Jiamin Qiu

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.

Echinococcosis on the Tibetan Plateau: prevalence and risk factors for cystic and alveolar echinococcosis in Tibetan populations in Qinghai Province, China

Infections by larval stages of tapeworms of the genus Echinococcus (echinococcosis or hydatid disease) are zoonotic infections of major public health importance throughout much of the world. Humans become infected through accidental ingestion of eggs passed in faeces of canid definitive hosts. Tibetan populations of China have some of the highest documented levels of infections by both Echinococcus granulosus and E. multilocularis, the causes of cystic and alveolar echinococcosis, respectively. In this study we measured the prevalence of cystic (CE) and alveolar (AE) echinococcosis disease in Tibetan communities in Qinghai, Province, China, and identified putative risk factors for both infections in these communities. 3703 volunteers in three predominately Tibetan counties of Qinghai were surveyed between June 1997 and June 1998. Parasitic lesions were diagnosed by imaging of characteristic space-occupying lesions in abdominal organs (ultrasound) or the lungs (radiographs). Specific serodiagnostic assays (Dot-ELISA and Em2-ELISA) were performed on sera of positively imaged subjects to further distinguish the disease agent. All participants completed a questionnaire documenting age, sex, education level, occupation, lifestyle (nomadic or settled), slaughter practices, drinking water source, hygienic practice and association with dogs. Data were analyzed using SAS version 8. 6.6% of the volunteers had image-confirmed infection with E. granulosus (CE) and 0.8% had E. multilocularis (AE) infection. The significant univariate factors for echinococcal infection (both CE and AE) included livestock ownership, Tibetan ethnicity, female gender, low income, herding occupation, limited education, water source, age greater than 25 years old, poor hygienic practices, offal disposal practices and dog care. Multivariate analysis revealed that livestock ownership was a significant risk factor for both forms of the disease, as well as age greater than 25 years, female gender, herding occupation, and being nomadic (vs semi-nomadic or settled). No additional significant risk factors were identified among the 344 nomadic participants. Being female and being older than 25 years of age were significant factors among the 1906 semi-nomadic participants. Among the 1445 settled participants, allowing dogs to sleep indoors was statistically significant. Issues such as inadequate assessment of animal ownership, selection bias, disease misclassification, and loss of information may have led to reduction in strength of some risk factor associations and need to be addressed in future epidemiologic analysis of echinococcosis in this population.

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.

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.

Role of dog behaviour and environmental fecal contamination in transmission of Echinococcus multilocularis in Tibetan communities

On the Eastern Tibetan Plateau region (Sichuan province, China) dogs are regarded as important definitive hosts of Echinococcus multilocularis. We studied dog spatial behaviour in 4 Tibetan villages in order to determine the role of dogs in environmental contamination and their potential interactions with small mammal intermediate hosts. We identified definitive host species and Echinococcus spp. infection status of feces collected in the field by PCR methods and analysed the spatial distribution of canid feces. Nocturnal space utilization of GPS collared dogs in and around villages was also undertaken. E. multilocularis DNA was amplified in 23% of dog feces (n=142) and in 15% of fox feces (n=13) but this difference was not significant. However, dog feces were more frequently observed (78% of collected feces) than fox feces and are therefore assumed to largely contribute to human environment contamination. Feces were mainly distributed around houses of dog owners (0-200 m) where collared dogs spent the majority of their time. Inside villages, the contamination was aggregated in some micro-foci where groups of dogs defecated preferentially. Finally, small mammal densities increased from the dog core areas to grasslands at the periphery of villages occasionally used by dogs; male dogs moving significantly farther than females. This study constitutes a first attempt to quantify in a spatially explicit way the role of dogs in E. multilocularis peri-domestic cycles and to identify behavioural parameters required to model E. multilocularis transmission in this region.

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.

Specific IgG responses to recombinant antigen B and Em18 in cystic and alveolar echinococcosis in China

ABSTRACT An understanding of the correlation of the specific antibody responses and the disease phase is essential in evaluating diagnostic values of immunological tests in human echinococcosis. In this study, 422 echinococcosis patients diagnosed by ultrasonography, including 246 with cystic echinococcosis (CE), 173 with alveolar echinococcosis (AE), and 3 with dual infection, were tested for specific IgG in sera against recombinant AgB (rAgB) and recombinant Em18 (rEm18) in an enzyme-linked immunosorbent assay. As a result, rAgB-specific antibody was detected in 77.6% of CE and 86.1% of AE patients, while rEm18-specific antibody was present in 28.9% of CE and 87.3% of AE patients. Additionally, all three patients with dual infection exhibited specific antibodies responding to rAgB and rEm18. Further analysis revealed that rAgB-specific antibody was elevated in a significantly greater proportion (87.3%) of CE patients with cysts at active or transitional stages (CE1, CE2, or CE3), compared to 54.8% of other patients with cysts at an early or an inactive stage (CL or CE4 or CE5). Furthermore, rAgB-specific antibody was detected in 95.6% of CE2 cases, which was statistically greater than that (73.7%) in CE1 patients. Although rEm18-specific antibody was elevated in 28.9% of CE patients, the positive reaction was much weaker in CE than in AE cases. Serum levels and concentrations of rEm18-specific antibody were further indicated to be strongly disease phase correlated in AE patients, with positive rates of 97.4% in cases with alveolar lesions containing central necrosis and 66.7% in patients with early alveolar lesions that measured ≤5 cm.

Reinfection studies of canine echinococcosis and role of dogs in transmission of Echinococcus multilocularis in Tibetan communities, Sichuan, China

SUMMARY In the eastern Tibetan plateau both human cystic and alveolar echinococcosis (AE) caused by infection with Echincoccus granulosus or Echinococcus multilocularis, respectively are highly endemic. The domestic dog plays a key role in zoonotic transmission in this region. Our primary objective was to investigate the role of domestic dogs in maintaining transmission of E. multilocularis in Shiqu county, Sichuan. A cohort of 281 dogs was followed up over one year after a single treatment with praziquantel followed by re-infection surveillance at 2, 5 and 12 months post-treatment. Faecal samples were tested by an Echinococcus genus-specific coproantigen ELISA and two species-specific copro-PCR tests. Total Echinococcus coproantigen prevalence in Shiqu at baseline was 21% and 9·6% after 2 months. E. multilocularis copro-PCR was positive in 11·2% of dogs before treatment (vs 3·6% with E. granulosus copro-DNA), 2·9% at 2 months post-treatment, and 0% at 5 month and 12 months. The results suggest that dogs may have the potential to maintain E. multilocularis transmission within local pastoral communities, and thus dog dosing could be an effective strategy to reduce transmission of E. multilocularis as well as E. granulosus in these co-endemic Tibetan communities.

A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas.