Evaluation of sensitivity and specificity of Kato-Katz and circulating cathodic antigen in terms of Schistosoma japonicum using latent class analysis.

Schistosoma japonicum is endemic in the Philippines. The Kato-Katz (KK) method was used to diagnose S. japonicum. This is impractical, particularly when the sample size is limited. Knowledge on point-of-care circulating cathodic antigen (CCA) test performance for S. japonicum is limited. Determining the sensitivity and specificity of new diagnostics is difficult when the gold standard test is less effective or absent. Latent class analysis (LCA) can address some limitations. A total of 484 children and 572 adults from the Philippines were screened for S. japonicum. We performed Bayesian LCA to estimate the infection prevalence, sensitivity and specificity of each test by stratifying them into two age groups. Observed prevalence assessed by KK was 50.2% and 31.8%, and by CCA was 89.9% and 66.8%, respectively. Using Bayesian LCA, among children, the sensitivity and specificity of CCA were 94.8% (88.7-99.4) and 21.5% (10.5-36.1) while those of KK were 66.0% (54.2-83.3) and 78.1% (61.1-91.3). Among adults, the sensitivity and specificity of CCA were 86.4% (76.6-96.9) and 62.8% (49.1-81.1) while those of KK were 43.6% (35.1-53.9) and 85.5% (75.8-94.6). Overall, CCA was more sensitive than KK, regardless of the age group at diagnosis, as KK was more specific. KK and CCA have different diagnostic performance, which should inform their use in the planning and implementation of S. japonicum control programs.

The Impact of Prenatal Alcohol Exposure on Longitudinal Growth, Nutritional Status, and Insulin-Like Growth Factor 1 in Early Childhood in Leyte, the Philippines.

OBJECTIVE: To assess the impact and potential mechanistic pathways of prenatal alcohol exposure (PAE) on longitudinal growth and nutritional status in early childhood. STUDY DESIGN: A cohort of 296 mother-infant dyads (32% with PAE vs 68% unexposed) were recruited in Leyte, the Philippines, and followed from early gestation through 24 months of age. PAE was assessed using serum phosphatidylethanol (PEth) captured twice prenatally and in cord blood and supplemented with self-reported alcohol consumption. Linear mixed models were used to examine longitudinal effects of PAE on growth from birth through 2 years including key potential mediating factors (placental histopathology, and infant serum leptin and Insulin-like Growth Factor 1 [IGF-1]). RESULTS: After adjusting for potential confounders, we found that PAE was significantly associated with a delayed blunting of linear growth trajectories (height-for-age z-score, body length) and weight (weight-for-age z-score, body weight) that manifested between 4 and 6 months and continued through 12-24 months. PAE was also associated with a decreased rate of mid-upper-arm circumference growth from birth to 12 months, and a lower mean IGF-1 levels at birth and 6 months. CONCLUSION: This study demonstrates a delayed impact of PAE on growth that manifested around 6 months of age, underscoring the importance of routine clinical monitoring in early childhood. Furthermore, the findings supported prior animal model findings that suggest a mechanistic role for IGF-1 in PAE-induced growth delay.

Comparison of self-reported and observed water contact measures in Schistosoma japonicum-endemic villages in Leyte, The Philippines.

BACKGROUND: We compared individuals' self-reported water contact from a questionnaire to direct observation of water contact. Questionnaires that accurately capture water contact are necessary to risk-stratify individuals and communities at high risk for schistosomiasis. METHODS: Individuals (N=677) ages 7-30 y were included from three Schistosoma japonicum-endemic villages in Leyte, The Philippines. Each individual was observed for 12 d over the course of the 18-month study and the questionnaire was administered six times. A questionnaire index was derived that captured the number of self-reported contacts with water bodies for any purpose. An exposure index was created based on the sum of contacts that was weighted by the percentage of body surface area (BSA) exposed and exposure duration. RESULTS: Of 16 water contact activities, only bathing and washing clothes exhibited a significant, positive correlation between self-reported contacts and the observed exposure index related to those contacts. CONCLUSIONS: We found that only the reported frequencies of bathing and washing clothes were significantly related to an individual's overall observed exposure index, while use of all reported contacts was not related to the observed exposure. This study further supports the need for questionnaires to be augmented by some measure of how much BSA is exposed and/or time is spent in the water on average for a specific activity.

High prevalence of Schistosoma japonicum by perfusion in naturally exposed water buffalo in a region of the Philippines endemic for human schistosomiasis.

