Low Sensitivity of the Formol-Ethyl Acetate Sedimentation Concentration Technique in Low-Intensity Schistosoma japonicum Infections

Background

The endemic countries are in a diagnostic dilemma concerning Schistosoma japonicum with increasing difficulties in diagnosing the infected individuals. The formol-ethyl acetate sedimentation concentration technique is preferred by many clinical microbiology laboratories for the detection of parasites in stool samples. It is potentially more sensitive than the diagnostic methods traditionally used.

Methodology/Principal Findings

We evaluated the technique for detection of low-intensity S. japonicum infections in 106 stool samples from China and used a commercial kit, Parasep Midi Faecal Parasite Concentrator. One stool sample and one serum sample were collected from each person. As reference standard we used persons positive by indirect hemagglutination in serum and positive by Kato-Katz thick smear microscopy (three slides from a single stool), and/or the hatching test. We found the sedimentation technique to have a sensitivity of only 28.6% and specificity of 97.4%.

Conclusion/Significance

This study indicates that the sedimentation technique has little to offer in the diagnosis of low-intensity S. japonicum infections, at least when only a single stool sample is examined.     

A piezoelectric immunosensor using hybrid self-assembled monolayers for detection of Schistosoma japonicum

Background

The parasite Schistosoma japonicum causes schistosomiasis disease, which threatens human life and hampers economic and social development in some Asian countries. An important lesson learned from efforts to reduce the occurrence of schistosomiasis is that the diagnostic approach must be altered as further progress is made towards the control and ultimate elimination of the disease.

Methodology/Principal Findings

Using mixed self-assembled monolayer membrane (mixed SAM) technology, a mixture of mercaptopropionic acid (MPA) and mercaptoethanol (ME) was self-assembled on the surface of quartz crystals by gold-sulphur-bonds. Soluble egg antigens (SEA) of S. japonicum were then cross-linked to the quartz crystal using a special coupling agent. As compared with the traditional single self-assembled monolayer immobilization method, S. japonicum antigen (SjAg) immobilization using mixed self-assembled monolayers exhibits much greater immunoreactivity. Under optimal experimental conditions, the detection range is 1∶1500 to 1∶60 (infected rabbit serum dilution ratios). We measured several infected rabbit serum samples with varying S. japonicum antibody (SjAb) concentrations using both immunosensor and ELISA techniques and then produced a correlation analysis. The correlation coefficients reached 0.973.

Conclusions/Significance

We have developed a new, simple, sensitive, and reusable piezoelectric immunosensor that directly detects SjAb in the serum. This method may represent an alternative to the current diagnostic methods for S. japonicum infection in the clinical laboratory or for analysis outside the laboratory.     

Spatial Distribution of Human Schistosoma japonicum Infections in the Dongting Lake Region,China

Background

The aim of this study was to spatially model the effect of demographic, reservoir hosts and environmental factors on human Schistosoma japonicum infection prevalence in the Dongting Lake area of Hunan Province, China and to determine the potential of each indicator in targeting schistosomiasis control.

Methodology/Principal Findings

Cross-sectional serological, coprological and demographic data were obtained from the 2004 nationwide periodic epidemiologic survey for Hunan Province. Environmental data were downloaded from the USGS EROS data centre. Bayesian geostatistical models were employed for spatial analysis of the infection prevalence among study participants. A total of 47,139 participants from 47 administrative villages were selected. Age, sex and occupation of residents and the presence of infected buffaloes and environmental factors, i.e. NDVI, distance to the lake and endemic type of setting, were significantly associated with S. japonicum infection prevalence. After taking into account spatial correlation, however, only demographic factors (age, sex and occupation) and the presence of infected buffaloes remained significant indicators.

Conclusions/Significance

Long established demographic factors, as well presence of host reservoirs rather than environmental factors are driving human transmission. Findings of this work can be used for epidemiologic surveillance and for the future planning of interventions in the Dongting Lake area of Hunan Province.     

