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BACKGROUND: Giardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified. METHODS: This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages. RESULTS: Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages. CONCLUSIONS: The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.
Assuntos
Antiprotozoários , Variações do Número de Cópias de DNA , Resistência a Medicamentos , Giardia lamblia , Giardíase , Metronidazol , Giardia lamblia/genética , Giardia lamblia/efeitos dos fármacos , Metronidazol/farmacologia , Resistência a Medicamentos/genética , Humanos , Giardíase/parasitologia , Giardíase/tratamento farmacológico , Antiprotozoários/farmacologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Protozoários/genéticaRESUMO
Diet impacts human health, influencing body adiposity and the risk of developing cardiometabolic diseases. The gut microbiome is a key player in the diet-health axis, but while its bacterial fraction is widely studied, the role of micro-eukaryotes, including Blastocystis, is underexplored. We performed a global-scale analysis on 56,989 metagenomes and showed that human Blastocystis exhibits distinct prevalence patterns linked to geography, lifestyle, and dietary habits. Blastocystis presence defined a specific bacterial signature and was positively associated with more favorable cardiometabolic profiles and negatively with obesity (p < 1e-16) and disorders linked to altered gut ecology (p < 1e-8). In a diet intervention study involving 1,124 individuals, improvements in dietary quality were linked to weight loss and increases in Blastocystis prevalence (p = 0.003) and abundance (p < 1e-7). Our findings suggest a potentially beneficial role for Blastocystis, which may help explain personalized host responses to diet and downstream disease etiopathogenesis.
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The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Surtos de Doenças , Cryptosporidium parvum/genética , Estados Unidos/epidemiologia , Europa (Continente)/epidemiologia , Humanos , Criptosporidiose/parasitologia , Criptosporidiose/epidemiologia , Animais , Genômica/métodos , Polimorfismo de Nucleotídeo Único , Filogenia , Sequenciamento Completo do Genoma/métodos , Genoma de Protozoário , China/epidemiologia , Egito/epidemiologiaRESUMO
While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.
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Cryptosporidium parvum , Cryptosporidium , Oocistos , Cryptosporidium/genética , Fatores de Risco , Saúde Pública , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.
Assuntos
Equinococose , Echinococcus multilocularis , Animais , Echinococcus multilocularis/genética , Filogenia , Equinococose/epidemiologia , Equinococose/veterinária , Equinococose/parasitologia , Europa (Continente)/epidemiologia , Zoonoses , Raposas/parasitologia , Variação GenéticaRESUMO
Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.
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Giardia lamblia , Giardíase , Humanos , Giardia lamblia/genética , Giardíase/parasitologia , Giardia/genética , GenômicaRESUMO
Cryptosporidium parvum is a globally distributed zoonotic pathogen and a major cause of diarrhoeal disease in humans and ruminants. The parasite's life cycle comprises an obligatory sexual phase, during which genetic exchanges can occur between previously isolated lineages. Here, we compare 32 whole genome sequences from human- and ruminant-derived parasite isolates collected across Europe, Egypt and China. We identify three strongly supported clusters that comprise a mix of isolates from different host species, geographic origins, and subtypes. We show that: (1) recombination occurs between ruminant isolates into human isolates; (2) these recombinant regions can be passed on to other human subtypes through gene flow and population admixture; (3) there have been multiple genetic exchanges, and most are probably recent; (4) putative virulence genes are significantly enriched within these genetic exchanges, and (5) this results in an increase in their nucleotide diversity. We carefully dissect the phylogenetic sequence of two genetic exchanges, illustrating the long-term evolutionary consequences of these events. Our results suggest that increased globalization and close human-animal contacts increase the opportunity for genetic exchanges between previously isolated parasite lineages, resulting in spillover and spillback events. We discuss how this can provide a novel substrate for natural selection at genes involved in host-parasite interactions, thereby potentially altering the dynamic coevolutionary equilibrium in the Red Queens arms race.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Animais , Humanos , Cryptosporidium parvum/genética , Criptosporidiose/parasitologia , Cryptosporidium/genética , Filogenia , RuminantesRESUMO
BACKGROUND: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. METHODS: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. RESULTS: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. CONCLUSION: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.
