Corynebacterium diphtheriae and Corynebacterium ulcerans: development of EUCAST methods and generation of data on which to determine breakpoints.

BACKGROUND: Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. OBJECTIVES: We aimed to evaluate broth microdilution and disc diffusion methods and create a dataset of MIC values and inhibition zone diameters (ZDs) from which breakpoints could be determined. METHODS: We included 400 recent clinical isolates equally distributed by species (Corynebacterium diphtheriae and Corynebacterium ulcerans) and by national surveillance programmes (France and Germany). Non-duplicate toxigenic and non-toxigenic isolates were chosen to enable the inclusion of a diversity of susceptibility levels for the 13 agents tested. Broth microdilution and disc diffusion, using EUCAST methodology for fastidious organisms, were used. RESULTS: The distributions of MIC and ZD values were largely in agreement among methods and countries. Breakpoints to allow categorization of WT isolates as susceptible, i.e. susceptible (S) or susceptible, increased exposure (I) were determined for 12 agents. The data supported a breakpoint for benzylpenicillin and amoxicillin of resistant (R) > 1 mg/L since WT isolates were inhibited by 1 mg/L or less. WT isolates were categorized as I (S ≤ 0.001 mg/L) for benzylpenicillin, emphasizing the need for increased exposure, and S (S ≤ 1 mg/L) for amoxicillin. Erythromycin breakpoints were set at S ≤ 0.06 mg/L and R > 0.06 mg/L. The corresponding ZD breakpoints were determined for all agents except amoxicillin, for which categorization was based on benzylpenicillin results. CONCLUSIONS: This work provided a large set of antimicrobial susceptibility data for C. diphtheriae and C. ulcerans, using a harmonized methodology. The dataset allowed EUCAST and experts in the diphtheria field to develop evidence-based breakpoints in January 2023.

Management and outcome of cutaneous diphtheria in adolescent refugees in Germany, June 2022 - October 2023.

OBJECTIVES: From September 2022 an increase in Corynebacterium diphtheriae (C. diphtheriae) infections was reported in Europe. Our study focuses on 31 adolescent and young adult refugees with cutaneous C. diphtheriae infections detected in Germany. We examined treatment regimens and outcomes to provide targeted insights into the management of this infection. METHODS: We distributed a standardized survey, focused on children and adolescents presenting to paediatric clinics through the German Paediatric Infectious Diseases Society (DGPI) and additional professional contacts in Germany. Data were extracted from routine medical documentation and reported anonymously. RESULTS: A total of 31 individuals with cutaneous C. diphtheriae infection were reported by 9 centres. Two of these showed diphtheria toxin (DT) related systemic symptoms and four exhibited systemic inflammation requiring complex management. The remaining 25 cases, with exclusively cutaneous manifestations, were afebrile. Treatment with topical antiseptics and systemic antibiotics, mainly aminopenicillin/beta-lactamase inhibitors (BLI) (35%) or clindamycin (25%), achieved eradication in all but two cases treated with aminopenicillin/BLI. Treatment duration varied between 5 and 17 days. CONCLUSIONS: In refugees presenting with chronic skin wounds, C. diphtheriae should be included into the differential diagnosis. Fever seems to be a valuable marker to differentiate severe cases with potentially DT-mediated sequelae from exclusively cutaneous diphtheria (CD). For afebrile CD, topical antiseptics and oral antibiotic therapy with clindamycin for 7 days, followed by clinical surveillance appears to be a safe treatment regimen. Patients with CD who present with fever or pharyngitis should be thoroughly investigated including blood and pharyngeal swab cultures.

A high fidelity approach to assembling the complex Borrelia genome.

BACKGROUND: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. RESULTS: Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. CONCLUSION: Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Analysis of seven SARS-CoV-2 rapid antigen tests in detecting omicron (B.1.1.529) versus delta (B.1.617.2) using cell culture supernatants and clinical specimens.

PURPOSE: Omicron is rapidly spreading as a new SARS-CoV-2 variant of concern (VOC). The question whether this new variant has an impact on SARS-CoV-2 rapid antigen test (RAT) performance is of utmost importance. To obtain an initial estimate regarding differences of RATs in detecting omicron and delta, seven commonly used SARS-CoV-2 RATs from different manufacturers were analysed using cell culture supernatants and clinical specimens. METHODS: For this purpose, cell culture-expanded omicron and delta preparations were serially diluted in Dulbecco's modified Eagle's Medium (DMEM) and the Limit of Detection (LoD) for both VOCs was determined. Additionally, clinical specimens stored in viral transport media or saline (n = 51) were investigated to complement in vitro results with cell culture supernatants. Ct values and RNA concentrations were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: The in vitro determination of the LoD showed no obvious differences in detection of omicron and delta for the RATs examined. The LoD in this study was at a dilution level of 1:1,000 (corresponding to 3.0-5.6 × 106 RNA copies/mL) for tests I-V and at a dilution level of 1:100 (corresponding to 3.7-4.9 × 107 RNA copies/mL) for tests VI and VII. Based on clinical specimens, no obvious differences were observed between RAT positivity rates when comparing omicron to delta in this study setting. Overall positivity rates varied between manufacturers with 30-81% for omicron and 42-71% for delta. Test VII was only conducted in vitro with cell culture supernatants for feasibility reasons. In the range of Ct < 23, positivity rates were 50-100% for omicron and 67-93% for delta. CONCLUSION: In this study, RATs from various manufacturers were investigated, which displayed no obvious differences in terms of analytical LoD in vitro and RAT positivity rates based on clinical samples comparing the VOCs omicron and delta. However, differences between tests produced by various manufacturers were detected. In terms of clinical samples, a focus of this study was on specimens with high virus concentrations. Further systematic, clinical and laboratory studies utilizing large datasets are urgently needed to confirm reliable performance in terms of sensitivity and specificity for all individual RATs and SARS-CoV-2 variants.

