Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance.

BACKGROUND NlmCategory: BACKGROUND content: "Benzimidazole resistance is associated with isotype-1 \xCE\xB2-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta." - Label: METHODS NlmCategory: METHODS content: "The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 \xCE\xB2-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L." - Label: RESULTS NlmCategory: RESULTS content: "The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r\xE2\x80\x89=\xE2\x80\x890.929, P\xE2\x80\x89<\xE2\x80\x890.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r\xE2\x80\x89>\xE2\x80\x890.720, P\xE2\x80\x89<\xE2\x80\x890.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P\xE2\x80\x89<\xE2\x80\x890.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT." - Label: CONCLUSIONS NlmCategory: CONCLUSIONS content: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

[EN] BACKGROUND: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P  0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcinctaSIThis study was funded by the Spanish “Ramón y Cajal” Programme of the Ministry of Economy and Competitiveness (MMV, RYC‑2015‑18368), and the Cooperativa Bajo Duero, COBADU. EVG was funded by FPU16/03536, JG by Ramon Areces Foundation, VCGA by Junta de Castilla y León and Fondo Social Europeo (LE082‑18), MCP by the Stopping Transmission Of intestinal Parasites (STOP) project (EDCTP2 programme; RIA2017NCT‑1845) and MMV by the Spanish “Ramon y Cajal” Programme (RYC‑2015‑18368

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

Background: There is an urgent need for an extensive evaluation of benzimidazole efcacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequenc‑ ing, and standardized it for large-scale benzimidazole efcacy screening use. Methods: Three diferent protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by fotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at diferent proportions, simulating diferent resistance levels. These mixtures were used to compare previ‑ ously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defned, the utility of the protocols was assessed by measur‑ ing the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results: The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previ‑ ously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequenc‑ ing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample

Towards soil-transmitted helminths transmission interruption: The impact of diagnostic tools on infection prediction in a low intensity setting in Southern Mozambique

Copyright: © 2021 Grau-Pujol et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.[EN] World Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of moderate and heavy infections. Some regions are reaching WHO goals, but transmission could rebound if strategies are discontinued without an epidemiological evalu-ation. For that, sensitive diagnostic methods to detect low intensity infections and localiza-tion of ongoing transmission are crucial. In this work, we estimated and compared the STH infection as obtained by different diagnostic methods in a low intensity setting. We conducted a cross-sectional study enrolling 792 participants from a district in Mozambique. Two stool samples from two consecutive days were collected from each participant. Samples were analysed by Telemann, Kato-Katz and qPCR for STH detection. We evaluated diagnostic sensitivity using a composite reference standard. By geostatistical methods, we estimated neighbourhood prevalence of at least one STH infection for each diagnostic method. We used environmental, demographical and socioeconomical indicators to account for any existing spatial heterogeneity in infection. qPCR was the most sensitive technique compared to composite reference standard: 92% (CI: 83%– 97%) for A. lumbricoides, 95% (CI: 88%– 98%) for T. trichiura and 95% (CI: 91%– 97%) for hookworm. qPCR also estimated the highest neighbourhood prevalences for at least one STH infection in a low intensity set-ting. While 10% of the neighbourhoods showed a prevalence above 20% when estimating with single Kato-Katz from one stool and Telemann from one stool, 86% of the neighbour-hoods had a prevalence above 20% when estimating with qPCR. In low intensity settings, STH estimated prevalence of infection may be underestimated if based on Kato-Katz. qPCR diagnosis outperformed the microscopy methods. Thus, implementation of qPCR based predictive maps at STH control and elimination programmes would disclose hidden transmission and facilitate targeted interventions for transmission interruption.SIBGP and JM received financial support for this study from Mundo Sano Foundation (www. mundosano.org). JG was personally supported at the beginning of the work by the Ramo´n Areces Foundation and is now funded by the Spanish ‘Juan de la Cierva’ Programme, Ministry of Economy and Competitiveness (FJC-2018-38305). MMV is personally supported by the Spanish ‘Ramo´n y Cajal’ Programme, Ministry of Economy and Competitiveness (RYC-2015-18368). MCP is personally supported by Junta de Castilla y Leo´n and Fondo Social Europeo (LE-135-19). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID). Prof. Dr. P.C. Flu Foundation also founded this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Novel compound shows in vivo anthelmintic activity in gerbils and sheep infected by Haemonchus contortus

