Tereza Rojko

Epidemiological, clinical and laboratory characteristics of patients with human granulocytic anaplasmosis in Slovenia

SummaryHuman granulocytic anaplasmosis (HGA) has been recently recognized as an emerging tick-borne disease. Several reports indicate the presence of infection with Anaplasma phagocytophilum in Europe. Between January 1996 and December 2004, 24 adult patients with proven HGA were identified in a prospective study conducted at the Department of Infectious Diseases, University Medical Center Ljubljana, Slovenia, on the etiology of febrile illnesses occurring within 30 days after a tick bite. The diagnosis of acute HGA was established from seroconversion in 18 (75%) patients or at least a four-fold increase in antibody titers to A. phagocytophilum antigens in six (25%) patients and molecular identification of ehrlichial organisms in 15 (62.5%) patients. Clinical characteristics and laboratory findings were similar to those reported from the other European countries. All the patients had an acute febrile illness with headache, malaise, myalgia and/or arthralgia. Leukopenia was found in 16 (66.7%) patients, thrombocytopenia in 20 (83.3%), abnormal liver function test results in 23 (95.8%), elevated erythrocyte sedimentation rates in 18 (75%), and elevated concentration of C-reactive protein in 23 (95.8%). The disease course was relatively mild; none of the patients died and no long-term sequelae were found during a follow-up of one year even though only 15 (62.5%) were treated with doxycycline. At the examination one year after the first visit, 16/24 (66.7%) patients tested seropositive (≥1 : 256) for A. phagocytophilum antibody, and two years after the first visit positive titers were still present in 10/18 (55.6%) patients.

Prevalence and incidence of Lyme borreliosis among Slovene forestry workers during the period of tick activity.

SummaryOBJECTIVES: To establish the prevalence and incidence of symptomatic and asymptomatic infection with Borrelia burgdorferi sensu lato during the period of tick activity, to compare the risk of infection with B. burgdorferi s.l. for forestry workers and indoor workers in Slovenia, and to compare the outcome of an in-house immunofluorescent assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA). METHODS: The study included 122 forestry workers; the control group consisted of 93 indoor workers. All participants were examined twice in 2002: before the beginning of tick activity (March) and at the end of tick activity (November). At each examination, principal demographic and epidemiological data were collected and a blood sample taken for serological analysis. Specific IgM and IgG antibodies against B. burgdorferi s.l. in the paired sera were determined with an in-house IFA and a commercially available ELISA flagellin test (DAKO). RESULTS: 9.8% of the forestry workers and 4.3% of the indoor workers tested positive for IgG with the IFA (p = 0.26); 23.8% of the forestry workers and 9.7% of the indoor workers tested positive for IgG with the ELISA (p = 0.02). During the study period the incidence of symptomatic Lyme borreliosis was 2.3% and the rate of IgG and/or IgM seroconversion of 10.2% was the same with both tests. CONCLUSIONS: The seroprevalence of antibodies against B. burgdorferi s.l. among the Slovene forestry workers was greater than among the indoor workers, but the difference between the two groups was not significant when the IFA was used. The incidence of Lyme borreliosis during the period of tick activity was lower than we expected, with a large proportion of seroconversions being asymptomatic.

Clinical Characteristics Associated with Borrelia burgdorferi Sensu Lato Skin Culture Results in Patients with Erythema Migrans

Clinical characteristics associated with isolation of Borrelia burgdorferi sensu lato from skin have not been fully evaluated. To gain insight into predictors for a positive EM skin culture, we compared basic demographic, epidemiologic, and clinical data in 608 culture-proven and 501 culture-negative adult patients with solitary EM. A positive Borrelia spp. skin culture was associated with older age, a time interval of >2 days between tick bite and onset of the skin lesion, EM ≥5 cm in diameter, and location of the lesion on the extremities, whereas several other characteristics used as clinical case definition criteria for the diagnosis of EM (such as tick bite at the site of later EM, information on expansion of the skin lesion, central clearing) were not. A patient with a 15-cm EM lesion had almost 3-fold greater odds for a positive skin culture than patients with a 5-cm lesion. Patients with a free time interval between the tick bite and onset of EM had the same probability of a positive skin culture as those who did not recall a tick bite (OR=1.02); however, the two groups had >3-fold greater odds for EM positivity than patients who reported a tick bite with no interval between the bite and onset of the lesion. In conclusion, several yet not all clinical characteristics used in EM case definitions were associated with positive Borrelia spp. skin culture. The findings are limited to European patients with solitary EM caused predominantly by B. afzelii but may not be valid for other situations.

