Human Co-Infections between Borrelia burgdorferi s.l. and Other Ixodes-Borne Microorganisms: A Systematic Review.

When it comes to tick-borne diseases, co-infections are often mentioned. This concept includes several entities. On the one hand, tick vectors or vertebrate reservoir host can harbor several microorganisms that can be pathogenic for humans. On the other hand, human co-infections can also be understood in different ways, ranging from seropositivity without clinical symptoms to co-disease, i.e., the simultaneous clinical expression of infections by two tick-borne microorganisms. The latter, although regularly speculated, is not often reported. Hence, we conducted a systematic review on co-infections between B. burgdorferi s.l., the etiological agent of Lyme borreliosis, and other microorganisms potentially transmitted to humans by Ixodes spp. ticks. A total of 68 relevant articles were included, presenting 655 cases of possible co-infections. Most cases of co-infections corresponded to patients with one tick-borne disease and presenting antibody against another tick-borne microorganism. Co-disease was particularly frequent in two situations: patients with clinical symptoms of high fever and erythema migrans (EM), and patients with neurological symptoms linked to the TBEv or a neuroborreliosis. No impact on severity was evidenced. Further studies are needed to better appreciate the frequency and the impact of co-infections between several tick-borne microorganisms.

Impact of Different Anthropogenic Environments on Ticks and Tick-Associated Pathogens in Alsace, a French Region Highly Endemic for Tick-Borne Diseases.

Ticks and tick-borne diseases have spread over the last decades. In parallel, the incidence in humans, accidental hosts for most of these zoonotic diseases, has increased. This epidemiological intensification can be associated with anthropogenic alterations of forest ecosystems and animal biodiversity, but also with socioeconomic changes. Their proliferation is largely due to human-induced effects on the factors that favor the circulation of these infectious agents. We selected different types of anthropogenic environments in Alsace, a region endemic for tick-borne diseases in France, to better understand the impact of human interventions on tick populations and tick-borne disease incidence. Ticks were collected in one golf course, three urban parks, one mid-mountain forest, and one alluvial forest that is currently part of a protected natural area. Ixodes ricinus was found primarily in humid vegetation, which is favorable for tick survival, such as grounds populated with trees and covered with leaf litter. We also observed that reforestation and high animal biodiversity in a protected area such as the alluvial forest led to a greater number of ticks, including both Ixodes ricinus and Dermacentor reticulatus, as well as to a higher prevalence of pathogens such as Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Borrelia miyamotoi, and Rickettsia raoulti.

Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing.

Antimicrobial susceptibility testing of anaerobes is challenging. Because MIC determination is recommended by both CLSI and EUCAST, commercial broth microdilution and diffusion strip tests have been developed. The reliability of broth microdilution methods has not been assessed yet using the agar dilution reference method. In this work, we evaluated two broth microdilution kits (MICRONAUT-S Anaerobes® MIC and Sensititre Anaerobe MIC®) and one gradient diffusion strip method (Liofilchem®) for antimicrobial susceptibility testing of 47 Clostridiales isolates (Clostridium, Clostridioides and Hungatella species) using the agar dilution method as a reference. The evaluation focused on comparing six antimicrobial molecules available in both microdilution kits. Analytical performances were evaluated according to the Food and Drug Administration (FDA) recommendations. Essential agreements (EA) and categorical agreements (CA) varied greatly according to the molecule and the evaluated method. Vancomycin had values of essential and categorical agreements above 90% for the three methods. The CA fulfilled the FDA criteria for three major molecules in the treatment of Gram-positive anaerobic infections (metronidazole, piperacillin/tazobactam and vancomycin). The highest rate of error was observed for clindamycin. Multicenter studies are needed to further validate these results.

The emerging tick-borne pathogen Neoehrlichia mikurensis: first French case series and vector epidemiology.

