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Abstract
Introduction Neoehrlichia mikurensis is a tick-borne bacterium that primarily causes disease in immunocompromised patients. The bacterium has been detected in ticks throughout Europe, with a 0%–25% prevalence. N. mikurensis infection presents unspecific symptoms, which can easily be mistaken for inflammatory disease activity. We aimed to determine the prevalence of N. mikurensis in rheumatological patients receiving tumour necrosis factor inhibitors (TNFi) and a cohort of healthy individuals.
Materials and methods This retrospective cohort study included 400 rheumatological patients treated with TNFi and 400 healthy blood donors. Plasma samples were retrieved from the Danish Rheumatological Biobank and the Danish Blood Donor Study between 2015 and 2022. Age, sex, diagnosis and duration of TNFi treatment were recovered from the Danish Rheumatological Database, DANBIO. Data on age and sex were available for the blood donors. One plasma sample per individual was tested for N. mikurensis DNA-specific real-time PCR targeting the groEL gene.
Results In the rheumatological patients, the median age was 61 years (IQR 55–68 years), 62% were women, and 44% had a diagnosis of seropositive rheumatoid arthritis. In total, 54% of the patients were treated with infliximab. The median time from TNFi initiation to blood sampling was 20 months (IQR, 5–60 months). N. mikurensis DNA was not detected in any samples from patients or blood donors.
Conclusion N. mikurensis infection does not appear to represent a prevalent risk in Danish rheumatological patients receiving TNFi or in blood donors.
- Tumor Necrosis Factor Inhibitors
- Rituximab
- Arthritis, Rheumatoid
Data availability statement
Data are available upon reasonable request.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Neoehrlichia mikurensis is a tick-borne bacterium that can cause subclinical disease, which may go unnoticed in immunocompromised individuals.
WHAT THIS STUDY ADDS
This study investigates the prevalence of N. mikurensis in a large population of rheumatological patients receiving tumour necrosis factor inhibitors and a cohort of blood donors.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
N. mikurensis infection does not represent a prevalent risk in rheumatological patients receiving tumour necrosis factor inhibitors or in blood donors.
Introduction
The tick-transmitted bacterium Neoehrlichia mikurensis is prevalent in ticks across Europe and Asia.1–4 In Europe, N. mikurensis has been detected in ticks in 20 countries, including Norway, Sweden and Denmark, with a prevalence ranging up to 25%.1 3 5–7
N. mikurensis can cause the disease neoehrlichiosis in humans, and approximately 200 cases of neoehrlichiosis have been published.3 5 8–10 Detecting the bacterium can be difficult because N. mikurensis does not grow in standard blood cultures, and a serological assay is not yet available.11 N. mikurensis is detected only by a specific real-time PCR or 16S ribosomal RNA PCR (16S PCR).12 The clinical presentation of neoehrlichiosis varies from asymptomatic infection to severe life-threatening disease.13–16 Commonly described clinical features of neoehrlichiosis include fatigue, nightly sweats, fever, rash and vascular or thromboembolic events.5 17 High age, splenectomy, haematological malignancy, systemic autoimmune disease and B-cell depleting therapy are all known risk factors for severe neoehrlichiosis.5
Biological therapies, such as tumour necrosis factor inhibitors (TNFi), are widely used to treat autoimmune diseases.18 TNFi has led to improved disease control and a reduction in comorbidities and mortality.19 However, TNFi therapy has also been associated with an increased risk of infections in patients with rheumatoid arthritis.20 Specifically, reactivation of tuberculosis has led to recommendations for pretreatment screening for latent infection.21 22 Other infectious complications include non-tuberculous mycobacteriosis, histoplasmosis, listeriosis, aspergillosis and nocardiosis.23 24 It is uncertain whether N. mikurensis can cause infectious complications or undetected infection in TNFi-treated patients.
In Denmark, N. mikurensis is still a largely unknown pathogen among physicians, and diagnostic availability is limited. To date, clinically symptomatic cases of neoehrlichiosis in Denmark have exclusively been found among patients treated with the B-cell-depleting drug rituximab for a haematological, neurological or rheumatological disease.14 25 26 A recent Norwegian study found a prevalence of detectable N. mikurensis DNA in 4.7% of patients treated with rituximab and 10% of patients treated with the TNFi infliximab,13 selected regardless of symptoms. This observation indicates a significant prevalence of N. mikurensis among TNFi-treated patients, raising concerns about the potential for symptom development and infectious complications. No previous studies explore whether patients receiving TNFi are at higher risk of N. mikurensis infection than healthy controls. Often unspecific symptoms caused by neoehrlichiosis, such as fever, night sweats, fatigue and unspecific rash, can be challenging due to the overlap in symptoms with inflammatory rheumatological disease.
