Biology subjects

Roose, S.

Publications and source records attributed to Roose, S..

3 recordsLinked to original sources

bioRxiv2026

Development, field-testing and optimization of tools to quantify soil-transmitted helminths in fecal sludge from school pit latrines in Ethiopia

BackgroundSurveys to monitor large-scale deworming programs against soil-transmitted helminthiases (STH) involve examination of stool samples from schoolchildren. These surveys are resource demanding and impact school activities. A potentially cost-saving alternative that does not involve children is to process fecal sludge samples from school pit latrines. To provide a proof-of-principle of latrine-based monitoring of STH programs, we optimized tools to collect fecal sludge and to quantify STH eggs in the samples. MethodsFirst, we designed, developed and field-tested three locally made fecal sludge sampling prototypes. Second, we developed a modified egg-counting method and conducted spiking experiments to explore its analytical performance. Third, we estimated the variation in egg counts in fecal sludge samples collected from six primary schools in Ethiopia at different pit latrine depths used by boys and girls and by repeatedly examining samples. Finally, field data were used to inform an egg count simulation model to quantify this variation in egg counts and to determine the sampling and analysis strategies that resulted in surveys as precise as a stool-based survey. ResultsThe modified fecal sludge sampling prototypes were generally successful, except for a few pit latrines with dried/solid sludge types and insufficient sludge volume. The egg-counting method had moderately high analytical sensitivity that varied across the consistency of the samples. The variation in egg counts was mainly explained by differences between squat holes followed by repeated fecal sludge sample processing. Latrine-based surveys were as precise as stool-based surveys only for Ascaris and when the intensity of infections was low. ConclusionsWe developed a sampling and diagnostic strategy that we will use in a follow-up study. This study will be conducted in Jimma Zone across 25 schools (52 children per school) and will compare the mean fecal sludge egg counts at school level with the STH prevalence in children. Author summaryProgress of large-scale deworming programs are currently monitored through screening individual stool samples of schoolchildren. A latrine-based program monitoring, without active participation of schoolchildren and interruption of routine school activities, is a potentially cost-effective alternative. As a proof-of-concept, we developed a method to quantify worm eggs in fecal sludge samples, conducted spiking experiments to determine its analytical performance and applied the method across schools in Ethiopia to explore variation in egg counts (e.g., squat holes, depth of sample collection, and repeated analyses). Based on our findings, we then determined the sample collection and analysis strategy that results a latrine-based survey as precise as a survey based on screening individual stool samples. The analytical performance was moderately high but varied across the consistency of the samples. The variation in egg counts was mainly driven by variation between squat holes on the same pit latrine, and thus it is better to sample more squat holes (at least 3) than repeatedly processing the same fecal sludge sample (one examination is sufficient). We will now apply this sample collection and analysis across 25 schools (52 children per school) in Ethiopia and compare it with a survey based on screening individual stool samples.

microbiology↗

bioRxiv2025

Over-expression and increased copy numbers of a cytochrome P450 and two UDP-glucuronosyltransferase genes in macrocyclic lactone resistant Psoroptes ovis of cattle

