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IntroductionOverdiagnosis is the diagnosis of a disease that would never have caused any symptom or problem. It is a harmful side effect of screening and may lead to unnecessary treatment, costs and emotional drawbacks. Doctors and other healthcare professionals (HCPs) have the opportunity to mitigate these consequences, not only by informing their patients or the public but also by adjusting screening methods or even by refraining from screening. However, it is unclear to what extent HCPs are fully aware of overdiagnosis and whether it affects their screening decisions. With this systematic review, we aim to synthesise all available research about what HCPs know and think about overdiagnosis, how it affects their position on screening policy and whether they think patients and the public should be informed about it.Methods and analysisWe will systematically search several databases (MEDLINE, Embase, Web of Science, Scopus, CINAHL and PsycArticles) for studies that directly examine HCPs' knowledge and subjective perceptions of overdiagnosis due to health screening, both qualitatively and quantitatively. We will optimise our search by scanning reference and citation lists, contacting experts in the field and hand searching abstracts from the annual conference on 'Preventing Overdiagnosis'. After selection and quality appraisal, we will analyse qualitative and quantitative findings separately in a segregated design for mixed-method reviews. The data will be examined and presented descriptively. If the retrieved studies allow it, we will review them from a constructivist perspective through a critical interpretive synthesis.Ethics and disseminationFor this type of research, no ethical approval is required. Findings from this systematic review will be published in a peer-reviewed journal and presented at the annual congress of 'Preventing Overdiagnosis'. In addition, the results will serve as guidance for further research on this topic.PROSPERO registration numberCRD42021244513.

ObjectiveTo estimate the rate and type of downstream activities (DAs) after laboratory testing in primary care, with a specific focus on check-up laboratory panels, and to explore the effect of a clinical decision support system (CDSS) for laboratory ordering on these DAs.DesignCluster randomised clinical trial.Setting72 primary care practices in Belgium, with 272 general practitioners (GPs), randomly assigned to the intervention arm or the control arm.ParticipantsThe study included 10 270 lab panels from 9683 primary care patients (women 55.1%, mean age 56.5). All adult patients who consulted one of the participating GPs during the trial period and needed a laboratory exam were eligible for participation.InterventionsGPs in the intervention group used a CDSS integrated into their online laboratory ordering system, while GPs in the control arm used their lab ordering system as usual. The trial duration was 6 months, with another 6 months follow-up.Main outcome measuresThis publication reports on the exploratory outcome of DAs after an initial laboratory exam and the effect of the CDSS on these DAs.Results19.7% of all laboratory panels resulted in further diagnostic procedures (95% CI 18.9% to 20.5%) and 19% (95% CI 18.2% to 19.7%) in treatment changes. Check-up laboratory exams showed similar rates of DAs, with 17.5% (95% CI 13.8% to 21.2%) diagnostic DAs and 18.9% (95% CI 13.9% to 23.9%) treatment changes. Using the CDSS resulted in a significant reduction in downstream referrals (−2.4%; 95% CI −4.2% to −0.6%; p=0008), imaging and endoscopies (−0.9%; 95% CI −1.6% to −0.1%; p=0026) and treatment changes (−5.4%; 95% CI −9.5% to −1.2%; p=0.01).ConclusionThis is the largest study so far to examine DAs after laboratory testing. It shows that almost one in three laboratory exams leads to further DAs, even in check-up panels. Using a CDSS for laboratory orders may reduce the rate of some DAs.Trial registration numberNCT02950142.

