Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver.

BACKGROUND: In the last years, the number of notified hepatitis E cases in humans has continuously increased in Europe. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Undercooked liver and raw sausages containing the liver of pigs and wild boar are at high risk of containing HEV. However, so far, no standardized method for the detection of HEV-RNA in pig liver is available. METHODS: An international collaborative study on method reproducibility involving 11 laboratories was performed for an HEV-RNA detection method, which consists of steps of sample homogenization, RNA extraction and real-time RT-PCR detection, including a process control. Naturally contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. RESULTS: Valid results were retrieved from 10 laboratories. A specificity of 100% and a sensitivity of 79% were calculated for the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. CONCLUSIONS: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, therefore, be applied to routine food control as well as in monitoring studies.

Hepatitis A Outbreak in the General Population due to a MSM-Associated HAV Genotype Linked to a Food Handler, November 2017-February 2018, Germany.

Hepatitis A (HAV) is a viral infection causing a range of symptoms, sudden onset of fever, malaise, diarrhea, and jaundice. It is mostly transmitted fecal-oral through contaminated food, with immediate household and sexual contacts having a higher risk of infection. Since 2016 an increased number of HAV infections, mostly affecting men who have sex with men (MSM) have been noticed worldwide, with three main genotypes circulating. We report here on the first spillover outbreak of the MSM-associated HAV genotype RIVM-HAV16-090 in the German general population in November 2017-February 2018. In total, twelve cases could be attributed to the outbreak with the index case and a coworker in a butchers shop being the most probable source of the outbreak. The identical HAV genotype was detected in two environmental samples in the premises of the butchers shop and in nine cases. Outbreak control measures included detailed contact tracing and stool examinations, several environmental investigations, thorough cleaning, and disinfection of the premises of the butchers shop. Post-exposure vaccination was recommended to all unprotected contacts during the investigation. Furthermore, although hand-washing facilities were in accordance with the required law, additional installment of soap and disinfectant dispensers and contactless faucets has been recommended.

Interlaboratory Validation of a Method for Hepatitis E Virus RNA Detection in Meat and Meat Products.

Increasing numbers of hepatitis E cases are currently recognized in many European countries. The zoonotic hepatitis E virus (HEV) genotype 3 mainly circulates in domestic pigs and wild boars, and can be transmitted to humans via consumption of insufficiently heated meat or meat products produced from those animals. Here, a detailed protocol for detection of HEV RNA in meat products is provided, which is based on the method originally described by Szabo et al. (Intl J Food Microbiol 215:149-156, 2015). It consists of a TRI Reagent®/chloroform-based food matrix homogenization, a silica bead-based RNA extraction and a real-time RT-PCR-based RNA detection. The method was further validated in a ring trial with nine independent laboratories using pork liver sausage samples artificially contaminated with different amounts of HEV. The results indicate sufficient sensitivity, specificity, and accuracy of the method for its broad future use in survey studies, routine food control or outbreak investigations.

Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates from Milk Samples of Dairy Cows with Mastitis in Bavaria, Germany.

The aim of this study was to determine the rate of extended-spectrum ß-lactamase (ESBL)-producing microorganisms among Escherichia coli isolates causing bovine mastitis, including molecular characterization of these isolates. Therefore, a total of 490 bovine E. coli isolates from milk samples of dairy cows with mastitis were investigated for ESBL production by antimicrobial susceptibility testing, PCR-based detection, and sequencing of ESBL encoding genes, which were identified in 22 isolates (4.5%). Moreover, resistance to the fluoroquinolones enrofloxacin and marbofloxacin occurred in 15 of 22 ESBL-producing isolates (68.2%). All ESBL-producing isolates carried a blaCTX-M-like gene, with blaCTX-M-14 (n = 10) as the most prevalent type. Seven isolates producing CTX-M-14 and belonging to phylogenetic group A were further investigated for genetic relatedness by multilocus sequence typing. Five of them could be assigned to four different sequence types (STs): ST10 (n = 2), ST167 (n = 1), ST410 (n = 1), and ST744 (n = 1), whereas the remaining two isolates could not be assigned. To conclude, the rate of ESBL-producing E. coli associated with cattle mastitis was 4.5%. Furthermore, a high proportion of fluoroquinolone coresistance could be detected. Therefore, careful and continuous surveillance of ESBL-producing E. coli in cattle and consequent implementation of prevention measures are needed to avoid a further spread of these multidrug-resistant bacteria.