Neoehrlichia mikurensis is an intracellular bacterium transmitted in Europe and Asia by ticks of the Ixodes ricinus complex. Interest in this bacterium has increased since it was demonstrated to be responsible for febrile syndromes in patients. To date, most clinical cases have been reported in northern Europe, but case series have also been described in central Europe and China. Notably, thrombotic events occurred during the course of the disease. We investigated the presence of N. mikurensis in 10,885 I. ricinus nymphs in two regions of France (Alsace and Brittany) collected between 2013 and 2020 and in 934 patients suspected of human granulocytic anaplasmosis in Alsace, an endemic area for Lyme borreliosis, using a specific PCR assay. N. mikurensis was detected in 5.42% of the ticks from Alsace, whereas only one (0.03%) tick was found to be positive in Brittany. Spatiotemporal disparities were also noticed within the Alsace region over the four collection sites investigated, and a significant increase in the prevalence of nymphs carrying N. mikurensis was also observed in the last three years of collection. Four out of 934 screened patients were found to be positive for N. mikurensis. Two had malignancies, and the other two were apparently immunocompetent. Superficial thrombosis was noticed in one patient, and long-lasting bacteremia was noted in another patient. These four patients are the first clinical cases of neoehrlichiosis described in France. We suggest including N. mikurensis in the differential diagnosis of post-tick bite febrile syndromes to treat patients and prevent the occurrence of thrombotic complications.

First Case of Bacteraemia Due to Carbapenem-Resistant Bacteroides faecis.

Multidrug resistant (MDR) bacteria are increasingly observed in nosocomial and community-acquired settings. Anaerobes are no exception to this rule, but there are fewer reports of MDR in the scientific literature on anaerobes than there are for other bacteria. In this short case report, we describe the first case of bacteraemia caused by a multidrug-resistant Bacteroides faecis, which produces a carbapenemase encoded by the blaCfiA gene. This bacteraemia followed a digestive surgery operation. Surprisingly, these findings did not lead to a change in antibiotic therapy, probably because the patient's clinical state had improved. Nevertheless this report calls for better knowledge of anaerobic bacteria and for a systematic antimicrobial stewardship procedure following bacteraemia.

The Population Structure of Borrelia lusitaniae Is Reflected by a Population Division of Its Ixodes Vector.

Populations of vector-borne pathogens are shaped by the distribution and movement of vector and reservoir hosts. To study what impact host and vector association have on tick-borne pathogens, we investigated the population structure of Borrelia lusitaniae using multilocus sequence typing (MLST). Novel sequences were acquired from questing ticks collected in multiple North African and European locations and were supplemented by publicly available sequences at the Borrelia Pubmlst database (accessed on 11 February 2020). Population structure of B. lusitaniae was inferred using clustering and network analyses. Maximum likelihood phylogenies for two molecular tick markers (the mitochondrial 16S rRNA locus and a nuclear locus, Tick-receptor of outer surface protein A, trospA) were used to confirm the morphological species identification of collected ticks. Our results confirmed that B. lusitaniae does indeed form two distinguishable populations: one containing mostly European samples and the other mostly Portuguese and North African samples. Of interest, Portuguese samples clustered largely based on being from north (European) or south (North African) of the river Targus. As two different Ixodes species (i.e., I. ricinus and I. inopinatus) may vector Borrelia in these regions, reference samples were included for I. inopinatus but did not form monophyletic clades in either tree, suggesting some misidentification. Even so, the trospA phylogeny showed a monophyletic clade containing tick samples from Northern Africa and Portugal south of the river Tagus suggesting a population division in Ixodes on this locus. The pattern mirrored the clustering of B. lusitaniae samples, suggesting a potential co-evolution between tick and Borrelia populations that deserve further investigation.

Assessment of Borrelia miyamotoi in febrile patients and ticks in Alsace, an endemic area for Lyme borreliosis in France.

