Association of Antibodies to Helminth Defense Molecule 1 With Inflammation, Organomegaly, and Decreased Nutritional Status in Schistosomiasis Japonica.

Immunomodulation enhances parasite fitness by reducing inflammation-induced morbidity in the mammalian host, as well as by attenuating parasite-targeting immune responses. Using a whole-proteome differential screening method, we identified Schistosoma japonicum helminth defense molecule 1 (SjHDM-1) as a target of antibodies expressed by S. japonicum-resistant but not S. japonicum-susceptible individuals. In a longitudinal cohort study (n = 644) conducted in a S. japonicum-endemic region of the Philippines, antibody levels to SjHDM-1 did not predict resistance to reinfection but were associated with increased measures of inflammation. Individuals with high levels of anti-SjHDM-1 immunoglobulin G had higher levels of C-reactive protein than those with low anti-SjHDM-1. High anti-SjHDM-1 immunoglobulin G responses were also associated with reduced biomarkers of nutritional status (albumin), as well as decreased anthropometric measures of nutritional status (weight-for-age and height-for-age z scores) and increased measures of hepatomegaly. Our results suggest that anti-SjHDM-1 responses inhibit the immunomodulatory function of SjHDM-1, resulting in increased morbidity rates.

A review of health education activities targeting schoolchildren for the control of soil-transmitted helminthiasis in Southeast Asia, with emphasis upon the Magic Glasses approach.

Soil-transmitted helminths continue to be a serious problem causing disease and morbidity globally. Children, mostly school-aged, are more at risk of these infections. The main strategy for control remains to be the mass drug administration (MDA) of antihelminthic drugs. With the limitation of MDA to prevent re-infection, the need for additional approaches such as hygiene education and improvements in water, sanitation and hygiene (WASH) infrastructure are required. Although the importance of health education as a crucial component of an integrated approaches to STH control is highlighted, this component has often been disregarded because the other more complex solutions have been the focus of most studies and programmes. We performed literature searches from four bibliographic databases - Scopus, PubMed, Web of Science and Cochrane Library - to determine availability of studies on the impact of health education interventions targeting STH infections on schoolchildren in Southeast Asia. Our review found only three studies that evaluated health education interventions targeting children. The current lack of evidence in this area suggests the need for more studies assessing the impact of health education intervention for STH control. A successful health education programme for STH called "The Magic Glasses" has been developed targeting schoolchildren in China and the Philippines. This public health intervention displayed significant impact in terms of improving knowledge, attitude and practices, reducing prevalence of STH infections in schoolchildren and encouraging compliance to MDA. This article details the successes and benefits of the Magic Glasses programme as a promising control tool for STH in the Southeast Asian region.

Comparative assessment of the SjSAP4-incorporated gold immunochromatographic assay for the diagnosis of human schistosomiasis japonica.

Background: Schistosomiasis, a disease caused by parasites of the genus Schistosoma, remains a global public health threat. This study aimed to validate the diagnostic performance of a recently developed gold immunochromatographic assay (GICA) for the detection of S. japonicum infection in a rural endemic area of the Philippines. Methods: Human clinical samples were collected from 412 subjects living in Laoang and Palapag municipalities, Northern Samar, the Philippines. The presence of Schistosoma-specific antibodies in serum samples was tested with the SjSAP4-incorporated GICA strips and the results were converted to fully quantitative data by introducing an R value. The performance of the established GICA was further compared with other diagnostic tools, including the Kato-Katz (KK) technique, point-of-care circulating cathodic antigen (POC-CCA), droplet digital (dd) PCR, and enzyme-linked immunosorbent assays (ELISAs). Results: The developed GICA strip was able to detect KK positive individuals with a sensitivity of 83.3% and absolute specificity. When calibrated with the highly sensitive faecal ddPCR assay, the immunochromatographic assay displayed an accuracy of 60.7%. Globally, the GICA assay showed a high concordance with the SjSAP4-ELISA assay. The schistosomiasis positivity rate determined by the GICA test was similar to those obtained with the SjSAP4-ELISA assay and the ddPCR assay performed on serum samples (SR_ddPCR), and was 2.3 times higher than obtained with the KK method. Conclusion: The study further confirms that the developed GICA is a valuable diagnostic tool for detecting light S. japonicum infections and implies that this point-of-care assay is a viable solution for surveying endemic areas of low-intensity schistosomiasis and identifying high-priority endemic areas for targeted interventions.

Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis.

BACKGROUND: Schistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis. METHODS: We developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda. FINDINGS: The S. japonicum SHERLOCK assay achieved 93-100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples. INTERPRETATION: These schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases. FUNDING: Australian Infectious Diseases Research Centre seed grant (2022) and National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).

Development and assessment of a novel gold immunochromatographic assay for the diagnosis of schistosomiasis japonica.

Background: The neglected zoonosis, schistosomiasis japonica, remains a major public health problem in the Philippines. The current study aims to develop a novel gold immunochromatographic assay (GICA) and evaluate its performance in the detection of Schistosoma japonicum infection. Methods: A GICA strip incorporating a S. japonicum saposin protein, SjSAP4 was developed. For each GICA strip test, diluted serum sample (50 µl) was loaded and strips were scanned after 10 min to convert the results into images. ImageJ was used to calculate an R value, which was defined as the signal intensity of the test line divided by the signal intensity of the control line within the cassette. After determination of optimal serum dilution and diluent, the GICA assay was evaluated with sera collected from non-endemic controls (n = 20) and individuals living in schistosomiasis-endemic areas of the Philippines (n = 60), including 40 Kato Katz (KK)-positive participants and 20 subjects confirmed as KK-negative and faecal droplet digital PCR assay (F_ddPCR)-negative at a dilution of 1:20. An ELISA assay evaluating IgG levels against SjSAP4 was also performed on the same panel of sera. Results: Phosphate-buffered saline (PBS) and 0.9% NaCl were determined as optimal dilution buffer for the GICA assay. The strips tested with serial dilutions of a pooled serum sample from KK-positive individuals (n = 3) suggested that a relatively wide range of dilutions (from 1:10 to 1:320) can be applied for the test. Using the non-endemic donors as controls, the GICA strip showed a sensitivity of 95.0% and absolute specificity; while using the KK-negative and F_ddPCR-negative subjects as controls, the immunochromatographic assay had a sensitivity of 85.0% and a specificity of 80.0%. The SjSAP4-incorperated GICA displayed a high concordance with the SjSAP4-ELISA assay. Conclusions: The developed GICA assay exhibited a similar diagnostic performance with that of the SjSAP4-ELISA assay, yet the former can be performed by local personnel with minimal training with no requirement for specialised equipment. The GICA assay established here represents a rapid, easy-to-use, accurate and field-friendly diagnostic tool for the on-site surveillance/screening of S. japonicum infection.

Cost analysis for "The Magic Glasses Philippines" health education package to prevent intestinal worm infections among Filipino schoolchildren.

Background: Soil-transmitted helminth (STH) infections are a significant public health problem affecting over 900 million people globally. Health education has been shown to complement mass drug administration (MDA) for the control of these intestinal worms. We reported recently results of a cluster randomised control trial (RCT) showing the positive impact of the "The Magic Glasses Philippines (MGP)" health education package in reducing STH infections among schoolchildren in intervention schools with ≤15% STH baseline prevalence in Laguna province, the Philippines. To inform decision making on the economic implications of the MGP, we evaluated the in-trial costs and then quantified the costs of scaling up the intervention both regionally and nationally. Methods: Costs were determined for the MGP RCT conducted in 40 schools in Laguna province. We estimated the total cost and the costs incurred per student for the actual RCT and the total costs for regional and national scale-up in all schools regardless of STH endemicity. The costs associated with the implementation of standard health education (SHE) activities and mass drug administration (MDA) were determined with a public sector perspective. Findings: The cost per participating student in the MGP RCT was Php 58.65 (USD 1.15) but if teachers instead of research staff had been involved, the estimated cost would have been considerably lower at Php 39.45 (USD 0.77). Extrapolating the costs for regional scale-up, the costs per student were estimated to be Php 15.24 (USD 0.30). As it is scaled up at the national level to include more schoolchildren, the estimated cost was increased at Php 17.46 (USD 0.34). In scenario 2 and 3, consistently, labour/salary costs associated with the delivery of the MGP contributed most to overall programme expenditure. Furthermore, the estimated average cost per student for SHE and MDA were Php 117.34 (USD 2.30) and Php 58.17 (USD 1.14), respectively. Using national scale up estimates, the cost of combining the MGP with SHE and MDA was Php 192.97 (USD 3.79). Interpretation: These findings suggest that the integration of MGP into the school curriculum would be an affordable and scalable approach to respond to the continuous burden of STH infection among schoolchildren in the Philippines. Funding: National and Medical Research Council, Australia, and the UBS-Optimus Foundation, Switzerland.

