Expression profile analysis of the transient receptor potential (TRPM) channel, a possible target of praziquantel in Schistosoma japonicum.

The WHO considers schistosomiasis, which is controlled by the mass administration of the drug praziquantel (PZQ), to be a neglected tropical disease. Despite its clinical use for over four decades, PZQ remains the only choice of chemotherapy against this disease. Regarding the previous studies that demonstrated that PZQ activates the transient receptor potential (TRP) channel in Schistosoma mansoni (Sm.TRPMPZQ), the expression profile of the ortholog of this channel gene (Smp_246790.5) in S. japonicum (EWB00_008853) (Sj.TRPMPZQ) was analyzed. The relative expression of this gene in various stages of the parasite lifecycle was analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR), and the expression of Sj.TRPMPZQ was observed by immunohistochemical staining using anti-serum against the recombinant Sj.TRPMPZQ protein. qRT-PCR revealed the significantly lower mRNA expression in the snail stage in comparison to other stages (p < 0.01). The relative quantity of the Sj.TRPMPZQ expression for paired females, unpaired males, and eggs was 60%, 56%, and 68%, respectively, in comparison to paired males that showed the highest expression (p < 0.05). Interestingly, immunostaining demonstrated that Sj.TRPMPZQ is expressed in the parenchyma which contains muscle cells, neuronal cells and tegument cells in adult worms. This may support the two major effects of PZQ-worm paralysis and tegument disruption-induced by channel activation. Moreover, the channel was expressed in both the eggshell and the miracidia inside, but could not be observed in sporocyst. These results suggest that the expression of Sj.TRPMPQZ corresponds to the known sensitivity of S. japonicum to PZQ.

Evaluation of Crude and Recombinant Antigens of Schistosoma japonicum for the Detection of Schistosoma mekongi Human Infection.

Asian schistosomiasis caused by the blood fluke Schistosoma mekongi is endemic in northern Cambodia and Southern Lao People's Democratic Republic. The disease is mainly diagnosed by stool microscopy. However, serodiagnosis such as enzyme-linked immunosorbent assay (ELISA) with soluble egg antigen (SEA), has been shown to have better sensitivity compared to the stool examination, especially in the settings with a low intensity of infection. To date, no recombinant antigen has been assessed using ELISA for the detection of S. mekongi infection, due to the lack of genome information for this schistosome species. Thus, the objective of this study is to evaluate several recombinant S. japonicum antigens that have been developed in our laboratory for the detection of S. mekongi infection. The crude antigen SjSEA and recombinant antigens Sj7TR, SjPCS, SjPRx-4, and SjChi-3 were evaluated in ELISA using serum samples positive for S. mekongi infection. The cross-reaction was checked using sera positive for Ophistorchis viverrini. ELISA results showed that S. japonicum SEA at low concentrations showed better diagnostic performance than the recombinant antigens tested using the archived serum samples from Cambodia. However, further optimization of the recombinant antigens should be conducted in future studies to improve their diagnostic performance for S. mekongi detection.

Cloning, Expression and Evaluation of Thioredoxin Peroxidase-1 Antigen for the Serological Diagnosis of Schistosoma mekongi Human Infection.

Schistosoma mekongi, a blood fluke that causes Asian zoonotic schistosomiasis, is distributed in communities along the Mekong River in Cambodia and Lao People's Democratic Republic. Decades of employing numerous control measures including mass drug administration using praziquantel have resulted in a decline in the prevalence of schistosomiasis mekongi. This, however, led to a decrease in sensitivity of Kato-Katz stool microscopy considered as the gold standard in diagnosis. In order to develop a serological assay with high sensitivity and specificity which can replace Kato-Katz, recombinant S. mekongi thioredoxin peroxidase-1 protein (rSmekTPx-1) was expressed and produced. Diagnostic performance of the rSmekTPx-1 antigen through ELISA for detecting human schistosomiasis was compared with that of recombinant protein of S. japonicum TPx-1 (rSjTPx-1) using serum samples collected from endemic foci in Cambodia. The sensitivity and specificity of rSmekTPx-1 in ELISA were 89.3% and 93.3%, respectively, while those of rSjTPx-1 were 71.4% and 66.7%, respectively. In addition, a higher Kappa value of 0.82 calculated between rSmekTPx-1 antigen ELISA and Kato-Katz confirmed better agreement than between rSjTPx-1 antigen ELISA and Kato-Katz (Kappa value 0.38). These results suggest that ELISA with rSmekTPx-1 antigen can be a potential diagnostic method for detecting active human S. mekongi infection.

