Goran Stevanovic

Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients.

Seven strains of Epstein–Barr virus (EBV) are defined based on C‐terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30‐bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C‐terminal region of LMP1 in Serbian isolates. This study included 53 EBV‐DNA‐positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506‐bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non‐deleted (66%), and the rest had 30‐bp, rare 69‐bp, or yet unknown 27‐bp deletions, which were not related to malignant or non‐malignant isolate origin. However, the majority of 69‐bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33‐bp repeats were found in the majority of non‐deleted isolates (68.6%), whereas most 69‐bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95‐8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical‐associated characteristics in the LMP1 C terminus of investigated isolates. J. Med. Virol. 84:632–642, 2012.

Imported malaria in Belgrade, Serbia, between 2001 and 2009

ZusammenfassungSeit 2000 steigt die Zahl serbischer Bürger, die Reisen in die Tropen machen, langsam aber ständig. Um die epidemiologischen und klinischen Charakteristika der nach Serbien verschleppten Malaria zu erfassen, analysierten wir die klinische Anamnese aller Reisenden, die sich nach der Rückkehr aus tropischen oder subtropischen Gebieten zwischen 2001 und 2009 am Institut für Infektionen und Tropenkrankheiten in Belgrad vorstellten. Insgesamt wurden 2981 Reisende erfasst, von denen 847 gesundheitliche Probleme hatten. Eine Malaria wurde bei 102 (3,4 % aller Reisenden; 12 % aller Reisenden mit Fieberschüben) diagnostiziert. Mit einer Inzidenzrate von 6-16 Fälle pro Jahr wurde die Malaria hauptsächlich aus Afrika (92,2 %), vor allem Äquatorial Guinea (38,2 %) und Nigeria (15,7 %) eingeschleppt. Der häufigste Grund für die Reisen war geschäftlich, beziehungsweise beruflich. Die Patienten waren zumeist Männer (87,3 %) in einem Alter großteils zwischen 40 und 59 Jahren (66,7 %). Insgesamt nahmen 15 der erkrankten Personen (14,7 %) irgendeine Form einer Malariaprophylaxe ein. Plasmodium (P) falciparum (78), alleine (70) oder gemischt mit P. vivax (5) war die häufigste ursächliche Spezies. P. vivax, P. ovale und P. malariae konnten in 11, 1 und 1 der Fälle als alleiniger Erreger nachgewiesen werden. Von den 11 Fällen, in denen der Parasit nicht gefunden wurde, schienen sechs echte submikroskopische Fälle zu sein. Der klinische Verlauf war bei 13 Patienten (alle mit P. falciparum infiziert) schwer. Drei dieser Patienten (2,9 %) verstarben. Screening auf Malaria sollte in Serbien für in endemische Regionen Reisende, die sich mit Fieber präsentieren, (unabhängig davon, ob sie eine Chemoprophylaxe einnehmen oder nicht), verpflichtend sein. Nicht ausreichende Sensitivität der konventionellen Methoden (wie wir sie bei den submikroskopischen Fällen beobachtet haben) erfordert die Einführung molekularer Diagnostik in die Routinepraxis.SummarySince 2000, travel of Serbian citizens to tropical areas has been slowly but steadily increasing. To determine the epidemiological and clinical characteristics of imported malaria in Serbia, we analyzed clinical history data of all travelers who presented at the Clinic for Infectious and Tropical Diseases in Belgrade after their return from tropical and subtropical areas between 2001 and 2009. The study series involved a total of 2981 travelers, and included both those with (847) and without (2134) health problems. Malaria was diagnosed in 102 cases (3.4% of all travelers; 12.0% of travelers with febrile episodes). Occurring at a rate of 6 to 16 cases per year, it was predominantly imported from Africa (92.2%), particularly from Equatorial Guinea (38.2%) and Nigeria (15.7%). The most frequent reason for travel was work/business. Patients were predominantly (87.3%) male, and the majority (66.7%) was between 40 and 59 years of age. A total of 15 (14.7%) patients took some form of anti-malarial chemoprophylaxis. The dominant causative species was Plasmodium falciparum (78), alone (70) or in mixed infection with P. vivax (5) and P. malariae (3). P. vivax, P. ovale and P. malariae as single agents were each identified in 11, 1 and 1 cases, respectively. Of the 11 cases in which the parasite was not detected, six appeared to be true submicroscopic cases. The clinical course of the disease was severe in 13 patients, all with falciparum malaria, of which three (2.9%) died. Rather than for all travelers, in Serbia screening for malaria should be mandatory in all travelers to endemic regions who present with fever irrespective of chemoprophylaxis history. Inadequate sensitivity of conventional diagnostic methods, illustrated by the cases of submicroscopic malaria, requires introduction of molecular diagnosis in routine practice.