In the past decade, ecological surveys emphasized rats and dogs as the most significant animal reservoirs for Schistosoma japonicum (S.j) in the Philippines. However, recent studies demonstrated 51-91% prevalence of schistosomiasis among water buffalo using qPCR in the Sj endemic regions in the Philippines. In order to resolve the inconsistency of reported surveys regarding Sj endemicity among carabao, a domestic water buffalo that is the most important draught animal, we introduced 42 schistosome negative water buffalo to Macanip, Jaro municipality, Leyte, the Philippines, a subsistence rice-farming village that has been the focus of schistosomiasis japonica studies of our group for the past 20 years. We conducted perfusion to the remaining 34 buffalo that survived 10 months of nature exposure and Typhoon Haiyan. Thirty-three water buffalo were found to be positive with at least 1 pair of worms from the mesenteric vein. The infection rate is 97%, with the worm burden of 94 (95% confidence interval, 49-138 worms) worms. To our knowledge, this is the first report about S. japonicum worm burden in naturally infected water buffalo in the Philippines. The fact that with less than one-year of exposure, in this human schistosomiasis endemic area, only 1 out of 34 water buffalo was uninfected is striking. Urgent attention is needed for a cost-effective technique for monitoring Sj infection in animals and humans. Meanwhile, intervention implementation, including water buffalo treatment and vaccination, should be taken into consideration.

Effect of maternal praziquantel treatment for Schistosoma japonicum infection on the offspring susceptibility and immunologic response to infection at age six, a cohort study.

In areas endemic to schistosomiasis, fetal exposure to schistosome antigens prime the offspring before potential natural infection. Praziquantel (PZQ) treatment for Schistosoma japonicum infection in pregnant women has been demonstrated to be safe and effective. Our objectives were to evaluate whether maternal PZQ treatment modifies the process of in utero sensitization to schistosome antigens potentially impacting later risk of infection, as well as immune response to S. japonicum. We enrolled 295 children at age six, born to mothers with S. japonicum infection who participated in a randomized control trial of PZQ versus placebo given at 12-16 weeks gestation in Leyte, The Philippines. At enrollment, we assessed and treated current S. japonicum infection and measured serum cytokines. During a follow-up visit four weeks later, we assessed peripheral blood mononuclear cell (PBMC) cytokine production in response to soluble worm antigen preparation (SWAP) or soluble egg antigen (SEA). Associations between maternal treatment group and the child's S. japonicum infection status and immunologic responses were determined using multivariate linear regression analysis. PZQ treatment during pregnancy did not impact the prevalence (P = 0.12) or intensity (P = 0.59) of natural S. japonicum infection among children at age six. Among children with infection at enrollment (12.5%) there were no significant serum cytokine concentration differences between maternal treatment groups. Among children with infection at enrollment, IL-1 production by PBMCs stimulated with SEA was higher (P = 0.03) in the maternal PZQ group compared to placebo. Among children without infection, PBMCs stimulated with SEA produced greater IL-12 (P = 0.03) and with SWAP produced less IL-4 (P = 0.01) in the maternal PZQ group compared to placebo. Several cytokines produced by PBMCs in response to SWAP and SEA were significantly higher in children with S. japonicum infection irrespective of maternal treatment: IL-4, IL-5, IL-10, and IL-13. We report that maternal PZQ treatment for S. japonicum shifted the PBMC immune response to a more inflammatory signature but had no impact on their offspring's likelihood of infection or serum cytokines at age six, further supporting the safe use of PZQ in pregnant women. Trial Registration: ClinicalTrials.gov NCT00486863.

Whole-Proteome Differential Screening Identifies Novel Vaccine Candidates for Schistosomiasis japonica.

Schistosomiasis remains a leading cause of chronic morbidity in endemic regions despite decades of widespread mass chemotherapy with praziquantel. Using our whole proteome differential screening approach, and plasma and epidemiologic data from a longitudinal cohort of individuals living in a Schistosoma japonicum-endemic region of the Philippines, we interrogated the parasite proteome to identify novel vaccine candidates for Schistosoma japonicum. We identified 16 parasite genes which encoded proteins that were recognized by immunoglobulin G or immunoglobulin E antibodies in the plasma of individuals who had developed resistance to reinfection, but were not recognized by antibodies in the plasma of individuals who remained susceptible to reinfection. Antibody levels to Sj6-8 and Sj4-1 measured in the entire cohort (N = 505) 1 month after praziquantel treatment were associated with significantly decreased risk of reinfection and lower intensity of reinfection over 18 months of follow-up.

