (G1) and E. canadensis, respectively. Of the 9 CE cases caused by E. granulosus s. s. (G1) with positive serology, 5 were females. Of the 12 CE cases caused by E. canadensis (G6/7) with positive serology, 6 were females. CE cases with the highest absorbance values (>1. 200) for E. canadensis (G6/7) were male children, whereas cases infected with E. granulosus s. s. (G1) were all adults (Table 1).
  Molecular identification of the causative species is important for epidemiological studies on CE. Use of rAgB serology for preoperative and postoperative diagnoses of CE may be a good complement to diagnostic imaging. Further studies are needed to explore the age variation in CE cases caused by E. granulosus s. s. versus E. canadensis in Mongolia and all other countries where both species are co-distributed. Additional studies on human CE cases, intermediate hosts, and definitive hosts are needed to better evaluate the epidemiology of the circulating Echinococcus species as well as risk factors for infection.
summary: Cystic echinococcosis (CE) is a parasitic zoonosis with a cosmopolitan distribution. Molecular analysis was carried out on 43 hepatic CE cysts from 43 cases confirmed histopathologically at the NCP, Mongolia. Molecular analysis revealed two species, Echinococcus canadensis and Echinococcus granulosus s. s. Twenty three haplotypes of the cox1 gene were identified. All pediatric cases (n = 18) were by E. canadensis. Sixteen of 39 CE cases with data on age, sex and province of residence were from UB (41. 0%), and 13 of these 16 cases were caused by E. canadensis (81. 3%). Among the 13 cases from UB, nine were children (69. 2%). A total of 31 serum samples from these 43 cases were analyzed for antibody response to rAgB with 22 (71. 0%) samples positive by ELISA to rAgB. Thirteen of 20 E. canadensis (G6/7) (65%) and nine of 10 E. granulosus s. s. (90%) were seropositive. CE cases by E. granulosus s. s. showed a higher absorbance value than cases by E. canadensis (p = 0. 0137). This is the first study to evaluate age distribution of and antibody responses to rAgB in CE cases caused by the two species in Mongolia. It remains unknown why E. canadensis appears to be more common in pediatric cases.
keywords: infectious diseases
  medicine and health sciences
  diagnostic medicine
  genetics
  biology and life sciences
  immunology
  microbiology
  surgical and invasive medical procedures
  pediatrics
title: Cystic Echinococcoses in Mongolia: Molecular Identification, Serology and Risk Factors