A Comprehensive Analysis of Renal and Endothelium Dysfunction Markers Fourteen Years after Hemorrhagic Fever with Renal Syndrome Contraction.

While the pathology of acute hemorrhagic fever with renal syndrome (HFRS) has been widely researched, details on the chronic HFRS sequelae remain mainly unexplored. In this study, we analyzed the clinical and laboratory characteristics of 30 convalescent HFRS patients 14 years after the disease contraction, mainly emphasizing several endothelial dysfunction parameters. Convalescent HFRS patients exhibited significantly higher serum levels of erythrocyte sedimentation rate, von Willebrand factor, uric acid, C-reactive protein and immunoglobulin A when compared to healthy individuals. Furthermore, 24 h urine analyses revealed significantly lower sodium and potassium urine levels, as well as significantly higher proteinuria, microalbumin levels and ß2-microglobulin levels when compared to healthy individuals. First morning urine analysis revealed significantly higher levels of hematuria in convalescent HFRS patients. None of the additional analyzed endothelium dysfunction markers were significantly different in post-HFRS patients and healthy individuals, including serum and urine P-selectin, E-selectin, soluble intercellular adhesion molecule 1, vascular intercellular adhesion molecule 1 (sVCAM-1) and vascular endothelial growth factor (VEGF). However, binary logistic regression revealed a weak association of serum sVCAM-1 and urine VEGF levels with HFRS contraction. Generally, our findings suggest mild chronic inflammation and renal dysfunction levels in convalescent HFRS patients 14 years after the disease contraction.

Uroflowmetry in Non-Monosymptomatic Nocturnal Enuresis in Children of Coastal Region of Croatia.

PURPOSE: The aim of the study was to describe clinical characteristics and bladder assessment in children with Non-Monosymptomatic Nocturnal Enuresis (NMNE) in coastal region of Croatia. MATERIALS AND METHODS: Records on 85 patients with NMNE were retrospectively reviewed. Bladder assessments were performed in all children. In this research we: (i) compare clinical characteristics and features of bladder assessment: uroflowmetry, post void residuals (PVR) and bladder wall thickness between boys and girls with NMNE and we compare (ii) clinical characteristics and bladder assessment between children with primary and secondary NMNE. RESULTS: There were 46 girls and 39 boys. The total of 59 children had primary NMNE and 26 children had secondary NMNE. Uroflow pattern was abnormal in 42% of all children with NMNE. Abnormal uroflow pattern in children with NMNE was more often in girls than in boys (P < 0.05) and in children with secondary than in children with primary NMNE (P < 0.05). Ultrasound evidence of bladder wall thickness was more frequent in boys than in girls. Girls were more likely to have dysfunctional voiding and larger residual urinary volume than boys. CONCLUSIONS: Abnormal uroflow pattern in children with NMNE was more often in girls than boys and in children with secondary than in children with primary NMNE.

Clinical, Virological, and Immunological Findings in Patients with Toscana Neuroinvasive Disease in Croatia: Report of Three Cases.

Toscana virus (TOSV) is an arthropod-borne virus, transmitted to humans by phlebotomine sandflies. Although the majority of infections are asymptomatic, neuroinvasive disease may occur. We report three cases of neuroinvasive TOSV infection detected in Croatia. Two patients aged 21 and 54 years presented with meningitis, while a 22-year old patient presented with meningoencephalitis and right-sided brachial plexitis. Cerebrospinal fluid (CSF), serum, and urine samples were collected and tested for neuroinvasive arboviruses: tick-borne encephalitis, West Nile, Usutu, TOSV, Tahyna, and Bhanja virus. In addition, CSF and serum samples were tested for the anti-viral cytokine response. High titers of TOSV IgM (1000-3200) and IgG (3200-10,000) antibodies in serum samples confirmed TOSV infection. Antibodies to other phleboviruses (sandfly fever Sicilian/Naples/Cyprus virus) were negative. CSF samples showed high concentrations of interleukin 6 (IL-6; range 162.32-2683.90 pg/mL), interferon gamma (IFN-γ; range 110.12-1568.07 pg/mL), and IL-10 (range 28.08-858.91 pg/mL), while significantly lower cytokine production was observed in serum. Two patients recovered fully. The patient with a brachial plexitis improved significantly at discharge. The presented cases highlight the need of increasing awareness of a TOSV as a possible cause of aseptic meningitis/meningoencephalitis during summer months. Association of TOSV and brachial plexitis with long-term sequelae detected in one patient indicates the possibility of more severe disease, even in young patients.

