Leptospirosis and characterization of Leptospira isolates from patients in Koprivnica-Krizevci County, Croatia from 2000-2004.

Introduction: Leptospirosis, caused by spirochetes of the genus Leptospira , is present in the Koprivnica-Krizevci County area, Croatia. Clinical manifestation can range from asymptomatic, short-term mild, non-specific febrile disease, to severe forms with high mortality rates. Aim: The aim of the study was to valuate culture in front of microscopic agglutination test (MAT) for diagnosis of infection, and to evaluate clinical and laboratory features of the disease. Moreover, we want to characterize Leptospira strains involved in infection in Koprivnica-Krizevci County, Croatia. Methods: We included 68 patients with clinical presentation consistent with leptospirosis collected in a 5-year period (2000-2004). Clinical samples (blood, urine and cerebrospinal fluid, CSF) were inoculated in Kolthoff's medium; species of isolated Leptospira strains was determined with Tm of real-time PCR, serogroup/serovar with MAT and NotI-RFLP analysis. Demonstration of specific antibodies in patients' sera was done using microscopic agglutination test. Results: Leptospira was isolated from the blood of 14/51(27.5 %) patients and the most often identified serogroup/serovar was Icterohaemorrhagiae (8/10, 80%) followed by Grippotyphosa (10%). Regarding to species level, 8/10 isolated belonged to L. interrogans sensu stricto and one to L. kirschneri species. MAT was carried out on 51 patients with suspected leptospirosis, and was positive in 11/51(21.5 %) patients. Most of our patients presented with moderate severe symptoms, were hospitalized from August to October, and were infected mainly during the work or recreation in our county. The frequency of particular clinical features and pathological laboratory findings correlated with the severity of the clinical condition. Conclusions: Leptospirosis can be confirmed microbiologically, culture and MAT contributed almost equally to the diagnosis of infection. Serovar Icterohaemorrhagiae was found as the dominant one, and L. interrogans sensu stricto as dominant species in our county. Epidemiological data shown that leptospirosis occurs seasonally, affects the rural population, and most commonly is presented with moderate severe clinical course.

Epidemiological and clinical features of leptospirosis in a highly endemic area over three time periods.

OBJECTIVES: To present the features of human leptospirosis over three time periods (1970-1975; 2000-2005; 2010-2015), to compare the collected data and to determine whether the incidence, seasonal and spatial distribution, prevalence of presumptive infective serogroups and clinical features have changed over the last 50 years. METHODS: Epidemiological and clinical data obtained from patients hospitalised and treated in a well-known endemic focus of leptospirosis, Koprivnica-Krizevci County in Croatia, were analysed. RESULTS: We observed a steady decline in the overall incidence of leptospirosis and a change in the patient age distribution, with the age ratio changing in favour of middle-aged and older patients. Although leptospirosis was most frequently diagnosed in August in all time periods, the number of cases increased in autumn. The most prevalent serogroup during the first and the second time period was Icterohaemorrhagiae, while in the third time period, the serogroup Australis prevailed. We also noted an increase in the number of severe clinical manifestations. CONCLUSIONS: This retrospective research demonstrates a continuous decline in the incidence of human leptospirosis in Croatia. The pattern of disease has changed from predominantly mild clinical forms observed in children to more severe clinical forms observed in middle-aged to older patients, especially those working in agriculture. Additional epidemiological changes included an increase in the number of cases during the autumn months and changes in prevailing serogroups. Statistical analysis revealed a significant relationship between the severity of the clinical picture, patient age and presumed sources of infection.

[Post-encephalitic syndrome in patients with tick-borne encephalitis]. / Postencefaliticki sindrom u bolesnika s krpeljnim meningoencefalitisom.