Circulating microRNAs as Biomarkers of Hepatic Fibrosis in Schistosomiasis Japonica Patients in the Philippines.

Host-derived microRNAs (miRNAs) play important regulatory roles in schistosomiasis-induced hepatic fibrosis. This study analyzed selected serum miRNAs among Filipino schistosomiasis japonica patients with ultrasound (US)-detectable hepatic fibrosis. A prospective cohort study design with convenience sampling was employed from 2017 to 2019. The study sites were eight endemic barangays in Leyte, Philippines. Eligible chronic schistosomiasis patients with varying severities of hepatic fibrosis were enrolled in the cohort and serially examined at 6, 12, and 24 months from baseline. Baseline serum miR-146a-5p, let-7a-5p, miR-150-5p, miR-122-5p, miR-93-5p, and miR200b-3p were measured using RT-qPCR. A total of 136 chronic schistosomiasis patients were included in this prospective cohort study. Approximately, 42.6% had no fibrosis, 22.8% had mild fibrosis, and 34.6% had severe fibrosis at baseline The serum levels of the antifibrotic miR-146a (p < 0.0001), miR-150 (p = 0.0058), and let-7a (p < 0.0001) were significantly lower in patients with hepatic fibrosis while the profibrotic miR-93 (p = 0.0024) was elevated. miR-146a-5p (AUC = 0.90, 95% CI [0.84, 0.96], p < 0.0001) has the most promising potential to differentiate patients with (n = 78) versus without (n = 58) hepatic fibrosis. The baseline level of serum miR-146-5p was significantly different in patients with progressive fibrosis (n = 17) compared to those who never developed fibrosis (n = 30, p < 0.01) or those who had fibrosis reversal (n = 20, p < 0.01) after 24 months. These findings demonstrate the potential utility of serum miRNAs, particularly of miR-146a, as a supplementary tool for assessing hepatic fibrosis in chronic schistosomiasis japonica patients.

Diagnostic Performance of Parasitological, Immunological, Molecular, and Ultrasonographic Tests in Diagnosing Intestinal Schistosomiasis in Fieldworkers From Endemic Municipalities in the Philippines.

Schistosomiasis remains to ha/ve a significant public health impact in the Philippines. The Kato-Katz (K-K) technique is the reference standard and most used technique for definitive diagnosis of intestinal schistosomiasis for control programs in endemic regions. However, this has a very low sensitivity when applied in areas of low endemicity and patients with light infection. Hence, this study determined the diagnostic performance of immunological, molecular, parasitological, and ultrasonographic tests in diagnosing intestinal schistosomiasis in endemic municipalities in the Philippines. We performed a community-based cross-sectional study to determine the positivity of schistosomiasis in Leyte, Philippines. The diagnostic performance of five different detection techniques: (1) three stool K-K with duplicate smears; (2) soluble egg antigen IgG ELISA; (3) urine point-of-care circulating cathodic antigen (POC-CCA) test; (4) detection of Schistosoma japonicum circulating DNA (SjcDNA) in serum and urine samples; (5) focused abdominal ultrasound (US), were also obtained in this study. Multiple stool examinations enhanced the sensitivity of K-K from 26.2% (95% CI [16.4, 38.8]) with single stool to 53.8% (95% CI [41.1, 66.1]) and 69.2% (95% CI [56.4, 80.0]) with two and three stools from consecutive days, respectively. Among the SjcDNA nucleic acid amplification test (NAAT)-based detection assays, loop-mediated isothermal amplification (LAMP) PCR using sera had the highest sensitivity at 92.3% (95% CI [82.2, 97.1]) with LAMP consistently identifying more positive cases in both serum and urine samples. This study showed that single stool K-K, which remains the only diagnostic test available in most endemic areas in the Philippines, had low sensitivity and failed to identify most patients with light infection. SjcDNA detection assay and POC-CCA urine test were more sensitive than stool microscopy in detecting schistosomiasis. On the other hand, US was less sensitive than the widely utilized K-K technique in diagnosing schistosomiasis. This study emphasizes the need to revisit the use of single stool K-K in the surveillance and case detection of schistosomiasis in endemic areas of the Philippines. The availability of advanced and more sensitive diagnostic tests will help better control, prevent, and eliminate schistosomiasis in the country.

