A significant global problem is the microbiological contamination of foods and water. The microorganisms associated with about half of the foodborne disease outbreaks still go unrecognized, primarily as a result of inadequate diagnostic methods and sampling. A significant amount of food- and waterborne diseases are associated with viruses, information that has been obtained only in recent years. Improved diagnostic methods have established that caliciviruses are the most important non-bacterial pathogens associated with food- and waterborne outbreaks, and are the major cause of seafood-associated gastroenteritis.
A food-borne outbreak of gastroenteritis with more than 650 suspected cases occurred in April 2016 in Sollentuna, Sweden. It originated in a school kitchen serving a total of 2,700 meals daily. Initial microbiological testing (for Campylobacter, Salmonella, Shigella, Yersinia, Giardia, Cryptosporidium, Entamoeba histolytica, adeno-, astro-, noro-, rota- and sapovirus) of stool samples from 15 symptomatic cases was negative, despite a clinical presentation suggestive of calicivirus. Analyses of the findings from both the Sollentuna municipality environmental team and a web-based questionnaire suggested that the source of the outbreak was the salad buffet served on 20 April, although no specific food item could be identified. Subsequent electron microscopic examination of stool samples followed by whole genome sequencing revealed a variant of sapovirus genogroup V. The virus was not detected using standard PCR screening. This paper describes the epidemiological outbreak investigation and findings leading to the discovery.
Conventional reverse transcription-polymerase chain reaction (Con RT-PCR) assay to detect norovirus is a complex multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the results.
To develop and evaluate a multiplex real time RT-PCR (Mrt RT-PCR) assay that detect and quantify norovirus GI and GII with a single amplification and detection step.
The primers and TaqMan probes for the Mrt RT-PCR were selected from the ORF1-ORF2 junction region. A total of 97 stools from 41 gastroenteritis outbreaks and 726 stools from children with sporadic diarrhoea were used for this study.
For the 97 outbreak samples, norovirus were detected in 61 of the 69 previously tested positive and 11 of the 28 previously tested negative samples. Eight samples that tested positive for GII by Con RT-PCR but negative by the Mrt RT-PCR also tested negative by a Light Cycler RT-PCR assay. Eighty-two GII and two GI were detected in the 726 sporadic samples. Random primers were more sensitive than specific primers in the cDNA synthesis. The two-step assay using the random primers in RT reaction was 100 times more sensitive than the one-step assay. The Mrt RT-PCR had the same sensitivity as that using two real time RT-PCR for separate detection of GI and GII. A wide dynamic range was obtained with the two-step assay, detecting from 3000 to 3x10(11) of copies RNA/g stool. Very good precision was observed with no cross-reaction with other enteric viruses. The new assay is able to detect both GI and GII in one reaction and brings a cost reduction of approximately 40% compared to separate reactions for GI and GII.
The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test.
The norovirus group (NVG) of caliciviruses are the etiological agents of most institutional outbreaks of gastroenteritis in North America and Europe. Identification of NVG is complicated by the non-culturable nature of this virus, and the absence of a diagnostic gold standard makes traditional evaluation of test characteristics problematic.
We evaluated 189 specimens derived from 440 acute gastroenteritis outbreaks investigated in Ontario in 2006-07. Parallel testing for NVG was performed with real-time reverse-transcriptase polymerase chain reaction (RT2-PCR), enzyme immunoassay (EIA) and electron microscopy (EM). Test characteristics (sensitivity and specificity) were estimated using latent class models and composite reference standard methods. The practical implications of test characteristics were evaluated using binomial probability models.
Latent class modelling estimated sensitivities of RT2-PCR, EIA, and EM as 100%, 86%, and 17% respectively; specificities were 84%, 92%, and 100%; estimates obtained using a composite reference standard were similar. If all specimens contained norovirus, RT2-PCR or EIA would be associated with > 99.9% likelihood of at least one test being positive after three specimens tested. Testing of more than 5 true negative specimens with RT2-PCR would be associated with a greater than 50% likelihood of a false positive test.
Our findings support the characterization of EM as lacking sensitivity for NVG outbreaks. The high sensitivity of RT2-PCR and EIA permit identification of NVG outbreaks with testing of limited numbers of clinical specimens. Given risks of false positive test results, it is reasonable to limit the number of specimens tested when RT2-PCR or EIA are available.
Clean drinking water is essential for public health. The cause of waterborne outbreaks is most often faecal contamination of water from animals or humans. The objective of this resarch was to collect available information on waterborne outbreaks in Iceland for the twenty year period, 1998-2017. Incident of faecal and pathogenic pollution in samples where also collected even though rarely followed by registered outbreak.
Data are obtained from laboratory databases, the Directorate of Health, reports and interviews with the relevant surveillance authorities and epidemiologists.
The results show that for the period investigated fifteen waterborne outbreaks were registered, all in small water supplies, many of which served transitent population, tourists and summerhouse dwellers. About 500 illnesses were confirmed and 8000 people affected. Other research have shown that around 10% of illnesses in waterborne outbreaks are registered so it can be estimated that on average 250 people have been taken ill every year because of contaminated drinking water. Analysis of monitoring water quality data show that on average 50 water supplies, or about 5% of the Icelandic registered water supplies have contained faecal matter every year. The most frequent cause of waterborne outbreak were poor design and inadequate maintainance of water intakes.
It is likely that waterborne outbreaks are more numerous than are registered in official reports, especially concerning small water supplies. It also seems that the local heath authorities are often not informed of incidents of non-compliance. It is important to improve registration, information exchange between parties, epidemiological surveys and follow up of outbreaks due to drinking water to gather lessons learned. Water quality at small water supplies needs to be improved with risk-based approach and risk management.