Ten years of detecting Neoehrlichia mikurensis infections in Sweden: demographic, clinical and inflammatory parameters.

PURPOSE: To increase knowledge about the varied clinical manifestations of human infection with the emerging tick-borne pathogen Neoehrlichia mikurensis. METHODS: All patients diagnosed in Sweden with N. mikurensis infection during a 10-year period (2013-2023) were investigated regarding their demographic factors, risk factors, comorbidities, clinical signs and symptoms, and laboratory results. Multivariate models were generated using "Orthogonal Projections to Latent Structures-Discriminant Analysis" to identify clinical and immune parameters associated with N. mikurensis infection. RESULTS: During the 10-year period, 134 patients were diagnosed with N. mikurensis infection, 102 of whom were included in this study. Most of the patients (79%) were immunosuppressed. The main comorbidities were malignant B-cell lymphomas, multiple sclerosis, and rheumatoid arthritis. Rituximab therapy (59%) and splenectomy (14%) featured prominently. All patients resided in the southern tick-endemic part of Sweden, yet one-third of them were diagnosed in wintertime when ticks are inactive. Two asymptomatically infected blood donors were identified but transfusion-transmitted infection was not confirmed. Increased levels of C-reactive protein, orosomucoid, and total IgM in serum were associated with neoehrlichiosis. Previously unreported symptoms such as ankle edema, neck pain, numbness, and sudden deafness were detected in some patients. One case of aplastic anemia partially improved after eradication of the infection. CONCLUSIONS: Neoehrlichiosis is a multi-faceted emerging infectious disease.

Infection with Neoehrlichia mikurensis promotes the development of malignant B-cell lymphomas.

The tick-borne pathogen Neoehrlichia (N.) mikurensis is implicated in persistent infection of the vascular endothelium. B cells are crucial for the host defence to this infection. Chronic stimulation of B cells may result in B-cell transformation and lymphoma. Five patients with malignant B-cell lymphoma and concomitant N. mikurensis infection were investigated regarding clinical picture, lymphoma subtype, B-cell lymphoma immunophenotype and IGHV (variable region of the immunoglobulin heavy) gene repertoire. Three of the five patients improved markedly and ceased lymphoma treatment after doxycycline treatment to eliminate N. mikurensis. Sequencing the B-cell lymphoma IGHV genes revealed preferred usage of the IGHV1 (IGHV1-2, and -69) and IGHV3 (IGHV3-15, -21, -23) families. In conclusion, N. mikurensis infection may drive the development of malignant B-cell lymphomas. Eradication of the pathogen appears to induce remission with apparent curing of the lymphoma in some cases.

Spiroplasma ixodetis Infections in Immunocompetent and Immunosuppressed Patients after Tick Exposure, Sweden.

We report 2 cases of Spiroplasma ixodetis infection in an immunocompetent patient and an immunocompromised patient who had frequent tick exposure. Fever, thrombocytopenia, and increased liver aminotransferase levels raised the suspicion of anaplasmosis, but 16S rRNA PCR and Sanger sequencing yielded a diagnosis of spiroplasmosis. Both patients recovered after doxycycline treatment.

Vasculitis due to Candidatus Neoehrlichia mikurensis: A Cohort Study of 40 Swedish Patients.

BACKGROUND: Candidatus (Ca.) Neoehrlichia (N.) mikurensis is an emerging tick-borne pathogen of humans that is closely related to Ehrlichia and Anaplasma species. This strict intracellular bacterium escapes detection by routine microbiologic diagnostic methods such as blood culture, leading to considerable under-diagnosis of the infectious disease it causes, neoehrlichiosis. METHODS: Here, we describe the vascular and thromboembolic events afflicting a series of 40 patients diagnosed with neoehrlichiosis in Sweden during a 10-year period (2009-2019). RESULTS: The majority of the patients (60%) developed vascular events ranging from repeated thrombophlebitis, deep vein thrombosis, pulmonary embolism, transitory ischemic attacks, to arteritis. Younger age was a risk factor for vascular complications. In contrast, there was no difference in the incidence of vascular events between immunosuppressed and immunocompetent patients. However, there were qualitative differences, such that deep vein thrombosis exclusively afflicted the immunosuppressed patients, whereas arteritis was restricted to the immunocompetent persons. We also present the case histories of two patients who developed vasculitis mimicking polyarteritis nodosa and giant cell arteritis. Both were cured by doxycycline treatment. CONCLUSIONS: Ca. N. mikurensis infection should be considered in patients living in tick-endemic areas of Europe and northern Asia who present with atypical vascular and/or thromboembolic events. Early diagnosis and antibiotics targeting this emerging infectious agent can eradicate the infection and prevent the development of new vascular events.

