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BackgroundSoil-transmitted helminth (STH) control programs currently lack evidence-based recommendations for cost-efficient survey designs for monitoring and evaluation. Here, we present a framework to provide evidence-based recommendations, using a case study of therapeutic drug efficacy monitoring based on the examination of helminth eggs in stool.MethodsWe performed an in-depth analysis of the operational costs to process one stool sample for three diagnostic methods (Kato-Katz, Mini-FLOTAC and FECPAKG2). Next, we performed simulations to determine the probability of detecting a truly reduced therapeutic efficacy for different scenarios of STH species (Ascaris lumbricoides, Trichuris trichiura and hookworms), pre-treatment infection levels, survey design (screen and select (SS); screen, select and retest (SSR) and no selection (NS)) and number of subjects enrolled (100-5,000). Finally, we integrated the outcome of the cost assessment into the simulation study to estimate the total survey costs and determined the most cost-efficient survey design.Principal findingsKato-Katz allowed for both the highest sample throughput and the lowest cost per test, while FECPAKG2 required both the most laboratory time and was the most expensive. Counting of eggs accounted for 23% (FECPAKG2) or ≥80% (Kato-Katz and Mini-FLOTAC) of the total time-to-result. NS survey designs in combination with Kato-Katz were the most cost-efficient to assess therapeutic drug efficacy in all scenarios of STH species and endemicity.Conclusions/significanceWe confirm that Kato-Katz is the fecal egg counting method of choice for monitoring therapeutic drug efficacy, but that the survey design currently recommended by WHO (SS) should be updated. Our generic framework, which captures laboratory time and material costs, can be used to further support cost-efficient choices for other important surveys informing STH control programs. In addition, it can be used to explore the value of alternative diagnostic techniques, like automated egg counting, which may further reduce operational costs.Trial registrationClinicalTrials.gov NCT03465488.

BackgroundStudies have implicated schistosomiasis as a cause of intestinal barrier disruption, a salient feature of environmental enteric dysfunction (EED), as eggs translocate from the sterile bloodstream through the gut wall. We examined the longitudinal impact of praziquantel (PZQ) treatment on a) EED biomarkers and b) Insulin growth factor I (IGF-1), a key driver of childhood linear growth, since EED has been implicated in linear growth stunting.Methodology290 children infected with S. mansoni in Brazil were treated with PZQ at baseline. EED biomarkers lipopolysaccharide (LPS) and intestinal fatty acid binding-protein (I-FABP) were measured, as well as IGF-1 at baseline, 6 and 12-months. Multivariate regression analysis was applied to assess associations between S. mansoni intensity and plasma biomarkers (LPS, I-FABP, and IGF-1), controlling for potential confounding variables.Principal findingsAt baseline, S. mansoni infection intensities were 27.2% light, 46.9% moderate, and 25.9% heavy. LPS concentrations were significantly reduced at the 12-month visit compared to baseline (p = 0.0002). No longitudinal changes were observed for I-FABP or IGF-1 in the 6- or 12-month periods following baseline treatment. After 6-months, I-FABP concentration was significantly higher in high vs low intensity (p = 0.0017). IGF-1 concentrations were significantly lower among children with high and moderate vs low intensity infections at each study visit.Conclusions/significanceWe report that S. mansoni infection impacts LPS, I-FABP and IGF-1. These findings suggest a mechanistic role for EED in schistosomiasis-related morbidities, particularly linear growth.

BackgroundAlbendazole (ALB) is administered annually to millions of children through global deworming programs targeting soil-transmitted helminths (STHs: Ascaris lumbricoides, Trichuris trichiura and hookworms, Necator americanus and Ancylostoma duodenale). However, due to the lack of large individual patient datasets collected using standardized protocols and the application of population-based statistical methods, little is known about factors that may affect individual responses to treatment.Methodology/principal findingsWe re-analyzed 645 individual patient data from three standardized clinical trials designed to assess the efficacy of a single 400 mg oral dose of ALB against STHs in schoolchildren from different study sites, each with varying history of drug pressure based on duration of mass drug administration programs: Ethiopia, low; Lao People's Democratic Republic (PDR), moderate; Pemba Island (Tanzania), high. Using a Bayesian statistical modelling approach to estimate individual responses (individual egg reduction rates, ERRi), we found that efficacy was lower in Pemba Island, particularly for T. trichiura. For this STH, the proportion of participants with a satisfactory response (ERRi ≥50%), was 65% in Ethiopia, 61% in Lao PDR but only 29% in Pemba Island. There was a significant correlation between ERRi and infection intensity prior to drug administration (ERRi decreasing as a function of increasing infection intensity). Individual age and sex also affected the drug response, but these were of negligible clinical significance and not consistent across STHs and study sites.Conclusions/significanceWe found decreased efficacy of ALB against all the STHs analyzed in Pemba Island (Tanzania), an area with high drug pressure. This does not indicate causality, as this association may also be partially explained by differences in infection intensity prior to drug administration. Notwithstanding, our results indicate that without alternative treatment regimens, program targets will not be achievable on Pemba Island because of inadequate efficacy of ALB.Trial registrationThe study was registered on Clinicaltrials.gov (ID: NCT03465488) on March 7, 2018.

