Shigella spp. and entero-invasive Escherichia coli:diagnostics, clinical implications and impact on public health

The bacteria Shigella spp. and entero-invasive Escherichia coli (EIEC) cause a watery or bloody diarrhea in humans. This disease is known as dysentery or shigellosis. Shigella spp. and EIEC are related to that extent that distinction with culture or molecular methods at laboratories is challenging. As in many other countries, the Netherlands regulates only the control of infections with Shigella spp. through laws and guidelines. However, the problematic distinction of Shigella spp. and EIEC hampers the compliance to these regulations. The RIVM, UMCG and Certe collaborated to investigate the diagnostics, the clinical implications of Shigella spp. and EIEC and their impact on public health in the Netherlands. These studies are described in this thesis. First, different opportunities for the improvement of current diagnostics were examined. Unfortunately, it appeared that distinction of Shigella spp. and EIEC remained difficult, even with modern methods. Second, a study was conducted in cooperation with multiple health care institutions in which the consequences of infections with Shigella spp. and EIEC were assessed. The results indicated that there were no differences in severity of disease caused by these bacteria. Third, it was demonstrated that the detailed assessment of the genomes of the bacteria improved the determination of both the similarity of bacteria isolated from different patients and the source of the infections. Finally, suggestions for improvement of the shigellosis guidelines were made, which are now part of a reconsideration of these guidelines

MALDI-TOF MS Using a Custom-Made Database, Biomarker Assignment, or Mathematical Classifiers Does Not Differentiate Shigella spp. and Escherichia coli

Shigella spp. and E. coli are closely related and cannot be distinguished using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) with commercially available databases. Here, three alternative approaches using MALDI-TOF MS to identify and distinguish Shigella spp., E. coli, and its pathotype EIEC were explored and evaluated using spectra of 456 Shigella spp., 42 E. coli, and 61 EIEC isolates. Identification with a custom-made database resulted in >94% Shigella identified at the genus level and >91% S. sonnei and S. flexneri at the species level, but the distinction of S. dysenteriae, S. boydii, and E. coli was poor. With biomarker assignment, 98% S. sonnei isolates were correctly identified, although specificity was low. Discriminating markers for S. dysenteriae, S. boydii, and E. coli were not assigned at all. Classification models using machine learning correctly identified Shigella in 96% of isolates, but most E. coli isolates were also assigned to Shigella. None of the proposed alternative approaches were suitable for clinical diagnostics for identifying Shigella spp., E. coli, and EIEC, reflecting their relatedness and taxonomical classification. We suggest the use of MALDI-TOF MS for the identification of the Shigella spp./E. coli complex, but other tests should be used for distinction

Increased antimicrobial resistance among non-typhoidal Salmonella infections in international travellers returning to the Netherlands

Highlights: Increased fluoroquinolone resistance in the two most common non-typhoidal Salmonella (NTS) serotypes among travellers returning to the Netherlands. Resistant Salmonella Enteritidis infections are most likely to be acquired abroad, specifically outside Europe. This study highlights the importance of travel history when patients with NTS infections require empiric antimicrobial treatment

Genome-wide association studies of Shigella spp. and Enteroinvasive Escherichia coli isolates demonstrate an absence of genetic markers for prediction of disease severity

BACKGROUND: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control. RESULTS: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used. CONCLUSIONS: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC

Changing epidemiology of Salmonella Enteritidis human infections in the Netherlands and Belgium, 2006 to 2019: a registry-based population study.

BackgroundSalmonellosis remains the second most common zoonosis in the European Union despite a long-term decreasing trend. However, this trend has been reported to have stagnated in recent years, particularly for Salmonella enterica serotype Enteritidis (SE).AimTo describe temporal changes in the incidence of SE human infections, and in its associated factors between 2006 and 2019. In addition, we aim to determine which factors influenced the stagnated trend seen in recent years.MethodsData on culture-confirmed SE human infections from national surveillance registries in the Netherlands and Belgium between 2006 and 2019 were analysed using multivariable negative-binomial regression models with restricted cubic splines.ResultsSE incidence was significantly higher in summer and autumn than winter, in persons aged 0-4 years and 5-14 years than in persons ≥ 60 years, and increased with increasing proportions of travel-related and resistant SE infections. SE incidence decreased significantly in both countries until 2015, followed by an increasing trend, which was particularly pronounced in the Netherlands. Potential SE outbreaks in both countries and invasive infections in the Netherlands also increased after 2015.ConclusionThe increase in potential outbreaks and invasive infections since 2015 may partially explain the observed reversal of the decreasing trend. While these results provide insights into the possible causes of this trend reversal, attention should also be given to factors known to influence SE epidemiology at primary (animal) production and pathogen genomic levels