Diagnostic performance of a urine-based ELISA assay for the screening of human schistosomiasis japonica: A comparative study.

The current study developed and evaluated the performance of a urine-based enzyme-linked immunosorbent assay (ELISA) for the screening of Schistosoma japonicum infection in a human cohort (n = 412) recruited from endemic areas, Northern Samar, the Philippines. The diagnostic performance of the urine ELISA assay was further compared with the Kato-Katz (KK) technique, serum-based ELISA assays, point-of-care circulating cathodic antigen (POC-CCA) urine cassette test, and droplet digital (dd)PCR assays performed on feces, serum, urine, and saliva samples, which were designated as F_ddPCR, SR_ddPCR, U_ddPCR, and SL_ddPCR, respectively. When urine samples concentrated 16× were assessed, the SjSAP4 + Sj23-LHD-ELISA (U) showed sensitivity/specificity values of 47.2/93.8% for the detection of S. japonicum infection in KK-positive individuals (n = 108). The prevalence of S. japonicum infection in the total cohort determined by the urine ELISA assay was 48.8%, which was lower than that obtained with the F_ddPCR (74.5%, p < 0.001), SR_ddPCR (67.2%, p < 0.001), and SjSAP4 + Sj23-LHD-ELISA (S) (66.0%, p < 0.001), but higher than that determined by the Sj23-LHD-ELISA (S) (24.5%, p < 0.001), POC-CCA assay (12.4%, p < 0.001), and SL_ddPCR (25.5%, p < 0.001). Using the other diagnostic tests as a reference, the urine ELISA assay showed a sensitivity between 47.2 and 56.9%, a specificity between 50.7 and 55.2%, and an accuracy between 49.3 and 53.4%. The concentrated urine SjSAP4 + Sj23-LHD-ELISA developed in the current study was more sensitive than both the KK test and POC-CCA assay, and showed a comparable level of diagnostic accuracy to that of the U_ddPCR. However, its diagnostic performance was less robust than that of the F_ddPCR, SR_ddPCR, and SjSAP4 + Sj23-LHD-ELISA (S) assays. Although they are convenient and involve a highly acceptable non-invasive procedure for clinical sample collection, the insufficient sensitivity of the three urine-based assays (the urine ELISA assay, the U_ddPCR test, and the POC-CCA assay) will limit their value for the routine screening of schistosomiasis japonica in the post mass drug administration (MDA) era, where low-intensity infections are predominant in many endemic areas.

Identification of a linear B-cell epitope on the Schistosoma japonicum saposin protein, SjSAP4: Potential as a component of a multi-epitope diagnostic assay.

BACKGROUND: Schistosoma japonicum is one of three major species of blood flukes causing schistosomiasis, a disease, which continues to be a major public health issue in the Philippines. SjSAP4, a member of a multigene family of saposin-like proteins, is a recognized immunodiagnostic biomarker for schistosomiasis japonica. This study aimed to identify linear B-cell epitopes on SjSAP4 and to validate their potential as components of a multi-epitope assay for the serological diagnosis of schistosomiasis japonica. METHODOLOGY: SjSAP4-derived peptides were expressed as GST-peptide-fused proteins and these were Western blot probed with human serum samples from S. japonicum Kato-Katz (KK)-positive individuals and uninfected controls. A core epitope was further identified by Western blotting through probing a series of truncated peptides with the schistosomiasis patient sera. The diagnostic performance of the core epitope-containing peptides and the full-length SjSAP4 was evaluated by enzyme-linked immunosorbent assay (ELISA) using a panel of sera collected from subjects resident in a schistosomiasis-endemic area of the Philippines. MAIN FINDINGS: As a result of the peptide mapping, one peptide (P15) was found to be highly immunogenic in the KK-positive individuals. We subsequently showed that -S163QCSLVGDIFVDKYLD178- is a core B-cell epitope of P15. Subsequent ELISAs incorporating SjSAP4, SjSAP4-Peptide and SjSP-13V2-Peptide showed a sensitivity of 94.0%, 46.0% and 74.0%, respectively, and a specificity of 97.1%, 100% and 100%, respectively. Notably, complementary recognition of the B-cell epitopes (SjSAP4-Peptide and SjSP-13V2-Peptide) was observed in a subset of the KK-positive individuals. A dual epitope-ELISA (SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA) showed a diagnostic sensitivity of 84.0% and a specificity of 100%. CONCLUSIONS/SIGNIFICANCE: In this study, -S163QCSLVGDIFVDKYLD178- was identified as a dominant linear B-cell epitope on SjSAP4. This peptide and the complementary recognition of other B-cell epitopes using sera from different KK-positive individuals can provide the basis of developing a multi-epitope assay for the serological diagnosis of schistosomiasis.