BackgroundEfforts to control soil-transmitted helminth (STH) infections have intensified over the past decade. Field-survey data on STH prevalence, infection intensity and drug efficacy is necessary to guide the implementation of control programs and should be of the best possible quality.MethodologyDuring four clinical trials designed to evaluate the efficacy of albendazole against STHs in Brazil, Ethiopia, Lao PDR and Tanzania, quality control (QC) was performed on the duplicate Kato-Katz thick smears and the data entry. We analyzed datasets following QC on both fecal egg counts (FECs) and data entry, and compared the prevalence of any STH infection and moderate-to-heavy intensity (MHI) infections and the drug efficacy against STH infections.ResultsAcross the four study sites, a total of 450 out of 4,830 (9.3%) Kato-Katz thick smears were re-examined. Discrepancies in FECs varied from ~3% (hookworms) to ~6.5% (Ascaris lumbricoides and Trichuris trichiura). The difference in STH prevalence and prevalence of MHI infections using the datasets with and without QC of the FECs did not exceed 0.3%, except for hookworm infections in Tanzania, where we noted a 2.2 percentage point increase in MHI infections (pre-QC: 1.6% vs. post-QC: 3.8%). There was a 100% agreement in the classification of drug efficacy of albendazole against STH between the two datasets. In total, 201 of the 28,980 (0.65%) data entries that were made to digitize the FECs were different between both data-entry clerks. Nevertheless, the overall prevalence of STH, the prevalence of MHI infections and the classification of drug efficacy remained largely unaffected.Conclusion/significanceIn these trials, where staff was informed that QC would take place, minimal changes in study outcomes were reported following QC on FECs or data entry. Nevertheless, imposing QC did reduce the number of errors. Therefore, application of QC together with proper training of the personnel and the availability of clear standard operating procedures is expected to support higher data quality.

BackgroundIn view of the potential immunosuppressive and regenerative properties of mesenchymal stem cells (MSC), we investigated whether transplantation of adipose tissue-derived stem cells (ASC) could be used to control the granulomatous reaction in the liver of mice infected with Schistosoma mansoni after Praziquantel (PZQ) treatment.Methodology/prinicpal findingsC57BL/6 mice infected with S. mansoni were treated with PZQ and transplanted intravenously with ASC from uninfected mice. Liver morpho-physiological and immunological analyses were performed. The combined PZQ/ASC therapy significantly reduced the volume of hepatic granulomas, as well as liver damage as measured by ALT levels. We also observed that ASC accelerated the progression of the granulomatous inflammation to the advanced/curative phase. The faster healing interfered with the expression of CD28 and CTLA-4 molecules in CD4+ T lymphocytes, and the levels of IL-10 and IL-17 cytokines, mainly in the livers of PZQ/ASC-treated mice.ConclusionsOur results show that ASC therapy after PZQ treatment results in smaller granulomas with little tissue damage, suggesting the potential of ASC for the development of novel therapeutic approaches to minimize hepatic lesions as well as a granulomatous reaction following S. mansoni infection. Further studies using the chronic model of schistosomiasis are required to corroborate the therapeutic use of ASC for schistosomiasis.

More than 40 agencies that fund health research capacity strengthening in low- and middle-income countries (LMICs) participate in the ESSENCE Health Research initiative, which has established a mechanism for reviewing and coordinating their funding. Taken together, the expected outcomes of implementation of the review mechanism are increases in the efficiency and equity in health research capacity strengthening activities with decreased duplication of efforts. The overall goal is increased support of research on national health priorities as well as improved pandemic preparedness in LMICs, and, eventually, fewer countries with very limited research capacity.