BACKGROUND: It was 55 years ago when the first patients with tick-borne encephalitis (TBE) were diagnosed in the Koprivnica-Krizevci County. Since then, we have acquired some new knowledge about the disease. TBE is an endemic disease and the second most common tick-borne disease following Lyme borreliosis in our country, with an average morbidity rate of 12 patients per year and predominance of male individuals older than 50. There are no specific risk groups because such patients have been continuously vaccinated for the past 27 years. In 88.0% of patients, the infection is manifested as aseptic meningitis and meningoencephalitis with a biphasic course. As opposed to detailed descriptions of acute morbidity, there are few reports on the course of disease and its outcome. OBJECTIVES: The aim of the study was to assess the presence of post-encephalitic syndrome (PES) in patients with TBE, to evaluate its incidence and demonstrate its characteristics. PATIENTS AND METHODS: This prospective study was conducted from 1995 to 2008 and enrolled PES patients treated at Department of Infectious Diseases, Dr. Tomislav Bardek General Hospital in Koprivnica during the study period. The study included patients of both sexes older than 14 years with recent TBE virus infection, patients with clear temporal correlation between acute morbidity and PES onset, and patients where any other cause of PES was ruled out. The immunoenzyme linked assay (ELISA) was used for detection of serum IgM and IgG antibodies. Recent TBE virus infection was detected in 133 patients. Nine of these patients refused further cooperation, and the remaining 124 patients, 80 male (64.5%) and 44 female (35.4%), aged 16-76, were included in the study. We longitudinally examined the manifestation and characteristics of PES in each patient during a 3-year period (and longer if necessary). Study patients were divided into three groups of mild, moderate and severe PES based on data collected and entered into specially prepared questionnaire and by qualitative analysis of PES effect on their daily habits and activities. RESULTS: Out of 124 patients included in the study, 60 (48.3%) had no symptoms/signs of PES, or these were mild and of short duration 15 (12.0%). The remaining 49 (39.5%) patients developed moderate (30/47.0%) or severe (19/30.0%) PES lasting for 3-18 months, with significant impact on their daily habits and activities requiring some adjustment. The main characteristics of PES were mental disorders, balance and movement coordination disorders, headache, general malaise, and reduced working ability. PES was recorded in 35 (28.2%) patients with meningoencephalitis and 14 (11.2%) patients with meningoencephalomyelitis. Permanent sequels were left over in 11 (17.1%) patients: spinal nerve paresis in five (4.0%), hearing impairment in six (5.6%), dysarthria in two (1.6%) patients, and severe mental disorder in one (0.8%) patient. In three patients we recorded simultaneous permanent spinal nerve paralysis and permanent deafness. During our longitudinal study three (2.5%) patients died. CONCLUSION: The study undoubtedly confirmed the presence of PES in our patients with TBE. Moderate and severe PES has a significant impact on the patient quality of life, demands patient adjustment, and increases expenses of long-term sick-leave and rehabilitation.

[Human anaplasmosis (ehrlichiosis)]. / Humana anaplazmoza (erlihioza)--prikaz bolesnika.