A simple and efficient miracidium hatching technique for preparing a single-genome DNA sample of Schistosoma japonicum.

In this study, a simple and efficient miracidium hatching technique (MHT) protocol for preparing a single-genome DNA of Schistosoma japonicum was proposed. The protocol was designed with 96-well plates to collect a miracidium for single-genome DNA preparation, and the effects of lighting conditions on hatching rates were evaluated. The highest hatching rate was recorded under sunlight (92.4%), followed by fluorescent light (88.0%), and the lowest rate was recorded under the dark condition (4.7%). The results suggested for the first time, to our knowledge, that sunlight was efficient for this simple MHT protocol. Successful amplification of microsatellite marker genes using DNA isolated from a single miracidium also confirmed the quality of the single-genome DNA for subsequent applications.

Field Evaluation of Recombinant Antigen ELISA in Detecting Zoonotic Schistosome Infection Among Water Buffaloes in Endemic Municipalities in the Philippines.

In this study, we investigated the use of recombinant antigens thioredoxin peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.

Analyses of the expression, immunohistochemical properties and serodiagnostic potential of Schistosoma japonicum peroxiredoxin-4.

BACKGROUND: Schistosoma japonicum, which inhabits the mesenteric vein of the mammalian hosts for about 20 to 30 years, is subjected to the oxidative stresses from the host defense mechanism during their intra-mammalian stages. To counteract this host immune attack, the parasite utilizes their antioxidant system for survival inside the host. Peroxiredoxins (Prxs), thiol-specific antioxidant proteins, play an essential role for protecting the parasite against oxidative stress by reducing hydrogen peroxide to water. Only three types of 2-Cys Prxs have been previously characterized in S. japonicum whereas a fourth Prx has been identified for Schistosoma mansoni as Prx-4. A sequence coding homologous to this gene in the S. japonicum database was identified, characterized and expressed as recombinant SjPrx-4 protein (rSjPrx-4). Furthermore, rSjPrx-4 was evaluated in this study for its diagnostic potentials in detecting S. japonicum infection in humans. RESULTS: The gene found in the parasite genome contained 2 active-site cysteines with conserved sequences in the predicted amino acid (AA) sequence and showed 75% identity with that of the previously characterized Prx (TPx-1) of S. japonicum. The gene was expressed in different stages of schistosome life-cycle with highest transcription level in the adult male. The gene was cloned into a plasmid vector and then transfected into Escherichia coli for expression of rSjPrx-4. Anti-rSjPrx-4 mouse sera recognized native SjPrx-4 in egg and adult worm lysate by western blotting. The result of a mixed function oxidation assay in which rSjPrx-4 prevented the nicking of DNA from hydroxyl radicals confirmed its antioxidant activity. Subsequently, immunolocalization analysis showed the localization of SjPrx-4 inside the egg, on the tegument and in the parenchyma of the adult worm. Enzyme-linked immunosorbent assay results showed that rSjPrx-4 has 83.3% sensitivity and 87.8% specificity. Its diagnostic potential was further evaluated in combination with recombinant SjTPx-1 protein, yielding an improved sensitivity and specificity of 90% and 92.7%, respectively. CONCLUSIONS: These results suggest that SjPrx-4 plays a role as an antioxidant dealing with oxidative stresses of S. japonicum, and its diagnostic potential improved by coupling it with SjTPx-1 is a proof for developing a serological test with better diagnostic performance for human schistosomiasis.

Detection of canine Schistosoma japonicum infection using recombinant thioredoxin peroxidase-1 and tandem repeat proteins.