Serum-based diagnosis of Pneumocystis pneumonia by detection of Pneumocystis jirovecii DNA and 1,3-ß-D-glucan in HIV-infected patients: a retrospective case control study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) is one of the most common HIV-related opportunistic infections. The diagnosis of PCP is based on analyses from respiratory tract specimens which may require the invasive procedure of a diagnostic bronchoscopy. The objective of this study was to evaluate the diagnostic potential of Pneumocystis jirovecii PCR in serum combined with the 1,3-ß-D-glucan (betaglucan) test for the diagnosis of PCP in HIV-infected patients. METHODS: This was a retrospective case-control study including serum samples from 26 HIV-infected patients with PCP collected within 5 days prior to the start of PCP treatment, 21 HIV-infected control subjects matched by blood CD4+ cell counts, and 18 blood donors. The serum samples were analyzed for Pneumocystis jirovecii PCR and betaglucan. The reference standard for PCP was based on previously described microbiological and clinical criteria. RESULTS: All patients with PCP had detectabe Pneumocystis jirovecii DNA in serum yielding a sensitivity for the Pneumocystis jirovecii PCR assay in serum of 100%. All blood donors had negative Pneumocystis PCR in serum. The specificity when testing HIV-infected patients was 71%, but with a PCR Cycle threshold (Ct) value of 34 as cut-off the specificity was 90%. At a putative pretest probaility of 20%, the negative and positive predictive value for the Pneumocystis PCR assay in serum was 0.99 and 0.71, respectively. Betaglucan with cut-off level 200 pg/ml combined with a positive Pneumocystis jirovecii PCR result had sensitivity and specificity of 92 and 90%, respectively. The concentration of Pneumocystis jirovecii DNA in serum samples, expressed by the PCR Ct values, correlated inversely to the betaglucan levels in serum. CONCLUSION: In this case-control study including 70% of all HIV-infected patients with PCP treated at Sahlgrenska University Hospital during a time period of 13 years, Pneumocystis PCR analysis on serum samples had a very high sensitivity and negative predictive value for the diagnosis of PCP in HIV-infected patients. A serum-based diagnostic procedure either based on Pneumocystis jirovecii PCR alone or in combination with betaglucan analysis may thus be feasible and would facilitate the care of HIV-infected patients with suspected PCP.

Serological reactivity to Anaplasma phagocytophilum in neoehrlichiosis patients.

The tick-borne bacterium Candidatus (Ca.) Neoehrlichia (N.) mikurensis is a cause of "fever of unknown origin" because this strict intracellular pathogen escapes detection by routine blood cultures. Case reports suggest that neoehrlichiosis patients may display serological reactivity to Anaplasma (A.) phagocytophilum. Since Anaplasma serology is part of the diagnostic work-up of undetermined fever in European tick-exposed patients, we wanted to investigate (1) the prevalence of A. phagocytophilum seropositivity among neoehrlichiosis patients, (2) the frequency of misdiagnosed neoehrlichiosis patients among A. phagocytophilum seropositive patients, and (3) the frequency of A. phagocytophilum and Ca. N. mikurensis co-infections. Neoehrlichiosis patients (n = 18) were analyzed for A. phagocytophilum IgM and IgG serum antibodies by indirect immunofluorescence assay. Serum samples from suspected anaplasmosis patients (n = 101) were analyzed for bacterial DNA contents by singleplex PCR specific for A. phagocytophilum and Ca. N. mikurensis, respectively. One fifth of the neoehrlichiosis patients (4/18) were seropositive for IgM and/or IgG to A. phagocytophilum at the time of diagnosis. Among the patients with suspected anaplasmosis, 2% (2/101) were positive for Ca. N. mikurensis by PCR whereas none (0/101) had detectable A. phagocytophilum DNA in the serum. To conclude, patients with suspected anaplasmosis may in fact have neoehrlichiosis. We found no evidence of A. phagocytophilum and Ca. N. mikurensis co-infections in humans with suspected anaplasmosis or confirmed neoehrlichiosis.

