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Human bocavirus is a recently described respiratory pathogen. A case of a life-threatening human bocavirus infection of a previously healthy pediatric patient is described. An initial clinical presentation of acute bronchiolitis developed into an extremely severe course of disease characterized by pneumothorax, pneumomediastinum, and acute respiratory failure with pronounced air-leak syndrome.

Ixodes ricinus is a vector of pathogens that cause many diseases in Europe and Slovenia: tick-borne encephalitis (TBE), anaplasmosis, borreliosis, babesiosis and others. The risk for contracting these diseases depends strongly on the density of the infected questing ticks and many studies have investigated tick population dynamics and the parameters affecting them. They have shown a clear influence of climatic and landscape arrangements in the microhabitat on tick abundance and dynamics and therefore on transmission of pathogens important in human and veterinary medicine. In our study we assessed the influence of climatic factors on questing activity of ticks over a three-year period at 7 locations in Slovenia. Locations were selected in endemic foci of TBE with different intensity, which were identified according to the presence of human disease. Sites differ according to various abiotic and biotic factors, such as climate, amount of rain, height above sea level, vegetation and wildlife. All three stages of ticks were collected monthly over a three-year period (2005-2007). Temperature, humidity and precipitation data were collected for these years. The purpose of our study was to relate observed differences in I. ricinus ticks questing activity to local climate. We found a correlation between the decrease of questing ticks in the summer and the combination of air temperatures and humidity in the form of saturation deficit.

Aim: Urinary incontinence is a classical sign of childbirth-related perineal trauma, the prevalence of which is estimated, depending on age, at between 10% and 50%. Pelviperineal rehabilitation offers an excellent opportunity to prevent pelviperineal disorders. The aim of the present study was to evaluate the prevalence of urinary incontinence and pelviperineal disorders in the local context, and also that of pelviperineal rehabilitation., Methods: Of the 1793 women who gave birth in Udine in 2006, 900 primipara and secondipara Caucasians with single term pregnancies dated ultrasonically to be within 20 weeks of gestation, were selected. A total of 602 of them were contacted by telephone and two questionnaires were administered., Results: The prevalence of pelviperineal rehabilitation in our population was 4.49%, while that of pelviperineal disorders was much higher, at around 40.20%. The prevalence of urinary incontinence was due in 27.57% of cases to stress and in 14.45% to urgency, with an overlap in 9.8% of cases and a story of prior incontinence in 9.97%. The prevalence of urinary urgency in women subjected to rehabilitation is significantly lower than in those not treated (P=0.004). Dyspareunia represents 16.11% of cases, coital incontinence 0.33%. One case of gas incontinence emerged but there was no case of faecal incontinence., Conclusions: Pelviperineal rehabilitation in the observed population has a very low prevalence, especially if compared with the high prevalence of disturbances related to dysfunctions of the pelvic floor during postpartum.

The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF) endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti) isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01). This suggests that distinct virus strains can coexist in highly endemic areas.

We used quantitative real-time reverse transcription-PCR to measure viral load in serum from 24 patients in Kosovo who had acute Crimean-Congo hemorrhagic fever. Viral load correlated with clinical disease and antibodies and could be used as a predictor of disease outcome.

Anaplasma phagocytophilum is a widely distributed tick-borne pathogen of humans, livestock, and companion animals. We used in silico methods to identify 10 variable-number tandem-repeat (VNTR) loci within the genome sequence of the A. phagocytophilum HZ strain and used these data to develop a multilocus VNTR-based typing scheme for the species. Having confirmed the stability of four of the loci in replicates of the A. phagocytophilum strain that had been subjected to different numbers of passages through cell cocultures in vitro, we then used this typing scheme to discriminate between 20 A. phagocytophilum strains of diverse geographical and host provenances. Extensive diversity was found at each of the four loci studied, with total allele numbers ranging from 13 to 18 and Hunter-Gaston discriminatory index values ranging from 0.93 to 0.99. Only 2 of the 20 strains examined shared alleles at all four loci. The discriminatory power of VNTR analysis was found to be greater than that of either partial msp4 or 16S rRNA gene sequence comparison. The extremely high sensitivity of this novel approach to the genetic fingerprinting of A. phagocytophilum strains should serve well in molecular epidemiological studies of infection transmission, particularly when fine-scale strain delineation is required.

