Potential drivers of vector-borne pathogens in urban environments: European hedgehogs (Erinaceus europaeus) in the spotlight.

Vector-borne diseases (VBDs) are considered as (re-)emerging, but information on the transmission cycles and wildlife reservoirs is often incomplete, particularly with regard to urban areas. The present study investigated blood samples from European hedgehogs (Erinaceus europaeus) presented at wildlife rehabilitation centres in the region of Hanover. Past exposure to B. burgdorferi sensu lato (s.l.) and tick-borne encephalitis virus (TBEV) was assessed by serological detection of antibodies, while current infections with Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Neoehrlichia mikurensis, Bartonella spp., Babesia spp. and Spiroplasma ixodetis were investigated by (q)PCR. Of 539 hedgehogs tested for anti-Borrelia antibodies, 84.8% (457/539) were seropositive, with a higher seropositivity rate in adult than subadult animals, while anti-TBEV antibodies were detected in one animal only (0.2%; 1/526). By qPCR, 31.2% (168/539) of hedgehog blood samples were positive for Borrelia spp., 49.7% (261/525) for A. phagocytophilum, 13.0% (68/525) for Bartonella spp., 8.2% for S. ixodetis (43/525), 8.0% (42/525) for Rickettsia spp. and 1.3% (7/525) for Babesia spp., while N. mikurensis was not detected. While further differentiation of Borrelia spp. infections was not successful, 63.2% of the A. phagocytophilum infections were assigned to the zoonotic ecotype I and among Rickettsia spp. infections, 50.0% to R. helvetica by ecotype- or species-specific qPCR, respectively. Sequencing revealed the presence of a Rickettsia sp. closely related to Rickettsia felis in addition to a Bartonella sp. previously described from hedgehogs, as well as Babesia microti and Babesia venatorum. These findings show that hedgehogs from rehabilitation centres are valuable sources to identify One Health pathogens in urban areas. The hedgehogs are not only exposed to pathogens from fleas and ticks in urban areas, but they also act as potent amplifiers for these vectors and their pathogens, relevant for citizens and their pets.

An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

Unprecedented genetic variability of PFam54 paralogs among Eurasian Lyme borreliosis-causing spirochetes.

Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere caused by spirochetes belonging to the Borrelia burgdorferi sensu lato (Bbsl) complex. Borrelia spirochetes circulate in obligatory transmission cycles between tick vectors and different vertebrate hosts. To successfully complete this complex transmission cycle, Bbsl encodes for an arsenal of proteins including the PFam54 protein family with known, or proposed, influences to reservoir host and/or vector adaptation. Even so, only fragmentary information is available regarding the naturally occurring level of variation in the PFam54 gene array especially in relation to Eurasian-distributed species. Utilizing whole genome data from isolates (n = 141) originated from three major LB-causing Borrelia species across Eurasia (B. afzelii, B. bavariensis, and B. garinii), we aimed to characterize the diversity of the PFam54 gene array in these isolates to facilitate understanding the evolution of PFam54 paralogs on an intra- and interspecies level. We found an extraordinarily high level of variation in the PFam54 gene array with 39 PFam54 paralogs belonging to 23 orthologous groups including five novel paralogs. Even so, the gene array appears to have remained fairly stable over the evolutionary history of the studied Borrelia species. Interestingly, genes outside Clade IV, which contains genes encoding for proteins associated with Borrelia pathogenesis, more frequently displayed signatures of diversifying selection between clades that differ in hypothesized vector or host species. This could suggest that non-Clade IV paralogs play a more important role in host and/or vector adaptation than previously expected, which would require future lab-based studies to validate.

Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato.

Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l. Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays. The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays. Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.

Longitudinal analysis of 20 Years of external quality assurance schemes for PCR/NAAT-based bacterial genome detection in diagnostic testing.

