Dan-Dan Lin

Serodiagnosis of Schistosoma japonicum infection: genome-wide identification of a protein marker, and assessment of its diagnostic validity in a field study in China

BACKGROUND Schistosomiasis remains a highly prevalent and serious parasitic disease. A major factor preventing its effective management is the scarcity of effective diagnostic tools. We did a genome-wide identification of diagnostic protein markers for schistosome infection and assessed their diagnostic validity in a field study. METHODS We predicted putative secreted proteins of Schistosoma japonicum (SjSPs) and expressed them as glutathione S-transferase (GST)-fusion proteins. The fusion proteins were arrayed on glutathione (GSH)-immobilised microplates and screened with serum samples from patients with schistosomiasis diagnosed by the Kato-Katz method. We further assessed an identified protein marker for sensitivity and specificity, first in infected serum samples collected from Jiangxi and Hunan Provinces, China, and then through a field study, done in two villages located in a high schistosomiasis-endemic area of the southeast of China. FINDINGS Of 204 recombinant proteins, 35 yielded seropositive reactions, eight showed strong immunoreactivity, and only one (SjSP-13) reacted to the entire panel of 14 archived samples. The reactivity of SjSP-13 to 476 serum samples showed 90·4% (95% CI 86·5-93·5) sensitivity and 98·9% (95% CI 95·9-99·9) specificity. Of 1371 residents enrolled in a field study from Dec 6, 2010, to June 23, 2011, only 74 individuals were identified as being egg-positive, whereas 465 were diagnosed as positive by the SjSP-13-based ELISA kit (rSP13-ELISA). Of the 394 individuals found egg-negative but rSP13-ELISA-positive, 363 (92·4%) were confirmed to be positive for schistosome infection by PCR detection of S japonicum SjR2 retrotransposon. INTERPRETATION The application of this sensitive, specific, and affordable rSP13-ELISA method should help reduce schistosomiasis transmission through targeted treatment of individuals, particularly with low intensity infections, and therefore support schistosomiasis control and elimination strategies. FUNDING National 973 project in China.

Immune Events Associated with High Level Protection against Schistosoma japonicum Infection in Pigs Immunized with UV-Attenuated Cercariae

Background The vaccination of radiation-attenuated Schistosoma japonicum cercariae can induce effective protection in artiodactyl, but the immune events related to protective immunity are not fully understood. To provide a paradigm for a human recombinant antigen vaccine, we have undertaken a vaccination and challenge experiment in pigs, which was recognized as an appropriate animal model in this type of study because of their similarity to human in immunology, and investigated the relative immune events induced by the radiation-attenuated S. japonicum cercariae. Methods and Findings We found that pigs immunized once with 400 µw UV-irradiated cercariae exhibited 63.84% and 71.82% reductions in worm burden and hepatic eggs respectively. Protective immunity in vaccinated pigs was associated with high level productions of IgM, total IgG, IgG1 and IgG2; IgG2 was significantly increased in the acute infection. IFN-γ levels could be elicited by immunization. At week 6 post-infection, IFN-γ, IL-4 and IL-10 levels also showed a dramatic rise synchronously in vaccinated pigs. Moreover, the granzyme b, nk-lysin, ifnγ, il4 and il10 mRNA levels in early skin-draining lymph nodes of immunized pigs were higher than those in pigs with non-irradiated cercariae infection. In addition, cytotoxicity-related genes in the mesenteric lymph nodes were significantly upregulated in vaccinated pigs in the acute infection. Conclusion/Significance Our results demonstrated that IFN-γ and IgG2 antibody production, as well as genes related to cytotoxicity are associated with the high level protection induced by UV-irradiated Schistosoma japonicum vaccine. These findings indicated that optimal vaccination against S. japonicum required the induction of IFN-γ, IgG2 antibody related to Th1 responses and cytotoxicity effect.