Campylobacter fetus infection presenting with bacteremia and cellulitis in a 72-year-old man with an implanted pacemaker: a case report.

INTRODUCTION: Campylobacter is an important causative agent of intestinal infections in humans. Bacteremia is detected in less than 1% of patients, mainly in immunocompromised patients and in extreme age groups. Cellulitis is a relatively common manifestation of Campylobacter infection, but concomitant bacteremia is a rare event. Infections of the pacemaker area are caused primarily by staphylococci, followed by fungi, streptococci and Gram-negative rods. To the best of our knowledge, this is the first case report of pacemaker pocket infection and bacteremia caused by Campylobacter fetus. CASE PRESENTATION: A 72-year-old Croatian Caucasian man with myelodysplasia, impaired fasting glucose levels and a recently implanted permanent pacemaker was admitted to hospital after six days of fever, development of red swelling of the pacemaker pocket area and worsening of his general condition. No antibiotic therapy was introduced in the outpatient setting. He denied any recent gastrointestinal disturbances. With the exception of an elevated leukocyte count, erythrocyte sedimentation rate, and C-reactive protein and blood glucose levels, other laboratory findings were normal. Treatment with vancomycin plus netilmicin was introduced, and a surgical incision with drainage of the pacemaker pocket was performed. The entire pacemaker system was removed and a new one re-implanted after 14 days of antibiotic therapy. Transesophageal echocardiography showed no pathological findings. Three subsequent blood cultures obtained on admission as well as swab culture of the incised pacemaker area revealed Campylobacter fetus; stool and pacemaker lead cultures were negative. According to the microbiological results, antibiotic therapy was changed to ciprofloxacin plus netilmicin. A clinical examination and the results of a laboratory analysis performed after two weeks of therapy were within normal limits. CONCLUSION: Myelodysplasia, impaired fasting glucose levels and older age could be contributing factors for the development of bacteremic Campylobacter fetus cellulitis. Emergent surgical and antibiotic treatment are mandatory and provide the optimal outcome for such types of pacemaker pocket infection.

Zoonoses and vector-borne diseases in Croatia - a multidisciplinary approach.

Emerging and re-emerging infectious diseases create constant and serious concerns for public health. The majority of emerging infectious diseases (EID) are wildlife zoonotic diseases and vector-borne diseases. Croatia has a long tradition in the control, management and research of EID zoonotic diseases and vector-borne diseases. There has also been a long and advantageous tradition in the collaboration of different experts and professionals in EID research in Croatia involving physician clinicians in infectious diseases, microbiologists, pathologists, veterinarians and animal scientists, ecologists, forestry experts, wildlife scientists, public health specialists and epidemiologists and laboratory scientists. The University Hospital for Infectious Diseases in Zagreb established the Centre for Emerging and Re-emerging Infectious Diseases in liaison with national and international partners from Europe and the United States. This Centre is working in line with the 'One Health initiative' which recognises the inter-relationships between human, animal and environmental health.

Hla-Cw7 allele as predictor of favorable therapeutic response to interferon-alpha in patients with chronic hepatitis C.

AIM: To evaluate the association between human leukocyte antigens (HLA) class I and therapeutic response to interferon-alpha in Croatian patients with chronic hepatitis C. METHODS: HLA-A, -B, and -C genotyping was performed in 55 patients with sustained virological response and in 57 patients without sustained virological response to interferon-alpha therapy. Patients were treated in the period from 1998-2001 with interferon-alpha at a dose of 3 million units three times a week. Patients who became negative for hepatitis C virus RNA after 12 weeks of therapy completed 48 weeks of therapy. RESULTS: There was no association between therapeutic outcome and frequency of HLA-A, as well as of HLA-B alleles. HLA-Cw7 was significantly more frequent in patients with than those without sustained virological response (27.0% vs 6.7%; P=0.011). CONCLUSION: In Croatian patients with chronic hepatitis C, HLA-Cw7 is the predictor of sustained virological response to interferon-alpha therapy.

Chronic fatigue syndrome after Q fever.