Humans and dogs live very close together and share various pathogens causing zoonotic parasitoses like schistosomiasis. A previous population genetics study done for schistosomes in the Philippines suggested that there is a high transmission level of Schistosoma japonicum among humans and dogs proving that the latter are important reservoirs for this zoonotic parasite. A more sensitive and specific test detecting schistosome infection in dogs will therefore strengthen the zoonotic surveillance, which might help in the possible elimination of this ancient disease. In this study, recombinant thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) previously tested on human and water buffalo samples were used to assess its diagnostic applicability to dogs. Fifty-nine dog serum and stool samples were collected in the schistosomiasis-endemic municipalities of Calatrava, Negros Occidental and Catarman, Northern Samar in the Philippines and examined using the ELISA as compared to microscopy and fecal sample-based PCR. Samples positive for Babesia gibsoni and Dirofilaria immitis were also used to check for cross-reaction. Results showed that SjTPx-1 (80% sensitivity, 92.3% specificity) and Sj7TR (73.3% sensitivity, 92.3% specificity) have good potentials for diagnosing S. japonicum infection in dogs. These diagnostic antigens will therefore improve the surveillance in the transmission of the parasites from dogs to humans.

Schistosoma japonicum cathepsin B as potential diagnostic antigen for Asian zoonotic schistosomiasis.

In this study, the diagnostic value of Schistosoma japonicum cathepsin B (SjCatB) was evaluated as an antigen for the early detection of S. japonicum infection. SjCatB is a key protease used by the cercaria to penetrate the intact skin of the host for transdermal infection. The early exposure of the host's immune system to this enzyme may elicit early production of antibodies against this molecule. Therefore, the recombinant SjCatB (rSjCatB) was expressed in Escherichia coli with N-terminal 6xHis-tag. rSjCatB was tested for its performance as a diagnostic antigen using indirect enzyme-linked immunosorbent assay (ELISA) with sera from experimentally infected mice collected at > 8 weeks post-infection. Showing 100% sensitivity and 95.0% specificity in the ELISA, rSjCatB was then evaluated with sera from experimentally infected mice collected at 1-7 weeks post-infection to determine how early the antibodies can be detected. Results showed that as early as 6 weeks post-infection, 2 of the 3 infected mice were found to be positive with the antibodies against SjCatB. Furthermore, the potential of the recombinant antigen in detecting human schistosomiasis was evaluated with archived serum samples collected from individuals who had been diagnosed with S. japonicum infection by stool examination. Results showed 86.7% sensitivity and 96.7% specificity suggesting its high diagnostic potential for human schistosomiasis. In addition, SjCatB showed minimal cross-reaction with the sera collected from patients with other parasitic diseases. In conclusion, the results of this study suggest that SjCatB will be useful in the development of a sensitive and specific early detection test for S. japonicum infection.

Elimination of Schistosomiasis Mekongi from Endemic Areas in Cambodia and the Lao People's Democratic Republic: Current Status and Plans.

The areas endemic for schistosomiasis in the Lao People's Democratic Republic and in Cambodia were first reported 50 and 60 years ago, respectively. However, the causative parasite Schistosoma mekongi was not recognized as a separate species until 1978. The infection is distributed along a limited part of the Mekong River, regulated by the focal distribution of the intermediate snail host Neotricula aperta. Although more sensitive diagnostics imply a higher figure, the current use of stool examinations suggests that only about 1500 people are presently infected. This well-characterized setting should offer an exemplary potential for the elimination of the disease from its endemic areas; yet, the local topography, reservoir animals, and a dearth of safe water sources make transmission control a challenge. Control activities based on mass drug administration resulted in strong advances, and prevalence was reduced to less than 5% according to stool microscopy. Even so, transmission continues unabated, and the true number of infected people could be as much as 10 times higher than reported. On-going control activities are discussed together with plans for the future.

Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar.

