Potential molecular tools for assessing the public health risk associated with waterborne Cryptosporidium oocysts

Kothavade, R. J.

doi:10.1099/jmm.0.043158-0

Cited by 33 publications

(14 citation statements)

References 87 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, without assays determining the species/genotype, viability or infectivity, all oocysts detected by microscopy must be assumed to present a public health risk (Figure 1). Viability and infectivity assays have been reviewed recently by Kothavade, who highlights the difficulties in applying these to the small numbers of oocysts often present in water samples, lack of interlaboratory trials and validation and difficulties in interpretation of the data [55]. In contrast, some genotyping assays have been standardised and applied to counted oocysts from microscope slides; thus both sets of data are collected: the oocyst count and the species, improving the data for assessment of risk to public health.…”

Section: Application Of Molecular Assays For Species Determination Anmentioning

confidence: 99%

“…Other methods, such as laser capture microscopy and reverse line hybridization are at developmental stages, and loop-mediated isothermal amplification have yet to be validated independently [55]. The benchmark assay is a specialist test requiring equipment and skill outwith the scope of most routine diagnostic or detection laboratories.…”

Section: Application Of Molecular Assays For Species Determination Anmentioning

confidence: 99%

See 1 more Smart Citation

Waterborne outbreaks of cryptosporidiosis

Chalmers

2012

Annali Dell'Istituto Superiore Di Sanità

102

View full text Add to dashboard Cite

Abstract. Water is the most commonly reported vehicle of transmission in Cryptosporidium outbreaks. While mains drinking water quality is highly regulated in industrialised countries, treated recreational water venues remain highly variable and these have emerged as important settings in the transmission of cryptosporidiosis. Epidemiological investigations of outbreaks benefit from supplementary microbiological evidence and, more recently, the application of molecular typing data to link isolates from cases to each other and to suspected sources. This article documents how waterborne Cryptosporidium outbreaks are identified and reported, how such outbreaks have acted as drivers of regulatory change, and some of the recent developments in the detection and investigation of these outbreaks and their spread, especially the application of molecular typing assays.Key words: Cryptosporidium, genotyping, recreational, drinking, water.Riassunto (Epidemie di cryptosporidiosi trasmesse con le acque). L'acqua è il più comune veicolo di trasmissione di epidemie dovute a Cryptosporidium. Mentre la qualità delle acque potabili distribuite da acquedotti è fortemente regolamentata nei paesi industrializzati, la qualità delle acque trattate di ambienti ricreativi chiusi è fortemente variabile e questi ambienti si sono rivelati importanti nella trasmissione della criptosporidiosi. Le indagini epidemiologiche sulle epidemie traggono beneficio dalle evidenze microbiologiche e, più recentemente, dall'applicazione dei dati di tipizzazione molecolare per collegare i ceppi isolati con i casi d'infezione e con le fonti di contagio sospette. Quest'articolo documenta come vengono individuate e notificate le epidemie di Cryptosporidium trasmesse con le acque, come tali epidemie hanno agito da guida per migliorare la normativa, ed alcuni recenti sviluppi nella rilevazione e nelle indagini di queste epidemie e della loro diffusione, in particolare l'applicazione dei saggi di tipizzazione molecolare.Parole chiave: Cryptosporidium, genotipizzazione, acqua ad uso ricreativo, acqua potabile.

show abstract

Section: Application Of Molecular Assays For Species Determination Anmentioning

confidence: 99%

Section: Application Of Molecular Assays For Species Determination Anmentioning

confidence: 99%

Waterborne outbreaks of cryptosporidiosis

Chalmers

2012

Annali Dell'Istituto Superiore Di Sanità

102

View full text Add to dashboard Cite

show abstract

“…Cryptosporidium species are commonly monitored in waters using standard detection methodologies (e.g., EPA Method 1622 and Method 1623). However, these methods provide no information on either the infectivity or identity of the detected oocyst, providing little information on the relative health risk posed to humans (9,10).…”

mentioning

confidence: 99%

“…Recently, intensive efforts have been made to examine source waters destined for potable consumption, as well as wastewaters intended for reuse, using genotyping assays and, to a lesser extent, infectivity assays (9)(10)(11)(12)(13). The information from these assays provides an indication of the risk to of human health (13).…”

mentioning

confidence: 99%

Integrated Cryptosporidium Assay To Determine Oocyst Density, Infectivity, and Genotype for Risk Assessment of Source and Reuse Water

King

Fanok

Phillips

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures.C ryptosporidium is ubiquitous in source waters and wastewaters, presenting a treatment challenge on account of its small size, resistance to chlorine disinfection, and the absence of more easily measured surrogates to allow treatment performance validation (1, 2). These characteristics make Cryptosporidium problematic for the water industry, and the ever-present threat from this pathogen requires sound characterization and management of risks, including validation and monitoring of critical control points (3-5). Removal of Cryptosporidium by treatment processes can be highly variable (6), so validation and monitoring of individual processes are important to ensure appropriate performance. Even for validated systems, treatment failure is possible under highly adverse conditions, and management of this risk may require significant capital expenditure to provide sufficient risk mitigation for extreme events. However, risk assessments frequently utilize historical monitoring data based on total oocyst numbers, not considering either oocyst infectivity or species/genotype, resulting in possible overestimation of risk.Of the greater than 26 species or genotypes of Cryptosporidium that might be detected in the environment, only C. parvum and C. hominis common...

show abstract

“…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).…”

mentioning

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

View full text Add to dashboard Cite

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...

show abstract

Potential molecular tools for assessing the public health risk associated with waterborne Cryptosporidium oocysts

Cited by 33 publications

References 87 publications

Waterborne outbreaks of cryptosporidiosis

Waterborne outbreaks of cryptosporidiosis

Integrated Cryptosporidium Assay To Determine Oocyst Density, Infectivity, and Genotype for Risk Assessment of Source and Reuse Water

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

Contact Info

Product

Resources

About