Tiaoying Li

Genetic polymorphisms of Echinococcus tapeworms in China as determined by mitochondrial and nuclear DNA sequences

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 Tajimas D and Fus 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 founder effects arose in E. granulosus and E. multilocularis after introducing particular individuals into the endemic areas by anthropogenic movement or natural migration of host mammals, and (ii) the ancestor of E. shiquicus was segregated into the Tibetan Plateau by colonising alpine mammals and its mitochondrial locus has evolved without bottleneck effects.

Evaluation of a loop-mediated isothermal amplification method using fecal specimens for differential detection of Taenia species from humans.

ABSTRACT We compared the performance of loop-mediated isothermal amplification (LAMP) with that of a multiplex PCR method for differential detection of human Taenia parasites in fecal specimens from taeniasis patients. The LAMP method, with no false positives, showed a higher sensitivity (88.4%) than the multiplex PCR (37.2%). Thus, it is expected that the LAMP method has a high value for molecular diagnosis of taeniasis.

Widespread co-endemicity of human cystic and alveolar echinococcosis on the eastern Tibetan Plateau, northwest Sichuan/southeast Qinghai, China

Cystic echinococcosis (CE) or hydatid disease is known to be cosmopolitan in its global distribution, while alveolar echinococcosis (AE) is a much rarer though more pathogenic hepatic parasitic disease restricted to the northern hemisphere. Both forms of human echinococcosis are known to occur on the Tibetan Plateau, but the epidemiological characteristics remain poorly understood. In our current study, abdominal ultrasound screening programs for echinococcosis were conducted in 31 Tibetan townships in Ganze and Aba Tibetan Autonomous Prefectures of northwest Sichuan Province during 2001-2008. Hospital records (1992-2006) in a major regional treatment centre for echinococcosis in Sichuan Province were also reviewed. Of 10,186 local residents examined by portable ultrasound scan, 645 (6.3%) were diagnosed with echinococcosis: a prevalence of 3.2% for CE, 3.1% for AE and 0.04% for dual infection (both CE and AE). Human cystic and alveolar echinococcosis in pastoral areas was highly co-endemic, in comparison to much lower prevalences in semi-pastoral or farming regions. The high ultrasound prevalence in these co-endemic areas in northwest Sichuan Province was also reflected in the hospital study, and hospital records furthermore indicated another possible highly co-endemic focus in Guoluo Prefecture of Qinghai Province, located at the border of northwest Sichuan. These chronic cestode zoonoses constitute an unparalleled major public health problem for pastoral Tibetan communities, and pose great difficulties for adequate treatment access and effective transmission control in such remote regions.

Transmission ecosystems of Echinococcus multilocularis in China and Central Asia.

SUMMARY From continental to regional scales, the zoonosis alveolar echinococcosis (AE) (caused by Echinococcus multilocularis) forms discrete patches of endemicity within which transmission hotspots of much larger prevalence may occur. Since the late 80s, a number of hotspots have been identified in continental Asia, mostly in China, wherein the ecology of intermediate host communities has been described. This is the case in south Gansu, at the eastern border of the Tibetan plateau, in south Ningxia, in the western Tian Shan of Xinjiang, and in the Alay valley of south Kyrgyzstan. Here we present a comparative natural history and characteristics of transmission ecosystems or ecoscapes. On this basis, regional types of transmission and their ecological characteristics have been proposed in a general framework. Combining climatic, land cover and intermediate host species distribution data, we identified and mapped 4 spatially distinct types of transmission ecosystems typified by the presence of one of the following small mammal ‘flagship’ species: Ellobius tancrei, Ochotona curzoniae, Lasiopodomys brandtii or Eospalax fontanierii. Each transmission ecosystem had its own characteristics which can serve as a reference for further in-depth research in the transmission ecology of E. multilocularis. This approach may be used at fine spatial scales to characterize other poorly known transmission systems of the large Eurasian endemic zone, and help in consideration of surveillance systems and interventions.

Drivers of Echinococcus multilocularis transmission in China: small mammal diversity, landscape or climate?