Background Inappropriate laboratory test ordering poses an important burden for healthcare. Clinical decision support systems (CDSS) have been cited as promising tools to improve laboratory test ordering behavior. The objectives of this study were to evaluate the effects of an intervention that integrated a clinical decision support service into a computerized physician order entry (CPOE) on the appropriateness and volume of laboratory test ordering, and on diagnostic error in primary care. Methods This study was a pragmatic, cluster randomized, open-label, controlled clinical trial. Setting Two hundred eighty general practitioners (GPs) from 72 primary care practices in Belgium. Patients Patients aged ≥ 18 years with a laboratory test order for at least one of 17 indications: cardiovascular disease management, hypertension, check-up, chronic kidney disease (CKD), thyroid disease, type 2 diabetes mellitus, fatigue, anemia, liver disease, gout, suspicion of acute coronary syndrome (ACS), suspicion of lung embolism, rheumatoid arthritis, sexually transmitted infections (STI), acute diarrhea, chronic diarrhea, and follow-up of medication. Interventions The CDSS was integrated into a computerized physician order entry (CPOE) in the form of evidence-based order sets that suggested appropriate tests based on the indication provided by the general physician. Measurements The primary outcome of the ELMO study was the proportion of appropriate tests over the total number of ordered tests and inappropriately not-requested tests. Secondary outcomes of the ELMO study included diagnostic error, test volume, and cascade activities. Results CDSS increased the proportion of appropriate tests by 0.21 (95% CI 0.16–0.26, p < 0.0001) for all tests included in the study. GPs in the CDSS arm ordered 7 (7.15 (95% CI 3.37–10.93, p = 0.0002)) tests fewer per panel. CDSS did not increase diagnostic error. The absolute difference in proportions was a decrease of 0.66% (95% CI 1.4% decrease–0.05% increase) in possible diagnostic error. Conclusions A CDSS in the form of order sets, integrated within the CPOE improved appropriateness and decreased volume of laboratory test ordering without increasing diagnostic error. Trial registration ClinicalTrials.gov Identifier: NCT02950142, registered on October 25, 2016

Coagulase-negative staphylococci (CNS) are abundantly present in the dairy farm environment and on bovine skin and mucosae. They are also the most prevalent bacteria causing bovine intramammary infections (IMI). Reservoirs and transmission routes of CNS are not yet fully unraveled. The objectives of this study were to explore the distribution of CNS in parlor-related extramammary niches and to compare it to the distributions of CNS causing IMI in those herds. Niches that were targeted in this study were cows' teat apices, milking machine unit liners, and milker's skin or gloves. Each of the three herds had its own CNS microbiota in those niches. The most prevalent species in the parlor-related extramammary niches were Staphylococcus cohnii, S. fleurettii, and S. equorum in the first, second, and third herd, respectively, whereas S. haemolyticus and S. sciuri were found in all herds. S. cohnii and S. fleurettii, as well as S. haemolyticus, which was present in each herd, were also frequently found in milk samples. By contrast, S. chromogenes, S. simulans, and S. xylosus favored the mammary gland, whereas S. equorum was more common in the parlor-associated niches. Within each herd, species distribution was similar between teat apices and milking machine unit liners. In conclusion, some of the extramammary niches related to the milking process might act as infection sources for IMI-causing CNS. This study provides further evidence that the group of CNS species is comprised of environmental, opportunistic and host-adapted species which differ in ecology.

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.

Coagulase-negative staphylococci (CNS) are a major cause of intramammary infections (IMI) in dairy cows and they colonize the teat skin. Staphylococcus haemolyticus, one of the more common CNS, has been identified as a highly versatile opportunistic species. The aim of the present study was to gain better insight into the adaptation of S. haemolyticus subtypes to the udder ecosystem with respect to IMI development. During a longitudinal observational study conducted over 13 mo on 6 Flemish dairy herds, S. haemolyticus isolates were recovered from milk and teat apices. A total of 44 S. haemolyticus isolates originating from milk (24 isolates) and teat apices (20 isolates) of 6 selected udder quarters were singled out and analyzed using a combined methodology of (GTG)5-PCR and amplified fragment length polymorphism (AFLP) fingerprinting to determine intraspecies differences. Combining both fingerprinting methods, 4 S. haemolyticus subtypes were obtained (I to IV). Subtypes I, II, and IV were recovered from both milk and teat apex samples and were found to be associated with persisting IMI. Subtype III, not apparently related to IMI, was isolated solely from teat apices and not from milk. In general, S. haemolyticus subtypes found in milk from infected quarters could be recovered from the corresponding teat apices, although the latter could be colonized with up to 3 different subtypes. Comparing subtypes from milk and teat apices indicates that the IMI-causing agent likely originates from the teat skin.