Rodents, collected in three zoogeographical regions across Slovenia, were tested for the presence of bartonellae using direct PCR-based amplification of 16S/23S rRNA gene intergenic spacer region (ITS) fragments from splenic DNA extracts. Bartonella DNA was detected in four species of rodents, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius and Clethrionomys glareolus, in all three zoogeographic regions at an overall prevalence of 40.4%. The prevalence of infection varied significantly between rodent species and zoogeographical regions. Comparison of ITS sequences obtained from bartonellae revealed six sequence variants. Four of these matched the ITS sequences of the previously recognized species, Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and Bartonella birtlesii, but one was new. The identity of the bartonellae from which the novel ITS sequences was obtained were further assessed by sequence analysis of cell division protein-encoding gene (ftsZ) fragments. This analysis demonstrated that the strain is most likely a representative of possible new species within the genus.

In Slovenia, the co-existence of Dobrava and Puumala (PUUV) hantaviruses in a single endemic region has been demonstrated. This study presents selected Slovenian HFRS cases caused by PUUV combined with genetic analysis of viral genome sequences recovered from clinical specimens and tissue samples of Clethrionomys glareolus (bank voles). Serum samples from nine HFRS patients were included in the study. Rodents study sites were selected with regard to the HFRS cases. Partial S segment sequences were recovered from all nine patients and partial M segment sequences could be recovered from seven. Partial S and M segments sequences were also recovered from five C. glareouls captured at three different study sites. The sequences from Slovenian clinical specimens and rodent tissue samples belonged to the PUUV genotype and formed a distinct genetic lineage of PUUV. Human and rodent PUUV sequences located in the closest proximity to each other on the phylogenetic trees suggest genetic links between the human cases and the hantaviral strains circulating in natural foci of this zoonotic infection. Analysis of the complete S segment sequences recovered for two wild-type PUUV strains confirmed the existence of a distinct genetic lineage and also indicated a possible quasispecies type of Slovenian PUUV.

Human 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 (> or =1 : 256) for A. phagocytophilum antibody, and two years after the first visit positive titers were still present in 10/18 (55.6%) patients.

We report five consecutive cases of Anaplasma (A.) phagocytophilum infection (the causative agent of human granulocytic anaplasmosis (HGA)) from western Austria. All infections were acquired between June and August in 2003 and 2004 in the Inn valley (Tyrol, Austria). Four patients required hospitalisation, one patient was treated as an outpatient. During the acute stage of illness, laboratory findings included thrombocytopenia (5/5), elevated C-reactive protein (5/5), elevated neopterin (5/5), elevated lactate dehydrogenase (4/5), and elevation of liver enzymes (4/5). Leukopenia (3/5) and elevated procalcitonin (2/5) were less frequently observed. All patients were treated with tetracyclines, which led to prompt improvement of the clinical conditions. Anti-platelet antibodies were observed in one of four patients, but remained unchanged after complete covalescence.

Early and accurate diagnosis of Crimean-Congo hemorrhagic fever (CCHF) is essential for the treatment and outcome of the disease and prevention of its further transmission. Molecular-based diagnostic assays now serve as the front-line tool in the diagnosis of CCHF. However, the development of real-time RT-PCR assay for the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) has been hampered by a virus strain variation. The development of a one-step real-time RT-PCR assay for the detection of CCHFV is described herein. The technique is based on the fluorescence resonance energy transfer probe technology employing the endonuclease activity of Taq polymerase enzyme. The assay was designed to detect specifically the strains from a phylogenetic cluster of CCHFV which encompasses the known CCHFV strains circulating in the Balkan region. The detection system was tested using CCHFV strain Kosovo Hoti, clinical serum samples and ticks. The real-time assay described is rapid, specific and sensitive. Since the Balkan peninsula is also an endemic region for hemorrhagic fever with renal syndrome (HFRS), this method is suggested as convenient for early differential diagnosis of suspected viral hemorrhagic fever patients.

