The effect of changing diagnostic method from culture to PCR on the number of episodes of human campylobacteriosis in Denmark: a retrospective study (2015-2022).

IMPORTANCE: This study is important because it shows the potential epidemiological silence associated with the use of culture as the primary diagnostic method for the laboratory identification of human campylobacteriosis. Also, we show how polymerase chain reaction methods are associated with a systematic increase in the number of human campylobacteriosis episodes as reported by routine disease surveillance. These findings are operationally relevant and have public health implications because they tell how crucial it is to consider changes in diagnostic methods, e.g., in the epidemiological analysis of historical data and in the interpretation of future data in light of the past. We also believe that this study highlights how the synergy between microbiology and epidemiology is essential for disease surveillance.

LiSEQ - whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe.

We present the LiSEQ (Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.

Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics?

In microbial food safety, molecular methods such as quantitative PCR (qPCR) and next-generation sequencing (NGS) of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample preparation for NGS-based shotgun metagenomics with a diagnostic focus. This was used on human fecal samples spiked with 10³ or 106 colony-forming units (CFU)/g Campylobacter jejuni, as well as porcine fecal samples spiked with 10³ or 106 CFU/g Salmonella typhimurium. DNA was extracted from the samples using variations of two widely used kits. The following quality parameters were measured: DNA concentration, qPCR, DNA fragmentation during library preparation, amount of DNA available for sequencing, amount of sequencing data, distribution of data between samples in a batch, and data insert size; none showed any correlation with the target ratio of the spiking organism detected in sequencing data. Surprisingly, diagnostic metagenomics can have better detection sensitivity than qPCR for samples spiked with 10³ CFU/g C. jejuni. The study also showed that qPCR and sequencing results may be different due to inhibition in one of the methods. In conclusion, qPCR cannot uncritically be used as an indicator for the optimization of sample preparation for diagnostic metagenomics.

PFGE standard operating procedures for Listeria monocytogenes: harmonizing the typing of food and clinical strains in Europe.

Listeria monocytogenes is a foodborne pathogen responsible for a severe disease known as listeriosis. The European Centre for Disease Prevention and Control (ECDC) coordinates a network of national public health laboratories (NPHLs) in charge of typing clinical strains. In food, it is the European Union Reference Laboratory for L. monocytogenes (EURL Lm), which manages a network of National Reference Laboratories (NRLs). A pulsed-field gel electrophoresis (PFGE) standard operating procedure (EURL SOP) has been used routinely at the EURL Lm since 2007. The EURL Lm has recommended that NRLs use the EURL SOP, whereas the Statens Serum Institut (SSI), under contract for ECDC, requested that NPHLs use Halpins' SOP (HSOP) published in 2010 for the PulseNet USA network. An update of Halpins' SOP (uHSOP) was published in 2013. To facilitate the exchange of profiles among human and food European reference laboratories, it is crucial to ensure that the PFGE profiles obtained with these different SOPs are comparable. The aim here was to compare the EURL SOP with HSOP and uHSOP. The panel comprised 114 well-characterized SSI/EURL strains. All were characterized at the EURL using both the EURL SOP and uHSOP. Seventy of the 114 strains were also characterized at the SSI using HSOP. The EURL SOP and uHSOP produced indistinguishable combined (ApaI/AscI) profiles for the 114 strains tested. The EURL SOP and HSOP produced indistinguishable combined profiles for 69 of the 70 strains tested. One strain displayed for the AscI profile an additional low-intensity band at 184 kbp with HSOP. For this strain, SSI and EUR Lm had already observed the same profile from NPHLs and NRLs. However, this deviation is minor as it accounted for about 1% of all the 114 combined profiles. This study should facilitate the exchange of reproducible PFGE profiles among human and food reference laboratories.

Occurrence of Campylobacter jejuni in pets living with human patients infected with C. jejuni.

Campylobacter jejuni was recovered from four dogs (11%) and four cats (33%) living with Danish human patients infected with C. jejuni. Pulsed-field gel electrophoresis (PFGE) analysis revealed the occurrence of the same quinolone-resistant strain in a girl and her dog. C. jejuni isolates with closely related (>95% similarity) PFGE profiles occurred in humans and pets from different Danish counties.

Water-borne Campylobacter jejuni infection in a Danish town---a 6-week continuous source outbreak.

OBJECTIVE: To determine the cause and characteristics of illness of a Campylobacter jejuni outbreak in Denmark in 1995--96. METHODS: A retrospective follow-up study was designed for culture-confirmed cases and for residents without a bacteriologic diagnosis. Stored clinical and environmental isolates were analyzed by serotyping and genotyping with restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE) and ribotyping. RESULTS: Campylobacter jejuni was isolated from 110 residents and visitors to the area. However, an estimate based on a telephone survey indicated that some 2400 people were affected by the outbreak. Water samples obtained from the community waterworks contained Campylobacter jejuni serotype O2, the same serotype as in all but one of the 30 stored isolates from the outbreak. The water and clinical isolates also showed the same DNA profile, except for the single strain showing the distinct serotype. The contamination of the water supply was traced back to contamination of ground water due to a break in a sewage pipe. CONCLUSIONS: A retrospective and demographic epidemiologic investigation of both culture-confirmed and non-culture-confirmed cases in the town combined with typing of the isolates was crucial in defining the extent and cause of the outbreak.