Sigurdur Skarphedinsson

Survey of tickborne infections in Denmark

We conducted a study of the distribution and prevalence of tickborne infections in Denmark by using roe deer as sentinels. Blood samples from 237 roe deer were collected during the 2002–2003 hunting season. Overall, 36.6% of deer were Borrelia seropositive, while 95.6% were Anaplasma phagocytophilum positive; all animals were negative for Bartonella quintana and B. henselae by indirect immunofluorescence assay. When a hemagglutination-inhibition test was used, 8.7% of deer were found positive for tickborne encephalitis (TBE)-complex virus. A total of 42.6% were found positive by polymerase chain reaction (PCR) for A. phagocytophilum with significant seasonal variation. All were PCR negative for Rickettsia helvetica. PCR and sequencing also showed a novel bacterium in roe deer previously only found in ticks. The study showed that the emerging pathogen A. phagocytophilum is widely distributed and that a marked shift has occurred in the distribution of TBE-complex virus in Denmark. This finding supports studies that predict alterations in distribution due to climatic changes.

Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks

Borreliosis is an endemic infection in Denmark. Recent serosurveys have indicated that human anaplasmosis may be equally common. The aim of this study was to look for Anaplasma phagocytophilum and related pathogens in Ixodes ricinus ticks and estimate their prevalence, compared to Borrelia, using PCR. Ticks were collected from three locations in Denmark: Jutland, Funen, and Bornholm. Ticks from Jutland and Funen were analysed individually, ticks from Bornholm were analysed in pools of 20. A. phagocytophilum was found in ticks from all areas. A. phagocytophilum was found in 23.6% of ticks from Jutland and Funen, while 11% were positive for Borrelia burgdorferi. The Borrelia genotype B. afzelii was most prevalent, followed by B. valaisiana, B. burgdorferi s.s. and B. garinii.A. phagocytophilum was found in 14.5% of nymphs and 40.5% of adult ticks, while Borrelia was found in 13% of nymphs and 8% of adult ticks. The difference in prevalence between Anaplasma and Borrelia in adult ticks supports the idea that their maintenance cycles in nature may be different. Ticks were also infected with Rickettsia helvetica. Our study indicates that A. phagocytophilum prevalence in ticks in Denmark is as high as Borrelia prevalence and that human anaplasmosis may be unrecognized.

Seroprevalence of Human Granulocytic Ehrlichiosis in High-risk Groups in Denmark

The aim of this study was to evaluate the seroprevalence of human granulocytic ehrlichiosis (HGE) among 300 residents in the county of Funen, Denmark. All of these people had either suspected or confirmed borreliosis. Two hundred control sera were included in the study. Samples were submitted by general practitioners and by hospital departments. An indirect immunofluorescence assay was used to identify sera reactive to HGE and an enzyme-linked immunosorbent assay was used to analyse Borrelia burgdorferi antibodies. There were 63 (21%) HGE-positive sera, 53 of which came from Borrelia-seropositive patients. Among patients with negative Borrelia serology, but with clinical suspicion of borreliosis, 14.3% were HGE-positive (n = 70). Of the 200 control sera, 3.5% were HGE-positive and 10.5% were Borrelia-positive. No HGE-positive samples were found among subjects < 20 y of age, wheras 20.4% of Borrelia seropositive samples where from subjects < 20 y of age. No mortality was observed in the HGE-positive group and the percentage of serum samples positive for both Borrelia and HGE did not differ significantly between hospitalized and non-hospitalized patients. Our study indicates that HGE infection with or without concomitant or previous Borrelia burgdorferi infection is common in tick-exposed individuals > 20 y old in the county of Funen, Denmark.The aim of this study was to evaluate the seroprevalence of human granulocytic ehrlichiosis (HGE) among 300 residents in the county of Funen, Denmark. All of these people had either suspected or confirmed borreliosis. Two hundred control sera were included in the study. Samples were submitted by general practitioners and by hospital departments. An indirect immunofluorescence assay was used to identify sera reactive to HGE and an enzyme-linked immunosorbent assay was used to analyse Borrelia burgdorferi antibodies. There were 63 (21%) HGE-positive sera, 53 of which came from Borrelia-seropositive patients. Among patients with negative Borrelia serology, but with clinical suspicion of borreliosis, 14.3% were HGE-positive (n = 70). Of the 200 control sera, 3.5% were HGE-positive and 10.5% were Borrelia-positive. No HGE-positive samples were found among subjects < 20 y of age, wheras 20.4% of Borrelia seropositive samples where from subjects < 20 y of age. No mortality was observed in the HGE-positive group and the percentage of serum samples positive for both Borrelia and HGE did not differ significantly between hospitalized and non-hospitalized patients. Our study indicates that HGE infection with or without concomitant or previous Borrelia burgdorferi infection is common in tick-exposed individuals > 20 y old in the county of Funen, Denmark.