OBJECTIVES: Schistosomiasis is an important disease in Madagascar, and several studies on the disease have focused on the occurrence of the parasite in humans. However, the range of the pathogen in the environment and its impact on human infection is difficult to predict. An environmental DNA (eDNA) detection system for Schistosoma mansoni was developed to improve schistosomiasis eco-epidemiology studies. METHODS: Primers and probes were designed and tested in experimental biotopes. The field study was conducted in Maevatanana District of Madagascar. Seven water sources with human use were sampled, with a total of 21 water samples collected. Snails were collected, and patients were examined by ultrasound to determine the occurrence of schistosomiasis in the study area. RESULTS: One water source with active transmission was identified through the detection of S. mansoni eDNA in the water and the intermediate host Biomphalaria pfeifferi collected from the same water source. People with clinical schistosomiasis were found in the area, reinforcing the findings. CONCLUSIONS: The application of eDNA in eco-epidemiology enables the determination of hot spots and safe spots in endemic areas, constituting an alternative ecological tool for follow-up and monitoring of control programs for schistosomiasis, and contributing information on water safety for improving the standard of living of the people in endemic areas.

Utilization of real time PCR for the assessment of egg burden in the organs of Schistosoma japonicum experimentally infected mice.

Schistosoma japonicum, causing zoonotic intestinal schistosomiasis, is found in China, the Philippines and parts of Indonesia. Severe disease manifestations are basically due to the deposition of eggs in some vital organs such as the liver, spleen and brain. Traditionally, histopathological microscopic examination of the egg burden was used to evaluate the intensity of infection in the affected organs. However, this technique is laborious, time-consuming and requires trained personnel. In this study, real time PCR targeting the mitochondrial NADH dehydrogenase I gene was used to compare with microscopic examination of tissue sections in evaluating the egg burdens in different affected organs. Livers, spleens and brains of the S. japonicum infected mice after 8 and 18 weeks post-infection (p.i) were harvested and examined. Results showed that there were statistically significant correlations between the egg burden evaluated by tissue section examination, and the Ct values of the real time PCR of livers with heavy egg burden at 8 (r = -0.81) and 18 (r = -0.80) weeks p.i. Furthermore, a correlation (r = -0.56) between the egg burden assessed by the microscopic examination and Ct value of the real time PCR of spleens with moderate egg burden after 18 weeks p.i and not 8 weeks p.i was also observed. Brains with low egg burden showed no schistosome eggs in the microscopic examination, however one sample tested positive by real time PCR. These results suggested that real time PCR is useful in evaluating schistosome egg burden in the organs of the experimentally infected mice model that will give further insights into the pathology of schistosomiasis.

Evaluation of Schistosoma japonicum thioredoxin peroxidase-1 as a potential circulating antigen target for the diagnosis of Asian schistosomiasis.

Asian schistosomiasis caused by Schistosoma japonicum is a serious zoonotic disease endemic in China, the Philippines and parts of Indonesia. Mass drug administration in endemic areas resulted to decline in disease severity and intensity. The low intensity of infection limits the use of current parasitological methods for schistosomiasis diagnosis. Detection of parasite circulating antigens might provide more informative result as it may indicate the true status of infection. In this study, S. japonicum thioredoxin peroxidase-1 (SjTPx-1) a 22 kDa secreted antioxidant enzyme expressed throughout the life stages of the parasite was evaluated for its potential use as a biomarker for schistosomiasis japonica infection. Rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) were raised against the recombinant SjTPx-1 (rSjTPx-1). The antibodies produced against the recombinant antigen was confirmed to detect the native SjTPx-1 in crude adult worm lysate. Likewise, the specific binding of mAbs to parasite TPx-1 and not to mammalian peroxiredoxin-1 orthologues was also confirmed. The double antibody sandwich ELISA developed in this study was able to detect at least 1 ng/ml of rSjTPx-1. In addition, this method was able to detect the antigen from all serum samples of experimentally infected rabbit and mice. The diagnostic potential of SjTPx-1 in human clinical samples was also evaluated, in which 4 out of 10 stool-confirmed serum samples had detectable levels of the antigen. The results suggest that SjTPx-1 can be a potential biomarker for Asian zoonotic schistosomiasis.

Geographic strain differentiation of Schistosoma japonicum in the Philippines using microsatellite markers.

