[Propagating the Culture of Schistosomiasis Control and Promoting Its Sustainable Development in China].

Great achievements have been gained in schistosomiasis control in China due to the last half-century endeavor. These achievements not only indicate the success in disease control, but also represent a particularly successful social practice with a unique cultural property. Inspite of the accumulating reports on schistosomiasis prevention and control experience, there is a need to sublimate the experience to the "cultural" level, as historically the value of cultural and spiritual merits goes far beyond that of the achievements. Although the strategy and technology will be enriched with time, the cultural spirit remains especially important for the long- term promotion of schistosomiasis control. This paper materially dissected the meaning of "China's culture for schistosomiasis control", i.e., people-first, government-oriented, science-respected, collective wisdom and efforts, and diversity-embracing.

[A historical reflect on the disciplinary development of human parasitology].

The authors, in this paper, has briefly looked back the developmental history of human parasitology, which, as an independent discipline, was established in late 19th century and early 20th century. In the process, it underwent an early height of development, then met with setback and relative decline. Since 70s-80s of last century, the introduction and application of new theory of modern biology, especially advanced biotechniques to parasitology has led to a striking development in many fields of the discipline, leading to deeper understanding of parasite-host interplay as well as providing new ideals and tools for disease control. The authors also stressed that nowadays the discipline is still relatively isolated from the mainstream of modern biologic research and is still neglected by scientific community and medical education in the world, though it still is one of the major problems in public health, particularly in developing world including China. To argue the currently neglected situation of parasitology, especially in medical education, the authors emphasized the continuing requirements for the discipline and reflected on the developmental strategy of parasitology to meet the coming challenges and opportunities for further development.

Evaluation of IgG-ELISA for the diagnosis of Schistosoma japonicum in a high prevalence, low intensity endemic area of China.

Antibody-based diagnostic methods for detecting infection with Schistosoma japonicum have been developed and integrated into the national control program in China; however, the utility of these methods compared with conventional coprological methods remains unclear. In two consecutive years, we compared the performance characteristics of Kato-Katz with a commercially available enzyme-linked immunosorbent assay (ELISA) that detects anti-egg antigen IgG antibodies in a high prevalence, low intensity village in China (1025 subjects in 2005 and 652 subjects in 2006). In comparison with Kato-Katz based on duplicate stool specimens, each read in triplicate, the sensitivity of IgG-ELISA was high, ranging from 79.3% to 87.4% but with a relatively low specificity of 38.9% to 53.5%. The positive predictive value ranged from 20.8% to 24.6% while the negative predictive value ranged from 93.1% to 94.4%. When analyzed as continuous variables, there was a poor correlation between EPG (eggs per gram feces) and antibody level in both years (r(2005)=0.23 and r(2006)=0.41). We detected a trend toward reduced sensitivity at lower infection intensity as measured by Kato-Katz in 2005 (P=0.262) and 2006 (P=0.287). We evaluated changes in antibody levels and the prevalence of positive antibody in the cohort of subjects examined in both 2005 and 2006 (n=565). The prevalence of positive antibody but not the continuous antibody level, decreased in individuals who were uninfected at both time points or who transitioned from infected to uninfected as assessed by Kato-Katz. In this cohort, the distribution of antibody levels measured in 2006 among individuals who were positive by Kato-Katz in 2006 broadly overlapped with the distribution of antibody levels in individuals who were negative by Kato-Katz in both 2005 and 2006. Our results indicate fairly poor performance characteristics of the anti-egg antigen IgG-ELISA for the detection of active infection with S. japonicum in our community based sample and are in contrast with other reports based on more selected populations. The high prevalence but low intensity of S. japonicum in our study community reflects the evolving epidemiology of schistosomiasis in communities receiving intermittent treatment with praziquantel in China. We suggest marked caution in implementing anti-egg antigen IgG-ELISA based diagnosis for either individual level diagnosis or population-based targeting for national control programs.