Background Human alveolar echinococcocosis (AE) is a highly pathogenic zoonotic disease caused by the larval stage of the cestode E. multilocularis. Its life-cycle includes more than 40 species of small mammal intermediate hosts. Therefore, host biodiversity losses could be expected to alter transmission. Climate may also have possible impacts on E. multilocularis egg survival. We examined the distribution of human AE across two spatial scales, (i) for continental China and (ii) over the eastern edge of the Tibetan plateau. We tested the hypotheses that human disease distribution can be explained by either the biodiversity of small mammal intermediate host species, or by environmental factors such as climate or landscape characteristics. Methodology/findings The distributions of 274 small mammal species were mapped to 967 point locations on a grid covering continental China. Land cover, elevation, monthly rainfall and temperature were mapped using remotely sensed imagery and compared to the distribution of human AE disease at continental scale and over the eastern Tibetan plateau. Infection status of 17,589 people screened by abdominal ultrasound in 2002–2008 in 94 villages of Tibetan areas of western Sichuan and Qinghai provinces was analyzed using generalized additive mixed models and related to epidemiological and environmental covariates. We found that human AE was not directly correlated with small mammal reservoir host species richness, but rather was spatially correlated with landscape features and climate which could confirm and predict human disease hotspots over a 200,000 km2 region. Conclusions/Significance E. multilocularis transmission and resultant human disease risk was better predicted from landscape features that could support increases of small mammal host species prone to population outbreaks, rather than host species richness. We anticipate that our study may be a starting point for further research wherein landscape management could be used to predict human disease risk and for controlling this zoonotic helminthic.

Species identification of human echinococcosis using histopathology and genotyping in northwestern China.

Human cystic echinococcosis, caused by infection with the larval stage of Echinococcus granulosus, and alveolar echinococcosis, caused by the larval form of E. multilocularis, are known to be important public health problems in western China. Echinococcus shiquicus is a new species of Echinococcus recently described in wildlife hosts from the eastern Tibetan plateau and its infectivity and/or pathogenicity in humans remain unknown. In the current study, parasite tissues from various organs were collected post-operatively from 68 echinococcosis patients from Sichuan and Qinghai provinces in eastern China. The tissues were examined by histopathology and genotyped using DNA sequencing and PCR-RFLP. Histopathologically, 38 human isolates were confirmed as E. granulosus and 30 as E. multilocularis. Mitochondrial cob gene sequencing and PCR-RFLP with rrnL as the target gene confirmed 33 of 53 of the isolates to have the G1 genotype of sheep/dog strain of E. granulosus as the only source of infection, while the remaining 20 isolates were identified as E. multilocularis. No infections were found to be caused by E. shiquicus. Additionally, 5 of 20 alveolar echinococcosis patients were confirmed to have intracranial metastases from primary hepatic alveolar echinococcosis lesions. All these cases originated from four provinces or autonomous regions but most were distributed in Sichuan and Qinghai provinces, where high prevalence rates of human alveolar echinococcosis and cystic echinococcosis were previously documented.

Post-treatment follow-up study of abdominal cystic echinococcosis in Tibetan communities of northwest Sichuan Province, China

