Epidemiology and transmission characteristics of early COVID-19 cases, 20 January-19 March 2020, in Bavaria, Germany.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) led to a significant disease burden and disruptions in health systems. We describe the epidemiology and transmission characteristics of early coronavirus disease 2019 (COVID-19) cases in Bavaria, Germany. Cases were reverse transcription polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infections, reported from 20 January-19 March 2020. The incubation period was estimated using travel history and date of symptom onset. To estimate the serial interval, we identified pairs of index and secondary cases. By 19 March, 3546 cases were reported. A large proportion was exposed abroad (38%), causing further local transmission. Median incubation period of 256 cases with exposure abroad was 3.8 days (95%CI: 3.5-4.2). For 95% of infected individuals, symptom onset occurred within 10.3 days (95%CI: 9.1-11.8) after exposure. The median serial interval, using 53 pairs, was 3.5 days (95%CI: 3.0-4.2; mean: 3.9, s.d.: 2.2). Travellers returning to Germany had an important influence on the spread of SARS-CoV-2 infections in Bavaria in early 2020. Especially in times of low incidence, public health agencies should identify holiday destinations, and areas with ongoing local transmission, to monitor potential importation of SARS-CoV-2 infections. Travellers returning from areas with ongoing community transmission should be advised to quarantine to prevent re-introductions of COVID-19.

Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi.

BACKGROUND: Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology. RESULTS: Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids. CONCLUSION: Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

Impact of blanching, sweating and drying operations on pungency, aroma and color of Piper borbonense.

Low pungency, high aromatic potential and red color, give to Piper borbonense its originality when compared to Piper nigrum. Effects of blanching, sweating and drying on these characteristics were assessed. The three operations had no impact on the concentration of piperine and essential oil but affected the composition of essential oil slightly and considerably affected the color of the pepper. The "wet process", including blanching, sweating and drying, had the largest impact on the composition of aroma, increasing para-cymene content by 89% and reducing safrole content by 33% in dried pepper compared to fresh. Blanching increased the drying rate thus reducing drying time. Drying had a major impact on color, which changed from red to brown. The biggest differences observed led to reductions of 2.2, 7.9 and 8.4units in L∗, a∗ and b∗ values, when chromatic values measured in fresh pepper were compared to those of dried pepper.

Outbreak investigation for toxigenic Corynebacterium diphtheriae wound infections in refugees from Northeast Africa and Syria in Switzerland and Germany by whole genome sequencing.

Toxigenic Corynebacterium diphtheriae is an important and potentially fatal threat to patients and public health. During the current dramatic influx of refugees into Europe, our objective was to use whole genome sequencing for the characterization of a suspected outbreak of C. diphtheriae wound infections among refugees. After conventional culture, we identified C. diphtheriae using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and investigated toxigenicity by PCR. Whole genome sequencing was performed on a MiSeq Illumina with >70×coverage, 2×250 bp read length, and mapping against a reference genome. Twenty cases of cutaneous C. diphtheriae in refugees from East African countries and Syria identified between April and August 2015 were included. Patients presented with wound infections shortly after arrival in Switzerland and Germany. Toxin production was detected in 9/20 (45%) isolates. Whole genome sequencing-based typing revealed relatedness between isolates using neighbour-joining algorithms. We detected three separate clusters among epidemiologically related refugees. Although the isolates within a cluster showed strong relatedness, isolates differed by >50 nucleotide polymorphisms. Toxigenic C. diphtheriae associated wound infections are currently observed more frequently in Europe, due to refugees travelling under poor hygienic conditions. Close genetic relatedness of C. diphtheriae isolates from 20 refugees with wound infections indicates likely transmission between patients. However, the diversity within each cluster and phylogenetic time-tree analysis suggest that transmissions happened several months ago, most likely outside Europe. Whole genome sequencing offers the potential to describe outbreaks at very high resolution and is a helpful tool in infection tracking and identification of transmission routes.

Trans-Atlantic exchanges have shaped the population structure of the Lyme disease agent Borrelia burgdorferi sensu stricto.

