Prevalence of tick-borne encephalitis virus in questing Ixodes ricinus nymphs in southern Scandinavia and the possible influence of meteorological factors.

Ixodes ricinus ticks are Scandinavia's main vector for tick-borne encephalitis virus (TBEV), which infects many people annually. The aims of the present study were (i) to obtain information on the TBEV prevalence in host-seeking I. ricinus collected within the Øresund-Kattegat-Skagerrak (ØKS) region, which lies in southern Norway, southern Sweden and Denmark; (ii) to analyse whether there are potential spatial patterns in the TBEV prevalence; and (iii) to understand the relationship between TBEV prevalence and meteorological factors in southern Scandinavia. Tick nymphs were collected in 2016, in southern Scandinavia, and screened for TBEV, using pools of 10 nymphs, with RT real-time PCR, and positive samples were confirmed with pyrosequencing. Spatial autocorrelation and cluster analysis was performed with Global Moran's I and SatScan to test for spatial patterns and potential local clusters of the TBEV pool prevalence at each of the 50 sites. A climatic analysis was made to correlate parameters such as minimum, mean and maximum temperature, relative humidity and saturation deficit with TBEV pool prevalence. The climatic data were acquired from the nearest meteorological stations for 2015 and 2016. This study confirms the presence of TBEV in 12 out of 30 locations in Denmark, where six were from Jutland, three from Zealand and two from Bornholm and Falster counties. In total, five out of nine sites were positive from southern Sweden. TBEV prevalence of 0.7%, 0.5% and 0.5%, in nymphs, was found at three sites along the Oslofjord (two sites) and northern Skåne region (one site), indicating a potential concern for public health. We report an overall estimated TBEV prevalence of 0.1% in questing I. ricinus nymphs in southern Scandinavia with a region-specific prevalence of 0.1% in Denmark, 0.2% in southern Sweden and 0.1% in southeastern Norway. No evidence of a spatial pattern or local clusters was found in the study region. We found a strong correlation between TBEV prevalence in ticks and relative humidity in Sweden and Norway, which might suggest that humidity has a role in maintaining TBEV prevalence in ticks. TBEV is an emerging tick-borne pathogen in southern Scandinavia, and we recommend further studies to understand the TBEV transmission potential with changing climate in Scandinavia.

Invasive meningococcal disease in Norway in the two decades before the COVID-19 pandemic.

OBJECTIVES: Disease caused by the bacterium Neisseria meningitidis remains a worldwide public health challenge, despite the steadily decreasing incidence in Western countries. The objective of this study was to explore the epidemiology of invasive meningococcal disease in Norway over the last two decades. DESIGN: All isolates sent to the National Reference Laboratory from patients with invasive meningococcal disease between the years 2000 and 2019 were analyzed using whole genome sequencing (total: 625). RESULTS: A five-fold decrease in case numbers occurred over this period, and the situation has gone from being dominated by serogroup B to one where serogroups Y and W are more prevalent. Concurrently, the mean age at infection has increased from 18 to 33 years. Among the 350 serogroup B isolates, 87% were an exact match or cross-reactive with one or both the currently available serogroup B vaccines, but the proportion decreased in the past decade. Core genome analyses revealed a high variation in the number of allelic differences accumulated in epidemiologically linked isolates to the point that near-identical isolates were found several years apart. CONCLUSION: Allelic distance is an imprecise metric for the degree of epidemiologic linkage between isolates in N. meningitidis.

Fourier transform infrared spectroscopy; can it be used as a rapid typing method of Neisseria gonorrhoeae?

