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

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

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

Prevalence and Spatial Distribution of Heterophyidiasis in Southern Philippines

Heterophyidiasis is a fish-borne zoonotic disease that is considered to be an emerging public health problem in the Philippines. This study was carried out to determine the spatial distribution and risk factors of heterophyidiasis in five selected villages in New Corella; Davao del Norte in Southern Mindanao. Of the 1,101 individuals examined; 26 (2.36% overall prevalence rate; 95% CI 1.46–3.25) were positive for heterophyid eggs. Higher infection rate was observed in males (3.85%; 95% CI 2.27–5.43) than females (0.76%; 95% CI 0.02–1.5). Mapping of cases was done to show the spatial distribution of heterophyidiasis in New Corella. Multiple logistic regression analysis showed that gender; raw freshwater fish consumption; undercooked grilled fish consumption and proximity to rivers or creeks are the risk factors significantly associated with heterophyid infection. This study confirmed the presence of heterophyid infection in humans in the surveyed villages in New Corella in Southern Philippines

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

Current status of neglected tropical diseases (NTDs) in the Philippines

In the Philippines, ten NTDs are prevalent, but only six namely LF, schistosomiasis, STH, food-borne trematodiases, rabies and leprosy are considered to be of public health importance. The 81 provinces in the country are endemic for at least one of these NTDs. Others may be endemic for two or even more of these diseases. Since 2000, after the Philippines accepted and implemented the WHO guidelines for NTDs prevention, control and elimination, significant progress has been achieved in reducing the magnitude of NTDs endemic in the country. Since 2009, out of 46 filariasis-endemic provinces, the number of provinces that has eliminated LF has progressively increased so that by 2015, 76% are already LF-free. By 2019, only four provinces remain endemic for LF. For schistosomiasis, as of 2012, report from the Department of Health (DOH) put the number of high endemic provinces at 10, moderately endemic at 6 and low to elimination levels at 12. For STH, results of the National Parasite Survey in the Philippines among school-aged children conducted in 2015 by the Research Institute for Tropical Medicine, the research arm of the Philippine DOH, however, showed that the overall cumulative prevalence was 28.4% with a prevalence range between 7.1% and 67.4%. The figures are way above the \u3c20% prevalence standard set by the World Health Organization. Control and prevention efforts for FBTs just gained traction with the call of WHO for elimination of NTDs in 2015. There is an urgent need to update information by an intensive national baseline survey that can validate previous data as well as generate new information on the magnitude of the FBT problem in the Philippines. For leprosy, elimination activities have been intensified in high prevalence areas and from 2009 to 2013, case detection and prevalence were sustained at \u3c1.0 per 10,000 population. Rabies elimination activities have been effective that by 2011, only five regions out of 16 remained with the highest number of rabies cases. In a period of seven years from 2010 to 2017, the number of rabies-free provinces and municipalities increased from 3 to 49. Problems continue to hound the NTD programmes in the Philippines as priorities shift to more urgent health problems in a country that is weighed down not only by the triple burden of disease but serious health consequences of emergencies and disasters and the fast-growing population itself. Paradigm shifts are suggested to replace the traditional and conventional perspectives of control. These include change from disease approach to intervention approach to allow for integration of strategies targeting several NTDs and multisectoral, multidisciplinary approach requiring strong, viable and sustainable partnerships involving various agencies of the government, public and private sector, pharmaceuticals, academe, researchers, local government units and the endemic communities themselves

Oncomelania hupensis quadrasi: Snail intermediate host of Schistosoma japonicum in the Philippines

Oncomelania hupensis quadrasi is the snail intermediate host of Schistosoma japonicum in the Philippines. It was discovered by Dr. Marcos Tubangui in 1932 more than two decades after the discovery of the disease in the country in 1906. This review, the first for O. h. quadrasi, presents past and present works on the taxonomy, biology, ecology, control, possible paleogeographic origin of the snail intermediate host and future in research, control and surveillance of the snail. Extensive references are made of other subspecies of O. hupensis such as the subspecies in China for which majority of the advances has been accomplished. Contrasting views on whether the snail is to be considered an independent species of Oncomelania or as one of several subspecies of Oncomelania hupensis are presented. Snail control methods such as chemical methods using synthetic and botanical molluscicides, environmental manipulation and biological control are reviewed. Use of technologies such as Remote Sensing, Geographical Information System and landscape genetics is stressed for snail surveillance. Control and prevention efforts in the Philippines have consistently focused on mass drug administration which has proved inadequate in elimination of the disease. An integrated approach that includes snail control, environmental sanitation and health education has been proposed. Population movement such as migration for employment and economic opportunities and ecotourism and global climate change resulting in heavy rains and flooding challenge the gains of control and elimination efforts. Concern for possible migration of snails to non-endemic areas is expressed given the various changes both natural and mostly man-made favoring habitat expansion