Multiple vaccinations with UV- attenuated cercariae in pig enhance protective immunity against Schistosoma japonicum infection as compared to single vaccination

BackgroundSchistosomiasis japonica is a major public health problem in the endemic areas of China, the Philippines, and Indonesia. To date, a vaccine has not been developed against this disease but immunization with UV-attenuated cercariae can induce a high level of protective immunity in Landrace/Yorkshire/Duroc crossbred pigs. To compare the efficacy of a single vaccination and multiple vaccinations with UV-attenuated Schistosoma japonicum cercariae, two groups of pigs received either one or three exposures to 10,000 cercariae attenuated with 400 μw UV.ResultsPigs with a single immunization had a 59.33% reduction in adult worm burden, a 89.87% reduction in hepatic eggs and a 86.27% reduction in fecal eggs at eight weeks post-challenge (P < 0.01). After three immunizations, protection increased to 77.62%, 88.8% and 99.78% reduction in adult worms, hepatic eggs and fecal eggs, respectively (P < 0.01). Humoral and cellular immunological parameters measured indicated that schistosome-specific IgG1 and IgG2 levels in the vaccinated groups were higher than in the infection-control group. Triple vaccinations resulted in higher levels of antibodies, especially IgG2, compared with a single vaccination and IFN-γ levels increased with repeated immunization with UV-irradiated cercariae.ConclusionThe high levels of protection against S. japonicum infection can be achieved with a UV-attenuated vaccine in pigs, and that three vaccinations were possibly more effective than a single vaccination. Moreover, triple vaccinations evoked a more vigorous IFN-γ response and a stronger antibody-mediated response, especially an increase in the levels of IgG2 antibodies.

Enhancing collaboration between China and African countries for schistosomiasis control

Schistosomiasis remains an important public health issue, with a large number of cases reported across sub-Saharan Africa, and parts of Asia and Latin America. China was once highly endemic, but has made substantial progress and is moving towards elimination of schistosomiasis. Meanwhile, despite long-term, repeated, school-based chemotherapy in many African countries, more than 90% of all schistosomiasis cases are concentrated in Africa, and hence, this continent constitutes the key challenge for schistosomiasis control. Opportunities and issues for international collaboration in the fight against schistosomiasis are outlined with a focus on Chinas experiences, including the role of public health authorities and intersectoral collaboration, use of new and effective snail control approaches and diagnostic tools adapted to the specific stage of control, as well as the strengthening of risk mapping and surveillance-response mechanisms. Training courses targeting African governmental officials and professionals, coupled with field visits of African scientists and control programme managers to China, and vice versa, are considered important for improved schistosomiasis control and elimination. The crucial question remains whether the Chinese experience can be translated and applied in African countries to improve the effectiveness of health interventions and scale-up.

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 Schistosoma japonicum self-cure phenomenon in water buffaloes: Potential impact on the control and elimination of schistosomiasis in China

Schistosomiasis japonica, caused by Schistosoma japonicum, is an important zoonotic disease in China, the Philippines and small pockets of Indonesia. In addition to infecting people, S. japonicum can infect over 40 species of wild and domestic animals which have varying impacts on human infection. It is now generally accepted that bovines, particularly water buffaloes, are the major reservoir for human infection in China as they are naturally infected with schistosomes and deposit more eggs into the environment than humans or any other animal host. This complicates control efforts and the economic burden associated with schistosomiasis morbidity and mortality has taken its toll on both human and livestock populations. Over the last 50years, the schistosomiasis control program in China has made great strides in reducing prevalence and morbidity, and the Chinese authorities now aim to eliminate the disease nationwide in the next decade. Current Chinese control strategies place particular importance on interventions targeting bovines including: praziquantel treatment, barrier farming to prevent grazing in transmission areas, their replacement with mechanized tractors and possible bovine vaccination. A number of studies have shown that in the period following S. japonicum infection, the worm burden drops sharply in water buffaloes and some other animal hosts such as pigs. This is due to a self-cure phenomenon whereby there is parasite clearance by both immune and non-immune factors. Here we review studies investigating the self-cure effect, paying particular attention to S. japonicum infection in water buffaloes, and discuss its potential impact on the future schistosomiasis control and elimination efforts in China. Further understanding of the mechanism of self-cure in water buffaloes could be important for future schistosome vaccine design and delivery.

Immune responses result in misdiagnosis of Schistosoma japonicum by immunodiagnosis kits in egg-positive patients living in a low schistosomiasis transmission area of China.