Comparing Multiple Locus Variable-Number Tandem Repeat Analyses with Whole-Genome Sequencing as Typing Method for Salmonella Enteritidis Surveillance in The Netherlands, January 2019 to March 2020

In the Netherlands, whole-genome sequencing (WGS) was implemented as routine typing tool for Salmonella Enteritidis isolates in 2019. Multiple locus variable-number tandem repeat analyses (MLVA) was performed in parallel. The objective was to determine the concordance of MLVA and WGS as typing methods for S. Enteritidis isolates. We included S. Enteritidis isolates from patients that were subtyped using MLVA and WGS-based core-genome Multilocus Sequence Typing (cgMLST) as part of the national laboratory surveillance of Salmonella during January 2019 to March 2020. The concordance of clustering based on MLVA and cgMLST, with a distance of ≤5 alleles, was assessed using the Fowlkes-Mallows (FM) index, and their discriminatory power using Simpson's diversity index. Of 439 isolates in total, 404 (92%) were typed as 32 clusters based on MLVA, with a median size of 4 isolates (range:2 to 141 isolates). Based on cgMLST, 313 (71%) isolates were typed as 48 clusters, with a median size of 3 isolates (range:2 to 39 isolates). The FM index was 0.34 on a scale from 0 to 1, where a higher value indicates greater similarity between the typing methods. The Simpson's diversity index of MLVA and cgMLST was 0.860 and 0.974, respectively. The median cgMLST distance between isolates with the same MLVA type was 27 alleles (interquartile range [IQR]:17 to 34 alleles), and 2 alleles within cgMLST clusters (IQR:1-5 alleles). This study shows the higher discriminatory power of WGS over MLVA and a poor concordance between both typing methods regarding clustering of S. Enteritidis isolates. IMPORTANCE Salmonella is the most frequently reported agent causing foodborne outbreaks and the second most common zoonoses in the European Union. The incidence of the most dominant serotype Enteritidis has increased in recent years. To differentiate between Salmonella isolates, traditional typing methods such as pulsed-field gel electrophoresis (PFGE) and multiple locus variable-number tandem repeat analyses (MLVA) are increasingly replaced with whole-genome sequencing (WGS). This study compared MLVA and WGS-based core-genome Multilocus Sequence Typing (cgMLST) as typing tools for S. Enteritidis isolates that were collected as part of the national Salmonella surveillance in the Netherlands. We found a higher discriminatory power of WGS-based cgMLST over MLVA, as well as a poor concordance between both typing methods regarding clustering of S. Enteritidis isolates. This is especially relevant for cluster delineation in outbreak investigations and confirmation of the outbreak source in trace-back investigations

A Multifactorial Approach for Surveillance of Shigella spp. and Entero-Invasive Escherichia coli Is Important for Detecting (Inter)national Clusters

Shigella spp. and entero-invasive Escherichia coli (EIEC) can cause mild diarrhea to dysentery. In Netherlands, although shigellosis is a notifiable disease, there is no laboratory surveillance for Shigella spp. and EIEC in place. Consequently, the population structure for circulating Shigella spp. and EIEC isolates is not known. This study describes the phenotypic and serological characteristics, the phenotypic and genetic antimicrobial resistance (AMR) profiles, the virulence gene profiles, the classic multi-locus sequence types (MLST) and core genome (cg)MLST types, and the epidemiology of 414 Shigella spp. and EIEC isolates collected during a cross-sectional study in Netherlands in 2016 and 2017. S. sonnei (56%), S. flexneri (25%), and EIEC (15%) were detected predominantly in Netherlands, of which the EIEC isolates were most diverse according to their phenotypical profile, O-types, MLST types, and cgMLST clades. Virulence gene profiling showed that none of the isolates harbored Shiga toxin genes. Most S. flexneri and EIEC isolates possessed nearly all virulence genes examined, while these genes were only detected in approximately half of the S. sonnei isolates, probably due to loss of the large invasion plasmid upon subculturing. Phenotypical resistance correlated well with the resistant genotype, except for the genes involved in resistance to aminoglycosides. A substantial part of the characterized isolates was resistant to antimicrobials advised for treatment, i.e., 73% was phenotypically resistant to co-trimoxazole and 19% to ciprofloxacin. AMR was particularly observed in isolates from male patients who had sex with men (MSM) or from patients that had traveled to Asia. Furthermore, isolates related to international clusters were also circulating in Netherlands. Travel-related isolates formed clusters with isolates from patients without travel history, indicating their emergence into the Dutch population. In conclusion, laboratory surveillance using whole genome sequencing as high-resolution typing technique and for genetic characterization of isolates complements the current epidemiological surveillance, as the latter is not sufficient to detect all (inter)national clusters, emphasizing the importance of multifactorial public health approaches

