High infection rate of bank voles (Myodes glareolus) with Puumala virus is associated with a winter outbreak of haemorrhagic fever with renal syndrome in Croatia.

An outbreak of haemorrhagic fever with renal syndrome (HFRS) started on Medvednica mountain near Zagreb in January 2012. In order to detect the aetiological agent of the disease in small rodents and to make the link with the human outbreak, rodents were trapped at four different altitudes. Using nested RT-PCR, Puumala virus (PUUV) RNA was detected in 41/53 (77·4%) bank voles (Myodes glareolus) and Dobrava virus (DOBV) RNA was found in 6/61 (9·8%) yellow-necked mice (Apodemus flavicollis). Sequence analysis of a 341-nucleotide region of the PUUV S segment, obtained from all infected bank voles and five HFRS patients, showed 98·8-100% sequence similarity, indicating that the patients were probably exposed to PUUV on Medvednica mountain. A very large bank-vole population combined with an extremely high infection rate of PUUV was responsible for this unusual winter outbreak of HFRS in Croatia.

Clinical and laboratory findings in patients with oliguric and non-oliguric hantavirus haemorrhagic fever with renal syndrome: an analysis of 128 patients.

Patients with haemorrhagic fever with renal syndrome (HFRS) may present without significant oliguria. We compared different initial clinical symptoms and laboratory findings in patients who developed oliguric acute renal failure (ARF) with those in patients who did not develop oliguric ARF. Overall, 128 patients with serologically confirmed HFRS were hospitalized at the University Hospital for Infectious Disease, Zagreb, Croatia between January 1999 and December 2010. Clinical signs and laboratory findings were extracted from medical charts, and were assessed for their relationship to the development of oliguric ARF. Puumala virus infection was diagnosed in 101 (79%) patients, and Dobrava-Belgrade virus infection in 27 (21%). Oliguria or anuria developed in 30% of patients. We identified the following risk factors for the development of oliguria and anuria on multivariable analysis: conjunctival hyperaemia or bleeding (relative risk (RR) 1.84, 95% CI 1.09-3.10; p 0.023), diarrhoea (RR 1.45, 95% CI 1.07-1.97; p 0.017), serum sodium of ≤133 mM (RR 2.21, 95% CI 1.34-3.64; p 0.002), and dipstick protein value of >1.5 g/L (RR 1.59, 95% CI 1.09-2.33; p 0.016), as well as hiking in the forest (RR 1.92, 95% CI 1.13-3.26; p 0.016). Our findings may help physicians in the earlier identification of patients with a more severe form of HFRS caused by Puumala and Dobrava-Belgrade viruses. Particular attention should be given to findings such as conjunctival hyperaemia or bleeding, diarrhoea, a low serum sodium level, and proteinuria.

Superoxide dismutase, copper and zinc concentrations in platelet-rich plasma of pneumonia patients.

BACKGROUND: The aim of this study was to analyse platelet superoxide dismutase (SOD) activities (total SOD, manganese SOD and copper zinc SOD) and copper (Cu) and zinc (Zn) concentrations during the course of community-acquired pneumonia (CAP), and to compare them between patients with normal platelet count and those who have developed reactive thrombocytosis (RT). METHODS: Platelet count, SOD activities and Cu and Zn concentrations in platelet-rich plasma were measured in patients with CAP on admission and at discharge. RESULTS: Post-therapeutic platelet count increased significantly from the value recorded on admission. By the end of treatment, 42% of patients developed RT. All platelet SOD activities as well as Cu concentration were significantly lower in CAP patients than in control subjects. The initial Zn concentration was greater in CAP patients compared with controls and showed a decrease at discharge. On admission, there was no difference in all SOD activities between either subgroup with normal platelet count or subgroup with RT. At discharge all SOD activities were significantly lower in patients with RT. Also, catalytic activities of those enzymes were significantly lower in both subgroups in comparison with the initial values. Post-therapeutic Cu value was lower in patients with RT in comparison with patients having normal platelet count. Zn concentration decreased significantly at discharge when compared with the initial values only in patients with RT. CONCLUSION: The pattern of changes might be indicative of a certain role of platelets in antioxidant response during treatment in CAP patients.