OBJECTIVE: The north part of Croatia, especially the Koprivnica-Krizevci County is a well known endemic area of several tick-transmitted diseases including tick-borne encephalitis, Lyme borreliosis and tularemia. Each summer numerous patients develop a nonspecific febrile illness after tick bite, etiology of which is sometimes never established. PURPOSE OF WORK: The aim of this prospective study was to determine whether the infection with Anaplasma phagocytophilum was associated with febrile illnesses occurring after tick bite. The epidemiological, clinical and laboratory characteristics, and diagnostic procedures in patients suffering from confirmed and probable anaplasmosis are presented. PATIENTS AND METHODS: A prospective study to assess the potential ehrlichial etiology of febrile illnesses after tick bite was conducted at the Department of Infectious Disease, General Hospital, Koprivnica, Croatia, during the period from January 1998 to August 2004. The study included patients of all age groups with acute febrile illness (temperature higher than 38 degrees C) manifesting within 30 days of a tick bite, in whom a recent infection with Anaplasma phagocytophilum was established serologically by seroconversion or at least 4-fold increase in antibody titer to anaplasma antigen (defined as confirmed cases of anaplasmosis), or by the presence of positive IFA titer > or = 1: 256 in acute and convalescent phase serum samples without demonstrating a 4-fold titer change (defined as probable cases of anaplasmosis), and with no alternative explanation for the acute febrile illness. Thorough medical histories were collected, physical examinations performed, and several laboratory tests carried out. Giemsa-stained peripheral blood smears were examined by high microscopy for the presence of ehrlichial morulae within leukocytes. Acute and convalescent phase serum samples were tested by use of an indirect immunofluorescence assay (IFA) for the presence of specific IgG antibodies to Anaplasma phagocytophilum antigen. At the same time all serum samples were tested for other tick borne agents including tick-borne encephalitis virus, Borrelia burgdorferi sensu lato and Ehrlichia chaffeensis. Serological tests were performed at the Institute of Microbiology and Immunology, School of Medicine, University of Ljubljana, Ljubljana, Slovenia. Oral consent was obtained from all patients enrolled in the study. RESULTS: Of 132 patients included in the study during the 7-year period, eight (6%) patients were diagnosed with anaplasmosis, without any additional diagnosis, and were included in the analysis. Three patients fulfilled the case definition criteria for confirmed anaplasmosis, while five had probable anaplasmosis (three men and five women aged 17-43). Clinically, the disease presented as a nonspecific, flu-like febrile illness with leukopenia, thrombocytopenia and moderately elevated aminotransferases. Intracytoplasmic morulae were not seen in leukocytes in blood smears. Specific antimicrobial therapy was administered in four patients. All patients recovered rapidly and no long-term consequences were found during the one-year follow-up. CONCLUSION: We report on the first patients meeting the case definition criteria for confirmed and probable anaplasmosis. The frequency and new concepts in the disease were analyzed.

[Hepatitis caused by tick-borne meningoencephalitis virus (TBEV)--a rare clinical manifestation outside the central nervous system involvement]. / Hepatitis uzrokovan virusom krpeljnog meningoencefalitisa--rijetka klinicka manifestacija izvan sredisnjeg zivcanog sustava.

OBJECTIVE: North part of Croatia, especially the Koprivnica-Krizevci County has been a well-known endemic area of tick-borne encephalitis (TBE) for more than 50 years. To date, this disease caused by tick-borne encephalitis virus (TBEV) has retained the leading position among inflammatory diseases of the central nervous system (CNS), with an average morbidity of 19 patients per year. In 88% of patients the infection manifested with signs of meningitis or meningoencephalitis with a biphasic course. TBE may take a more severe course with different neurologic dysfunction or even lethal outcome. There have been isolated reports on some rare clinical manifestations outside CNS involvement caused by TBEV, including transient hepatitis, pancreatitis, and myocarditis. These manifestations have been most frequently detected during the initial stage of disease but also as a complication, or as an individually separate clinical entity. The incidence rate of these manifestations is not known. Until now, such clinical manifestations outside CNS have not been reported in the county. It was the reason to present the characteristics of liver failure in our patients suffering from TBE. Purpose of Work: To determine the frequency of elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in patients with TBEV infection, to note the time from the detection to disappearance of elevated AST and to investigate and establish whether the degree of liver failure influences the course and outcome of TBE. PATIENTS AND METHODS: The study was conducted from January 1991 until September 2004, and included patients with TBEV infection and elevated AST and ALT, examined at the Department of Infectious Diseases, Koprivnica General Hospital. Study patients were of all age groups and occupation. On their first examination detailed epidemiologic and patient history data were collected, with special reference to the history of alcohol abuse, disease of the liver or biliary tract, or any other apparent cause of liver failure. Clinical examination and laboratory tests were performed. Patients were classified into four groups as suffering from meningitis, meningoencephalitis, meningoencephalomyelitis, or abortive form of disease. The acute stage of meningoencephalitis was classified as mild, moderate or severe, depending on meningeal symptoms and severity of clinical signs of encephalitis, and presence of focal CNS signs. ELISA test was used to detect specific IgM and IgG antibodies. Serum samples were tested for other tick-borne diseases, including human monocyte and granulocyte ehrlichiosis. Oral consent was obtained from all patients enrolled in the study. RESULTS AND DISCUSSION: During the study, recent infection with TBEV was proven in 261 patients. Liver function tests were done in 115 (44%) patients. Elevated AST and ALT activity was found in 25 (22%) patients, without bilirubin and alkaline phosphatase increase. All these were County residents, with a male sex predominance, aged 28-70 (median 41) years, and none had received TBE vaccination. Elevated AST and ALT activities were detected during the first stage of TBE in 16 (64%) and during the second stage of meningoencephalitis in 9 (36%) patients suffering from TBE. Elevated activity of AST was observed in 76% and of ALT in 96% of study patients. The most frequently observed elevated AST and ALT activity was two- or threefold the usual normal values. The time of the first detection of elevated AST and ALT activity could not be precisely determined, since all laboratory findings were performed during the first examination of patients. In the initial stage of disease, patients were mostly tested between 2-5 days from the onset of disease, and during the first week in the second stage of meningoencephalitis. Elevated AST and ALT activities were transient and normalized in 3-4 weeks. The importance of elevated AST and ALT activity for the course and prognosis of TBE was not verified in this study. CONCLUSION: The first patients with hepatitis as one of the possible manifestation outside CNS involvement caused by TBEV are described. The prevalence and new concepts on this type of the disease were investigated in the study.