Hence, this retrospective cohort study aimed to assess the occurrence of N. mikurensis among rheumatological patients undergoing TNFi treatment. A comparison was made with a randomly selected group of blood donors. We hypothesised a higher prevalence of N. mikurensis in patients treated with TNFi compared with healthy individuals (blood donors), posing an increased risk of severe N. mikurensis infections in TNFi-treated patients.
Materials and methods
Study design and participants
This study was a retrospective cohort study. A search in the Danish nationwide Rheumatological Database (DANBIO)27 was made applying the following inclusion criteria: adult patients (≥18 years); monitored in the DANBIO database between May 2015 and September 2021; currently receiving TNFi therapy and minimum for 3 months; an available plasma sample in the Danish Rheumatologic Biobank.28 29 In total, 400 participants were identified. Age, sex, sample date, TNFi initiation date, end date, and diagnosis were captured from DANBIO.
Pseudonymised plasma samples from 400 healthy blood donors were randomly retrieved from the Danish Blood Donor Study.30 The samples were collected between February 2016 and August 2022. Data on age, sex and donation date were available.
DNA purification
DNA was purified from EDTA plasma using the DNeasy Blood and Tissue Kit (Qiagen, Germany) following the manufacturer’s instructions. As internal PCR amplification and purification control, 10 µL Phocine Herpes virus was added before purification. A total volume of 200 µL plasma was used for patients on TNFi inhibitor treatment, whereas 400 µL plasma was used for the blood donor samples due to expected lower bacterial gene copies in healthy individuals.31 Purified DNA was stored at −20°C for later analyses.
Real-Time PCR
Real-time PCR runs were performed on an Agilent Aria Mx. We used a specific TaqMan probe-based real-time PCR targeting the 169 bp segment of the groEL gene of the N. mikurensis bacterium. The method, including primers and probe, has been described previously12 and accommodated our setup. Amplifications were performed using 5 µL template DNA in a reaction mixture of 22.5 µL. The reaction mixture included 12.5 µL Platinum Quantitative PCR SuperMix-UDG (Invitrogen, USA), 1 µM of each primer, 0.1 µM probe, 1 µM MgCl2, 6.3 µL DNAse free H2O. Reaction conditions were 95°C for 10 min, followed by 45 cycles at 95°C for 15 s, and a final cycle at 54°C for 1 min. A synthetic plasmid containing the 169 bp sequence cloned into a pUC57 vector (Genscript, Piscataway, New Jersey) was used to estimate bacterial gene copies with a lower limit of detection of 2.5×102 gene copies. The assay was tested with known positive samples, which have been verified by sequencing and known negative patient samples. All samples were run in singlets. The positive control consisted of a positive patient sample, where DNA was extracted using the smallest volume (200 µL). The positive and negative controls were included in all runs. A positive real-time PCR was defined as a cycle threshold (Ct) value of ≤36 combined with a proper sigmoid curve.
Statistical analyses
A power calculation was based on the published prevalence of N. mikurensis infection in different patient cohorts.13 32 N. mikurensis was detected in 10% of Norwegian patients receiving TNFi.13 However, as the prevalence of N. mikurensis in Danish ticks is lower than in ticks from Norway,1 6 7 33 34 we anticipated an incidence of 4%, which, with a sample size of 400 samples, would allow the detection of a 1% or higher prevalence of microbial DNA with reasonable power (80%) and a confidence level of 95%.35 Mann-Whitney U test was used to compare the characteristics of the two cohorts. A p value <0.05 was considered significant. All descriptive statistical analyses were performed in SAS Enterprise guide 7.1.
Results
Participants
Characteristics of the 400 rheumatological patients receiving TNFi and 400 blood donors are summarised in table 1.
Characteristics of rheumatological patients receiving TNFi and blood donors at time of plasma sampling
Rheumatological patients receiving TNFi
The median age was 61 years with an IQR of 55–68 years, and the majority was women (62%). All patients received TNFi; the median time from treatment initiation to blood sampling was 20 months (IQR, 5–60 months). Most of the patients received infliximab (54%) and had seropositive rheumatoid arthritis (44%) (table 1). The samples were collected throughout the year, with a slight majority collected during autumn.