Background: Quality control (QC), quality assurance, and standardization are crucial for modern diagnostic testing in the field of medical microbiology. The need for efficient QC to ensure accurate laboratory results, treatment, and infection prevention has led to significant efforts in standardizing assay reagents and workflows. External quality assessment (EQA) schemes, like those offered by INSTAND, play a vital role in evaluating in-house and commercial routine diagnostic assays, regarded as mandatory by national and global guidelines. The recent impact of polymerase chain reaction/nucleic acid amplification technology (PCR/NAAT) assays in medical microbiology requires that high-performing assays be distinguished from inadequately performing ones, especially those made by inexperienced suppliers. Objectives: The study assesses the evolving diagnostic performance trends over 2 decades for the detection of EHEC/STEC, Borrelia (B.) burgdorferi, and MRSA/cMRSA. It explores the historical context of assay utilization, participant engagement, and rates of correct results in EQA schemes. The research seeks to identify patterns in assay preferences, participant proficiency, and the challenges encountered in detecting emerging variants or clinical strains. Results: The study highlights the decline in in-house PCR assay usage, the emergence of new diagnostic challenges, and educational aspects within EQA schemes. Specific examples, such as the inclusion, in certain EQA surveys, of EHEC strains carrying stx-2f or B. miyamotoi, highlight the role of EQAs in increasing awareness and diagnostic capabilities. Advancements in MRSA detection, especially through the adoption of commercial assays, demonstrate the impact that technology evolution has had on diagnostic performance. Conclusion: Achieving excellence in diagnostic molecular microbiology involves a multifaceted approach, including well-evaluated assays, careful instrumentation selection, and structured training programs. EQA schemes contribute significantly to this pursuit by providing insights into the evolving diagnostic landscape and identifying areas for improvement in the diagnostic workflow as well as in PCR/NAAT assay design.

Serological evidence of louse-borne relapsing fever in northern Kenya.

BACKGROUND: Tick- and louse-borne relapsing fever are highly-neglected, vector-borne diseases caused by diverse Borrelia species. Presently, there are no data available on the endemicity of tick- and louse-borne relapsing fever spirochetes in Kenya. Here, we present data of a retrospective study on the seroprevalence of louse-borne relapsing fever (LBRF) in northern Kenya. METHODS: A novel immunoassay, recently established for the diagnosis of LBRF was utilized to screen 2005 blood samples collected from individuals with fever without a source in Turkana County, Kenya between May 2009 and November 2010 for anti-LBRF antibodies. RESULTS: Out of the 2005 sera analyzed, 287 samples (14.3 %) were considered anti-LBRF IgG positive. Subsequent analyses revealed that 87 out of 152 sera randomly selected from these 2005 samples were tested positive (57.2 %) for anti-LBRF IgM antibodies. Most of the IgG and IgM positive samples were from individuals living in northern regions of Turkana County. CONCLUSION: Our serological finding provides strong evidence for the occurrence of LBRF in Kenya.

Frequency of Anaplasma phagocytophilum, Borrelia spp., and coinfections in Ixodes ricinus ticks collected from dogs and cats in Germany.

BACKGROUND: Changing geographical and seasonal activity patterns of ticks may increase the risk of tick infestation and tick-borne pathogen (TBP) transmission for both humans and animals. METHODS: To estimate TBP exposure of dogs and cats, 3000 female I. ricinus from these hosts were investigated for Anaplasma phagocytophilum and Borrelia species. RESULTS: qPCR inhibition, which was observed for ticks of all engorgement stages but not questing ticks, was eliminated at a template volume of 2 µl. In ticks from dogs, A. phagocytophilum and Borrelia spp. prevalence amounted to 19.0% (285/1500) and 28.5% (427/1500), respectively, while ticks from cats showed significantly higher values of 30.9% (464/1500) and 55.1% (827/1500). Accordingly, the coinfection rate with both A. phagocytophilum and Borrelia spp. was significantly higher in ticks from cats (17.5%, 262/1500) than dogs (6.9%, 104/1500). Borrelia prevalence significantly decreased with increasing engorgement duration in ticks from both host species, whereas A. phagocytophilum prevalence decreased only in ticks from dogs. While A. phagocytophilum copy numbers in positive ticks did not change significantly over the time of engorgement, those of Borrelia decreased initially in dog ticks. In ticks from cats, copy numbers of neither A. phagocytophilum nor Borrelia spp. were affected by engorgement. Borrelia species differentiation was successful in 29.1% (365/1254) of qPCR-positive ticks. The most frequently detected species in ticks from dogs were B. afzelii (39.3% of successfully differentiated infections; 70/178), B. miyamotoi (16.3%; 29/178), and B. valaisiana (15.7%; 28/178), while B. afzelii (40.1%; 91/227), B. spielmanii (21.6%; 49/227), and B. miyamotoi (14.1%; 32/227) occurred most frequently in ticks from cats. CONCLUSIONS: The differences in pathogen prevalence and Borrelia species distribution between ticks collected from dogs and cats may result from differences in habitat overlap with TBP reservoir hosts. The declining prevalence of A. phagocytophilum with increasing engorgement duration, without a decrease in copy numbers, could indicate transmission to dogs over the time of attachment. The fact that this was not observed in ticks from cats may indicate less efficient transmission. In conclusion, the high prevalence of A. phagocytophilum and Borrelia spp. in ticks collected from dogs and cats underlines the need for effective acaricide tick control to protect both animals and humans from associated health risks.

