An interlaboratory study on the detection methods for enterotoxigenic Escherichia coli in vegetables using enterotoxin gene screening and selective agars for ETEC-specific isolation.

Enterotoxigenic Escherichia coli (ETEC) causes acute diarrhea and is transmitted through contaminated food and water; however, systematic procedures for its specific detection in foods have not been established. To establish an efficient detection method for ETEC in food, an interlaboratory study using ETEC O148 and O159 as representative serogroups was first conducted with 13 participating laboratories. A series of tests including enrichment, real-time PCR assays, plating on selective agars, and concentration by immunomagnetic separation followed by plating onto selective agar (IMS-plating methods) were employed. This study particularly focused on the detection efficiencies of real-time PCR assays for enterotoxin genes (sth, stp, and lt), IMS-plating methods, and direct plating onto sorbitol MacConkey agar and CHROMagar STEC medium, supplemented with tobramycin, which is a novel modification in the preparation of a selective agar. Cucumber and leek samples inoculated with ETEC O148 and O159, either at 4-7 CFU/25 g (low levels) or at 21-37 CFU/25 g (high levels) were used as samples with uninoculated samples used as controls. At high inoculation levels, the sensitivities of sth, stp, and lt detection, direct-plating, and IMS-plating methods in cucumber inoculated with O148 and in both foods inoculated with O159 were 100%. In leek inoculated with high levels of O148, the sensitivities of sth, stp, and lt detection, direct-plating, and the IMS-plating method were 76.9%, 64.1%, and 74.4%, respectively. At low inoculation levels, the sensitivities of sth, stp, and lt detection, direct plating, and IMS-plating method in cucumber inoculated with O148 and in both foods inoculated with O159 were in the range of 87.2-97.4%. In leek inoculated with low levels of O148, the sensitivities of sth, stp, and lt detection, direct plating, and the IMS-plating method were 59.0%, 33.3%, and 38.5%, respectively. Thus, ETEC in food contaminated with more than 21 CFU/25 g were detected at high rate (over 74%) using real-time PCR assays and IMS-plating onto selective agar. Therefore, screening sth, stp, and lt genes followed by isolation of STEC using the IMS-plating method may be an efficient method for ETEC detection.

Inter-Laboratory Study to Validate New Rapid Screening Methods for Kudoa septempunctata.

Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is time-consuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification (LAMP) method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method (10(5) spores/g olive flounder), have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 10(4) spores/g; however, these methods were able to detect more than 10(5) spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.

First report on invasion of yellow fever mosquito, Aedes aegypti, at Narita International Airport, Japan in August 2012.

The invasion of the yellow fever mosquito Aedes aegypti at Narita International Airport, Japan was detected for the first time. During the course of routine vector surveillance at Narita International Airport, 27 Ae. aegypti adults emerged from larvae and pupae collected from a single larvitrap placed near No. 88 spot at passenger terminal 2 on August 8, 2012. After the appearance of Ae. aegypti in the larvitrap, we defined a 400-m buffer zone and started an intensive vector survey using an additional 34 larvitraps and 15 CO2 traps. International aircraft and passenger terminal 2 were also inspected, and one Ae. aegypti male was collected from the cargo space of an international aircraft from Darwin via Manila on August 28, 2012. Larvicide treatment with 1.5% fenitrothion was conducted in 64 catch basins and one ditch in the 400-m buffer zone. Twenty-four large water tanks were also treated at least once with 0.5% pyriproxyfen, an insect growth regulator. No Ae. aegypti eggs or adults were found during the 1-month intensive vector survey after finding larvae and pupae in the larvitrap. We concluded that Ae. aegypti had failed to establish a population at Narita International Airport.

Estimating the burden of acute gastroenteritis and foodborne illness caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus by using population-based telephone survey data, Miyagi Prefecture, Japan, 2005 to 2006.

Most cases of acute gastroenteritis and foodborne disease are not ascertained by public health surveillance because the ill person does not always seek medical care and submit a stool sample for testing, and the laboratory does not always test for or identify the causative organism. We estimated the total burden of acute gastroenteritis in Miyagi Prefecture, Japan, using data from two 2-week cross-sectional, population-based telephone surveys conducted in 2006 and 2007. To estimate the number of acute gastroenteritis illnesses caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus in Miyagi Prefecture, we determined the number of cases for each pathogen from active laboratory-based surveillance during 2005 to 2006 and adjusted for seeking of medical care and submission of stool specimens by using data from the population-based telephone surveys. Monte Carlo simulation was used to incorporate uncertainty. The prevalence of acute gastroenteritis in the preceding 4 weeks was 3.3% (70 of 2,126) and 3.5% (74 of 2,121) in the winter and summer months, yielding an estimated 44,200 episodes of acute gastroenteritis each year in this region. Among people with acute gastroenteritis, the physician consultation rate was 32.0%, and 10.9% of persons who sought care submitted a stool sample. The estimated numbers of Campylobacter-, Salmonella-, and V. parahaemolyticus -associated episodes of acute gastroenteritis were 1,512, 209, and 100 per 100,000 population per year, respectively, in this region. These estimates are significantly higher than the number of reported cases in surveillance in this region. Cases ascertained from active surveillance were also underrepresented in the present passive surveillance, suggesting that complementary surveillance systems, such as laboratory-based active surveillance in sentinel sites, are needed to monitor food safety in Japan.

The human health burden of foodborne infections caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus in Miyagi Prefecture, Japan.

To estimate the human health burden of foodborne infections caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus in Japan, an epidemiological study was conducted in Miyagi Prefecture. Laboratory-confirmed infections among patients with diarrhea caused by the three pathogens were ascertained from two clinical laboratories in the prefecture from April 2005 to March 2006. To estimate the number of ill persons who were not laboratory-confirmed, we estimated physician-consultation rates for patients with acute diarrhea by analyzing foodborne outbreak investigation data for each pathogen and the frequency at which stool specimens were submitted from a physician survey. Each factor was added to a Monte-Carlo simulation model as a probability distribution, and the number of laboratory-confirmed cases was extrapolated to estimate the total number of ill persons. The estimated incidence of foodborne infections per 100,000 per year in this region estimated by this model was 237 cases for Campylobacter, 32 cases for Salmonella, and 15 cases for V. parahaemolyticus. Simulated results indicate a significant difference between our estimated incidence and the reported cases of food poisoning in this region. An enhanced surveillance system is needed to complement the present passive surveillance on foodborne illnesses in Japan to identify food safety issues more precisely, and to monitor the effectiveness of risk management options.