Spatial and seasonal determinants of Lyme borreliosis incidence in France, 2016 to 2021.

BackgroundLyme borreliosis (LB) is the most widespread hard tick-borne zoonosis in the northern hemisphere. Existing studies in Europe have focused mainly on acarological risk assessment, with few investigations exploring human LB occurrence.AimWe explored the determinants of spatial and seasonal LB variations in France from 2016 to 2021 by integrating environmental, animal, meteorological and anthropogenic factors, and then mapped seasonal LB risk predictions.MethodsWe fitted 2016-19 LB national surveillance data to a two-part spatio-temporal statistical model. Spatial and temporal random effects were specified using a Besag-York-Mollie model and a seasonal model, respectively. Coefficients were estimated in a Bayesian framework using integrated nested Laplace approximation. Data from 2020-21 were used for model validation.ResultsA high vegetation index (≥ 0.6) was positively associated with seasonal LB presence, while the index of deer presence (> 60%), mild soil temperature (15-22 °C), moderate air saturation deficit (1.5-5 mmHg) and higher tick bite frequency were associated with increased incidence. Prediction maps show a higher risk of LB in spring and summer (April-September), with higher incidence in parts of eastern, midwestern and south-western France.ConclusionWe present a national level spatial assessment of seasonal LB occurrence in Europe, disentangling factors associated with the presence and increased incidence of LB. Our findings yield quantitative evidence for national public health agencies to plan targeted prevention campaigns to reduce LB burden, enhance surveillance and identify further data needs. This approach can be tested in other LB endemic areas.

Seroprevalence for Borrelia burgdorferi sensu lato and tick-borne encephalitis virus antibodies and associated risk factors among forestry workers in northern France, 2019 to 2020.

BackgroundLyme borreliosis (LB) is the most common tick-borne disease (TBD) in France. Forestry workers are at high risk of TBD because of frequent exposure to tick bites.AimWe aimed to estimate the seroprevalence of Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV) antibodies among forestry workers in northern France. We compared seroprevalence by geographical area and assessed factors associated with seropositivity.MethodsBetween 2019 and 2020, we conducted a randomised cross-sectional seroprevalence survey. Borrelia burgdorferi sl seropositivity was defined as positive ELISA and positive or equivocal result in western blot. Seropositivity for TBEV was defined as positive result from two ELISA tests, confirmed by serum neutralisation. We calculated weighted seroprevalence and adjusted prevalence ratios to determine association between potential risk factors and seropositivity.ResultsA total of 1,778 forestry workers participated. Seroprevalence for B. burgdorferi sl was 15.5% (95% confidence interval (CI): 13.9-17.3), 3.5 times higher in the eastern regions than in the western and increased with seniority and with weekly time in a forest environment. Seroprevalence was 2.5 times higher in forestry workers reporting a tick bite during past years and reporting usually not removing ticks rapidly. Seroprevalence for TBEV was 0.14% (95% CI: 0.05-0.42).ConclusionWe assessed for the first time seroprevalence of B. burgdorferi sl and TBEV antibodies among forestry workers in northern France. These results will be used, together with data on LB and tick-borne encephalitis (TBE) incidence and on exposure to tick-bites, to target prevention programmes.

Novel Chronic Anaplasmosis in Splenectomized Patient, Amazon Rainforest.

We report a case of unusual human anaplasmosis in the Amazon rainforest of French Guiana. Molecular typing demonstrated that the pathogen is a novel Anaplasma species, distinct to all known species, and more genetically related to recently described Anaplasma spp. causing infections in rainforest wild fauna of Brazil.

Value of PCR, Serology, and Blood Smears for Human Granulocytic Anaplasmosis Diagnosis, France.

We prospectively examined the effectiveness of diagnostic tests for anaplasmosis using patients with suspected diagnoses in France. PCR (sensitivity 0.74, specificity 1) was the best-suited test. Serology had a lower specificity but higher sensitivity when testing acute and convalescent samples. PCR and serology should be used in combination for anaplasmosis diagnosis.

[Epidemiology of Lyme borreliosis in France - both certainties and uncertainties]. / Épidémiologie de la borréliose de Lyme en France : entre incertitudes et certitudes.

Epidemiological evidence for tick-borne infections, particularly those related to Lyme borreliosis, is heterogeneous. Lyme borreliosis is a tick-born zoonosis transmitted by ticks of the genus Ixodes ricinus. After tick bite, the risk of transmission of an infectious agent remains low, most often represented by Borrelia burgdorferi sensu lato; co-infections in Humans by several different infectious agents (bacterial, viral or parasitic) are possible but a priori rare. In addition, besides well-known tick-borne pathogens, new species or gender of micro-organisms are regularly described in ticks but their pathogenicity in human pathology is not described or not yet established. The clinical presentation of Lyme borreliosis is varied, with localized and disseminated forms occurring long ago after tick bite, making diagnosis sometimes difficult. The natural course of Lyme borreliosis is insufficiently known because of recommendations of antibiotherapy in case of illness; however, some historical studies seem reassuring with possible spontaneous healing and seemingly minor sequelae. The diagnosis of disseminated forms requires paraclinical examinations, in first place serology, whose sensitivity increases with time of evolution of borreliosis; this is all the more interesting as the disseminated forms are of more difficult clinical diagnosis. After antibiotherapy, the clinical course is good, the sequelae remain possible especially in case of late diagnosis or late disseminated form; however, their frequency remains unknown.

Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.

Discovery and targeted proteomics on cutaneous biopsies infected by borrelia to investigate lyme disease.

Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge with insufficient means of reliable diagnosis. Skin is rarely investigated in proteomics but constitutes in the case of Lyme disease the key interface where the pathogens can enter, persist, and multiply. Therefore, we investigated proteomics on skin samples to detect Borrelia proteins directly in cutaneous biopsies in a robust and specific way. We first set up a discovery gel prefractionation-LC-MS/MS approach on a murine model infected by Borrelia burgdorferi sensu stricto that allowed the identification of 25 Borrelia proteins among more than 1300 mouse proteins. Then we developed a targeted gel prefractionation-LC-selected reaction monitoring (SRM) assay to detect 9/33 Borrelia proteins/peptides in mouse skin tissue samples using heavy labeled synthetic peptides. We successfully transferred this assay from the mouse model to human skin biopsies (naturally infected by Borrelia), and we were able to detect two Borrelia proteins: OspC and flagellin. Considering the extreme variability of OspC, we developed an extended SRM assay to target a large set of variants. This assay afforded the detection of nine peptides belonging to either OspC or flagellin in human skin biopsies. We further shortened the sample preparation and showed that Borrelia is detectable in mouse and human skin biopsies by directly using a liquid digestion followed by LC-SRM analysis without any prefractionation. This study thus shows that a targeted SRM approach is a promising tool for the early direct diagnosis of Lyme disease with high sensitivity (<10 fmol of OspC/mg of human skin biopsy).

The BioFilm Ring Test: a Rapid Method for Routine Analysis of Pseudomonas aeruginosa Biofilm Formation Kinetics.

Currently, few techniques are available for the evaluation of bacterial biofilm adhesion. These detection tools generally require time for culture and/or arduous handling steps. In this work, the BioFilm Ring Test (BRT), a new technology, was used to estimate the biofilm formation kinetics of 25 strains of Pseudomonas aeruginosa, isolated from the sputum of cystic fibrosis (CF) patients. The principle of the new assay is based on the mobility measurement of magnetic microbeads mixed with a bacterial suspension in a polystyrene microplate. If free to move under the magnetic action, particles gather to a visible central spot in the well bottom. Therefore, the absence of spot formation in the plate reflects the bead immobilization by a biofilm in formation. The BRT device allowed us to classify the bacterial strains into three general adhesion profiles. Group 1 consists of bacteria, which are able to form a solid biofilm in <2 h. Group 2 comprises the strains that progressively set up a biofilm during 24 h. Lastly, group 3 includes the strains that stay in a planktonic form. The grouping of our strains did not differ according to culture conditions, i.e., the use of different sets of beads or culture media. The BRT is shown to be an informative tool for the characterization of biofilm-forming bacteria. Various application perspectives may be investigated for this device, such as the addition of antibiotics to the bacterial suspension to select which would have the ability to inhibit the biofilm formation.

Ixodes tick saliva suppresses the keratinocyte cytokine response to TLR2/TLR3 ligands during early exposure to Lyme borreliosis.

Ixodes hard tick induces skin injury by its sophisticated biting process. Its saliva plays a key role to enable an efficient blood meal that lasts for several days. We hypothesized that this feeding process may also be exploited by pathogens to facilitate their transmission, especially in the context of arthropod-borne diseases. To test this, we used Lyme borreliosis as a model. This bacterial infection is caused by Borrelia burgdorferi sensu lato transmitted by Ixodes. We co-incubated Borrelia with human keratinocytes in the presence of poly (I: C), a dsRNA TLR3 agonist generated by skin injury. This induced a strong cytokine response from human primary keratinocytes that was much greater than that induced by Borrelia alone. OspC, a TLR2/1 agonist and a major surface lipoprotein of Borrelia also amplified the process. Interestingly, tick saliva inhibited cytokine responses by keratinocytes to these TLR agonists. We propose that Borrelia uses the immunoprivileged site produced by tick saliva to facilitate its transmission.

Expanding the clinicopathological spectrum of late cutaneous Lyme borreliosis (acrodermatitis chronica atrophicans [ACA]): A prospective study of 20 culture- and/or polymerase chain reaction (PCR)-documented cases.