Background Human cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, with the liver as the most frequently affected organ, is known to be highly endemic in Tibetan communities of northwest Sichuan Province. Antiparasitic treatment with albendazole remains the primary choice for the great majority of patients in this resource-poor remote area, though surgery is the most common approach for CE therapy that has the potential to remove cysts and lead to complete cure. The current prospective study aimed to assess the effectiveness of community based use of cyclic albendazole treatment in Tibetan CE cases, and concurrently monitor the changes of serum specific antibody levels during treatment. Methodology/Principal Findings Ultrasonography was applied for diagnosis and follow-up of CE cases after cyclic albendazole treatment in Tibetan communities of Sichuan Province during 2006 to 2008, and serum specific IgG antibody levels against Echinococcus granulosus recombinant antigen B in ELISA was concurrently monitored in these cases. A total of 196 CE cases were identified by ultrasound, of which 37 (18.9%) showed evidence of spontaneous healing/involution of hepatic cyst(s) with CE4 or CE5 presentations. Of 49 enrolled CE cases for treatment follow-up, 32.7% (16) were considered to be cured based on B-ultrasound after 6 months to 30 months regular albendazole treatment, 49.0% (24) were improved, 14.3% (7) remained unchanged, and 4.1% (2) became aggravated. In general, patients with CE2 type cysts (daughter cysts present) needed a longer treatment course for cure (26.4 months), compared to cases with CE1 (univesicular cysts) (20.4 months) or CE3 type (detached cyst membrane or partial degeneration of daughter cysts) (9 months). In addition, the curative duration was longer in patients with large (>10 cm) cysts (22.3 months), compared to cases with medium (5–10 cm) cysts (17.3 months) or patients with small (<5 cm) cysts (6 months). At diagnosis, seven (53.8%) of 13 cases with CE1 type cysts without any previous intervention showed negative specific IgG antibody response to E. granulosus recombinant antigen B (rAgB). However, following 3 months to 18 months albendazole therapy, six of these 7 initially seronegative CE1 cases sero-converted to be specific IgG antibody positive, and concurrently ultrasound scan showed that cysts changed to CE3a from CE1 type in all the six CE cases. Two major profiles of serum specific IgG antibody dynamics during albendazole treatment were apparent in CE cases: (i) presenting as initial elevation followed by subsequent decline, or (ii) a persistent decline. Despite a decline, however, specific antibody levels remained positive in most improved or cured CE cases. Conclusions This was the first attempt to follow up community-screened cystic echinococcosis patients after albendazole therapy using ultrasonography and serology in an endemic Tibetan region. Cyclic albendazole treatment proved to be effective in the great majority of CE cases in this resource-poor area, but periodic abdominal ultrasound examination was necessary to guide appropriate treatment. Oral albendazole for over 18 months was more likely to result in CE cure. Poor drug compliance resulted in less good outcomes. Serology with recombinant antigen B could provide additional limited information about the effectiveness of albendazole in CE cases. Post-treatment positive specific IgG antibody seroconversion, in initially seronegative, CE1 patients was considered a good indication for positive therapeutic efficacy of albendazole.