Infections with Candidatus Neoehrlichia mikurensis and Cytokine Responses in 2 Persons Bitten by Ticks, Sweden.

The prevalence of Candidatus Neoehrlichia mikurensis infection was determined in 102 persons bitten by ticks in Sweden. Two infected women had erythematous rashes; 1 was co-infected with a Borrelia sp., and the other showed seroconversion for Anaplasma phagocytophilum. Both patients had increased levels of Neoehrlichia DNA and serum cytokines for several months.

Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

"Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n = 9), the Czech Republic (n = 2), and Germany (n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

Infections with the tick-borne bacterium "Candidatus Neoehrlichia mikurensis" mimic noninfectious conditions in patients with B cell malignancies or autoimmune diseases.

BACKGROUND: Candidatus Neoehrlichia mikurensis is a newly discovered noncultivatable bacterium spread among ticks and rodents in Europe and Asia that can infect humans, particularly immunocompromised patients. METHODS: We compiled clinical and laboratory data from 11 patients with hematological malignances or autoimmune diseases who were diagnosed with Candidatus N. mikurensis infection in Europe 2010-2013. Both published (6) and unpublished cases (5) were included. RESULTS: The patients had a median age of 67, were mostly male (8/11), and resided in Sweden, Switzerland, Germany, and the Czech Republic. All but one had ongoing or recent immune suppressive treatment and a majority were splenectomized (8/11). Less than half of them recalled tick exposure. The most frequent symptoms were fever (11/11), localized pain afflicting muscles and/or joints (8/11), vascular and thromboembolic events (6/11), that is, deep vein thrombosis (4), transitory ischemic attacks (2), pulmonary embolism (1), and arterial aneurysm (1). Typical laboratory findings were elevated C-reactive protein, leukocytosis with neutrophilia, and anemia. Median time from onset of symptoms to correct diagnosis was 2 months. In at least 4 cases, the condition was interpreted to be due to the underlying disease, and immunosuppressive therapy was scheduled. All patients recovered completely when doxycycline was administered. CONCLUSIONS: Candidatus N. mikurensis is an emerging tick-borne pathogen that may give rise to a systemic inflammatory syndrome in persons with hematologic or autoimmune diseases that could be mistaken for recurrence of the underlying disease and/or unrelated arteriosclerotic vascular events. Awareness of this new pathogen is warranted among rheumatologists, hematologists, oncologists, and infectious disease specialists.

Calprotectin levels in amniotic fluid in relation to intra-amniotic inflammation and infection in women with preterm labor with intact membranes: A retrospective cohort study.

OBJECTIVE: To evaluate the concentrations of calprotectin in amniotic fluid with respect to intra-amniotic inflammation and infection and to assess the presence or absence of bacteria in the amnio-chorionic niche with respect to presence or absence of intra-amniotic inflammation. STUDY DESIGN: Seventy-nine women with singleton pregnancies and preterm labor with intact membranes (PTL) were included in the study. Amniotic fluid was collected at the time of admission by amniocentesis and calprotectin levels were analyzed from frozen/thawed samples using ELISA. Interleukin (IL)-6 concentration was measured by point-of-care test. Samples from amniotic fluid and the amnio-chorionic niche (space between amniotic and chorionic membranes) were microbiologically analyzed. Microbial invasion of the amniotic cavity (MIAC) was diagnosed based on a positive PCR result for Ureaplasma species, Mycoplasma hominis, 16S rRNA or positive culture. Intra-amniotic inflammation (IAI) was defined as amniotic fluid point-of-care IL-6 concentration ≥ 745 pg/mL. The cohort of included women was divided into 4 subgroups based on the presence or absence of IAI/MIAC; i) intra-amniotic infection, ii) sterile IAI, iii) intra-amniotic colonization and iv) neither MIAC nor IAI. RESULTS: Women with intra-amniotic infection had a significantly higher intra-amniotic calprotectin concentration (median; 101.6 µg/mL) compared with women with sterile IAI (median; 9.2 µg/mL), women with intra-amniotic colonization (median; 2.6 µg/mL) and women with neither MIAC nor IAI (median 4.6 µg/mL) (p = 0.001). Moreover, significantly higher amniotic fluid calprotectin concentration was seen in women who delivered within 7 days (p = 0.003). A significant negative correlation was found between amniotic fluid calprotectin and gestational age at delivery (rho = 0.32, p = 0.003). Relatively more bacteria in the amnio-chorionic niche were found in the sterile IAI group compared with the other groups. CONCLUSIONS: Calprotectin concentrations in amniotic fluid were significantly higher in the intra-amniotic infection group compared with the other groups. Moreover, the bacterial presence in the amnio-chorionic niche was higher in IAI group.