A preliminary evaluation of a locally produced biotinylated polyclonal anti-rabies antibody for direct rapid immunohistochemical test (DRIT) in the Philippines.

Rabies is a fatal zoonotic disease endemic in developing countries of Asia and Africa. Recently, the direct rapid immunohistochemical test (DRIT) was recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE) as a diagnostic test for rabies. Therefore, a biotinylated polyclonal antibody (pAb) against the rabies lyssavirus (RABV) nucleoprotein was developed using a plasmid cDNA vaccine derived from a challenge virus standard 11 strain. A preliminary evaluation on the efficacy of this reagent in recognizing the Philippine RABV strain was tested using banked canine hippocampal tissue samples with DRIT and the results were compared to dFAT. The effects of acetone and formalin fixation on DRIT were also assessed through immunoreactivity scores of the specimens. Of the 142 samples examined, 104 tested positive and 38 negative using both dFAT and DRIT, showing 100% agreement between the two diagnostic procedures. Moreover, no false positive or false negative results were observed using acetone and formalin fixation. Thus, locally prepared biotinylated pAb from plasmid cDNA can be used for DRIT, especially in resource-limited laboratories in the Philippines. However, these results should be confirmed with a more thorough evaluation of this technique, and the range of detection needs to be further evaluated in a larger panel of animal samples and on other lyssaviruses.

Intron sequence of the taurocyamine kinase gene as a marker to investigate genetic variation of Paragonimus species in Japan and the origins of triploidy in P. westermani.

BACKGROUND: Paragonimiasis is a foodborne parasitic infection caused by lung flukes of the genus Paragonimus. Several species of Paragonimus are endemic in Japan: P. westermani (diploid and triploid) P. miyazakii, P. ohirai and P. iloktsuenensis. The taxonomic status and genetic variability of these lung flukes remains poorly understood. METHODS: The second intron of domain 1 of the taurocyamine kinase gene (TKD1int2) region was used to explore genetic variation and differentiation of diploid and triploid P. westermani, as well as P. miyazakii, P. ohirai and P. iloktsuenensis originating from Japan. RESULTS: We found high levels of intraspecific variation in P. westermani, but only low levels of variation within the other species studied. Haplotype network and phylogenetic tree analyses demonstrated the sister-group relationship of P. ohirai and P. iloktsuenensis and the phylogenetically distant relationship of P. westermani with the other species. All individuals except for triploid P. westermani were homozygous. Each triploid contained at least one allele similar to that seen in most diploids from Chiba and one allele resembling that seen in diploids from Oita. One triploid contained three different sequences. CONCLUSIONS: Our findings suggested that the TKD1int2 region is a suitable marker for use in studying the genetic variation and phylogenetics of Paragonimus species, as well as providing clues to the origins of triploidy in P. westermani.

Evaluation of Direct Rapid Immunohistochemical test (DRIT) of Canis lupus familiaris hippocampal touch impression smears using a monospecific polyclonal antibody for rabies virus detection

@#<p style="text-align: justify;"><strong>BACKGROUND:</strong> Rabies is an important zoonotic disease that needs to be eradicated worldwide. It is still prevalent in the Philippines, thus development of a relatively affordable but still accurate and rapid post-mortem detection test for the virus is desired, especially in regional laboratories.<br /><strong>METHODS:</strong>The study evaluated the Direct Rapid Immunohistochemical Testing (DRIT) of hippocampal touch impressions of suspected rabid Canis lupus familiaris using monospecific N protein polyclonal antibody developed by the Research Institute for Tropical Medicine (RITM). One hundred sixty (160) acetone-fixed hippocampal touch impressions were subjected DRIT.<br /><strong>RESULTS:</strong> One hundred thirteen (70.6%) out of 160 samples tested positive for rabies viral antigen (RVA) and 47 (29.4%) out of 160 samples tested negative for RVA. No false positive and false negative results were obtained. The results agree with the gold standard, dFAT.<br /><strong>CONCLUSION:</strong> DRIT was able to detect low to high concentrations of RVA in the hippocampal touch impressions based on the grading distribution. DRIT had 100% sensitivity, specificity and over-all accuracy using monospecific polyclonal antibodies, which suggests its use as a more affordable alternative to the gold standard dFAT.</p>

Development of Paramyosin as a Vaccine Candidate for Schistosomiasis.