Tick-borne diseases in the North Sea region-A comprehensive overview and recommendations for diagnostics and treatment.

As part of the NorthTick project, co-funded by the European Union through the European Regional Development Fund and the North Sea Region Programme, specialists in the field of tick-borne diseases from seven North Sea countries co-operated with patient organisations and governmental health care institutions to provide this comprehensive overview of diagnostics and treatment recommendations in the region for Lyme borreliosis, Borrelia miyamotoi infection, tick-borne encephalitis, human granulocytic anaplasmosis, rickettsiosis, neoehrlichiosis and babesiosis. The main conclusion is that the recommendations in these northern countries are essentially the same, with very few differences. This overview presents the current diagnostics and provides useful clinical guidance.

Evidence for Bartonella quintana in Lice Collected from the Clothes of Ethiopian Homeless Individuals.

Human lice, Pediculus humanus, can transmit various pathogens, including Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii. Xenosurveillance is an epidemiological approach to assessing human infection risks performed by screening vectors of infectious disease agents. In the proof-of-principle study reported herein, the DNA of 23 human lice was collected from the clothes of 30 homeless Ethiopian individuals. These samples were assessed using 16S rRNA gene-specific pan-eubacterial PCR for screening, followed by Bartonella genus 16S-23S internal transcribed spacer (ITS) sequence-specific PCR, Bartonella genus gltA gene-specific PCR, and 16S rRNA gene PCR with specificity for relapsing-fever-associated Borrelia spp. with subsequent sequencing of the amplicons. In one sample, the pan-eubacterial 16S rRNA gene-specific screening PCR, the Bartonella genus 16S-23S ITS sequence-specific PCR, and the Bartonella genus gltA gene-specific PCR allowed for the sequencing of B. quintana-specific amplicons. In two additional samples, Bartonella genus gltA gene-specific PCR also provided sequences showing 100% sequence identity with B. quintana. In total, 3/23 (13.0%) of the assessed lice were found to be positive for B. quintana. Correlating clinical data were not available; however, the assessment confirmed the presence of B. quintana in the local louse population and thus an associated infection pressure. Larger-sized cross-sectional studies seem advisable to more reliably quantify the infection risk of lice-infested local individuals. The need for prevention by providing opportunities to maintain standard hygiene for Ethiopian homeless individuals is stressed by the reported findings, especially in light of the ongoing migration of refugees.

Genome diversity of Borrelia garinii in marine transmission cycles does not match host associations but reflects the strains evolutionary history.

Borrelia burgdorferi sensu lato is a species complex of spirochetal bacteria that occupy different ecological niches which is reflected in their reservoir host- and vector-associations. Borrelia genomes possess numerous linear and circular plasmids. Proteins encoded by plasmid genes play a major role in host- and vector-interaction and are important for Borrelia niche adaptation. However, the plasmid composition and therewith the gene repertoire may vary even in strains of a single species. Borrelia garinii, one of the six human pathogenic species, is common in Europe (vector Ixodes ricinus), Asia (vector Ixodes persulcatus) and in marine birds (vector Ixodes uriae). For the latter, only a single culture isolate (Far04) and its genome were previously available. The genome was rather small containing only one circular and six linear plasmids with a notable absence of cp32 plasmids. To further investigate B. garinii from marine transmission cycles and to explore i) whether the small number of plasmids found in isolate Far04 is a common feature in B. garinii from marine birds and presents an adaptation to this particular niche and ii) whether there may be a correlation between genome type and host species, we initiated in vitro cultures from live I. uriae collected in 2017 and 2018 from marine avian hosts and their nests. Hosts included common guillemots, Atlantic Puffin, razorbill, and kittiwake. We obtained 17 novel isolates of which 10 were sequenced using Illumina technology, one also with Pacific Bioscience technology. The 10 genomes segregated into five different genome types defined by plasmid types (based on PFam32 loci). We show that the genomes of seabird associated B. garinii contain fewer plasmids (6-9) than B. garinii from terrestrial avian species (generally ≥10), potentially suggesting niche adaptation. However, genome type did not match an association with the diverse avian seabird hosts investigated but matched the clonal complex they originated from, perhaps reflecting the isolates evolutionary history. Questions that should be addressed in future studies are (i) how is plasmid diversity related to host- and/or vector adaptation; (ii) do the different seabird species differ in reservoir host competence, and (iii) can the genome types found in seabirds use terrestrial birds as reservoir hosts.

