Background: With a prevalence of 4.7–13% in Danish
Rickettsioses; Tick-borne infections; Tick-borne pathogens; Co-infection; Lyme neuroborreliosis; Neuroinfection
With an annual incidence of over 3/100,000, Lyme neuroborreliosis (LNB) is the commonest bacterial neuroinfection in Denmark [[
The Rickettsia genus consists of small, aerobic, obligately intracellular Gram-negative organisms and can be divided into two serotypically distinct groups: the spotted fever group (SFG) and the typhus group (TG). Members of the SFG rickettsiae can be found across the globe and are known for causing infections of varying pathogenicity, ranging from the mild African tick-bite fever (Rickettsia africae), endemic in sub-Saharan Africa and the West Indies, to the more severe and potentially fatal Rocky Mountain spotted fever (Rickettsia rickettsii) in the Americas. Of the two Rickettsia spp. existing endemically in Denmark, R. helvetica is the most prevalent and is also one of the most frequently encountered tick-borne organisms in the country [[
Because R. helvetica is transmitted by the same tick vector as the Bbsl complex, co-transmission of both agents is a possibility and has been shown to occur in humans [[
As Rickettsia spp. are intrinsically resistant to penicillin, the treatment of choice for Lyme borreliosis, the clinical significance and incidence of co-transmission needs to be further examined in a Danish population. The aim of this study was threefold; 1) to investigate the seroprevalence of antibodies against SFG rickettsia in patients investigated for LNB 2) to examine the presence of Rickettsia DNA in the CSF and sera of same patients, and 3) to examine the clinical significance of such a finding.
The study cohort consisted of archival samples of paired sera and CSF from patients tested for LNB using the intrathecal anti-Borrelia (IgM/IgG) antibody index (AI) test. One-hundred and four paired samples were selected from AI positive patients, indicating certain prior exposure to I. ricinus ticks, and 110 paired samples were selected from AI negative patients. As some of the archival samples only had small remaining volumes, insufficient for further testing, some of them had to be discarded. A total of 99 sera and 87 CSF samples from AI positive patients, and 101 sera and 103 CSF samples from AI negative patients, remained for inclusion in this study after duplicates and samples containing insufficient volumes had been removed (Fig. 1).
All samples were collected in the months of June to November during the years 2011–2015 and have been stored at the State Serum Institute (SSI) at − 20 °C. The serological analysis for LNB was during these years mostly centralized at SSI and the samples thus represent patients from across the whole country.
The intrathecal anti-Borrelia antibody index test had been performed using an in-house capture enzyme-linked immunosorbent assay (ELISA) that uses native purified flagellum from B. afzelii (strain DK1) as antigen [[
The sera were examined for antibodies against SFG rickettsiae using a commercially available indirect immunofluorescense assay (IFA) (IF0100G, Rickettsia IFA IgG; Focus Diagnostics, Inc., Cypress, CA, USA). Inactivated R. rickettsii (RR) was used as antigen to detect SFG rickettsiae. The samples were screened at dilutions of 1:64 (IgM) and 1:128 (IgG) and were titrated to end-point fluorescence in two-fold dilutions. A titer of 1:64 or above was considered positive for IgM. The cut-off for IgG had previously, for diagnostic purposes, been raised to 1:512, following testing of healthy Danish blood donors, to achieve a specificity of 95% and reduce cross-reactivity with other bacteria [[
All of the CSF samples and all, but six, of the sera were examined using real-time PCR for the presence of Rickettsia DNA. All samples were centrifuged for 10 min at 15,000 g, after which part of the supernatant was discarded leaving a volume of 200 μl along with the pellet. Extraction of DNA was achieved using the Qiagen QIAamp DNA Mini Kit (Qiagen Danmark, Copenhagen, Denmark), and DNA was eluted into a final volume of 75 μl. All extractions were performed in a hooded bench with laminar air flow and caution was exercised to prevent contamination. A genus-specific real-time PCR with a reported sensitivity of one copy of Rickettsia DNA per reaction, was used to amplify a 74 bp fragment of the R. rickettsii citrate synthase (CS) gene (gltA) using the primers CS-F and CS-R and the probe CS-P [[
Table 1 Sample characteristics of negative control group ( n = 103) consisting of CSF, with and without pleocytosis, from patients examined for other neuroinfections than LNB
Control samples (n = 103) Median age (IQR, range): 51 (33–63, 0–92) CSF pleocytosis: ≤ 5 × 106/L, n: 82 > 5 × 106/L, n (median, IQR, range): 21 (14, 9–65, 6–4248) CSF protein: ≤ 0.50 g/L, n: 64 > 0.50 g/L, n (median, IQR, range): 39 (0.69, 0.56–0.99, 0.51–2.30)
IQR interquartile range, CSF cerebrospinal fluid
Statistical significance was calculated with Fisher's exact test (two-tailed) and the two-sample Wilcoxon rank sum test using the statistical software R Studio (v1.0.153) [[
Antibody titers for the AI positive patients, AI negative patients and healthy blood donors are presented in Table [
Table 2 Antibody titers against SFG rickettsiae in the AI positive group (a), the AI negative group (b), and the healthy blood donors (c)
Antibody index positive (n = 99) a. 1:64 1:128 1:256 1:512 1:1024 1:2048 1:4096 RR IgM 1 0 0 1 0 0 0 RR IgG – 12 14 7 5 0 0 b. Antibody index negative (n = 101) RR IgM 0 2 0 0 0 0 0 RR IgG – 9 13 1 1 2 0 c. Blood donors (n = 111/60) RR IgM 1 0 0 1 0 0 0 RR IgG – 18 10 4 1 0 0
When using the routine, diagnostic cut-off of 1:512 [[
At the genus-level, Rickettsia DNA was identified in eight of 190 (4%) archival CSF samples, with cycle threshold (Ct) values ranging between 39 and 44. Compared with the 103 negative controls, this is a non-significant difference (p = 0.054). However, five of these patients were AI positive, reflecting a significantly increased probability of detecting Rickettsia DNA in this subgroup compared with the controls (p = 0.02). Sequencing was attempted but failed on all eight samples. None of these patients displayed anti-Rickettsia antibodies in the corresponding sera. No CSF in the negative control group, regardless of biochemical findings, showed evidence of Rickettsia DNA and no Rickettsia DNA could be detected in the sera. All internal negative controls remained negative and all positive controls displayed positive reactions.
