Molecular Analysis of Rabies Virus Using RNA Extracted from Used Lateral Flow Devices.

Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; P < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.

From Perpetual Wetness to Soil Chemistry: Enumerating Environmental and Physicochemical Factors Favoring Oncomelania hupensis quadrasi Snail Presence in the Municipality of Gonzaga, Cagayan, Philippines.

Snail control to complement mass drug administration is being promoted by the World Health Organization for schistosomiasis control. Oncomelania hupensis quadrasi, the snail intermediate host of Schistosoma japonicum in the Philippines, has a very focal distribution; thus, scrutinizing baseline data and parameters affecting this distribution is very crucial. In this study in Gonzaga, Cagayan, Philippines, snail habitats were surveyed, and the various factors affecting the existence of the snails were determined. Malacological surveys and the mapping of sites of perpetual wetness in five endemic and five neighboring non-endemic barangays were conducted. Environmental and physicochemical factors were also examined. Maps of both snail and non-snail sites were generated. Of the fifty sites surveyed, O. h. quadrasi were found in twelve sites, and two sites yielded snails that were infected with S. japonicum cercariae. Factors such as silty loam soil, proximity to a snail site, water ammonia, and soil attributes (organic matter, iron, and pH) are all significantly associated with the presence of snails. In contrast, types of habitats, temperatures, and soil aggregation have no established association with the existence of snails. Mapping snail sites and determining factors favoring snail presence are vital to eliminating snails. These approaches will significantly maximize control impact and minimize wasted efforts and resources, especially in resource-limited schistosomiasis endemic areas.

Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis.

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3-98.8%) and 100% (95% CI, 92.0-100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.

Immunohistochemical detection of virus antigen in the nasal planum of rabid dogs.

The rabies virus is one of the most neurotropic of all viruses infecting mammals. During the terminal phases of infection, the virus spreads to peripheral tissues, including the skin. The external skin of the nose, called the nasal planum, is a sensory organ where numerous nerve bundles and terminal nerves are distributed. Therefore, the nasal planum is expected to serve as a postmortem diagnostic material. However, the distribution of rabies virus antigens in the nasal planum in rabid animals has not yet been studied. In this study, the nasal planum was obtained from 45 rabid dogs. In all rabid dogs, the viral antigen was detected in the peripheral nerve tissues, Merkel cells, and squamous cells. The viral antigen in the epidermis exhibited three patterns: first, a diffuse positive pattern from the basal layer to the squamous layer; second, a reticular positive pattern along the cell membrane in the squamous layer; and third, a basal layer pattern of the epidermis. In the dermis, viral antigens were detected more often in lamellated corpuscles just beneath the rete pegs. These results suggest that the nasal planum could serve as a useful alternative source for postmortem diagnosis in rabies endemic countries.

Validation of serum apolipoprotein A1 in rabies virus-infected mice as a biomarker for the preclinical diagnosis of rabies.

Rabies is a type of acute fetal encephalitis caused by rabies virus (RABV). While it becomes incurable after symptom onset, it can be prevented by post-exposure prophylaxis (PEP) during the long incubation period. While preclinical diagnosis aids the appropriate PEP administration, it is mostly nonfeasible owing to the absence of viremia or a specific antibody response during the incubation period. Here, an attempt was made to identify a serum biomarker for the preclinical diagnosis of rabies. Using the serum from a mouse inoculated intramuscularly (i.m.) with 5 × 105 focus-forming units (FFU) of recombinant RABV expressing red firefly luciferase (1088/RFLuc) immediately before symptom onset, two-dimensional differential gel electrophoresis was conducted, followed by mass spectrometry, and it was confirmed that apolipoprotein A1 (ApoA1) was up-regulated. ELISA showed that the serum ApoA1 and specific antibody levels increased during the incubation period and on the day of symptom onset. Since a lower infectious dose can be used to induce the unstable and long incubation period generally observed in natural infection, the ApoA1 level in mice inoculated i.m. with 103 FFU of 1088/RFLuc was examined by monitoring viral dynamics using in vivo imaging. The serum ApoA1 and specific antibody levels were up-regulated in 50% and 58.3% of mice exhibiting robust RABV replication, respectively, but not in mice exhibiting weak RABV replication. In addition, it was reported that ApoA1 was found to be a biomarker for neuronal damage. Additional biomarker candidates will be needed for the effective preclinical diagnosis of rabies.