Assuntos
Giardia lamblia , Giardíase , Animais , Humanos , Giardíase/parasitologia , Tipagem de Sequências Multilocus , Filogenia , Genótipo , Fezes/parasitologia , Mamíferos/genéticaRESUMO
Cryptosporidium is a leading global cause of waterborne disease, with many reported outbreaks related to main water supplies. In August 2019, an outbreak of cryptosporidiosis involving 80 cases occurred among 114 vacationers in a small municipality located in the Tuscan-Emilian Apennines, north-eastern Italy. After excluding a potential food-borne outbreak, the epidemiological investigation focussed on the hypothesis of a waterborne outbreak. This was confirmed by the finding of Cryptosporidium oocysts in stools of the cases and in water samples from the municipal water network. Molecular characterisation revealed the zoonotic species Cryptosporidium parvum as the causative agent. A single subtype (IIdA25G1) was found among all cases, and in one of two positive water samples. The municipality's water supply used spring water that only received a disinfection treatment insufficient to inactivate the parasite. Possible entry means into the water mains were found through further environmental investigations. As these types of water supplies are particularly vulnerable to various environmental factors, a control system based on the risk assessment of each phase of the water supply chain is required to guarantee water safety. Effective methods for detection of protozoan pathogens, which are generally excluded from routine water supply analysis, should be applied.
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Criptosporidiose , Cryptosporidium , Água Potável , Criptosporidiose/diagnóstico , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/genética , Surtos de Doenças , Humanos , Abastecimento de ÁguaRESUMO
Large-scale comparative genomics- and population genetic studies generate enormous amounts of polymorphism data in the form of DNA variants. Ultimately, the goal of many of these studies is to associate genetic variants to phenotypes or fitness. We introduce VIVID, an interactive, user-friendly web application that integrates a wide range of approaches for encoding genotypic to phenotypic information in any organism or disease, from an individual or population, in three-dimensional (3D) space. It allows mutation mapping and annotation, calculation of interactions and conservation scores, prediction of harmful effects, analysis of diversity and selection, and 3D visualization of genotypic information encoded in Variant Call Format on AlphaFold2 protein models. VIVID enables the rapid assessment of genes of interest in the study of adaptive evolution and the genetic load, and it helps prioritizing targets for experimental validation. We demonstrate the utility of VIVID by exploring the evolutionary genetics of the parasitic protist Plasmodium falciparum, revealing geographic variation in the signature of balancing selection in potential targets of functional antibodies.
Assuntos
Genômica , Software , Genômica/métodos , Genótipo , Fenótipo , Polimorfismo GenéticoRESUMO
Cryptosporidiosis is a major global health problem and a primary cause of diarrhea, particularly in young children in low- and middle-income countries (LMICs). The zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis cause most human infections. Here, we present a comprehensive whole-genome study of C. hominis, comprising 114 isolates from 16 countries within five continents. We detect two lineages with distinct biology and demography, which diverged circa 500 years ago. We consider these lineages two subspecies and propose the names C. hominis hominis and C. hominis aquapotentis (gp60 subtype IbA10G2). In our study, C. h. hominis is almost exclusively represented by isolates from LMICs in Africa and Asia and appears to have undergone recent population contraction. In contrast, C. h. aquapotentis was found in high-income countries, mainly in Europe, North America, and Oceania, and appears to be expanding. Notably, C. h. aquapotentis is associated with high rates of direct human-to-human transmission, which may explain its success in countries with well-developed environmental sanitation infrastructure. Intriguingly, we detected genomic regions of introgression following secondary contact between the subspecies. This resulted in high diversity and divergence in genomic islands of putative virulence genes, including muc5 (CHUDEA2_430) and a hypothetical protein (CHUDEA6_5270). This diversity is maintained by balancing selection, suggesting a co-evolutionary arms race with the host. Finally, we find that recent gene flow from C. h. aquapotentis to C. h. hominis, likely associated with increased human migration, maybe driving the evolution of more virulent C. hominis variants.