Sensitive and specific target sequences selected from retrotransposons of Schistosoma japonicum for the diagnosis of schistosomiasis

Background

Schistosomiasis japonica is a serious debilitating and sometimes fatal disease. Accurate diagnostic tests play a key role in patient management and control of the disease. However, currently available diagnostic methods are not ideal, and the detection of the parasite DNA in blood samples has turned out to be one of the most promising tools for the diagnosis of schistosomiasis. In our previous investigations, a 230-bp sequence from the highly repetitive retrotransposon SjR2 was identified and it showed high sensitivity and specificity for detecting Schistosoma japonicum DNA in the sera of rabbit model and patients. Recently, 29 retrotransposons were found in S. japonicum genome by our group. The present study highlighted the key factors for selecting a new perspective sensitive target DNA sequence for the diagnosis of schistosomiasis, which can serve as example for other parasitic pathogens.

Methodology/Principal Findings

In this study, we demonstrated that the key factors based on the bioinformatic analysis for selecting target sequence are the higher genome proportion, repetitive complete copies and partial copies, and active ESTs than the others in the chromosome genome. New primers based on 25 novel retrotransposons and SjR2 were designed and their sensitivity and specificity for detecting S. japonicum DNA were compared. The results showed that a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon (SjCHGCS19) had high sensitivity and specificity. The 303-bp target sequence was amplified from the sera of rabbit model at 3 d post-infection by nested-PCR and it became negative at 17 weeks post-treatment. Furthermore, the percentage sensitivity of the nested-PCR was 97.67% in 43 serum samples of S. japonicum-infected patients.

Conclusions/Significance

Our findings highlighted the key factors based on the bioinformatic analysis for selecting target sequence from S. japonicum genome, which provide basis for establishing powerful molecular diagnostic techniques that can be used for monitoring early infection and therapy efficacy to support schistosomiasis control programs.     

Surveillance of Schistosoma japonicum infection in domestic ruminants in the Dongting Lake region, Hunan province, China

Background

Schistosomiasis japonica is prevalent in Asian countries and it remains a major public health problem in China. The major endemic foci are the marsh and lake regions of southern China, particularly the Dongting Lake region bordering Hunan and Hubei provinces, and the Poyang Lake region in Jiangxi province. Domestic ruminants, especially bovines, have long been considered to play a major role in the transmission of Schistosoma japonicum to humans.

Methods and Findings

A miracidial hatching technique was used to investigate the prevalence of S. japonicum infections in domestic ruminants and field feces collected from two towns located to the south and east of Dongting Lake, Hunan province, between 2005 and 2010. The overall prevalence of infection was not significantly reduced from 4.93% in 2005 to 3.64% in 2008, after which it was maintained at this level. Bovines comprised 23.5–58.2% of the total infected ruminants, while goats comprised 41.8–76.5%. Infection rates in cattle and goats were significantly higher than those found in buffalo in most study years. The prevalence in buffalo younger than three years was significantly higher than that in those aged over three years. All the positive field samples of feces were derived from bovines in Nandashan. In Matang Town, 61.22% of the positive field feces were from bovines, while the rest were from goats. The positive rates for field feces were approximately the same in April and November/October.

Conclusions

The present study found that bovines and goats are major sources of S. japonicum infection in the Dongting lake region and there was age-related resistance in buffalo. Both bovines and goats should be treated equally when controlling S. japonicum infections in the Dongting lake region. It is essential to conduct an additional mass treatment in late March or early April, in addition to the original treatment scheme.     

Assessing the impact of misclassification error on an epidemiological association between two helminthic infections

Background

Polyparasitism can lead to severe disability in endemic populations. Yet, the association between soil-transmitted helminth (STH) and the cumulative incidence of Schistosoma japonicum infection has not been described. The aim of this work was to quantify the effect of misclassification error, which occurs when less than 100% accurate tests are used, in STH and S. japonicum infection status on the estimation of this association.