Schistosomiasis, caused by three principal species of diecious trematodes (flatworms), currently afflicts over 250 million individuals, results in an estimated 2-15% chronic disability, and contributes to poor health and economic stagnation in endemic areas. Although schistosomiasis is effectively treated with praziquantel, rapid reinfection with rebound morbidity precludes effective control based on chemotherapy alone and justifies current efforts to develop vaccines for these parasites. Paramyosin (Pmy), an invertebrate muscle-associated protein, has emerged as a promising vaccine candidate for both Schistosoma mansoni and Schistosoma japonicum. Herein, we discuss the discovery of Pmy, its development as a vaccine candidate in rodents and bovines, as well as studies of naturally occurring immune responses to Pmy in prospective, observational human studies. We conclude with a proposed developmental plan to move Pmy toward Phase I clinical trials.

Phosphagen kinase in Schistosoma japonicum: II. Determination of amino acid residues essential for substrate catalysis using site-directed mutagenesis.

Phosphagen kinases (PKs) play major roles in the regulation of energy metabolism in animals. Creatine kinase (CK) is the sole PK in vertebrates, whereas several PKs are present in invertebrates. We previously identified a contiguous dimer taurocyamine kinase (TK) from the trematode Schistosoma japonicum (Sj), a causative agent of schistosomiasis. SjTK contiguous dimer is comprised of domain 1 (D1) and domain 2 (D2). In this study, we used SjTK contiguous dimer (SjTKD1D2) or truncated single-domain constructs (SjTKD1 or SjTKD2) and employed site-directed mutagenesis to investigate the enzymatic properties of TK mutants. Mutation in SjTKD1 or SjTKD2 (D1E222G or D2E225G) caused complete loss of activity for the substrate taurocyamine. Likewise, a double mutant (D1E222GD2E225G) in the contiguous dimer (D1D2) exhibited complete loss of activity for the substrate taurocyamine. However, catalytic activity in the contiguous dimer remained in both of D1 inactive mutant (D1D2D1E222G) and D2 inactive mutant (D1D2D2E225G), suggesting that efficient catalysis of SjTKD1D2 is dependent on the activity of D1 and D2. The catalytic efficiency of the mixture of both single domains (WTD1+WTD2) showed same enzymatic properties (Km(Tauro)=0.68;Vmax/Km(Tauro)=137.04) to WTD1D2 (Km(Tauro)=0.47; Vmax/Km(Tauro)=144.30). This result suggests that the contiguous dimeric structure is not essential for the catalytic efficiencies of both domains of SjTK. Vmax/Km(Tauro) of the mixture of wild-type and inactivated domains (78.02 in WTD1+D2E225G and 128.24 in D1E222G+WTD2) were higher than the corresponding mutants (47.25 in D1D2D1E222G and 46.77 in D1D2D2E225G). To identify amino acid residues that are critical for taurocyamine binding, we performed alanine scanning mutagenesis at positions 57-63 on the guanidino specificity (GS) region of the SjTKD1, which is considered to be involved in guanidino-substrate recognition. R63A and R63Y mutants lost activity for taurocyamine, suggesting that these residues are associated with taurocyamine binding. In addition, we investigated the role of Tyr84 in D1 and found an association with substrate alignment. The Y84 residue was replaced with R, H, K, I, A, and G. Although the activities of each mutant were decreased (Vmax=2.36-67.50µmolPi/min/mgprotein), Y84 mutants possess binding affinity for taurocyamine (Km(Tauro)=3.19-10.04mM). The D1Y84R, D1Y84H, D1Y84K, and D1Y84A mutants exhibited low activity for taurocyamine, whereas the D1Y84I and D1Y84G mutants exhibited slightly decreased activity compared with the other Y84 mutants. The D1Y84K mutant lost substrate synergy between taurocyamine and ATP, suggesting that this mutation moves the position of the GS loop, similar to that of lombricine kinase (LK), and interferes with taurocyamine binding. This is the first comprehensive investigation of essential amino acid residues for substrate catalysis in trematode TK.

Genetic structure inferred from mitochondrial 12S ribosomal RNA sequence of Oncomelania quadrasi, the intermediate snail host of Schistosoma japonicum in the Philippines.