Seroprevalence, seroconversion and seroreversion of Borrelia burgdorferi-specific IgG antibodies in two population-based studies in children and adolescents, Germany, 2003 to 2006 and 2014 to 2017.

BackgroundLyme borreliosis (LB), caused by Borrelia burgdorferi (Bb), is the most common tick-borne infection in Germany. Antibodies against Bb are prevalent in the general population but information on temporal changes of prevalence and estimates of seroconversion (seroincidence) and seroreversion are lacking, especially for children and adolescents.AimWe aimed at assessing antibodies against Bb and factors associated with seropositivity in children and adolescents in Germany.MethodsWe estimated seroprevalence via two consecutive cross-sectional surveys (2003-2006 and 2014-2017). Based on a longitudinal survey component, we estimated annual seroconversion/seroreversion rates.ResultsSeroprevalence was 4.4% (95% confidence interval (CI): 3.9-4.9%) from 2003 to 2006 and 4.1% (95% CI: 3.2-5.1%) from 2014 to 2017. Seroprevalence increased with age, was higher in male children, the south-eastern regions of Germany and among those with a high socioeconomic status. The annual seroconversion rate was 0.3% and the annual seroreversion rate 3.9%. Males were more likely to seroconvert compared with females. Low antibody levels were the main predictor of seroreversion.ConclusionWe did not detect a change in seroprevalence in children and adolescents in Germany over a period of 11 years. Potential long-term changes, for example due to climatic changes, need to be assessed in consecutive serosurveys. Seroconversion was more likely among children and adolescents than among adults, representing a target group for preventive measures. Seroreversion rates are over twice as high in children and adolescents compared with previous studies among adults. Thus, seroprevalence estimates and seroconversion rates in children are likely underestimated.

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.

German Ixodes inopinatus samples may not actually represent this tick species.

Ticks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species. Ixodes inopinatus is considered a sister species and highly similar to Ixodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent of I. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified as I. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) and whole genome sequencing of specific ticks (n = 8), we were able to determine that German samples, morphologically identified as I. inopinatus, genetically represent I. ricinus regardless of previous morphological identification, and most likely are not I. ricinus/I. inopinatus hybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish between I. ricinus and I. inopinatus. Our results suggest that I. inopinatus is geographically isolated as a species (northern Africa and potentially southern Spain and Portugal) and brings into question whether I. inopinatus exists in central Europe. Our results highlight the probable existence of I. inopinatus and the power of utilizing genomic data in answering questions regarding tick taxonomy.

[Bacterial zoonoses of public health importance in Germany-incidence, distribution, and modes of transmission]. / Bakterielle Zoonosen mit Bedeutung für den öffentlichen Gesundheitsschutz in Deutschland ­ Vorkommen, Verbreitung und Übertragungswege.

Bacterial zoonotic pathogens are often the cause of diseases, sometimes with severe outcomes. They are mutually transferable between animals (both wild and domestic) and humans. The transmission paths are very variable and include oral intake via food, respiratory infection via droplets and aerosols, or infections via vectors such as tick bites or rodent contact. Furthermore, the emergence and spread of antibiotic-resistant bacterial pathogens is of paramount public health concern.The likelihood of further spread is influenced by various factors. These include the increase in international trade, the endangerment of animal habitats, and the increasingly closer contact between humans and wild animals. Additionally, changes in livestock and climate change may also contribute. Therefore, research into zoonoses serves to protect human and animal health and is of particular social, political, and economic importance.The aim of this review article is to present the range of infectious diseases caused by bacterial zoonotic pathogens in order to provide a better understanding of the important work in public health services, animal health services, and food safety control. The different transmission routes, epidemic potentials, and epidemiological measures of the exemplary selected diseases show the challenges for the public health system to monitor and control the spread of these bacterial pathogens in order to protect the population from disease.