Background: Tick-borne encephalitis virus (TBEV) is one of the most important causes of human viral infections of the central nervous system in Europe. Currently, the diagnosis of TBE is based on the demonstration of specific antibodies in patient's serum, which appear only several weeks after the infection., Objective: To determine how successfully can viral RNA be detected by RT-PCR in the samples of body fluids of patients with TBE prior to and after the appearance of antibodies., Study Design: Serum, whole blood and CSF samples from 34 patients with a serologically confirmed TBE were collected. Samples were tested for the presence of TBEV RNA by using RT-PCR method., Results: Viral RNA was detected in all blood and serum samples collected before the development of antibodies. After the appearance of IgM antibodies, the number of positive samples dropped by at least one third. After the development of IgG antibodies, only 3% of serum and 16% of blood samples tested positive for viral RNA. Samples of cerebrospinal fluid were shown to be inappropriate for the molecular diagnosis of TBE using this assay, since only one sample (10%) that was collected in the sero-negative phase of disease was found positive by the PCR assay., Conclusions: RT-PCR is an efficient method for an early detection of TBEV in blood and serum samples collected prior to the appearance of antibodies. This method can be of valuable use for a differential diagnosis of TBEV infection in patients with febrile illness after a tick bite, particularly in regions where more than one tick-transmitted diseases are endemic.

We describe cervids as potential reservoir hosts of Babesia EU1 and B. divergens. Both babesial parasites were found in roe deer. Sequence analysis of 18S rRNA showed 99.7% identity of roe deer Babesia EU1 with the human EU1 strain. B. divergens detected in cervids was 99.6% identical to bovine B. divergens.

New cases of human babesiosis were recently reported in Europe. The etiological agent was identified as Babesia EU1, a zoonotic pathogen with previously unreported molecular characteristics. On the basis of a comparison of the complete babesial 18S rRNA gene, we have generated strong molecular evidence that Ixodes ricinus ticks from Slovenia are infected with EU1.

We report a patient with febrile illness and epidemiologic and clinical findings consistent with human granulocytic ehrlichiosis and tickborne encephalitis, in whom infection with Anaplasma phagocytophilum was demonstrated by polymerase chain reaction and seroconversion. Tickborne encephalitis virus infection was established by serum immunoglobulin (Ig) M and IgG antibodies.

Canine babesiosis, caused by intraerythrocytic Babesia spp., is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Slovenia. Therefore, 238 dogs admitted to the Small animal clinic in Ljubljana from the years 2000 to 2002 were tested for the presence of babesial parasites in the blood. Based on clinical, microscopic and molecular investigations, 14 dogs (5.9%) were determined as being infected with babesiae. Clinical signs relating to acute haemolysis, fever, anorexia, depression and haematological abnormalities such as anaemia and thrombocytopenia were noticed in most of the 14 infected dogs. The morphology of the parasites was indicative of Babesia canis infection. Two subspecies were detected, namely B. canis canis (11 dogs, 4.6%) and B. canis vogeli (3 dogs, 1.3%) using PCR and subsequent sequence analysis of portions of nns rRNA gene. In addition, based on nucleotide sequence analysis, the 11 isolates of B. c. canis could be subdivided into three groups, whereas the three B. c. vogeli isolates were genetically identical. The results of this study demonstrate the presence of canine babesiosis due to B. c. canis and B. c. vogeli in Slovenia., (Copyright 2004 INRA, EDP Sciences)

In order to assess the validity of definitions for human granulocytic ehrlichiosis (HGE), epidemiological, clinical and laboratory findings for 13 patients with confirmed HGE and 21 patients fulfilling criteria for probable HGE were compared. The patients were identified during a prospective study on the aetiology of febrile illness occurring after a tick bite, performed in Slovenia from 1995 to 2002. Significant differences between the 2 groups were found for age of patients, duration of fever before the first examination, and several clinical and laboratory parameters including the occurrence of chills, myalgia, arthralgia, leukopenia, thrombocytopenia, abnormal liver function tests results, and elevated concentration of C-reactive protein. All the abnormalities were established more often in patients with confirmed HGE than in those with probable HGE. Patients with confirmed HGE exhibited higher geometric mean titre values and higher peak antibody titres to Anaplasma phagocytophilum that developed later in the course of their illness. The differences indicate that in some patients fulfilling criteria for probable HGE the signs and symptoms most probably are not the result of a recent infection with A. phagocytophilum.