The bioeconomy is fueled by Biological Resource Centers, which play a vital role in harnessing and preserving the world’s biodiversity [1]. MIRRI (the Microbial Resource Research Infra- structure: www.mirri.org) is an EU-project involving a total of 33 partners, aiming to provide facilitated access to microbial resources, associated data and expertise, and promote knowledge transfer and foster innovation. One crucial step is to properly define our stakeholder community and identify their current and future needs, and match them to our offer. In order to achieve these goals, MIRRI surveyed both its part- ners, and current and potential users of microbial resources and services. Here we present and analyze some of the results of this survey, focusing on training and education. GENERAL BIOTECHNOLOGY Despite current trends and benefits in increased use and produc- tion of contents in new formats (e.g. video, interactive), and use of new tools and technologies (e.g. e-learning, b-learning), train- ing within MIRRI still has an overwhelming dominance of classic content types and delivery methodologies. Also, we identified a much wider untapped market for education and training within our customer base, and we estimate a spike in demand of training from culture collections, particularly from the profit sector. Additional efforts are clearly necessary in adjusting our offer, adapting contents and content delivery and focusing on cost- efficiency and proper advertising to increase visibility, and better serve the needs of our customers.

Campylobacter is the most commonly reported gastrointestinal bacterial pathogen in humans in many developed countries. During slaughter of broiler flocks, it is difficult to avoid contamination of broiler carcasses. This study aimed to quantify Campylobacter contamination on broiler carcasses at 5 points in the slaughter processing during the slaughter of a Campylobacter-colonized flock by real-time PCR and conventional enumeration. In addition, the decontamination effect of neutral electrolyzed oxidizing (EO) water and 1.5% lactic acid (pH 2.0) were evaluated. During processing, the Campylobacter counts on the carcasses declined toward the end of the processing line. The log counts on the carcasses as determined by quantitative real-time PCR (qPCR), decreased from 9.37 after scalding to 8.08 after the last cooling step. Enumeration of the campylobacters on plates revealed the same trend, although the counts per carcass were generally 3 logs lower. After scalding, a mean of 6.86 log cfu/carcass were counted, which decreased to 4.83 log cfu/carcass after the last cooling step. Submerging carcasses after scalding in EO water gave a significant reduction of 1.31 log cfu/carcass by enumeration on plates and a not significant reduction of 0.53 log cfu/carcass by qPCR. Treatment of the carcasses after the inside-outside bird washer led to reductions from 0.09 to 0.91 log cfu/carcass, although not significant. After submerging the carcasses in a 1.5% lactic acid solution, significant reductions of 1.62 and 1.24 log cfu/carcass by qPCR and enumeration, respectively, were observed. Spraying the carcasses with lactic acid led to nonsignificant reductions of 0.68 log cfu/carcass determined by qPCR and 0.26 log cfu/carcass by enumeration. Both EO water and lactic acid seem promising for implementation in poultry processing plants.

Swab samples (n=72) obtained from the teat apex of lactating dairy cows without visual signs of inflammation (n=18) were gathered on 2 well-managed Flemish dairy herds (herds 1 and 2) during the same month to assess the bacterial diversity of teat apices before milking. A combination of both culture-dependent [plating and (GTG)(5)-PCR fingerprinting of the colonies] and culture-independent [denaturing gradient gel electrophoresis (PCR-DGGE)] techniques indicated that the teat apices contain a wide diversity of bacterial genera. Despite a low bacterial load, 20 bacterial genera of 3 phyla (Actinobacteria, Firmicutes, and Proteobacteria) were present. The most prevalent bacteria were the coagulase-negative staphylococci (CNS), encompassing a total of 15 species, which were identified to the species level using a combination of (GTG)(5)-PCR fingerprinting, gene sequencing (16S ribosomal RNA and rpoB genes), and a novel PCR-DGGE technique based on the tuf-PCR amplicon. Overall bacterial diversity did not differ significantly between the herds or between noninfected and subclinically infected quarters in herd 1. In herd 1, borderline significant lower CNS species diversity was found on teat apices of noninfected quarters compared with subclinically infected quarters. The most prevalent CNS species were Staphylococcus haemolyticus and Staphylococcus equorum in both herds and Staphylococcus carnosus in herd 2.

