Differentiating Lyme arthritis: a case-based review.

The incidence or prevalence of Lyme arthritis (LA) in Denmark is unknown and assumed very low. No published cases of polymerase chain reaction (PCR)-confirmed LA from Denmark exist. Clinically, LA does not differ from other rheumatic oligoarthritic disorders posing a differential diagnostic challenge. To review the incidence and prevalence of LA to our knowledge and to present a case series of PCR-confirmed LA cases from Denmark. We conducted a systematic literature review via MEDLINE and EMBASE to explore incidence and prevalence rates of LA. Additionally, we present six cases of patients diagnosed with LA in Denmark. Our literature review identified 23 studies reporting prevalence or incidence, yet only ten studies provided estimates ranging from 1.1 to 280/100.000 in the general population. Our case series identified six patients with LA from a localized region in Southern Denmark; all confirmed by Borrelia-specific real-time PCR from synovial fluid. The diagnostic delay was up to 38 months. All patients except one had a history of previous tick bites; none had erythema migrans lesions. All presented with recurrent arthritis in the knee joint, and two had arthritis in the wrist. The literature review showed an incidence of LA ranging from 1.1 to 15.8 per 100.000 in Europe. Our case series suggests a potentially higher prevalence of LA in Denmark than previously believed. Lack of tick exposure history, antibody assessments and test of Borrelia burgdorferi sensu lato DNA in synovial fluid might lead to misdiagnosed cases potentially explaining the assumed low incidence of LA in Denmark.

An intervention in general practice to improve the management of Lyme borreliosis in Denmark.

BACKGROUND: Our objectives were to improve the following outcomes in patients with Lyme borreliosis (LB) through an educational intervention in general practice: (i) increase the number of hospital referrals on suspicion of LB, (ii) increase the number of cerebrospinal fluid (CSF) tests examined for Borrelia burgdorferi antibody index, (iii) decrease the number of serum-B. burgdorferi antibody tests ordered, (iv) shorten delay from symptom onset to hospital in Lyme neuroborreliosis (LNB) patients, (v) increase LB knowledge among general practitioners. METHODS: A prospective non-blinded non-randomized intervention trial on the island of Funen, Denmark. The intervention included oral and written education about LB and was carried out in areas with an LNB incidence ≥4.7/100.000 between 22 January 2019 and 7 May 2019. Results were compared between the intervention group (49 general practices) and the remaining general practices in Funen (71 practices) 2 years before and after the intervention. RESULTS: In the study period, 196 patients were referred on suspicion of LB, a 28.9% increase in the intervention group post-intervention, 59.5% increase in the control group (P = 0.47). The number of CSF-Borrelia-antibody index tests increased 20.8% in the intervention group, 18.0% in the control group (P = 0.68), while ordered serum-B. burgdorferi antibody tests declined 43.1% in the intervention group, 34.5% in the control group (P = 0.30). 25.1% had the presence of serum-B. burgdorferi antibodies. We found no difference in LNB pre-hospital delay before and after intervention or between groups (P = 0.21). The intervention group performed significantly better on a follow-up questionnaire (P = 0.02). CONCLUSION: We found an overall improvement in LB awareness and referrals among general practitioners but could not show any effect of the intervention on clinical outcomes of LNB.

Characteristics and Clinical Outcome of Lyme Neuroborreliosis in a High Endemic Area, 1995-2014: A Retrospective Cohort Study in Denmark.

BACKGROUND: Despite a well-described symptomatology, treatment delay and sequelae are common in patients with Lyme neuroborreliosis (LNB). The aim of this study was to contribute to the knowledge about the symptomatology and epidemiology of LNB. METHODS: We conducted a retrospective study of all LNB cases verified by a positive Borrelia intrathecal antibody index test performed at the Department of Microbiology, Odense University Hospital, Denmark, from 1995 through 2014. RESULTS: The study included 431 patients; 126 were children. The mean incidence was 4.7 per 100 000 inhabitants per year. The median delay from neurological symptom debut to first hospital contact was 20 days and significantly longer for patients with symptom debut in the winter/early spring. The most common clinical symptoms were painful radiculitis (65.9%), cranial nerve palsy (43.4%), and headache (28.3%). A total of 30.6% were seen in >1 hospital department, and 85.6% were admitted during their course of treatment. Serum Borrelia immunoglobulin M and immunoglobulin G at the time of positive Borrelia intrathecal antibody index test were negative in 67 patients (15.5%). We found a median treatment delay of 24 days, with no improvement in our 20-year study period. Residual symptoms following treatment were found in 28.1% of patients, and risk of residual symptoms was significantly associated with delay from symptom debut to initiation of treatment. CONCLUSIONS: The association between treatment delay and residual symptoms and the lack of improvement in treatment delay during the study period highlight the need for standardized diagnostic routines and a better follow-up for LNB patients. Our findings disprove that all patients with LNB develop positive serum Borrelia antibodies within 6 weeks after infection.

