Introduction: Acute gastroenteritis is a common illness in humans. It has a high morbidity worldwide and in the United States (U.S); a sizeable mortality is reported in developing countries. Viruses are a common cause of acute gastroenteritis and noroviruses are the leading cause of non-bacterial gastroenteritis (1). Fomites play an important role in the infection cycle of norovirus. This study explored the comparability between a quantitative microbial risk assessment (QMRA) model to predict the probability of illness from norovirus due to environmental contamination with actual observed attack rates, the impact of appropriate cleaning procedures on fomites contamination, and the role of fomites in the transmission of norovirusMethod: Data from three different norovirus illness outbreaks occurring from 2004 to 2005 in Arizona in different venues were used. Questionnaires were used to determine demographics, symptoms associated with gastrointestinal illness, medical visits, and potential exposures such as food items consumed. When available, stool samples were collected from ill participants. Fomites samples were also gathered from the different venues; houseboats, college summer camp living spaces, and a restaurant. These samples were tested for norovirus using reverse transcriptase polymerase chain reaction (RT-PCR). Viral concentration on fomites was calculated using the most probable number methodology.Results: Norovirus was found on fomites from all three outbreaks. Improper cleaning of fomites increased the proportion of norovirus positive fomites from 40 percent to 70 percent in contrast, after cleaning and disinfection with 5000 mg/L free chloride, the proportion of norovirus positive fomites decreased to 33%. The predicted values from the quantitative microbial risk assessment model were consistent with the observed epidemiological attack rates.Conclusion: This study showed unequivocally that fomites play an important role in the transmission cycle of norovirus, and that improper cleaning and disinfection procedures only serve to spread the virus to previously uncontaminated fomites. In the case of outbreaks where people are not always available to answer questionnaires, QMRA should be considered as a worthwhile alternative to a full-scale epidemiological study.

There is a growing demand for safe and convenient meat products. The effect of natural spice extracts incorporated with other hurdles for controlling pathogenic bacteria and extending the shelf life of RTE doner kebab were investigated. A comprehensive literature review was undertaken to establish the status of microbial risk, use of additives, knowledge on oxidative deterioration and HACCP associated with meat products. The in vitro antioxidant and antibacterial activities of spice extracts were screened and compared. Cinnamon, clove, and sumac alcoholic extracts demonstrated strong antimicrobial effect, however, rosemary proved effective as antioxidant in a lamb fat model. An accelerated shelf life study on a model system indicated that storage temperature was the most critical factor affecting lipid oxidation, which was effectively delayed by vacuum packaging and rosemary extracts. The effects of spice extracts, packaging and storage time on physiochemical, microbiological, and sensory attributes of doner kebab were evaluated. Application of rosemary and cinnamon extracts significantly reduced TVC, inhibited LAB, and retarded lipid oxidation rate. Sensory evaluation by a consumer panel indicated that only taste and spiciness perception was significantly different between treatments. A challenge test against Listeria monocytogenes showed significant differences between control and spice treatments at day 28. Strong inhibitory effects were associated to high levels of cinnamon particularly when applied after cooking. The effect of heat treatment and sumac (Rhus coriaria) on Bacillus cereus and Clostridium perfringens inactivation was evaluated on a doner kebab prototype. Addition of sumac significantly reduced D-values and z-values for both organisms in comparison to the control. The investigation of the effect of spice extracts, and environmental conditions on changes in growth kinetic parameters for L. monocytogenes and Salmonella Typhimurium showed that spice extracts are highly significant. For both microorganisms, Mumax was reduced as salt and spice concentrations increased, and pH levels decreased. This study shows that spice extracts incorporated with other hurdles can help to maintain safe and good quality RTE doner kebab.