The origin and population structure of Borrelia burgdorferi sensu stricto (s.s.), the agent of Lyme disease, remain obscure. This tick-transmitted bacterial species occurs in both North America and Europe. We sequenced 17 European isolates (representing the most frequently found sequence types in Europe) and compared these with 17 North American strains. We show that trans-Atlantic exchanges have occurred in the evolutionary history of this species and that a European origin of B. burgdorferi s.s. is marginally more likely than a USA origin. The data further suggest that some European human patients may have acquired their infection in North America. We found three distinct genetically differentiated groups: i) the outgroup species Borrelia bissettii, ii) two divergent strains from Europe, and iii) a group composed of strains from both the USA and Europe. Phylogenetic analysis indicated that different genotypes were likely to have been introduced several times into the same area. Our results demonstrate that irrespective of whether B. burgdorferi s.s. originated in Europe or the USA, later trans-Atlantic exchange(s) have occurred and have shaped the population structure of this genospecies. This study clearly shows the utility of next generation sequencing to obtain a better understanding of the phylogeography of this bacterial species.

Clinical symptoms cannot predict influenza infection during the 2013 influenza season in Bavaria, Germany.

For influenza surveillance and diagnosis typical clinical symptoms are traditionally used to discriminate influenza virus infections from infections by other pathogens. During the 2013 influenza season we performed a multiplex assay for 16 different viruses in 665 swabs from patients with acute respiratory infections (ARIs) to display the variety of different pathogens causing ARI and to test the diagnostic value of both the commonly used case definitions [ARI, and influenza like illness (ILI)] as well as the clinical judgement of physicians, respectively, to achieve a laboratory-confirmed influenza diagnosis. Fourteen different viruses were identified as causing ARI/ILI. Influenza diagnosis based on clinical signs overestimated the number of laboratory-confirmed influenza cases and misclassified cases. Furthermore, ILI case definition and physicians agreed in only 287/651 (44%) cases with laboratory confirmation. Influenza case management has to be supported by laboratory confirmation to allow evidence-based decisions. Epidemiological syndromic surveillance data should be supported by laboratory confirmation for reasonable interpretation.

A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato.

PURPOSE: For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. METHODS: Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. RESULTS: The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. CONCLUSION: The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

Possible human-to-human transmission of toxigenic Corynebacterium ulcerans.

Toxigenic Corynebacterium ulcerans is an emerging cause of diphtheria. In contrast to the classical diphtheria pathogen C. diphtheriae, human-to-human transmission of this primarily zoonotic pathogen has not been clearly documented. Here we report on a two-person cluster suggesting an initial zoonotic and a subsequent human-to-human transmission event.

Corynebacterium species nasal carriage in pigs and their farmers in Bavaria, Germany: implications for public health.

Reports on cases of human diphtheria caused by toxigenic Corynebacterium ulcerans that were linked to occupational swine contact as well as isolation of C ulcerans from wild boars have suggested that pigs might serve as reservoir for human infections. Therefore, a prevalence study on Corynebacterium species nasal carriage in pigs and their farmers was performed between August 1 and December 31, 2009, in 41 swine farms from Bavaria, Germany. All 411 asymptomatic pigs and 29 of 30 healthy farmers were colonised with Corynebacterium strains of up to 11 different species. No potentially toxigenic Corynebacterium strain was isolated either from the pigs or from their farmers, respectively. The patterns of the species composition in the pigs and the farmers were very similar, suggesting a potential transmission of strains between animals and humans.

Two cases of cutaneous diphtheria associated with occupational pig contact in Germany.

In 2010, two independent cases of cutaneous diphtheria caused by toxigenic C. ulcerans were identified in Germany. Both patients had intense occupational contact with pigs. Diagnostic work-up comprising biochemical differentiation, rpoB sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis, real-time tox PCR and Elek test as well as public health measures including an intensified source tracing involving 83 asymptomatic pigs of an associated pig farm are presented.

'Not Always a Baker's Cyst' - An Unusual Presentation of a Central Voluminous Postero-Medial Meniscal Cyst.

Meniscal cysts are rare and often are a result of extrusion of synovial fluid through a tear of the meniscus, resulting in a one-way valve effect of the tear. Arthroscopic partial meniscectomy of the meniscus with intra-articular cyst drainage has become the standard of care. We report a case of an unusually large symptomatic medial meniscal cyst, situated postero-medially and pressing on the posterior cruciate ligament, which was initially clinically misdiagnosed as a Baker's cyst. The patient had difficulty and pain on squatting. He was successfully treated with arthroscopic debridement and needle decompression; a rarity in literature for such a voluminous perilabral cyst tenting the posterior cruciate ligament. This procedure has the advantage of being able to obtain the cystic fluid for histological and cytological analysis before debridement. This case also highlights the importance of the use of Magnetic Resonance Imaging (MRI) to accurately diagnose a central, posterior knee swelling.

Fatal anthrax infection in a heroin user from southern Germany, June 2012.