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Criptosporidiose , Cryptosporidium , Criança , Pré-Escolar , Criptosporidiose/epidemiologia , Criptosporidiose/genética , Cryptosporidium/genética , DNA de Protozoário/genética , Genoma , Genótipo , Humanos , MetagenômicaRESUMO
Giardiasis, the disease caused by the flagellate Giardia duodenalis (syn. G.lamblia, G. intestinalis), is the most commonly reported among the five food- and waterborne parasitic diseases under mandatory surveillance in 24 EU countries. From November 2018 to April 2019, an outbreak of giardiasis occurred in a municipality of the Bologna province, in north-eastern Italy. Microscopy and immunochromatography identified cysts and antigens, respectively, of the parasite in stool samples of 228 individuals. Molecular typing of 136 stool samples revealed a vast predominance (95%) of G. duodenalis assemblage B. Investigations into potential sources indicated tap water as the most likely vehicle of infection, although cysts were not detected in water samples. Control measures mostly aimed at preventing secondary transmission by informing citizens about the outbreak, and by treatment of patients with anti-parasitic drugs. This is the first documented human outbreak of giardiasis in Italy; its investigation has highlighted the difficulties in the timely detection and management of this parasite, which is often overlooked as a cause of human gastroenteritis. The long and variable incubation time, absence of specific symptoms and a general lack of awareness about this pathogen contributed to delay in diagnosis.
Assuntos
Giardia lamblia , Giardíase , Surtos de Doenças , Fezes , Genótipo , Giardia/genética , Giardia lamblia/genética , Giardíase/diagnóstico , Giardíase/epidemiologia , HumanosRESUMO
Parasites often have complex developmental cycles that account for their presence in a variety of difficult-to-analyze matrices, including feces, water, soil, and food. Detection of parasites in these matrices still involves laborious methods. Untargeted sequencing of nucleic acids extracted from those matrices in metagenomic projects may represent an attractive alternative method for unbiased detection of these pathogens. Here, we show how publicly available metagenomic datasets can be mined to detect parasite specific sequences, and generate data useful for environmental surveillance. We use the protozoan parasite Cryptosporidium parvum as a test organism, and show that detection is influenced by the reference sequence chosen. Indeed, the use of the whole genome yields high sensitivity but low specificity, whereas specificity is improved through the use of signature sequences. In conclusion, querying metagenomic datasets for parasites is feasible and relevant, but requires optimization and validation. Nevertheless, this approach provides access to the large, and rapidly increasing, number of datasets from metagenomic and meta-transcriptomic studies, allowing unlocking hitherto idle signals of parasites in our environments.
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To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.
Assuntos
Cyclospora/crescimento & desenvolvimento , Echinococcus multilocularis/crescimento & desenvolvimento , Fast Foods/parasitologia , Contaminação de Alimentos/análise , Frutas/parasitologia , Animais , Mirtilos Azuis (Planta)/parasitologia , Cyclospora/genética , Cyclospora/isolamento & purificação , Echinococcus multilocularis/genética , Echinococcus multilocularis/isolamento & purificação , Itália , México , Oocistos/genética , Oocistos/isolamento & purificação , Rubus/parasitologia , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/isolamento & purificaçãoRESUMO
BACKGROUND: Cryptosporidiosis has been identified as one of the major causes of diarrhea and diarrhea-associated deaths in young children in sub-Saharan Africa. This study traces back Cryptosporidium-positive children to their human and animal contacts to identify transmission networks. METHODS: Stool samples were collected from children < 5 years of age with diarrhea in Gabon, Ghana, Madagascar, and Tanzania. Cryptosporidium-positive and -negative initial cases (ICs) were followed to the community, where stool samples from households, neighbors, and animal contacts were obtained. Samples were screened for Cryptosporidium species by immunochromatographic tests and by sequencing the 18S ribosomal RNA gene and further subtyped at the 60 kDa glycoprotein gene (gp60). Transmission clusters were identified and risk ratios (RRs) calculated. RESULTS: Among 1363 pediatric ICs, 184 (13%) were diagnosed with Cryptosporidium species. One hundred eight contact networks were sampled from Cryptosporidium-positive and 68 from negative ICs. Identical gp60 subtypes were detected among 2 or more contacts in 39 (36%) of the networks from positive ICs and in 1 contact (1%) from negative ICs. In comparison to Cryptosporidium-negative ICs, positive ICs had an increased risk of having Cryptosporidium-positive household members (RR, 3.6 [95% confidence interval {CI}, 1.7-7.5]) or positive neighboring children (RR, 2.9 [95% CI, 1.6-5.1]), but no increased risk of having positive animals (RR, 1.2 [95% CI, .8-1.9]) in their contact network. CONCLUSIONS: Cryptosporidiosis in rural sub-Saharan Africa is characterized by infection clusters among human contacts, to which zoonotic transmission appears to contribute only marginally.