A avaliação quantitativa de risco microbiológico é o processo utilizado para estimar a probabilidade de infecção, doença ou morte após exposição a microrganismos patogênicos presente em águas, biossólidos, alimentos e ar. Essa pesquisa tem como objetivo descrever o estado da arte da avaliação qualitativa de risco microbiológico associadas a águas e biossólidos, bem como descrever as abordagens e etapas utilizadas na condução do processo, relatar a aplicabilidade e discutir as dificuldades e necessidades na condução da AQRM. Sendo uma pesquisa de caráter descritivo-explicativo, realizou-se revisão de literatura sobre a temática nas seguintes bases de dados: Scielo, LILACS, DEDALUS, MEDLINE e PUBMED e nos documentos produzidos pela Organização Mundial de Saúde e U.S. Environmental Protect Agency. Na literatura consultada verificou-se que a AQRM vem sendo utilizada desde o início da década de 1980 para estimar os riscos à saúde humana. Existem diferentes abordagens utilizadas na condução da ferramenta, diferem na forma de organizar e sistematizar as informações, porém são similares. A abordagem mais utilizada é a proposta pela NRC e consta de quatro etapas: identificação do perigo, avaliação de exposição, avaliação de dose-resposta e caracterização do risco. Constatou-se que a avaliação de exposição apresentase como a etapa mais complexa da AQRM, devido a: i) limitações metodológicas na determinação da concentração e viabilidade dos patógenos em águas e biossólidos e ii) escassez de dados de exposição e de consumo. Verificou-se que a utilização da ferramenta é proeminente na avaliação dos riscos decorrentes da exposição à patógenos presentes em águas de consumo, recreacionais e residuárias, bem como os decorrentes da exposição a solos e cultivos agrícolas fertilizados com biossólidos. É uma ferramenta que assume relevância no cenário internacional vem se consolidando no estabelecimento de valores-limite de patógenos presentes em diversas fontes, no desenvolvimento de normas, guias e legislações, bem como para discussões e implementação de planos de segurança da água e alimentar. Porém, é uma ferramenta pouco conhecida e empregada em nosso país que pode vir a atender às demandas atuais relacionadas a águas e biossólidos, tais como: i) estabelecimento de valores-limite e risco tolerável para patógenos, ii) proposição de métodos de tratamento e controle, iii) criação e revisão de normas, regulamentações e leis e iv) implementação de políticas públicas que visem a promoção e proteção da saúde humana / The quantitative microbial risk assessment is the process used to estimate the probability of infection, disease or death after exposure to pathogenic microorganisms present in water, biosolids, food and air. This research aims to describe the state of the art of quantitative microbial risk assessment associated with water and biosolids, and to describe the approaches and steps used in conducting the proceedings, report the applicability and discuss the difficulties and needs in the conduct of QMRA. As a search with a descriptive-explanatory character, there was a literature review on the subject on the following databases: SciELO, LILACS, DEDALUS, MEDLINE, PUBMED and in documents produced by the World Health Organization and the U.S. Environmental Protection Agency. In literature it was found that the QMRA has been used since the early 1980s to estimate the risk to human health. There are different approaches used in the conduct of the tool, they differ in the way of organizing and systematizing the information, but they are similar. The most used approach is the one proposed by the NRC and consists of four steps: hazard identification, exposure assessment, doseresponse assessment and risk characterization. It was found that the exposure assessment is presented as the most complex steps of QMRA due to: i) methodological limitations in determining the concentration and viability of pathogens in water and biosolids, and ii) lack of exposure data and consumption. It was verified that the use of the tool is prominent in assessing the risks from exposure to pathogens in drinking water, recreational water and wastewater, as well as from exposure to soils and crops fertilized with biosolids. It is a tool that is relevant in the international arena and has been consolidated in the establishment of limit values of pathogens present in different sources, in developing standards, guidelines and laws, as well as for discussion and implementation of plans for water and food security. However, it is a relatively unfamiliar tool used in our country that can come to attend current demands related to water and biosolids, such as: i) establishment of limit values for pathogens and tolerable risk, ii) proposition of methods of treatment and control, iii) creation and revision of standards, regulations and laws and iv) implementation of public policies for promotion and protection of human health

