Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool

Background : A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. Methodology and principal findings : In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNA later and were stored at 4 degrees C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. Conclusions : The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs

An Analysis of Genetic Diversity and Inbreeding in Wuchereria bancrofti: Implications for the Spread and Detection of Drug Resistance

Estimates of genetic diversity in helminth infections of humans often have to rely on genotyping (immature) parasite transmission stages instead of adult worms. Here we analyse the results of one such study investigating a single polymorphic locus (a change at position 200 of the β-tubulin gene) in microfilariae of the lymphatic filarial parasite Wuchereria bancrofti. The presence of this genetic change has been implicated in benzimidazole resistance in parasitic nematodes of farmed ruminants. Microfilariae were obtained from patients of three West African villages, two of which were sampled prior to the introduction of mass drug administration. An individual-based stochastic model was developed showing that a wide range of allele frequencies in the adult worm populations could have generated the observed microfilarial genetic diversity. This suggests that appropriate theoretical null models are required in order to interpret studies that genotype transmission stages. Wright's hierarchical F-statistic was used to investigate the population structure in W. bancrofti microfilariae and showed significant deficiency of heterozygotes compared to the Hardy-Weinberg equilibrium; this may be partially caused by a high degree of parasite genetic differentiation between hosts. Studies seeking to quantify accurately the genetic diversity of helminth populations by analysing transmission stages should increase their sample size to account for the variability in allele frequency between different parasite life-stages. Helminth genetic differentiation between hosts and non-random mating will also increase the number of hosts (and the number of samples per host) that need to be genotyped, and could enhance the rate of spread of anthelmintic resistance

The Fire and Smoke Model Evaluation Experiment - A plan for integrated, large fire-atmosphere field campaigns

The Fire and Smoke Model Evaluation Experiment (FASMEE) is designed to collect integrated observations from large wildland fires and provide evaluation datasets for new models and operational systems. Wildland fire, smoke dispersion, and atmospheric chemistry models have become more sophisticated, and next-generation operational models will require evaluation datasets that are coordinated and comprehensive for their evaluation and advancement. Integrated measurements are required, including ground-based observations of fuels and fire behavior, estimates of fire-emitted heat and emissions fluxes, and observations of near-source micrometeorology, plume properties, smoke dispersion, and atmospheric chemistry. To address these requirements the FASMEE campaign design includes a study plan to guide the suite of required measurements in forested sites representative of many prescribed burning programs in the southeastern United States and increasingly common high-intensity fires in the western United States. Here we provide an overview of the proposed experiment and recommendations for key measurements. The FASMEE study provides a template for additional large-scale experimental campaigns to advance fire science and operational fire and smoke models

The Fire and Smoke Model Evaluation Experiment—A Plan for Integrated, Large Fire–Atmosphere Field Campaigns

The Fire and Smoke Model Evaluation Experiment (FASMEE) is designed to collect integrated observations from large wildland fires and provide evaluation datasets for new models and operational systems. Wildland fire, smoke dispersion, and atmospheric chemistry models have become more sophisticated, and next-generation operational models will require evaluation datasets that are coordinated and comprehensive for their evaluation and advancement. Integrated measurements are required, including ground-based observations of fuels and fire behavior, estimates of fire-emitted heat and emissions fluxes, and observations of near-source micrometeorology, plume properties, smoke dispersion, and atmospheric chemistry. To address these requirements the FASMEE campaign design includes a study plan to guide the suite of required measurements in forested sites representative of many prescribed burning programs in the southeastern United States and increasingly common high-intensity fires in the western United States. Here we provide an overview of the proposed experiment and recommendations for key measurements. The FASMEE study provides a template for additional large-scale experimental campaigns to advance fire science and operational fire and smoke models

The optimal timing of post-treatment sampling for the assessment of anthelminthic drug efficacy against Ascaris infections in humans