Methodology/Principal Findings

Longitudinal data from 2276 participants in 50 villages in Samar province, Philippines treated at baseline for S. japonicum infection and followed for one year, served as the basis for this analysis. Participants provided 1–3 stool samples at baseline and 12 months later (2004–2005) to detect infections with STH and S. japonicum using the Kato-Katz technique. Variation from day-to-day in the excretion of eggs in feces introduces individual variations in the sensitivity and specificity of the Kato-Katz to detect infection. Bayesian logit models were used to take this variation into account and to investigate the impact of misclassification error on the association between these infections. Uniform priors for sensitivity and specificity of the diagnostic test to detect the three STH and S. japonicum were used. All results were adjusted for age, sex, occupation, and village-level clustering. Without correction for misclassification error, the odds ratios (ORs) between hookworm, Ascaris lumbricoides, and Trichuris trichiura, and S. japonicum infections were 1.28 (95% Bayesian credible intervals: 0.93, 1.76), 0.91 (95% BCI: 0.66, 1.26), and 1.11 (95% BCI: 0.80, 1.55), respectively, and 2.13 (95% BCI: 1.16, 4.08), 0.74 (95% BCI: 0.43, 1.25), and 1.32 (95% BCI: 0.80, 2.27), respectively, after correction for misclassification error for both exposure and outcome.

Conclusions/Significance

The misclassification bias increased with decreasing test accuracy. Hookworm infection was found to be associated with increased 12-month cumulative incidence of S. japonicum infection after correction for misclassification error. Such important associations might be missed in analyses which do not adjust for misclassification errors.     

Real-time PCR Demonstrates High Prevalence of Schistosoma japonicum in the Philippines: Implications for Surveillance and Control

Background

The Philippines has a population of approximately 103 million people, of which 6.7 million live in schistosomiasis-endemic areas with 1.8 million people being at risk of infection with Schistosoma japonicum. Although the country-wide prevalence of schistosomiasis japonica in the Philippines is relatively low, the prevalence of schistosomiasis can be high, approaching 65% in some endemic areas. Of the currently available microscopy-based diagnostic techniques for detecting schistosome infections in the Philippines and elsewhere, most exhibit varying diagnostic performances, with the Kato-Katz (KK) method having particularly poor sensitivity for detecting low intensity infections. This suggests that the actual prevalence of schistosomiasis japonica may be much higher than previous reports have indicated.

Methodology/Principal Findings

Six barangay (villages) were selected to determine the prevalence of S. japonicum in humans in the municipality of Palapag, Northern Samar. Fecal samples were collected from 560 humans and examined by the KK method and a validated real-time PCR (qPCR) assay. A high S. japonicum prevalence (90.2%) was revealed using qPCR whereas the KK method indicated a lower prevalence (22.9%). The geometric mean eggs per gram (GMEPG) determined by the qPCR was 36.5 and 11.5 by the KK. These results, particularly those obtained by the qPCR, indicate that the prevalence of schistosomiasis in this region of the Philippines is much higher than historically reported.

Conclusions/Significance

Despite being more expensive, qPCR can complement the KK procedure, particularly for surveillance and monitoring of areas where extensive schistosomiasis control has led to low prevalence and intensity infections and where schistosomiasis elimination is on the horizon, as for example in southern China.     

The synergistic effect of concomitant schistosomiasis, hookworm, and trichuris infections on children's anemia burden

Objective

To estimate the degree of synergism between helminth species in their combined effects on anemia.

Methods

Quantitative egg counts using the Kato–Katz method were determined for Ascaris lumbricoides, hookworm, Trichuris trichiura, and Schistosoma japonicum in 507 school-age children from helminth-endemic villages in The Philippines. Infection intensity was defined in three categories: uninfected, low, or moderate/high (M+). Anemia was defined as hemoglobin <11 g/dL. Logistic regression models were used to estimate odds ratios (OR), 95% confidence intervals (CI), and synergy index for pairs of concurrent infections.

Results

M+ co-infection of hookworm and S. japonicum (OR = 13.2, 95% CI: 3.82–45.5) and of hookworm and T. trichiura (OR = 5.34, 95% CI: 1.76–16.2) were associated with higher odds of anemia relative to children without respective M+ co-infections. For co-infections of hookworm and S. japonicum and of T. trichiura and hookworm, the estimated indices of synergy were 2.9 (95% CI: 1.1–4.6) and 1.4 (95% CI: 0.9–2.0), respectively.

Conclusion

Co-infections of hookworm and either S. japonicum or T. trichiura were associated with higher levels of anemia than would be expected if the effects of these species had only independent effects on anemia. This suggests that integrated anti-helminthic treatment programs with simultaneous deworming for S. japonicum and some geohelminths could yield a greater than additive benefit for reducing anemia in helminth-endemic regions.     