The Golden Agers and Tick-borne encephalitis. Conference report and position paper of the International Scientific Working Group on Tick-borne encephalitis.

The 7th meeting of the ISW TBE had the main topic "Tick-borne encephalitis in the Golden Agers". Data from 14 European countries were presented about incidence and clinical course of Tick borne encephalitis (TBE) in general and especially in the population over 50 years of age. With age immunity is impaired quantitatively and qualitatively, the reactions to vaccinations are generally slower, antibody titres reach lower values and decrease earlier. The incidence of the disease is increasing with age, also the clinical course is more severe, they suffer significantly more sequelae, need a longer rehabilitation and have a higher case fatality. Vaccination as the only efficient protection is needed in endemic areas, considering that mobility has increased very much. For the age group over 50 years regular booster vaccinations according to the recommended vaccination intervals or even shorter intervals are most important.

[An abortive type of tick-borne meningoencephalitis]. / Abortivni oblik krpeljnog meningoencefalitisa.

OBJECTIVE: Tick-borne encephalitis (TBE) was detected in the Koprivnica-Krizevci County 50 years ago. To date, it has retained the leading position among inflammatory diseases of the central nervous system (CNS), with an average morbidity of 20 patients per year. In 88% of patients, the infection manifested with signs of meningitis or biphasic meningoencephalitis (ME). Monophasic course was recorded in only 12% of patients of older age, during which the first initial stage remained inapparent, and the disease manifested only with ME stage. An abortive type of TBE virus infection with fever, headache and other general symptoms of infection is presented. The initial stage of the disease, without subsequent CNS involvement, is rarely described. PURPOSE OF WORK: To determine the frequency and present epidemiological, clinical and laboratory characteristics as well as diagnostic procedures in patients suffering from abortive type of TBE. PATIENTS AND METHODS: A prospective study was conducted in the period from 1997-2002, and included patients with possible tick-borne disease, examined at the Infectious Disease Department of the Koprivnica General Hospital. According to the set criteria, the study included patients of all age groups, sex and occupation, patients with febrile disease (T 38 degrees C) that developed within 6 weeks from the tick bite, patients who showed no signs of ME on their first examination, and patients in whom serologic analysis revealed recent infection with TBE virus. On admission to the hospital, detailed epidemiological and history data were collected, and clinical examination and laboratory testing were performed. ELISA test was used to detect specific IgM and IgG antibodies. At the same time all serum samples were tested for other tick-borne diseases, including Lyme disease and human monocyte and granulocyte ehrlichiosis. Oral consent was obtained from all patients enrolled in the study. RESULTS: A total of 127 patients met the study criteria. Specific antibodies to TBE virus were detected in 49 (38.5%) patients. After the latency period, 43 (87.7%) patients developed the second stage of ME disease. An abortive type of infection with TBE virus, without subsequent CNS involvement, was recorded in 6 (12.2%) patients. The diagnosis was confirmed in the first initial stage in 12% of patients and during the second ME stage in the remaining 37 (75.5%) patients. Active immunization against TBE was not conducted in any patient. CONCLUSION: We report on the first patients diagnosed with an abortive type of TBE virus infection in the Koprivnica-Krizevci County. The frequency of and new knowledge about this type of disease were assessed in the study.