BACKGROUND: Q fever is a common and acute but rare chronic zoonosis caused by Coxiella burnetii. Its acute form manifests as atypical pneumonia, flu-like syndrome, or hepatitis. Some authors observed symptoms of chronic fatigue in a small number of patients after the acute phase of Q fever; in many cases serological assay confirmed the activity of Coxiella burnetii infection. The effect of antibiotic therapy on post-Q-fever fatigue syndrome has not been studied in south-east Europe thus far. CASE REPORTS: Three patients are presented with post-Q-fever fatigue syndrome. All fulfilled the CDC criteria for chronic fatigue syndrome. IgA antibodies to phase I of the growth cycle of Coxiella burnetii were positive in two patients and negative in one. Two patients were treated with doxycycline for two weeks in the acute phase of illness and one with a combination of erythromycin and gentamycin. After 4-12 months they developed post-Q-fever fatigue syndrome and were treated with intracellular active antibiotics (fluoroquinolones and tetracycline) for 3-12 months. Efficacy of the treatment was observed in two patients, but in one patient the results were not encouraging. CONCLUSIONS: These results suggest the possibility of the involvement of Coxiella burnetii infection in the evolution of chronic fatigue syndrome. This is the first report on post-Q-fever fatigue syndrome in Mediterranean countries. Evidence of IgA antibodies to phase I of the growth cycle of Coxiella burnetii is not a prerequisite for establishing a diagnosis of CFS. The recommendation of antibiotic treatment in post-Q-fever fatigue syndrome requires further investigation.

[Haemophilus parainfluenzae--a rare cause of endocarditis]. / Haemophilus parainfluenzae--rijedak uzrocnik endokarditisa.

Haemophilus parainfluenzae endocarditis is a rare acute or subacute disease. It is usually associated with dental and surgical procedures in the oral cavity. In a 23-year-old athlete admitted to Department of Infectious Diseases, Split University Hospital, the diagnosis of infective endocarditis was established based on Duke's criteria. The patient was not exposed to risky medical procedures nor he had a predisposing heart disease. The course of illness was characterized by peripheral embolizations (splinter hemorrhages and petechiae) and vegetation on the dorsal mitral valve. After seven days of incubation, blood cultures revealed Haemophilus parainfluenzae. The patient was treated intravenously with amoxicillin-clavulanic acid plus gentamicin for four weeks. Peroral therapy was continued with amoxicillin-clavulanic acid alone for two weeks. By the end of treatment, heart ultrasound showed disappearance of endocardial vegetation. Echocardiographic and clinical examinations performed at 3, 6 and 12 months of therapy showed no pathological aberrations.

[Lymphoid form of intestinal tuberculosis with miliary dissemination: case report]. / Limfoidni oblik crijevne tuberkuloze s milijarnim rasapom--prikaz bolesnika.

Tuberculosis with the incidence 28-29/100000 residents still presents a major public health problem in Croatia. Miliary tuberculosis is uncommon cause of fever of unknown origin. Intestinal tuberculosis pose as diagnostic problem that can be identified by colonoscopy and/or explorative laparatomy involving histopathology and microbiology. A case is reported of a 40-year-old HIV negative patient admitted to the Department of Infectious Diseases after two weeks of fever, diarrhea, abdominal pain and weight loss. Biochemistry testing showed mild elevation of the erythrocyte sedimentation rate and increased serum aminotransferases. On admission, chest x-ray was normal and tuberculin skin test was negative. Crohn's disease was suspected. Computed tomography of the abdomen revealed solid infiltrative mass located retroperitoneally, along with enlarged lymph nodes. Explorative laparoscopy was necessary to confirm the diagnosis. Intraoperative specimens were referred for histopathologic and microbiologic examination, which proved the existence of granulomatous inflammation of the areas with caseous necrosis. Direct microscopy of the periappendicular abscess and Ziehl-Neelsen staining of a lymph node specimen confirmed the presence of an acidoresistant bacillus. The specimen culture on solid egg based agar (Löwenstein Jensen) and liquid broth (MGIT) showed the growth of Mycobacterium tuberculosis. Then the causative agent was cultured from all specimens: sputum, stool and urine. Repeat cheast x-ray, performed on day 30 of hospitalization, showed miliary dissemination to the lungs. The patient was treated with four antituberculotics (streptomycin, isoniazide, rifampin, ethambutol) and methylprednisolone for one month, then with isoniazide, rifampin and for 11 months ethambutol. Therapy led to a decrease of abdominal lymph nodes and absence of miliary lesions on chest radiography after two months of treatment. Intestinal tuberculosis has been almost forgotten in Croatia. The latest published cases referred to HIV infected patients. In less than 50% of patients with intestinal tuberculosis the lungs are also affected, which poses a diagnostic problem. Crohn's disease is the most common diagnostic problem. Histopathology of a specimen obtained on colonoscopy and/or explorative laparoscopy can often solve the dilemma, as also confirmed in our patient. Of diagnostic studies, computed tomography has the advantage of evaluating intestinal wall involvement, which is important for the early diagnosis of intestinal tuberculosis. Enteroclysis and irrigography provide diagnostic information in the advanced stage of intestinal tuberculosis. In a patient with fever, abdominal disorders and parameters which implicate granulomatosis hepatitis or Crohn's disease, the existence of abdominal tuberculosis is also possible. Computed tomography and biopsy obtained on colonoscopy for microbiology can help in making the diagnosis and initiating appropriate treatment.

