Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis.

Epidemiological investigation of a tularaemia outbreak after a hare hunt in Bavaria, Germany, 2018.

In November 2018, a tularaemia outbreak occurred in Bavaria, Germany, among participants of a hare hunt and butchery employees handling the hares. We conducted an epidemiological outbreak investigation, including a retrospective cohort study among hunting participants, to identify likely transmission routes and activities associated with infection. Twelve of 41 participants were antibody-positive for Francisella (F.) tularensis (attack rate: 29%). Cases reported influenza-like symptoms (n = 11), lymphadenopathy (n = 1) and conjunctivitis (n = 1). Infection only occurred in those hunting participants present while hares were processed, while risk of infection was highest when directly involved (RR = 10.0; 95%CI: 2.6-392). F. tularensis was isolated from 1/4 hares. Only two individuals reported using some of the recommended personal protective equipment (PPE). Occurrence of mainly non-specific symptoms, likely due to early treatment, was not indicative of a specific transmission route. Transmissions via direct (skin/mucosa) contact and by inhalation of contaminated aerosols seem plausible. Promoting and increasing appropriate use of PPE among people processing hares is crucial to prevent future outbreaks.

Outbreak of Tularemia in a Group of Hunters in Germany in 2018-Kinetics of Antibody and Cytokine Responses.

In November 2018, an outbreak of tularemia occurred among hare hunters in Bavaria, Germany. At least one infected hare was confirmed as the source of infection. A number of hunting dogs showed elevated antibody titers to Francisella tularensis, but the absence of titer increases in subsequent samples did not point to acute infections in dogs. Altogether, 12 persons associated with this hare hunt could be diagnosed with acute tularemia by detection of specific antibodies. In nine patients, the antibody and cytokine responses could be monitored over time. Eight out of these nine patients had developed detectable antibodies three weeks after exposure; in one individual the antibody response was delayed. All patients showed an increase in various cytokines and chemokines with a peak for most mediators in the first week after exposure. Cytokine levels showed individual variations, with high and low responders. The kinetics of seroconversion has implications on serological diagnoses of tularemia.

Francisella tularensis Subspecies holarctica and Tularemia in Germany.

Tularemia is a zoonotic disease caused by Francisella tularensis a small, pleomorphic, facultative intracellular bacterium. In Europe, infections in animals and humans are caused mainly by Francisella tularensis subspecies holarctica. Humans can be exposed to the pathogen directly and indirectly through contact with sick animals, carcasses, mosquitoes and ticks, environmental sources such as contaminated water or soil, and food. So far, F. tularensis subsp. holarctica is the only Francisella species known to cause tularemia in Germany. On the basis of surveillance data, outbreak investigations, and literature, we review herein the epidemiological situation-noteworthy clinical cases next to genetic diversity of F. tularensis subsp. holarctica strains isolated from patients. In the last 15 years, the yearly number of notified cases of tularemia has increased steadily in Germany, suggesting that the disease is re-emerging. By sequencing F. tularensis subsp. holarctica genomes, knowledge has been added to recent findings, completing the picture of genotypic diversity and geographical segregation of Francisella clades in Germany. Here, we also shortly summarize the current knowledge about a new Francisella species (Francisella sp. strain W12-1067) that has been recently identified in Germany. This species is the second Francisella species discovered in Germany.

Assessment of biorisk management systems in high containment laboratories, 18 countries in Europe, 2016 and 2017.

Europe-wide activities to improve biosafety and biosecurity performed within the frameworks of the European Union (EU)-funded Joint Actions EMERGE and QUANDHIP led to the development of an Integrated European Checklist for Laboratory Biorisk Management (ECL).To better understand different approaches shaping biorisk management (BRM) systems on an operational level in high containment laboratories, the ECL was used to map the implementation of BRM in 32 high containment laboratories in 18 countries in Europe. The results suggest that the BRM elements referring to standard microbiological working practices and the handling of infectious material were fulfilled particularly well. The elements safety exercises involving internal and external emergency responders, and appropriate decommissioning plans were not fulfilled particularly well. BRM in Biosafety Level (BSL) 4 laboratories handling Risk Group (RG) 4 viruses appear to vary among each other less than BSL3 laboratories handling RG 3 bacteria. It is important to agree on comparable regulations in Europe as high containment laboratories are indispensable for a safe, quick and effective response to public health threats. As high containment laboratories may also present a public health risk it is crucial to have robust BRM on organisational and operational levels.

Serological evidence for human exposure to Bacillus cereus biovar anthracis in the villages around Taï National Park, Côte d'Ivoire.