Enumeration of haemagglutinin-specific CD8+ T cells after influenza vaccination using MHC class I peptide tetramers.

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

Outcome of influenza vaccination in combat-related post-traumatic stress disorder (PTSD) patients.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that can occur after exposure to extreme traumatic experience such as war trauma, and is accompanied by fear, helplessness or horror. Exposure to trauma can result in immune dysregulation and influence susceptibility to infectious disease as well as vaccine efficacy. The aim of the study was to determine the relation of psychological stress and the immune response to influenza vaccination in combat-related PTSD patients (n = 28). Detection of anti-viral antibody titre was performed by inhibition of haemagglutination assay. Ex vivo tetramer staining of CD8(+) T lymphocytes was used to monitor T cells specific for human leucocyte antigen (HLA)-A*0201-restricted influenza A haemagglutinin antigens before and after vaccination. Twenty patients showed a fourfold antibody titre increase to one or both influenza A viral strains, and 18 of them showed the same response for both influenza B viral strains. Ten of 15 healthy controls showed a fourfold rise in antibody titre to both influenza A viral strains and eight of them showed the same response for both influenza B viral strains. HLA-A*0201(+) PTSD patients (n = 10) showed a significant increase of influenza-specific CD8 T cells after vaccination. Although those PTSD patients had a lower number of influenza-specific CD8(+) T cells before vaccination compared to HLA-A*0201(+) healthy controls (n = 6), there was no difference in influenza A antibody titre between PTSD patients and control subjects before vaccination. The generated humoral and cellular immune response in PTSD patients argues against the hypothesis that combat-related PTSD in war veterans might affect protection following influenza vaccination.

Chlamydia pneumoniae and Mycoplasma pneumoniae pneumonia: comparison of clinical, epidemiological characteristics and laboratory profiles.

The purpose of our retrospective 3-year study was to analyse and compare clinical and epidemiological characteristics in hospitalized patients older than 6 years with community-acquired pneumonia (CAP) caused by Chlamydia pneumoniae (87 patients) and Mycoplasma pneumoniae (147 patients). C. pneumoniae and M. pneumoniae infection was confirmed by serology. C. pneumoniae patients were older (42.12 vs. 24.64 years), and were less likely to have a cough, rhinitis, and hoarseness (P<0.001). C. pneumoniae patients had higher levels of C-reactive protein (CRP), and aspartate aminotransferase (AST) than M. pneumoniae patients (P<0.001). Pleural effusion was recorded more frequently in patients with M. pneumoniae (8.84 vs. 3.37%). There were no characteristic epidemiological and clinical findings that would distinguish CAP caused by M. pneumoniae from C. pneumoniae. However, some factors are indicative for C. pneumoniae such as older age, lack of cough, rhinitis, hoarseness, and higher value of CRP, and AST.

Electrocardiographic changes and myocarditis in trichinellosis: a retrospective study of 154 patients.

The frequencies of electrocardiographic (ECG) abnormalities and myocarditis were determined, retrospectively, among 154 cases of trichinellosis [101 males and 53 females, with a mean (S.D.) age of 35.60 (14.64) years] who were hospitalized at the University Hospital for Infectious Diseases in Zagreb, Croatia, over a 5-year period. Eighty-seven (56%) of the patients, most of them in the invasive phase of infection with Trichinella spiralis, were found to have abnormalities when examined by 12-lead, resting electrocardiography. The ECG disorder most frequently observed was a non-specific ventricular repolarization disturbance (with ST-T wave changes), followed by bundle-branch conduction disturbances, and sinus tachycardia. The other ECG disorders recorded, during various phases of the infection, were sinus bradycardia, right bundle-branch block, supraventricular and ventricular extrasystoles, low-voltage QRS complexes in standard limb leads, first-degree atrio-ventricular block, and atrial fibrillation. Eighteen (12%) of the patients were identified as cases of myocarditis (13 in the invasive phase and five in the convalescent) and two (1.3%) as cases of myopericarditis. One patient developed acute myocardial infarction 28 days after the onset of disease and died soon thereafter; an autopsy revealed multiple necroses and fibroses of the myocardium and thrombus of a coronary artery. Although ECG abnormalities appear to be a common feature of trichinellosis, especially during the invasive phase of the disease, they are rarely associated with a poor prognosis. A transient, non-specific, ventricular-repolarization disturbance is the abnormality most commonly observed.