A child with Bartonella henselae osteomyelitis of the right humerus.

We present a case of a 22-month-old child with swollen upper part of the right arm and osteolytic lesion of the right humerus, which resembled a neoplastic process. Epidemiological history revealed no scratch marks on the skin or cutaneous papule or pustule. Presumptive diagnosis of hematogenous osteomyelitis was established, but treatment with fusidic acid was unsuccessful. Histological examination of the bioptic specimen of the soft tissue swelling showed a lymph node morphology, with numerous granulomas with central stellate necrosis. Indirect immunofluorescence assay for Bartonella henselae yielded positive results. New treatment included 15 days of trimetoprime and sulfamethoxazole, followed by azithromycin for 5 days. Four months later, swelling resolved and osteolytic lesion almost completely healed with formation of surrounding sclerosis. In conclusion, cat-scratch disease without positive epidemiological history and primary cutaneous papule or pustule may be a serious diagnostic problem, but can be solved by serological and histological examination.

First isolation of Rickettsia conorii from human blood in Croatia.

AIM: To detect and isolate rickettsial strains from blood samples of patients with presumptive diagnosis of Mediterranean spotted fever (MSF) in the coastal region of south Croatia, and to compare the results with routine serology. METHODS: A "suicide" polymerase chain reaction (PCR), and a shell vial culture were done on samples of ethylenediamine tetra-acetic acid (EDTA) and citrate-anticoagulated blood samples. Indirect immunofluorescence was performed on sera collected from 17 patients clinically diagnosed with MSF during summer in three consecutive years, from 1998 to 2000. RESULTS: The primers used in PCR amplified the expected part of the rickettsia genomic DNA and Rickettsia conorii grew from the shell vial-cultured blood of a single patient. In 13 patients, the diagnosis was confirmed serologically by paired sera, whereas in 4 patients the diagnosis remained presumptive, since no paired sera were available. Analyzing sequences of the ompA and citrate synthase gene, respectively, derived from the shell vial isolate, a 100% similarity with Rickettsia conorii, strain Seven (Malish), was found. CONCLUSION: To the best of our knowledge, this is the first isolation of Rickettsia conorii from a human sample in Croatia, and the first proof of a causative agent of MSF in the country. Beside PCR-based methods and isolation, correct diagnosis of MSF could be still routinely reached by serology.

[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.

[Is permanent renal function damage possible after hemorrhagic fever with renal syndrome?]. / Je li moguce trajno ostecenje bubrezne funkcije nakon preboljele hemoragijske vrucice s bubreznim sindromom?

