Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati.

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15­120 min), and different concentrations of malachite green dye (0.004­0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10­100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.

An immunomics approach to schistosome antigen discovery: antibody signatures of naturally resistant and chronically infected individuals from endemic areas.

Schistosomiasis is a neglected tropical disease that is responsible for almost 300,000 deaths annually. Mass drug administration (MDA) is used worldwide for the control of schistosomiasis, but chemotherapy fails to prevent reinfection with schistosomes, so MDA alone is not sufficient to eliminate the disease, and a prophylactic vaccine is required. Herein, we take advantage of recent advances in systems biology and longitudinal studies in schistosomiasis endemic areas in Brazil to pilot an immunomics approach to the discovery of schistosomiasis vaccine antigens. We selected mostly surface-derived proteins, produced them using an in vitro rapid translation system and then printed them to generate the first protein microarray for a multi-cellular pathogen. Using well-established Brazilian cohorts of putatively resistant (PR) and chronically infected (CI) individuals stratified by the intensity of their S. mansoni infection, we probed arrays for IgG subclass and IgE responses to these antigens to detect antibody signatures that were reflective of protective vs. non-protective immune responses. Moreover, probing for IgE responses allowed us to identify antigens that might induce potentially deleterious hypersensitivity responses if used as subunit vaccines in endemic populations. Using multi-dimensional cluster analysis we showed that PR individuals mounted a distinct and robust IgG1 response to a small set of newly discovered and well-characterized surface (tegument) antigens in contrast to CI individuals who mounted strong IgE and IgG4 responses to many antigens. Herein, we show the utility of a vaccinomics approach that profiles antibody responses of resistant individuals in a high-throughput multiplex approach for the identification of several potentially protective and safe schistosomiasis vaccine antigens.

Spatial patterns of intestinal parasite infections among children and adolescents in some indigenous communities in Argentina.

Argentina has a heterogeneous prevalence of infections by intestinal parasites (IPs), with the north in the endemic area, especially for soil-transmitted helminths (STHs). We analyzed the spatial patterns of these infections in the city of Tartagal, Salta province, by an observational, correlational, and cross-sectional study in children and adolescents aged 1 to 15 years from native communities. One fecal sample per individual was collected to detect IPs using various diagnostic techniques: Telemann sedimentation, Baermann culture, and Kato-Katz. Moran's global and local indices were applied together with SaTScan to assess the spatial distribution, with a focus on cluster detection. The extreme gradient boosting (XGBoost) machine-learning model was used to predict the presence of IPs and their transmission pathways. Based on the analysis of 572 fecal samples, a prevalence of 78.3% was found. The most frequent parasite was Giardia lamblia (30.9%). High- and low-risk clusters were observed for most species, distributed in an east-west direction and polarized in two large foci, one near the city of Tartagal and the other in the km 6 community. Spatial XGBoost models were obtained based on distances with a minimum median accuracy of 0.69. Different spatial patterns reflecting the mechanisms of transmission were noted. The distribution of the majority of the parasites studied was aligned in a westerly direction close to the city, but the STH presence was higher in the km 6 community, toward the east. The purely spatial analysis provides a different and complementary overview for the detection of vulnerable hotspots and strategic intervention. Machine-learning models based on spatial variables explain a large percentage of the variability of the IPs.

Molecular mechanisms of hookworm disease: stealth, virulence, and vaccines.

Hookworms produce a vast repertoire of structurally and functionally diverse molecules that mediate their long-term survival and pathogenesis within a human host. Many of these molecules are secreted by the parasite, after which they interact with critical components of host biology, including processes that are key to host survival. The most important of these interactions is the hookworm's interruption of nutrient acquisition by the host through its ingestion and digestion of host blood. This results in iron deficiency and eventually the microcytic hypochromic anemia or iron deficiency anemia that is the clinical hallmark of hookworm infection. Other molecular mechanisms of hookworm infection cause a systematic suppression of the host immune response to both the parasite and to bystander antigens (eg, vaccines or allergens). This is achieved by a series of molecules that assist the parasite in the stealthy evasion of the host immune response. This review will summarize the current knowledge of the molecular mechanisms used by hookworms to survive for extended periods in the human host (up to 7 years or longer) and examine the pivotal contributions of these molecular mechanisms to chronic hookworm parasitism and host clinical outcomes.