Evaluation of mammalian and intermediate host surveillance methods for detecting schistosomiasis reemergence in southwest China

Background

Schistosomiasis has reemerged in China, threatening schistosomiasis elimination efforts. Surveillance methods that can identify locations where schistosomiasis has reemerged are needed to prevent the further spread of infections.

Methods and Principal Findings

We tested humans, cows, water buffalo and the intermediate host snail, Oncomelania hupensis, for Schistosoma japonicum infection, assessed snail densities and extracted regional surveillance records in areas where schistosomiasis reemerged in Sichuan province. We then evaluated the ability of surveillance methods to identify villages where human infections were present. Human infections were detected in 35 of the 53 villages surveyed (infection prevalence: 0 to 43%), including 17 of 28 villages with no prior evidence of reemergence. Bovine infections were detected in 23 villages (infection prevalence: 0 to 65%) and snail infections in one village. Two common surveillance methods, acute schistosomiasis case reports and surveys for S. japonicum-infected snails, grossly underestimated the number of villages where human infections were present (sensitivity 1% and 3%, respectively). Screening bovines for S. japonicum and surveys for the presence of O. hupensis had modest sensitivity (59% and 69% respectively) and specificity (67% and 44%, respectively). Older adults and bovine owners were at elevated risk of infection. Testing only these high-risk human populations yielded sensitivities of 77% and 71%, respectively.

Conclusions

Human and bovine schistosomiasis were widespread in regions where schistosomiasis had reemerged but acute schistosomiasis and S. japonicum-infected snails were rare and, therefore, poor surveillance targets. Until more efficient, sensitive surveillance strategies are developed, direct, targeted parasitological testing of high-risk human populations should be considered to monitor for schistosomiasis reemergence.     

Spatio-temporal Transmission and Environmental Determinants of Schistosomiasis Japonica in Anhui Province,China

Background

Schistosomiasis japonica still remains of public health and economic significance in China, especially in the lake and marshland areas along the Yangtze River Basin, where the control of transmission has proven difficult. In the study, we investigated spatio-temporal variations of S. japonicum infection risk in Anhui Province and assessed the associations of the disease with key environmental factors with the aim of understanding the mechanism of the disease and seeking clues to effective and sustainable schistosomiasis control.

Methodology/Principal Findings

Infection data of schistosomiasis from annual conventional surveys were obtained at the village level in Anhui Province, China, from 2000 to 2010 and used in combination with environmental data. The spatio-temporal kriging model was used to assess how these environmental factors affected the spatio-temporal pattern of schistosomiasis risk. Our results suggested that seasonal variation of the normalized difference vegetation index (NDVI), seasonal variation of land surface temperature at daytime (LSTD), and distance to the Yangtze River were negatively significantly associated with risk of schistosomiasis. Predictive maps showed that schistosomiasis prevalence remained at a low level and schistosomiasis risk mainly evolved along the Yangtze River. Schistosomiasis risk also followed a focal spatial pattern, fluctuating temporally with a peak (the largest spatial extent) in 2005 and then contracting gradually but with a scattered distribution until 2010.

Conclusion

The fitted spatio-temporal kriging model can capture variations of schistosomiasis risk over space and time. Combined with techniques of geographic information system (GIS) and remote sensing (RS), this approach facilitates and enriches risk modeling of schistosomiasis, which in turn helps to identify prior areas for effective and sustainable control of schistosomiasis in Anhui Province and perhaps elsewhere in China.     

Microarray Analysis of Gene Expression Profiles of Schistosoma japonicum Derived from Less-Susceptible Host Water Buffalo and Susceptible Host Goat

Background

Water buffalo and goats are natural hosts for S. japonicum in endemic areas of China. The susceptibility of these two hosts to schistosome infection is different, as water buffalo are less conducive to S. japonicum growth and development. To identify genes that may affect schistosome development and survival, we compared gene expression profiles of schistosomes derived from these two natural hosts using high-throughput microarray technology.

