The detection of Cryptosporidium and the resolution of mixtures of species and genotypes from water

Ruecker, Norma J.; Matsune, Joanne C.; Lapen, David R.; Topp, Edward; Edge, Thomas A.; Neumann, Norman F.

doi:10.1016/j.meegid.2012.09.009

Cited by 32 publications

(16 citation statements)

References 16 publications

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“…have been conducted with the goal of better understanding the environmental epidemiology of this parasite. Many studies describe the occurrence of C. meleagridis, C. hominis, C. parvum and C. andersoni in water samples (ARAÚJO et al, 2011;LEE et al, 2013;RUECKER et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

Almeida

Martins

Neto

et al. 2015

Rev. Bras. Parasitol. Vet.

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The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.

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Section: Discussionmentioning

confidence: 99%

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

Almeida

Martins

Neto

et al. 2015

Rev. Bras. Parasitol. Vet.

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“…Molecular characterization of the species and genotypes of Cryptosporidium and Giardia spp. found in environmental samples is necessary to track the origin of contamination, so that it is possible to establish the sources and routes of transmission and to determine the importance of this form of contamination in public health, which is reinforced with the information available about the species identified in the animals inhabiting the region (ROBINSON et al, 2011;RUECKER et al, 2013).…”

Section: Molecular Identification For Characterization Of Infection Smentioning

confidence: 99%

“…Low amounts of oocysts are often identified in environmental samples compared to fecal samples, involving only one round of PCR with reduced capacity to identify protozoa and species diversity as well as the genotypes present in only one sample (NICHOLS et al, 2010;RUECKER et al, 2013;DURIGAN et al, 2014). A way to improve performance in identifying these protozoa is to perform nested PCR repetitions on the same sample, followed by DNA sequencing (RUECKER et al, 2011).…”

Section: Molecular Identification For Characterization Of Infection Smentioning

confidence: 99%

“…A way to improve performance in identifying these protozoa is to perform nested PCR repetitions on the same sample, followed by DNA sequencing (RUECKER et al, 2011). Ruecker et al (2013) analyzed 5 repetitions of nested PCR from the material extracted from the same water sample and observed improvement in the identification of the species and genotypes of Cryptosporidium spp. in a single sample, which favored source tracking and risk evaluation.…”

Section: Molecular Identification For Characterization Of Infection Smentioning

confidence: 99%

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Waterborne Giardia and Cryptosporidium: contamination of human drinking water by sewage and cattle feces

Toledo

Martins

Freire

2017

SCA

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Water is an important vehicle for pathogens such as Cryptosporidium spp. and Giardia duodenalis. In fact, these organisms are responsible for almost 58% and 38% of the waterborne outbreaks reported in ~60 years. Characteristics related to the environmental phase of these protozoa and the different types of hosts are important factors related to environmental contamination. In cryptosporidiosis and giardiasis, outbreaks caused by waterborne pathogens are identified as major risk factors for contamination of water resources by untreated sewage and the entrainment of cattle feces in rainwater. Further, this review covers taxonomic, biologic, and epidemiologic aspects such as prevalence, risk factors, and molecular characterization of these protozoa observed in humans and cattle in order to elucidate the role of these hosts in environmental pollution, and consequently, as a source of infection for susceptible humans, especially in rural areas. Alternative water resources such as wells and springs are used without water treatment and potability monitoring. Epidemiological data are useful, but insufficient to characterize the source of infection, thereby requiring the use of appropriate molecular methods for subtyping the protozoa detected in environmental and feces samples as well as for assessing public health risk. Key words: Sanitation. Giardia duodenalis. Cryptosporidium parvum. Zoonosis. Rural areas. Molecular characterization. ResumoA água é um importante veículo de transmissão de patógenos, entre eles Cryptosporidium spp e Giardia duodenalis, respectivamente responsáveis por 58% e 38% dos surtos de veiculação hídrica registrados em cerca de 60 anos. Características relacionadas às fases ambientais destes protozoários e a grande quantidade de hospedeiros são importantes fatores relacionados à contaminação ambiental. Em surtos de criptosporidiose e giardíase por veiculação hídrica são apontados como principais fatores de risco a contaminação dos mananciais, utilizados para a dessedentação humana, por esgoto não tratado e o desague de fezes de animais devido ao carreamento por água da chuva. Além destes fatores, esta revisão aborda aspectos taxonômicos, biológicos e epidemiológicos, como prevalência, fatores de risco e caracterização molecular destes protozoários em humanos e bovinos como forma de elucidar a importância destes hospedeiros na contaminação ambiental e, consequentemente, como fonte de infecção para humanos suscetíveis, especialmente em áreas rurais. Nestas áreas são frequentemente utilizadas fontes alternativas de abastecimento de água, como poços e nascentes, sem tratamento e monitoramento de potabilidade. Dados epidemiológicos auxiliam, porém não são suficientes para caracterizar fontes de infecção, sendo necessário o uso de adequadas ferramentas de caracterização molecular para subtipagem de protozoários identificados em fezes e amostras ambientais e avaliação do risco de saúde pública.

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“…Traditional microscopy-based detection tools such as U.S. Environmental Protection Agency (EPA) Method 1622/1623 cannot differentiate Cryptosporidium species. Thus, genotyping tools are increasingly used in the assessment of the human-infective potential and source of Cryptosporidium oocysts in source or finished water (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”

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confidence: 99%

Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water

Neumann

Ruecker³

et al. 2015

Appl Environ Microbiol

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jThe occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples. Waterborne Cryptosporidium spp. present a serious threat to human health because of the ubiquitous presence of Cryptosporidium spp. in water and resistance of the oocysts to environmental conditions, various disinfectants, and many treatment practices (1). As watersheds are vulnerable to contamination with both human-pathogenic and nonpathogenic species, sensitive detection of Cryptosporidium oocysts in water and correct identification of oocysts to the species/genotype level are essential for source water management and risk assessment. Traditional microscopy-based detection tools such as U.S. Environmental Protection Agency (EPA) Method 1622/1623 cannot differentiate Cryptosporidium species. Thus, genotyping tools are increasingly used in the assessment of the human-infective potential and source of Cryptosporidium oocysts in source or finished water (2-12).Currently, numerous molecular techniques have been developed for Cryptosporidium detection and genotyping, based mostly on PCR (single-round PCR, nested PCR, real-time PCR, and multiplex PCR, etc.) followed by restriction fragment length polymorphism (RFLP) analysis, DNA sequencing, melt curve analysis, or single-strand conformation polymorphism (SSCP) analysis (13). Among these methods, the real-time PCR technique with melt curve analysis has recently evolved as a sensitive, specific, and time-saving tool for the detection and genotyping of Cryptosporidium spp. in clinical and environmental samples, relying on several gene targets, including the small-subunit (SSU) rRNA, the 70-kDa heat shock protein (hsp70), the Cryptosporidium oocyst wall protein (COWP), the 60-kDa glycoprotein (gp60), and others (14-21). The SSU rRNA gene is the mo...

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The detection of Cryptosporidium and the resolution of mixtures of species and genotypes from water

Cited by 32 publications

References 16 publications

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

Waterborne Giardia and Cryptosporidium: contamination of human drinking water by sewage and cattle feces

Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water

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