[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

Detection of diphtheria toxin production by toxigenic corynebacteria using an optimized Elek test.

PURPOSE: Diphtheria, still present in many countries of the world, is caused by toxigenic strains of species of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and the emerging zoonotic pathogen C. ulcerans. The immunoprecipitation test according to Elek is the gold standard for detection of the major virulence factor diphtheria toxin (DT) in toxigenic corynebacteria. Due to its sophisticated methodological requirements, the classical Elek test is performed mainly by specialized reference laboratories. It was revealed that the current modification of the Elek test does not detect the toxin in weakly toxigenic isolates. Therefore, a more robust method for detecting free DT is urgently needed, especially for toxigenic C. ulcerans strains which are known to produce often much lower amounts of DT than C. diphtheriae. METHODS: Thirty-one tox-positive C. ulcerans isolates with a negative standard Elek test result previously determined as NTTB (non-toxigenic tox bearing) were re-analyzed in this study using a modified immunoprecipitation method optimized regarding different parameters including type and concentration of antitoxin, medium volume, inoculum distance from the antitoxin disk and position of controls. RESULTS: All 31 C. ulcerans strains tested positive in the optimized Elek test. CONCLUSION: Only with a reliable and easy-to-handle method for detecting the toxigenicity of C. ulcerans, it is possible to assess the etiological role of this emerging zoonotic bacterium in human pathology.

Molecular SARS-CoV-2 surveillance in Bavaria shows no Omicron transmission before the end of November 2021.

BACKGROUND: Five SARS-CoV-2 variants are currently considered as variants of concern (VOC). Omicron was declared a VOC at the end of November 2021. Based on different diagnostic methods, the occurrence of Omicron was reported by 52 countries worldwide on December 7 2021. First notified by South Africa with alarming reports on increasing infection rates, this new variant was soon suspected to replace the currently pre-dominating Delta variant leading to further infection waves worldwide. METHODS: Using VOC PCR screening and Next Generation Sequencing (NGS) analysis of selected samples, we investigated the circulation of Omicron in the German federal state Bavaria. For this, we analyzed SARS-CoV-2 surveillance data from our laboratory generated from calendar week (CW) 01 to 49/2021. RESULTS: So far, we have detected 69 Omicron cases in our laboratory from CW 47-49/2021 using RT-qPCR followed by melting curve analysis. The first 16 cases were analyzed by NGS and all were confirmed as Omicron. CONCLUSION: Our data strongly support no circulation of the new Omicron variant before CW 47/2021.

Outbreak of imported diphtheria with Corynebacterium diphtheriae among migrants arriving in Germany, 2022.

From July 2022, cases of imported diphtheria with toxigenic Corynebacterium diphtheriae remarkably increased among migrants arriving in Germany. Up to 30 September 2022, 44 cases have been reported to the national public health institute, all laboratory-confirmed, male, and mainly coming from Syria (n = 21) and Afghanistan (n = 17). Phylogeny and available journey information indicate that most cases (n = 19) were infected along the Balkan route. Active case finding, increased laboratory preparedness and epicentre localisation in countries along this route are important.

Widespread Disease in Hedgehogs (Erinaceus europaeus) Caused by Toxigenic Corynebacterium ulcerans.

Toxin-producing Corynebacterium ulcerans, a causative agent of diphtheria in humans, was isolated from 53 hedgehogs in Belgium during the spring of 2020. Isolates showed low levels of acquired antimicrobial drug resistance. Strain diversity suggests emergence from an endemic situation. These findings stress the need for raising public awareness and improved wildlife disease surveillance.

Weekly SARS-CoV-2 Sentinel Surveillance in Primary Schools, Kindergartens, and Nurseries, Germany, June‒November 2020.

We investigated severe acute respiratory syndrome coronavirus 2 infections in primary schools, kindergartens, and nurseries in Germany. Of 3,169 oropharyngeal swab specimens, only 2 were positive by real-time reverse transcription PCR. Asymptomatic children attending these institutions do not appear to be driving the pandemic when appropriate infection control measures are used.