New insights into the epidemiology of Listeria monocytogenes – A cross-sectoral retrospective genomic analysis in the Netherlands (2010–2020)

IntroductionListeriosis, caused by infection with Listeria monocytogenes (Lm), is a relatively rare but severe disease with one of the highest mortality rates among bacterial foodborne illnesses. A better understanding on the degree of Lm clustering, the temporal distribution of the clusters, and their association with the various food sources is expected to lead to improved source tracing and risk-based sampling.MethodsWe investigated the genomic epidemiology of Lm in the Netherlands between 2010 and 2020 by analyzing whole-genome-sequencing (WGS) data of isolates from listerioss patients and food sources from nationwide integrated surveillance and monitoring. WGS data of 756 patient and 770 food/environmental isolates was assessed using core-genome multi-locus sequence typing (cgMLST) with Hamming distance as measure for pairwise distances. Associations of genotype with the epidemiological variables such as patient’s age and gender, and systematic use of specific drugs were tested by multinomial logistic regressions. Genetic differentiation of the Lm within and between food categories was calculated based on allele frequencies at the 1701 cgMLST loci in each food category.ResultsWe confirmed previous results that some clonal complexes (CCs) are overrepresented among clinical isolates but could not identify any epidemiological risk factors. The main findings of this study include the observation of a very weak attribution of Lm types to food categories and a much better attribution to the producer level. In addition, we identified a high degree of temporal persistence of food, patient and mixed clusters, with more than half of the clusters spanning over more than 1 year and up to 10  years.DiscussionTaken together this would indicate that identifying persistent contamination in food production settings, and producers that process a wide variety of raw food produce, could significantly contribute to lowering the Lm disease burden

Meta-analysis of the clinical performance of commercial SARS-CoV-2 nucleic acid and antibody tests up to 22 August 2020

Background: Reliable testing for SARS-CoV-2 is key for the management of the COVID-19 pandemic. Aim: We estimate diagnostic accuracy for nucleic acid and antibody tests 5 months into the COVID-19 pandemic, and compare with manufacturer-reported accuracy. Methods: We reviewed the clinical performance of SARS-CoV-2 nucleic acid and antibody tests based on 93,757 test results from 151 published studies and 20,205 new test results from 12 countries in the European Union and European Economic Area (EU/ EEA). Results: Pooling the results and considering only results with 95% confidence interval width ≤ 5%, we found four nucleic acid tests, including one pointof- care test and three antibody tests, with a clinical sensitivity ≥ 95% for at least one target population (hospitalised, mild or asymptomatic, or unknown). Nine nucleic acid tests and 25 antibody tests, 12 of them point-of-care tests, had a clinical specificity of ≥ 98%. Three antibody tests achieved both thresholds. Evidence for nucleic acid point-of-care tests remains scarce at present, and sensitivity varied substantially. Study heterogeneity was low for eight of 14 sensitivity and 68 of 84 specificity results with confidence interval width ≤ 5%, and lower for nucleic acid tests than antibody tests. Manufacturer-reported clinical performance was significantly higher than independently assessed in 11 of 32 and four of 34 cases, respectively, for sensitivity and specificity, indicating a need for improvement in this area. Conclusion: Continuous monitoring of clinical performance within more clearly defined target populations is needed.</p

MALDI-TOF MS Using a Custom-Made Database, Biomarker Assignment, or Mathematical Classifiers Does Not Differentiate Shigella spp. and Escherichia coli

Shigella spp. and E. coli are closely related and cannot be distinguished using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) with commercially available databases. Here, three alternative approaches using MALDI-TOF MS to identify and distinguish Shigella spp., E. coli, and its pathotype EIEC were explored and evaluated using spectra of 456 Shigella spp., 42 E. coli, and 61 EIEC isolates. Identification with a custom-made database resulted in &gt;94% Shigella identified at the genus level and &gt;91% S. sonnei and S. flexneri at the species level, but the distinction of S. dysenteriae, S. boydii, and E. coli was poor. With biomarker assignment, 98% S. sonnei isolates were correctly identified, although specificity was low. Discriminating markers for S. dysenteriae, S. boydii, and E. coli were not assigned at all. Classification models using machine learning correctly identified Shigella in 96% of isolates, but most E. coli isolates were also assigned to Shigella. None of the proposed alternative approaches were suitable for clinical diagnostics for identifying Shigella spp., E. coli, and EIEC, reflecting their relatedness and taxonomical classification. We suggest the use of MALDI-TOF MS for the identification of the Shigella spp./E. coli complex, but other tests should be used for distinction