The association between the genetic polymorphism of HLA-DQA1, DQB1, and DRB1 and serum alanine aminotransferase levels in chronic hepatitis C in the Chinese population.

BACKGROUND AND AIM: To investigate a possible association between HLA genes with serum alanine aminotransferase (ALT) levels and evaluate whether the HLA-DQA1, DQB1, and DRB1 genes could influence the development of liver damage in chronic hepatitis C. METHODS: A total of 145 patients with chronic hepatitis C virus (HCV) infection (36 patients with persistently normal ALT values; 109 patients with elevated ALT levels) and 160 uninfected healthy controls were examined for HLA-DQA1, DQB1, and DRB1 molecules by using polymerase chain reaction-sequencing based typing (PCR-SBT). RESULTS: Among the patients chronically infected with HCV, the frequencies of DQA1*0501, DQB1*0301, and DRB1*0401 alleles were significantly increased in the normal ALT group compared with those with abnormal ALT levels, whereas that of DQB1*0201 was significantly lower. As compared to uninfected healthy controls, DQA1*0501, DQB1*0301, and DRB1*0401 allele frequencies were also statistically higher in the normal ALT group, whereas that of DQB1*0201 was the inverse. The haplotype frequencies of DQA1*0301-DQB1*0301, DQA1*0501-DQB1*0301, and DRB1*1101-DQB1*0301 were found to be significantly higher in the normal ALT group. Multivariate logistic regression indicated that female sex, and the DQB1*0301 allele and DRB1*0401 allele were independently associated with normal ALT values, whereas DQB1*0201 allele was the inverse. CONCLUSIONS: These results suggest that particular HLA alleles may have an influence on the serum ALT level of chronic HCV infection as a host genetic factor in the Chinese population. The DQA1*0501, DQB1*0301, and DRB1*0401 alleles, and the DQA1*0301-DQB1*0301, DQA1*0501-DQB1*0301, and DRB1*1101-DQB1*0301 haplotypes seem to be associated with low hepatitis activity; whereas DQB1*0201 allele is closely correlated with the progression of liver injury in chronic HCV infection.

Routine Kato-Katz technique underestimates the prevalence of Schistosoma japonicum: a case study in an endemic area of the People's Republic of China.

There is an evidence that the Kato-Katz technique lacks sensitivity and may hence be an unsuitable method for the assessment of the 'real infection status' in community with low-intensity infections. In this study, six Kato-Katz thick smears (examination of two stool samples with three thick smears each) were used as the diagnostic 'gold' standard for estimating the prevalence of Schistosoma japonicum infection and the results were compared with results based on fewer Kato-Katz thick smear readings. A total of 1055 individuals in 2005 and 725 in 2006 from an endemic village were recruited for the study. The observed prevalence increased gradually with the number of Kato-Katz thick smears examined, and hence the rate of underestimation decreased accordingly. The prevalence based on single Kato-Katz thick smear readings was significantly lower than that obtained using five or six thick smears. The rate of underestimation based on using two and three Kato-Katz thick smears, a typical diagnostic effort in the national schistosomiasis control programme, was about 36.0% (28.4-48.9%) and 25.0% (15.9-40.7%). The number of Kato-Katz thick smears required to secure detection of a S. japonicum infection varies with the infection intensity level. Indeed, examination of a single thick smear was sufficient when the geometric mean of the fecal content of eggs per gram (EPG) was 250 or higher in infected individuals, while six Kato-Katz thick smears were required when the EPG score was lower than 10. In conclusion, our results confirm that the prevalence of S. japonicum infection in a community is generally considerably "underestimated". Moreover, our findings provide a benchmark for the proper application of the Kato-Katz technique and the rational evaluation of the epidemic situation, as well as a scientific basis for constructing a mathematic diagnostic model.

Epidemiology of schistosomiasis in the People's Republic of China, 2004.