Morphological anomalies in Ixodes ricinus and Ixodes inopinatus collected from tick-borne encephalitis natural foci in Central Europe.

In 2015, 0.02% nymphs displayed anomalies (ectromely) out of the 6744 collected ticks as part of a tick-borne encephalitis program in Germany. In 2016, questing Ixodes ricinus (n = 14,527) and Ixodes inopinatus (n = 75) ticks were collected by flagging as part of a tick-borne encephalitis program in Germany, Slovakia and Denmark. A total of 278 (1.9%) out of 14,602 nymph and adult ticks showed morphological anomalies. The anomalies were divided into general anomalies (body asymmetry) and local anomalies (anomalies of appendages, malformation of capitulum, exoskeleton anomalies and anal groove deformation) with nymphs being the most affected life stage. Most important, leg atrophy was the most common anomaly (209 nymphs, 11 females and three males) followed by asymmetry (10 nymphs, five females and a male) and ectromely (nine nymphs). Two females and one male displayed multiple anomalies on legs, palps and exoskeleton. Anal groove deformation was observed in three females and three nymphs. In 2016, the frequency of anomalies in I. inopinatus was found five times higher (9.3%) than in I. ricinus (1.9%). This is the first report of anomaly (ectromely, leg atrophy, idiosoma deformation) in flagged I. inopinatus and the first report of schizomely in I. ricinus.

Rickettsiosis in Denmark: A nation-wide survey.

Rickettsia helvetica has been reported at varying prevalences in Danish and other European Ixodes ricinus populations. Though apparently widespread and with reported cases of human infection, the significance of the bacteria as a threat to public health remains unclear. We present a nation-wide survey of rickettsia in ticks, roe deer and humans in Denmark. Ticks were collected by flagging and screened for presence of rickettsial DNA by polymerase chain reaction. Sera from roe deer, hunters, neuroborreliosis patients and blood donors were analyzed for presence of anti-R. helvetica and Rickettsia felis antibodies by immunofluorescence microscopy. The Rickettsia minimum infection rate in ticks was 4.9 % (367/973 pools positive, 7510 ticks in total), with 3.9 % in nymphs and 9.3 % in adults. Rickettsia helvetica accounted for 4.17 % and Rickettsia monacensis for 0.03 %, 0.6 % comprised non-differentiable rickettsial DNA. The prevalence of antibodies against R. helvetica was 2.8 % (9/319) in roe deer, while no hunters (n = 536) or blood donors (n = 181) were positive. The prevalence of anti-R. helvetica antibodies among Lyme neuroborreliosis patients was 6 % (3/47), where it co-occurred with Anaplasma phagocytophilum. Based on our study autochthonous rickettsiosis is of limited concern to the public health in Denmark, but our finding of R. monacensis for the first time in Denmark illustrates the dynamic nature of tick-borne pathogens, emphasizing that continuous surveillance is necessary.

Tick-borne encephalitis.

Tick-borne encephalitis (TBE) is a viral tick-borne infection occurring in many parts of Europe and Asia as described in this review. Increasing TBE case numbers have been reported over recent decades. In Denmark the infection is rare (1-14 annual cases). The rise in TBE in Denmark is mainly driven by microfoci outside of Bornholm, primarily North Zealand. Clinical illness has a bi-phasic presentation: "summer-flu" which may be followed by a neuroinfection. No specific treatment exists, and mortality is less-than 1%. A considerable percentage of patients may experience neurological sequelae. TBE is preventable through vaccination.

Detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid samples following pre-enrichment culture.

Molecular methods for diagnosing Lyme neuroborreliosis (LNB) have shown suboptimal diagnostic sensitivities. The objective of this study was to improve the clinical sensitivity of PCR detection of Borrelia burgdorferi sensu lato spirochetes by inoculating cerebrospinal fluid (CSF) from patients suspected of LNB directly into culture medium at the time of lumbar puncture, with this pursuing enrichment of Borrelia spirochetes before PCR analysis. Adult patients with symptoms suggestive of LNB were prospectively enrolled at two hospitals in the Region of Southern Denmark. The CSF-culture samples were incubated for at least eight weeks. During this period, culture sample aliquots were analysed for the presence of Borrelia DNA by separate PCR protocols in two independent clinical laboratories. The included patients were diagnosed with definite (n=12) or possible (n=2) LNB, and non-LNB (n=171) based on clinical and paraclinical findings. Patients in the LNB and the non-LNB group had a median duration from symptom onset to lumbar puncture of 40 days (IQR [23-90] days) and 120 days (IQR [32-365] days), respectively. Pre-enrichment growth of Borrelia spirochetes was accomplished from three patients (21 %) in the LNB group. The positive culture samples were confirmed by both the digital droplet PCR and the real-time PCR methods employed. All CSF samples were PCR negative in the non-LNB group. The results of this study do not support the use of Borrelia-specific PCR as a general routine diagnostic tool in adults. Still, they suggest it may prove of additional value in selected patients with a limited time from symptom onset to sample collection.