A avaliação quantitativa de risco microbiológico é o processo utilizado para estimar a probabilidade de infecção, doença ou morte após exposição a microrganismos patogênicos presente em águas, biossólidos, alimentos e ar. Essa pesquisa tem como objetivo descrever o estado da arte da avaliação qualitativa de risco microbiológico associadas a águas e biossólidos, bem como descrever as abordagens e etapas utilizadas na condução do processo, relatar a aplicabilidade e discutir as dificuldades e necessidades na condução da AQRM. Sendo uma pesquisa de caráter descritivo-explicativo, realizou-se revisão de literatura sobre a temática nas seguintes bases de dados: Scielo, LILACS, DEDALUS, MEDLINE e PUBMED e nos documentos produzidos pela Organização Mundial de Saúde e U.S. Environmental Protect Agency. Na literatura consultada verificou-se que a AQRM vem sendo utilizada desde o início da década de 1980 para estimar os riscos à saúde humana. Existem diferentes abordagens utilizadas na condução da ferramenta, diferem na forma de organizar e sistematizar as informações, porém são similares. A abordagem mais utilizada é a proposta pela NRC e consta de quatro etapas: identificação do perigo, avaliação de exposição, avaliação de dose-resposta e caracterização do risco. Constatou-se que a avaliação de exposição apresentase como a etapa mais complexa da AQRM, devido a: i) limitações metodológicas na determinação da concentração e viabilidade dos patógenos em águas e biossólidos e ii) escassez de dados de exposição e de consumo. Verificou-se que a utilização da ferramenta é proeminente na avaliação dos riscos decorrentes da exposição à patógenos presentes em águas de consumo, recreacionais e residuárias, bem como os decorrentes da exposição a solos e cultivos agrícolas fertilizados com biossólidos. É uma ferramenta que assume relevância no cenário internacional vem se consolidando no estabelecimento de valores-limite de patógenos presentes em diversas fontes, no desenvolvimento de normas, guias e legislações, bem como para discussões e implementação de planos de segurança da água e alimentar. Porém, é uma ferramenta pouco conhecida e empregada em nosso país que pode vir a atender às demandas atuais relacionadas a águas e biossólidos, tais como: i) estabelecimento de valores-limite e risco tolerável para patógenos, ii) proposição de métodos de tratamento e controle, iii) criação e revisão de normas, regulamentações e leis e iv) implementação de políticas públicas que visem a promoção e proteção da saúde humana / The quantitative microbial risk assessment is the process used to estimate the probability of infection, disease or death after exposure to pathogenic microorganisms present in water, biosolids, food and air. This research aims to describe the state of the art of quantitative microbial risk assessment associated with water and biosolids, and to describe the approaches and steps used in conducting the proceedings, report the applicability and discuss the difficulties and needs in the conduct of QMRA. As a search with a descriptive-explanatory character, there was a literature review on the subject on the following databases: SciELO, LILACS, DEDALUS, MEDLINE, PUBMED and in documents produced by the World Health Organization and the U.S. Environmental Protection Agency. In literature it was found that the QMRA has been used since the early 1980s to estimate the risk to human health. There are different approaches used in the conduct of the tool, they differ in the way of organizing and systematizing the information, but they are similar. The most used approach is the one proposed by the NRC and consists of four steps: hazard identification, exposure assessment, doseresponse assessment and risk characterization. It was found that the exposure assessment is presented as the most complex steps of QMRA due to: i) methodological limitations in determining the concentration and viability of pathogens in water and biosolids, and ii) lack of exposure data and consumption. It was verified that the use of the tool is prominent in assessing the risks from exposure to pathogens in drinking water, recreational water and wastewater, as well as from exposure to soils and crops fertilized with biosolids. It is a tool that is relevant in the international arena and has been consolidated in the establishment of limit values of pathogens present in different sources, in developing standards, guidelines and laws, as well as for discussion and implementation of plans for water and food security. However, it is a relatively unfamiliar tool used in our country that can come to attend current demands related to water and biosolids, such as: i) establishment of limit values for pathogens and tolerable risk, ii) proposition of methods of treatment and control, iii) creation and revision of standards, regulations and laws and iv) implementation of public policies for promotion and protection of human health