Clinical efficacy and safety of a short regimen of azithromycin sequential therapy vs standard cefuroxime sequential therapy in the treatment of community-acquired pneumonia: an international, randomized, open-label study.

An international, randomized, open-label, comparative study was undertaken in order to assess the efficacy and safety of azithromycin and cefuroxime, short sequential vs standard sequential therapy, respectively, in the treatment of patients with community-acquired pneumonia (CAP). 180 adult patients were included in the study. 89 patients received azithromycin 500 mg intravenously (i.v.) once daily for 1-4 days followed by azithromycin 500 mg orally once daily for 3 days. 91 patients received cefuroxime 1.5 g i.v. three times daily for 1-4 days followed by cefuroxime axetil 500 mg orally twice daily for 7 days. Clinical efficacy was achieved in 67/82 (81.7%) patients treated with azithromycin, and in 73/89 (82.0%) patients treated with cefuroxime. The mean duration of total (i.v. and oral) therapy was significantly shorter for the azithromycin group than for the cefuroxime group (6.2 days vs 10.1 days). Adverse events were recorded in 38.2% of patients treated with azithromycin, and in 29.7% of patients treated with cefuroxime (p = 0.20). Shorter sequential i.v.-to-oral azithromycin therapy of patients with CAP was as effective as standard sequential i.v.-to-oral cefuroxime therapy.

Characteristics of Puumala and Dobrava infections in Croatia.

In this study, two different hantaviruses, Puumala virus (PUUV) and Dobrava virus (DOBV), were demonstrated for the first time to coexist and cause hemorrhagic fever with renal syndrome (HFRS) in Croatia. Phylogenetic analysis showed some differences among the nucleotide sequences of PUUV originating from Dinara mountain, which was more closely related to Austrian PUUV than other Croatian PUUV from Mala Kapela mountain. More consistency was found among the Croatian DOBV. HFRS was verified in 85 of 201 suspected cases recorded in 1995 during the largest HFRS outbreak in Croatia. Most of these cases were soldiers. With the exception of the coastal region and islands, all of Croatia was found to be an area endemic for HFRS. A statistically significantly higher proportion of DOBV-infected patients had acute renal failure, visual disturbance, severe thrombocytopenia, and elevated levels of nonsegmented leukocytes, creatine, and total bilirubin. The prevalence of gastrointestinal and electrocardiography disorders also was greater in DOBV-infected patients. Interestingly, significantly more PUUV-infected patients had elevated systolic blood pressure on admission to the hospital. Further prospective studies are necessary to shed more light on differences in HFRS severity associated with PUU and DOB viruses.

Failure of azithromycin in treatment of Brill-Zinsser disease.

Two patients suffering from Brill-Zinsser disease were treated with azithromycin, which did not prove effective. Rickettsia prowazekii, the agent causing Brill-Zinsser disease, cannot be treated with azithromycin. Both patients had epidemiological features consistent with and a clinical course typical of the disease. The diagnosis of Brill-Zinsser disease was serologically confirmed.

Azithromycin: single 1.5 g dose in the treatment of patients with atypical pneumonia syndrome--a randomized study.