"The Magic Glasses Philippines": a cluster randomised controlled trial of a health education package for the prevention of intestinal worm infections in schoolchildren.

BACKGROUND: Soil-transmitted helminths (STH) cause substantial disease and disability globally. Health education has proven complementary to school-based drug administration programs for STH control. We determined the generalizability of the impact of "The Magic Glasses" health education package for STH prevention in schoolchildren in Laguna province, the Philippines, having previously shown its positive impact in China. METHODS: We conducted a cluster-randomised controlled trial, in schoolchildren, aged 9-10 years, across 40 schools over one year. Schools were randomly assigned either to the "Magic Glasses Philippines" health education intervention package (consisting of a cartoon video, classroom discussions, drawing and essay competition) complementing the standard health education activities of the Philippines Departments of Health and Education, or to a control group, which involved only the standard health education activities. The primary trial outcomes were the proportion of STH infected schoolchildren and their knowledge, attitude and behaviour of STH assessed in both groups at baseline and through two follow-up surveys undertaken immediately prior to the semi-annual national mass administration of albendazole. The outcomes between the study arms were compared using generalized estimating equation models, accounting for clustering at the school level. The trial is registered with Australian New Zealand Clinical Trials Registry number: ACTRN12616000508471. FINDINGS: At follow-up assessments, the mean knowledge and behaviour scores in the intervention group were, respectively, 5·3 (95% confidence interval [CI]:4·2-6·5; p=<0.001) and 1·1 (95% CI: 0·4-1·7; p=0.002) percentage points higher than the control group. There was no overall effect on helminth infections (any STH; adjusted odds ratio [aOR]:1·0; 95% CI: 0·8-1·3; p=0·856), Ascaris lumbricoides; aOR:1·0; 95% CI: 0·7-1·6; p=0·894, or Trichuris trichiura; aOR:1·7; 95% CI: 0·9-1·6; p=0·315) but sub-group analysis showed a 60% reduction in the odds of any STH infection resulting from the "Magic Glasses" intervention in schools with a baseline prevalence ≤15% (aOR: 0·4; 95% CI: 0·2-0·7; p=0·001). INTERPRETATION: The health-education package demonstrated a modest but statistically significant impact on the students' overall STH knowledge and changes in their behaviour but was only effective in preventing STH infections in intervention schools where the baseline prevalence was ≤15%. FUNDING: National Health and Medical Research Council, Australia, and the UBS-Optimus Foundation, Switzerland.

Soil-transmitted helminth infections and nutritional indices among Filipino schoolchildren.

BACKGROUND: Soil-transmitted helminth (STH) infections are still prevalent among schoolchildren in the Philippines. We evaluated the risk factors associated with STH and the relationship between STH and nutritional indices among schoolchildren aged 9-10 years in Laguna province, the Philippines. METHODS: We used the baseline data from 40 schools enrolled in a randomised controlled trial of the Magic Glasses Philippines health education package. Data on demographic and socio-economic variables, and STH related knowledge, attitudes and practices, were obtained through a questionnaire. Stool samples were collected and assessed for STH egg presence using the Kato-Katz technique. Haemoglobin levels and height and weight of study participants were also determined. The generalized estimating equations approach was used to construct logistic regression models to assess STH-associated risk factors, and the association between any STH infection and anaemia, child stunting, wasting and being underweight. The trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12616000508471). FINDINGS: Among 1,689 schoolchildren, the prevalence of any STH was 23%. The prevalence of anaemia, stunting, being underweight and wasting was 13%, 20.2%, 19% and 9.5%, respectively. Age, socio-economic status, rural/urban classification of schools and knowledge of STH were significant risk factors for acquiring a STH infection. Moreover, infections with any STH were significantly associated with stunting (P = <0.001) and being underweight (P = <0.003), but not wasting (P = 0.375) or anaemia (P = 0.462) after controlling for confounding covariates. CONCLUSION: The study findings emphasise the need for sustainable deworming in tandem with other measures such as the provision of health education, improvements in sanitation and hygiene, and nutritional programs in order to control STH infections and improve morbidity outcomes in schoolchildren. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ACTRN12616000508471).