Quantitative microbial risk assessment (QMRA) is being increasingly used to support decision-making for food safety issues. Decision-makers need to know whether these QMRA results can be trusted, especially when urgent and important decisions have to be made. This can be achieved by setting up a quality assurance (QA) framework for QMRA. A Belgian risk assessment project (the METZOON project) aiming to assess the risk of human salmonellosis due to the consumption of fresh minced pork meat was used as a case study to develop and implement QA methods for the evaluation of the quality of input data, expert opinion, model assumptions, and the quality of the QMRA model (the METZOON model). The first part of this thesis consists of a literature review of available QA methods of interest in QMRA (chapter 2). In the next experimental part, different QA methods were applied to the METZOON model. A structured expert elicitation study (chapter 4) was set up to fill in missing parameters for the METZOON model. Judgements of experts were used to derive subjective probability density functions (PDFs) to quantify the uncertainty on the model input parameters. The elicitation was based on Cookes classical model (Cooke, 1991) which aims to achieve a rational consensus about the elicitation protocol and allowed comparing different weighting schemes for the aggregation of the experts PDFs. Unique to this method was the fact that the performance of experts as probability assessors was measured by the experts ability to correctly and precisely provide estimates for a set of seed variables (=variables from the experts area of expertise for which the true values were known to the analyst). The weighting scheme using the experts performance on a set of calibration variables was chosen to obtain the combined uncertainty distributions of lacking parameters for the METZOON model. A novel method for the assessment of data quality, known as the NUSAP (Numeral Unit Spread Assessment Pedigree) system (chapter 5) was tested to screen the quality of the METZOON input parameters. First, an inventory with the essential characteristics of parameters including the source of information, the sampling methodology and distributional characteristics was established. Subsequently the quality of these parameters was evaluated and scored by experts using objective criteria (proxy, empirical basis, methodological rigour and validation). The NUSAP method allowed to debate on the quality of the parameters within the members of the risk assessment team using a structured format. The quality evaluation was supported by graphical representations which facilitated decisions on the inclusion or exclusion of inputs into the model. It is well known that assumptions and subjective choices can have a large impact on the output of a risk assessment. To assess the value-ladenness (degree of subjectivity) of assumptions in the METZOON model a structured approach based on the protocol by Kloprogge et al. (2005) was chosen (chapter 6). The key assumptions for the METZOON model were first identified and then evaluated by experts in a workshop using four criteria: the influence of situational limitations, the plausibility, the choice space and the agreement among peers. The quality of the assumptions was graphically represented (using kite diagrams, pedigree charts and diagnostic diagrams) and allowed to identify assumptions characterised by high degree of subjectivity and high expected influence on the model results, which can be considered as weak links in the model. The quality assessment of the assumptions was taken into account to modify parts of the METZOON model, and allows to increase the transparency in the QMRA process. In a last application of a QA method, a quality audit checklist (Paisley, 2007) was used to critically review and score the quality of the METZOON model and to identify its strengths and weaknesses (chapter 7). A high total score (87%) was obtained by reviewing the METZOON model with the Paisley-checklist. A higher score would have been obtained if the model was subjected to external peer review, if a sensitivity analysis, validation of the model with recent data, updating/replacing expert judgement data with empirical data was carried out. It would also be advisable to repeat the NUSAP/Pedigree on the input data and assumptions of the final model. The checklist can be used in its current form to evaluate QMRA models and to support model improvements from the early phases of development up to the finalised model for internal as well as for external peer review of QMRAs. The applied QA methods were found useful to improve the transparency in the QMRA process and to open the debate about the relevance (fitness for purpose) of a QMRA. A pragmatic approach by combining several QA methods is recommendable, as the application of one QA method often facilitates the application of another method. Many QA methods (NUSAP, structured expert judgement, checklists) are however not yet or insufficiently described in QMRA related guidelines (at EFSA and WHO level). Another limiting factor is the time and resources which need to be taken into account as well. To understand the degree of quality required from a QMRA a clear communication with the risk managers is required. It is therefore necessary to strengthen the training in QA methods and in the communication of its results. Understanding the usefulness of these QA methods could improve among the risk analysis actors when they will be tested in large number of QMRAs.