Utilization of serology for the diagnosis of suspected Lyme borreliosis in Denmark: Survey of patients seen in general practice

BackgroundSerological testing for Lyme borreliosis (LB) is frequently requested by general practitioners for patients with a wide variety of symptoms.MethodsA survey was performed in order to characterize test utilization and clinical features of patients investigated for serum antibodies to Borrelia burgdorferi sensu lato. During one calendar year a questionnaire was sent to the general practitioners who had ordered LB serology from patients in three Danish counties (population 1.5 million inhabitants). Testing was done with a commercial ELISA assay with purified flagella antigen from a Danish strain of B. afzelii.ResultsA total of 4,664 patients were tested. The IgM and IgG seropositivity rates were 9.2% and 3.3%, respectively. Questionnaires from 2,643 (57%) patients were available for analysis. Erythema migrans (EM) was suspected in 38% of patients, Lyme arthritis/disseminated disease in 23% and early neuroborreliosis in 13%. Age 0-15 years and suspected EM were significant predictors of IgM seropositivity, whereas suspected acrodermatitis was a predictor of IgG seropositivity. LB was suspected in 646 patients with arthritis, but only 2.3% were IgG seropositive. This is comparable to the level of seropositivity in the background population indicating that Lyme arthritis is a rare entity in Denmark, and the low pretest probability should alert general practitioners to the possibility of false positive LB serology. Significant predictors for treating the patient were a reported tick bite and suspected EM.ConclusionsA detailed description of the utilization of serology for Lyme borreliosis with rates of seropositivity according to clinical symptoms is presented. Low rates of seropositivity in certain patient groups indicate a low pretest probability and there is a notable risk of false positive results. 38% of all patients tested were suspected of EM, although this is not a recommended indication due to a low sensitivity of serological testing.

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.

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.

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.

Rickettsioses in Denmark: A retrospective survey of clinical features and travel history

Rickettsia spp. can be found across the globe and cause disease of varying clinical severity, ranging from life-threatening infections with widespread vasculitis to milder, more localized presentations. Vector and, to some degree, reservoir are hematophagous arthropods, with most species harboured by ticks. In Denmark, rickettsiae are known as a cause of imported travel-related infections, but are also found endemically in ticks across the country. Data are, however, lacking on the geographical origin and clinical features of diagnosed cases. In this study, we have examined the travel history and clinical features of two groups of patients; 1) hospital-patients diagnosed with rickettsioses in the years 2010-2015 and 2) patients from primary health care (PHC) centers in Denmark having demonstrated anti-rickettsia antibodies in the years 2012-2015. The patients were identified using the Danish National Patient Registry (DNPR) and through the serological database at the State Serum Institute, where the laboratory diagnosis of rickettsioses is currently centralized. Data were collected for 86 hospital patients and 26 PHC center patients by reviewing hospital medical records and performing telephone interviews with PHC centers. Of the hospital patients, 91% (78/86) had a history of international travel 14 days prior to symptom start, with most having imported their infection from southern Africa, South Africa in particular (65%), and presenting with a clinical picture most compatible with African tick-bite fever caused by R. africae. Only two patients presented with a CRP > 100 mg/L and no mortalities were reported. At the PHC centers, most patients presented with mild flu-like symptoms and had an unknown (50%) or no history (19%) of international travel, raising the possibility of endemic rickettsioses. In view of our findings, rickettsioses do not appear to constitute a major public health problem in Denmark, with most cases being imported infections and potential endemic cases presenting as mild infections.