Novel real-time PCR assays for the specific detection of human infective Cryptosporidium species

Cryptosporidium is a protozoan parasite causing gastrointestinal illness. Drinking waterborne outbreaks have been caused by C. hominis, C. parvum and C. cuniculus. Molecular detection techniques already exist for Cryptosporidium and usually target housekeeping genes. We set ourselves the task to identify species-specific genes. These genes are likely to be involved in host parasite interaction and virulence. Three subtelomeric species-specific putative virulence factor genes (Cops-2, Chos-1 and Chos-2) were identified in silico and used to develop novel real-time PCR assays. Our results show that Chos-2 is a suitable target for probe-based assays for the specific detection of C. hominis and C. cuniculus (two very closely related species) and that Cops-2 is a suitable target for specific detection of C. parvum

The genetic basis of host range and virulence in human-infective Cryptosporidium spp.

Cryptosporidium is a leading cause of diarrhoeal disease worldwide. 26 species and more than 70 genotypes have been characterised to date, but only two species, Cryptosporidium hominis and Cryptosporidium parvum, account for a majority of human infections. Despite a close genomic relationship between these species (>96.0% nucleotide identities) the former propagates in a predominantly anthroponotic transmission cycle, while the latter exhibits a broad zoonotic host range and appears well-adapted to both humans and cattle. Intra-C. parvum host range differences have also been demonstrated through the existence of an anthroponotic subtype commonly referred to as ‘IIc’. Using a novel dataset of diverse C. hominis and C. parvum whole genome sequences to perform comparative genomics analyses between zoonotic and anthroponotic subtypes, a number of divergent loci are herein identified that are subject to heightened polymorphism and selective pressure in both C. hominis and C. parvum subtype IIc. Glycoproteins, multi-copy gene families and secreted proteins are predominant, and the subtelomeres houses a significant majority of these highly-variable putative virulence factors. A concatenated phylogeny of novel WGS reveals the existence of an anthroponotic subspecies within the C. parvum species clade, warranting re-classification of zoonotic versus anthroponotic strains as C. parvum parvum and C. parvum anthroponosum, respectively. The concatenated phylogeny also demonstrates a hybrid-type IIcA5G3j isolate, which contains a IIc-type GP60 locus but an otherwise predominantly C. parvum parvum-type genome. Recombination analyses between this hybrid strain and two common zoonotic (IIa-type) and anthroponotic (IIc-type) C. parvum WGS and a C. hominis (Ib-type) WGS shows extensive genome-wide recombination between all of these strains, and divergence dating using recombination data reveals a relatively recent estimated time of divergence (<1,000 years ago) between these species and subspecies. Population genetics analyses of C. hominis GP60 sequences reveals inter-family disparities in allelic divergence and selective pressure, with subtype famailies Ia and Id exhibiting significantly higher rates of diversification. Evidence of a recent population expansion event is also identified for these two subtypes in C. hominis populations from Central Asia and Sub-Saharan Africa. An updated look at whole genome divergence between a ‘virulent’ Ib-type and ‘avirulent’ Ia-type C. hominis WGS reveals structural changes at the subtelomeres resulting in subtype-specific coding sequences, and identifies the location, type and degree of polymorphism and selective pressure that accompanies lineage-specific strain emergence and evolution. Finally, the development of a novel In Situ PCR/Fluorescence In Situ Hybridization technique for sensitive and specific detection of C. p. parvum oocysts is hereby presented. This demonstrates how in silico findings from preceding chapters identified a suitable subspecies-specific target, and were subsequently translated into an applied molecular method for discrimination of lineages at a subspecific level. Taken together, the results from this thesis have contributed significantly towards increasing our understanding of the genomic factors driving evolution and adaptation in human-infective Cryptosporidium spp

Improved subtyping affords better discrimination of Trichomonas gallinae strains and suggests hybrid lineages

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates

Evolutionary genomics of anthroponosis in Cryptosporidium

Human cryptosporidiosis is the leading protozoan cause of diarrhoeal mortality worldwide, and a preponderance of infections is caused by Cryptosporidium hominis and C. parvum. Both species consist of several subtypes with distinct geographical distributions and host preferences (that is, generalist zoonotic and specialist anthroponotic subtypes). The evolutionary processes that drive the adaptation to the human host and the population structures of Cryptosporidium remain unknown. In this study, we analyse 21 whole-genome sequences to elucidate the evolution of anthroponosis. We show that Cryptosporidium parvum splits into two subclades and that the specialist anthroponotic subtype IIc-a shares a subset of loci with C. hominis that is undergoing rapid convergent evolution driven by positive selection. C. parvum subtype IIc-a also has an elevated level of insertion and deletion mutations in the peri-telomeric genes, which is also a characteristic of other specialist subtypes. Genetic exchange between Cryptosporidium subtypes plays a prominent role throughout the evolution of the genus. Interestingly, recombinant regions are enriched for positively selected genes and potential virulence factors, which indicates adaptive introgression. Analysis of 467 gp60 sequences collected from locations across the world shows that the population genetic structure differs markedly between the main zoonotic subtype (isolation-by-distance) and the anthroponotic subtype (admixed population structure). We also show that introgression between the four anthroponotic Cryptosporidium subtypes and species included in this study has occurred recently, probably within the past millennium

Exposure to natural environments during pregnancy and birth outcomes in 11 European birth cohorts