Seroprevalence of human anaplasmosis in slovene forestry workers.

Abstract:  The aim of the present study was to establish the prevalence and incidence of symptomatic and asymptomatic infection with Anaplasma phagocytophilum during the period of tick activity and to compare the risk of infection for forestry workers and indoor workers in Slovenia.

Pathogenetic implications of the age at time of diagnosis and skin location for acrodermatitis chronica atrophicans

BACKGROUND The pathogenesis of acrodermatitis chronica atrophicans (ACA) is not well understood. OBJECTIVE The purpose of this study was to gain a better understanding of ACA by utilizing a large data set of adult Slovenian patients with Lyme borreliosis. METHODS The age of 590 ACA patients was compared with that of patients with other manifestations of Lyme borreliosis. The location of the ACA lesion on the body was compared with that of erythema migrans (EM). RESULTS Patients diagnosed with ACA were on average 14.3 years older than patients with EM (p<0.001). ACA patients were also significantly older than patients with Lyme neuroborreliosis or Lyme arthritis (p<0.001). The average delay in diagnosis of ACA was 1.6 years (range 0.1-20 years). For 572 (96.9%) of the ACA patients, the site of the skin lesion(s) was confined to an extremity vs. 79.6% for patients with EM, p<0.001. For the 20 ACA patients who reported a preceding untreated EM lesion at the same body site, the mean time between the development of the EM and the onset of ACA was 3.0±4.4 (median 1.3, range 0.1-15.0) years. CONCLUSIONS ACA is more likely to be diagnosed in older individuals than any other manifestation of Lyme borreliosis. ACA is more likely than EM to be localized anatomically to the extremities. Available data favor the hypothesis that ACA occurs most often on the extremities of older individuals because of predisposing age-related anatomic or physiologic changes, but more data are needed to define the latency period and other aspects of the pathogenesis of this skin condition.

Oral doxycycline versus intravenous ceftriaxone for treatment of multiple erythema migrans: an open-label alternate-treatment observational trial

Background Several guidelines advocate the same treatment approaches for both early disseminated Lyme borreliosis, manifested as multiple erythema migrans (EM), and early localized Lyme borreliosis, manifested as solitary EM. Methods Oral doxycycline (100 mg q12h) was compared on a non-inferiority premise with intravenous ceftriaxone (2 g q24h) for 14 days in 200 adult European patients with multiple EM in an open-label alternate-treatment observational trial performed in a single-centre university hospital. Treatment outcome was assessed at 14 days and at 2, 6 and 12 months post-enrolment. Non-specific symptoms in patients and 192 control subjects without a history of Lyme borreliosis were evaluated and compared. This trial was registered at http://clinicaltrials.gov (identifier NCT01163994). Results At the 12 month visit, 4/82 (4.9%) multiple EM patients prescribed doxycycline and 6/88 (6.8%) multiple EM patients prescribed ceftriaxone showed incomplete response manifested predominantly as post-Lyme symptoms (1.9% difference, upper limit of 95% CI 5.1%). The upper limit of 95% CI for the difference in proportion of patients with incomplete response between doxycycline and ceftriaxone groups did not exceed the predetermined non-inferiority margin of 10%. The frequency of non-specific symptoms in patients was similar to that in controls. Conclusions The 14 day oral doxycycline was not inferior to the 14 day intravenous ceftriaxone in treatment of adult European patients with early disseminated Lyme borreliosis manifested as multiple EM. The frequency of non-specific symptoms in patients was similar to that in controls without a history of Lyme borreliosis.