Comparative Genomics of Clinical Isolates of the Emerging Tick-Borne Pathogen Neoehrlichia mikurensis.

Tick-borne 'Neoehrlichia (N.) mikurensis' is the cause of neoehrlichiosis, an infectious vasculitis of humans. This strict intracellular pathogen is a member of the family Anaplasmataceae and has been unculturable until recently. The only available genetic data on this new pathogen are six partially sequenced housekeeping genes. The aim of this study was to advance the knowledge regarding 'N. mikurensis' genomic relatedness with other Anaplasmataceae members, intra-species genotypic variability and potential virulence factors explaining its tropism for vascular endothelium. Here, we present the de novo whole-genome sequences of three 'N. mikurensis' strains derived from Swedish patients diagnosed with neoehrlichiosis. The genomes were obtained by extraction of DNA from patient plasma, library preparation using 10× Chromium technology, and sequencing by Illumina Hiseq-4500. 'N. mikurensis' was found to have the next smallest genome of the Anaplasmataceae family (1.1 Mbp with 27% GC contents) consisting of 845 protein-coding genes, every third of which with unknown function. Comparative genomic analyses revealed that 'N. mikurensis' was more closely related to Ehrlichia chaffeensis than to Ehrlichia ruminantium, the opposite of what 16SrRNA sequence-based phylogenetic analyses determined. The genetic variability of the three whole-genome-sequenced 'N. mikurensis' strains was extremely low, between 0.14 and 0.22‰, a variation that was associated with geographic origin. No protein-coding genes exclusively shared by N. mikurensis and E. ruminantium were identified to explain their common tropism for vascular endothelium.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Cultivation of the causative agent of human neoehrlichiosis from clinical isolates identifies vascular endothelium as a target of infection.

Candidatus (Ca.) Neoehrlichia mikurensis is the cause of neoehrlichiosis, an emerging tick-borne infectious disease characterized by fever and vascular events. The bacterium belongs to the Anaplasmataceae, a family of obligate intracellular pathogens, but has not previously been cultivated, and it is uncertain which cell types it infects. The goals of this study were to cultivate Ca. N. mikurensis in cell lines and to identify possible target cells for human infection. Blood components derived from infected patients were inoculated into cell lines of both tick and human origin. Bacterial growth in the cell cultures was monitored by real-time PCR and imaging flow cytometry. Ca. N. mikurensis was successfully propagated from the blood of immunocompromised neoehrlichiosis patients in two Ixodes spp. tick cell lines following incubation periods of 7-20 weeks. Human primary endothelial cells derived from skin microvasculature as well as pulmonary artery were also susceptible to infection with tick cell-derived bacteria. Finally, Ca. N. mikurensis was visualized within circulating endothelial cells of two neoehrlichiosis patients. To conclude, we report the first successful isolation and propagation of Ca. N. mikurensis from clinical isolates and identify human vascular endothelial cells as a target of infection.

[Fever of unknown origin in a multiple sclerosis patient on immunomodulatory therapy was due to neoehrlichiosis]. / Oklar feber hos patient med MS och rituximab­behandling var neoehrlichios - Ny fästingburen infektion som är svår att diagnostisera.

Fever of unknown origin in a multiple sclerosis patient on immunomodulatory therapy was due to neoehrlichiosis The emerging tick-borne bacterium Candidatus Neoehrlichia mikurensis is the etiologic agent of neoehrlichiosis, a febrile illness that may be accompanied by vascular complications. Severe cases of neoehrlichiosis have been described in patients with hematologic malignancies and systemic rheumatic diseases. We present the first case of neoehrlichiosis in a patient with multiple sclerosis undergoing rituximab therapy.