An open comparative study was undertaken in order to assess the efficacy and safety of a single dose of azithromycin in the treatment of community-acquired atypical pneumonia. A total of 100 adult patients with atypical pneumonia syndrome were randomized to receive 1.5 g of azithromycin as a single dose, or 500 mg once daily for 3 days. The presence of Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci, Coxiella burnetii, and Legionella pneumophila infection was diagnosed by serological tests. Control clinical examinations were performed 72 h, 10-12 days and 4 weeks after treatment initiation. Among 96 patients (48 in each group) who were evaluable for clinical efficacy M. pneumoniae infection was confirmed in 24, C. pneumoniae in nine, C. psittaci in five, C. burnetii in six, and L. pneumophila in five. Forty-seven patients (97.9%) in each group were cured. Side effects were observed in two patients in the single-dose group, and one patient in the 3-day group. In conclusion, a single 1.5 g dose of azithromycin may be an alternative to the standard 3-day azithromycin regimen in the treatment of outpatients with atypical pneumonia syndrome.

Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS).

Hantaviruses cause an important human illness, HFRS. Blood samples from 22 HFRS-positive, six seronegative patients and 15 healthy controls were examined in 1995, during the largest HFRS epidemic in Croatia. Results of double- and triple-colour immunofluorescence analysis showed an increased percentage of cytotoxic T cells (CD3+CD8+) in seropositive patients compared with seronegatives and healthy controls. The majority of seropositive HFRS patients expressed activation and memory antigens on T and B lymphocytes. The percentage of CD23+ and CD21+ B lymphocytes was lower in seropositive patients. HFRS patients had elevated levels of sCD23 and five had elevated total IgE. The increased expression of both early and late T cell activation antigens, e.g. CD25, CD71 and HLA-DR, memory cells and sCD23 positively correlated with biochemical parameters (AST, ALT, urea, alpha2-globulin) during the acute phase of HFRS. The phenotypic changes observed, especially early and late T cell activation markers, as well as memory cells, could be useful parameters in the evaluation of HFRS course, and prognostic factors of HFRS severity. Additional attention should be paid to liver involvement in the pathogenesis of HFRS.

[An epidemic of hemorrhagic fever with renal syndrome in Croatia in 1995]. / Epidemija hemoragijske vrucice s bubreznim sindromom u Hrvatskoj 1995. godine.

In the spring of 1995, the largest outbreak of hemorrhagic fever with renal syndrome (HFRS) so far was recorded in Croatia. There were 125 patients reported to the National Croatian Institute of Public Health, Department of Epidemiology. The disease occurred simultaneously in several localities, some of them close to the previously known natural foci (Mala Kapela, western Slavonia); the focus on Dinara was newly discovered. War circumstances in Croatia were closely related to this outbreak. There were 50 patients hospitalized in the University Hospital of Infectious Diseases in Zagreb; 5 of them were civilians from Zagreb area and 45 soldiers (Mala Kapela 33, Dinara 7, western Slavonia 5). In all patients the disease was serologically proven (in 6 by indirect immunofluorescence method and in 44 by ELISA-test). Both previously known types of viruses--Hantaan and Puumala were diagnosed in each locality. In general, the illness was more severe in patients with Hantaan virus infection. Two patients died, the illness was severe in 25, moderately severe in 11 and mild in 12 patients. For the first time inflammatory lung changes were recorded in 13 out of 37 (35.1%) patients who were examined by X-ray in the early stage of the disease.

[Legionnaires' disease is not uncommon in Croatia]. / Legionarska bolest u nas nije rijetka.

Legionella pneumophila has been relatively frequent pathogen of pneumonias worldwide. It causes 2-15% of pneumonias requiring hospitalization. It appears sporadically and in outbreaks, and frequently as hospital-acquired infection. The main aim of this study is to show the permanent presence of Legionnaires' disease in our country. In the 9-year period (1986-94), 42 patients were treated in the University Hospital of Infectious Diseases "Dr. Fran Mihaljevic" in Zagreb. The disease was diagnosed every year, and only one patient came from an outbreak, while all other cases of pneumonia were sporadic ones of pneumonia from the community. There were 40 males and only two women. Most of the patients (14) were in aged 30-39 yrs. There were six times more patients during summer and fall than in winter and spring time (36:6), and the highest number was in July (10). In 17 (40.5%) patients exposure to some potential sources of infection was recorded. Provocative factors i.e. underlying chronic diseases have been reported in 13 patients. There were 33 (78.6%) smokers, and 15 patients consumed greater quantities of alcoholic drinks. Legionnaires' disease affects also previously healthy persons as well as those without recognized provocative factors.