BACKGROUND: Borrelia miyamotoi is a relapsing fever Borrelia species transmitted by ticks of the Ixodes ricinus complex. Human disease caused by B. miyamotoi was first described in Russia and later in the USA and Japan. Additionally, five cases of meningoencephalitis in immunocompromised patients and one case in an apparently immunocompetent patient were described. METHODS: We investigated the presence of B. miyamotoi in I. ricinus nymphs and in patients suspected of human granulocytic anaplasmosis, in Alsace (France), an endemic area for I. ricinus ticks and Lyme borreliosis, using direct (PCR) and indirect diagnosis (glycerophosphoryldiester-phosphodiesterase (GlpQ) serology). RESULTS: Borrelia miyamotoi was found in 2.2% of 4354 ticks collected between 2013 and 2016. None of the 575 blood samples, collected from the patients suspected of HGA, was found positive for B. miyamotoi by PCR. Acute and late sera from 138 of these 575 patients were available. These paired sera were tested for IgM and IgG antibodies against the B. miyamotoi GlpQ antigen. A total of 14 out of 138 patients had at least one positive parameter (i.e. anti-GlpQ IgG and/or IgM). One patient seroconverted for IgG, and three had isolated IgM in the acute serum. These three patients were treated with doxycycline which could have prevented seroconversion. After reviewing clinical data and other biological tests performed, co-exposure among different microorganisms vectored by ticks or serological cross-reactivity could not be ruled out in these different cases. One patient had persistent IgG, which strongly suggests previous exposure to B. miyamotoi. CONCLUSIONS: Humans can be exposed to B. miyamotoi through tick bites in Alsace. We present serological data for possible B. miyamotoi exposure or infection of patients with fever after tick bite. Future studies should determine the incidence, clinical course and burden of this emerging tick-borne disease in other parts of Western Europe.

Identification of closely related Ixodes species by protein profiling with MALDI-TOF mass spectrometry.

Ticks are vectors of infectious diseases of major importance in human and veterinary medicine. For epidemiological studies, accurate identification of ticks is crucial to define their potential role as vectors and to develop control and prevention strategies. Although morphological and molecular methods are widely used to identify ticks, an innovative approach using MALDI-TOF MS technology recently emerged as an alternative tool. Previous works showed that MALDI-TOF MS was highly effective in identifying ticks, but these works mainly tested tick specimens of different genera. To confirm the accuracy of this new tool for tick identification, nine closely related tick species belonging to the Ixodes genus were analysed, specimens of the Dermacentor reticulatus species were also included in the analysis as an outer group. Three of the species used for the present study belonged to the I. ricinus species complex, which are known to transmit Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. A total of 246 tick specimens were submitted to MALDI-TOF MS analysis, and two body parts (half-idiosoma and four legs) were individually investigated. For each body part, intraspecies reproducibility and interspecies specificity of the MS profiles were determined. The profile analysis revealed that the main determinant for spectra clustering was the tick species for both legs and half-idiosoma. For each body part, a reference database of spectra was set up including 2 to 5 specimens per species randomly selected, and genotyped using 16s rDNA and COI genes to confirm their morphological identification. Both created spectral databases were individually blind tested with their respective body part using the remaining specimens, which were correctly identified in 98.5% of the cases. MALDI-TOF MS is a reliable tool for tick identification, including specimens belonging to closely related species and hardly distinguishable using morphology. The 4-legs as well as the half-idiosoma of ticks can now be applied for specimen identification using two different databases. The combined use of these two body parts improves the rate of tick identification and their confidence level.

Multilocus sequence typing of clinical Borreliella afzelii strains: population structure and differential ability to disseminate in humans.

BACKGROUND: Lyme borreliosis in humans results in a range of clinical manifestations, thought to be partly due to differences in the pathogenicity of the infecting strain. This study compared European human clinical strains of Borreliella afzelii (previously named Borrelia afzelii) using multilocus sequence typing (MLST) to determine their spatial distribution across Europe and to establish whether there are associations between B. afzelii genotypes and specific clinical manifestations of Lyme borreliosis. For this purpose, typing was performed on 63 strains, and data on a further 245 strains were accessed from the literature. RESULTS: All 308 strains were categorized into 149 sequence types (STs), 27 of which are described here for the first time. Phylogenetic and goeBURST analyses showed short evolutionary distances between strains. Although the main STs differed among the countries with the largest number of strains of interest (Germany, the Netherlands, France and Slovenia), the B. afzelii clinical strains were less genetically structured than those previously observed in the European tick population. Two STs were found significantly more frequently in strains associated with clinical manifestations involving erythema migrans, whereas another ST was found significantly more frequently in strains associated with disseminated manifestations, especially neuroborreliosis. CONCLUSIONS: The MLST profiles showed low genetic differentiation between B. afzelii strains isolated from patients with Lyme borreliosis in Europe. Also, clinical data analysis suggests the existence of lineages with differential dissemination properties in humans.