BACKGROUND: Typing of Neisseria gonorrhoeae is necessary for epidemiologic surveillance, while time consuming and resource intensive. Fourier transform infrared (FTIR) spectroscopy has shown promising results when typing several bacterial species. This study investigates whether FTIR spectroscopy can be used as a rapid method for typing clinical N. gonorrhoeae isolates, comparing FTIR spectroscopy to multi locus sequence typing (MLST), N. gonorrhoeae multi antigen sequence typing (NG-MAST) and whole genome sequencing (WGS). METHODS: Sixty consecutive isolates from a venereology clinic and three isolates from an outbreak were included. Isolates were analysed with FTIR spectroscopy on the IR Biotyper system (Bruker Daltonik) with the IR Biotyper software (version 2.1) with default analysis settings (spectral range 1300-800 cm-1). Four technical replicates of each isolate were analysed in three different runs. The output was a hierarchical cluster analysis (HCA) presented as a dendrogram; a tree-like overview of how closely different isolates are related. FTIR spectroscopy was compared to MLST, NG-MAST and WGS to see if the FTIR spectroscopy-dendrogram grouped the isolates in the same clusters. RESULTS: Fifty-one out of 60 isolates, and the three outbreak isolates, produced at least one spectrum in each run and were included. No agreement between FTIR spectroscopy and MLST or NG-MAST or WGS was shown. The FTIR spectroscopy-dendrogram failed to cluster the outbreak isolates. CONCLUSION: FTIR spectroscopy (spectral range 1300-800 cm-1) is not yet suitable for epidemiologic typing of N. gonorrhoeae. Absence of a capsule as well as phase- and antigenic variation of carbohydrate surface structures of the gonococcal cell wall may contribute to our findings. Future studies should include analysis of a wider range of the spectrum recorded (4000-500 cm-1), and should also explore further mathematical analytic approaches of the similarity between spectra.

Naturally occurring Neisseria gonorrhoeae can have large deletions in housekeeping gene abcZ, making them untypable with multilocus sequence typing.

The abcZ gene is an essential housekeeping gene in all the Neisseria species. It is one of the seven genes used for multilocus sequence typing (MLST) this genus. It encodes the cytosolic component of an ATP-binding cassette (ABC) transporter complex of unknown function. We report here the finding of a strain of Neisseria gonorrhoeae with a 485 base pair deletion in the 5' region of the abcZ gene that truncates the protein product from 636 amino acids to 89 amino acids. A second open reading frame (ORF), encoding the latter 388 amino acids of the abcZ gene, was predicted downstream. The deletion will affect MLST profiling; interrogation of genomic sequences from PubMLST revealed that this isolate is not an anomaly. Deletions in abcZ were identified in 256 Neisseria genomes, roughly 0.6% of isolates. Furthermore, these deletions could leave the abcZ gene in a pseudogenized state. Our strain, isolated from a patient with symptoms of gonorrheal infection, nevertheless behaved normal in terms of growth and in vitro phenotypic properties.

Tracing and tracking the emergence, epidemiology and dispersal of dengue virus to Africa during the 20th century.

The four mosquito-borne dengue virus serotypes (DENV1-DENV4) cause a high burden of disease throughout the tropical and sub-tropical regions of the world. Nevertheless, their precise epidemiological history in Africa, including when and where they originated and were distributed during the 20th century, remains unclear stressing the need for One Health focused research. Accordingly, we conducted a time-scaled molecular epidemiological reconstruction using publicly available and newly sequenced dengue virus genomes of African origin representing all four serotypes to deduce the most likely temporal and spatial transmission routes of each DENV serotype from their ancestral regions to, within and from Africa. Our analyses suggest that during the 20th century, serotypes DENV1-DENV3 were introduced to Africa from South East Asia on multiple occasions. The earliest evidence recorded indicates introduction of DENV2 during the early-1940s and of DENV1 during the mid-1940s to Western Africa from South East Asia. The analysis also implies an early introduction of DENV4 during the mid-1940s to Western Africa, alongside DENV1, probably originating in South East Asia. Establishment of DENV3 in Africa appears to have occurred later in the 1960s, apparently originating from South East Asia. However, with the re-establishment of DENV in the Americas, following the cessation of the PAHO mosquito control programme during the mid-20th century, evidence of introductions of DENV1 and DENV2 from the Americas to Western Africa was also observed. The data also identify intra-regional circulation of DENV, but also inter-regional dispersal of all four serotypes within Africa, which has led to a high degree of geographical overlap among serotypes. It is also noteworthy that DENV from both Eastern and Western Africa, have been introduced into Central Africa but there is no support for the converse relationship. For serotypes DENV1-DENV3, we observed probable exports from within established African DENV clusters (≥2 sequences) primarily to Eastern and Southern Asia. Collectively, our findings support the view that all DENV serotypes, apart from DENV4, have been introduced on multiple occasions to Africa, primarily originating from South East Asia, and subsequently to neighbouring regions within Africa.