Results from the third nationwide cluster sampling survey on the epidemiology of schistosomiasis in the People's Republic of China, conducted by the Ministry of Health in 2004, are presented. A stratified cluster random sampling technique was used, and 239 villages were selected in 7 provinces where Schistosoma japonicum remains endemic. A total of 250,987 residents 6-65 years of age were included in the survey. Estimated prevalence rates in the provinces of Hunan, Hubei, Jiangxi, Anhui, Yunnan, Sichuan, and Jiangsu were 4.2%, 3.8%, 3.1%, 2.2%, 1.7%, 0.9%, and 0.3%, respectively. The highest prevalence rates were in the lake and marshland region (3.8%) and the lowest rates were in the plain region with waterway networks (0.06%). Extrapolation to all residents in schistosome-endemic areas indicated 726,112 infections. This indicates a reduction of 16.1% compared with a nationwide survey conducted in 1995. However, human infection rates increased by 3.9% in settings where transmission is ongoing.

CagA+ H pylori infection is associated with polarization of T helper cell immune responses in gastric carcinogenesis.

AIM: To characterize the immune responses including local and systemic immunity induced by infection with H pylori, especially with CagA+ H pylori strains and the underlying immunopathogenesis. METHODS: A total of 711 patients with different gastric lesions were recruited to determine the presence of H pylori infection and cytotoxin associated protein A (CagA), the presence of T helper (Th) cells and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMCs), expression of plasma cytokines, and RNA and protein expression of IFN-gamma and IL-4 in gastric biopsies and PBMCs were determined by rapid urease test, urea [(14)C] breath test, immunoblotting test, flow cytometry , real time RT-PCR and immunohistochemistry. RESULTS: Of the patients, 629 (88.47%) were infected with H pylori; 506 (71.16%) with CagA+ and 123 (17.30%) with CagA- strains. Among patients infected with CagA+ H pylori strains, Th1-mediated cellular immunity was associated with earlier stages of gastric carcinogenesis, while Th2-mediated humoral immunity dominated the advanced stages and was negatively associated with an abundance of Treg cells. However, there was no such tendency in Th1/Th2 polarization in patients infected with CagA- H pylori strains and those without H pylori infection. CONCLUSION: Polarization of Th cell immune responses occurs in patients with CagA+ H pylori infection, which is associated with the stage and severity of gastric pathology during the progression of gastric carcinogenesis. This finding provides further evidence for a causal role of CagA+ H pylori infection in the immunopathogenesis of gastric cancer.

[Current status and prospects on candidate vaccines against Schistosoma japonicum].

Great progress has been made on vaccine research for schistosomiasis, including those on immune mechanism and Schistosoma genome which have made active effect to vaccine development. This paper reviews the progress on the candidate vaccine antigens including protein vaccine, DNA vaccine and multivalent vaccine against Schistosoma japonicum.

Protective immunity induced by phage displayed mitochondrial related peptides of Schistosoma japonicum.

New antigens and strategies are necessary for vaccine development against schistosomiasis japonica. Using a pool of 43 high titred anti-SWA sera from individuals residing in an Schistosoma japonicum endemic area of China, we have cloned a S. japonicum gene by cDNA library screening. The recombinant Sj338 protein has 44-46% identity to a mitochondrial precursor receptor protein of humans and rats. Immunization of mice with the recombinant Sj338 conferred 27-32% (p<0.01) reduction in worm burdens following cercarial challenge. In an effort to identify protective epitopes in Sj338 and increase the level of protection, we screened a random 12-mer peptide library constructed in M13 using a polyspecific anti-Sj338 rabbit serum. After five rounds of biopanning, we identified 30 reactive clones consisting of 11 distinct peptide sequences. These clones shared limited primary sequence homology with the recombinant Sj338 protein. Anti-sera raised against these phage clones recognized recombinant Sj338 and SWAP by Western blot. In murine vaccination experiments using whole recombinant phage without adjuvant, four of these clones demonstrated worm reductions of 11.6-25.1% (p=ns - 0.05) compared to M13 vaccinated animals. Animals vaccinated with all four of these phage demonstrated 34.2% (p<0.01) worm reduction compared to controls vaccinated with M13 clone. These data suggest that mimotope peptides are potential vaccine candidates for S. japonicum.