Generalized urticaria induced by the Na-ASP-2 hookworm vaccine: implications for the development of vaccines against helminths.

BACKGROUND: Necator americanus Ancylostoma-secreted protein 2 (Na-ASP-2) is secreted by infective hookworm larvae on entry into human hosts. Vaccination of laboratory animals with recombinant Na-ASP-2 provides significant protection against challenge infections. In endemic areas antibodies to Na-ASP-2 are associated with reduced risk of heavy N americanus infections. OBJECTIVE: To assess the safety and immunogenicity of recombinant Na-ASP-2 adjuvanted with Alhydrogel in healthy Brazilian adults previously infected with N americanus. METHODS: Participants were randomized to receive Na-ASP-2 or hepatitis B vaccine. Major IgG and IgE epitopes of the Na-ASP-2 molecule were mapped by using sera from these same subjects. Seroepidemiologic studies in adults and children residing in hookworm-endemic areas were conducted to assess the prevalence of IgE responses to Na-ASP-2. RESULTS: Vaccination with a single dose of Na-ASP-2 resulted in generalized urticarial reactions in several volunteers. These reactions were associated with pre-existing Na-ASP-2-specific IgE likely induced by previous hookworm infection. Surveys revealed that a significant proportion of the population in hookworm-endemic areas had increased levels of IgE to Na-ASP-2. Epitope mapping demonstrated sites on the Na-ASP-2 molecule that are uniquely or jointly recognized by IgG and IgE antibodies. CONCLUSION: Infection with N americanus induces increased levels of total and specific IgE to Na-ASP-2 that result in generalized urticaria on vaccination with recombinant Na-ASP-2. These data advance knowledge of vaccine development for helminths given their propensity to induce strong T(H)2 responses. Study data highlight the important differences between the immune responses to natural helminth infection and to vaccination with a recombinant helminth antigen.

The Relationship Between Soil-Transmitted Helminth Infections and Environmental Factors in Puerto Iguazú, Argentina: Cross-Sectional Study.

BACKGROUND: Soil-transmitted helminths (STHs) are widely distributed throughout the world. Various factors, including the environment, socioeconomic characteristics, and access to water and sanitation, play an important role in the spread and persistence of these parasites within communities. They, in turn, affect the growth and development of members of the community, especially children. Studies in the northern provinces of Argentina have shown variable prevalence of STHs, but the factors associated with their presence have not been completely elucidated. OBJECTIVE: This cross-sectional study aimed to identify the socioeconomic and environmental factors related to STH infection in indigenous villages located in Puerto Iguazú (Misiones), Argentina. METHODS: Between 2018 and 2019, stool samples were collected from individuals ≥1 year residing in 3 villages: Mini-Marangatú, Yriapú, and Fortín Mbororé. Standard parasitological methods were used to determine STH prevalence. Standardized questionnaires were used to assess participants' habits, customs, and household characteristics, and environmental data were obtained through satellite imagery. Multilinear regression with Akaike information criterion stepwise variables was used to explore relevant associations. RESULTS: A total of 342 individuals from the 3 villages participated in this study. The prevalence of STHs varied across villages: 89.6% (43/48), in Mini-Marangatú, 80.8% (101/125) in Yriapú, and 68.5% (115/169) in Fortín Mbororé. Notably, there was a significant difference in hookworm infection among the villages (P=.02). The analysis highlighted the significant influence of specific environmental factors on STH presence and spatial distribution, particularly in relation to hookworm infection. Vegetation patterns represented by the Vegetation Heterogeneity Index, created ad hoc for this study, emerged as a critical factor, with 2 significant predictors related to it (P=.002 and P=.004) alongside impervious surface density with a significant predictor (P<.001). The multilinear regression model yielded a high F test score (F108=4.75, P<.001), indicating a strong fit (R2=0.5465). Furthermore, socioeconomic factors, including walking barefoot in houses with dirt floors and overcrowding, were significantly correlated with hookworm infection intensity (P<.001 and P=.001, respectively). We also used the multilinear regression model to calculate hookworm infection intensity (F110=21.15, P<.001; R2=0.4971). CONCLUSIONS: Our study underscores the complexity of STH transmission, as villages with similar living conditions and environmental characteristics displayed varied STH prevalence and spatial distribution. Specific environmental factors, such as vegetation pattern and impervious surface density, played major roles in STH presence, demonstrating the crucial relationship between environmental factors and hookworm infection distribution. Moreover, our findings emphasize the significant influence of socioeconomic factors on hookworm infection intensity. By gaining insights into this complex interplay, our research contributes to a better understanding of STH transmission characteristics, thereby informing targeted public health interventions for effective control.