The egg reduction rate (ERR) is the current standard mean to assess the efficacy of drugs against human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworm). Although the timing of post-treatment sampling is pivotal for a readily interpretation of drug efficacy, there is lack empirical data that allows recommending the optimal time point for a follow-up egg counting. In the present study, we re-analyzed both the kinetics of worm expulsion and egg output for Ascaris lumbricoides following a single oral dose of albendazole in a series of studies previously conducted in Kenyan communities. The results indicate that it takes up to 10 days post-treatment before the expulsion of both adult male and female Ascaris worms is completed, approximately 20% of the worms being expelled between day 7 and 10 post-treatment. The sequential analysis of the egg out put, indicated a poor ERR (89.4%) at day 7 post-treatment, but a 100% ERR at day 14 and 21 post-treatment. Based on our findings we recommend to wait at least 14 days after an albendazole treatment before conducting the follow-up egg count. Any sampling before this time point may result in biased ERR estimates, due the release of residual eggs from moribund or degenerating worms

Assays to Detect β-Tubulin Codon 200 Polymorphism in Trichuris trichiura and Ascaris lumbricoides

The soil-transmitted helminths Ascaris lumbricoides and Trichuris trichiura are gastrointestinal nematodes causing many disabilities in tropical parts of the developing world. Control programs, such as “The Focussing Resources on Effective School Health” (FRESH) Partnership, have been implemented to remove human soil-transmitted nematodes through large-scale use of benzimidazole anthelmintic drugs for school-aged children in developing countries. The benzimidazole drugs albendazole and mebendazole are commonly used as a single annual treatment in areas where the burden is high. In veterinary nematodes, repeated use of these anthelmintics has selected for resistant populations. Resistance to benzimidazoles is commonly associated with a single amino acid substitution from phenylalanine to tyrosine in the β-tubulin gene at position 200. In this study, we have developed pyrosequencing assays for codon 200 in A. lumbricoides and T. trichiura to screen for this single nucleotide polymorphism (SNP) in β-tubulin. The 200Tyr SNP was detected at low frequency in T. trichiura from non-treated people from Kenya and at high frequency in T. trichiura from treated people from Panama. The presence of the resistance-associated SNP may play a role in the sometimes low and variable efficacy of benzimidazole anthelmintics against T. trichiura

Modeling regional-scale wildland fire emissions with the wildland fire emissions information system

As carbon modeling tools become more comprehensive, spatial data are needed to improve quantitative maps of carbon emissions from fire. The Wildland Fire Emissions Information System (WFEIS) provides mapped estimates of carbon emissions from historical forest fires in the United States through a web browser. WFEIS improves access to data and provides a consistent approach to estimating emissions at landscape, regional, and continental scales. The system taps into data and tools developed by the U.S. Forest Service to describe fuels, fuel loadings, and fuel consumption and merges information from the U.S. Geological Survey (USGS) and National Aeronautics and Space Administration on fire location and timing. Currently, WFEIS provides web access to Moderate Resolution Imaging Spectroradiometer (MODIS) burned area for North America and U.S. fire-perimeter maps from the Monitoring Trends in Burn Severity products from the USGS, overlays them on 1-km fuel maps for the United States, and calculates fuel consumption and emissions with an open-source version of the Consume model. Mapped fuel moisture is derived from daily meteorological data from remote automated weather stations. In addition to tabular output results, WFEIS produces multiple vector and raster formats. This paper provides an overview of the WFEIS system, including the web-based system functionality and datasets used for emissions estimates. WFEIS operates on the web and is built using open-source software components that work with open international standards such as keyhole markup language (KML). Examples of emissions outputs from WFEIS are presented showing that the system provides results that vary widely across the many ecosystems of North America and are consistent with previous emissions modeling estimates and products

Model of success : World Association for the Advancement of Veterinary Parasitology African Foundation (1997-2019)

No abstract available.http://www.jsava.co.za/index.php/jsavapm2020Veterinary Tropical Disease

Onchocerciasis Control: Vision for the Future from a Ghanian perspective

Since 1987 onchocerciasis control has relied on the donation of ivermectin (Mectizan®, Merck & Co., Inc.) through the Mectizan Donation Programme. Recently, concern has been raised over the appearance of suboptimal responses to ivermectin in Ghana – highlighting the potential threat of the development of resistance to ivermectin. This report summarises a meeting held in Ghana to set the research agenda for future onchocerciasis control. The aim of this workshop was to define the research priorities for alternative drug and treatment regimes and control strategies to treat populations with existing evidence of suboptimal responsiveness and define research priorities for future control strategies in the event of the development of widespread ivermectin resistance