[The genetic polymorphism of HLA-DQA1 and HLA-DQB1 genes of Chinese Han population in Jiangsu area is studied by PCR-sequence-based typing].

OBJECTIVE: To investigate the polymorphism of HLA-DQA1 and DQB1 genes of Han population in Jiangsu of China. METHODS: The alleles and haplotypes frequencies of HLA-DQA1 and DQB1 genes in 100 unrelated healthy individuals were analyzed by using polymerase chain reaction-sequence-based typing (PCR-SBT). RESULTS: Among the 7 DQA1 alleles detected, the most common allele was DQA1*0301/02/03 with a frequency of 29.5%, which was followed by DQA1*0501, DQA1*0102 and DQA1*0201 with frequencies of 18.5%, 17.0% and 12.5%, respectively. Of the 13 DQB1 alleles detected, DQB1*0201/02 allele (21.5%) was the most frequent allele, followed by DQB1*0301/09 (14.5%), DQB1*0303 (13.5%) and DQB1*0603 (11.5%). The most common DQA1 vs DQB1 haplotype was DQA1*0301/02/03 vs DQB1*0303 with a frequency of 12.5%, which was followed by the DQA1*0201-DQB1*0201/02 (10.5%),DQA1*0501-DQB1*0201/02 (9.5%) and DQA1*0501-DQB1*0301/09 (7.0%). CONCLUSION: The distribution of HLA-DQ alleles and haplotypes in Jiangsu Han population shares some genetic characteristics with other population in northern of China, but has its own characteristics. The data will provide useful information for anthropology, organ transplantation and disease association studies.

Evaluation on the applied value of the dot immunogold filtration assay (DIGFA) for rapid detection of anti-Schistosoma japonicum antibody.

The dot immunogold filtration assay (DIGFA) is a rapid technique for the detection of anti-Schistosoma japonicum antibody. Its sensitivity with regard to sera obtained from patients with acute or chronic schistosomiasis was shown to be 100 and 96.9%, respectively. The specificity when using sera of people living in an area non-endemic for schistosomiasis japonica was 100%. Cross-reaction rates for paragonimiasis and clonorchiasis patients were 14.3% and 0%, respectively. Parallel serum tests of 1091 residents from an area endemic for S. japonicum by means of DIGFA, enzyme-linked immunosorbent assay and indirect haemagglutination test resulted in positive rates of 9.3%, 11.5% and 11.0%, respectively. Thus, there was a high level of agreement between the sets of results (P>0.05). In conclusion, DIGFA holds considerable promise for rapid and accurate diagnosis of S. japonicum, as it does not require any specific instruments and can be applied with ease. DIGFA has therefore several advantages over conventional diagnostic approaches and is useful not only for screening and sero-epidemiological surveys in the field, but also in clinical settings.

Human leukocyte antigen class II DQB1*0301, DRB1*1101 alleles and spontaneous clearance of hepatitis C virus infection: a meta-analysis.