Intestinal parasitic infections in a community from Pampa del Indio, Chaco (Argentina) and their association with socioeconomic and environmental factors.

Neglected tropical diseases are a group of 20 disabling diseases, which, in particular, are the most common chronic infections in the most vulnerable people. This study aimed to characterize the infection by intestinal parasites (IPs) in dwellings from a peri-urban neighborhood in Pampa del Indio, Chaco (Argentina), and its association with socioeconomic and environmental variables. Single stool samples were collected from all individuals older than 1 year through household visits and processed using coprological sedimentation and flotation techniques. Standardized questionnaires were used at the household level to collect socio-economic information. Environmental variables were obtained from the Planetscope image, Landsat 8 images and remote sensors, while land-use layers were obtained through the use of a maximum likelihood algorithm. Stool samples were provided by 314 individuals. The prevalence of IPs found was 30.6% (n = 96), with a predominance of Giardia lamblia (12.7%, n = 40) and Hymenolepis nana (7.6%, n = 24). The only soil-transmitted helminth found was Strongyloides stercoralis with a 2.5% prevalence (n = 8). Individuals of adult age (> 18 years) were 0.65 times less likely to present parasitic infections with respect to children and adolescents. The only environmental variable that was closely associated with the presence of IPs, was the Normalized Difference Water Index (NDWI), a measure of humidity; being higher around houses with positive individuals. Most of the IPs found in this study were of water-borne transmission and those transmitted directly from person-to-person, therefore fecal contamination is present. We believe that the low prevalence of STH in this area, which requires a passage through the soil, is related to the environmental characteristics, which are unsuitable for the development/permanence of the infective stages of these parasites. The geospatial data and tools used herein proved to be useful for the study of the relationship between the different factors that influence the presence of IPs in a community, from an eco-health approach.

Risk Stratification to Guide Prevention and Control Strategies for Arboviruses Transmitted by Aedes aegypti.

Strategies for the prevention of arboviral diseases transmitted by Aedes aegypti have traditionally focused on vector control. This remains the same to this day, despite a lack of documented evidence on its efficacy due to a lack of coverage and sustainability. The continuous growth of urban areas and generally unplanned urbanization, which favor the presence of Ae. aegypti, demand resources, both material and human, as well as logistics to effectively lower the population's risk of infection. These considerations have motivated the development of tools to identify areas with a recurrent concentration of arboviral cases during an outbreak to be able to prioritize preventive actions and optimize available resources. This study explores the existence of spatial patterns of dengue incidence in the locality of Tartagal, in northeastern Argentina, during the outbreaks that occurred between 2010 and 2020. Approximately half (50.8%) of the cases recorded during this period were concentrated in 35.9% of the urban area. Additionally, an important overlap was found between hotspot areas of dengue and chikungunya (Kendall's W = 0.92; p-value < 0.001) during the 2016 outbreak. Moreover, 65.9% of the cases recorded in 2022 were geolocalized within the hotspot areas detected between 2010 and 2020. These results can be used to generate a risk map to implement timely preventive control strategies that prioritize these areas to reduce their vulnerability while optimizing the available resources and increasing the scope of action.

Environmental Toxocara spp. presence in crowded squares and public parks from San Juan Province, Argentina: A call for a "One Health" approach.

