Epidemiology and detection as options for control of viral and parasitic foodborne disease.

Human enteric viruses and protozoal parasites are important causes of emerging food and waterborne disease. Epidemiologic investigation and detection of the agents in clinical, food, and water specimens, which are traditionally used to establish the cause of disease outbreaks, are either cumbersome, expensive, and frequently unavailable or unattempted for the important food and waterborne enteric viruses and protozoa. However, the recent introduction of regulatory testing mandates, alternative testing strategies, and increased epidemiologic surveillance for food and waterborne disease should significantly improve the ability to detect and control these agents. We discuss new methods of investigating foodborne viral and parasitic disease and the future of these methods in recognizing, identifying, and controlling disease agents

Evaluation of a novel chlorine dioxide-based packaging technology to reduce human enteric virus contamination on refrigerated tomatoes and blueberries

IntroductionChlorine dioxide (ClO2) is a promising antimicrobial with various food applications, one of those being inclusion in packaging. The purpose of this study was to evaluate a novel ClO2-based antimicrobial packaging system (InvisiShield™) for its efficacy against human norovirus (hNoV) and hepatitis A virus (HAV) in refrigerated fresh produce.MethodsGrape tomatoes or blueberries were placed in polypropylene trays and selectively inoculated with 6.0 log10 hNoV Genome Equivalent Copies (GEC; 20% stool suspension) or 6.2 log10 HAV GEC (cell culture lysate). Trays were heat sealed with a three-phase polymer film consisting of a base, channeling agent, and the ClO2 active (treatment); or control (no active) film and stored at 7°C for 24, 48 h, and 7 days. At each timepoint, the product was collected and processed for virus concentration using the sequential steps of elution and polyethylene glycol precipitation. Viruses in extracts were quantified using RNase-RT-qPCR.Results and discussionLog10 reductions (LR) in hNoV GEC for tomatoes were 2.2 ± 1.3, 2.9 ± 0.7, and 3.6 ± 0.3, after 24, 48 h and 7 days, respectively. For blueberries, hNoV LR were 1.4 ± 0.7, 1.7 ± 0.5, and 2.7 ± 0.2 GEC, respectively. Hepatitis A virus GEC LR were 0.4 ± 0.2, 1.0 ± 0.1, and 2.1 ± 0.7 for tomatoes, and 0.1 ± 0.2, 1.2 ± 0.4, and 3.2 ± 0.2 for blueberries, after 24, 48 h and 7 days, respectively. Position of the fruit in the tray did not affect inactivation (p > 0.05). Sensory analysis on the treated tomato products revealed no statistically significant difference in appearance, flavor and texture attributes compared to the control. This novel ClO2-based antimicrobial packaging system effectively reduced concentrations of hNoV and HAV, as evaluated using reduction in GEC as proxy for infectivity, on grape tomatoes and blueberries after one day, with efficacy improving over 7 days of refrigerated storage. This technology shows promise as an antiviral treatment as applied to refrigerated fresh produce items

Contamination of Fresh Produce by Microbial Indicators on Farms and in Packing Facilities: Elucidation of Environmental Routes

ABSTRACT To improve food safety on farms, it is critical to quantify the impact of environmental microbial contamination sources on fresh produce. However, studies are hampered by difficulties achieving study designs with powered sample sizes to elucidate relationships between environmental and produce contamination. Our goal was to quantify, in the agricultural production environment, the relationship between microbial contamination on hands, soil, and water and contamination on fresh produce. In 11 farms and packing facilities in northern Mexico, we applied a matched study design: composite samples (n � 636, equivalent to 11,046 units) of produce rinses were matched to water, soil, and worker hand rinses during two growing seasons. Microbial indicators (coliforms, Escherichia coli, Enterococcus spp., and somatic coliphage) were quantified from composite samples. Statistical measures of association and correlations were calculated through Spearman’s correlation, linear regression, and logistic regression models. The concentrations of all microbial indicators were positively correlated between produce and hands ( � range, 0.41 to 0.75; P � 0.01). When E. coli was present on hands, the handled produce was nine times more likely to contain E. coli (P � 0.05). Similarly, when coliphage was present on hands, the handled produce was eight times more likely to contain coliphage (P � 0.05). There were relatively low concentrations of indicators in soil and water samples, and a few sporadic significant associations were observed between contamination of soil and water and contamination of produce. This methodology provides a foundation for future field studies, and results highlight the need for interventions surrounding farmworker hygiene and sanitation to reduce microbial contamination of farmworkers’ hands. IMPORTANCE This study of the relationships between microbes on produce and in the farm environment can be used to support the design of targeted interventions to prevent or reduce microbial contamination of fresh produce with associated reductions in foodborne illness. KEYWORDS environmental microbiology, food-borne pathogens, produc