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.

Performance of the point-of-care circulating cathodic antigen test in the diagnosis of schistosomiasis japonica in a human cohort from Northern Samar, the Philippines.

BACKGROUND: Zoonotic schistosomiasis, caused by Schistosoma japonicum, remains a major public health problem in the Philippines. This study aimed to evaluate the commercially available rapid diagnostic point-of-care circulating cathodic antigen (POC-CCA) test in detecting individuals infected with S. japonicum in a human cohort from an endemic area for schistosomiasis japonica in the Philippines. METHODS: Clinical samples were collectedin 18 barangays endemic for S. japonicum infection in Laoang and Palapag municipalities, Northern Samar, the Philippines, in 2015. The presence of CCA in filter-concentrated urine samples (n = 412) was evaluated using the commercial kits and the results were converted to images, which were further analyzed by ImageJ software to calculate R values. The diagnostic performance of the immunochromatographic POC-CCA test was compared using the Kato-Katz (KK) procedure, in-house enzyme-linked immunosorbent assays (ELISAs) and droplet digital (dd) PCR assays as reference. RESULTS: The POC-CCA test was able to detect S. japonicum-infected individuals in the cohort with an eggs per gram of faeces (EPG) more than or equal to 10 with sensitivity/specificity values of 63.3%/93.3%. However, the assay showed an inability to diagnose schistosomiasis japonica infections in all cohort KK-positive individuals, of which the majority had an extremely low egg burden (EPG: 1-9). The prevalence of S. japonicum infection in the total cohort determined by the POC-CCA test was 12.4%, only half of that determined by the KK method (26.2%). When compared with the ELISAs and ddPCR assays as a reference, the POC-CCA assay was further shown to be a test with low sensitivity. Nevertheless, the assay exhibited significant positive correlations with egg burden determined by the KK technique and the target gene copy number index values determined by the ddPCR assays within the entire cohort. CONCLUSIONS: By using in silico image analysis, the POC-CCA cassette test could be converted to a quantitative assay to avoid reader-variability. Because of its low sensitivity, the commercially available POC-CCA assay had limited potential for determining the status of a S. japonicum infection in the target cohort. The assay should be applied with caution in populations where schistosome parasites (especially S. japonicum) are present at low infection intensity.

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.

Molecular identification of Ancylostoma ceylanicum in the Philippines.

Hookworms are some of the most widespread of the soil-transmitted helminths (STH) with an estimated 438.9 million people infected. Until relatively recently Ancylostoma ceylanicum was regarded as a rare cause of hookworm infection in humans, with little public health relevance. However, recent advances in molecular diagnostics have revealed a much higher prevalence of this zoonotic hookworm than previously thought, particularly in Asia. This study examined the prevalence of STH and A. ceylanicum in the municipalities of Palapag and Laoang in the Philippines utilizing real-time polymerase chain reaction (PCR) on stool samples previously collected as part of a cross-sectional survey of schistosomiasis japonica. Prevalence of hookworm in humans was high with 52.8% (n = 228/432) individuals positive for any hookworm, 34.5% (n = 149/432) infected with Necator americanus, and 29.6% (n = 128/432) with Ancylostoma spp; of these, 34 were PCR-positive for A. ceylanicum. Considering dogs, 12 (n = 33) were PCR-positive for A. ceylanicum. This is the first study to utilize molecular diagnostics to identify A. ceylanicum in the Philippines with both humans and dogs infected. Control and elimination of this zoonotic hookworm will require a multifaceted approach including chemotherapy of humans, identification of animal reservoirs, improvements in health infrastructure, and health education to help prevent infection.

Population Pharmacokinetics of Praziquantel in Pregnant and Lactating Filipino Women Infected with Schistosoma japonicum.