Background and descriptive features of rabies-suspected animals in Central Luzon, Philippines.

BACKGROUND: The Philippines is one of the major endemic countries for canine rabies in Southeast Asia. However, detailed description and analysis of laboratory-confirmed animal rabies are limited. Highly accurate surveillance requires a thorough understanding of the target area-specific problems and obstacles. Therefore, we aim to describe and analyze the rabies suspect animals in Central Luzon, Philippines, to clarify the characteristics of management and clinical signs by conducting interviews with the owners. METHODS: We prospectively collected information on the rabies suspect animals submitted to the Regional animal laboratory in Central Luzon through passive laboratory-based rabies surveillance between 1st April 2019 and 30th September 2020. We performed active interviews directly or telephonically with the owner. The direct fluorescent antibody test was performed on the hippocampus, brain stem, and cerebellum for laboratory confirmation. Descriptive statistics were used to characterize the number of rabies cases according to management methods and characteristics of suspected animals during the observation period. Clinical symptoms of suspected rabid animals were analyzed by univariate logistic regression analysis. RESULTS: There were 292 sample submissions during the study period. Of these, 160 were positive for dFAT. Samples of pet animals (85.3%) provided by owners or their acquaintances (59.2%) accounted for the majority of laboratory confirmed cases. Case mapping showed that more rabies-suspected cases were sent from areas near the regional laboratory than from those far from the laboratory, despite the incidence of rabies being high in these areas. The management and clinical symptoms of 227 animal cases showed that most owners were managing their animals at home and were allowing them to roam outside (69.6%) and be unvaccinated (78.9%). Rabid animals were more likely to manifest aimless running, restlessness, and agitation. CONCLUSIONS: Our study provided some features of animals with laboratory-confirmed rabies in Central Luzon. However, most of the samples were submitted from areas near the rabies diagnosis laboratory, and the number of samples submitted from remote areas was low. To improve the surveillance capacity, it is necessary to increase sample submissions from remote areas.

Evaluation of the diagnostic accuracy of lateral flow devices as a tool to diagnose rabies in post-mortem animals.

Implementation of lateral flow devices (LFDs) for rabies antigen detection is expected to improve surveillance through the efficient detection of rabid animals in resource-limited settings; however, the use of LFDs for diagnosis remains controversial because some commercially available kits show low sensitivity. Therefore, we compared the diagnostic efficacy of three LFDs (ADTEC, Bionote, and Elabscience kits) paralleled with the direct fluorescent antibody test (dFAT) using fresh samples and investigated the diagnostic accuracies. To do so, we evaluated rabies-suspected samples submitted to the Regional Animal Disease Diagnostic Laboratory III, Philippines. Furthermore, we conducted real-time RT-PCR and sequencing to measure the accuracy of field laboratory diagnosis. The total number of animals submitted during this study period was 184 cases, including negative control samples. Of these, 53.9% (84 cases) were positive in the dFAT. Dogs were the most common rabies-suspected animal (n = 135). The sensitivities of the ADTEC and Bionote kits were 0.88 (74 cases) and 0.95 (80 cases), respectively. The specificity of both kits was 1.00 (100 cases). Furthermore, the sensitivity and specificity of the ADTEC kit after directly homogenizing the samples in assay buffer without dilution in phosphate-buffered saline (ADTEC kit DM) were 0.94 (79 cases) and 1.00 (100 cases), respectively. By contrast, there were no positive results using the Elabscience kit among all dFAT-positive samples. The sensitivity and specificity of LFDs make these tests highly feasible if properly used. Therefore, LFD tests can be used to strengthen the surveillance of rabies-infected animals in endemic and resource-limited settings.

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.

Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

Localization of the rabies virus antigen in Merkel cells in the follicle-sinus complexes of muzzle skins of rabid dogs.