Species and subspecies of the Oncomelania hupensis species complex are recognized as intermediate hosts of Schistosoma japonicum. Of these species and subspecies, O. quadrasi is distributed throughout the Philippines. This study used 12S ribosomal RNA sequences to explore the genetic structure of O. quadrasi populations in the Philippines. Three subspecies, O. h. hupensis, O. h. formosana, and O. h. chiui of this group were also examined. The phylogenetic tree and haplotypes network showed that O. quadrasi separated from the subspecies. Ten O. quadrasi haplotypes (Oq1-Oq10) clustered in relation to their geographic origin. Genetic differentiation (FST) and estimated gene flow (Nm) among populations showed significant differences, ranging from 0.556-1.000 to 0.00-0.74, respectively. Genetic differences among groups (FCT = 0.466), populations within a group (FSC = 0.727), and populations (FST = 0.854) were observed. These results indicate that the O. quadrasi populations in the Philippines have a substructure associated with their geographic origin.

Maternal infection with Schistosoma japonicum induces a profibrotic response in neonates.

The global burden of schistosomiasis is significant, with fibrosis a major associated morbidity and the primary cause of mortality. We have previously shown that schistosomiasis during pregnancy upregulates proinflammatory cytokines in the cord blood. In this study, we extend these findings to include a large panel of fibrosis-associated markers. We developed a multiplex bead-based assay to measure the levels of 35 proteins associated with fibrosis. Cord blood from 109 neonates born to mothers residing in an area of Schistosoma japonicum endemicity was assessed for these molecules. Ten mediators were elevated in the cord blood from schistosome-infected pregnancies, including insulin-like growth factor 1 (IGF-1), tumor growth factor ß1 (TGF-ß1), connective tissue growth factor (CTGF), procollagen I carboxy-terminal propeptide (PICP), amino-telopeptide of type 1 collagen (ICTP), collagen VI, desmosine, matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 1 (TIMP-1), and TIMP-4. Many of these were also positively correlated with preterm birth (PICP, ICTP, MMP-2, TGF-ß1, desmosine, CTGF, TIMP-1). In addition, birth weight was 168 g lower for infants with detectable levels of CTGF than for those with CTGF levels below the level of detection. Maternal schistosomiasis results in upregulation of fibrosis-associated proteins in the cord blood of the neonate, a subset of which are also associated with adverse birth outcomes. As the first report of fibrosis-associated molecules altered in the newborn of infected mothers, this study has broad implications for the health of the fetus, stretching from gestation to adulthood.

Schistosomiasis japonica during pregnancy is associated with elevated endotoxin levels in maternal and placental compartments.

Schistosomiasis affects approximately 40 million women of reproductive age and has been linked to elevated levels of circulating endotoxin in nonpregnant individuals. We have evaluated endotoxin levels in maternal, placental, and newborn blood collected from women residing in Leyte, Philippines. Endotoxin levels in both maternal and placental compartments in pregnant women with schistosomiasis were 1.3- and 2.4-fold higher, respectively, than in uninfected women. In addition, higher concentrations of endotoxin in placental blood were associated with premature birth, acute chorioamnionitis, and elevated proinflammatory cytokines. By promoting endotoxemia, schistosomiasis may exert additional, maladaptive influences on pregnancy outcomes.

Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK) constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK) of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK) gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart, creatine.

The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase.

The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km(Tc) and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.

Gene structure of the two-domain taurocyamine kinase from Paragonimus westermani: evidence for a distinct lineage of trematode phosphagen kinases.

Taurocyamine kinase (TK) is an enzyme that catalyzes the reversible transfer of a phosphate between ATP and taurocyamine. Annelid TKs were suggested to have evolved from a CK ancestor. However, TKs from the lung fluke Paragonimus westermani comprised another lineage. Construction of phylogenetic tree and comparison of exon/intron organization showed that P. westermani TK and other trematode TKs evolved from a molluscan arginine kinase (AK) gene. Exon shuffling probably caused the changes in amino acid sequence thereby changing the affinity from AK to TK. The present study provides new insights on the evolution of phosphagen kinases found in trematodes.

Phosphagen kinase in Schistosoma japonicum: characterization of its enzymatic properties and determination of its gene structure.

