Background The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs. Methodology and principal findings First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. Significance This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Background We previously demonstrated that serology holds promise as an alternative diagnostic tool to copromicroscopy to monitor and evaluate deworming programs targeting soil-transmitted helminths (STHs). Here we explored the dynamics of anti-Ascaris antibodies (Ab) and evaluated the Ab-isotype of choice to assess the longitudinal exposure to Ascaris in Ethiopian school children. Methodology Between October 2018 and February 2020, stool and blood samples were collected every four months from school children (4 to 6 years of age). Stool samples were analyzed by duplicate Kato-Katz to assess the presence and intensity of any STH infection. Plasma Ab-responses against the total extract of Ascaris suum lung third stage larvae were measured through in-house Ab-ELISA’s for seven different Ab-isotypes. Principal findings At baseline, 42.4% of the 66 children were excreting eggs of any STH, Trichuris (37.9%) being the most prevalent. The cumulative prevalence (proportion of children tested that positive at least once over the entire study period) was 56.1% for Trichuris and 31.8% for Ascaris. For Ascaris, re-infections were frequently observed, whereas for Trichuris, children often remained excreting eggs following drug administration. When measuring anti-Ascaris Ab-levels, the cumulative seroprevalence was generally higher (IgG4: 60.6%; IgG1: 50.0%; IgE: 36.4%). The individual anti-Ascaris IgG4 levels at baseline were positively associated with the fecal egg counts averaged over the study period, the rate of egg-appearance and the number of positive test results. There was no apparent cross-reactivity between the anti-Ascaris IgG4 Ab-ELISA and Trichuris. Conclusions/Significance We demonstrate that the children are exposed to STH before the age of four and that both the exposure to Ascaris, highlighting that the exposure to disease is underestimated when measured with copromicroscopy. Compared to other Ab-isotypes, IgG4 is the Ab-isotype of choice to measure Ascaris exposure in STH endemic settings. Finally, the results also highlight that measuring anti-Ascaris IgG4 levels holds promise as a tool to identify individuals at higher risk for continued exposure to this STH.

Background WHO recommends periodical assessment of the prevalence of any soil-transmitted helminth (STH) infections to adapt the frequency of mass drug administration targeting STHs. Today, detection of eggs in stool smears (Kato-Katz thick smear) remains the diagnostic standard. However, stool examination (coprology) has important operational drawbacks and impedes integrated surveys of multiple neglected tropical diseases. Therefore, the aim of the present study was to assess the potential of applying serology instead of coprology in STH control program decision-making. Methodology An antibody-ELISA based on extract of Ascaris lung stage larvae (AsLungL3-ELISA) was applied in ongoing monitoring activities of the Ethiopian national control program against schistosomiasis and soil-transmitted helminthiasis. Blood and stool samples were collected from over 6,700 students (median age: 11) from 63 schools in 33 woredas (districts) across the country. Stool samples of two consecutive days were analyzed applying duplicate Kato-Katz thick smear. Principal findings On woreda level, qualitative (seroprevalence) and quantitative (mean optical density ratio) serology results were highly correlated, and hence seroprevalence was chosen as parameter. For 85% of the woredas, prevalence based on serology was higher than those based on coprology. The results suggested cross-reactivity of the AsLungL3-ELISA with Trichuris. When extrapolating the WHO coproprevalence thresholds, there was a moderate agreement (weighted κ = 0.43) in program decision-making. Using the same threshold values would predominantly lead to a higher frequency of drug administration. Significance This is the first time that serology for soil-transmitted helminthiasis is applied on such large scale, thereby embedded in a control program context. The results underscore that serology holds promise as a tool to monitor STH control programs. Further research should focus on the optimization of the diagnostic assay and the refinement of serology-specific program decision-making thresholds.