BackgroundIn recent field surveys, we failed to detect the presence of specific antibody against Schistosoma japonicum in some egg-positive patients by commonly used immunodiagnostic kits. To find out whether low levels of specific antibody truly exist among egg-positive individuals and elucidate the underlying immune mechanisms, we carried out a cross-sectional epidemiologic study in a S. japonicum low transmission endemic area of Poyang Lake region, China and compared the humoral and cellular immune characteristics between S. japonicum high and low antibody responders.MethodsKato–Katz thick smear assay was used to determine the schistosomiasis status of 3,384 participants residing in two Poyang Lake region villages, Jiangxi, China. Among the 142 stool egg-positive participants, we identified low and high S. japonicum antibody responders with soluble egg antigen (SEA) and adult worm antigen (AWA) specific IgG levels by adopting ROC curve analysis. To compare the humoral and cellular immune responses between high and low S. japonicum antibody responders, serum specific antibody levels as well as the percentage of T lymphocyte subpopulation in PMBC, and cell stimulated cytokines (IFN- gamma and interlukin-10) were detected.ResultsEight S. japonicum egg-positive participants were defined as low antibody responders. Although the percentage of CD3+T cells in low responders was slightly higher and the percentage of CD4+ T cells, CD8+ T cells, the ratio of CD4+/CD8+ and CD4+ CD25+ Treg cells were lower than those in high responders, the differences between the two groups were not significant (P > 0.05). AWA -stimulated interlukin-10 level was significantly higher in high responders, while other cytokines did not show differences between two groups. For antibody profiles, except AWA specific IgA, significant differences of each antibody isotype between low and high responders were detected (P < 0.05).ConclusionsOur study confirmed that there are S. japonicum antibody low responders among schistosome egg-positive residents in S. japonicum low-transmission areas in China. Thus, mis-diagnosis using immune-diagnosis kits do exist. Significant differences of responding antibody levels between low and high responders were detected, while no major cellular response changes were observed.

Village-based spatio-temporal cluster analysis of the schistosomiasis risk in the Poyang Lake Region, China

BackgroundThe Poyang Lake Region, one of the major epidemic sites of schistosomiasis in China, remains a severe challenge. To improve our understanding of the current endemic status of schistosomiasis and to better control the transmission of the disease in the Poyang Lake Region, it is important to analyse the clustering pattern of schistosomiasis and detect the hotspots of transmission risk.ResultsBased on annual surveillance data, at the village level in this region from 2009 to 2014, spatial and temporal cluster analyses were conducted to assess the pattern of schistosomiasis infection risk among humans through purely spatial (Local Moran’s I, Kulldorff and Flexible scan statistic) and space-time scan statistics (Kulldorff). A dramatic decline was found in the infection rate during the study period, which was shown to be maintained at a low level. The number of spatial clusters declined over time and were concentrated in counties around Poyang Lake, including Yugan, Yongxiu, Nanchang, Xingzi, Xinjian, De’an as well as Pengze, situated along the Yangtze River and the most serious area found in this study. Space-time analysis revealed that the clustering time frame appeared between 2009 and 2011 and the most likely cluster with the widest range was particularly concentrated in Pengze County.ConclusionsThis study detected areas at high risk for schistosomiasis both in space and time at the village level from 2009 to 2014 in Poyang Lake Region. The high-risk areas are now more concentrated and mainly distributed at the river inflows Poyang Lake and along Yangtze River in Pengze County. It was assumed that the water projects including reservoirs and a recently breached dyke in this area were partly to blame. This study points out that attempts to reduce the negative effects of water projects in China should focus on the Poyang Lake Region.

Elevated serum antibody against Schistosoma japonicum HSP60 as a promising biomarker for liver pathology in schistosomiasis

The pathology associated with Schistosoma japonicum (S. japonicum) infection in humans is attributed to parasite egg-induced granulomatous inflammation and fibrosis in the host liver. Currently, a marker that is reliable, cheap, less device-dependent, and can be easily and repeatedly used on a large scale to monitor the progression of liver pathology in schistosomiasis japonica endemic areas is lacking. The levels of serum S. japonicum heat shock protein 60 (SjHSP60)-specific IgG and its subtype antibodies in animals (mice and rabbits) or patients with schistosomiasis were measured by ELISA. Liver pathologies in mice and rabbits were evaluated by gross pathology and histopathology, and hepatic fibrosis in patients was examined with ultrasound imaging. The results revealed that the titers of the total IgG and subtype IgG1 anti-SjHSP60 antibodies were positively correlated with the severity of liver pathology after S. japonicum infection. Our findings indicate that the SjHSP60 IgG and IgG1 antibody levels can be used as potential candidate biomarkers for evaluation of liver pathology in schistosomiasis; however, validation remains to be explored in further work.