Biology subjects

Mekonnen, Z.

Publications and source records attributed to Mekonnen, Z..

5 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

Enrichment of helminth mitochondrial genomes from faecal samples using hybridisation capture

New approaches are urgently needed to enrich rare or low-abundant DNA in complex samples. Soil-transmitted helminths (STHs) inhabit heterogeneous environments, including the gastrointestinal tract of their host as adults and are excreted as eggs and larvae in faeces, complicating our understanding of their biology and the use of genetic tools for species monitoring and population tracking. We have developed a hybridisation capture approach to enrich mitochondrial genome sequences of two STH species, the roundworm Ascaris lumbricoides and whipworm Trichuris trichiura, from extracted DNA from faecal material and worm specimens. Employing [~]1000 targeted probes, we achieved >6,000 and >12,000 fold enrichment for A. lumbricoides and T. trichiura, respectively, relative to direct whole genome shotgun (WGS) sequencing. Sequencing coverage was highly concordant with probe targets and correlated with the number of eggs per gram (EPG) of parasites present, from which DNA from as few as 336 EPG for Ascaris and 48 EPG for Trichuris were efficiently captured and sufficient to provide effective mitochondrial genome data. Finally, allele frequencies were highly concordant between WGS and hybridisation capture, suggesting little genetic information is lost with additional sample processing required for enrichment. Our hybridisation capture design and approach enables sensitive and scalable STH mitochondrial genome sampling from faecal DNA extracts and paves the way for broader hybridisation capture-based genome-wide applications and molecular epidemiology studies of STHs.

genomics↗

bioRxiv2024

Global diversity of soil-transmitted helminths reveals population-biased genetic variation that impacts diagnostic targets

Soil-transmitted helminths (STHs) are intestinal pathogens affecting over a billion people worldwide. STH control relies on microsopy-based diagnostics to monitor parasite prevalence and enable post-treatment surveillance, however, molecular diagnostics are rapidly being developed due to increased sensitivity, particularly in low-STH-prevalence settings. The genetic diversity of helminths and its potential impact on molecular diagnostics remains unclear. Using low-coverage genome sequencing, we assessed the presence of STHs in worm, faecal and purified egg samples from 27 countries, identifying differences in the genetic connectivity and diversity of STH-positive samples across regions and cryptic diversity between closely related human- and pig-infective species. We defined substancial copy number and sequence variants in current diagnostic targets and validated the impact of genetic variants on qPCR diagnostics using in vitro assays. Our study provides insights into the diversity and genomic epidemiology of STHs, highlighting challenges and opportunities for developing molecular diagnostics needed to support STH control efforts.

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↗

bioRxiv2021

Population genomics of ancient and modern Trichuris trichiura

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

genomics↗