[EN] The control of gastrointestinal nematodes in livestock is becoming increasingly difficult due to the limited number of available drugs and the rapid development of anthelmintic resistance. Therefore, it is imperative to develop new anthelmintics that are effective against nematodes. Under this context, we tested the potential toxicity of three compounds in mice and their potential anthelmintic efficacy in Mongolian gerbils infected with Haemonchus contortus. The compounds were selected from previous in vitro experiments: two diamine (AAD-1 and AAD-2) and one benzimidazole (2aBZ) derivatives. 2aBZ was also selected to test its efficacy in sheep. In Mongolian gerbils, the benzimidazole reduced the percentage of pre-adults present in the stomach of gerbils by 95% at a dose of 200 mg/kg. In sheep, there was a 99% reduction in the number of eggs shed in faeces after 7 days at a dose of 120 mg/kg and a 95% reduction in the number of worm adults present in the abomasum. In conclusion, 2aBZ could be considered a promising candidate for the treatment of helminth infections in small ruminantsSIFinancial support came from MINECO: RETOS (AGL2016-79813-C2-1R/2R) and MICINN/AEI (PID2020- 119035RB-100). EVG was funded by FPU17/00627, FPU17/05346; VCGA, MAB, MCP and LGP are recipients of Junta de Castilla y León (JCyL) (LE082-18, LE051-18, LE135-19, LE096-20, respectively) and MMV by the Spanish “Ramon y Cajal” Programme (Ministerio de Economía y competitividad; MMV, RYC-2015-18368

An adaptive phase II/III safety and efficacy randomized controlled trial of single day or three-day fixed-dose albendazole-ivermectin co-formulation versus albendazole for the treatment of Trichuris trichiura and other STH infections. ALIVE trial protocol

Background: Soil-transmitted helminths (STH) are targeted for control through mass drug-administration campaigns to prevent morbidity affecting at-risk groups in endemic regions. Although broadly successful, the use of albendazole and mebendazole achieved variable progress, with deficiencies against Trichuris trichiura and a predictable low efficacy against Strongyloides stercoralis. Novel drug combinations offer a potential solution, providing they can be delivered safely and maintain efficacy against all STH species. Here we present the protocol of a clinical trial to evaluate a fixed-dose combination (FDC) tablet containing albendazole and ivermectin that will be compared against albendazole against STH. Methods: An adaptive phase II/III randomized controlled trial will be undertaken in STH endemic sites in Ethiopia, Kenya and Mozambique to evaluate an oral FDC of 400 mg albendazole and either 9- or 18 mg ivermectin. FDC will be administered as a single dose or single doses over three-consecutive days and assessed against a single dose of 400 mg albendazole. In the phase II trial, 126 T. trichiura-infected children weighting 15 to 45 kg will be treated in a dose-escalation manner to determine safety objectives. In the phase III trial, 1097 participants aged 5 to 18 years old infected with T. trichiura, hookworm and S. stercoralis will be recruited to determine safety and efficacy. The trial will be open-label with blinded outcome assessors. Cure rate measured 21-days after-treatment in duplicate Kato-Katz is the primary efficacy outcome. Secondary objectives include efficacy evaluation by quantitative polymerase chain reaction (PCR) as an outcome measurement, description of pharmacokinetic parameters, palatability and acceptability evaluations, and monitoring of anthelmintic resistance. Conclusions: This trial with registrational goals seeks to evaluate an innovative fixed-dose combination of albendazole and ivermectin co-formulated tablets, with the goal of providing an anthelmintic regimen with improved efficacy and spectrum of coverage against STH. ClinicalTrials.gov registration: NCT05124691 (18/11/2021)

Zoonotic Implications of Onchocerca Species on Human Health

The genus Onchocerca includes several species associated with ungulates as hosts, although some have been identified in canids, felids, and humans. Onchocerca species have a wide geographical distribution, and the disease they produce, onchocerciasis, is generally seen in adult individuals because of its large prepatency period. In recent years, Onchocerca species infecting animals have been found as subcutaneous nodules or invading the ocular tissues of humans; the species involved are O. lupi, O. dewittei japonica, O. jakutensis, O. gutturosa, and O. cervicalis. These findings generally involve immature adult female worms, with no evidence of being fertile. However, a few cases with fertile O. lupi, O. dewittei japonica, and O. jakutensis worms have been identified recently in humans. These are relevant because they indicate that the parasite&rsquo;s life cycle was completed in the new host&mdash;humans. In this work, we discuss the establishment of zoonotic Onchocerca infections in humans, and the possibility of these infections to produce symptoms similar to human onchocerciasis, such as dermatitis, ocular damage, and epilepsy. Zoonotic onchocerciasis is thought to be an emerging human parasitic disease, with the need to take measures such as One Health Strategies, in order to identify and control new cases in humans

Light-dependent inhibition of clathrin-mediated endocytosis in yeast unveils conserved functions of the AP2 complex

Summary: Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-β-adaptin/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the β-adaptin/β-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved

A novel benzimidazole derivative shows in vivo anthelmintic activity against in gerbils and sheep

Trabajo presentado al: COMBAR meeting (Combatting Anthelmintic Resistance in Ruminants). Atenas (Grecia). Febrero. 2022