BACKGROUND: Microsatellites have been found to be useful in determining genetic diversities of various medically-important parasites which can be used as basis for an effective disease management and control program. In Asia and Africa, the identification of different geographical strains of Schistosoma japonicum, S. haematobium and S. mansoni as determined through microsatellites could pave the way for a better understanding of the transmission epidemiology of the parasite. Thus, the present study aims to apply microsatellite markers in analyzing the populations of S. japonicum from different endemic areas in the Philippines for possible strain differentiation. METHODOLOGY/ PRINCIPAL FINDINGS: Experimental mice were infected using the cercariae of S. japonicum collected from infected Oncomelania hupensis quadrasi snails in seven endemic municipalities. Adult worms were harvested from infected mice after 45 days of infection and their DNA analyzed against ten previously characterized microsatellite loci. High genetic diversity was observed in areas with high endemicity. The degree of genetic differentiation of the parasite population between endemic areas varies. Geographical separation was considered as one of the factors accounting for the observed difference between populations. Two subgroups have been observed in one of the study sites, suggesting that co-infection with several genotypes of the parasite might be present in the population. Clustering analysis showed no particular spatial structuring between parasite populations from different endemic areas. This result could possibly suggest varying degrees of effects of the ongoing control programs and the existing gene flow in the populations, which might be attributed to migration and active movement of infected hosts from one endemic area to another. CONCLUSIONS/ SIGNIFICANCE: Based on the results of the study, it is reasonable to conclude that genetic diversity could be one possible criterion to assess the infection status in highly endemic areas. Genetic surveillance using microsatellites is therefore important to predict the ongoing gene flow and degree of genetic diversity, which indirectly reflects the success of the control program in schistosomiasis-endemic areas.

Molecular characterization of sympatrically distributed Neotricula aperta-like snails in the Mekong River, Kratie, Cambodia.

Fifty-six samples of Neotricula aperta-like snails were collected from six locations in Cambodia. Their mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences were examined using haplotype network and neighbor-joining (NJ) tree analysis. Twenty-seven haplotypes (H1-H27) were observed and were divided into two different groups/lineages. Of 27, 17 haplotypes (H11-H27) were clustered with the reference samples of the γ-race N. aperta. The remaining 10 haplotypes (H1-H10) were clustered in a separate group/lineage, differing from the reference samples of the α-, ß-, and γ-race N. aperta, suggesting a new lineage belonging the genus Neotricula. Our results show that both the γ-race and a new lineage were sympatrically present approximately 60 km upstream of the Mekong River near the Kratie port, Cambodia. Further morphological and molecular studies are required to confirm the taxonomic status of this new, unidentified lineage.

Development and optimization of cocktail-ELISA for a unified surveillance of zoonotic schistosomiasis in multiple host species.

The zoonotic characteristic of the human parasite Schistosoma japonicum infecting a significant number of wild and domestic animals highlights the need to develop a unified surveillance in multiple host species for a strengthened schistosomiasis control. It has been shown in several studies that water buffaloes and dogs are considered important reservoirs in the transmission of the schistosome parasite to humans. Recombinant antigens like thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj7TR) have been shown to be good diagnostic antigens individually in humans, water buffaloes, and dogs in previous studies. Mixing these antigens together in a cocktail-ELISA might not only improve their diagnostic potentials but rather produce a multi-host species detection means for zoonotic schistosomiasis. In this study, we aimed to develop and optimize cocktail-ELISA by testing different combinations of these recombinant antigens in humans, water buffaloes, and dogs. As compared with the diagnostic potential calculated for each of the three recombinant antigens used, their combination has presented improved specificities, positive predictive values, and kappa values. Using samples collected from various endemic areas in the Philippines, results showed that the combination of SjTPx-1/Sj7TR/Sj1TR has the highest sensitivity in humans (84.1 %), water buffaloes, and dogs (80 %) and specificity (100 %) in all host species. This study therefore suggests the use of cocktail-ELISA in improving the zoonotic surveillance in schistosomiasis endemic areas.

Detection of active schistosome infection by cell-free circulating DNA of Schistosoma japonicum in highly endemic areas in Sorsogon Province, the Philippines.

The current status of schistosomiasis in highly endemic areas is difficult to determine by ovum detection because of the superficially low parasite load after mass drug administration, whereas the parasite transmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing in the host's body fluids. We conducted population-based studies to test the presence of cell-free schistosome DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and urine of Kato-Katz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA was also detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA was found to be network echogenic pattern (NW)-positive (serum 53.3%, urine 42.9%) or NW-negative (serum 25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%) or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is a promising diagnostic marker for active schistosome infection in the case of light infection.