Assuntos
Criptosporidiose , Cryptosporidium , Animais , Criança , Pré-Escolar , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Fezes , Gabão , Genótipo , Gana , Humanos , Madagáscar , TanzâniaRESUMO
Although Giardia duodenalis is recognized as one of the leading causes of parasitic human diarrhea in the world, knowledge of the mechanisms of infection is limited, as the pathophysiological consequences of infection remain incompletely elucidated. Similarly, the reason for and consequences of the very specific genome-organization in this parasite with 2 active nuclei is only partially known. Consistent with its tradition, the 7th International Giardia and Cryptosporidium Conference (IGCC 2019) was held from June 23 to 26, 2019, at the Faculty of Medicine and Pharmacy of the University of Rouen-Normandie, France, to discuss current research perspectives in the field. This renowned event brought together an international delegation of researchers to present and debate recent advances and identify the main research themes and knowledge gaps. The program for this interdisciplinary conference included all aspects of host-parasite relationships, from basic research to applications in human and veterinary medicine, as well as the environmental issues raised by water-borne parasites and their epidemiological consequences. With regard to Giardia and giardiasis, the main areas of research for which new findings and the most impressive communications were presented and discussed included: parasite ecology and epidemiology of giardiasis, Giardia-host interactions, and cell biology of Giardia, genomes and genomic evolution. The high-quality presentations discussed at the Conference noted breakthroughs and identified new opportunities that will inspire researchers and funding agencies to stimulate future research in a "one health" approach to improve basic knowledge and clinical and public health management of zoonotic giardiasis.
TITLE: Mise à jour sur Giardia et la giardiase : faits saillants de la Septième Conférence Internationale sur Giardia et Cryptosporidium. ABSTRACT: Bien que Giardia duodenalis soit reconnu comme l'une des principales causes de diarrhée parasitaire humaine dans le monde, la connaissance des mécanismes de l'infection est limitée, car ses conséquences physiopathologiques restent incomplètement élucidées. De même, la raison et les conséquences de l'organisation génomique très spécifique de ce parasite à deux noyaux actifs ne sont que partiellement connues. Conformément à sa tradition, la 7ème Conférence internationale sur Giardia et Cryptosporidium (IGCC 2019) s'est tenue du 23 au 26 juin 2019, à la Faculté de médecine et de pharmacie de l'Université de Rouen-Normandie, France, pour discuter des perspectives de recherche actuelles dans ce champ. Cet événement de renom a réuni une délégation internationale de chercheurs pour présenter et débattre des avancées récentes et identifier les principaux thèmes de recherche et les lacunes dans les connaissances. Le programme de cette conférence interdisciplinaire comprenait tous les aspects des relations hôtes-parasites, de la recherche fondamentale aux applications en médecine humaine et vétérinaire, ainsi que les problèmes environnementaux soulevés par les parasites d'origine hydrique et leurs conséquences épidémiologiques. En ce qui concerne Giardia et la giardiase, les principaux domaines de recherche pour lesquels de nouvelles découvertes et les communications les plus impressionnantes ont été présentées et discutées comprenaient : l'écologie parasitaire et l'épidémiologie de la giardiase, les interactions Giardia-hôte, la biologie cellulaire de Giardia, les génomes et l'évolution génomique. Les présentations de haute qualité discutées lors de la conférence ont noté des avancées et identifié de nouvelles opportunités qui inspireront les chercheurs et les agences de financement à stimuler la recherche future dans une approche « une seule santé ¼ pour améliorer les connaissances de base et la gestion clinique et de santé publique de la giardiase zoonotique.