BackgroundPreventive chemotherapy (PC) with benzimidazole drugs is the backbone of soil-transmitted helminth (STH) control programs. Over the past decade, drug coverage has increased and with it, the possibility of developing anthelmintic resistance. It is therefore of utmost importance to monitor drug efficacy. Currently, a variety of novel diagnostic methods are available, but it remains unclear whether they can be used to monitor drug efficacy. In this study, we compared the efficacy of albendazole (ALB) measured by different diagnostic methods in a head-to-head comparison to the recommended single Kato-Katz.MethodsAn ALB efficacy trial was performed in 3 different STH-endemic countries (Ethiopia, Lao PDR and Tanzania), each with a different PC-history. During these trials, stool samples were evaluated with Kato-Katz (single and duplicate), Mini-FLOTAC, FECPAKG2, and qPCR. The reduction rate in mean eggs per gram of stool (ERR) and mean genome equivalents / ml of DNA extract (GERR) were calculated to estimate drug efficacy.Principal findings and conclusionsThe results of the efficacy trials showed that none of the evaluated diagnostic methods could provide reduction rates that were equivalent to a single Kato-Katz for all STH. However, despite differences in clinical sensitivity and egg counts, they agreed in classifying efficacy according to World Health Organization (WHO) guidelines. This demonstrates that diagnostic methods for assessing drug efficacy should be validated with their intended-use in mind and that other factors like user-friendliness and costs will likely be important factors in driving the choice of diagnostics. In addition, ALB efficacy against STH infections was lower in sites with a longer history of PC. Yet, further research is needed to identify factors that contribute to this finding and to verify whether reduced efficacy can be associated with mutations in the β-tubulin gene that have previously been linked to anthelmintic resistance.Trial registrationClinicalTrials.gov NCT03465488.

BackgroundBecause the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs).MethodologyWe compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAKG2) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs.Principal findingsAll diagnostic methods showed a clinical sensitivity of ≥90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAKG2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration.Conclusions/significanceOur results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAKG2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program.Trial registrationClinicalTrials.gov NCT03465488.

BACKGROUND:To work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs. METHODS:A series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAKG2) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the β-tubulin gene. These SNPs are known to be linked to AR in animal STHs. DISCUSSION:The results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the β-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs. TRIAL REGISTRATION:The trial was retrospectively registered the 7th of March 2018 on Clinicaltrials.gov (ID: NCT03465488).

BACKGROUND: New interventions tools are a priority for schistosomiasis control and elimination, as the disease is still highly prevalent. The identification of proteins associated with active infection and protective immune response may constitute the basis for the development of a successful vaccine and could also indicate new diagnostic candidates. In this context, post-genomic technologies have been progressing, resulting in a more rational discovery of new biomarkers of resistance and antigens for diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: Two-dimensional electrophoresed Schistosoma mansoni adult worm protein extracts were probed with pooled sera of infected and non-infected (naturally resistant) individuals from a S. mansoni endemic area. A total of 47 different immunoreactive proteins were identified by mass spectrometry. Although the different pooled sera shared most of the immunoreactive protein spots, nine protein spots reacted exclusively with the serum pool of infected individuals, which correspond to annexin, major egg antigen, troponin T, filamin, disulphide-isomerase ER-60 precursor, actin and reticulocalbin. One protein spot, corresponding to eukaryotic translation elongation factor, reacted exclusively with the pooled sera of non-infected individuals living in the endemic area. Western blotting of two selected recombinant proteins, major egg antigen and hemoglobinase, showed a similar recognition pattern of that of the native protein. CONCLUDING/SIGNIFICANCE: Using a serological proteome analysis, a group of antigens related to the different infection status of the endemic area residents was identified and may be related to susceptibility or resistance to infection.

BACKGROUND: The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL). METHODOLOGY/PRINCIPAL FINDINGS: A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression. CONCLUSIONS/SIGNIFICANCE: These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.