The impact of global lineage dynamics, border restrictions, and emergence of the B.1.1.7 lineage on the SARS-CoV-2 epidemic in Norway.

As the COVID-19 pandemic swept through an immunologically naïve human population, academics and public health professionals scrambled to establish methods and platforms for genomic surveillance and data sharing. This offered a rare opportunity to study the ecology and evolution of SARS-CoV-2 over the course of the ongoing pandemic. Here, we use population genetic and phylogenetic methodology to characterize the population dynamics of SARS-CoV-2 and reconstruct patterns of virus introductions and local transmission in Norway against this backdrop. The analyses demonstrated that the epidemic in Norway was largely import driven and characterized by the repeated introduction, establishment, and suppression of new transmission lineages. This pattern changed with the arrival of the B.1.1.7 lineage, which was able to establish a stable presence concomitant with the imposition of severe border restrictions.

Spatiotemporal and seasonal dynamics in the microbial communities of a landfill-leachate contaminated aquifer.

The microbiome of an aquifer contaminated by landfill leachate and undergoing intrinsic remediation was characterised using 16S rRNA metabarcoding. The archaeal/bacterial V3-V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq, and multivariate statistics were applied to make inferences. Results indicate that the aquifer recharge and aquifer sediment samples harbour different microbial communities compared to the groundwater samples. While Proteobacteria dominated both the recharge and groundwater samples, Acidobacteria dominated the aquifer sediment. The most abundant genera detected from the contaminated aquifer were Polynucleobacter, Rhodoferax, Pedobacter, Brevundimonas, Pseudomonas, Undibacterium, Sulfurifustis, Janthinobacterium, Rhodanobacter, Methylobacter and Aquabacterium. The result also shows that the microbial communities of the groundwater varied spatially, seasonally and interannually, although the interannual variation was significant for only one of the wells. Variation partitioning analysis indicates that water chemistry and well distance are intercorrelated and they jointly accounted for most of the variation in microbial composition. This implies that the species composition and water chemistry characteristics have a similar spatial structuring, presumably caused by the landfill leachate plume. The study improves our understanding of the dynamics in subsurface microbial communities in space and time.

Retrospective meta-transcriptomic identification of severe dengue in a traveller returning from Africa to Sweden, 1990.

Pathogens associated with haemorrhagic fever commonly have zoonotic origins. The first documented imported case of likely viral severe haemorrhagic fever in Sweden occurred in 1990. Despite extensive study, no aetiological agent was identified. Following retrospective investigation with total RNA-sequencing of samples collected between 7 and 36 days from onset of symptoms we identified dengue virus 3 (DENV-3) and a human pegivirus (HPgV). We conclude that the patient likely suffered from haemorrhagic symptoms due to an atypical severe and undiagnosed dengue infection.

Antibiotic Treatment Regimes as a Driver of the Global Population Dynamics of a Major Gonorrhea Lineage.