Results

The worm recovery rate was lower and the length and width of worms from water buffalo were smaller compared to those from goats following S. japonicum infection for 7 weeks. Besides obvious morphological difference between the schistosomes derived from the two hosts, differences were also observed by scanning and transmission electron microscopy. Microarray analysis showed differentially expressed gene patterns for parasites from the two hosts, which revealed that genes related to lipid and nucleotide metabolism, as well as protein folding, sorting, and degradation were upregulated, while others associated with signal transduction, endocrine function, development, immune function, endocytosis, and amino acid/carbohydrate/glycan metabolism were downregulated in schistosomes from water buffalo. KEGG pathway analysis deduced that the differentially expressed genes mainly involved lipid metabolism, the MAPK and ErbB signaling pathways, progesterone-mediated oocyte maturation, dorso-ventral axis formation, reproduction, and endocytosis, etc.

Conclusion

The microarray gene analysis in schistosomes derived from water buffalo and goats provide a useful platform to disclose differences determining S. japonicum host compatibility to better understand the interplay between natural hosts and parasites, and identify schistosome target genes associated with susceptibility to screen vaccine candidates.     

Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum

Background

Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia.

Methods and Findings

After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.

Conclusions

Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.     

Population Genetics of Schistosoma japonicum within the Philippines Suggest High Levels of Transmission between Humans and Dogs

Background

Schistosoma japonicum, which remains a major public health problem in the Philippines and mainland China, is the only schistosome species for which zoonotic transmission is considered important. While bovines are suspected as the main zoonotic reservoir in parts of China, the relative contributions of various non-human mammals to S. japonicum transmission in the Philippines remain to be determined. We examined the population genetics of S. japonicum in the Philippines in order to elucidate transmission patterns across host species and geographic areas.

Methodology/Principal Findings

S. japonicum miracidia (hatched from eggs within fecal samples) from humans, dogs, pigs and rats, and cercariae shed from snail-intermediate hosts, were collected across two geographic areas of Samar Province. Individual isolates were then genotyped using seven multiplexed microsatellite loci. Wright''s F_ST values and phylogenetic trees calculated for parasite populations suggest a high frequency of parasite gene-flow across definitive host species, particularly between dogs and humans. Parasite genetic differentiation between areas was not evident at the definitive host level, possibly suggesting frequent import and export of infections between villages, although there was some evidence of geographic structuring at the snail–intermediate host level.

Conclusions/Significance

These results suggest very high levels of transmission across host species, and indicate that the role of dogs should be considered when planning control programs. Furthermore, a regional approach to treatment programs is recommended where human migration is extensive.     

Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice

Background

The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4⁺ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4⁺ T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection.

Methodology

Levels of Th17 and the other three CD4⁺ T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice.

Conclusions

Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology.     

Diversification of Schistosoma japonicum in Mainland China revealed by mitochondrial DNA

Background

Schistosoma japonicum still causes severe parasitic disease in mainland China, but mainly in areas along the Yangtze River. However, the genetic diversity in populations of S. japonicum has not been well understood across its geographical distribution, and such data may provide insights into the epidemiology and possible control strategies for schistosomiasis.

Methodology/Principal Findings

In this study infected Oncomelania snails were collected from areas in the middle and lower (ML) reaches of the Yangtze River, including Hubei, Hunan, Anhui, Jiangxi and Jiangsu provinces, and in the upper reaches of the river, including Sichuan and Yunnan provinces in southwest (SW) China. The adult parasites obtained from experimentally infected mice using isolated cercariae were sequenced individually for several fragments of mitochondrial regions, including Cytb-ND4L-ND4, 16S-12S and ND1. Populations in the ML reaches exhibited a relatively high level of diversity in nucleotides and haplotypes, whereas a low level was observed for populations in the SW, using either each single fragment or the combined sequence of the three fragments. Pairwise analyses of F-statistics (Fst) revealed a significant genetic difference between populations in the ML reaches and those in the SW, with limited gene flow and no shared haplotypes in between. It is rather obvious that genetic diversity in the populations of S. japonicum was significantly correlated with the geographical distance, and the geographical separation/isolation was considered to be the major factor accounting for the observed difference between populations in the ML reaches and those in the SW in China.

Conclusions

S. japonicum in mainland China exhibits a high degree of genetic diversity, with a similar pattern of genetic diversity as observed in the intermediate host snails in the same region in China.