Blood donors
In total, 400 blood donors were included. The median age was 34, with an IQR of 25–45 years, and 52% were women. Samples were collected throughout the year, with the majority collected during autumn (table 1).
Detection of N. mikurensis DNA
None of the analysed plasma samples was positive for N. mikurensis DNA.
Comparison of the rheumatological cohort and the blood donors
The age was significantly higher in the rheumatological cohort compared with the blood donor cohort (p<0.0001). Further, significantly more women were included in the rheumatological cohort than in the blood donor cohort (p<0.0043).
Discussion
Contrary to our hypothesis, N. mikurensis DNA was not detected in any plasma samples from 400 rheumatological patients receiving TNFi nor in plasma samples from 400 blood donors. The results indicate that the prevalence of N. mikurensis may be low in both groups and that patients receiving TNFi are not at significant risk of N. mikurensis infection in Denmark. Our findings suggest a prevalence below 1% among TNFi-treated rheumatological patients in Denmark.
Designing the study, a higher incidence of N. mikurensis was anticipated among Danish rheumatological patients receiving TNFi. This assumption was based on two previous studies. First, a recent study from neighbouring country Norway reported a relatively high incidence of 7.4% (12 of 163) among patients receiving biological treatment. Interestingly, subgroup analyses suggested the infection to be more prevalent among patients receiving infliximab (TNFi); 10% (10 of 100), compared with those treated with rituximab; 4.7% (2 of 43), although numbers were low.13 Second, recent findings from our group demonstrated the presence of N. mikurensis in 1.3% (3 of 239) of Danish immunocompromised patients, primarily treated with the B-cell-depleting drug, rituximab, and none in the 192 blood donors.26 Thus, a sample size of 400 was considered adequate.
The relatively high N. mikurensis incidence of 10% (10 of 100) documented in the prospective study from South-Eastern Norway was performed during tick-season (September to December 2018 and March to May 2019).13 Of the 10 patients with confirmed infection, most (6 of 10) received TNFi for inflammatory bowel disease, whereas only four had a rheumatologic condition. Most had received infliximab for over 2 years, similar to our study population, with a median time on TNFi of 20 months. Another study from the same geographical area estimated the incidence of N. mikurensis in blood to be 10% (7 of 70).36 This was among symptomatic immunocompetent individuals with a tick-bite history, presenting with erythema migrans and flu-like symptoms, suggesting that the area is endemic for N. mikurensis.
Our present study included patients independent of symptoms, although retrospectively and throughout the year, compared with a seasonal prospective inclusion in the Norwegian studies.13 36 However, considering most of our samples (331/400) were collected in spring, summer and autumn, we do not consider seasonality to have a major influence on results. The analytical approach in this study was largely identical to these two prior studies, although we used plasma for DNA extraction instead of plasma with buffy coat/pellet. PCR detection based on DNA extraction from plasma is the standard in most clinical laboratories working with N. mikurensis (C. Wennerås, personal communication). Thus, we do not anticipate differences in analytical approach to have affected results noteworthy.
Taken together, we interpret the difference in incidences among patients receiving TNFi in the two countries to reflect the epidemiology accurately, probably due to higher tick infestation and higher prevalence of N. mikurensis among ticks in Norway (up to 25%) compared with Denmark (up to 13%).10
The frequency of N. mikurensis among blood donors has only been scarcely studied. We found no positive samples in the cohort of 400 blood donors. A Swedish study estimated the prevalence to be 0.7% (7/1006) of blood donors with N. mikurensis.32 Here, the PCR analysis was based on whole blood, which has been reported to be less sensitive than plasma.36 We increased the volume of plasma used for DNA extraction to account for a lower bacterial load among healthy individuals than immunocompromised.31
Safety is a concern in the use of biological treatment. Biological therapy suppresses different immune system pathways and may leave the patient vulnerable to infections. Treatment with TNFi is well known to increase the risk of bacterial infections,37 specifically during the first 6–12 months of treatment.19 38 The overall infection risk in rheumatoid arthritis patients appears higher during treatment with rituximab compared with other biological therapies, although confounding by indication should be kept in mind.39
Diagnosing neoehrlichiosis can be challenging because of the general vague symptoms, such as fever, night sweats, unintended weight loss, fatigue, myalgia and arthralgia.5 17 40 Due to the overlap with symptoms often experienced in patients with inflammatory diseases combined with rheumatological patients’ tolerance of their regular symptoms may increase the risk of missing or delaying a diagnosis of neoehrlichiosis.13 17 The fact that N. mikurensis can persist in blood for extended periods of time32 and the limited awareness of the infection among physicians, added to our concern that the infection could go unnoticed in this population.