Introduction: Canine soil-transmitted helminth (cSTH) parasites need specific environmental conditions to complete their life cycle. Toxocara canis and T. cati are the most important zoonotic cSTH, since they are the causal agents of human toxocariasis. Canine STHs are dispersed in feces from infected domestic and wildlife canines. In this study, the presence of STH in canine feces was evaluated in 34 crowded public parks and squares from San Juan Province (Argentina). Methods: Fecal samples were collected during different seasons in 2021-2022 and analyzed by standard coprological methods, including Sheather and Willis flotation and Telemann sedimentation. InfoStat 2020, OpenEpi V. 3.01 and R and RStudio® were used for statistical analysis and QGIS 3.16.10 for mapping. Results: From a total of 1,121 samples collected, 100 (8.9%) were positive for at least one intestinal parasite (IP) and three cSTH species were detected: Toxocara spp., Toxascaris leonina and Trichuris vulpis. The most prevalent cSTH species was T. vulpis (64/1121; 0.057%), while the least prevalent was Toxocara spp. (19/1121; 0.017%). The detection of Toxocara spp. eggs was significantly different depending on the season. The geo-spatial variation of each cSTH per season is described. Discussion: This is the first study in San Juan Province to identify environmental contamination of cSTHs in public areas. The specific localization of areas with the presence of cSTH eggs could provide information to guide strategies to reduce the cSTH infection burden in dogs and promote serological screening of the human population for Toxocara spp. Given the zoonotic nature of Toxocara spp. We hope this information will help to reinforce activities of control programs, focusing on the "One Health" approach.

Chagas prevention and control in an endemic area from the Argentinian Gran Chaco Region: Data from 14 years of uninterrupted intervention.

BACKGROUND: Chagas Disease (ChD) is a Neglected Tropical Disease (NTD) affecting 6 to 7 million people worldwide, mostly from Latin America. In Argentina, a national control program has been implemented since 1962, yet there are still an estimated 1.6 million infected individuals. Control programs were based almost exclusively on entomological surveillance and chemical control of households and were not continuous given a lack of coordination and resources. Argentina´s ChD program was originally vertical and centralized; later, it was partially and, in general, unsuccessfully transferred to the provinces. Herein, we describe the implementation of a control program for ChD with an ecohealth approach in rural settlements around the city of Añatuya, Santiago del Estero. METHODS: The program included yearly household visits for entomological surveillance and control, health promotion workshops, and structural house improvements. Improved structures included internal and external walls and roofs, as well as the construction of water wells and latrines, and the organization and improvement of peri-domestic structures. Activities were carried out by specifically trained personnel except for house improvements, which were performed by the community, under technical guidance and provision of materials. Data was collected using standardized questionnaires for household characterization, entomological infestation status and chemical control activities. RESULTS: This program was continuously implemented since 2005 with high community participation and adherence, incorporating 13 settlements and 502 households. During the surveillance phase, 4,193 domiciliary inspections were performed, and both the intra- and peri-domestic infestation rate were reduced from 17.9% to 0.2% (P < 0.01) and from 20.4% to 3%, respectively. Additionally, 399 households were structurally improved. CONCLUSION: The program is still ongoing and, after 14 years of implementation, has built social networks and collaboration between implementers and beneficiaries with a reduction of T. infestans infestation in the intra- and peri-domicile. This reduction, especially inside the household, has enabled access to diagnosis and treatment of the population, with minimal risk of re-infection.

A history of hookworm vaccine development.

The human hookworms Necator americanus and Ancylostoma duodenale remain among the most common infections of humans in areas of rural poverty in the developing regions of the world, with an estimated 1 billion people infected with one or more of these parasites. Herein, we review the nearly 100 years of research, development, animal testing, and fieldwork that have led to our current progress in recombinant hookworm vaccines. We begin with the identification of hookworm at the start of the 20th century in Southern US, then discuss the progress in developed countries to eliminate human hookworm infection, and then the industrial development and field use in the 1970s a canine hookworm vaccine(Ancylostoma caninum), and finally our progress to date in the development and clinical testing of an array of recombinant antigens to prevent human hookworm disease from N. americanus infection. Special attention is given to the challenges faced in the development of a vaccine against a blood-feeding nematode, including the epidemiology of infection (high prevalence of infection), pathogenesis (chronic infection that increases with the age of the host), and a robust immune response that fails to confer the protection in the host and a concomitant absence of correlates of protection by a successful vaccine could be developed and tested. Finally, we provide the optimal and acceptable profiles of a human hookworm vaccine, including the proposed indication, target population, and route of administration, as developed by the Human Hookworm Vaccine Initiative, the only group currently working on vaccines targeting this parasite.