An estimated 40 million women of reproductive age are infected with one of three species of the waterborne parasite Schistosoma spp. Treatment with praziquantel (PZQ) via mass drug administration (MDA) campaigns is the mainstay of schistosomiasis control for populations living in areas of endemicity. The World Health Organization recommends that pregnant and lactating women be included in schistosomiasis MDA programs, and several recent studies have evaluated the safety and efficacy of PZQ use during pregnancy. To date, there are no data describing PZQ pharmacokinetics (PK) during pregnancy or among lactating postpartum women. As part of a randomized controlled trial investigating the safety and efficacy of PZQ during human pregnancy, we examined the PK of this therapeutic drug among three distinct cohorts of women infected with S. japonicum in Leyte, Philippines. Specifically, we studied the PK properties of PZQ among early- and late-gestation pregnant women (n = 15 each) and lactating postpartum women (n = 15) with schistosomiasis. We found that women in early pregnancy had increased apparent clearance and lower area-under-the-curve (AUC0-24) values that may be related to physiological changes in drug clearance and/or changes in oral bioavailability. There was no relationship between body weight and apparent clearance. The mean ± standard deviation partition ratio of plasma to breast milk was 0.36. ± 0.13. The estimated median infant PZQ daily dose would be 0.037 mg/kg of body weight ingested from breast milk, which is significantly lower than the dosage required for antischistosomal activity and not known to be harmful to the infant. Our PK data do not support the suggestion to delay breastfeeding 72 h after taking PZQ. Results can help inform future drug efficacy studies in pregnant and lactating women with schistosomiasis.

Determining the Impact of a School-Based Health Education Package for Prevention of Intestinal Worm Infections in the Philippines: Protocol for a Cluster Randomized Intervention Trial.

BACKGROUND: Repeated mass drug administration (MDA) of antihelminthics to at-risk populations is still the main strategy for the control of soil-transmitted helminth (STH) infections. However, MDA, as a stand-alone intervention, does not prevent reinfection. Accordingly, complementary measures to prevent STH reinfection, such as health education and improved sanitation, as part of an integrated control approach, are required to augment the effectiveness of MDA for optimal efficiency and sustainability. OBJECTIVE: The aim of this study is to determine the impact and generalizability of a school-based health education package entitled The Magic Glasses for STH prevention in the Philippines. METHODS: We conducted a cluster randomized controlled intervention trial, involving 2020 schoolchildren aged 9-10 years, in 40 schools in Laguna Province, Philippines, to evaluate the impact of the school-based health education package for the prevention of STHs. The trial was conducted over the course of 1 year (June 2016 to July 2017). A total of 20 schools were randomly assigned to the intervention arm, in which The Magic Glasses Philippines health education package was delivered with the standard health education activities endorsed by the Philippines Department of Health (DOH) and the Department of Education (DepEd). The other 20 schools comprised the control arm of the study, where the DOH/DepEd's standard health education activities were done. At baseline, parasitological assessments and a knowledge, attitude, and practice survey were carried out in all schools. In addition, height, weight, and hemoglobin levels were obtained from each child (after parental consent), and their school attendance and academic performance in English and mathematics were accessed from the school records. The baseline and 2 follow-up surveys were completed using the same study measurements and quality-control assessments. RESULTS: Key results from this cluster randomized intervention trial will shed light on the impact that The Magic Glasses health education package will have against STH infections in schoolchildren in the province of Laguna, located on the Island of Luzon, in the Calabarzon Region of the Philippines. CONCLUSIONS: The results of the trial will be used to assess the generalizability of the impact of The Magic Glasses health education package in different epidemiological and cultural settings, providing evidence for translation of this health education package into public health policy and practice in the Asian region and beyond. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry number ACTRN12616000508471; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=368849. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/18419.

Serum Exosomal miRNAs for Grading Hepatic Fibrosis Due to Schistosomiasis.