Azithromycin in the treatment of pneumonias caused by Chlamydia spp: a retrospective study.

A retrospective study was undertaken in order to compare the efficacy and safety of azithromycin and doxycycline in the treatment of pneumonias caused by Chlamydia spp. Patients with radiologically confirmed pneumonia and positive complement fixation test for chlamydial infection who were hospitalized in the University Hospital of Infectious Diseases, Zagreb during the years 1989-1992 were reviewed. Among them, 83 were treated with azithromycin, given in a total dose of 1.5 g over 5 days (500 mg once daily at day 1 followed by 250 mg at days 2-5, 60 patients) or 3 days (500 mg once daily, 23 patients). Twenty-two patients were treated with doxycycline (100 mg b.i.d. for 10 days). Treatment groups were comparable with respect to age, sex, and severity of signs and symptoms of illness. All the patients were cured. There were no differences in duration of fever after treatment initiation between patients treated with azithromycin (whether pretreated with beta-lactam antibiotics or not) and doxycycline (p > 0.05). In addition, 3- and 5-day azithromycin courses were equally effective (p > 0.05). Both drugs were well tolerated, and only two patients treated with azithromycin reported nausea. It may be concluded that in the treatment of pneumonias caused by Chlamydia spp. azithromycin is as effective and well tolerated as doxycycline.

Azithromycin for treatment of community acquired pneumonia caused by Legionella pneumophila: a retrospective study.

A clinical, retrospective and non-comparative study was undertaken to assess the clinical efficacy and tolerability of azithromycin in the treatment of community-acquired pneumonia caused by Legionella pneumophila. A total of 16 patients with a serologically confirmed diagnosis of Legionnaires' diseases were included. Azithromycin was administered orally at a total dose of 1.5 g for either 3 or 5 days. All patients were no side-effects requiring discontinuation of the treatment. Further increase of abnormal baseline liver function was recorded in 2 patients and in 1 patient mild, transient eosinophilia. Equal clinical efficacy and tolerability were observed with the 3- and 5-day dosage regimen. These results indicate that azithromycin given at a standard dose of 1.5 g is effective and well tolerated in the treatment of Legionnaires' disease.

[Clinical and epidemiologic features of acute respiratory infection caused by Mycoplasma pneumoniae]. / Klinicke i epidemioloske znacajke akutnih respiratornih infekcija izazvanih mikoplazmom pneumonije.

The results of clinical and laboratory analysis and treatment, as well as epidemiological features of acute respiratory infections caused by Mycoplasma pneumoniae in 390 patients have been reported. The patients were treated at the University Hospital of Infectious Diseases "Dr. Fran Mihaljevic", Zagreb, between January 1, 1980, and December 31, 1985. The diagnosis was established by the serologic method of complement fixation, on the grounds of fourfold increase or decrease of antibody titer in paired sera. Mycoplasma pneumoniae was the most frequently proved causative agent of acute respiratory infections in our admitted patients. There were 315 patients with pneumonia what makes 13.28% of all pneumonias, respectively 25.08% of nonbacterial pneumonias. Its participation in febrile respiratory catarrh syndrome was only 5.75%. Pneumonia occurred in schoolchildren most frequently, especially in those aged 10-14 years in whom 65.52% of nonbacterial pneumonias were connected with Mycoplasma pneumoniae. Men (230) were affected more frequently than women (160). The main symptoms of pneumonia were temperature, headache and cough. Inflammatory infiltrates were mostly interstitial and located unilaterally in the lower lobes of the lungs. Pleural effusion was recorded in 24 patients (7.62%). Doxycycline appeared to be the most effective antibiotic, but erythromycin and midecamycin yielded good results, as well.