Environmental characteristics around the household and their association with hookworm infection in rural communities from Bahir Dar, Amhara Region, Ethiopia.

Soil-Transmitted Helminths (STH) are highly prevalent Neglected Tropical Disease in Ethiopia, an estimated 26 million are infected. Geographic Information Systems and Remote Sensing (RS) technologies assist data mapping and analysis, and the prediction of the spatial distribution of infection in relation to environmental variables. The influence of socioeconomic, environmental and soil characteristics on hookworm infection at the individual and household level is explored in order to identify spatial patterns of infection in rural villages from Zenzelema (Amhara region). Inhabitants greater than 5 years old were recruited in order to assess the presence of STH. Socioeconomic and hookworm infection variables at the household level and environmental variables and soil characteristics using RS were obtained. The dominant STH found was hookworm. Individuals which practiced open defecation and those without electricity had a significant higher number of hookworm eggs in their stool. Additionally, adults showed statistically higher hookworm egg counts than children. Nonetheless, the probability of hookworm infection was not determined by socioeconomic conditions but by environmental characteristics surrounding the households, including a combination of vigorous vegetation and bare soil, high temperatures, and compacted soils (high bulk density) with more acidic pH, given a pH of 6.0 is optimal for hatching of hookworm eggs. The identification of high-risk environmental areas provides a useful tool for planning, targeting and monitoring of control measures, including not only children but also adults when hookworm is concerned.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples.

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

Impact of a public health intervention for active surveillance and mitigation of SARS-CoV-2 in a district from Buenos Aires province, Argentina: a descriptive epidemiological study.

BACKGROUND: The first case of SARS-CoV-2 was reported in Argentina on 3 March 2020. Measures to limit the spread of the virus were implemented, including complete lockdown (26 March). Nonetheless, the virus spread throughout the country, with a first peak of almost a million cases in October. On 30 November, the government's recommendation switched from social, preventive and compulsory isolation, to social, preventive and compulsory distancing. OBJECTIVES: To describe a tailored public health strategy to mitigate the spread of SARS-CoV-2 and determine its behaviour in San Antonio de Areco district from Buenos Aires province (Argentina) through a private-public association. DESIGN, SETTING AND PARTICIPANTS: Surveillance of the virus was performed with the local healthcare system, through early identification of cases and the systematic study of each infected individual and contact, regardless of symptomatology, using telemedicine and a COVID-19-specific outpatient clinic. Real-time PCR was used for detection using both individual and pooled samples, with a 12-hour turnaround time. RESULTS: Up to 30 November, a total of 2426 suspected cases were analysed and 578 were confirmed. Surveillance of health personnel and at-risk populations proved effective, mitigating viral spread. Pooling samples allowed reduction of operator time, helped reduce costs, and allowed detection of both symptomatic and asymptomatic cases. CONCLUSION: After 8 months of protocol implementation, the strategy to intensively survey groups at higher epidemiological risk and the systematic search for asymptomatic cases with the incorporation of pooled PCR for diagnosis, in combination with individual testing, is an efficient and viable option in populations with similar characteristics, in the frame of social isolation.

Correction: Safety and immunogenicity of the Na-GST-1 hookworm vaccine in Brazilian and American adults.

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

Prevalence of intestinal parasites and the absence of soil-transmitted helminths in Añatuya, Santiago del Estero, Argentina.