The high incidence of pathogens is one of the main causes for impaired surface water quality designations in the United States. Pathogen presence in fresh water is monitored through the detection of indicator bacteria. Indicator bacteria concentrations, spatial and temporal variability, and microbial risks were evaluated in two rural watersheds, the Bosque and Leon Rivers, and one predominantly urban watershed, the San Jacinto River, all in Texas. Human health risk was predicted from contaminated waters as indicated by ingestion of Escherichia coli found in surface water for contact recreation scenarios. The watersheds were chosen because many segments were previously placed on the 303 (d) list (published by the TCEQ) for failing the indicator bacteria standards. Predominantly urban areas of the San Jacinto River and rural portions of the Bosque and Leon Rivers, where Concentrated Animal Feeding Operations (CAFOs) are numerous, were compared to relatively pristine rural watersheds. Spatial analysis of the watersheds with E.coli concentrations exceeding the single sample (394 MPN/100mL) and the geometric mean standards (126 MPN/100mL) indicated that land use is a significant factor influencing the incidence of bacterial concentrations. Non-agricultural rural areas of the watersheds, such as forests and rangelands, had significantly lower E.coli concentrations compared to the agricultural areas and urban land uses. Human health risk due to ingestion of E.coli as an indicator organism indicated a similar pattern to that of their concentrations in that urban and agricultural areas had a greater risk compared to the other rural areas of the watersheds. The risk estimate for urban and agricultural areas exceeded the acceptable limit of one in ten thousand (10^-4), indicating a potential for adverse health effects to humans. Temporal variability in the watersheds as a function of streamflow, rainfall, and temperature indicated a positive correlation between bacterial concentration and high streamflow, rainfall and temperature. The positive correlation for these effects was greater in the rural areas compared to urban areas, indicating the presence of multiple factors responsible for E.coli concentrations in urban areas. Thus, land use was confirmed to be a major factor contributing to the presence of indicator bacteria in surface waters.

Water supply systems aim, among other objectives, to protect public health by reducing the concentration of, and potentially eliminating, microorganisms pathogenic to human beings. Yet, because water supply systems are engineered systems facing variable conditions, such as raw water quality or treatment process performance, the quality of the drinking water produced also exhibits variability. The reliability of a treatment system is defined in this context as the probability of producing drinking water that complies with existing microbial quality standards. This thesis examines the concept of reliability for two physicochemical treatment technologies, conventional rapid granular filtration and ultrafiltration, used to remove the protozoan pathogen Cryptosporidium parvum from drinking water. First, fault tree analysis is used as a method of identifying technical hazards related to the operation of these two technologies and to propose ways of minimizing the probability of failure of the systems. This method is used to compile operators’ knowledge into a single logical diagram and allows the identification of important processes which require efficient monitoring and maintenance practices. Second, an existing quantitative microbial risk assessment model is extended to be used in a reliability analysis. The extended model is used to quantify the reliability of the ultrafiltration system, for which performance is based on full-scale operational data, and to compare it with the reliability of rapid granular filtration systems, for which performance is based on previously published data. This method allows for a sound comparison of the reliability of the two technologies. Several issues remain to be addressed regarding the approaches used to quantify the different input variables of the model. The approaches proposed herein can be applied to other water treatment technologies, to aid in prioritizing interventions to improve system reliability at the operational level, and to determine the data needs for further refinements of the estimates of important variables.

Water supply systems aim, among other objectives, to protect public health by reducing the concentration of, and potentially eliminating, microorganisms pathogenic to human beings. Yet, because water supply systems are engineered systems facing variable conditions, such as raw water quality or treatment process performance, the quality of the drinking water produced also exhibits variability. The reliability of a treatment system is defined in this context as the probability of producing drinking water that complies with existing microbial quality standards. This thesis examines the concept of reliability for two physicochemical treatment technologies, conventional rapid granular filtration and ultrafiltration, used to remove the protozoan pathogen Cryptosporidium parvum from drinking water. First, fault tree analysis is used as a method of identifying technical hazards related to the operation of these two technologies and to propose ways of minimizing the probability of failure of the systems. This method is used to compile operators’ knowledge into a single logical diagram and allows the identification of important processes which require efficient monitoring and maintenance practices. Second, an existing quantitative microbial risk assessment model is extended to be used in a reliability analysis. The extended model is used to quantify the reliability of the ultrafiltration system, for which performance is based on full-scale operational data, and to compare it with the reliability of rapid granular filtration systems, for which performance is based on previously published data. This method allows for a sound comparison of the reliability of the two technologies. Several issues remain to be addressed regarding the approaches used to quantify the different input variables of the model. The approaches proposed herein can be applied to other water treatment technologies, to aid in prioritizing interventions to improve system reliability at the operational level, and to determine the data needs for further refinements of the estimates of important variables.