Evaluation of molecular-based methods for the detection and quantification of Cryptosporidium spp. in wastewater.

Cryptosporidium poses significant public health risks as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized wastewater surveillance methods for Cryptosporidium spp. challenges implementation design and the comparability between studies. Thus, this study compared and contrasted Cryptosporidium wastewater surveillance methods for concentrating wastewater oocysts, extracting oocyst DNA, and detecting Cryptosporidium genetic markers. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome Particles, with and without additional immunomagnetic separation purification (except for the Nanotrap Microbiome Particles). Oocyst DNA extraction by either the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit were evaluated and the impact of bead beating and freeze-thaw pretreatments on DNA recoveries was assessed. Genetic detection via qPCR assays targeting either the Cryptosporidium 18S rRNA gene or the Cryptosporidium oocyst wall protein gene were tested. Oocyst recovery percentages were highest for centrifugation (39-77 %), followed by the Nanotrap Microbiome Particles (24 %), electronegative filtration with a PBST elution (22 %), and Envirocheck HV capsule filtration (13 %). Immunomagnetic separation purification was found to be unsuitable due to interference from the wastewater matrix. Bead-beating pretreatment enhanced DNA recoveries from both the DNeasy Powersoil Pro kit (314 gc/µL DNA) and the QIAamp DNA Mini kit (238 gc/µL DNA). In contrast, freeze-thaw pretreatment reduced DNA recoveries to under 92 gc/µL DNA, likely through DNA degradation. Finally, while both qPCR assays were specific to Cryptosporidium spp., the 18S rRNA assay had a 5-fold lower detection limit and could detect a wider range of Cryptosporidium spp. than the Cryptosporidium oocyst wall protein assay.

Comparison of survey methods in norovirus outbreak investigation, Oregon, USA, 2009.

We compared data from an Internet-based survey and a telephone-based survey during a 2009 norovirus outbreak in Oregon. Survey initiation, timeliness of response, and attack rates were comparable, but participants were less likely to complete Internet questions. Internet-based surveys permit efficient data collection but should be designed to maximize complete responses.

Shigellosis from an interactive fountain: implications for regulation.

In July 2003, the authors investigated an outbreak of Shigella sonnei infections in Marion County, Oregon. Nineteen confirmed and 37 presumptive cases, mostly young children, were identified. A case-control study implicated play in an interactive fountain in a city park (matched odds ratio undefined; p < .002). The association was confirmed by a cohort study among local schoolchildren (RR [relative risk] = 12.6, p < .001) that allowed the authors to estimate that > 500 persons became ill. Fountain design flaws and inadequate maintenance set the stage for the outbreak. In 2007, the authors surveyed state health departments to assess rules and regulations governing interactive fountains. Thirty of 48 states responding (62%) reported public health regulation of fountains; standards and enforcement capacity varied. Regulation is a relatively new phenomenon; only 13 states (27%) had rules before 2000. A lack of enforceable design and maintenance standards increases the risk of enteric disease transmission at these increasingly popular venues.

Case Finding Using Syndromic Surveillance Data During an Outbreak of Shiga Toxin-Producing Escherichia coli O26 Infections, Oregon, 2015.

Shiga toxin-producing Escherichia coli (STEC) causes an estimated 265 000 infections in the United States annually. Of emerging non-O157:H7 STEC serotypes, O26 is the most commonly recognized. During an outbreak of STEC O26 in Oregon in 2015, we used syndromic surveillance data to supplement case finding by laboratory reporting. From 157 records retrieved by querying syndromic surveillance data, we detected 4 confirmed and 5 suspected cases. However, none of the suspected cases were confirmed by stool culture, and by the time that the data were being analyzed, the confirmed cases were already known to investigators. Syndromic surveillance data can potentially supplement case finding during outbreaks of foodborne disease. To be an effective case-finding strategy, timely completion of all steps, including collecting specimens from suspected cases, should be performed in real time.