Research suggests that maternal exposure to natural environments (i.e., green and blue spaces) promotes healthy fetal growth. However, the available evidence is heterogeneous across regions, with very few studies on the effects of blue spaces. This study evaluated associations between maternal exposure to natural environments and birth outcomes in 11 birth cohorts across nine European countries. This study, part of the LifeCycle project, was based on a total sample size of 69,683 newborns with harmonised data. For each participant, we calculated seven indicators of residential exposure to natural environments: surrounding greenspace in 100m, 300m, and 500m using Normalised Difference Vegetation Index (NDVI) buffers, distance to the nearest green space, accessibility to green space, distance to the nearest blue space, and accessibility to blue space. Measures of birth weight and small for gestational age (SGA) were extracted from hospital records. We used pooled linear and logistic regression models to estimate associations between exposure to the natural environment and birth outcomes, controlling for the relevant covariates. We evaluated the potential effect modification by socioeconomic status (SES) and region of Europe and the influence of ambient air pollution on the associations. In the pooled analyses, residential surrounding greenspace in 100m, 300m, and 500m buffer was associated with increased birth weight and lower odds for SGA. Higher residential distance to green space was associated with lower birth weight and higher odds for SGA. We observed close to null associations for accessibility to green space and exposure to blue space. We found stronger estimated magnitudes for those participants with lower educational levels, from more deprived areas, and living in the northern European region. Our associations did not change notably after adjustment for air pollution. These findings may support implementing policies to promote natural environments in our cities, starting in more deprived areas

Green spaces and respiratory, cardiometabolic, and neurodevelopmental outcomes:An individual-participant data meta-analysis of &gt;35.000 European children

Studies evaluating the benefits and risks of green spaces on children's health are scarce. The present study aimed to examine the associations between exposure to green spaces during pregnancy and early childhood with respiratory, cardiometabolic, and neurodevelopmental outcomes in school-age children. We performed an Individual-Participant Data (IPD) meta-analysis involving 35,000 children from ten European birth cohorts across eight countries. For each participant, we calculated residential Normalized Difference Vegetation Index (NDVI) within a 300 m buffer and the linear distance to green spaces (meters) during prenatal life and childhood. Multiple harmonized health outcomes were selected: asthma and wheezing, lung function, body mass index, diastolic and systolic blood pressure, non-verbal intelligence, internalizing and externalizing problems, and ADHD symptoms. We conducted a two-stage IPD meta-analysis and evaluated effect modification by socioeconomic status (SES) and sex. Between-study heterogeneity was assessed via random-effects meta-regression. Residential surrounding green spaces in childhood, not pregnancy, was associated with improved lung function, particularly higher FEV1 (β = 0.06; 95 %CI: 0.03, 0.09 I2 = 4.03 %, p &lt; 0.001) and FVC (β = 0.07; 95 %CI: 0.04, 0.09 I2 = 0 %, p &lt; 0.001) with a stronger association observed in females (p &lt; 0.001). This association remained robust after multiple testing correction and did not change notably after adjusting for ambient air pollution. Increased distance to green spaces showed an association with lower FVC (β = −0.04; 95 %CI: −0.07, −0.02, I2 = 4.8, p = 0.001), with a stronger effect in children from higher SES backgrounds (p &lt; 0.001). No consistent associations were found between green spaces and asthma, wheezing, cardiometabolic, or neurodevelopmental outcomes, with direction of effect varying across cohorts. Wheezing and neurodevelopmental outcomes showed high between-study heterogeneity, and the age at outcome assessment was only associated with heterogeneity in internalizing problems. This large European meta-analysis suggests that childhood exposure to green spaces may lead to better lung function. Associations with other respiratory outcomes and selected cardiometabolic and neurodevelopmental outcomes remain inconclusive.</p

Molecular Characterization of Cryptosporidium Species and Giardia duodenalis from Symptomatic Cambodian Children

Background: In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays. Methodology/Principal Findings: Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1–10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67–55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79–11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16–7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58–0.95). Conclusions/Significance: This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites

Associations of Maternal Educational Level, Proximity to Green Space During Pregnancy, and Gestational Diabetes With Body Mass Index From Infancy to Early Adulthood:A Proof-of-Concept Federated Analysis in 18 Birth Cohorts

International sharing of cohort data for research is important and challenging. We explored the feasibility of multicohort federated analyses by examining associations between 3 pregnancy exposures (maternal education, exposure to green vegetation, and gestational diabetes) and offspring body mass index (BMI) from infancy to age 17 years. We used data from 18 cohorts (n = 206,180 mother-child pairs) from the EU Child Cohort Network and derived BMI at ages 0-1, 2-3, 4-7, 8-13, and 14-17 years. Associations were estimated using linear regression via 1-stage individual participant data meta-analysis using DataSHIELD. Associations between lower maternal education and higher child BMI emerged from age 4 and increased with age (difference in BMI z score comparing low with high education, at age 2-3 years = 0.03 (95% confidence interval (CI): 0.00, 0.05), at 4-7 years = 0.16 (95% CI: 0.14, 0.17), and at 8-13 years = 0.24 (95% CI: 0.22, 0.26)). Gestational diabetes was positively associated with BMI from age 8 years (BMI z score difference = 0.18, 95% CI: 0.12, 0.25) but not at younger ages; however, associations attenuated towards the null when restricted to cohorts that measured gestational diabetes via universal screening. Exposure to green vegetation was weakly associated with higher BMI up to age 1 year but not at older ages. Opportunities of cross-cohort federated analyses are discussed.</p