BackgroundHookworms survive for several years (5 to 7 years) in the host lumen, inducing a robust but largely ineffective immune response. Among the most striking aspects of the immune response to hookworm (as with many other helminths) is the ablation of parasite-specific T cell proliferative response (hyporesponsiveness). While the role of the adaptive immune response in human helminth infection has been well investigated, the role of the innate immune responses (e.g., dendritic cells and eosinophils) has received less attention and remains to be clearly elucidated.Methodology/principal findingsWe report on the differentiation/maturation of host dendritic cells in vitro and the eosinophil activation/function associated with human hookworm infection. Mature DCs (mDCs) from Necator americanus (Necator)-infected individuals showed an impaired differentiation process compared to the mDCs of non-infected individuals, as evidenced by the differential expression of CD11c and CD14. These same hookworm-infected individuals also presented significantly down-regulated expression of CD86, CD1a, HLA-ABC, and HLA-DR. The lower expression of co-stimulatory and antigen presentation molecules by hookworm-infected-derived mDCs was further evidenced by their reduced ability to induce cell proliferation. We also showed that this alternative DC differentiation is partially induced by excreted-secreted hookworm products. Conversely, eosinophils from the same individuals showed a highly activated status, with an upregulation of major cell surface markers. Antigen-pulsed eosinophils from N. americanus-infected individuals induced significant cell proliferation of autologous PBMCs, when compared to non-infected individuals.ConclusionChronic N. americanus infection alters the host's innate immune response, resulting in a possible modulation of the maturation process of DCs, a functional change that may diminish their ability for antigen presentation and thus contribute to the ablation of the parasite-specific T cell proliferative response. Interestingly, a concomitant upregulation of the major cell surface markers of eosinophils was observed in hookworm-infected individuals, indicative of antigen-specific immune responses, especially antigen presentation. We showed that in addition to the postulated role of the eosinophils as effector cells against helminth infection, activated cells may also be recruited to sites of inflammation and contribute to the immune response acting as antigen presenting cells.

BackgroundRecombinant Schistosoma mansoni Tetraspanin-2 formulated on Alhydrogel (Sm-TSP-2/Alhydrogel) is being developed to prevent intestinal and hepatic disease caused by S. mansoni. The tegumentary Sm-TSP-2 antigen was selected based on its unique recognition by cytophilic antibodies in putatively immune individuals living in areas of ongoing S. mansoni transmission in Brazil, and preclinical studies in which vaccination with Sm-TSP-2 protected mice following infection challenge.MethodsA randomized, observer-blind, controlled, Phase 1b clinical trial was conducted in 60 healthy adults living in a region of Brazil with ongoing S. mansoni transmission. In each cohort of 20 participants, 16 were randomized to receive one of two formulations of Sm-TSP-2 vaccine (adjuvanted with Alhydrogel only, or with Alhydrogel plus the Toll-like receptor-4 agonist, AP 10-701), and 4 to receive Euvax B hepatitis B vaccine. Successively higher doses of antigen (10 μg, 30 μg, and 100 μg) were administered in a dose-escalation fashion, with progression to the next dose cohort being dependent upon evaluation of 7-day safety data after all participants in the preceding cohort had received their first dose of vaccine. Each participant received 3 intramuscular injections of study product at intervals of 2 months and was followed for 12 months after the third vaccination. IgG and IgG subclass antibody responses to Sm-TSP-2 were measured by qualified indirect ELISAs at pre- and post-vaccination time points through the final study visit.ResultsSm-TSP-2/Alhydrogel administered with or without AP 10-701 was well-tolerated in this population. The most common solicited adverse events were mild injection site tenderness and pain, and mild headache. No vaccine-related serious adverse events or adverse events of special interest were observed. Groups administered Sm-TSP-2/Alhydrogel with AP 10-701 had higher post-vaccination levels of antigen-specific IgG antibody. A significant dose-response relationship was seen in those administered Sm-TSP-2/Alhydrogel with AP 10-701. Peak anti-Sm-TSP-2 IgG levels were observed approximately 2 weeks following the third dose, regardless of Sm-TSP-2 formulation. IgG levels fell to low levels by Day 478 in all groups except the 100 μg with AP 10-701 group, in which 57% of subjects (4 of 7) still had IgG levels that were ≥4-fold higher than baseline. IgG subclass levels mirrored those of total IgG, with IgG1 being the predominant subclass response.ConclusionsVaccination of adults with Sm-TSP-2/Alhydrogel in an area of ongoing S. mansoni transmission was safe, minimally reactogenic, and elicited significant IgG and IgG subclass responses against the vaccine antigen. These promising results have led to initiation of a Phase 2 clinical trial of this vaccine in an endemic region of Uganda.Trial registrationNCT03110757.

[This corrects the article DOI: 10.1371/journal.pntd.0005574.].

[This corrects the article DOI: 10.1371/journal.pntd.0006655.].