[First Danish case of tick-borne encephalitis virus vaccine failure].

Tick-borne encephalitis virus (TBEV) is spreading geographically, and new risk areas are expected throughout Denmark. In this case report, we describe the first Danish case of vaccine breakthrough tick-borne encephalitis (TBE) in a 76-year old female suffering from severe symptoms with need of intensive-care therapy. We want to draw attention to TBE as a differential diagnosis in all undiagnosed patients with symptoms of viral encephalitis, regardless of travel history and vaccine status. TBEV can cause severe disease, especially in the elderly. Patients with vaccine breakthrough infection may develop a more severe disease than non-vaccinated.

Comparison of six commercially available SARS-CoV-2 antibody assays-Choice of assay depends on intended use.

OBJECTIVES: Evaluate six commercial serological assays for detection of IgA, IgM or IgG SARS-CoV-2 antibodies in different disease severities. METHODS: Three lateral flow tests (LFTs) (Acro IgM/IgG, CTK IgM/IgG, Livzon IgM/IgG) and three ELISA assays (Euroimmun IgA and IgG, Wantai IgM) were included. Application was evaluated using samples from 57 patients with a positive SARS-CoV-2 reverse transcription polymerase chain reaction, stratified according to disease severity. Specificity was assessed using historical samples from 200 blood donors. RESULTS: While IgM LFTs failed to detect SARS-CoV-2 antibodies in 37-84% of non-hospitalised patients, the Wantai IgM ELISA detected antibodies in 79%. The Euroimmun IgG ELISA detected antibodies in 95% of non-hospitalised patients. IgA, IgM and IgG ELISA levels were initially low, increased over time, and correlated with disease severity. LFT sensitivity declined in samples taken >28 days after symptom onset/resolution. The Livzon IgG LFT had the highest specificity (98.5%), followed by the Euroimmun IgG ELISA (96.2%). The specificity for Euroimmun IgA ELISA improved (≥97.5%) using a custom cut-off value (4.0). CONCLUSIONS: The sensitive and semi-quantitative ELISA assays are most appropriate for serologic detection of SARS-CoV-2 infection in mild cases. Livzon LFT and Euroimmun ELISA had the highest specificity among the IgG assays, making them most suitable for seroprevalence studies.

Continued expansion of tick-borne pathogens: Tick-borne encephalitis virus complex and Anaplasma phagocytophilum in Denmark.

Tick-borne encephalitis virus (TBEV) is a tick-transmitted flavivirus within the tick-borne encephalitis (TBE) complex. The TBE complex is represented by both TBEV and louping ill virus (LIV) in Denmark. Anaplasma phagocytophilum is also transmitted by ticks and is believed to play an essential role in facilitating and aggravating LIV infection in sheep. This study aimed to describe the distribution of TBE complex viruses in Denmark, to establish the possible emergence of new foci and their association with the distribution of A. phagocytophilum. We performed a nationwide seroprevalence study of TBE complex viruses using roe deer (Capreolus capreolus) as sentinels and determined the prevalence of A. phagocytophilum in roe deer. Danish hunters obtained blood samples from roe deer during the hunting season of 2013-14. The samples were examined for TBEV-specific antibodies by virus neutralization tests (NT). A. phagocytophilum infection was assessed by specific real-time-PCR. The overall seroprevalence of the TBE complex viruses in roe deer was 6.9% (51/736). The positive samples were primarily obtained from a known TBE endemic foci and risk areas identified in previous sentinel studies. However, new TBE complex risk areas were also identified. The overall prevalence of A. phagocytophilum was 94.0% (173 PCR-positive of 184 roe deer), which is twice the rate observed ten years ago. These results point to an expansion of these tick-borne diseases geographically and within reservoir populations and, therefore, rationalize the use of sentinel models to monitor changes in transmission of tick-borne diseases and development of new risk areas. We found no association between TBE complex-positive roe deer and the prevalence of A. phagocytophilum, as almost all roe deer were infected. Based on our findings we encourage health care providers to be attentive to tick-borne illnesses such as TBE when treating patients with compatible symptoms.

Phylogenetic characterization of tick-borne encephalitis virus from Bornholm, Denmark.