The direct fluorescent antibody test (dFAT) on fresh brain tissues is the gold standard for rabies virus antigen detection in dogs. However, this method is laborious and holds a high risk of virus exposure for the experimenter. Skin biopsies are useful for the diagnosis of humans and animals. In mammals, the tactile hair, known as the follicle-sinus complex (FSC), is a specialized touch organ that is abundant in the muzzle skin. Each tactile hair is equipped with more than 2,000 sensory nerve endings. Therefore, this organ is expected to serve as an alternative postmortem diagnostic material. However, the target cells and localization of rabies virus antigen in the FSCs remain to be defined. In the present study, muzzle skins were obtained from 60 rabid dogs diagnosed with rabies by dFAT at the Research Institute of Tropical Medicine in the Philippines. In all dogs, virus antigen was clearly detected in a part of the outer root sheath at the level of the ring sinus of the FSCs, and the majority of cells were positive for the Merkel cell (MC) markers cytokeratin 20 and CAM5.2. Our results suggest that MCs in the FSCs of the muzzle skin are a target for virus replication and could serve as a useful alternative specimen source for diagnosis of rabies.

Molecular and mathematical modeling analyses of inter-island transmission of rabies into a previously rabies-free island in the Philippines.

Rabies is endemic in the Philippines and dog bites are a major cause of rabies cases in humans. The rabies control program has not been successful in eliminating rabies because of low vaccination coverage among dogs. Therefore, more effective and feasible strategies for rabies control are urgently required in the country. To control rabies, it is very important to know if inter-island transmission can occur because rabies can become endemic once the virus is introduced in areas that previously had no reported cases. Our molecular epidemiological study suggests that inter-island transmission events can occur; therefore, we further investigated these inter-island transmission using phylogenetic and modeling approaches. We investigate inter-island transmission between Luzon and Tablas Islands in the Philippines. Phylogenetic analysis and mathematical modeling demonstrate that there was a time lag of several months to a year from rabies introduction to initial case detection, indicating the difficulties in recognizing the initial rabies introductory event. There had been no rabies cases reported in Tablas Island; however, transmission chain was sustained on this island after the introduction of rabies virus because of low vaccination coverage among dogs. Across the islands, a rabies control program should include control of inter-island dog transportation and rabies vaccination to avoid viral introduction from the outside and to break transmission chains after viral introduction. However, this program has not yet been completely implemented and transmission chains following inter-island virus transmission are still observed. Local government units try to control dog transport; however, it should be more strictly controlled, and a continuous rabies control program should be implemented to prevent rabies spread even in rabies-free areas.

Phylogeographic analysis of rabies viruses in the Philippines.

Rabies still remains a public health threat in the Philippines. A significant number of human rabies cases, about 200-300 cases annually, have been reported, and the country needs an effective strategy for rabies control. To develop an effective control strategy, it is important to understand the transmission patterns of the rabies viruses. We conducted phylogenetic analyses by considering the temporal and spatial evolution of rabies viruses to reveal the transmission dynamics in the Philippines. After evaluating the molecular clock and phylogeographic analysis, we estimated that the Philippine strains were introduced from China around the beginning of 20th century. Upon this introduction, the rabies viruses evolved within the Philippines to form three major clades, and there was no indication of introduction of other rabies viruses from any other country. However, within the Philippines, island-to-island migrations were observed. Since then, the rabies viruses have diffused and only evolved within each island group. The evolutionary pattern of these viruses was strongly shaped by geographical boundaries. The association index statistics demonstrated a strong spatial structure within the island group, indicating that the seas were a significant geographical barrier for viral dispersal. Strong spatial structure was also observed even at a regional level, and most of the viral migrations (79.7% of the total median number) in Luzon were observed between neighboring regions. Rabies viruses were genetically clustered at a regional level, and this strong spatial structure suggests a geographical clustering of transmission chains and the potential effectiveness of rabies control that targets geographical clustering. Dog vaccination campaigns have been conducted independently by local governments in the Philippines, but it could be more effective to implement a coordinated vaccination campaign among neighboring areas to eliminate geographically-clustered rabies transmission chains.

Genetic diversity and geographic distribution of genetically distinct rabies viruses in the Philippines.