BACKGROUND:Standard diagnosis of human soil-transmitted helminth (STH) infections is based on the microscopic detection of helminth eggs in stool and supports programmatic decision making in control programs. However, the current standard diagnostic techniques still show a number of limitations. Recently, the FECPAKG2 method was developed to detect helminth infections and asses drug efficacy in sheep or cattle. It includes a device that takes digital images of helminth eggs that have been concentrated into one microscopic field of view and stores these images online for future evaluation. The goal of this study was to introduce a standard operating procedure (SOP) for the detection and quantification of human STH eggs using the FECPAKG2 and to optimize 2 crucial steps of the protocol, namely the sedimentation step (aimed at separating sinking eggs from floating debris) and the accumulation step (aimed at concentrating the eggs by flotation). METHODOLOGY/PRINCIPAL FINDINGS:A total of 55 stool samples from naturally infected children were used from 4 different geographical areas (Ethiopia, Laos, Tanzania and Brazil). The results showed that Trichuris eggs generally moved slower than eggs of the other two STH species during both sedimentation in water in the FECPAKG2 sedimenter as during accumulation in flotation solution in the FECPAKG2 cassettes. The highest number of eggs were present in the slurry of the sedimenter after overnight sedimentation (Ascaris: 95.7%, Trichuris: 89.8% and hookworm: 94.2% of the eggs). A minimum of 24 minutes were needed to ensure the accumulation of at least 80% of the eggs from all three STH species in the FECPAKG2 cassette (Ascaris: 96.1%; Trichuris: 88.2% and hookworm: 87.6%). CONCLUSIONS/SIGNIFICANCE:This study introduces for the first time a SOP for the FECPAKG2 method. Different aspects of the method for diagnosing human STH infections were optimized. Our study forms the basis for a thorough and objective evaluation of the system as a diagnostic tool that could be implemented in STH control programs.

Necator americanus Glutathione-S-Transferase-1 (Na-GST-1) plays a role in the digestion of host hemoglobin by adult N. americanus hookworms. Vaccination of laboratory animals with recombinant Na-GST-1 is associated with significant protection from challenge infection. Recombinant Na-GST-1 was expressed in Pichia pastoris and adsorbed to aluminum hydroxide adjuvant (Alhydrogel) according to current Good Manufacturing Practice. Two Phase 1 trials were conducted in 142 healthy adult volunteers in the United States and Brazil, first in hookworm-naïve individuals and then in residents of a N. americanus endemic area in Brazil. Volunteers received one of three doses of recombinant Na-GST-1 (10, 30, or 100 μg) adjuvanted with Alhydrogel, adjuvanted with Alhydrogel and co-administered with an aqueous formulation of Glucopyranosyl Lipid A (GLA-AF), or the hepatitis B vaccine. Vaccinations were administered via intramuscular injection on days 0, 56, and 112. Na-GST-1/Alhydrogel was well tolerated in both hookworm-naïve and hookworm-exposed adults, with the most common adverse events being mild to moderate injection site pain and tenderness, and mild headache and nausea; no vaccine-related severe or serious adverse events were observed. Antigen-specific IgG antibodies were induced in a dose-dependent fashion, with increasing levels observed after each vaccination in both trials. The addition of GLA-AF to Na-GST-1/Alhydrogel did not result in significant increases in specific IgG responses. In both the US and Brazil studies, the predominant IgG subclass induced against Na-GST-1 was IgG1, with lesser amounts of IgG3. Vaccination of both hookworm-naïve and hookworm-exposed adults with recombinant Na-GST-1 was safe, well tolerated, and resulted in significant antigen-specific IgG responses. Based on these results, this vaccine will be advanced into clinical trials in children and eventual efficacy studies.ClinicalTrials.gov (NCT01261130 for the Brazil trial and NCT01385189 for the US trial).