BACKGROUND: The diagnosis of acrodermatitis chronica atrophicans (ACA), the late cutaneous manifestation of Lyme borreliosis, can be challenging. Histologic changes in ACA have been described in a few studies from endemic countries, relying on cases documented by serology only. OBJECTIVES: We sought to reassess the clinicopathological spectrum of ACA in a series of thoroughly documented cases. METHODS: Patients prospectively included in a national prospective study were selected on the basis of positive culture and/or polymerase chain reaction of a skin biopsy sample. The diagnosis of ACA was confirmed by reviewing the clinical and serologic data. Histopathological samples were carefully reviewed. RESULTS: Twenty patients were included. Unusual clinical features (ie, numerous small violaceous patches and equidistant small spinous papules with background faint erythema) were observed in 2 patients. Histopathological examination revealed a classic plasma cell-rich perivascular and interstitial pattern with telangiectases in 16 of 25 samples, whereas strikingly prominent granuloma annulare-like or lichenoid features were observed in 4 and 2 of 25 cases, respectively, and discrete nonspecific minor changes in 3 of 25 cases. LIMITATIONS: The small number of patients was a limitation. CONCLUSIONS: Genuine culture- and/or polymerase chain reaction-proven ACA can rarely present as numerous violaceous patches or cluster of spinous papules clinically, and as a granuloma annulare-like or lichenoid dermatosis histologically.

Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: relevance for Lyme vaccine design.

Lyme borreliosis is the most important vector-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC-MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild-type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT-PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 (http://proteomecentral.proteomexchange.org/dataset/PXD000876).

Implementation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis.

The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Staphylococcus aureus and 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 as S. aureus and 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized Staphylococcus species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis strains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified species S. pettenkoferi and putative virulent species, including S. lugdunensis.

Evolving epidemiology and antimicrobial resistance in spontaneous bacterial peritonitis: a two-year observational study.

BACKGROUND: Current recommendations for empirical antimicrobial therapy in spontaneous bacterial peritonitis (SBP) are based on quite old trials. Since microbial epidemiology and the management of patients have changed, whether these recommendations are still appropriate must be confirmed. METHODS: An observational study that exhaustively collected the clinical and biological data associated with positive ascitic fluid cultures was conducted in four French university hospitals in 2010-2011. RESULTS: Two hundred and sixty-eight documented positive cultures were observed in 190 cirrhotic patients (median age 61.5 years, 58.5% Child score C). Of these, 57 were classified as confirmed SBP and 140 as confirmed bacterascites. The predominant flora was Gram-positive cocci, whatever the situation (SBP, bacterascites, nosocomial/health-care related or not). Enteroccocci (27.7% E. faecium) were isolated in 24% of the episodes, and in 48% from patients receiving quinolone prophylaxis. E. coli were susceptible to amoxicillin-clavulanate and to third-generation cephalosporins in 62.5% and 89.5% of cases, respectively. No single antibiotic allowed antimicrobial coverage of more than 60%. Only combinations such as amoxicillin + third-generation cephalosporin or cotrimoxazole allowed coverage close to 75-80% in non-nosocomial episodes. Combinations based on broader spectrum antibiotics should be considered for empirical therapy of nosocomial infections. CONCLUSIONS: Our study confirmed the changing spectrum of pathogens in SBP and bacterascites, and the need for more complex antibiotic strategies than those previously recommended. Our findings also underline the need for new clinical trials conducted in the current epidemiological context.

Prevalence and factors associated with a prescription of a Lyme borreliosis serology for erythema migrans diagnosis in general practice: a study from the French sentinel network, 2009-2020.

BACKGROUND: Serological testing of patients consulting for typical erythema migrans (EM) is not recommended in European recommendations for diagnosis of Lyme borreliosis (LB). Little is known on the level of adherence of French general practitioners to these recommendations. The objectives were to estimate the proportion of Lyme borreliosis serological test prescription in patients with erythema migrans seen in general practice consultations in France, and to study the factors associated with this prescription. METHODS: LB cases with an EM reported by the French general practitioners (GPs) of the Sentinelles network between January 2009 and December 2020 were included. To assess the associations with a prescription of a serological test, multilevel logistic regression models were used. RESULTS: Among the 1,831 EM cases included, a prescription for a LB serological test was requested in 24.0% of cases. This proportion decreased significantly over the study period, from 46.8% in 2009 to 15.8% in 2020. A LB serological prescription was associated with patients with no reported tick bite (Odds Ratio (OR): 1.95; 95% confidence interval [1.23-3.09]), multiple EM (OR: 3.82 [1.63-8.92]), EM of five centimeters or more (OR: 4.34 [2.33-8.08]), and GPs having diagnosed less than one EM case per year during the study period (OR: 5.28 [1.73-16.11]). CONCLUSIONS: Serological testing of patients consulting for EM is not recommended in European recommendations for diagnosis of Lyme borreliosis. Therefore, the significant decrease in the rate of LB serological test for EM over the study period is encouraging. The factors identified in this study can be used to improve messaging to GPs and patients. Further efforts are needed to continue to disseminate diagnostic recommendations for LB to GPs, especially those who rarely see patients with EM.

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.