Chronic infection with Schistosoma japonicum or Schistosoma mansoni results in hepatic fibrosis of the human host. The staging of fibrosis is crucial for prognosis and to determine the need for treatment of patients with schistosomiasis. This study aimed to determine whether there is a correlation between the levels of serum exosomal micro-ribonucleic acids (miRNAs) (exomiRs) and fibrosis progression in schistosomiasis. Reference gene (RG) validation was initially carried out for the analysis of serum exomiRs expression in staging liver fibrosis caused by schistosome infection. The expression levels of liver fibrosis-associated exomiRs in serum were determined in a murine schistosomiasis model and in a cohort of Filipino schistosomiasis japonica patients (n = 104) with different liver fibrosis grades. Of twelve RG candidates validated, miR-103a-3p and miR-425-5p were determined to be the most stable genes in the murine schistosomiasis model and subjects from the schistosomiasis-endemic area, respectively. The temporal expression profiles of nine fibrosis-associated serum exomiRs, as well as their correlations with the liver pathologies, were determined in C57BL/6 mice during S. japonicum infection. The serum levels of three exomiRs (miR-92a-3p, miR-146a-5p and miR-532-5p) were able to distinguish subjects with fibrosis grades I-III from those with no fibrosis, but only the serum level of exosomal miR-146a-5p showed potential for distinguishing patients with mild (grades 0-I) versus severe fibrosis (grades II-III). The current data imply that serum exomiRs can be a supplementary tool for grading liver fibrosis in hepatosplenic schistosomiasis with moderate accuracy.

Helminths, polyparasitism, and the gut microbiome in the Philippines.

Polyparasitism, involving soil-transmitted helminths. and Schistosoma blood flukes, is common in low to middle income countries. These helminths impact on the gut environment and can cause changes to the gut microbiome composition. Here we examined the gut microbiome in individuals with polyparasitism from two human cohorts in the Philippines utilising DNA sequencing-based profiling. Multiple helminth species infections were high with 70.3% of study participants harbouring at least two parasite species, and 16% harbouring at least five species. Increased numbers of helminth co-infections, in particular with the gut-resident soil-transmitted helminths, were significantly associated with increased bacterial diversity; however no significant parasite-gut microbiome associations were evident for individuals infected only with Schistosoma japonicum. In general, a healthy gut is associated with high bacterial diversity, which in these human cohorts may be the result of helminth-mediated immune modulation, or due to changes in the gut environment caused by these parasitic helminths.

Parasite-derived circulating microRNAs as biomarkers for the detection of human Schistosoma japonicum infection.

Novel tools for early diagnosis and monitoring of schistosomiasis are urgently needed. This study aimed to validate parasite-derived miRNAs as potential novel biomarkers for the detection of human Schistosoma japonicum infection. A total of 21 miRNAs were initially validated by real-time-polymerase chain reaction (RT-PCR) using serum samples of S. japonicum-infected BALB/c mice. Of these, 6 miRNAs were further validated with a human cohort of individuals from a schistosomiasis-endemic area of the Philippines. RT-PCR analysis showed that two parasite-derived miRNAs (sja-miR-2b-5p and sja-miR-2c-5p) could detect infected individuals with low infection intensity with moderate sensitivity/specificity values of 66%/68% and 55%/80%, respectively. Analysis of the combined data for the two parasite miRNAs revealed a specificity of 77.4% and a sensitivity of 60.0% with an area under the curve (AUC) value of 0.6906 (P = 0.0069); however, a duplex RT-PCR targeting both sja-miR-2b-5p and sja-miR-2c-5p did not result in an increased diagnostic performance compared with the singleplex assays. Furthermore, the serum level of sja-miR-2c-5p correlated significantly with faecal egg counts, whereas the other five miRNAs did not. Targeting S. japonicum-derived miRNAs in serum resulted in a moderate diagnostic performance when applied to a low schistosome infection intensity setting.

Milestones of networking and global engagements for the Regional Network on Asian Schistosomiasis and other Helminthic Zoonoses (RNAS+).

This paper introduces the three stages of development of the Regional Network on Asian Schistosomiasis and other Helminthic Zoonoses (RNAS+), namely the preparatory stage, the strengthening stage and the expanding stage. Significant achievements have been made through the help of RNAS+, particularly on research on helminthiases. As scientists, researchers, academics work closer with control authorities, elimination of these diseases is slowly getting nearer and within reach. RNAS+, at present can boast of the following strengths: (i) strong collaboration in the regional research area with support from experts on research and control; (ii) presence of experts in various areas who can improve and promote both research and control; (iii) RNAS+ has been successful in translating research output to field application; (iv) setting up a platform that is capable of advancing the mechanisms of sharing information through its website, databases, publications and meetings; (v) RNAS+ has proved that it is capable of undertaking joint collaborative projects on operational research through multi-country, multi-disease using multi-disciplinary approach and involving different academic and research institutions in the region. Most importantly, experts who are members of RNAS+ are also linked with control programmes of the endemic member countries in the region.