Clinical and virological features of first human monkeypox cases in Germany.

BACKGROUND: Monkeypox is a zoonotic orthopoxvirus infection endemic in central and western Africa. In May 2022, human monkeypox infections including human-to-human transmission were reported in a multi-country outbreak in Europe and North America. CASE PRESENTATIONS: Here we present the first two cases of monkeypox infection in humans diagnosed in Germany. We present clinical and virological findings, including the detection of monkeypox virus DNA in blood and semen. The clinical presentation and medical history of our patients suggest close physical contact during sexual interactions as the route of infection. CONCLUSION: Monkeypox requires rapid diagnosis and prompt public health response. The disease should be considered in the current situation especially the differential diagnosis of vesicular or pustular rash, particularly in patients with frequent sexual contacts. Most importantly, it is essential to raise awareness among all health professionals for the rapid and correct recognition and diagnosis of this disease, which is probably still underreported in Europe (Adler et al. in Lancet Infect Dis https://doi.org/10.1016/s1473-3099(22)00228-6 , 2022).

Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

The zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

FlexTaxD: flexible modification of taxonomy databases for improved sequence classification.

SUMMARY: The Flexible Taxonomy Database framework provides a method for modification and merging official and custom taxonomic databases to create improved databases. Using such databases will increase accuracy and precision of existing methods to classify sequence reads. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://github.com/FOI-Bioinformatics/flextaxd and installable through Bioconda.

Sensitivity of two SARS-CoV-2 variants with spike protein mutations to neutralising antibodies.

SARS-CoV-2 infections elicit a humoral immune response capable of neutralising the virus. However, multiple variants have emerged with mutations in the spike protein amongst others, the key target of neutralising antibodies. We evaluated the neutralising efficacy of 89 serum samples from patients, infected with SARS-CoV-2 in the beginning of 2020, against two virus variants isolated from acutely infected patients and harbouring spike protein mutations. One isolate was assigned to lineage B.1.351 (MUC-IMB-B.1.351) whilst the other (MUC-484) was isolated from an immunocompromised patient, sharing some but not all mutations with B.1.351 and representing a transitional variant. Both variants showed a significant reduction in neutralisation sensitivity compared to wild-type SARS-CoV-2 with MUC-IMB-B.1.351 being almost completely resistant to neutralisation. The observed reduction in neutralising activity of wild-type-specific antibodies against both variants suggests that individual mutations in the spike protein are sufficient to confer a potent escape from the humoral immune response. In addition, the effect of escape mutations seems to accumulate, so that more heavily mutated variants show a greater loss of sensitivity to neutralisation up to complete insensitivity as observed for MUC-IMB-B.1.351. From a clinical point of view, this might affect the efficacy of (monoclonal) antibody treatment of patients with prolonged infections as well as patients infected with variants other than the donor. At the same, this could also negatively influence the efficacy of current vaccines (as they are based on wild-type spike protein) emphasising the need to thoroughly surveil the emergence and distribution of variants and adapt vaccines and therapeutics accordingly.

Whole genome sequencing and phylogenetic classification of Tunisian SARS-CoV-2 strains from patients of the Military Hospital in Tunis.

In the present work, two complete genome sequences of SARS-CoV-2 were obtained from nasal swab samples of Tunisian SARS-CoV-2 PCR-positive patients using nanopore sequencing. The virus genomes of two of the patients examined, a Tunisian soldier returning from a mission in Morocco and a member of another Tunisian family, showed significant differences in analyses of the total genome and single nucleotide polymorphisms (SNPs). Phylogenetic relationships with known SARS-CoV-2 genomes in the African region, some European and Middle Eastern countries and initial epidemiological conclusions indicate that the introduction of SARS-CoV-2 into Tunisia from two independent sources was travel-related.

A headache with surprising outcome: first case of brucellosis caused by Brucella suis biovar 1 in Germany.