Hospital medical records were reviewed for the eight patients in whom Rickettsia DNA was detected in the CSF. The clinical data are summarized in Table [
Table 3 Clinical data on patients with detected Rickettsia DNA in CSF
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Co-morbidity Asthma Esophageal hernia None None Colitis ulcerosa None None Spinal stenosis None Symptoms - Bilateral peripheral facial palsy - Radiculitis - Unilateral peripheral facial palsy - Headache - Unilateral peripheral facial palsy - Lower back pain - Radiculitis - Radiculitis - Erythema migrans - Myalgia - Radiculitis - Severe headache - Pain in neck and shoulder girdle - Fever - Photophobia - Transient hemiparesis - Sensorimotor polyneuropathy - Fever - Headache - Pain in neck and shoulder girdle - Rash Clinical diagnosis LNB LNB LNB LNB LNB Transient cerebral attack – Enteroviral meningitis (PCR-verified) SFG Rickettsia IFA NEG NEG NEG NEG NEG NEG NEG NEG Intrathecal Borrelia-specific antibody synthesis (index ≥ 0.3) YES YES YES YES YES NO NO NO CRP 5.3 < 1 < 1 6.7 < 1 1 2 130 CSF-leukocytes 34 100 863 192 300 2 1 133 CSF-protein 1.01 0.42 1.95 0.61 1.36 0.36 0.56 0.52 CSF-glucose 3.7 3.6 1.8 2.9 2.9 3.7 – 3.0 RT-PCR Ct value (CSF) 41 40 39.5 42 39.6 39 43.5 43.5 RT-PCR (sera) NEG NEG NEG NEG NEG NEG NEG NEG Antibiotic treatment IV ceftriaxone + IV acyclovir IV ceftriaxone IV ceftriaxone IV ceftriaxone IV ceftriaxone – – IV ceftriaxone + IV acyclovir Follow-up Asymptomatic 1 month post treatment Asymptomatic 1 month post treatment Clinical follow-up data missing. Well-being at hospital discharge. Responds to treatment. Slight headache and fatigue 1 month post hospital discharge. Remits after 36 h with full recovery of neurological function. Symptoms persistent for several years. Asymptomatic at hospital discharge
Abnormal values are printed in boldface. All patients with CSF pleocytosis had mostly mononuclear leukocytes. The cut-offs used for SFG Rickettsia IFA were 1:64 for IgM and 1:128 for IgG. Reference values: CRP: < 10 mg/L. CSF-leukocytes: < 5 × 10
One of the patients (Case 5) had, in the months prior to hospital admission, experienced a tick bite in Sweden. No other patients had any mention of international travel, leading up to the hospital admission. As R. helvetica is also the most common tick-borne rickettsia found in Sweden, all patients had thus most likely been infected with this species or possibly, R. monacensis [[
We found no significant difference in the seroprevalence of antibodies against SFG rickettsiae between patients investigated for LNB and healthy blood donors and no Rickettsia DNA in the tested sera, indicating low rates of exposure to SFG rickettsiae in Danish patients with Lyme borreliosis. Eight patients showed evidence of Rickettsia DNA in the CSF, five of whom were Borrelia AI positive, reflecting a significantly increased probability of detecting Rickettsia DNA in the AI positive subgroup compared with patients tested for other neuroinfections than LNB (p = 0.02). Despite this, the AI positive patients presented with typical clinical signs of LNB with no apparent alteration in the clinical manifestations of this infection.
The high seroprevalence of IgG antibodies against SFG rickettsiae among healthy blood donors is indicative of considerable non-specific background reactivity, revealed by the lowering of our titer cut-off for this study. Nevertheless, the absence of a significant difference in the seroprevalence of antibodies between healthy blood donors and patients examined for LNB, regardless of titer cut-off and Borrelia AI, is indicative of low rates of exposure in this group of patients.
