Dataset Information

Evaluation of Next-Generation Sequencing Applied to Cryptosporidium parvum and Cryptosporidium hominis Epidemiological Study.

ABSTRACT: Background. Nowadays, most of the C. parvum and C. hominis epidemiological studies are based on gp60 gene subtyping using the Sanger sequencing (SgS) method. Unfortunately, SgS presents the limitation of being unable to detect mixed infections. Next-Generation Sequencing (NGS) seems to be an interesting solution to overcome SgS limits. Thus, the aim of our study was to (i) evaluate the reliability of NGS as a molecular typing tool for cryptosporidiosis, (ii) investigate the genetic diversity of the parasite and the frequency of mixed infections, (iii) assess NGS usefulness in Cryptosporidium sp. outbreak investigations, and (iv) assess an interpretation threshold of sequencing data. Methods. 108 DNA extracts from positive samples were sequenced by NGS. Among them, two samples were used to validate the reliability of the subtyping obtained by NGS and its capacity to detect DNA mixtures. In parallel, 106 samples from French outbreaks were used to expose NGS to epidemic samples. Results. NGS proved suitable for Cryptosporidium sp. subtyping at the gp60 gene locus, bringing more genetic information compared to SgS, especially by working on many samples simultaneously and detecting more diversity. Conclusions. This study confirms the usefulness of NGS applied to C. hominis and C. parvum epidemiological studies, especially aimed at detecting minority variants.

SUBMITTER: Bailly E

PROVIDER: S-EPMC9414878 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Evaluation of Next-Generation Sequencing Applied to Cryptosporidium parvum and Cryptosporidium hominis Epidemiological Study.

Bailly Eloïse E Valot Stéphane S Vincent Anne A Duffourd Yannis Y Grangier Nadège N Chevarin Martin M Costa Damien D Razakandrainibe Romy R Favennec Loïc L Basmaciyan Louise L Dalle Frédéric F

Pathogens (Basel, Switzerland) 20220818 8

Background. Nowadays, most of the C. parvum and C. hominis epidemiological studies are based on gp60 gene subtyping using the Sanger sequencing (SgS) method. Unfortunately, SgS presents the limitation of being unable to detect mixed infections. Next-Generation Sequencing (NGS) seems to be an interesting solution to overcome SgS limits. Thus, the aim of our study was to (i) evaluate the reliability of NGS as a molecular typing tool for cryptosporidiosis, (ii) investigate the ...[more]

PMID: 36015058

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Project description:BackgroundNon-human primates are often infected with human-pathogenic Cryptosporidium hominis subtypes, but rarely with Cryptosporidium parvum. In this study, 1452 fecal specimens were collected from farmed crab-eating macaques (Macaca fascicularis) in Hainan, China during the period April 2016 to January 2018. These specimens were analyzed for Cryptosporidium species and subtypes by using PCR and sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively.ResultsAltogether, Cryptosporidium was detected using 18S rRNA-based PCR in 132 (9.1%) sampled animals, with significantly higher prevalence in females (12.5% or 75/599 versus 6.1% or 43/706), younger animals (10.7% or 118/1102 in monkeys 1-3-years-old versus 4.0% or 14/350 in those over 3-years-old) and animals with diarrhea (12.6% or 46/365 versus 7.9% or 86/1087). Four Cryptosporidium species were identified, namely C. hominis, C. parvum, Cryptosporidium muris and Cryptosporidium ubiquitum in 86, 30, 15 and 1 animal, respectively. The identified C. parvum, C. hominis and C. ubiquitum were further subtyped by using gp60 PCR. Among them, C. parvum belonged to subtypes in two known subtype families, namely IIoA14G1 (in 18 animals) and IIdA19G1 (in 2 animals). In contrast, C. hominis mostly belonged to two new subtype families Im and In, which are genetically related to Ia and Id, respectively. The C. hominis subtypes identified included ImA18 (in 38 animals), InA14 (in six animals), InA26 (in six animals), InA17 (in one animal) and IiA17 (in three animals). The C. ubiquitum isolates belonged to subtype family XIId. By subtype, ImA18 and IIoA14G1 were detected in animals with diarrhea whereas the remaining ones were mostly found in asymptomatic animals. Compared with C. parvum and C. muris, higher oocyst shedding intensity was observed in animals infected with C. hominis, especially those infected with the Im subtype family.ConclusionsData from the study suggest that crab-eating macaques are infected with diverse C. parvum and C. hominis subtypes. The C. parvum IIo subtype family previously seen in rodents in China has apparently expanded its host range.

Project description:BackgroundWhole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.ResultsThe IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.ConclusionThis represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

Dataset Information

Evaluation of Next-Generation Sequencing Applied to Cryptosporidium parvum and Cryptosporidium hominis Epidemiological Study.

Publications

Evaluation of Next-Generation Sequencing Applied to <i>Cryptosporidium parvum</i> and <i>Cryptosporidium hominis</i> Epidemiological Study.

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