Background: To establish the etiology in Slovenian children with febrile illnesses occurring after a tick bite., Methods: Eighty-six febrile patients younger than 15 years referred to our institution in 2001 with a history of a tick bite within 6 weeks before onset of the illness were included in this prospective study. Acute and convalescent serum samples were tested for the presence of antibodies to tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Rickettsia conorii, Babesia microti, Bartonella henselae, Bartonella quintana and Francisella tularensis. Cerebrospinal fluid was investigated in patients in whom meningeal involvement was clinically suspected. Blood and/or cerebrospinal fluid from the patients were cultured in modified Kelly-Pettenkofer medium. PCR was performed to detect ribosomal DNA of A. phagocytophilum and E. chaffeensis., Results: Of 86 patients 33 (38%) were excluded because a well-defined febrile illness not associated with tick bite was established. Tick-borne illness was diagnosed in 28 (53%) of the 53 remaining patients. The most common diagnosis was tick-borne encephalitis (64%), followed by Lyme borreliosis (46%), human monocytic ehrlichiosis and human granulocytic ehrlichiosis (serologic evidence of infection in 9 and 4%, respectively). In 6 (21%) patients there was evidence for infection with more than 1 tick-borne agent., Conclusions: Tick-borne illness was established in 53% of the patients younger than 15 years presenting with febrile illness occurring within 6 weeks after a tick bite. The most common identified illnesses were tick-borne encephalitis and Lyme borreliosis.

Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at -70 degrees C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 x 10(6) neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.

Several reports indicate the presence of infection with Anaplasma phagocytophila in Europe. The first case with confirmed human granulocytic ehrlichiosis was reported from Slovenia in 1997. By 2002, 13 patients have been discovered in a prospective study on the etiology of febrile illnesses occurring within six weeks following a tick bite, conducted at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia. The diagnosis was established by molecular identification of ehrlichial organisms and/or by seroconversion to A. phagocytophila. None of the patients had detectable morulae on blood smear examination. Clinical characteristics and laboratory findings were similar to those reported from the United States. The severity of the illness was mild to moderately severe. All patients recovered rapidly and without sequelae. HGE should be included in the differential diagnosis of febrile illnesses occurring after a tick bite in Slovenia.

Objective: To establish epidemiological, clinical and laboratory differences between adult patients with proven acute human granulocytic ehrlichiosis (HGE) and patients with the initial phase of tick-borne encephalitis (TBE)., Methods: Epidemiological, clinical and laboratory findings were compared in nine patients with acute HGE (established by the presence of specific DNA sequences of Ehrlichia phagocytophila in whole blood by polymerase chain reaction and/or by seroconversion or at least a four-fold change of serum antibody titres to E. phagocytophila by indirect immunofluorescence assay) and 29 patients with the initial phase of TBE (demonstrated in ELISA tests by the presence of serum IgM antibodies to TBE virus)., Results: Findings were similar for the majority of the general parameters examined, which included sex, age, incubation period, duration of fever, and frequency of additional tick bites. However, a statistically significant difference was found for the duration of fever before the first visit (median 7 days in patients with acute HGE and 4 days in patients with the initial phase of TBE; p = 0.0169), and for the frequency of hospitalisation, which was higher for patients with the initial phase of TBE (29/29) than for those with acute HGE (5/9; p = 0.0017). Comparison of clinical signs and symptoms and laboratory findings in the two groups revealed significant differences: in patients with acute HGE there was a higher frequency of chills (4/9 versus 3/29, respectively; p = 0.0407), myalgia (8/9 versus 13/29, respectively; p = 0.0263), and arthralgia (7/9 versus 3/29, respectively; p = 0.0003), and elevated values of lactate dehydrogenase (7/9 versus 3/29, respectively; p = 0.0003) and concentration of C-reactive protein (9/9 versus 6/29, respectively; p = 0.0000)., Conclusions: In a patient with febrile illness occurring after a tick bite, a clinical report of chills, myalgia and arthralgia, and laboratory findings of elevated values for lactate dehydrogenase and concentration of C-reactive protein direct towards a diagnosis of acute HGE rather than the initial phase of TBE.