A new generation of vaccines for the neglected tropical diseases (NTDs) have now advanced into clinical development, with the Na-GST-1/Alhydrogel Hookworm Vaccine already being tested in Phase 1 studies in healthy adults. The current manuscript focuses on the often overlooked critical aspects of NTD vaccine product development, more specifically, vaccine stability testing programs. A key measure of vaccine stability testing is "relative potency" or the immunogenicity of the vaccine during storage. As with most NTD vaccines, the Na-GST-1/Alhydrogel Hookworm Vaccine was not developed by attenuation or inactivation of the pathogen (Necator americanus), so conventional methods for measuring relative potency are not relevant for this investigational product. Herein, we describe a novel relative potency testing program and report for the first time on the clinical lot of this NTD vaccine during its first 60 months of storage at 2-8°C. We also describe the development of a complementary functional assay that measures the ability of IgG from animals or humans immunized with Na-GST-1/Alhydrogel to neutralize this important hookworm enzyme. While 90% inhibition of the catalytic activity of Na-GST-1 was achieved in animals immunized with Na-GST-1/Alhydrogel, lower levels of inhibition were observed in immunized humans. Moreover, anti-Na-GST-1 antibodies from volunteers in non-hookworm endemic areas were better able to inhibit catalytic activity than anti-Na-GST-1 antibodies from volunteers resident in hookworm endemic areas. The results described herein provide the critical tools for the product development of NTD vaccines.

Dilated cardiomyopathy, the most severe manifestation in chronic phase of Chagas disease, affects about 30% of patients and is characterized by myocardial dysfunction and interstitial fibrosis due to extracellular matrix (ECM) remodeling. ECM remodeling is regulated by proteolytic enzymes such as matrix metalloproteinases (MMPs) and cytokines produced by immune cells, including phagocytes. We evaluated by flow cytometry the expression of MMP-2, MMP-9, IL-1β, TNF-α, TGF-β and IL-10 by neutrophils and monocytes from patients with indeterminate (IND) and cardiac (CARD) clinical forms of Chagas disease and non-infected individuals (NI), before and after in vitro stimulation with Trypanosoma cruzi antigens. Our results showed an important contribution of neutrophils for MMPs production, while monocytes seemed to be involved in cytokine production. The results showed that neutrophils and monocytes from IND and CARD patients had higher intracellular levels of MMP-2 and MMP-9 than NI individuals. On the other hand, T. cruzi derived-antigens promote a differential expression of MMP-2 and MMP-9 in patients with Chagas disease and may regulate MMPs expression in neutrophils and monocytes, mainly when a cardiac alteration is not present. Our data also showed that in the presence of T. cruzi derived-antigens the production of cytokines by neutrophils and monocytes, but mainly by monocytes, may be intensified. Correlation analysis demonstrated that MMP-2 had a positive correlation with IL-10 and a negative correlation with IL-1β, whereas MMP-9 showed a negative correlation with IL-10. We also observed that IND patients presented a greater percentage of high producer cells of regulatory molecules when compared to CARD patients, indicating a different pattern in the immune response. Our data suggest that MMPs and cytokines produced by neutrophils and monocytes are important contributors for cardiac remodeling and may be an interesting target for new biomarker research.

Due to the recent increased use of the McMaster (MM) fecal egg counting method for assessing benzimidazole drug efficacy for treating soil-transmitted helminth (STH) infections, the aim of the current study was to determine the operational value of including the MM method alongside the Kato-Katz (KK) fecal thick smear to increase the diagnostic sensitivity when STHs are co-endemic with trematode helminths (e.g., Schistosoma mansoni).A cross-sectional study was conducted in school-aged children aged 4-18 years in the northeastern region of the State of Minas Gerais (Brazil), where Necator americanus, Ascaris lumbricoides, Trichuris trichiura, and S. mansoni are co-endemic. One fecal sample from each participant was collected and transported to the field laboratory for analysis. Coprological diagnosis was performed on each fecal sample by three different methods: Formalin-Ether Sedimentation (FES), KK and the MM technique. The diagnostic sensitivity and negative predictive value (NPV) of each technique was calculated using the combination of all three techniques as the composite standard. In order to determine the agreement between the three techniques Fleiss´ kappa was used. Both the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR) were calculated using the two quantification techniques (i.e., the MM and KK).Fecal samples from 1260 children were analyzed. The KK had higher diagnostic sensitivity than the MM for the detection of both A. lumbricoides (KK 97.3%, MM 69.5%) and hookworm (KK 95.1%, MM 80.8%). The CR of a single dose of mebendazole varied significantly between the KK and MM for both A. lumbricoides (p = 0.016) and hookworm (p = 0.000), with lower rates obtained with the KK. On the other hand, the FECR was very similar between both techniques for both A. lumbricoides and hookworm.The MM did not add any diagnostic value over the KK in areas where both STHs and trematodes were co-endemic. The lower sensitivity of the MM would have an important impact on the administration of selective school-based treatment in this area since if only the MM were used, 36 (13.9%) children diagnosed with A. lumbricoides would have gone untreated.