In July 2018, brucellosis was diagnosed in a German patient without a travel history to regions endemic for Brucella. Microbiological analysis, including whole-genome sequencing, revealed Brucella suis biovar 1 as the etiologic agent. Core-genome-based multilocus sequence-typing analysis placed the isolate in close proximity to strains originating from Argentina. Notably, despite a strong IgM response, the patient did not develop Brucella-specific IgG antibodies during infection. Here, we describe the clinical course of infection, the extensive epidemiological investigations, and discuss possible routes of transmission.

Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases.

BACKGROUND: Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970's. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively. RESULTS: The isolates were genetically characterized by PCR-based methods such as multilocus variable number of tandem repeat analysis (MLVA) and whole genome-sequence analysis (WGS). Phylogenetic comparison of the two strains HKI4363/88 and BA2968 by chromosomal single nucleotide polymorphism (SNP) analysis grouped these organisms within their relatives of the minor canonical A-branch canSNP-group A.Br.003/004 (A.Br.V770) or canonical B-branch B.Br.001/002, respectively. Strain HKI4363/88 clustered relatively closely with other members of the A.Br.003/004 lineage from Europe, South Africa, and South America. In contrast, strain BA2968 clearly constituted a new sublineage within B.Br.001/002 with its closest relative being HYO01 from South Korea. CONCLUSIONS: Our results suggest that Finland harbors both unique (autochthonous) and more widely distributed, common clades of B. anthracis. We suspect that members of the common clades such as strains HKI4363/88 have been introduced only recently by anthropogenic activities involving importation of contaminated animal products. On the other hand, autochthonous strains such as isolate BA2968 probably have an older history of their introduction into Finland as evidenced by a high number of single nucleotide variant sites in their genomes.

Molecular analysis of Coxiella burnetii in Germany reveals evolution of unique clonal clusters.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.

New Insights into the Phylogeny of the A.Br.161 ("A.Br.Heroin") Clade of Bacillus anthracis.

Bacillus anthracis is a rare but highly dangerous zoonotic bacterial pathogen. At the beginning of this century, a new manifestation of the disease, injectional anthrax, emerged as a result of recreational heroin consumption involving contaminated drugs. The organisms associated with this 13-year-lasting outbreak event in European drug consumers were all grouped into the canonical single-nucleotide polymorphism (canSNP) clade A-branch (A.Br.) 161 of B. anthracis. Related clade A.Br.161 strains of B. anthracis not associated with heroin consumption have also been identified from different countries, mostly in Asia. Because of inadvertent spread by anthropogenic activities, other strains of this A.Br.161 lineage were, however, isolated from several countries. Thus, without additional isolates from this clade, its origin of evolution or its autochthonous region remains obscure. Here, we genomically characterized six new A.Br.161 group isolates, some of which were from Iran, with others likely historically introduced into Germany. All the chromosomes of these isolates could be grouped into a distinct sub-clade within the A.Br.161 clade. This sub-clade is separated from the main A.Br.161 lineage by a single SNP. We have developed this SNP into a PCR assay facilitating the future attribution of strains to this group.

Comparative Analysis of Vaccine-Induced Neutralizing Antibodies against the Alpha, Beta, Delta, and Omicron Variants of SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 660 million people and caused nearly seven million deaths worldwide. During the pandemic, a number of SARS-CoV-2 vaccines were rapidly developed, and several are currently licensed for use in Europe. However, the optimization of vaccination regimens is still ongoing, particularly with regard to booster vaccinations. At the same time, the emergence of new virus variants poses an ongoing challenge to vaccine efficacy. In this study, we focused on a comparative analysis of the neutralization capacity of vaccine-induced antibodies against four different variants of concern (i.e., Alpha, Beta, Delta, and Omicron) after two and three doses of COVID-19 vaccine. We were able to show that both two (prime/boost) and three (prime/boost/boost) vaccinations elicit highly variable levels of neutralizing antibodies. In addition, we did not observe a significant difference in antibody levels after two and three vaccinations. We also observed a significant decrease in the neutralization susceptibility of all but one SARS-CoV-2 variants to vaccine-induced antibodies. In contrast, a SARS-CoV-2 breakthrough infection between the second and third vaccination results in overall higher levels of neutralizing antibodies with a concomitant improved neutralization of all virus variants. Titer levels remained highly variable across the cohort but a common trend was observed. This may be due to the fact that at the time of this study, all licensed vaccines were still based exclusively on wild-type SARS-CoV-2, whereas infections were caused by virus variants. Overall, our data demonstrate the importance of (booster) vaccinations, but at the same time emphasize the need for the continued adaptation of vaccines to induce a protective immune response against virus variants in order to be prepared for future (seasonal) SARS-CoV-2 outbreaks.