Objective: Human granulocytic ehrlichiosis (HGE) was recently recognized as an emerging tick-borne infection in Europe. The disease is caused by Anaplasma (previously Ehrlichia) phagocytophila. The first confirmed acute human disease caused by A. phagocytophila was reported from Slovenia in 1998. The tick Ixodes ricinus was identified as the likely vector for this pathogen of humans and animals in Europe. In order to assess the possibility that roe and red deer in Slovenia serve as potential reservoir hosts for A. phagocytophila, materials from both species were examined., Methods: Samples were obtained from 32 red deer (Cervus elaphus) and 56 roe deer (Capreolus capreolus). Polyvalent antibodies to the USG3 isolate of Anaplasma phagocytophila were detected by indirect immunofluorescence assay (IFA). DNA was extracted from spleen tissue. The 16S rRNA gene and a portion of the groESL heat shock operon were used for PCR detection and subsequent direct sequencing of amplified products., Results: Serological and PCR results indicated that high proportions of roe and red deer were infected with A. phagocytophila. Infection was confirmed in 74% of the animals by IFA and in 86% of animals by PCR. While similar prevalences by PCR were seen in the two species (approximately 86%), the prevalence of antibodies was much higher in roe deer (94% vs. 35% in red deer). Sequence analysis of a 1256-bp fragment of the groESL operon revealed genetic diversity among collected samples. Identity of sequences ranging from 98% to 100%. None of the A. phagocytophila groESL and 16S rRNA gene sequences from red or roe deer were identical to the sequences previously obtained from human patients with ehrlichiosis from Slovenia or elsewhere in the world. All red deer sequences clustered with those obtained from humans, whereas all but two sequences from roe deer clustered separately., Conclusions: The results of our study indicate that a high percentage of red deer and roe deer in Slovenia are infected with A. phagocytophila. Analysis of groESL and 16S rRNA gene sequences revealed two distinct genetic lineages. Among deer, one variant was primarily associated with roe deer. Although none of the sequences from red deer was identical to those found in humans, they were more closely related.

A randomized clinical trial was undertaken to compare clinical and serologic parameters of the efficacy of one-dose azithromycin in the prophylaxis of Mediterranean spotted fever (MSF) in 122 persons with a tick bite history. Antibodies to Rickettsia conorii (R. conorii) were detected by indirect immunofluorescence (IFA) assay in 55 (45.1%) of 122 subjects. Positive result was obtained in 19 (31.1%) of 61 subjects with azithromycin prophylaxis and in 36 (59.0%) of 61 subjects without prophylaxis. Clinical signs and symptoms of MSF were recorded in 6 (9.8%) and asymptomatic infection in 30 (49.2%) of 61 subjects without prophylaxis. In subjects with prophylaxis, clinical signs of the disease were not recorded at all, whereas asymptomatic infection was detected in 19 (31.1%) of 61 subjects. Based on the results obtained in our study, we assume that a single dose of azithromycin is promising in MSF prevention.

A total of 197 ticks belonging to four species (Haemaphysalis punctata, Hyalomma marginatum, Rhipicephalus bursa and Dermacentor marginatus) collected in October 2000 from domestic animals in southern Croatia were examined for the presence of rickettsiae by molecular techniques. Specific sequences of the rickettsiae were detected in 25 (12.7%) of ticks tested. The prevalence of infection in D. marginatus and H. marginatum ticks was 36.8 and 64.7%, respectively. None of the ticks belonging to the species H. punctata or Rh. bursa were infected. Sequence analysis of amplified products revealed that D. marginatus ticks are infected with Rickettsia slovaca, whereas H. marginatum are infected with R. aeschlimannii. The results of this study extend the knowledge of the geographic distribution of SFG rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in ticks in southern Croatia.