BACKGROUND: Rabies continues to be a major public health problem in the Philippines, where 200-300 human cases were reported annually between 2001 and 2011. Understanding the phylogeography of rabies viruses is important for establishing a more effective and feasible control strategy. METHODS: We performed a molecular analysis of rabies viruses in the Philippines using rabied animal brain samples. The samples were collected from 11 of 17 regions, which covered three island groups (Luzon, Visayas, and Mindanao). Partial nucleoprotein (N) gene sequencing was performed on 57 samples and complete glycoprotein (G) gene sequencing was performed on 235 samples collected between 2004 and 2010. RESULTS: The Philippine strains of rabies viruses were included in a distinct phylogenetic cluster, previously named Asian 2b, which appeared to have diverged from the Chinese strain named Asian 2a. The Philippine strains were further divided into three major clades, which were found exclusively in different island groups: clades L, V, and M in Luzon, Visayas, and Mindanao, respectively. Clade L was subdivided into nine subclades (L1-L9) and clade V was subdivided into two subclades (V1 and V2). With a few exceptions, most strains in each subclade were distributed in specific geographic areas. There were also four strains that were divided into two genogroups but were not classified into any of the three major clades, and all four strains were found in the island group of Luzon. CONCLUSION: We detected three major clades and two distinct genogroups of rabies viruses in the Philippines. Our data suggest that viruses of each clade and subclade evolved independently in each area without frequent introduction into other areas. An important implication of these data is that geographically targeted dog vaccination using the island group approach may effectively control rabies in the Philippines.

Seroprevalence of cysticercosis in children and young adults living in a helminth endemic community in leyte, the Philippines.

Cysticercosis is a significant public health problem in countries where pigs are raised for consumption and remains an important cause of neurological disease worldwide. The Philippines is considered an endemic area for cysticercosis because cases in both humans and pigs have been reported; however, epidemiologic information stays limited. We conducted a pilot survey of the seroprevalence of human cysticercosis in a village in Leyte, the Philippines, by measuring antibody specific for Taenia solium cyst-fluid antigen. There were 497 subjects aged 7-30 years in our study and most subjects were infected with one or more helminths. The overall cysticercosis seroprevalence in this population was 24.6% (95% CI: 20.82% ~ 28.58%) with no significant difference based on age, sex, or other helminth coinfection status. Although the sample may not be representative of the whole community, the findings suggest that cysticercosis is a significant, but underrecognized public health concern in the Philippines.

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

OBJECTIVE: To estimate the degree of synergism between helminth species in their combined effects on anemia. METHODS: Quantitative egg counts using the Kato-Katz method were determined for Ascaris lumbricoides, hookworm, Trichuris trichiura, and Schistosoma japonicum in 507 school-age children from helminth-endemic villages in The Philippines. Infection intensity was defined in three categories: uninfected, low, or moderate/high (M+). Anemia was defined as hemoglobin <11 g/dL. Logistic regression models were used to estimate odds ratios (OR), 95% confidence intervals (CI), and synergy index for pairs of concurrent infections. RESULTS: M+ co-infection of hookworm and S. japonicum (OR = 13.2, 95% CI: 3.82-45.5) and of hookworm and T. trichiura (OR = 5.34, 95% CI: 1.76-16.2) were associated with higher odds of anemia relative to children without respective M+ co-infections. For co-infections of hookworm and S. japonicum and of T. trichiura and hookworm, the estimated indices of synergy were 2.9 (95% CI: 1.1-4.6) and 1.4 (95% CI: 0.9-2.0), respectively. CONCLUSION: Co-infections of hookworm and either S. japonicum or T. trichiura were associated with higher levels of anemia than would be expected if the effects of these species had only independent effects on anemia. This suggests that integrated anti-helminthic treatment programs with simultaneous deworming for S. japonicum and some geohelminths could yield a greater than additive benefit for reducing anemia in helminth-endemic regions.

Pubertal development predicts resistance to infection and reinfection with Schistosoma japonicum.