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process

The Prevalence of Norovirus in returning international travelers with diarrhea

Background: There is a high incidence of diarrhea in traveling populations. Norovirus (NV) infection is a common cause of diarrhea and is associated with 7% of all diarrhea related deaths in the US. However, data on the overall prevalence of NV infection in traveling populations is limited. Furthermore, the prevalence of NV amongst travelers returning to Europe has not been reported. This study determined the prevalence of NV among international travelers returning to Germany from over 50 destinations in and outside Europe. Methods: Stool samples of a total of 104 patients with a recent (&lt; 14days) history of international travel (55 male, mean age 37 yrs.) were tested for the presence of NV genogroup (GG) I and II infection using a sensitive and well established quantitative RT PCR method. 57 patients experienced diarrhea at the time of presentation at the Department of Infectious Diseases & Tropical Medicine. The remaining 47 patients had no experience of diarrhea or other gastrointestinal symptoms for at least 14 days prior to their date of presentation at our institute. Results: In our cohort, NV infection was detected in 15.7% of returning travelers with diarrhea. The closer to the date of return symptoms appeared, the higher the incidence of NV, ranging as high as 21.2% within the first four days after return. Conclusions: In our cohort, NV infection was shown to be frequent among returning travelers especially in those with diarrhea, with over 1/5 of diarrhea patients tested positive for NV within the first four days after their return to Germany. Due to this prevalence, routine testing for NV infection and hygienic precautions may be warranted in this group. This is especially applicable to patients at an increased risk of spreading the disease, such as healthcare workers, teachers or food-handlers

Detection of human enteric viruses in oysters by in vivo and in vitro amplification of nucleic acids.

This study describes the detection of enteroviruses and hepatitis A virus in 31 naturally contaminated oyster specimens by nucleic acid amplification and oligonucleotide probing. Viruses were extracted by adsorption-elution-precipitation from 50-g oyster samples harvested from an area receiving sewage effluent discharge. Ninety percent of each extract was inoculated into primate kidney cell cultures for virus isolation and infectivity assay. Viruses in the remaining 10% of oyster extract that was not inoculated into cell cultures were further purified and concentrated by a procedure involving Freon extraction, polyethylene glycol precipitation, and Pro-Cipitate precipitation. After 3 to 4 weeks of incubation, RNA was extracted from inoculated cultures that were negative for cytopathic effects (CPE). These RNA extracts and the RNA from virions purified and concentrated directly from oyster extracts were subjected to reverse transcriptase PCR (RT-PCR) with primer pairs for human enteroviruses and hepatitis A virus. The resulting amplicons were confirmed by internal oligonucleotide probe hybridization. For the portions of oyster sample extracts inoculated into cell cultures, 12 (39%) were positive for human enteroviruses by CPE and 6 (19%) were positive by RT-PCR and oligoprobing of RNA extracts from CPE-negative cell cultures. For the remaining sample portions tested by direct RT-PCR and oligoprobing after further concentration, five (about 16%) were confirmed to be positive for human enteroviruses. Hepatitis A virus was also detected in RNA extracts of two CPE-positive samples by RT-PCR and oligoprobing. Combining the data from all three methods, enteric viruses were detected in 18 of 31 (58%) samples. Detection by nucleic acid methods increased the number of positive samples by 50% over detection by CPE in cell culture. Hence, nucleic acid amplification methods increase the detection of noncytopathic human enteric viruses in oysters