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Michael W. Sanderson / Shiga-toxin-producing Escherichia coli (STEC), of which enterohemorrhagic E. coli (EHEC) are a pathogenic sub-group, are foodborne pathogens of significant public health importance in the United States. STEC belong to the family Enterobacteriaceae commonly found in the large intestine of humans and other warm-blooded animals. EHEC harbors shiga toxin (stx1 and/or stx2) and eae genes which confers the ability to cause human illnesses. The U.S. Department of Agriculture Food Safety and Inspection Service declared seven STEC (O26, O45, O103, O111, O121, O145, and O157) as adulterants in ground beef and non-intact beef products to reduce/eliminate the burden of the pathogens in the beef production chain. STEC control efforts in the U.S. include the development of quantitative microbial risk assessment (QMRA) to identify mitigation strategies that are effective and economical in reducing exposure and reduce occurrence and public health risk from STEC in the beef chain. Collection of accurate and unbiased data is critical for the development of a QMRA that is valid for decision making. Determining the prevalence and concentration of the seven STEC in the different cattle types and seasons is valuable for the development a valid QMRA for STEC in beef production in the U.S. Our systematic review and meta-analysis study of the prevalence and concentration of E. coli O157 along the beef production chain indicated differences in the fecal prevalence of E. coli O157 among cattle types and seasons, revealed decreasing prevalence and concentration of E. coli O157 on cattle hides and carcass surfaces from pre-evisceration to the final chilled carcass stage, and identified study setting, detection method, hide or carcass swab area, and study design as significant sources of heterogeneity among studies reporting prevalence of E. coli O157 along the beef production chain. Bayesian estimation of the diagnostic performance of three laboratory methods (culture, conventional PCR [cPCR], and multiplex quantitative PCR [mqPCR]) used for the detection of the seven STEC in the feces of cattle is necessary to estimate true prevalence of EHEC in cattle. The analysis revealed highest sensitivity of mqPCR, followed by cPCR, and culture for the detection of E. coli O157; the cPCR and mqPCR had comparable specificity, but specificity of mqPCR method was heavily dependent on prior specification. The mqPCR method was the most sensitive for the detection O26, O45, and O103 serogroups. The cPCR method was more sensitive than the culture method for serogroups O26, and O121, but comparable for serogroups O45, O103, O111, and O145. The cPCR method showed higher specificity than mqPCR within serogroups O45, O121, and O145 but no apparent differences within serogroups O26, O103, and O111. A second order quantitative microbial risk assessment was developed to quantify the prevalence and concentration of the seven STEC on pre-evisceration beef carcasses and evaluate the impact of peri-harvest interventions. Simulation scenarios of current industry peri-harvest intervention practices showed variable effectiveness in reducing STEC contamination on pre-evisceration beef carcass, however, a scenario of increased adoption of peri-harvest interventions was more effective at reducing STEC contamination. Fecal-to-hide transfer and hide-to-carcass transfer had a large effect on prevalence and concentration of STEC on pre-evisceration carcasses.

Water supply systems aim, among other objectives, to protect public health by reducing the concentration of, and potentially eliminating, microorganisms pathogenic to human beings. Yet, because water supply systems are engineered systems facing variable conditions, such as raw water quality or treatment process performance, the quality of the drinking water produced also exhibits variability. The reliability of a treatment system is defined in this context as the probability of producing drinking water that complies with existing microbial quality standards. This thesis examines the concept of reliability for two physicochemical treatment technologies, conventional rapid granular filtration and ultrafiltration, used to remove the protozoan pathogen Cryptosporidium parvum from drinking water. First, fault tree analysis is used as a method of identifying technical hazards related to the operation of these two technologies and to propose ways of minimizing the probability of failure of the systems. This method is used to compile operators’ knowledge into a single logical diagram and allows the identification of important processes which require efficient monitoring and maintenance practices. Second, an existing quantitative microbial risk assessment model is extended to be used in a reliability analysis. The extended model is used to quantify the reliability of the ultrafiltration system, for which performance is based on full-scale operational data, and to compare it with the reliability of rapid granular filtration systems, for which performance is based on previously published data. This method allows for a sound comparison of the reliability of the two technologies. Several issues remain to be addressed regarding the approaches used to quantify the different input variables of the model. The approaches proposed herein can be applied to other water treatment technologies, to aid in prioritizing interventions to improve system reliability at the operational level, and to determine the data needs for further refinements of the estimates of important variables. / Applied Science, Faculty of / Civil Engineering, Department of / Graduate