AIM: To assess the associations of human leukocyte antigen (HLA) class II DQB1*0301 and/or DRB1*1101 allele with spontaneous hepatitis C virus (HCV) clearance by meta-analysis of individual dataset from all studies published till date. METHODS: To clarify the impact of HLA class II polymorphisms on viral clearance, we performed a meta-analysis of the published data from 11 studies comparing the frequencies of DQB1*0301 and DRB1*1101 alleles in individuals with spontaneous resolution to those with persistent infection. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. RESULTS: Meta-analyses yielded summary estimates-odds ratio (OR) of 2.36 [95%CI (1.62, 3.43), P<0.00001] and 2.02 [95%CI (1.56, 2.62), P<0.00001] for the effects of DQB1*0301 and DRB1*1101 alleles on spontaneous clearance of HCV, respectively. CONCLUSION: These results support the hypothesis that specific HLA class II alleles might influence the susceptibility or resistance to persistent HCV infection. Both DQB1*0301 and DRB1*1101 are protective alleles and present HCV epitopes more effectively to CD4(+)T lymphocytes than others, and subjects with these two alleles are at a lower risk of developing chronic HCV infection. Large, multi-ethnic confirmatory and well-designed studies are needed to determine the host genetic determinants of HCV infection.

[Purification and immunogenicity identification of the recombinant protein related with specific IgE against Schistosoma japonicum].

OBJECTIVE: To purify the specific IgE antibody-related recombinant protein of Schistosoma japonicum and to identify its immunogenicity. METHODS: The recombinant plasmid Sj43B/pGEX-6p-1 was expressed in E. coli BL 21. The inclusion body of the fusion protein was washed by TNMFX buffer and separated by FPLC. After renaturation, the fusion protein was used to vaccinate the mice. The specific IgG and IgE antibodies were detected by dot-ELISA and Western blotting analysis, respectively. RESULTS: Most of the proteins mixed with the inclusion body of the recombinant protein could be eliminated by washing with TNMFX buffer. The purified recombinant fusion protein could be obtained by FPLC separation. The experiment on mice immunized with the fusion protein showed that the specific IgE antibody was generated against the target part of the fusion protein, but not the specific IgG antibody. CONCLUSION: The fusion protein expressed by the recombinant plasmid Sj43B/pGEX-6p-1 could induce specific IgE response of the immunized mice.

[Identification of epitope of 22.6 kDa antigen of Schistosoma japonicum with phage-displayed random peptide library].

OBJECTIVE: To identify the epitopes of 22.6 kDa antigen of Schistosoma japonicum (Sj22.6). METHODS: A 12-mer library displayed on pIII of fd phage was used to screen the epitopes of Sj22.6 with the purified multiple clone IgG antibody against the Sj22.6 antigen. Five rounds of biopanning were carried out and fourteen clones from the fifth round biopanning were randomly selected and identified by Western blotting. The mice were immunized with the obtained positive clones and the antibody titers of sera from the mice were detected. The clones which could stimulate the mice to produce anti-Sj22.6 antibodies were sequenced and their amino acid sequences were compared with that of Sj22.6. RESULTS: Six clones selected from the fourteen ones could stimulate the mice to produce anti-Sj22.6 antibodies analysed by Western blotting. The amino acid sequence of one epitope showed high homology with Sj22.6. CONCLUSION: Four epitopes of Sj22.6 were obtained. One may be a structure epitope and the other three were mimic epitopes.

[Preliminary identification of T cell epitopes on 22.6 kDa antigen of Schistosoma japonicum].

OBJECTIVE: To identify the T cell epitopes on 22.6 kDa antigen of Schistosoma japonicum (Sj22.6). METHODS: The primary structure of Sj22.6 molecule was analysed using various predictive algorithms and a panel of 4 peptides were acquired. Their oligonucleotides were designed, synthesized and inserted into the multiple cloning site of plasmid pET-32c(+). The recombinant plasmids were transformed into E. coli BL21 and identified by endonuclease digestion and sequencing. The positive clones containing the recombinant plasmids could express specific fusion proteins (trx-epitope, MW approximately 20 kDa) induced by IPTG. The fusion protein with 6 x His could be coupled with NTA resin specifically, and purified by elusion of the column with buffer containing imidazole. The purified fusion proteins were incubated with splenocytes of C3H mice and then, the proliferation of splenocytes was determined by 3H-TdR incorporation assay. RESULTS: The recombinant plasmids were constructed successfully and the positive clones containing the recombinant plasmids expressed specific fusion proteins. Three of the purified fusion proteins (P4, P5, P6) could stimulate the lymphocyte proliferation. CONCLUSION: Three T cell epitopes on Sj22.6 antigen were identified.