BACKGROUND: In communities where Schistosoma species are endemic, the prevalence and intensity of schistosomiasis is disproportionately high among children, compared with adults. This epidemiologic pattern is consistent with either the slow development of resistance or the requirement of host developmental changes for the expression of resistance. METHODS: We enrolled 87 individuals aged 7-18 years who did not have Schistosoma japonicum infection and 641 individuals aged 7-30 years with S. japonicum infection, all of whom reside in 3 villages in Leyte, Philippines. At baseline, S. japonicum infection was assessed by Kato-Katz thick-smear stool examination, and the levels of the pubertal hormone dehydroepiandrosterone sulfate (DHEA-S) in serum were determined. Individuals with S. japonicum infection were treated with praziquantel, after which stool examination and DHEA-S level measurement were performed every 3 months for 18 months. RESULTS: In cross-sectional analyses, the intensity of infection among individuals with high DHEA-S levels was 43% lower (28 eggs per g, n = 243), compared with individuals with low DHEA-S levels (50 eggs per g, n = 242), even after adjusting for age, sex, and village (P = .01). Following praziquantel treatment, increased DHEA-S levels were associated with resistance to reinfection (P = .006). The intensity of reinfection among individuals with high DHEA-S levels was 42% lower, compared with individuals with low DHEA-S levels, even after adjusting for age, baseline intensity of S. japonicum infection, village, sex and water contact (P < .001). CONCLUSIONS: Increased DHEA-S levels in serum, a marker for adrenal development, is associated with reduced S. japonicum infection and reinfection, even after adjusting for age and, by proxy, cumulative exposure. These data suggest that an intrinsic property of host pubertal development mediates, in part, the resistance to infection observed in older individuals.

Schistosomiasis japonica, anemia, and iron status in children, adolescents, and young adults in Leyte, Philippines 1.

BACKGROUND: Observational and interventional evidence supports a relation between human schistosomiasis and anemia; however, the exact causal mechanisms remain unclear. Eggs translocating across the intestinal or bladder wall may result in extracorporeal blood loss with subsequent iron deficiency. Alternatively, anemia may result from cytokine-mediated dyserythropoiesis, as seen in anemia of inflammation. OBJECTIVES: By evaluating the cross-sectional relation between the intensity of Schistosoma japonicum infection, hemoglobin concentration, and iron status in 7-30-y-old persons from S. japonicum-endemic rice-farming villages in the province of Leyte, Philippines, we assessed the relative contribution of iron deficiency and anemia of inflammation to schistosomiasis-associated anemia. DESIGN: We enrolled 627 S. japonicum-infected and 111 S. japonicum-uninfected persons. We obtained stool samples to quantify S. japonicum infection and venous blood samples for hemograms and measures of iron status and inflammation. RESULTS: Intensity of S. japonicum infection was independently associated with hemoglobin (beta = -0.24; 95% CI: -0.31, -0.17). Persons with high-intensity infection had a greater risk of iron deficiency anemia (adjusted prevalence odds ratio: 6.6; 95% CI: 2.9, 14.7), but there was no evidence of this relation in low-intensity infections. In contrast, anemia without iron deficiency was prevalent across all intensities (adjusted prevalence odds ratio: 3.8; 95% CI: 1.5, 9.5). CONCLUSIONS: Storage iron deficiency is a major contributor to anemia in high-intensity S. japonicum infection. A high prevalence of anemia without iron deficiency, exclusion of other mechanisms of anemia, and the evidence of low bioavailable iron suggest that anemia of inflammation contributes to S. japonicum-associated anemia at all infection intensities.

T-helper-2 cytokine responses to Sj97 predict resistance to reinfection with Schistosoma japonicum.

