Ning Xiao

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.

Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis

Intraspecific genetic variation of Echinococcus multilocularis, the etiologic agent of human alveolar echinococcosis, has been evaluated among 76 geographic isolates from Europe, Asia and North America by using sequence data of mitochondrial and nuclear DNA. Relatively low genetic variation was found only in the mitochondrial DNA sequence consisting of 3 protein-coding genes. Pairwise divergence among the resultant 18 haplotypes ranged from 0.03 to 1.91%. Phylogenetic trees and parsimony network of these haplotypes depicted a geographic division into European, Asian and North American clades, but 1 haplotype from Inner Mongolia was unrelated to other haplotypes. The coexistence of the Asian and North American haplotypes could be seen, particularly on the St. Lawrence Island in the Bering Sea. These data suggest an evolutionary scenario in which distinct parasite populations derived from glacial refugia have been maintained by indigenous host mammals. The nuclear DNA sequence for the immunodominant B cell epitope region of ezrin/radixin/moesin-like protein (elp) was extremely conservative, indicating that the elp antigen is available for immunodiagnosis in any endemic areas.

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.

Molecular Cloning, Expression, and Serological Evaluation of an 8-Kilodalton Subunit of Antigen B from Echinococcus multilocularis

ABSTRACT Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH2-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.

Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) with Affinity-Purified Em18 and an ELISA with Recombinant Em18 for Differential Diagnosis of Alveolar Echinococcosis: Results of a Blind Test

ABSTRACT Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out “blindly” using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.

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.

Evaluation of Use of Recombinant Em18 and Affinity-Purified Em18 for Serological Differentiation of Alveolar Echinococcosis from Cystic Echinococcosis and Other Parasitic Infections

ABSTRACT To further evaluate recombinant Em18 antigen (rEm18) for immunodiagnosis of human alveolar echinococcosis, 208 serum samples were examined by enzyme-linked immunosorbent assay (ELISA). To comparatively assess the results of rEm18-ELISA, ELISA and immunoblot analysis with two affinity-purified native antigens were also performed with 45 selected serum samples. The results indicate that rEm18 is highly useful for serodiagnosis.

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.

Analysis of Malaria Epidemiological Characteristics in the People's Republic of China, 2004–2013

This study aims to explore and characterize the malaria-endemic situation and trends from 2004 to 2013, to provide useful evidence for subsequently more effective strategic planning of malaria elimination in China. A total of 256,179 confirmed malaria cases were recorded in this period, and 86.8% of them were reported during 2004-2008. Between 2004 and 2008, Plasmodium vivax was the major species (72.2%) of malaria parasite. Most cases (67.3%) were found in male, and mainly in the age group of 35-39 years. A total of 236 deaths resulting from malaria were reported and nearly half (45.3%) of them were in Yunnan province. In all, 204,760 local malaria (79.9%) and 51,419 imported malaria (20.1%) were observed during 2004-2013. However, afterward the proportion of imported malaria continuously increased from 2004 (16.2%) to 2013 (97.9%). Moreover, 9,285 imported malaria cases were recorded during 2011-2013 in China, of which 5,976 cases (64.4%) came back from Africa. Overall, China has made achievements in controlling malaria, the locally transmitted malaria significantly declined in the past decades, by which the incidence has achieved historically the lowest levels. On the other hand, imported malaria has increasingly become a severe threat to malaria elimination. Therefore, to prevent the reintroduction of malaria, surveillance systems need to be well planned and managed to ensure timely case detection and prompt response at the elimination stage.

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.