The Danish island of Bornholm in the Baltic Sea has been known as a tick-borne encephalitis (TBE) natural focus for more than 60 years. TBE in humans is diagnosed on a regular basis either in inhabitants or tourists of the island. Other areas in Denmark have been suggested as possible risk areas of TBE. Despite the long-known endemicity on Bornholm and the possibility of the virus circulating in other areas, no data on the prevalences of TBE virus (TBEV) in ticks, or adequate molecular characterization and phylogenetic studies are available for the circulating TBEV strains. This study aimed to detect TBEV in ticks collected on the island of Bornholm and other possible risk areas, with the attempt to isolate the circulating viruses for molecular and phylogenetic analysis and confirm the presence of virus in the predicted risk areas. From 2014 to 2016, 9321 I. ricinus (nymphs, females, and males) were collected by flagging 31 locations in Denmark. The ticks were pooled and tested for TBEV by qPCR. The envelope gene of the detected TBE virus strains was amplified and sequenced by RT-PCR. After successful virus isolation, whole genome sequencing was performed. Phylogenetic analysis of the obtained sequences was done by the Maximum Likelihood method. One pool of 11 females and one pool of eight males from a total of 34 tick pools collected from the northwestern shore of lake Rubinsøen on Bornholm tested positive, resulting in a local estimated point prevalence of 0.6% [CI95% 0,1-1.85%] in this microfocus. We were not successful in confirming any other of the predicted TBEV-endemic areas. Alignment of the two complete E genes from Bornholm revealed identical sequences. Virus isolation and whole genome sequencing were succeeded from one of the positive samples. Phylogenetic analysis showed that the isolated virus had the closest phylogenetic relationship to TBEV sequences detected in Eastern and Central Europe.

Reduction in human Lyme neuroborreliosis associated with a major epidemic among roe deer.

Lyme neuroborreliosis is the most severe clinical manifestation of Lyme borreliosis. In most of Denmark, and also Europe, the overall prevalence of Lyme borreliosis seems to be stabilising. This is not the case on the island of Funen, Denmark, where the number of human Lyme neuroborreliosis cases has markedly declined throughout the last decade. We propose the reason for the decline is a major epidemic among roe deer, killing almost half of their population, resulting in a reduction in the tick population which make it less likely to get a tick bite and therefore to contract Lyme neuroborreliosis. This is the first time such a relationship is described as a naturally occurring phenomenon in Europe.

[Tick-borne infections in Denmark].

The castor bean tick, Ixodes ricinus, is common in woodlands in most of Denmark. Besides Borrelia burgdorferi, it can harbour a number of pathogenic microorganisms such as tick-borne encephalitis virus, Anaplasma phagocytophilum, Rickettsia helvetica, Francisella tularensis, Candidatus Neoehrlichia mikurensis, Bartonella spp., Borrelia miyamotoi and Babesia spp. These tick-borne infections should be a differential diagnostic consideration during the tick season in Denmark. We review the distribution, clinical manifestations, diagnosis and treatment of these microorganisms.

Validity of the coding for herpes simplex encephalitis in the Danish National Patient Registry.

BACKGROUND: Large health care databases are a valuable source of infectious disease epidemiology if diagnoses are valid. The aim of this study was to investigate the accuracy of the recorded diagnosis coding of herpes simplex encephalitis (HSE) in the Danish National Patient Registry (DNPR). METHODS: The DNPR was used to identify all hospitalized patients, aged ≥15 years, with a first-time diagnosis of HSE according to the International Classification of Diseases, tenth revision (ICD-10), from 2004 to 2014. To validate the coding of HSE, we collected data from the Danish Microbiology Database, from departments of clinical microbiology, and from patient medical records. Cases were classified as confirmed, probable, or no evidence of HSE. We estimated the positive predictive value (PPV) of the HSE diagnosis coding stratified by diagnosis type, study period, and department type. Furthermore, we estimated the proportion of HSE cases coded with nonspecific ICD-10 codes of viral encephalitis and also the sensitivity of the HSE diagnosis coding. RESULTS: We were able to validate 398 (94.3%) of the 422 HSE diagnoses identified via the DNPR. Hereof, 202 (50.8%) were classified as confirmed cases and 29 (7.3%) as probable cases providing an overall PPV of 58.0% (95% confidence interval [CI]: 53.0-62.9). For "Encephalitis due to herpes simplex virus" (ICD-10 code B00.4), the PPV was 56.6% (95% CI: 51.1-62.0). Similarly, the PPV for "Meningoencephalitis due to herpes simplex virus" (ICD-10 code B00.4A) was 56.8% (95% CI: 39.5-72.9). "Herpes viral encephalitis" (ICD-10 code G05.1E) had a PPV of 75.9% (95% CI: 56.5-89.7), thereby representing the highest PPV. The estimated sensitivity was 95.5%. CONCLUSION: The PPVs of the ICD-10 diagnosis coding for adult HSE in the DNPR were relatively low. Hence, the DNPR should be used with caution when studying patients with encephalitis caused by herpes simplex virus.