[Studies on the characteristic of interferon-gamma mediating resistance in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the molecular characteristic of interferon-gamma mediating protective immunity against schistosomiasis japonica in mice. METHODS: CD4+ T cells were isolated from spleens of mice infected with Schistosoma japonicum at different time-points. The cDNA microarray technique combined with RT-PCR was used to explore IFN-gamma inducible GTPase family gene expression profile of CD4+ T cell. IGTP, a representative IFN-gamma, inducible GTPase having vital anti-infection activity, was amplified from spleen of BALB/c mice using RT-PCR, then cloned into pGEM(r)-T easy vector for sequencing. RESULTS: IFN-gamma inducible GTPase family had the similar characteristic over the course of S. japonicum infection. The gene expression of these members were up-regulated or had little change at 3 wk post-infection, then down-modulated from 6 wk to 13 wk post-infection, which was also confirmed by RT-PCR. As for IGTP, two inserts were identified after sequencing. One was 142 bp shorter than another, but the fragment was lost due to low annealing temperature. CONCLUSION: There is a dramatic inhibition of IFN-gamma pathway and IFN-gamma-dependent anti-infective immunity during the infection of S. japonicum.

[Effect of costimulatory signals in schistosome infection].

T-cell activation requires T-cell receptor (TCR) signaling and a second signal called costimulation. This article overviewes the research progress of the effect of costimulatory signals in schistosome infection.

Schistosoma japonicum soluble egg antigens attenuate IFN-γ-induced MHC class II expression in RAW 264.7 macrophages.

Innate immune response plays the key role in initiating and guiding the immune response. Elucidating the innate immune related molecular events involved in the interaction between the parasite and the host will aid in the development of an effective vaccine and anti-schistosome pharmaceuticals. In this study, we examined the regulatory effect of Schistosoma japonicum soluble egg antigen (SEA) on MHC class II expression in macrophage cell line RAW 264.7. We demonstrated that SEA possesses the ability to down-regulate IFN-γ-induced MHC class II expression in RAW 264.7 cells. The production of IL-10 and IL-6 in RAW 264.7 cells, induced by SEA, was responsible for mediating the down-regulation of MHC class II. Our findings suggest that in RAW 264.7 cells (1) IFN-γ provides a condition for lower concentrations of SEA to attenuate MHC class II expression; (2) SEA attenuated IFN-γ-induced MHC class II expression and the IL-10 and IL-6 production is mediated at least partly by the interaction of SEA with TLR4; and (3) SEA attenuated IFN-γ-induced MHC class II expression at the transcriptional level.

[Antibody responses to leucine aminopeptidase in Schistosoma japonicum infection].

OBJECTIVE: To investigate the antibody response to leucine aminopeptidase in the different stages of Schistosoma japonicum infection. METHODS: The expression product of SjLAP was identified by Western blot and further purified by using nickel column. The IgG levels in the response to SjLAP in murine and porcine sera were detected by ELISA at different time points after the infection of S. japonicum. RESULTS: SjLAP expressed by E. coli was recognized by anti-his monoclonal antibody and S. japonicum-infected mice sera by Western blot. The results of ELISA showed that IgG responses to SjLAP rose gradually and reached the peak at 4 weeks post-infection for pigs (P1) and 6 weeks post-infection for mice (P2). With the appearance of a large number of eggs in the tissue, SjLAP-specific IgG levels were significantly down-regulated ( P1 = 0. 0004, P2 = 0. 0001). CONCLUSION: SjLAP originated from the adult worm might become a potential target for early diagnosis of schistosomiasis japonica.