Herein we report on the first confirmed pediatric case of acute human granulocytic ehrlichiosis in Europe. Presentation in this 11-year-old girl was comparable to clinical findings seen in adult European patients with human granulocytic ehrlichiosis; i.e., she had self-limited febrile illness with leukopenia, thrombocytopenia, and elevated serum C-reactive protein concentration. It is of interest that the patient not only had a fourfold change in antibody titer to Ehrlichia phagocytophila but also developed antibodies to Ehrlichia chaffeensis and that her PCR test result was positive on the third as well as on the 22nd day after the onset of illness, that is, 16 days after spontaneous defervescence.

We report a case involving a 15-year-old girl with atypical, clinically unsuspected cat scratch disease (CSD) presenting as isolated posterior pancreatic duodenal lymphadenitis, fever, and abdominal pain. The serological, abdominal ultrasonographic, and CT findings, as well as clinical and epidemiological data, indicate that B. henselae was likely an etiologic agent of CSD in our patient.

A prospective study established the etiology of febrile illnesses in residents of Slovenia that occurred within 6 weeks after a tick bite. A combination of laboratory and clinical criteria identified 64 (49.2%) of 130 patients as having confirmed, probable, or possible cases of tickborne disease during 1995 and 1996. Of the 130 patients, 36 (27.7%) had laboratory evidence of tickborne encephalitis, all of whom had clinically confirmed disease. Evidence of infection with Borrelia burgdorferi sensu lato was identified in 26 patients; 10 (7.7%) had confirmed Lyme borreliosis. Of 22 patients with evidence of Ehrlichia phagocytophila infection, 4 (3.1%) had confirmed ehrlichiosis. Infection by multiple organisms was found in 19 (14.6%) of 130 patients. Patients with meningeal involvement (43 [72.3%] of 59) were more likely to have confirmed tickborne disease than were patients with illness of undefined localization (18 [26.5%] of 68; P<.0001). Tickborne viral and bacterial infections are an important cause of febrile illness in Slovenia.

Background: Little is known about the seroprevalence of ehrlichiosis in adults and much less about the same in children., Methods: One hundred and forty-three healthy children and young adults (6-24 years of age, male to female ratio, 1:1) were assessed for the presence of antibodies to the agents of human granulocytic ehrlichiosis (HGE), human monocytic ehrlichiosis (HME), Borrelia burgdorferi sensu stricto (BB), and tick-borne encephalitis (TBE) virus in Slovenia, where tick-related infections are endemic. Antibodies to HGE and HME agents were assayed by indirect immunofluorescence, and antibodies to BB and TBE by enzyme-linked immunosorbent assay. A questionnaire about tick exposure was answered by all subjects. In the event of a positive result, a detailed interview was conducted., Results: Of 143 study subjects, 22 (15.4%) had detectable antibodies to HGE agent, 22 (15.4%) were positive to BB, 18 (12.6%) were positive to TBE virus (12 of these were vaccinated) and 4 (2.8%) were positive to the HME agent. The history of persons seropositive to an HGE agent had been uneventful., Conclusions: Our study documents a high seroprevalence of HGE in children and young adults in Slovenia, similar to the seroprevalence of LB and higher than that of TBE and HME. Although the majority of these infections are probably asymptomatic or mild, active surveillance for acute HGE infections in children in areas endemic for tick-related infections is necessary.

Genetic analysis was performed of wild-type (wt) Dobrava hantavirus (DOB) strains from Slovenia, the country where the virus was first discovered and where it was found to cause haemorrhagic fever with renal syndrome (HFRS), with a fatality rate of 12%. Two hundred and sixty mice of the genus APODEMUS:, trapped in five natural foci of DOB-associated HFRS during 1990-1996, were screened for the presence of anti-hantavirus antibodies and 49 APODEMUS: flavicollis and four APODEMUS: agrarius were found to be positive. RT-PCR was used to recover partial sequences of the wt-DOB medium (M) and small (S) genome segments from nine A. flavicollis and one A. agrarius. Sequence comparison and phylogenetic analysis of the Slovenian wt-DOB strains revealed close relatedness of all A. flavicollis-derived virus sequences (nucleotide diversity up to 6% for the M segment and 5% for the S segment) and the geographical clustering of genetic variants. In contrast, the strain harboured by A. agrarius showed a high level of genetic diversity from other Slovenian DOB strains (14%) and clustered together on phylogenetic trees with other DOB strains harboured by A. agrarius from Russia, Estonia and Slovakia. These findings suggest that the DOB variants carried by the two species of APODEMUS: in Europerepresent two distinct genetic lineages.