Psoroptes ovis is a mite species that feeds on sheep, cattle, other ungulates, rabbits, and horses, which can develop into a severe exudative dermatitis known as psoroptic mange. The macrocyclic lactone (ML) family of acaricides are commonly used to control psoroptic mange. However, certain strains of cattle and sheep mites have developed resistance against MLs, which has led to reduced treatment efficacy and even treatment failure. Here we investigated the genetic basis of ML resistance in mites collected from cattle across Belgium. We compared gene expression between susceptible and resistant mites before and after exposure to ivermectin and genetic diversity between a single susceptible and resistant populations. We generated chromosomal genome assemblies of Psoroptes ovis derived from sheep and cattle respectively and correlated genomic diversity of susceptible and resistant mite populations sampled across Belgium. Gene expression data revealed constitutive over-expression of a cytochrome P450 monooxygenase (CYP) gene and two tandemly located UDP-glucuronosyltransferase (UGT) genes among others. On investigation of the genomic data, we observed copy number variation at both loci in population genomic data. The CYP gene is not amplified in the susceptible population but occurs in multiple copies in all resistant populations and is associated with a peak in Fst between resistant and susceptible populations indicative of selection. By contrast, the two UGT genes are massively and tandemly amplified in all populations including the susceptible dataset with a weaker associated signal of selection than the amplified CYP. Hence, distinct mechanisms of amplification and gene regulation are occurring at these putative resistance loci in P. ovis. Author SummaryFor the first time, we investigated the genetic basis of resistance in scab mites to a key family of drugs (Macrocyclic Lactones) used to control this parasite. Scab mite infestations can cause serious welfare issues in affected cattle and sheep with associated economic impact on production. To identify genes associated with resistance, we applied a combination of approaches including targeted sequencing of candidate genes, genome sequencing and gene expression comparisons of resistant mites with mites that are still susceptible to treatment. We were able to rule-out one family of candidate genes but identified very high expression of genes from two gene families that metabolise, and thereby detoxify, the treatment drug in resistant mites. On examination of the genomic context of these genes we found that the genes had undergone an increase in copy number in the resistant mites compared to the susceptible mites. With our now much increased understanding of resistance in scab mites we can track the spread of resistance using markers in these genes present in resistant mites. We can also now test the suitability of alternative treatments that restore the lethality of Macrocyclic Lactones in scab mites, mitigating the damaging effects of resistance in this species.

genomics↗

bioRxiv2024

The assessment of single nucleotide polymorphisms in the ss-tubulin genes in human soil-transmitted helminths exposed to different pressure with benzimidazole drugs

BackgroundWe aimed to gain insights into the role of known single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the {beta}-tubulin gene as markers for possible benzimidazole resistance in human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus and Ancylostsoma duodenale). MethodsFirstly, we determined the analytical performance of our PCR/pyrosequencing assays. Secondly, we applied them on stool samples collected during clinical trials in Ethiopia, Lao PDR, and Pemba Island (Tanzania) to assess any associations between the presence/ratio of mutant (MT): wild type (WT) SNPs and drug pressure history, individual drug response and time of sampling (baseline vs. follow-up sample). Principal findingsOverall, the limit of blank of our in-house PCR/pyrosequencing assays to detect MT SNPs was non-zero ([~]3.5%), and hence the limit of detection for MT SNPs was relatively high (2% - 7%). The assays systematically overestimated the true underlying ratio of MT:WT SNPS within sample, but we derived functions for more accurate estimates. The assays were more precise when the ratio MT:WT SNPs was high (>5%). No PCR amplicon was observed in 25% of the samples subjected to PCR. In the remaining samples, the presence of MT SNPs in codon 200 was detected in half of the analysed Trichuris samples, the proportion of the analysed samples containing MT SNPs did not exceed 14% for all other codons and STH species. Associations between drug pressure history, individual drug response and time of sampling, were not consistent across all codons and STHs. ConclusionWe could not provide compelling evidence for the role of the known SNPs in the {beta}-tubulin gene as markers for benzimidazole resistance. Our study also highlights that there is a need to assess the diagnostic performance of any assays in order to readily interpret and compare results. Further research should therefore also focus on genes other than the {beta}-tubulin genes. Author summaryAlthough large-sale deworming programs are reducing the morbidity caused by intestinal worms, widespread treatment of large populations for a long period of time may trigger drug resistance. An early detection of DNA mutations that may give rise to resistant worm population is therefore important. We evaluated the analytical performance of in-house assays to detect DNA mutations that are known to cause resistant intestinal worms of animals. Subsequently, we applied these assays on stool samples to verify (i) whether the mutations are more prevalent in areas were large proportions of children have been dewormed for a longer period, (ii) a poor individual drug response can be explained by higher frequency of the mutations. Our results indicate that comprehensive evaluation of the analytical performance of the genotyping tests was required to readily interpret the results. We did not find any compelling evidence that the presence of mutations was associated with either drug pressure or poor individual drug responses. This suggests that it is warranted to explore other mutations than those documented in animal worms.

molecular biology↗