Direct Sequencing of Cryptosporidium in Stool Samples for Public Health

Morris, Arthur; Robinson, Guy; Swain, Martin; Chalmers, Rachel M.

doi:10.3389/fpubh.2019.00360

Cited by 37 publications

(29 citation statements)

References 107 publications

(173 reference statements)

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“…The protozoan parasite Cryptosporidium is an emerging pathogen that causes gastrointestinal disease worldwide. Existing as an infectious, thick-walled oocyst in the environment, this enteric pathogen can infect a wide range of hosts, including wildlife, domestic livestock and humans ( Morris et al, 2019 ). This apicomplexan is transmitted via the fecal-oral route either directly from infected people or animals, or through contaminated water or food ( Vanathy et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

“…Two genetic loci are commonly used to characterize Cryptosporidium positive samples: the small subunit ribosomal RNA (SSU rRNA) gene (also known as the 18S rRNA gene), and the 60-kDa glycoprotein ( gp60 ) gene. The SSU rRNA gene contains variable regions interspersed amongst conserved regions, allowing for effective species and genotype identification ( Morris et al, 2019 ). This region is AT-rich, ranging from 57 to 61% ( Xiao et al, 1999 ).…”

Section: Introductionmentioning

confidence: 99%

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CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification

Yanta

Bessonov

Robinson

et al. 2021

Food and Waterborne Parasitology

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Cryptosporidium is a protozoan parasite that is transmitted to both humans and animals through zoonotic or anthroponotic means. When a host is infected with this parasite, it causes a gastrointestinal disease known as cryptosporidiosis. To understand the transmission dynamics of Cryptosporidium , the small subunit (SSU or 18S) rRNA and gp60 genes are commonly studied through PCR analysis and conventional Sanger sequencing. However, analyzing sequence chromatograms manually is both time consuming and prone to human error, especially in the presence of poorly resolved, heterozygous peaks and the absence of a validated database. For this study, we developed a Cryptosporidium genotyping tool, called CryptoGenotyper, which has the capability to read raw Sanger sequencing data for the two common Cryptosporidium gene targets (SSU rRNA and gp60 ) and classify the sequence data into standard nomenclature. The CryptoGenotyper has the capacity to perform quality control and properly classify sequences using a high quality, manually curated reference database, saving users' time and removing bias during data analysis. The incorporated heterozygous base calling algorithms for the SSU rRNA gene target resolves double peaks, therefore recovering data previously classified as inconclusive. The CryptoGenotyper successfully genotyped 99.3% (428/431) and 95.1% (154/162) of SSU rRNA chromatograms containing single and mixed sequences, respectively, and correctly subtyped 95.6% (947/991) of gp60 chromatograms without manual intervention. This new, user-friendly tool can provide both fast and reproducible analyses of Sanger sequencing data for the two most common Cryptosporidium gene targets.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification

Yanta

Bessonov

Robinson

et al. 2021

Food and Waterborne Parasitology

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“…Worldwide, Cryptosporidium is considered an important protozoan parasite that causes gastroenteritis in a wide range of animals, including humans [1][2][3][4]. Cryptosporidiosis can lead to severe problems in immunocompromised or young hosts, in both animals and humans [5]. In the livestock farming industry, cryptosporidiosis can cause great economic losses due to weight loss and delayed growth in young animals and decreased production in adult animals [1].…”

Section: Introductionmentioning

confidence: 99%

“…Currently, many species and genotypes of Cryptosporidium spp. have been described, in a wide range of animals, livestock, and wild animals [2,3,5,12]. It was previously thought that each species of Cryptosporidium had a limited host range and was able to infect only a single host or closely related host species [3].…”

Section: Introductionmentioning

confidence: 99%

Molecular Epidemiology of Cryptosporidium spp. in an Agricultural Area of Northern Vietnam: A Community Survey

Iwashita

Takemura²,

Tokizawa

et al. 2020

Preprint

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BackgroundCryptosporidium spp. is a protozoan parasite with worldwide distribution that causes cryptosporidiosis in humans and other animals. In the countryside of northern Vietnam, where free-roaming livestock are widespread, cryptosporidiosis is an important zoonotic disease. However, there have been few studies of cryptosporidiosis in Southeast Asia from the perspective of zoonotic disease epidemiology. The purpose of this study was to investigate the occurrence of Cryptosporidium infection in both humans and animals and to gain an awareness of the potential threat posed by this zoonotic infection in northern Vietnam.Methods We conducted a community survey to collect information about cases of diarrhea in an agricultural area of northern Vietnam. For this study, a total of 2715 samples (2120 human diarrheal samples, 471 non-diarrheal human samples, and 124 animal stool samples) were collected. A direct immunofluorescence assay (DFA) was used to detect Cryptosporidium spp. oocysts in concentrated stool samples by observation under a fluorescent microscope. DNA extraction, PCR amplification of the three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp.Results Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were detected; C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample); C. hominis (from four human diarrheal samples); and C. suis (from two pig samples). In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples.Conclusions Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. For example, zoonotic transmission of C. canis, between dogs and humans was suspected. Further studies are needed to assess the risk of oocyst contamination in the wider environment, including water, in this study area.

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“…Most of these loci do not evolve neutrally, and the rate of evolution may vary among the different species of the genus ( Sulaiman, Lal & Xiao, 2002 ). According to the above, phylogenetic inference based on analysis of one or a few genetic loci, some under selection pressure, might not reflect the real phylogenetic relationships at the whole genome level ( Sulaiman, Lal & Xiao, 2002 ; Morris et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Comparative genomic analysis of the principal Cryptosporidium species that infect humans

Arias-Agudelo

García-Montoya

Cabarcas

et al. 2020

PeerJ

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Cryptosporidium parasites are ubiquitous and can infect a broad range of vertebrates and are considered the most frequent protozoa associated with waterborne parasitic outbreaks. The intestine is the target of three of the species most frequently found in humans: C. hominis, C. parvum, and. C. meleagridis. Despite the recent advance in genome sequencing projects for this apicomplexan, a broad genomic comparison including the three species most prevalent in humans have not been published so far. In this work, we downloaded raw NGS data, assembled it under normalized conditions, and compared 23 publicly available genomes of C. hominis, C. parvum, and C. meleagridis. Although few genomes showed highly fragmented assemblies, most of them had less than 500 scaffolds and mean coverage that ranged between 35X and 511X. Synonymous single nucleotide variants were the most common in C. hominis and C. meleagridis, while in C. parvum, they accounted for around 50% of the SNV observed. Furthermore, deleterious nucleotide substitutions common to all three species were more common in genes associated with DNA repair, recombination, and chromosome-associated proteins. Indel events were observed in the 23 studied isolates that spanned up to 500 bases. The highest number of deletions was observed in C. meleagridis, followed by C. hominis, with more than 60 species-specific deletions found in some isolates of these two species. Although several genes with indel events have been partially annotated, most of them remain to encode uncharacterized proteins.

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Direct Sequencing of Cryptosporidium in Stool Samples for Public Health

Cited by 37 publications

References 107 publications

CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification

CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification

Molecular Epidemiology of Cryptosporidium spp. in an Agricultural Area of Northern Vietnam: A Community Survey

Comparative genomic analysis of the principal Cryptosporidium species that infect humans

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