The Neisseria gonorrhoeae multilocus sequence type (ST) 1901 is among the lineages most commonly associated with treatment failure. Here, we analyze a global collection of ST-1901 genomes to shed light on the emergence and spread of alleles associated with reduced susceptibility to extended-spectrum cephalosporins (ESCs). The genetic diversity of ST-1901 falls into a minor and a major clade, both of which were inferred to have originated in East Asia. The dispersal of the major clade from Asia happened in two separate waves expanding from ∼1987 and 1996, respectively. Both waves first reached North America, and from there spread to Europe and Oceania, with multiple secondary reintroductions to Asia. The ancestor of the second wave acquired the penA 34.001 allele, which significantly reduces susceptibility to ESCs. Our results suggest that the acquisition of this allele granted the second wave a fitness advantage at a time when ESCs became the key drug class used to treat gonorrhea. Following its establishment globally, the lineage has served as a reservoir for the repeated emergence of clones fully resistant to the ESC ceftriaxone, an essential drug for effective treatment of gonorrhea. We infer that the effective population sizes of both clades went into decline as treatment schemes shifted from fluoroquinolones via ESC monotherapy to dual therapy with ceftriaxone and azithromycin in Europe and the United States. Despite the inferred recent population size decline, the short evolutionary path from the penA 34.001 allele to alleles providing full ceftriaxone resistance is a cause of concern.

High-throughput sequencing of two European strains of tick-borne encephalitis virus (TBEV), Hochosterwitz and 1993/783.

Tick-borne encephalitis virus (TBEV) is a medically important arbovirus, widespread in Europe and Asia. The virus is primarily transmitted to humans and animals by bites from ticks and, in rare cases, by consumption of unpasteurized dairy products. The aim of this study was to sequence and characterize two TBEV strains with amplicon sequencing by designing overlapping primers. The amplicon sequencing, via Illumina MiSeq, covering nearly the entire TBEV genome, was successful: We retrieved and characterized the complete polyprotein sequence of two TBEV strains, Hochosterwitz and 1993/783 from Austria and Sweden, respectively. In this study the previous phylogenetic analysis of both strains was confirmed to be of the European subtypes of TBEV (TBEV-Eu) by whole genome sequencing. The Hochosterwitz strain clustered with the two strains KrM 93 and KrM 213 from South Korea, and the 1993/783 strain clustered together with the NL/UH strain from the Netherlands. Our study confirms the suitability and rapidness of the high-throughput sequencing method used to produce complete TBEV genomes from TBEV samples of high viral load giving high-molecular-weight cDNA with large overlapping amplicons.

Sudden emergence of a Neisseria gonorrhoeae clade with reduced susceptibility to extended-spectrum cephalosporins, Norway.

Neisseria gonorrhoeae multilocus sequence type (ST)-7827 emerged in a dramatic fashion in Norway in the period 2016-2018. Here, we aim to shed light on the provenance and expansion of this ST. ST-7827 was found to be polyphyletic, but the majority of members belonged to a monophyletic clade we termed PopPUNK cluster 7827 (PC-7827). In Norway, both PC-7827 and ST-7827 isolates were almost exclusively isolated from men. Phylogeographical analyses demonstrated an Asian origin of the genogroup, with multiple inferred exports to Europe and the USA. The genogroup was uniformly resistant to fluoroquinolones, and associated with reduced susceptibility to both azithromycin and the extended-spectrum cephalosporins (ESCs) cefixime and ceftriaxone. From a genetic background including the penA allele 13.001, associated with reduced ESC susceptibility, we identified repeated events of acquisition of porB alleles associated with further reduction in ceftriaxone susceptibility. Transmission of the strain was significantly reduced in Norway in 2019, but our results indicate the existence of a recently established global reservoir. The worrisome drug-resistance profile and rapid emergence of PC-7827 calls for close monitoring of the situation.

Genomic epidemiology and population structure of Neisseria gonorrhoeae in Norway, 2016-2017.