Co-Infection of Potential Tick-Borne Pathogens of the Order Rickettsiales and Borrelia burgdorferi s. l. and Their Link to Season and Area in Germany.

The prevalence of potential human pathogenic members of the order Rickettsiales differs between Borrelia burgdorferi sensu lato-positive and -negative tick microbiomes. Here, co-infection of members of the order Rickettsiales, such as Rickettsia spp., Anaplasma phagocytophilum, Wolbachia pipientis, and Neoehrlichia mikurensis as well as B. burgdorferi s.l. in the tick microbiome was addressed. This study used conventional PCRs to investigate the diversity and prevalence of the before-mentioned bacteria in 760 nucleic acid extracts of I. ricinus ticks detached from humans, which were previously tested for B. burgdorferi s.l.. A gltA gene-based amplicon sequencing approach was performed to identify Rickettsia species. The prevalence of Rickettsia spp. (16.7%, n = 127) and W. pipientis (15.9%, n = 121) were similar, while A. phagocytophilum was found in 2.8% (n = 21) and N. mikurensis in 0.1% (n = 1) of all ticks. Co-infection of B. burgdorferi s. l. with Rickettsia spp. was most frequent. The gltA gene sequencing indicated that Rickettsia helvetica was the dominant Rickettsia species in tick microbiomes. Moreover, R, monacensis and R. raoultii were correlated with autumn and area south, respectively, and a negative B. burgdorferi s. l. finding. Almost every fifth tick carried DNA of at least two of the human pathogenic bacteria studied here.

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.

15-year Borrelia prevalence and species distribution monitoring in Ixodes ricinus/inopinatus populations in the city of Hanover, Germany.

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato (s.l.) spirochaetes, is the most common tick-borne disease (TBD) in the Northern Hemisphere. Rising incidences indicate that its epidemiology may be affected by global changes. Therefore, the current study aimed to assess changes in tick infection rates with Borrelia spp. over a 15-year monitoring period in the city of Hanover, Germany, as a follow-up to previous prevalence studies (years 2005, 2010 and 2015). To assess the epidemiological risk, ticks of the Ixodes ricinus/inopinatus-complex were sampled from April to October 2020 by the flagging method at 10 frequently visited recreation areas in Hanover. Analysis by quantitative real-time PCR of 2100 individual ticks revealed an overall Borrelia prevalence of 25.5% (535/2100). Regarding different tick developmental stages, nymphs showed a significantly lower Borrelia prevalence (18.4% [193/1050]) than adult ticks (32.6% [342/1050]). Comparison with previous years revealed a stable total Borrelia prevalence along with consistent infection rates in the different developmental stages over the 15-year monitoring period. Borrelia species differentiation by Reverse Line Blot was successful in 67.3% of positive ticks collected in 2020, with B. afzelii being the dominating species (59.2% of the differentiated infections), besides B. burgdorferi sensu stricto (s.s.), B. garinii, B. valaisiana, B. spielmanii, B. bavariensis and B. bissettiae and the relapsing fever spirochaete B. miyamotoi. Additionally, the proportion of infections attributed to B. afzelii showed a significant increase in 2020 compared to 2005 and 2015 (59.2% vs. 37.6% and 32.0% of successfully differentiated infections, respectively). Coinfections with Anaplasma phagocytophilum and Rickettsia spp. stayed stable comparing 2020 with previous years. Therefore, although changes in the Borrelia prevalence in questing ticks were not observed throughout the 15-year monitoring period, shifts in Borrelia species distribution may alter the epidemiological risk.

Out of Asia? Expansion of Eurasian Lyme borreliosis causing genospecies display unique evolutionary trajectories.

Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host and vector shifts can lead to geographic expansion of infectious agents and the emergence of new diseases in susceptible individuals. Three bacterial genospecies (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus). Through these vectors, the bacteria can infect various vertebrate groups (e.g., rodents, birds) including humans where they cause Lyme borreliosis, the most common vector-borne disease in the Northern hemisphere. Yet, how and in which order the three Borrelia genospecies colonized each continent remains unclear including the evolutionary consequences of this geographic expansion. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we found evidence that the ancestors of each of the three genospecies probably have an Asian origin. Even so, each genospecies studied displayed a unique substructuring and evolutionary response to the colonization of Europe. The pattern of allele sharing between continents is consistent with the dispersal rate of the respective vertebrate hosts, supporting the concept that adaptation of Borrelia genospecies to the host is important for pathogen dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion with host association influencing their dispersal; further displaying the importance of host and vector association to the geographic expansion of vector-borne pathogens and potentially conditioning their capacity as emergent pathogens.

Novel approaches for the serodiagnosis of louse-borne relapsing fever.

Louse-borne relapsing fever (LBRF) caused by B. recurrentis is a poverty-related and neglected infectious disease with an endemic focus in the Horn of Africa. Re-emergence of the disease occurred in Europe during the refugee crisis in 2015 and sporadic outbreaks were frequently reported in Eastern Africa where poor settings lack affordable diagnostics. Currently, there are no validated in vitro assays available for the serodiagnosis of LBRF. The aim of this study was to develop novel and reliable immunoassays by investigating clinically suspected and culture-confirmed serum samples from LBRF patients and a broad panel of serum samples from patients with other spirochetal, bacterial, and parasitic diseases. We identified two immunoreactive antigens (complement-inhibiting protein CihC and the glycerophosphodiester phosphodiesterase GlpQ of B. recurrentis) as the most promising target candidates leading to the evaluation of two immunoassays (line immunoblot and ELISA) for IgM and IgG. To optimize the IgM immunoassay, we conducted a bioinformatic approach to localize the relevant immunogenic regions within CihC. By utilizing a N-terminal CihC fragment, the sensitivity and specificity of both immunoassays (CihC and GlpQ) were high (IgM: sensitivity 100%, specificity of 89.9%, IgG: sensitivity 100%, specificity 99.2%). In conclusion, our findings indicate the diagnostic potential of CihC and GlpQ as valuable markers for the serodiagnosis of LBRF even at early time points of infection. Here, we provide strong evidence for the utilization of these immunoassays as reliable tools in clinical practice.

Epidemiological and Serological Analysis of a SARS-CoV-2 Outbreak in a Nursing Home: Impact of SARS-CoV-2 Vaccination and Enhanced Neutralizing Immunity Following Breakthrough Infection.

Background: Despite a vaccination rate of 82.0% (n = 123/150), a SARS-CoV-2 (Alpha) outbreak with 64.7% (n = 97/150) confirmed infections occurred in a nursing home in Bavaria, Germany. Objective: the aim of this retrospective cohort study was to examine the effects of the Corminaty vaccine in a real-life outbreak situation and to obtain insights into the antibody response to both vaccination and breakthrough infection. Methods: the antibody status of 106 fully vaccinated individuals (54/106 breakthrough infections) and epidemiological data on all 150 residents and facility staff were evaluated. Results: SARS-CoV-2 infections (positive RT-qPCR) were detected in 56.9% (n = 70/123) of fully vaccinated, compared to 100% (n = 27/27) of incompletely or non-vaccinated individuals. The proportion of hospitalized and deceased was 4.1% (n = 5/123) among fully vaccinated and therewith lower compared to 18.5% (n = 5/27) hospitalized and 11.1% (n = 3/27) deceased among incompletely or non-vaccinated. Ct values were significantly lower in incompletely or non-vaccinated (p = 0.02). Neutralizing antibodies were detected in 99.1% (n = 105/106) of serum samples with significantly higher values (p < 0.001) being measured post-breakthrough infection. α-N-antibodies were detected in 37.7% of PCR positive but not in PCR negative individuals. Conclusion: Altogether, our data indicate that SARS-CoV-2 vaccination does provide protection against infection, severe disease progression and death with regards to the Alpha variant. Nonetheless, it also shows that infection and transmission are possible despite full vaccination. It further indicates that breakthrough infections can significantly enhance α-S- and neutralizing antibody responses, indicating a possible benefit from booster vaccinations.