AIM: To investigate the possibility of permanent renal function impairment and other organ lesions following hemorrhagic fever with renal syndrome (HFRS). METHODS: Data on 30/37 patients infected with HFRS, treated at the Department of Infectious Diseases, Split University Hospital, in 1995 were retrospectively analyzed. The data were collected three to six years following the appearance of HFRS. In 1998, 30/37 patients presented for control checkup, when their history data were collected, along with physical examination, hematology and biochemistry tests, and urinalysis. Creatinine clearance and sodium, potassium, chlorine, phosphorus, beta 2-microglobulin and N-acetyl-beta-D glucosaminase in 24-h urine were determined. In native urine, erythrocyturia was observed, with 10 erythrocytes per field were considered pathologic result. During the 1998-2001 period, renal scintigraphy by means of technetium labeled diethylene triaminopentacetic acid (99mTC DTPA) was performed in 13/30 patients. RESULTS: Of subjective discomforts, 29/30 (96.7%) patients reported lumbar pain. Elevated blood pressure was found in 9/30 (30.0%), erythrocyturia in 4/30 (13.3%) and hepatic lesion in 4/30 (13.3%) patients. Decreased creatinine clearance values (< 1.2 ml/s) were found in 4 and increased values (> 2.35 ml/s) in 10 patients. Increased sodium in 24-h urine was recorded in 10/23 and increased beta 2-microglobulin in 6/23 (26%) patients. Proteinuria exceeding 150 mg/day was detected in 11/23 (47.8%) patients. Scintigraphy of the kidneys demonstrated reduced glomerular filtration (< 100 ml/min/1.72 m2) in 3/13 patients. Prolonged mean times (> 5 minutes) of radiopharmaceutical passage through the renal parenchymae were found in 7/13 (53.8%) patients. DISCUSSION: Studies performed in 30 patients three years after they had recovered from HFRS revealed changes suggesting a mild to moderate impairment of the renal function. Hypertension found in 9/30 patients was a significant finding, considering the fact that all subjects were soldiers, thus having undergone through examinations to prove them completely healthy prior to joining army. Hypertension results were consistent with those reported from the USA. Although erythrocyturia points to urinary tract damage, its glomerular or postglomerular origin was not examined. Decreased creatinine clearance found in 4/23 patients suggested functional renal impairment. Increased natriuresis found in 10/23 patients implied tubular damage, i.e. reduced ability of tubular cells for sodium reabsorption from primary urine. Non-selective albuminuria detected in 11/23 patients indicated permanent lesion of the glomerular basal membrane. Increased beta 2-microglobulin found in 6/23 patients indicated that the lysosomal enzyme level was elevated only in the acute stage of the disease, but may have been an indicator of permanent lesion. No description of post-HFRS scintigraphic lesion of the kidneys was found in the literature. A decreased value of glomerular filtration, found in three patients, and especially the prolonged mean time of glomerular micropharmaceutical passage in 7/13 (53.8%) patients may have suggested glomerular damage. However, the possible reason may have also been a reduced passage of glomerular filtrate through the damaged lower parts of the nephrons. Transaminase increase during the acute stage of HFRS suggested the possible liver infection, maybe even hantavirus replication in hepatocytes. Even though biopsy confirmed the histologic picture of chronic hepatitis in one patient, the question remains whether it could have been caused by hantavirus. CONCLUSION: Studies performed in 30 patients with a history of HFRS revealed renal function impairment, along with hypertension and damage to the liver parenchyma in some patients. The results obtained showed that the HFRS infection in Croatia may have entailed chronic sequels. To confirm this hypothesis, additional studies including a control group of hantavirus negative persons are needed.

Dinara - new natural focus of hemorrhagic fever with renal syndrome in Croatia.

AIM: To investigate the characteristics and determine risk factors for hantanvirus infection in natural focus of hemorrhagic fever with renal syndrome (HFRS) on the Dinara Mountain, where outbreak of disease emerged among Croatian soldiers in 1995, and to describe the features of HFRS acquired on the Dinara mountain and determine the scale of the largest HFRS epidemic so far in Croatia. METHODS: During 1996, small mammals were captured in the region of Dinara Mountain where infected Croatian soldiers had sojourned. By taxonomic classification of 42 captured small mammals, three species were determined: 23 yellow-necked mouse, 9 wood mouse, and 5 bank vole. Hantavirus antigen was determined in the lungs of the captured animals by means of direct immunofluorescence assay. The most important features of HFRS were retrospectively determined in 37 soldiers with HFRS treated in the Department for Infectious Diseases of the Split University Hospital. The degree of inapparent exposure to infection was determined by indirect immunofluorescence in 103 soldiers sojourning in this region of natural focus with no apparent signs of HFRS. Epidemiological questionnaire included 50 soldiers with negative serum antibodies, as well as 33 available out of total 37 soldiers with HFRS. Chi-square test was used to determine risk factors. RESULTS: Hantavirus was found in the lungs of 5/42 (12%) captured animals. Mild form of the disease, with few hemorrhagic symptoms and pronounced renal insufficiency, was present in 19/37 patients. The epidemiological questionnaire determined the following risk factors for hantanvirus infection in this focus: service in artillery corps (p=0.040), sleep in wooden barracks (p=0.004), station in forest biotope (p=0.037), usage of natural camouflage (p=0.024), smoking (p=0.010), and the presence of rodents in the place of housing (p<0.001). CONCLUSION: A new natural focus of HFRS in Croatia, and the first one in Dalmatia, was defined by seroepidemiologic, mamologic, and virologic analysis. The risk factors for infection in the new focus have been identified. Our patients suffered from a mild form of HFRS, which predominates in south-eastern Europe, without lethal outcome.