BACKGROUND: Intestinal parasites (IP) have been reported in point studies from different provinces of Argentina. The presence of soil-transmitted helminths (STH) was detected in many of these studies, including varied prevalences of all five species of STH in the north were the climate is more appropriate for transmission. Nonetheless, Argentina lacks a comprehensive prevalence map of STH. Therefore, the objective of this study was to determine the prevalence of intestinal parasites, focusing on STH, in rural and peri-urban areas of Añatuya, Santiago del Estero Province and identifying risk factors for their transmission. METHODS: We conducted a cross-sectional survey in the entire population of three rural lots located on the outskirts of the city between March and June of 2015 and among children in a peri-urban neighbourhood of Añatuya city in July 2016. Socio-economic variables, characteristics of the house and stool samples were collected from each household. RESULTS: A total of 470 stool samples were analysed. The prevalence of STH was extremely low, with only 2 people being positive for hookworm and no detection of other STH. The prevalence of IP was 11.7% for protozoans and 11.1% for helminths. IP were significantly more prevalent in pre-school and school-aged children than in adults (P < 0.05). The level of education of adults was also significantly associated with infection (P = 0.001), as well as the practice of informal slaughter of animals (P = 0.002) and the presence of unimproved walls (P = 0.046) and unimproved floors (P = 0.021) in the household. Nonetheless, the only significant predictors of IP in the logistic regression analysis were age (P < 0.001) and main source of electricity (P = 0.026). CONCLUSIONS: The total prevalence of intestinal parasites in the study population was 22.6%. The intestinal parasites detected are transmitted mostly through water (Giardia lamblia) and close contact (Hymenolepis nana), evidencing the need to improve quality control in water facilities and access to improved sanitation to avoid contamination of stored water. The presence of IP was significantly associated with age (with children being more susceptible), households containing unimproved walls and those that did not have access to an electricity network.

Operational satellite-based temporal modelling of Aedes population in Argentina.

Aedes aegypti is a vector for Chikungunya, Dengue and Zika viruses in Latin America and is therefore a large public health problem for the region. For this reason, several inter-institutional and multidisciplinary efforts have been made to support vector control actions through the use of geospatial technologies. This study presents the development of an operational system for the application of free access to remotely sensed products capable of assessing the oviposition activity of Ae. aegypti in all of Argentina's northern region with the specific aim to improve the current Argentine National Dengue risk system. Temporal modelling implemented includes remotely sensed variables like the normalized difference vegetation index, the normalized difference water index, day and night land surface temperature and precipitation data available from NASA's tropical rainfall measuring mission and global precipitation measurement. As a training data set, four years of weekly mosquito oviposition data from four different cities in Argentina were used. A series of satellite-generated variables was built, downloading and resampling the these products both spatially and temporally. From an initial set of 41 variables chosen based on the correlation between these products and the oviposition series, a subset of 11 variables were preserved to develop temporal forecasting models of oviposition using a lineal multivariate method in the four cities. Subsequently, a general model was generated using data from the cities. Finally, in order to obtain a model that could be broadly used, an extrapolation method using the concept of environmental distance was developed. Although the system was oriented towards the surveillance of dengue fever, the methodology could also be applied to other relevant vector-borne diseases as well as other geographical regions in Latin America.

Advances in neglected tropical disease vaccines: Developing relative potency and functional assays for the Na-GST-1/Alhydrogel hookworm vaccine.

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.

Safety and immunogenicity of the Na-GST-1 hookworm vaccine in Brazilian and American adults.

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. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01261130 for the Brazil trial and NCT01385189 for the US trial).

Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt.

The free-living amoebae of the genus Acanthamoeba include non-pathogenic and pathogenic species and has been recently classified into 15 different genotypes, T1-T15. In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in freshwater sources associated with human activities in the Nile Delta region, Egypt. Identification of Acanthamoeba was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized using temperature and osmotolerance assays and PCR reactions with two primer pairs specific to Acanthamoeba pathogenesis. Isolates genotypes were also determined after ribosomal DNA sequencing. These data revealed that isolates belong to T1, T2, T3, T4 and T7 genotypes. As expected, T4 isolates exhibited the most pathogenic traits and were osmotolerant, temperature tolerant and expressed extracellular serine proteases. This is the first report presenting environmental distribution of Acanthamoeba genotypes in Egypt.