This study presents the nationwide epidemiology of Neisseria gonorrhoeae, using whole-genome sequencing of all culture-positive cases, which comprise roughly 40 % of all cases of gonorrhea reported in Norway from 2016 to 2017. Isolates were assigned to sequence types and Bayesian analysis clusters and variation in genes coding for antibiotic resistance was linked to phenotypic resistance data. The study also included isolates taken from the same patients from different anatomical sites at one or more time points. Comparing these isolates allows for observation of patterns of infections, i.e. multiple reinfections of genetically related clones vs. reinfections of genetically distant clones, and quantification of the genomic variation of closely related isolates from samples taken from a patient within the same day. Demographically, the patients in the study could be split into two groups; one group of patients from the capital with a high proportion of men who have sex with men (MSM), and another consisting of young adults with transmission primarily between males and females from outside the capital. Some clusters of N. gonorrhoeae were restricted to one of these two demographic groups. Pairwise comparison of multiple isolates from the same patients revealed that most were reinfected with different clones. Observations of frequent reinfections in patients is a concern and should be taken into account in the development of improved information and treatment guidelines.

Meta-transcriptomic identification of hepatitis B virus in cerebrospinal fluid in patients with central nervous system disease.

Determining the etiological basis of central nervous system (CNS) infections is inherently challenging, primarily due to the multi-etiological nature. Using RNA sequencing, we aimed to identify microbes present in cerebrospinal fluid (CSF) of two patients suffering CNS infection, previously diagnosed with Cryptococcus sp. and Streptococcus pneumoniae infection, respectively. After meta-transcriptomic analysis, and confirmation with real-time PCR, hepatitis B virus (HBV) was detected in the CSF of two patients diagnosed with CNS syndrome. Phylogenetic analysis of the partial HBV genomes from these patients showed that they belonged to genotypes B and C and clustered with other viruses of Asian origin. In countries with high levels of HBV endemicity, the virus is likely to be found in patients diagnosed with CNS infections, although whether it contributes to symptoms and pathology, or is simply a coincidental infection, is unknown and merits further investigation.

Whole genome sequencing of Salmonella Chester reveals geographically distinct clusters, Norway, 2000 to 2016.

IntroductionDuring summer 2016, Norway observed an increase in Salmonella enterica subsp. enterica serovar Chester cases among travellers to Greece.AimOur aim was to investigate genetic relatedness of S. Chester for surveillance and outbreak detection by core genome multilocus sequence typing (cgMLST) and compare the results to genome mapping.MethodsWe included S. Chester isolates from 51 cases of salmonellosis between 2000 and 2016. Paired-end sequencing (2 × 250 bp) was performed on Illumina MiSeq. Genetic relatedness by cgMLST for Salmonella enterica subsp. enterica, including 3,002 genes and seven housekeeping genes, was compared by reference genome mapping with CSI Phylogeny version 1.4 and conventional MLST.ResultsConfirmed travel history was available for 80% of included cases, to Europe (n = 13), Asia (n = 12) and Africa (n = 16). Isolates were distributed into four phylogenetic clusters corresponding to geographical regions. Sequence type (ST) ST411 and a single-locus variant ST5260 (n = 17) were primarily acquired in southern Europe, ST1954 (n = 15) in Africa, ST343 (n = 11) and ST2063 (n = 8) primarily in Asia. Part of the European cluster was further divided into a Greek (n = 10) and a Cypriot (n = 4) cluster. All isolates in the African cluster displayed resistance to ≥ 1 class of antimicrobials, while resistance was rare in the other clusters.ConclusionWhole genome sequencing of S. Chester in Norway showed four geographically distinct clusters, with a possible outbreak occurring during summer 2016 related to Greece. We recommend public health institutes to implement cgMLST-based real-time Salmonella enterica surveillance for early and accurate detection of future outbreaks and further development of cluster cut-offs.

Global expansion of Mycobacterium tuberculosis lineage 4 shaped by colonial migration and local adaptation.

On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.

Genome-based transmission modelling separates imported tuberculosis from recent transmission within an immigrant population.