Imported parasitic infections in Serbia

BACKGROUND Travel to the tropics is associated with a risk of parasitic infection, which is increasing in parallel with the rise in travel to these areas. We thus examined the prevalence and trend in the occurrence of parasitic infections in Serbian travelers. METHODS A retrospective analysis of the medical records of all travelers returning from tropical and subtropical areas, who presented at the Institute for Infectious and Tropical Diseases in Belgrade between January 2001 and January 2008, was performed. RESULTS Of a total of 2440 travelers, 169 (6.9%) were diagnosed with a parasitic infection, including malaria in 79, intestinal parasites in 84 (pathogenic species in 30 and non-pathogenic in 54), filariasis in four, and visceral leishmaniasis and fascioliasis in one patient each. Importantly, of the whole series only 583 (23.9%) were symptomatic, of which 19.4% were found to be infected with a parasite. The single pathogenic parasite occurring in asymptomatic patients was Giardia intestinalis. CONCLUSIONS Parasitic infection causing symptomatic disease among travelers returning from tropical areas to Serbia is not infrequent. In view of the expected increase in travel to the tropics, diagnostic protocols for tropical parasitic diseases should take these data into account.

The first outbreak of brucellosis in the region of Sabac.

BACKGROUND/AIM In Serbia brucellosis is a primary disease of the animals in the southern parts of the country. The aim of this study was to describe the first outbreak of human and animal brucellosis in the region of Sabac, Serbia. METHODS An epidemiological investigation was conducted to identify a source of outbreak and the ways of transmission of brucellosis infection in human population. A descriptive and analytical epidemiological methods (cohort study) were used. Additional data included monthly reports of the infectious diseases from the Institutes of Public Health and data from the Veterinary Specialistic Institute in Sabac. The serological tests for human brucellosis cases were performed in the Laboratory of the Military Medical Academy; laboratory confirmation of animal brucellosis cases was obtained from the reference laboratory of the Faculty of Veterinary Medicine, Belgrade. RESULTS Twelve cases of brucellosis were recorded from February 9 to September 1, 2004. Total attack rate was 8.1% (7.5% of males, 14.2% of females). Relative risk (RR) of milk consumption was 8.9 (95% confidence interval: 1.63-13.38), and RR for direct contact with animals was 14 (95% confidence interval: 3.5-55.6). The prevalence of seropositive animals in 33 villages of the Macva region accounted for 0.8%. Regarding animal species, sheep were predominant--264 (95.7%). Out of a total number of seropositive animals, ELISA results were positive in 228 (88.7%) of them. CONCLUSION As contact epidemics generally last longer, it is probable that the implemented measures of outbreak control did reduce the length of their duration.

Study toward resolving the controversy over the definition of allergic fungal rhinosinusitis