The first lethal case of Mediterranean spotted fever in Croatia is described. A previously healthy, 58-year-old man was admitted to the hospital with high fever and rash. Several days later, severe anemia, leukopenia and thrombocytopenia developed, and the patient died about eight weeks after the onset of disease. Bone marrow biopsy showed hypercellularity of the cortical and trabecular bone structures with morphologically normal cells of all three hematopoietic lineages that were reduced due to regular nodular infiltrates. Serologic findings indicated that Rickettsia conorii infection was the etiologic cause of the patient's death.

Thirty-four small mammals collected in the vicinity of Ljubljana were tested for the presence of Borrelia burgdorferi sensu lato by polymerase chain reaction (PCR) of urinary bladder tissues, using universal flagellin primers and species specific rRNA primers. Seventeen small mammals (50%) were found to be positive, and 7 small mammals were infected with two species of B. burgdorferi sensu lato simultaneously. The most commonly found species was B. afzelii (n = 14), followed by B. burgdorferi sensu stricto (n = 7) and B. garinii (n = 3), as determined by species-specific primers. We conclude that PCR is a rapid and reliable method to detect infection with B. burgdorferi sensu lato in small mammals.

Ehrlichioses are tick-transmitted diseases associated with illnesses of animals for decades, but recently recognised to be emerging human diseases. In the last ten years increasing number of cases of human infections caused by Ehrlichia chaffeensis and granulo-cytic ehrlichia were described in the United States. Several reports also indicate the presence of infection with the human granulocytic ehrlichiosis (HGE) agent in Europe. The first confirmed acute human disease caused by HGE agent was reported from Slovenia. Until 1997, five patients have been discovered in a prospective study on the etiology of febrile illnesses occurring within six weeks following a tick bite, conducted at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia. The diagnosis of acute HGE was established by seroconversion to the HGE agent and/or molecular identification of ehrlichial organisms. None of the patients had detectable morulae on blood smear examination. Clinical characteristics and laboratory findings were similar to those reported from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only three patients received antibiotic therapy (of whom only two were treated with doxycycline). Many ehrlichiosis cases could go undetected due to a lack of physician awareness, lack of public knowledge, or limited investigation. HGE should now be also included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

The authors have compared the results obtained using four main drugs (calcitonin, ipriflavone, transdermal estrogens, fluorine-calcium) actually employed for the treatment of post-menopausal osteoporosis, administered to four groups of fast loser patients (442) in natural (months mean 19.97 +/- 5.99) and surgical (months mean 16.94 +/- 4.29) menopause. After six months of treatment, the efficacy of therapy has been evaluated on the basis of BD (bone density) and osteoarticular pain changes. The BD results have been compared with those of 100 non-treated patients, in the same clinical conditions. The authors have noticed an increase in bone mass (from +0.47% to +1.59%) and a great improvement in osteoarticular pain with all therapeutical protocols used while in the control group there was a progressive decrease of BMC (-1.23%) and a worsening pain. Comparing the results obtained with different therapies, the difference of mean mineralometric gain is not particularly significant among several treatments; but this difference is very significant between treated and non-treated patients who have continued to lose bone mass. The collateral effects, observed during administration of different drugs, have been minimal and the suspension of therapy has been always associated to their disappearance. In the opinion of the authors the good results, achieved with different therapies, depend on the precocity of the treatment, but also on the fact that, in peri-menopausal period, their effect has been increased by the estrogens. Being osteoporosis a multifactorial pathology, a careful control of the risk factors is appropriate and needs to be enforced in order to carry out a precocious treatment with specific drugs on bone metabolism and try to balance the natural turnover with the loss of bone mass.