The long-term outcome of tick-borne encephalitis in Central Europe

BACKGROUND Information on the long-term outcome of tick-borne encephalitis (TBE) is limited. OBJECTIVES To assess the frequency and severity of post-encephalitic syndrome (PES) at different time points after TBE, and to determine the parameters associated with unfavourable outcome. METHODS Adult patients diagnosed with TBE in Slovenia in the period 2007-2012 were followed-up for 12 months and also examined 2-7 years after TBE. Each patient was asked to refer a person of similar age without a history of TBE to serve as control. RESULTS A total of 420 patients and 295 control persons participated in the study. The proportion of patients with PES (defined as the presence of ≥ 2 subjective symptoms that newly developed or worsened since the onset of TBE and which had no other known medical explanation, and/or ≥ 1 objective neurological sign) was higher (P < 0.001) at the follow-up visit 6 months after the acute illness (127/304, 42%, 95% CI: 36-47%) than at 12 months (68/207, 33%, 95% CI: 26-40%); the proportion at 12 months was the same as at 2-7 years after TBE (137/420, 33%, 95% CI: 28-37%). However, the proportion of severe PES at the last two time points differed (9.7% vs 4.3%, P = 0.008). Multivariate logistic regression showed that unfavourable outcome at 6 months was associated with CSF leukocyte count (OR = 1.003, 95% CI: 1.001-1.005%, P = 0.017), at 12 months with the disease outcome at 6 months (OR = 115.473, 95% CI: 26.009-512.667%, P < 0.001), and at the final visit with disease outcome at 6 months (OR = 3.808, 95% CI: 1.151-12.593%, P = 0.028) and 12 months (OR = 26.740, 95% CI: 8.648-82.680%, P < 0.001). Unspecific symptoms that occurred within the four weeks before the final examination were more frequent and more constant in patients than in the control group. CONCLUSIONS The frequency of PES diminished over time and stabilized 12 months after the acute illness, whereas the severity of PES continued to decline. Unfavourable outcomes at 12 months and at the final visit were strongly associated with the presence of PES at previous time points.

Comparison of MKP and BSK-H media for the cultivation and isolation of Borrelia burgdorferi sensu lato

The isolation of B. burgdorferi sensu lato requires the use of complex cultivation media. The aim of the study was to compare the usefulness of BSK-H (a commercial medium produced by HiMedia, India) and MKP medium. MKP and BSK-H media were prepared in accordance with the relevant protocols. Borrelia strains and skin culture biopsies were simultaneously inoculated into both media, incubated and checked for growth. Borrelial growth characteristics, isolation rates and characteristics of the isolated borreliae were analysed and compared. Initially, numbers of spirochaetes were higher in BSK-H than in MKP; however, in comparison with MKP, the strains subcultured in BSK-H medium were more frequently irregular, thin and non-motile, and rapidly died. In addition, the borrelial isolation rate from erythema migrans skin samples was higher in MKP than in BSK-H medium (108/171, 63.2% versus 70/171, 40.9%; p<0.0001). The far most frequently isolated species was Borrelia afzelii (92.9% and 97.2% strains isolated from BSK-H and MKP, respectively). Comparison of strains cultured from individual patients in both media showed differences in plasmid contents in 9/46 (19.6%) strain pairs, and protein profiles differed in 30/43 (69.8%) strain pairs, most often in the expression of OspC (in 27/28 patients OspC was expressed only in strains growing in MKP). BSK-H medium supports the growth of borrelial strains but MKP is superior with regard to the isolation rate, morphology and motility of strains. BSK-H medium supports fast initial growth of borreliae but this is followed by rapid deformation and death of the spirochaetes.

Are Patients with Erythema Migrans Who Have Leukopenia and/or Thrombocytopenia Coinfected with Anaplasma phagocytophilum or Tick-Borne Encephalitis Virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.

Impact of pre-existing treatment with statins on the course and outcome of tick-borne encephalitis

Objectives Although statins have anti-inflammatory and potentially also antimicrobial (including antiviral) activity, their therapeutic impact on infectious diseases is controversial. In this study, we evaluated whether pre-existing statin use influenced the course and outcome of tick-borne encephalitis. Methods To assess the influence of statin usage on the severity of acute illness and the outcome of tick-borne encephalitis, univariate and multivariable analyses were performed for 700 adult patients with tick-borne encephalitis of whom 77 (11%) were being treated with statins, and for 410 patients of whom 53 (13%) were receiving statins, respectively. Results Multivariable analyses found no statistically significant association between statin usage and having a milder acute illness. There was also no statistically significant benefit with respect to a favorable outcome defined by the absence of post-encephalitic syndrome (ORs for a favorable outcome at 6 months was 0.96, 95% CI: 0.46–2.04, P = 0.926; at 12 months 0.29, 95% CI: 0.06–1.33, P = 0.111; at 2–7 years after acute illness 0.44, 95% CI: 0.09–2.22, P = 0.321), by a reduction in the frequency of six nonspecific symptoms (fatigue, myalgia/arthralgia memory disturbances, headache, concentration disturbances, irritability) occurring during the 4 week period before the last examination, or by higher SF-36 scores in any of the eight separate domains of health as well as in the physical and mental global overall component. Furthermore, there were no significant differences between patients receiving statins and those who were not in the cerebrospinal fluid or serum levels for any of the 24 cytokines/chemokines measured. Conclusions In this observational study, we could not prove that pre-existing use of statins affected either the severity of the acute illness or the long-term outcome of tick-borne encephalitis.