In many countries the incidence of tuberculosis (TB) is low and is largely shaped by immigrant populations from high-burden countries. This is the case in Norway, where more than 80 % of TB cases are found among immigrants from high-incidence countries. A variable latent period, low rates of evolution and structured social networks make separating import from within-border transmission a major conundrum to TB control efforts in many low-incidence countries. Clinical Mycobacterium tuberculosis isolates belonging to an unusually large genotype cluster associated with people born in the Horn of Africa have been identified in Norway over the last two decades. We modelled transmission based on whole-genome sequence data to estimate infection times for individual patients. By contrasting these estimates with time of arrival in Norway, we estimate on a case-by-case basis whether patients were likely to have been infected before or after arrival. Independent import was responsible for the majority of cases, but we estimate that about one-quarter of the patients had contracted TB in Norway. This study illuminates the transmission dynamics within an immigrant community. Our approach is broadly applicable to many settings where TB control programmes can benefit from understanding when and where patients acquired TB.

A genomic view of experimental intraspecies and interspecies transformation of a rifampicin-resistance allele into Neisseria meningitidis.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P<0.001). The recombination events were generally observed to be mosaic, with donor sequences interspersed with recipient sequence. Here, we present four models to explain these observations, by fragmentation of the transformed DNA, by interruptions of the recombination mechanism, by secondary recombination of endogenous self-DNA, or by repair/replication mechanisms.

Modeling of the GC content of the substituted bases in bacterial core genomes.

BACKGROUND: The purpose of the present study was to examine the GC content of substituted bases (sbGC) in the core genomes of 35 bacterial species. Each species, or core genome, constituted genomes from at least 10 strains. We also wanted to explore whether sbGC for each strain was associated with the corresponding species' core genome GC content (cgGC). We present a simple mathematical model that estimates sbGC from cgGC. The model assumes only that the estimated sbGC is a function of cgGC proportional to fixed AT→GC (α) and GC → AT (ß) mutation rates. Non-linear regression was used to estimate parameters α and ß from the empirical data described above. RESULTS: We found that sbGC for each strain showed a non-linear association with the corresponding cgGC with a bias towards higher GC content for most core genomes (66.3% of the strains), assuming as a null-hypothesis that sbGC should be approximately equal to cgGC. The most GC rich core genomes (i.e. approximately %GC > 60), on the other hand, exhibited slightly less GC-biased sbGC than expected. The best fitted regression model indicates that GC → AT mutation rates ß = (1.91 ± 0.13) p < 0.001 are approximately (1.91/0.79) = 2.42 times as high, on average, as AT→GC α = (- 0.79 ± 0.25) p < 0.001 mutation rates. Whether the observed sbGC GC-bias for all but the most GC-rich prokaryotic species is due to selection, compensating for the GC → AT mutation bias, and/or selective neutral processes is currently debated. Residual standard error was found to be σ = 0.076 indicating estimated errors of sbGC to be approximately within ±15.2% GC (95% confidence interval) for the strains of all species in the study. CONCLUSION: Not only did our mathematical model give reasonable estimates of sbGC it also provides further support to previous observations that mutation rates in prokaryotes exhibit a universal GC → AT bias that appears to be remarkably consistent between taxa.

Re-visiting the evolution, dispersal and epidemiology of Zika virus in Asia.

Based on serological evidence and viral isolation, Zika virus (ZIKV) has circulated for many years relatively benignly in a sylvatic cycle in Africa and an urban cycle in South East Asia (SEA). With the recent availability of limited but novel Indian ZIKV sequences to add to the plethora of SEA sequences, we traced the phylogenetic history and spatio-temporal dispersal pattern of ZIKV in Asia prior to its explosive emergence in the Pacific region and the Americas. These analyses demonstrated that the introduction and dispersal of ZIKV on the Pacific islands were preceded by an extended period of relatively silent transmission in SEA, enabling the virus to expand geographically and evolve adaptively before its unanticipated introduction to immunologically naive populations on the Pacific islands and in the Americas. Our findings reveal new features of the evolution and dispersal of this intriguing virus and may benefit future disease control strategies.