Bacillus cereus biovar anthracis (Bcbva) is an untypical anthrax-causing pathogen responsible for high wildlife mortality in Taï National Park (TNP), Côte d'Ivoire. However, nothing is known about its effect on the rural population living in the region bordering TNP. Contact to bushmeat is a known risk factor for exposure to a variety of zoonotic pathogens, but no human infections with Bcbva were noted so far. Therefore, we performed a retrospective seroprevalence analysis with sera from 1,386 study volunteers. We used assays which detect antibodies against the protective antigen PA, which is synthesized by both Bcbva and classic B. anthracis, and against the recently described antigen pXO2-60, a 35-kDa protein only produced by Bcbva. We found a high seroprevalence (22.37%) of antibodies against PA, and approximately half of those sera (10.46%) were also positive for the Bcbva-specific antigen pXO2-60. All sera negative for PA were also negative for antibodies against pXO2-60, confirming specificity and suitability of the PA/pXO2-60 combined serological assay. The fact that a large fraction of sera was positive for PA but negative for pXO2-60 can most likely be explained by lower immunogenicity of pXO2-60, but exposure to classic B. anthracis cannot be excluded. As only Bcbva has been detected in the TNP area so far, exposure to Bcbva can be suspected from the presence of antibodies against PA alone. In a questionnaire, most study participants reported contact to bushmeat and livestock carcasses. Unfortunately, risk factor analysis indicated that neither animal contacts, sex, age, nor country of origin were significant predictors of Bcbva seroprevalence. Nevertheless, our study added to an assessment of the distribution of Bcbva and its impact on the human population, and our data can serve to raise awareness of anthrax in the affected regions.

Evaluation of Sporicidal Disinfectants for the Disinfection of Personal Protective Equipment During Biological Hazards.

A fast, effective, and safe disinfection of personal protective equipment (PPE) is vitally important for emergency forces involved in biological hazards. This study aimed to investigate a broad range of disinfectants to improve the established disinfection procedure. We analyzed the efficacy of chlorine-, peracetic acid-, and oxygen-based disinfectants against Bacillus spores on PPE. Therefore, spores of different Bacillus species were exposed to disinfectants on PPE material by using a standardized procedure covering the dried spores with disinfectants and applying mechanical distribution. Efficacy of disinfectants was quantified by determining the reduction factor (log10 levels) and number of viable spores left afterward. The chlorine-based granulate Hypochlorit CA G (2% chlorine) sufficiently inactivated Bacillus spores of risk groups 1 and 2, even with temperatures ranging from -20 to 35°C. Wofasteril® SC super (1.75% peracetic acid) achieved a reliable reduction of risk groups 1 and 2 and even fully virulent Bacillus spores by ≥5 log10 levels on PPE. With this, Hypochlorit-CA G and Wofasteril® SC super proved to be promising alternatives to the previously proven and widely used peracetic acid compound Wofasteril® (2% peracetic acid) for the disinfection of PPE when bacterial spores are known to be the contaminating agent. These results will help to improve the disinfection of PPE during biological hazards by providing new data on promising alternative compounds.

Genetic Diversity and Spatial Segregation of Francisella tularensis Subspecies holarctica in Germany.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%.

Geographical Variability Affects CCHFV Detection by RT-PCR: A Tool for In-Silico Evaluation of Molecular Assays.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient's travel history and geographic distribution of the different CCHFV strains.

[Four years after the Ebola crisis : Challenges, experiences, and implications in the German public health context]. / Vier Jahre nach der Ebolakrise : Herausforderungen, Erfahrungen und Schlussfolgerungen für den Öffentlichen Gesundheitsdienst in Deutschland.

The Ebola virus disease outbreak in West Africa in 2014/2015 was by far the biggest, most prolonged, and geographically most widespread outbreak of this disease since the discovery of the Ebola virus in 1976. Although no cases of Ebola virus disease were confirmed in Germany, a number of crisis management activities were initiated.Based on a combination of local, national, and international lessons learned, literature research, and a large number of discussions among German colleagues as well as German and foreign colleagues, the experiences of selected German public health actors as well as implications for health protection activities in Germany are presented.On the one hand, preparedness for managing unusual high consequence health events-caused by rare, highly pathogenic biological agents-including the provision of adequate material and personnel resources remains important in Germany. On the other hand, more German engagement in global health is necessary, because the dividing line between global health and local health is increasingly disappearing.

Screen for fitness and virulence factors of Francisella sp. strain W12-1067 using amoebae.

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.

Decontamination of Personal Protective Equipment.

Exploratory field analyses of the inactivation capacity of disinfectants on contaminated personal protective equipment (PPE) are required to select a suitable surrogate for biohazardous agents like spores of Bacillus anthracis. The objectives of our study were (1) the determination of an appropriate surrogate for the inactivation of spores of B. anthracis with peracetic acid (PAA), and (2) application of optimized inactivation conditions for an effective decontamination of PPE with PAA under field conditions. For inactivation studies, B. anthracis spores from different strains and B. thuringiensis spores were fixed by air drying on carriers prepared from PPE fabric. Time and concentration studies with PAA-based disinfectants revealed that the spores of the B. thuringiensis strain DSM 350 showed an inactivation profile comparable to that of the spores of the B. anthracis strain with the highest stability, implying that B. thuringiensis can serve as an appropriate surrogate. Rapid (3 to 5 minutes) and effective surface decontamination was achieved with 2% PAA/0.2% surfactant. In field studies, PPE contaminated with spores of B. thuringiensis was treated with the disinfectant. Optimizing the decontamination technique revealed that spraying in combination with brushing was effective within 5 minutes of exposure.