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n = 12) and a teat apex (n = 1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA–DNA hybridization with S. hyicus DSM 20459T and S. chromogenes DSM 20674T confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C15 : 0, ai-C15 : 0, i-C17 : 0 and C20 : 0 and the peptidoglycan type was A3α l-Lys–Gly5. Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4T ( = DSM 23656T = CCUG 59809T) is the type strain.

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant pathogens causing intramammary infections (IMI) in dairy cows. The cows' environment is thought to be a possible source for CNS mastitis and this was investigated in the present paper. A longitudinal field study was carried out in 6 well-managed dairy herds to determine the distribution and epidemiology of various CNS species isolated from milk, causing IMI and living freely in the cows' environment, respectively. In each herd, quarter milk samples from a cohort of 10 lactating cows and environmental samples from stall air, slatted floor, sawdust from cubicles, and sawdust stock were collected monthly (n = 13). Isolates from quarter milk samples (n = 134) and the environment (n = 637) were identified to species level using amplified fragment length polymorphism (AFLP) genotyping. Staphylococcus chromogenes, S. haemolyticus, S. epidermidis, and S. simulans accounted for 81.3% of all CNS milk isolates. Quarters were considered infected with CNS (positive IMI status) only when 2 out of 3 consecutive milk samples yielded the same CNS AFLP type. The species causing IMI were S. chromogenes (n = 35 samples with positive IMI status), S. haemolyticus (n = 29), S. simulans (n = 14), and S. epidermidis (n = 6). The observed persistent IMI cases (n = 17) had a mean duration of 149.4 d (range 63.0 to 329.8 d). The CNS species predominating in the environment were S. equorum, S. sciuri, S. haemolyticus, and S. fleurettii. Herd-to-herd differences in distribution of CNS species were observed in both milk and the environment, suggesting that herd-level factors are involved in the establishment of particular species in a dairy herd. Primary reservoirs of the species causing IMI varied. Staphylococcus chromogenes and S. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. For S. haemolyticus and S. simulans, the environment was found as a reservoir, suggesting that IMI withthese species were possibly environmental in origin.

Salmonella Enteritidis strains of egg- and non-egg-related origin were characterized molecularly to find markers correlated with the egg-contaminating property of Salmonella Enteritidis. Isolates were examined by random amplified polymorphic DNA (RAPD), plasmid profiling and phage typing. Furthermore, the presence of 30 virulence genes was tested by PCR. In genetic fingerprinting and gene content, only small differences between the strains were found and no correlation was observed with the origin (egg-related versus non-egg-related). A major RADP group was present in both egg- and non-egg-related strains, but other smaller RAPD groups were present as well in both categories of strains. Phage types PT4 and PT21 were predominant. Differential mRNA expression levels of fimA and agfA under conditions of growth simulating the conditions during egg formation were determined by real-time RT-PCR. Although differences in fimA and agfA expression levels were observed between the strains, these could not be correlated with the origin of the strains (egg-related versus non-egg-related). The highest expression levels of agfA and fimA were only found in two non-egg-related strains, which seemed to be correlated with the presence of a 93 kb plasmid instead of the 60 kb virulence plasmid. Our results seem to indicate only a limited role for at least type I fimbriae (encoded by fim operon) in egg contamination by Salmonella Enteritidis.