Spanish Outbreak Isolates Bridge Phylogenies of European and American Bacillus anthracis.

The geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.

Draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user.

We report the draft genome sequence of Bacillus anthracis UR-1, isolated from a fatal case of injectional anthrax in a German heroin user. Analysis of the genome sequence of strain UR-1 may aid in describing phylogenetic relationships between virulent heroin-associated isolates of B. anthracis isolated in the United Kingdom, Germany, and other European countries.

Draft genome sequence of Bacillus anthracis BF-1, isolated from Bavarian cattle.

Bacillus anthracis BF-1 was isolated from a cow in Bavaria (Germany) that had succumbed to anthrax. Here, we report the draft genome sequence of this strain, which belongs to the European B2 subclade of B. anthracis. The closest phylogenetic neighbor of strain BF-1 is a strain isolated from cattle in France.

Machine Learning Algorithms for Classification of MALDI-TOF MS Spectra from Phylogenetically Closely Related Species Brucella melitensis, Brucella abortus and Brucella suis.

(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of B. melitensis, B. suis and B. abortus to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised k-means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested k-fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance Brucella spp. classification using MALDI-TOF MS data.

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

Catching SARS-CoV-2 by Sequence Hybridization: a Comparative Analysis.

Controlling and monitoring the still ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic regarding geographical distribution, evolution, and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and sharing sequence data worldwide. Efficient sequencing strategies enable the retrieval of increasing numbers of high-quality, full-length genomes and are, hence, indispensable. Two opposed enrichment methods, tiling multiplex PCR and sequence hybridization by bait capture, have been established for SARS-CoV-2 sequencing and are both frequently used, depending on the quality of the patient sample and the question at hand. Here, we focused on the evaluation of the sequence hybridization method by studying five commercially available sequence capture bait panels with regard to sensitivity and capture efficiency. We discovered the SARS-CoV-2-specific panel of Twist Bioscience to be the most efficient panel, followed by two respiratory panels from Twist Bioscience and Illumina, respectively. Our results provide on the one hand a decision basis for the sequencing community including a computation for using the full capacity of the flow cell and on the other hand potential improvements for the manufacturers. IMPORTANCE Sequencing the genomes of the circulating SARS-CoV-2 strains is the only way to monitor the viral spread and evolution of the virus. Two different approaches, namely, tiling multiplex PCR and sequence hybridization by bait capture, are commonly used to fulfill this task. This study describes for the first time a combined approach of droplet digital PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were able to determine the most sensitive and efficient capture panel, distinguish the mode of action of the various bait panels, and compute the number of read pairs needed to recover a high-quality full-length genome. By calculating the minimum number of read pairs needed, we are providing optimized flow cell loading conditions for all sequencing laboratories worldwide that are striving for maximizing sequencing output and simultaneously minimizing time, costs, and sequencing resources.

Tick-Borne Encephalitis Vaccination Protects from Alimentary TBE Infection: Results from an Alimentary Outbreak.