Phosphagen kinases (PKs) play a major role in the regulation of energy metabolism in animals. Creatine kinase (CK) is the sole PK in vertebrates, whereas several PKs are present in invertebrates. Here, we report the enzymatic properties and gene structure of PK in the trematode Schistosoma japonicum (Sj). SjPK has a unique contiguous dimeric structure comprising domain 1 (D1) and domain 2 (D2). The three states of the recombinant SjPK (D1, D2, and D1D2) show a specific activity for the substrate taurocyamine. The comparison of the two domains of SjPK revealed that D1 had a high turnover rate (kcat=52.91) and D2 exhibited a high affinity for taurocyamine (Km(Tauro) =0.53±0.06). The full-length protein exhibited higher affinity for taurocyamine (Km(Tauro) =0.47±0.03) than the truncated domains (D1=1.30±0.10, D2=0.53±0.06). D1D2 also exhibited higher catalytic efficiency (kcat/Km(Tauro) =82.98) than D1 (40.70) and D2 (29.04). These results demonstrated that both domains of SjTKD1D2 interacted efficiently and remained functional. The three-dimensional structure of SjPKD1 was constructed by the homology modeling based on the transition state analog complex state of Limulus AK. This protein model of SjPKD1 suggests that the overall structure is almost conserve between SjPKD1 and Limulus AK except for the flexible loops, that is, particularly guanidino-specificity (GS) region, which is associated with the recognition of the corresponding guanidino substrate. The constructed NJ tree and the comparison of exon/intron organization suggest that SjTK has evolved from an arginine kinase (AK) gene. SjTK has potential as a novel antihelminthic drug target as it is absent in mammals and its strong activity may imply a significant role for this protein in the energy metabolism of the parasite.

High prevalence of Schistosoma japonicum infection in Carabao from Samar Province, the Philippines: implications for transmission and control.

Schistosoma japonicum is endemic in the Philippines, China and Indonesia, and infects more than 40 mammalian host species, all of which can act as reservoirs of infection. In China, water buffaloes have been shown to be major reservoirs of human infection. However, in the Philippines, carabao have not been considered important reservoir hosts for S. japonicum due to the low prevalence and infection intensities reported, the only exception being a qPCR-based study indicating 51% of carabao were S. japonicum-positive. However, the low prevalence found for the same animals when using conventional copro-parasitological techniques means that there is still confusion about the role of carabao in the transmission of schistosomiasis japonicum. To address this inconsistency, and to shed light on the potential role of carabao in the transmission of S. japonicum in the Philippines, we undertook a pilot survey, collecting fecal samples from animals in Western Samar Province and we used a combination of molecular and copro-parasitological techniques to determine the prevalence and intensity of S. japonicum. We found a high prevalence of S. japonicum in the carabao using a validated real-time PCR (qPCR) and a copro-parasitological tool, the formalin-ethyl acetate sedimentation (FEA-SD) technique. A much lower prevalence of S. japonicum was recorded for the same fecal samples using conventional PCR, the Kato-Katz technique and miracidial hatching. These results suggest that, due to their low diagnostic sensitivity, traditional copro-parasitological techniques underestimate infection in carabao. The use of FEA-SD and qPCR provides a more accurate diagnosis. Based on these findings, the role of bovines in the transmission of S. japonicum appears to be more important in the Philippines than previously recognized, and this may have significant implications for the future control of schistosomiasis there, particularly as, in contrast with previous surveys, we found an unprecedented high prevalence of S. japonicum in humans.

Tissue inhibitor of matrix-metalloprotease-1 predicts risk of hepatic fibrosis in human Schistosoma japonicum infection.

BACKGROUND: Schistosomes infect 200 million individuals annually and cause significant hepatic fibrosis in up to 20%. Little is known regarding the mechanisms of schistosome-associated hepatic fibrosis in humans, and few biomarkers for risk of fibrosis have been identified. METHODS: We treated 611 Schistosoma japonicum-infected Filipinos with praziquantel (PZQ) and performed ultrasound to quantify hepatic fibrosis at baseline and 12 months after PZQ treatment. We developed a multiplexed assay (FibroPlex) that quantifies predictors and effect modifiers of fibrosis. We measured FibroPlex analytes produced by peripheral blood mononuclear cells stimulated with schistosome egg antigen 4 weeks after PZQ treatment and related these levels to risk of fibrosis 1 year after PZQ treatment. RESULTS: After adjusting for potential confounders, including baseline grade of fibrosis, individuals with detectable tissue inhibitor of matrix-metalloprotease-1 (TIMP-1) had a 3.5-fold greater risk of fibrosis 1 year after PZQ treatment, compared with individuals with undetectable levels (odds ratio, 3.48; 95% confidence interval, 1.41-8.43; P = .007). DISCUSSION: Because TIMP-1 inhibits most matrix metalloproteases, which are responsible for collagen degradation, these data suggest that schistosome-associated hepatic fibrosis results, in part, from excessive inhibition of collagen remodeling. These data further suggest that TIMP-1 is a promising biomarker for assessing risk of hepatic fibrosis in schistosomiasis and, potentially, other infectious and noninfectious causes of liver disease.