In this paper, the Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), with the mandate to review helminthiases research and identify research priorities and gaps, focuses on the environmental, social, behavioural, and political determinants of human helminth infections and outlines a research and development agenda for the socioeconomic and health systems research required for the development of sustainable control programmes. Using Stockols' social-ecological approach, we describe the role of various social (poverty, policy, stigma, culture, and migration) and environmental determinants (the home environment, water resources development, and climate change) in the perpetuation of helminthic diseases, as well as their impact as contextual factors on health promotion interventions through both the regular and community-based health systems. We examine these interactions in regard to community participation, intersectoral collaboration, gender, and possibilities for upscaling helminthic disease control and elimination programmes within the context of integrated and interdisciplinary approaches. The research agenda summarises major gaps that need to be addressed.

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4(+)CD25(+)FOXP3(+) regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4(+)CD25(+)FOXP3(+) T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people.

Helminth co-infection in humans is common in tropical regions of the world where transmission of soil-transmitted helminths such as Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale as well as other helminths such as Schistosoma mansoni often occur simultaneously.We investigated whether co-infection with another helminth(s) altered the human immune response to crude antigen extracts from either different stages of N. americanus infection (infective third stage or adult) or different crude antigen extract preparations (adult somatic and adult excretory/secretory). Using these antigens, we compared the cellular and humoral immune responses of individuals mono-infected with hookworm (N. americanus) and individuals co-infected with hookworm and other helminth infections, namely co-infection with either A. lumbricoides, Schistosoma mansoni, or both. Immunological variables were compared between hookworm infection group (mono- versus co-infected) by bootstrap, and principal component analysis (PCA) was used as a data reduction method.Contrary to several animal studies of helminth co-infection, we found that co-infected individuals had a further downmodulated Th1 cytokine response (e.g., reduced INF-γ), accompanied by a significant increase in the hookworm-specific humoral immune response (e.g. higher levels of IgE or IgG4 to crude antigen extracts) compared with mono- infected individuals. Neither of these changes was associated with a reduction of hookworm infection intensity in helminth co-infected individuals. From the standpoint of hookworm vaccine development, these results are relevant; i.e., the specific immune response to hookworm vaccine antigens might be altered by infection with another helminth.

CD25(High) CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25(High)CD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25(High) CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections.

BACKGROUND: Schistosomiasis affects more than 200 million individuals worldwide, with a further 650 million living at risk of infection, constituting a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we report the cloning and characterization of a S. mansoni Stomatin-like protein 2 (SmStoLP-2). In silico analysis predicts three putative sites for palmitoylation (Cys11, Cys61 and Cys330), which could contribute to protein membrane association; and a putative mitochondrial targeting sequence, similar to that described for human Stomatin-like protein 2 (HuSLP-2). The protein was detected by Western blot with comparable levels in all stages across the parasite life cycle. Fractionation by differential centrifugation of schistosome tegument suggested that SmStoLP-2 displays a dual targeting to the tegument membranes and mitochondria; additionally, immunolocalization experiments confirm its localization in the tegument of the adult worms and, more importantly, in 7-day-old schistosomula. Analysis of the antibody isotype profile to rSmStoLP-2 in the sera of patients living in endemic areas for schistosomiasis revealed that IgG1, IgG2, IgG3 and IgA antibodies were predominant in sera of individuals resistant to reinfection as compared to those susceptible. Next, immunization of mice with rSmStoLP-2 engendered a 30%-32% reduction in adult worm burden. Protective immunity in mice was associated with specific anti-rSmStoLP-2 IgG1 and IgG2a antibodies and elevated production of IFN-gamma and TNF-alpha, while no IL-4 production was detected, suggesting a Th1-predominant immune response. CONCLUSIONS/SIGNIFICANCE: Data presented here demonstrate that SmStoLP-2 is a novel tegument protein located in the host-parasite interface. It is recognized by different subclasses of antibodies in patients resistant and susceptible to reinfection and, based on the data from murine studies, shows protective potential against schistosomiasis. These results indicate that SmStoLP-2 could be useful in a combination vaccine.