[The biggest epidemic of hemorrhagic fever with renal syndrome in Croatia]. / Najveca epidemija hemoragijske vrucice s bubreznim sindromom u Hrvatskoj.

INTRODUCTION: The largest outbreak of hemorrhagic fever with renal syndrome (HFRS) to date occurred in Croatia in the year 2002. The epidemic started in winter, lasted throughout spring to summer months, ending not earlier than November. AIM: The aim of this study was to investigate and analyze the basic epidemiologic and clinical features of HFRS in Croatia by uniform and standardized prospective-retrospective analysis of all patients affected by the epidemic. PATIENTS AND METHODS: When the epidemic started, a patient questionnaire with questions on the basic demographic data, site of infection and other epidemiologic characteristics, clinical symptoms, disease severity and laboratory results was designed. Data on 401 patient with a clinical diagnosis of HFRS were collected. The etiologic diagnosis of the disease was confirmed by ELISA, and in some patients by indirect immunofluorescence test (IFT). The results were analyzed using a descriptive statistical method. RESULTS: HFRS was clinically diagnosed in 401 patients from all over Croatia. A total of 320 (79.8%) cases were reported to the Epidemiology Service of the National Institute of Public Health. The majority of patients (n = 128) were registered in June. Males were three times more affected than females. Apart from its long duration, this epidemic was characteristic for the involvement of general population, with only a small number of the affected from the potential risk groups (forestry workers 28, soldiers 14, farmers 18). The epidemic spread almost throughout inland Coratia. At least 44 patients were infected in the Plitvice Lakes area, 32 in Slunj, 27 on Sljeme, 24 in Velika, and at least 19 in the area of Kutjevo. The youngest patient was aged 4 and the oldest 80 years. The majority of patients were treated in Zagreb (University Hospital for Infectious Diseases--110, Zagreb University Hospital Center--3), followed by Karlovac (71 inpatients and 39 outpatients), Pozega (n = 79), and Rijeka (n = 37). Serologic analysis (ELISA method) detected Puumala virus in 161 and Dobrava virus in only 17 patients. The disease was confirmed by immunofluorescence method in 53 patients (mostly in Kariovac). During the 2002 outbreak, HFRS clinically manifested mostly in a milder form with general symptoms and transitory renal insufficiency, while hemorrhages were rarely recorded. According to our disease severity score, a mild form of the disease was recorded in 65%, moderately severe in 28%, severe in 5% and extremely severe form in 2% of the patients. One patient died. Two thirds of the patients were hospitalized during the febrile stage of the disease. All patients had fever, whereas headache and pain in the lumbar region were recorded in more than 90% of cases, polyuria in 75%, oliguira and vomiting in approximately 50%, respiratory symptoms in 35%, and hemorrhages (mostly on the skin and mucous membranes), vision disturbances, conjunctivitis and diarrhea in approximately 25% of patients. ESR was elevated in 64% and CRP in 93% of patients. Leukocytosis was recorded in 25% and thrombocytopenia in 70% of patients. Increased values of urea and creatinine and signs of liver damage were recorded in approximately 50% of the patients. CONCLUSION: The largest outbreak of HFRS occurred in Croatia in 2002, with more than 400 diseased throughout Croatia. This epidemic confirmed our previous assumption that the whole Croatia, apart from its narrow coastline area and islands, is a natural focus of HFRS with different causative types of hantaviruses. Efforts should be made to conduct a comprehensive ecologic and mammologic study on hantaviruses and their biologic characteristics in these areas.