Echinococcoses and Tibetan Communities

To the Editor: The People’s Republic of China accounts for >500,000 cases of echinococcosis and more disability-associated life years (DALYs) lost because of this disease than any other world region (1,2). Hydatid cysts of Echinococcus granulosus (cystic echinococcosis [CE]), or the more pathogenic lesions with multiple vesicles caused by E. multilocularis infection (alveolar echinococcosis [AE]), usually grow slowly in the liver, so that severe illness and death may eventually occur in a high proportion of those with untreated infections (3,4). Apart from surgery, long-term anthelminthic therapy (>6 months) with the benzimidazole compound albendazole, although parasitostatic only, has a beneficial outcome in >50% of cases (5). To control the transmission of this zoonosis, veterinary public health measures must be emphasized (6). In 2004 the Chinese Ministry of Health (MoH) undertook a nationwide assessment of 8 parasitic diseases, including malaria, schistosomiasis, and echinococcosis. To identify echinococcosis, 7 provincial MoHs carried out a mass abdominal screening of 34,500 persons using portable ultrasound scanners. The overall prevalence (2.5%) was highest in Tibetan communities in the Tibet Autonomous Region and in northwestern Sichuan and Qinghai Provinces (these latter regions form part of the eastern Tibetan Plateau). Collaborative studies involving the Sichuan Center for Disease Control and Prevention (based in Chengdu) and an international consortium of research institutes partly funded by the US National Institutes of Health (Bethesda, MD, USA) have shown an increasingly serious public health problem at the village, township, and county levels. In Shiqu County of Ganze Tibetan Autonomous Prefecture, 414 (12.9%) of nearly 3,199 persons surveyed by ultrasound (with serologic confirmation) exhibited CE or AE, including 19% in this category (7). The effects of human echinococcosis are substantial, with >50,000 DALYs lost in a population of 63,000 in Shiqu County (8). Despite increased urbanization in China, >70% of Tibetans still live as seminomadic pastoralists on the high grasslands at an altitude >3,500 m. Most Tibetan herdsmen families keep at least 1 dog, and large numbers of ownerless stray dogs are tolerated by pastoralists and Buddhist monks. Risk factors for human echinococcosis (both CE and AE) in Tibetan communities usually include occupation, age (older persons are at higher risk), gender (higher risk for female), environment (pastoral landscapes), livestock ownership, and a history of dog ownership, as well as indicators of low socioeconomic status, including poor water quality and illiteracy (7,9) The prevalence levels of human AE in Ganze Tibetan Autonomous Prefecture (Sichuan Province) are among the highest recorded anywhere in the world. This situation presents a formidable challenge for early diagnosis, optimal affordable treatment, and prevention and control. Markham Hospital in Aba Tibetan Autonomous Prefecture (Sichuan) performed 1,200 operations for echinococcosis from 1992 through 2005, 20% for AE disease. For remote, high-altitude, pastoral Tibetan communities, however, long-term albendazole therapy is the only realistic treatment option, but regular follow-up of patients is difficult in these poorly accessible communities. To address the public health concerns and consider options for controlling hydatidosis/echinococcosis in the eastern Tibetan Plateau, an International Workshop on Treatment, Prevention and Control of Echinococcosis was held in Chengdu in May 2006 with support from the Sichuan Center for Disease Control and Prevention, MoH Beijing, the New Zealand International Aid and Development Agency [NZIAD]), Fogarty-National Institutes of Health (USA), Xinjiang Medical University, and the Boulder-Lhasa Sister City Project. Recommendations stressed the following public health needs: improved treatment centers within the known disease-endemic counties or prefectures for long-term follow-up of patients after surgery and chemotherapy for both CE and AE disease, a better understanding of the epidemiology and ecology of transmission, and planning for pilot control interventions against both CE and AE transmission. NZAID made a detailed report of the implementation and effects of a pilot echinococcosis control program (2000–2006) in Datangma County, Ganze Tibetan Autonomous Prefecture (Sichuan Province). Problems occurred chiefly because of poor intersectoral cooperation, difficult logistics, cultural antagonism, lack of participatory planning, difficult access, treatment of dogs (with praziquantel), vaccination of livestock with the new EG95 vaccine (6), and lack of adequate surveillance of dog and livestock infection levels. The report indicated how many of these difficulties could be overcome. Consequently, the People’s Republic of China MoH and provincial disease control networks approved funding in July 2006 to initiate pilot intervention programs against echinococcosis in 17 Tibetan autonomous counties of northwest Sichuan. Control options initially focused on regular supervised dosing of owned dogs and stray dogs (with praziquantel) by local operatives from district disease control centers and on improving health education at primary healthcare levels. Surveillance relies on measuring regularly the degree of Echinococcus infection in dogs by using a coproantigen test. Also, a specific age cohort of schoolchildren is monitored by ultrasound and serologic testing each year to determine changes in the prevalence of the 2 diseases. Albendazole is provided free, and the cost of surgery for hydatid disease is also subsidized through the new National Rural Cooperative Medical Insurance System. In addition to the major public health problem now being recognized for echinococcosis in Tibetan communities, their general health indices are low (higher prevalence of tuberculosis, bone diseases such as arthritis, and poorer health in general) because of living and working at altitudes >4,000 m, compared with those in most other areas of China. Access and outreach should be improved (in conjunction with animal health initiatives) (10) for effective delivery of treatment, vaccination, and health education packages to these largely scattered and marginalized pastoral communities.

Recent hybridization between Taenia asiatica and Taenia saginata

Five Taenia tapeworms collected from humans in Tibetan Plateau, Sichuan, China, where three species of human Taenia are sympatrically endemic, were examined for the mitochondrial cox1 gene and two nuclear genes, ef1 and elp. Phylogenetic analyses of these genes revealed that two adult worms showed nuclear-mitochondrial discordance, suggesting that they originated from hybridization between Taenia saginata and Taenia asiatica. One of two worms had T. asiatica-type mtDNA, whereas another worm had T. saginata-type mtDNA, indicating that reciprocal hybridization between T. saginata and T. asiatica could occur. The worm having T. asiatica-type mtDNA was heterozygous at both nuclear loci with T. saginata-type alleles and T. asiatica-type alleles. In another worm, the ef1 locus was heterozygous with a T. saginata-type alleles and T. asiatica-type alleles, while the elp locus was homozygous with T. saginata-type alleles. Self-fertilization is the main reproductive method of the genus Taenia. Since self-fertilization represents a type of inbreeding, each locus in the offspring would become homozygous over generations with genetic drift. The fact that some nuclear loci are still heterozygous means that hybridization might have occurred recently. Hybridization between T. asiatica and T. saginata is probably an ongoing event in many areas in which they are sympatrically endemic.

Cystic echinococcoses in Mongolia: molecular identification, serology and risk factors.

Background 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. Methodology/Principal Findings 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). Conclusion/Significance 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.