Aims: In this study, a real-time quantitative polymerase chain reaction (PCR) method was examined for its ability to quantify Campylobacter spp. in chicken carcass rinses and compared with bacteriological culturing. Methods and Results: The linearity of the real-time PCR quantification protocol was assessed on pure DNA. The amplification efficiency was 100% and the square regression coefficient (R 2 ) was 0AE998. Quantification was linear over at least 7 log units. Using a crude cell lysate gave the highest sensitivity and the detection limit of the method was 3AE3 log CFU per carcass. The statistical correlation between the bacteriological enumeration and the real-time quantitative (Q)-PCR determined using chicken carcasses sampled at the end of the slaughter line was 0AE733. The difference in detection levels was probably because of the detection of stressed, dead or viable but not culturable cells by Q-PCR. Conclusion: The real-time Q-PCR method described in this study is a powerful tool for determining the number of Campylobacter cells on carcasses. Significance and Impact of the Study: The real-time Q-PCR method is available to quantify the Campylobacter contamination at the slaughterhouse level and could be used to evaluate primary production.

A longitudinal study was carried out to detect intramammary infections caused by Klebsiella pneumoniae and to identify potential sources of this bacterial species in the environment of the cows. The study was performed in 6 well-managed Belgian dairy herds from May 2008 to May 2009. Monthly (n = 13), unused and used sawdust bedding samples as well as individual quarter milk and feces samples were collected from 10 randomly selected cohort cows in each herd. Cases of clinical mastitis of all lactating cows in the 6 herds were also sampled (n = 64). From the 3,518 collected samples, 153 K. pneumoniae isolates were obtained, of which 2 originated from milk (clinical mastitis cases). In feces (n = 728), used bedding (n = 73), and unused bedding (n = 73), respectively, 125 (17.2%), 20 (27.4%), and 6 (8.2%) isolates were found. The isolates were fingerprinted by means of pulsed field gel electrophoresis. In total, 109 different pulsotypes were differentiated, indicating a high degree of genetic diversity within the isolates. All isolates from unused bedding belonged to pulsotypes other than those from the other sources, suggesting that sources other than unused sawdust may introduce K. pneumoniae into the herd. Only 2 pulsotypes contained isolates originating from different sources. Pulsotype 10 was found in milk and used bedding and pulsotype 21 was found in feces and used bedding. The 2 milk isolates originated from 2 cows in the same herd but they belonged to a different pulsotype. The results indicate that K. pneumoniae can be prevalent in the environment without causing significant mastitis problems. Most cows were shedding K. pneumoniae in feces, substantiating findings under very different conditions (i.e., American dairy herds). Contamination of used bedding in the cubicles with K. pneumoniae from feces was confirmed, whereas unused bedding was not an important source of K. pneumoniae for the environment of the cows.

The aim of this study was to investigate whether the main coagulase-negative staphylococci (CNS) species involved in bovine intramammary infections (IMI) possess specific characteristics that promote colonization of the udder. Virulence markers associated with biofilm formation, antimicrobial resistance, and biocide tolerance were compared between typically contagious CNS species (Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans) and those rarely causing IMI (Staphylococcus sciuri, Staphylococcus equorum, and others) to find possible associations with pathogenicity. Coagulase-negative staphylococci isolates (n=366) belonging to 22 different species were analyzed by PCR for the presence of the biofilm-associated genes bap and icaA, and the methicillin resistance gene mecA. A selection of 82 isolates was additionally tested for their susceptibility to 5 antibiotics and 2 commercial teat dip products. Minimum inhibitory concentrations of antimicrobials were determined by Etest (AB bioMerieux, Marcy l'Etoile, France), and a microdilution method was optimized to determine minimum biocidal concentrations of teat dips. The bap, icaA, and mecA genes were detected significantly more in isolates from CNS species typically living in the cows' environment than in isolates from IMI-causing species. Antimicrobial resistance was mainly against erythromycin (23%) or oxacillin (16%), and was detected more often in the environmental species. The isolates least susceptible to the teat dips belonged to the IMI-causing species Staph. chromogenes and Staph. simulans. We concluded that carriage of biofilm genes and antimicrobial resistance were not associated with the ability to colonize the mammary gland because free-living CNS species constituted a more significant reservoir of biofilm and resistance determinants than did IMI-causing species. In contrast, increased tolerance to biocides may favor the establishment of bovine IMI by some CNS species.