Molecular identification of the source of an uncommon tularaemia outbreak, Germany, autumn 2016.

BackgroundIn 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.AimWe describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.MethodsWe incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case's lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.ResultsNo bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica, belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case's isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.ConclusionThe discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

Clinical characteristics in a sentinel case as well as in a cluster of tularemia patients associated with grape harvest.

BACKGROUND: Tularemia is caused by Francisella tularensis and can occasionally establish foodborne transmission. METHODS: Patients were identified by active case detection through contact with the treating physicians and consent for publication was obtained. Clinical data were accumulated through a review of the patient charts. Serology, culture, and PCR methods were performed for confirmation of the diagnosis. CASE CLUSTER: A 46-year-old patient was hospitalised in the University Hospital Frankfurt (a tertiary care hospital) for pharyngitis and cervical lymphadenitis with abscess. A diagnosis of tularemia was made serologically, but treatment with ciprofloxacin initially failed. F. tularensis was detected in pus from the lymph node using a specific real-time PCR. The use of RD1 PCR led to the identification of the subspecies holarctica. Antibiotic therapy with high-dose ciprofloxacin and gentamicin was administered and was subsequently changed to ciprofloxacin and rifampicin. During a must-tasting, five other individuals became infected with tularemia by ingestion of contaminated must. All patients required treatment durations of more than 14 days. CONCLUSIONS: Mechanically harvested agricultural products, such as wine must, can be a source of infection, probably due to contamination with animal carcasses. The clinical course of tularemia can be complicated and prolonged and requires differentiated antibiotic treatment.

Laboratory management of Crimean-Congo haemorrhagic fever virus infections: perspectives from two European networks.

BackgroundCrimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging infectious disease threat in the European Union. Since 2000, the incidence and geographic range of confirmed CCHF cases have markedly increased, following changes in the distribution of its main vector, Hyalomma ticks.AimsTo review scientific literature and collect experts' opinion to analyse relevant aspects of the laboratory management of human CCHF cases and any exposed contacts, as well as identify areas for advancement of international collaborative preparedness and laboratory response plans.MethodsWe conducted a literature review on CCHF molecular diagnostics through an online search. Further, we obtained expert opinions on the key laboratory aspects of CCHF diagnosis. Consulted experts were members of two European projects, EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level) and EVD-LabNet (Emerging Viral Diseases-Expert Laboratory Network).ResultsConsensus was reached on relevant and controversial aspects of CCHF disease with implications for laboratory management of human CCHF cases, including biosafety, diagnostic algorithm and advice to improve lab capabilities. Knowledge on the diffusion of CCHF can be obtained by promoting syndromic approach to infectious diseases diagnosis and by including CCHFV infection in the diagnostic algorithm of severe fevers of unknown origin.ConclusionNo effective vaccine and/or therapeutics are available at present so outbreak response relies on rapid identification and appropriate infection control measures. Frontline hospitals and reference laboratories have a crucial role in the response to a CCHF outbreak, which should integrate laboratory, clinical and public health responses.

Draft Genome Sequence of Bacillus anthracis Strain Sterne 09RA8929.

A Bacillus anthracis vaccine strain (Sterne), used as an attenuated laboratory comparative strain, was sequenced and analyzed. A comparison to assemblies of B. anthracis strain Sterne (NZ_CP009541 and NZ_CP009540) was performed. The lack of the pX02 plasmid and pX01 in approximately five copies was confirmed.

Status, quality and specific needs of Ebola virus diagnostic capacity and capability in laboratories of the two European preparedness laboratory networks EMERGE and EVD-LabNet.

From December 2013 to March 2016, West Africa experienced the largest Ebola virus (EBOV) outbreak to date, leading to a European-wide activation of laboratory preparedness and response. At the end of the outbreak, laboratories associated with the two European preparedness networks of expert laboratories EMERGE JA and EVD-LabNet were invited to participate in an assessment of the response of European laboratories to the EBOV outbreak, to identify learning points and training needs to strengthen future outbreak responses. Response aspects assessed included diagnostics, biorisk management and quality assurance. The overall coverage of EBOV diagnostics in the European Union/European Economic Area (EU/EEA) was found to be adequate although some points for quality improvement were identified. These included the need for relevant International Organization for Standardization (ISO) accreditation, the provision of EBOV external quality assessments (EQA) in periods where there is no emergency, facilitating access to controls and knowledge, biorisk management without compromising biosafety and a rapid public health response, and the need for both sustained and contingency funding for preparedness and response activities.