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. Using a longitudinal treatment-reinfection study design with 616 participants 7 to 30 years of age, we evaluated the relationship between cytokine responses to Schistosoma japonicum soluble adult worm extract (SWAP), Sj97, Sj22.6, and Sj67, measured 4 weeks after treatment with Praziquantel, and resistance to reinfection in a population from Leyte, The Philippines, where S. japonicum is endemic. S. japonicum transmission was high: 54.8% and 91.1% were reinfected within 6 and 18 months, respectively. A Th2 bias in the following cytokine ratios, interleukin-4 (IL-4)/IL-12, IL-5/IL-12, IL-13/IL-12, IL-4/gamma-IFN (IFN-gamma), IL-5/IFN-gamma, and IL-13/IFN-gamma, in response to SWAP predicted a 1.4- to 2.9-month longer time to reinfection (P < 0.05) and a 27 to 55% lower intensity of reinfection (P < 0.05). Similarly, a Th2 bias in response to Sj97 predicted a 1.6- to 2.2-month longer time to reinfection (P < 0.05) and a 30 to 41% lower intensity of reinfection (P < 0.05). Only a high IL-5/IL-10 ratio in response to Sj22.6 predicted a 3.0-month-longer time to reinfection (P = 0.03). Cytokine responses to Sj67 were not associated with protection. In a large population-based treatment-reinfection study we found that Th2 responses to SWAP and Sj97 consistently predicted resistance to reinfection. These findings underscore Th2-type immune responses as central in human resistance to S. japonicum and support Sj97 as a leading vaccine candidate for this parasite.

Nutritional status improves after treatment of schistosoma japonicum-infected children and adolescents.

Studies addressing the relation between chronic schistosomiasis japonica and nutritional status are limited. We conducted a longitudinal treatment-reinfection study in Leyte, the Philippines, among 477 Schistosoma japonicum-infected subjects aged 7-20 y, to evaluate changes in nutritional status after treatment with praziquantel. Stool, Tanner stage, anthropometric indices, and hemoglobin (Hb) were evaluated at baseline, 4 wk post-treatment, and every 3 mo for 18 mo. Height-for-age Z-score (HAZ) and BMI Z-score (BMIZ) were calculated. Change scores relative to baseline were created for all outcome measures. Multilevel repeated-measures analyses were used to adjust for socioeconomic status, sex, either pubertal status or age, and household-level clustering. Z-scores improved modestly but significantly over time. BMIZ in children wasted at baseline improved the most [0.41 (0.26-0.56) Z-score unit] and HAZ improved only in children stunted at baseline [0.17 (0.l2-0.21) Z-score unit]. Hb improvement peaked at 15 mo and occurred only in subjects that were anemic at baseline [peak improvement: 8.3 (6.0-10.6) g/L] and in males [peak improvement 4.7 (2.9-6.6) g/L]. Reinfection, evaluated as egg count over time and egg count 3 mo earlier to assess a delay in effect, was inversely associated with improvement in Hb (P = 0.06 and 0.004, respectively). High-intensity reinfection at 18 mo was associated with significantly less absolute growth from baseline compared with lower-intensity and no reinfection. Based on the peak Hb improvement at 15 mo post-treatment, annual treatment intervals are recommended to reduce schistosomiasis-associated nutritional morbidity.

Nutritional status and serum cytokine profiles in children, adolescents, and young adults with Schistosoma japonicum-associated hepatic fibrosis, in Leyte, Philippines.

In a cross-sectional study of 641 Schistosoma japonicum-infected individuals in Leyte, Philippines, who were 7-30 years old, we determined the grade of hepatic fibrosis (HF) by ultrasound and used anthropometric measurements and hemoglobin levels to assess nutritional status. Serum levels of interleukin (IL)-1, IL-6, and IL-10; tumor-necrosis factor (TNF)-alpha; soluble TNF- alpha receptor I; and C-reactive protein (CRP) were measured to examine the association between these markers of inflammation and HF grade. HF was present in 8.9% of the cohort; the majority of cases were mild (grade I), and severe (grade II or grade III) cases occurred only in male individuals. Compared with individuals without HF, those with severe HF--and, to a lesser degree, those with mild HF--had a significantly lower body-mass index (BMI) and BMI z-score, a higher prevalence of anemia, and a higher level of CRP and were more likely to produce IL-6; furthermore, those with severe HF had a significantly higher level of IL-1, compared with those either without HF or with mild HF. These findings suggest that even mild HF is associated with nutritional morbidity and underscore the importance of early recognition and treatment. In addition, our data are consistent with the hypothesis that, by systemically increasing the levels of the proinflammatory cytokines IL-1 and IL-6, HF causes undernutrition and anemia.