However, our results indicate that the infection is rare among rheumatological patients receiving TNFi in Denmark, and, therefore, routine screening of N. mikurensis is not recommended. However, persons who often reside in nature and present with unexplained unspecific symptoms testing for N. mikurensis should be considered.
Study strengths and limitations
The retrospective design has inherent limitations. The blood samples were not tied to specific symptom presentations, and the samples were collected throughout the year and not only in the tick season. However, this might be of minor importance because N. mikurensis can cause asymptomatic prolonged bacteremia in both immunocompetent and immunocompromised patients. A strength of the study is the well-defined cohort of rheumatological patients who were receiving or had received TNFi for a median time of 20 months; thus, the patients had been assumably immunocompromised for a longer period.
Conclusion
N. mikurensis was not detected in any of the plasma samples from the 400 rheumatological patients or the 400 blood donor samples, suggesting a very low prevalence in both groups and that patients receiving TNFi are not at significant risk of N. mikurensis infection in Denmark. Therefore, ad hoc testing of symptomatic individuals is preferred over screening in this population.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by The study protocol was approved by the Danish Regional Ethical Committee of the Capital Region (Region Hovedstaden) (journal number: H-20009322) and the Knowledge Center for Data Reviews (approval number: P-2021-364). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors would like to acknowledge the Bio- and Genome Bank Denmark (The Danish Rheumatologic Biobank) for generously providing the essential biological materials. Gratitude is also expressed to the Danish Rheumatology Quality Registry, DANBIO/DRQ, for their contribution of clinical information related to the rheumatological patients. The authors wish to acknowledge the Danish Blood Donor Study for its role in providing the essential blood donor samples that were used in this study. Special recognition is reserved for Britt Corfixen and her substantial contributions to this paper. In addition, the authors would like to acknowledge Stine Østergaard for her skilled assistance in the laboratory, which played an important role in the successful execution of this research. The collaborative efforts have been essential in realising this study, and the contributions mentioned above are gratefully acknowledged.
References
Footnotes
Contributors RG: conceptualisation, methodology, validation, formal analysis, investigation, writing—original draft, visualisation, project administration. MØ: conceptualisation, formal analysis, writing—review and editing. EH: conceptualisation, resources, writing—review and editing. MLH: resources, writing—review and editing. SRO: resources, writing—review and editing. LH: resources, writing—review and editing. BG: conceptualisation, visualisation, resources, writing—review and editing. A-ML: conceptualisation, methodology, investigation, writing—original draft, review and editing, visualisation, project administration, supervision, and funding acquisition. HM: conceptualisation, methodology, investigation, writing—original draft, review and editing, visualisation, project administration and supervision. RG and HM are the overall guarantors.
Competing interests This work was supported as part of NorthTick, an Interreg project supported by the North Sea Programme of the European Regional Development Fund of the European Union (Grant number 38-2-7-19). The funding bodies had no role in writing the manuscript. Outside of the present work: MØ has received an unrestricted research grant from Rigshospitalets Forskningspuljer. AML discloses speaker honoraria and advisory board activities from Gilead, ViiV/GSK, and Pfizer. AML reports a grant from The Lundbeck Foundation (R366-2021-127) and Aase and Ejnar Danielsen’s Foundation. BG discloses research grants (paid to institution) from AbbVie, Sandoz. MLH discloses research grants (paid to institution) from AbbVie, Biogen, BMS, Celltrion, Eli Lilly, Janssen Biologics B.V, Lundbeck Foundation, MSD, Pfizer, Roche, Samsung Biopsies, Sandoz and Novartis. MLH discloses honoraria (paid to institution) from Pfizer, Medac, and Sandoz. MLH has chaired the steering committee of the Danish Rheumatology Quality Registry (DANBIO, DRQ), which receives public funding from hospital owners and funding from pharmaceutical companies. MLH co-chairs EuroSpA, which generates real-world evidence of treatment of psoriatic arthritis and axial spondyloarthritis based on secondary data and is partly funded by Novartis. The other authors declare no conflict of interest.
Provenance and peer review Not commissioned; externally peer reviewed.