BACKGROUND: Obtaining informed consent for clinical trials is especially challenging when working in rural, resource-limited areas, where there are often high levels of illiteracy and lack of experience with clinical research. Such an area, a remote field site in the northeastern part of the state of Minas Gerais, Brazil, is currently being prepared for clinical trials of experimental hookworm vaccines. This study was conducted to assess whether special educational tools can be developed to increase the knowledge and comprehension of potential clinical trial participants and thereby enable them to make truly informed decisions to participate in such research. METHODOLOGY/PRINCIPAL FINDINGS: An informational video was produced to explain the work of the research team and the first planned hookworm vaccine trial, using a pedagogical method based on analogies. Seventy-two adults living in a rural community of Minas Gerais were administered a structured questionnaire that assessed their knowledge of hookworm, of research and of the planned hookworm vaccine trial, as well as their attitudes and perceptions about the researchers and participation in future vaccine trials. The questionnaire was administered before being shown the educational video and two months after and the results compared. After viewing the video, significant improvements in knowledge related to hookworm infection and its health impact were observed: using a composite score combining related questions for which correct answers were assigned a value of 1 and incorrect answers a value of 0, participants had a mean score of 0.76 post-video compared to 0.68 pre-video (p = 0.0001). Similar improvements were seen in understanding the purpose of vaccination and the possible adverse effects of an experimental vaccine. Although 100% of participants expressed a positive opinion of the researchers even before viewing the film and over 90% said that they would participate in a hookworm vaccine trial, an increase in the number who expressed fear of being vaccinated with a novel vaccine was seen after viewing the video (51.4% post-video versus 29.2% pre-video). Increases were also seen in the proportion who thought that participation in a vaccine trial would be inconvenient or disrupt their daily activities. CONCLUSIONS/SIGNIFICANCE: Even in rural, resource-limited populations, educational tools can be specially designed that significantly improve understanding and therefore the likelihood of obtaining truly informed consent for participation in clinical research. The observed changes in the knowledge and perceptions of the research participants about hookworm infection and the experimental hookworm vaccine demonstrate that the video intervention was successful in increasing understanding and that the subjects acquired knowledge pertinent to the planned research.

BACKGROUND: Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4(+) and CD8(+) T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4(+) and CD8(+) T lymphocytes; the pattern of intracellular cytokines (IFN-gamma, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA(high)) CD4(+) and CD8(+) T cells. However, statistical analysis of ex-vivo profiles of CD4(+) T cells showed that IND have lower percentage of CD45RA(high) in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO(high)) CD4(+) T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8(+)CD45RA(high) IFN-gamma-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens. CONCLUSIONS: Based on a correlation between the frequency of IFN-gamma producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-gamma. Furthermore, we showed that individuals from IND group presented more T(CM) CD4(+) T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.

BackgroundIndividuals living in areas endemic for helminths are commonly infected with multiple species. Despite increasing emphasis given to the potential health impacts of polyparasitism, few studies have investigated the relative importance of household and environmental factors on the risk of helminth co-infection. Here, we present an investigation of exposure-related risk factors as sources of heterogeneity in the distribution of co-infection with Necator americanus and Schistosoma mansoni in a region of southeastern Brazil.MethodologyCross-sectional parasitological and socio-economic data from a community-based household survey were combined with remotely sensed environmental data using a geographical information system. Geo-statistical methods were used to explore patterns of mono- and co-infection with N. americanus and S. mansoni in the region. Bayesian hierarchical models were then developed to identify risk factors for mono- and co-infection in relation to community-based survey data to assess their roles in explaining observed heterogeneity in mono and co-infection with these two helminth species.Principal findingsThe majority of individuals had N. americanus (71.1%) and/or S. mansoni (50.3%) infection; 41.0% of individuals were co-infected with both helminths. Prevalence of co-infection with these two species varied substantially across the study area, and there was strong evidence of household clustering. Hierarchical multinomial models demonstrated that relative socio-economic status, household crowding, living in the eastern watershed and high Normalized Difference Vegetation Index (NDVI) were significantly associated with N. americanus and S. mansoni co-infection. These risk factors could, however, only account for an estimated 32% of variability between households.ConclusionsOur results demonstrate that variability in risk of N. americanus and S. mansoni co-infection between households cannot be entirely explained by exposure-related risk factors, emphasizing the possible role of other household factors in the heterogeneous distribution of helminth co-infection. Untangling the relative contribution of intrinsic host factors from household and environmental determinants therefore remains critical to our understanding of helminth epidemiology.

The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the classification of M&HI infections. Validation studies are required before they can be recommended for general use in assessing M&HI infections in programmatic settings.