Relapsing Fevers: Neglected Tick-Borne Diseases.

Relapsing fever still remains a neglected disease and little is known on its reservoir, tick vector and physiopathology in the vertebrate host. The disease occurs in temperate as well as tropical countries. Relapsing fever borreliae are spirochaetes, members of the Borreliaceae family which also contain Lyme disease spirochaetes. They are mainly transmitted by Ornithodoros soft ticks, but some species are vectored by ixodid ticks. Traditionally a Borrelia species is associated with a specific vector in a particular geographical area. However, new species are regularly described, and taxonomical uncertainties deserve further investigations to better understand Borrelia vector/host adaptation. The medical importance of Borrelia miyamotoi, transmitted by Ixodes spp., has recently spawned new interest in this bacterial group. In this review, recent data on tick-host-pathogen interactions for tick-borne relapsing fevers is presented, with special focus on B. miyamotoi.

Assessment of MALDI-TOF MS biotyping for Borrelia burgdorferi sl detection in Ixodes ricinus.

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been demonstrated to be useful for tick identification at the species level. More recently, this tool has been successfully applied for the detection of bacterial pathogens directly in tick vectors. The present work has assessed the detection of Borrelia burgdorferi sensu lato in Ixodes ricinus tick vector by MALDI-TOF MS. To this aim, experimental infection model of I. ricinus ticks by B. afzelii was carried out and specimens collected in the field were also included in the study. Borrelia infectious status of I. ricinus ticks was molecularly controlled using half-idiosome to classify specimens. Among the 39 ticks engorged on infected mice, 14 were confirmed to be infected by B. afzelii. For field collection, 14.8% (n = 12/81) I. ricinus ticks were validated molecularly as infected by B. burgdorferi sl. To determine the body part allowing the detection of MS protein profile changes between non-infected and B. afzelii infected specimens, ticks were dissected in three compartments (i.e. 4 legs, capitulum and half-idiosome) prior to MS analysis. Highly reproducible MS spectra were obtained for I. ricinus ticks according to the compartment tested and their infectious status. However, no MS profile change was found when paired body part comparison between non-infected and B. afzelii infected specimens was made. Statistical analyses did not succeed to discover, per body part, specific MS peaks distinguishing Borrelia-infected from non-infected ticks whatever their origins, laboratory reared or field collected. Despite the unsuccessful of MALDI-TOF MS to classify tick specimens according to their B. afzelii infectious status, this proteomic tool remains a promising method for rapid, economic and accurate identification of tick species. Moreover, the singularity of MS spectra between legs and half-idiosome of I. ricinus could be used to reinforce this proteomic identification by submission of both these compartments to MS.

No evidence of Borrelia mayonii in an endemic area for Lyme borreliosis in France.

BACKGROUND: Borrelia mayonii is currently the latest species belonging to the Borrelia burgdorferi (sensu lato) complex to be discovered. Interestingly it is involved in human pathology causing a high fever. We looked for its presence in post- tick bite febrile patients as well as in Ixodes ricinus ticks in an endemic area of France. RESULTS: After ensuring that our molecular technics correctly detected B. mayonii, 575 patients and 3,122 Ixodes ricinus nymphs were tested. Neither B. mayonii nor another species of the B. burgdorferi (s.l.) complex previously not reported in Europe has been identified. CONCLUSIONS: For now, B. mayonii seems to be an epiphenomenon. However, its discovery broadens the etiology of post-Ixodes bite febrile syndromes.