Assuntos
Giardia , Giardíase , Animais , Criptosporidiose/epidemiologia , Cryptosporidium/genética , França , Giardia/genética , Giardíase/epidemiologia , Giardíase/parasitologia , HumanosRESUMO
Shotgun metagenomics with high-throughput sequencing (HTS) techniques is increasingly used for pathogen identification and characterization. While many studies apply targeted amplicon sequencing, here we used untargeted metagenomics to simultaneously identify protists and helminths in pre-diagnosed faecal and tissue samples. The approach starts from RNA and operates without an amplification step, therefore allowing the detection of all eukaryotes, including pathogens, since it circumvents the bias typically observed in amplicon-based HTS approaches. The generated metagenomics datasets were analysed using the RIEMS tool for initial taxonomic read assignment. Mapping analyses against ribosomal reference sequences were subsequently applied to extract 18S rRNA sequences abundantly present in the sequence datasets. The original diagnosis, which was based on microscopy and/or PCR, could be confirmed in nearly all cases using ribosomal RNA metagenomics. In addition to the pre-diagnosed taxa, we detected other intestinal eukaryotic parasites of uncertain pathogenicity (of the genera Dientamoeba, Entamoeba, Endolimax, Hymenolepis) that are often excluded from routine diagnostic protocols. The study clearly demonstrates the applicability of untargeted RNA metagenomics for the parallel detection of parasites.
Assuntos
Enteropatias Parasitárias/diagnóstico , Metagenômica , Técnicas de Diagnóstico Molecular/métodos , Parasitos/isolamento & purificação , Animais , Fezes/parasitologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Parasitos/classificação , Parasitos/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
Data sharing enables research communities to exchange findings and build upon the knowledge that arises from their discoveries. Areas of public and animal health as well as food safety would benefit from rapid data sharing when it comes to emergencies. However, ethical, regulatory and institutional challenges, as well as lack of suitable platforms which provide an infrastructure for data sharing in structured formats, often lead to data not being shared or at most shared in form of supplementary materials in journal publications. Here, we describe an informatics platform that includes workflows for structured data storage, managing and pre-publication sharing of pathogen sequencing data and its analysis interpretations with relevant stakeholders.
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Bases de Dados Factuais , Disseminação de Informação , Bactérias/classificação , Metagenômica , Filogenia , Interface Usuário-ComputadorRESUMO
The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12â¯Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1â¯ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.
Assuntos
DNA de Protozoário/genética , Giardia lamblia/genética , Giardíase/parasitologia , Pré-Escolar , Biologia Computacional , República Tcheca , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Feminino , Estudo de Associação Genômica Ampla , Variação Estrutural do Genoma , Humanos , Mutação , Técnicas de Amplificação de Ácido Nucleico , Fases de Leitura Aberta/genéticaRESUMO
The aim of this study was to evaluate the occurrence of Cryptosporidium oocysts in a drinking water treatment plant (DWTP) located in a rural area of northern Italy. Influent and effluent samples were collected at the DWTP over three years (2013-2016). In parallel, tap water samples from a public drinking fountain were collected as well. All samples were analyzed for the presence of Cryptosporidium spp. oocysts by a common method based on an immunomagnetic separation (IMS)/immunofluorescence assay (IFA), complemented by 4,6-diamidino-2-phenylindole (DAPI) staining. A reverse transcriptase-PCR (RT-PCR) protocol was added to evaluate oocyst viability. The results highlighted a high variability of oocyst concentrations across all samples (mean 4.3 ± 5.8/100 L) and a high variability in the percentage of DAPI-positive specimens (mean 48.2% ± 40.3%). Conversely, RT-PCR did not reveal the presence of viable C. parvum and C. hominis oocysts. A nested PCR targeting Cryptosporidium 18S ribosomal DNA, carried out in two water samples, confirmed the presence of a Cryptosporidium genotype associated with wild animals in the river and in tap water. The results obtained underline the vulnerability of the investigated surface water to Cryptosporidium spp. contamination. Although the recovered Cryptosporidium genotype is not a human pathogen, its presence demonstrates the existence of a potential pathogen Cryptosporidium spp. contamination risk. Moreover, these results underline the importance of also considering unconventional (not bacterial) biological contaminations (protozoa) in water resources in rural areas, including those of developed countries.