In May 2017, a hospitalized index case of tick-borne encephalitis (TBE) was confirmed by Serology. The case was linked to alimentary infection by raw milk from a goat farm in the region of Tübingen, Baden-Württemberg, Germany, where no previous TBE cases in the area had been reported before. The TBE focus was confirmed by isolation of the TBE virus from ticks and Serological confirmation of past infection in one of the five flock goats. Additional investigations by the local public health office identified 27 consumers of goat milk at the putative period of exposure. For 20/27 exposed persons, anamnestic information was gained by the local public health office. Twelve/fourteen exposed and non-vaccinated people developed clinical illness and were confirmed as TBE cases by Serology. Five/six vaccinated and exposed people did not develop the disease. The one exposed and vaccinated person had their last TBE vaccination booster more than 15 years ago, and therefore a booster was more than 10 years overdue. None of the regularly vaccinated and exposed persons developed clinical overt TBE infection. We report the first known TBE outbreak, during which, protection by TBE vaccination against alimentary TBE infection was demonstrated.

Rapid inactivation of SARS-CoV-2 with LED irradiation of visible spectrum wavelengths.

Difficulty in controlling SARS-CoV-2 transmission made the ability to inactivate viruses in aerosols and fomites to be an important and attractive risk reduction measure. Evidence that light frequencies have the ability to inhibit microorganisms has already been reported by many studies which, however, focused on ultraviolet (UV) wavelengths, which are known to induce potential injury in humans. In the present study, the effect on suspensions of SARS-CoV-2 of a Light Emitting Diode (LED) device capable of radiating frequencies in the non-hazardous visible light spectrum (VIS) was investigated. In order to evaluate the efficiency of viral inactivation, plaque assay and western blot of viral proteins were performed. The observed results showed a significant reduction in infectious particles that had been exposed to the LED irradiation of visible light. Furthermore, the analysis of the intracellular expression of viral proteins confirmed the inactivating effect of this irradiation technology. This in vitro study revealed for the first time the inactivation of SARS-CoV-2 through LED irradiation with multiple wavelengths of the visible spectrum. However additional and more in-depth studies can aim to demonstrate the data obtained during these experiments in different matrices, in mutable environmental conditions and on other respiratory viruses such as the influenza virus. The type of LED technology can decisively contribute on reducing virus transmission through the continuous sanitation of common environments without risks for humans and animals.

A Rare Glimpse into the Past of the Anthrax Pathogen Bacillus anthracis.

The bacterium Bacillus anthracis is the causative agent of the zoonotic disease anthrax. While genomics of extant B. anthracis isolates established in-depth phylogenomic relationships, there is scarce information on the historic genomics of the pathogen. Here, we characterized the oldest documented B. anthracis specimen. The inactive 142-year-old material originated from a bovine diseased in Chemnitz (Germany) in 1878 and is contemporary with the seminal studies of Robert Koch on B. anthracis. A specifically developed isolation method yielded high-quality DNA from this specimen for genomic sequencing. The bacterial chromosome featuring 242 unique base-characters placed it into a major phylogenetic clade of B. anthracis (B.Branch CNEVA), which is typical for central Europe today. Our results support the notion that the CNEVA-clade represents part of the indigenous genetic lineage of B. anthracis in this part of Europe. This work emphasizes the value of historic specimens as precious resources for reconstructing the past phylogeny of the anthrax pathogen.

Evaluation of a Highly Efficient DNA Extraction Method for Bacillus anthracis Endospores.

A variety of methods have been established in order to optimize the accessibility of DNA originating from Bacillus anthracis cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. Here, we started by systematically assessing the efficiencies of 20 DNA extraction kits for vegetative B. anthracis cells. Of these, the Epicentre MasterPure kit gave the best DNA yields and quality suitable for further genomic analysis. Yet, none of the kits tested were able to extract reasonable quantities of DNA from cores of the endospores. Thus, we developed a mechanical endospore lysis protocol, facilitating the extraction of high-quality DNA. Transmission electron microscopy or the labelling of spores with the indicator dye propidium monoazide was utilized to assess lysis efficiency. Finally, the yield and quality of genomic spore DNA were quantified by PCR and they were found to be dependent on lysis matrix composition, instrumental parameters, and the method used for subsequent DNA purification. Our final standardized lysis and DNA extraction protocol allows for the quantitative detection of low levels (<50 CFU/mL) of B. anthracis endospores and it is suitable for direct quantification, even under resource-limited field conditions, where culturing is not an option.