Blood cultures from a heroin user who died in June 2012, a few hours after hospital admission, due to acute septic disease, revealed the presence of Bacillus anthracis. This report describes the extended diagnosis by MALDI-TOF and real-time PCR and rapid confirmation of the anthrax infection through reference laboratories. Physicians and diagnostic laboratories were informed and alerted efficiently through the reporting channels of German public health institutions, which is essential for the prevention of further cases.

[Infections with pneumococci, menigococci, H. influenzae and diphtheria in Germany: the RKI Reference Network for Invasive Bacterial Infections (IBI) at the 5th Würzburg Workshop on Epidemiology, Prevention and Therapy for Invasive Meningococcal Diseases 2010 (meeting report)]. / Infektionen mit Pneumokokken, Meningokokken, H. influenzae und Diphtherie in Deutschland: das RKI Referenznetzwerk für Invasive Bakterielle Infektionen (IBI) beim 5. Würzburger Workshop zur Epidemiologie, Prävention und Therapie von invasiven Meningokokkenerkrankung 2010 (Tagungsbericht).

The surveillance and prevention of invasive bacterial infections requires flexible strategic coordination of all involved health-care professionals. For this purpose, the German National Reference Centres for Meningococci, Streptococci and the Consultant Laboratories for Haemophilus influenzae and diphtheria have formed the Reference Network for Invasive bacterial infections (IBI). The 5th Würzburg Workshop on Meningococcal Diseases 2010 provided the network with a forum for the interdisciplinary exchange between scientists, public health professionals, medical microbiologists and clinicians. The topics covered the analysis of surveillance data for meningococcal disease in the last decade, as well as methods to control for antibody response following vaccination, including a serum bactericidal antibody (SBA) assay, and the development of new vaccines that also include the most common serogroup B. The presentation on diphtheria showed that this rare disease in Germany has become a diagnostic challenge, and that apart from the classical pathogen also toxigenic C. ulcerans strains must be considered. Due to the successful vaccination against Hib, H. influenzae disease has changed from a classical childhood disease to an infection of elderly people mainly caused by unencapsulated strains. Following the introduction of vaccines, changes in the serotype distribution and antibiotic resistance profiles have become apparent for S. pneumoniae infections. The epidemiological data were complemented by clinical aspects concerning the vaccination of immunocompromised children.

Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp.

AIMS: The aim of this study was to develop a real-time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). METHODS AND RESULTS: A duplex real-time PCR specific for the genes encoding for ß-glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety-six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. CONCLUSIONS: The duplex real-time PCR assay was found to be simple, rapid, reliable and specific. SIGNIFICANCE AND IMPACT OF THE STUDY: If possible at all, delineation of so-called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno- and serotypes. This assay clearly simplifies the differentiation of both.

A multiplex one-step real-time RT-PCR assay for influenza surveillance.

For surveillance purposes real-time PCR assays for influenza viruses had to be adapted to the pandemic influenza A(H1N1)2009 strain. We combined published primers and probes for influenza A, influenza B and an internal amplification control with a detection system for influenza A(H1N1)2009 to set up a rapid, reliable, simple and cost-effective high-throughput multiplex one-step real-time RT-PCR. The workflow also includes automated sample preparation for high-throughput screening. The lower limit of detection of the multiplex assay was 3.5x10(2) RNA copies per PCR reaction. The diagnostic sensitivity of the multiplex assay was 87.7%, but increased to 99.4% for influenza-positive samples yielding C(t) values of less than 34 cycles in the respective diagnostic assay. High specificity was confirmed by sequencing and correct detection of 15 reference samples from two quality assurance studies. The multiplex PCR was introduced for surveillance of samples from a network of general practitioners and paediatricians in Bavaria, Germany during the influenza pandemic of 2009. Comparison with surveillance data from reported cases proved the reliability of the multiplex assay for influenza surveillance programmes.

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry as a tool for rapid diagnosis of potentially toxigenic Corynebacterium species in the laboratory management of diphtheria-associated bacteria.

The rapid identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis is essential for diagnosis and treatment of diphtheria and diphtheria-like diseases. We used matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDIT-OF MS) in comparison with classical microbiological and molecular methods on 116 Corynebacterium strains. All 90 potentially toxigenic Corynebacterium strains collected by the German National Consiliary Laboratory on Diphtheria in a period of more than ten years were correctly identified by MALDI-TOF MS. We propose an algorithm for fast and reliable diagnosis of diphtheria incorporating MALDI-TOF MS, real-time tox PCR and Elek testing.