Important caveats to be emphasized are that the precise time elapsed from being bitten to being tested is not known and that the precise kinetics of the human antibody response to R. helvetica and R. monacensis have not been studied, factors that could potentially impact the current results. However, considering that adult Danish patients with LNB are often diagnosed late in the course of their disease with a median delay of 20 days from the start of neurological symptoms until first hospital contact, one can assume that enough time had passed to allow seroconversion in most of the AI positive patients exposed to SFG rickettsiae [[
Lastly, R. rickettsii, and not R. helvetica, was used as IFA antigen in the current study. Rickettsia conorii is often used as antigen to screen for SFG rickettsiae and cross-reactivity between this species and R. helvetica has previously been demonstrated [[
Whether the detected Rickettsia DNA represents true neuroinfection or not can be discussed. All internal negative controls remained negative, making contamination less likely. Furthermore, Rickettsia DNA was not detected in the negative control group, consisting of patients tested for other neuroinfections than LNB and representing a range of varying biochemical values, reducing the chances of it being an artifact. Sequencing was attempted but failed on all eight samples, most likely due to the small amounts of bacterial DNA in the samples, as reflected in the high Ct values. However, the general lack of discerning clinical features in these cases and the apparent lack of pleocytosis and clinical signs of infection in two of the subjects necessitates cautious interpretation of these PCR findings. It is important to note that none of the patients received treatment with doxycycline (Table [
Interestingly, none of the patients exhibiting DNA in their CSF had detectable antibodies against SFG rickettsiae. Possible explanations could be that the patients were tested prior to the development of anti-rickettsia antibodies, a lack of sensitivity in the IFA assay or sample contamination with rickettsial DNA. It is possible that the lack of antibodies facilitated the detection of Rickettsia DNA by allowing the bacteria to survive and spread, as suggested by a recent study [[
The study is limited by its retrospective nature and its use of frozen archival specimens of variable volume, ill-suited for culture and lacking convalescent samples for serology. However, the use of archival material also allowed the inclusion of a greater number of patients with a confirmed anti-Borrelia AI, ascertaining prior tick exposure, than would have been possible in a reasonable time frame following a prospective study design. It is important to remember that all samples had initially been sent to a serological laboratory and have thus, not been handled in a clean working environment appropriate for PCR analysis, why potential sample contamination cannot be ruled out. This also applies to the negative CSF controls, many of which had previously been tested for HSV/VZV using an intrathecal antibody index test. However, contamination seems unlikely as we have never amplified or cultured rickettsiae in our serological laboratory and have only used commercial IFA kits with preformed slides, on which the antigen is already fixed, for serological analysis.
The effect of prolonged storage in − 20 °C freezers and multiple cycles of thawing-freezing on the quality and yield of potential bacterial DNA can also be discussed. The former was not considered a major problem as R. helvetica has previously been detected in CSF (using both culture and PCR) after having been stored for over a year in a − 20 °C freezers [[
Despite them being one of the commonest tick-borne organisms in Denmark, antibody reactivity against Rickettsia spp. was not significantly increased in patients investigated for LNB as compared with healthy blood donors. The same was true when looking at the subgroup of patients with a positive anti-Borrelia AI test, confirming a prior tick bite. These findings indicate low levels of SFG rickettsiae co-transmission in this group of patients.
Furthermore, eight patients displayed small amounts of Rickettsia DNA in the CSF. However, the lack of CSF pleocytosis in two of the patients, the complete or near-complete clinical recovery in most of the patients, despite being withheld doxycycline, and a general lack of clear, discerning clinical features in all of the patients, necessitates cautious interpretation of these results. Should this CSF data indicate true rickettsial infection, such occurrences appear to be rare and of uncertain significance to the patient.
The study was approved by the Regional Medical Ethics Committee (approval No. 53005) and the Danish Data Protection Agency (approval No. 15/09765). Exemption from informed consent was granted for all included patients.
Not applicable.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We would like to thank Gitte Jensen and the diligent laboratory staff at the department of Virus and Microbiological Special Diagnostics at SSI for their kind help with IFA and PCR. We would also like to thank Scandtick Innovation, an EU-Interreg project, for providing funding to perform this study.
This research has been conducted using the Danish National Biobank resource, supported by the Novo Nordisk Foundation, grant number 2010-11-12 and 2009-07-28. The study was partly funded by the Interreg V Program (the Scandtick Innovation project, grant number 20200422). The sponsor was not involved in the design and execution of the study, analysis of data or in the writing and submission of the manuscript.
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
LFO, RBD, AML and KAK were involved in the conception and design of the study. Data was collected and analysed by LFO. CSJ and RFP aided in the laboratory analysis of the samples. Substantial contributions to the writing of the manuscript have been made by all authors, all of whom have given final approval of the version to be published.
By Lukas Frans Ocias; Ram Benny Dessau; Anne-Mette Lebech; Charlotte Sværke Jørgensen; Randi Føns Petersen and Karen Angeliki Krogfelt