&NA; Dysbiosis of the microbiome on the airway mucosa leads to the development of chronic inflammatory and allergic disorders. The aim of this study was to consider the potential diagnostic criteria for allergic fungal rhinosinusitis (AFRS) and nonallergic fungal rhinosinusitis (FRS), and the role of fungal presence in an environment for the development of AFRS. In this study, 136 patients were divided into two groups: patients with positive specific immunoglobulin E (sIgE) and fungal finding (AFRS group), and patients with negative sIgE and positive fungal finding (FRS group). The study design included: anamnesis data, sIgE, eosinophil count and skin‐prick test, rhinology and computerized tomography (CT) observation and mycological finding. Our results showed: (i) the prevalence in Serbia is: AFRS 1.3%, FRS 2.8%; (ii) 30.4% patients with sIgE+ had more often severe and recurrent chronic rhinosinusitis (CRS) (P = .005) and the presence of polyps (P = .025); (iii) 46.4% patients with sIgE+ had positive fungi on the sinonasal mucosa and were considered as AFRS; (iv) patients with AFRS had more frequent asthma (P = .024) and chronicity of CRS >10 years (P = .000). The persistent fungal presence and prolonged duration of CRS could be a silent threat for the progression of inflammation and development of FRS. Lavage with hypertonic‐NaCl should be included in the everyday hygiene routine in an effort to decrease fungal load and antigenic exposure. The presence of allergological parameters and better response to corticosteroid therapy in AFRS patients should be considered as crucial diagnostic criteria for AFRS.

Antifungal activity of Myrtus communis against Malassezia sp. isolated from the skin of patients with pityriasis versicolor

The increasing incidence of fungal infections and antifungal resistance has prompted the search for novel antifungal drugs and alternative agents. We explored the antifungal activity of Myrtus communis essential oil (EO) against Malassezia sp. isolated from the skin of patients with pityriasis versicolor. These broad-spectrum antimicrobial activities of M. communis EO and its potent inhibiting activity on Malassezia growth deserve further research with aim to considerate this EO as candidate for topical use in treatment of skin diseases.

Safety and immunogenicity of a seasonal trivalent inactivated split influenza vaccine: a phase I randomized clinical trial in healthy Serbian adults

ABSTRACT This study was a phase I double-blind, randomized, placebo-controlled trial to evaluate the safety and immunogenicity of a Serbian-produced seasonal trivalent split, inactivated influenza vaccine in healthy adults. The vaccine was manufactured in eggs by the Torlak Institute of Virology, Vaccines and Sera, Belgrade, Serbia and contained A/H1N1, A/H3N2 and B viruses. The clinical trial took place at the Clinical Center of Serbia in Belgrade. Sixty healthy volunteers, aged 18–45 years, were enrolled in the trial. On the day of immunization, volunteers were randomly assigned to receive either a single dose of the trivalent seasonal influenza vaccine (15 μg of hemagglutinin per strain) or placebo (phosphate-buffered saline). Subjects were monitored for adverse events through a clinical history and physical examination, and blood was taken for testing at screening and on day 8 to assess vaccine safety. Serum samples obtained before and 21 days after immunization were tested for influenza antibody titers using hemagglutination-inhibition (HAI) and microneutralization (MN) tests. No serious adverse events were reported. Pain and tenderness at the injection site were the most commonly reported symptoms in both vaccine and placebo groups. Overall, serum HAI responses of fourfold or greater magnitude were observed to H1, H3, and B antigen in 80%, 75%, and 70% of subjects, respectively. Seroprotection rates as measured by HAI were also high (100%, 100% and 86.67%, respectively, for H1, H3 and B). Thus, Torlaks seasonal trivalent influenza vaccine was not associated with adverse events, was well-tolerated and immunogenic. It should be further evaluated in clinical trials to provide sufficient safety and immunogenicity data for licensing in Serbia.

Predictors of Hospitalization and Admission to Intensive Care Units of Influenza Patients in Serbia through Four Influenza Seasons from 2010/2011 to 2013/2014

A retrospective analysis of the surveillance data on laboratory confirmed cases of influenza in 4 post pandemic seasons in Serbia was performed to evaluate predictors of hospitalization and admission to intensive care units (ICU). The specimens, including nasal and throat swabs were tested for influenza. Univariate and multivariate logistic regression analyses were performed. Data of a total of 777 confirmed influenza cases were analyzed. Age > 65 years, the presence of any co-morbidity or the presence of ≥ 2 comorbidities, infection with influenza virus subtype A (H1) pdm09, and an interval greater than 3 days between symptom onset and the first physician visit, were independently associated with hospital admission. These variables, as well as infection with non-subtype influenza virus A, were predictors for ICU admission. Obesity and chronic neurological disease were independent predictors for ICU admission but not hospitalization. Overall, 41.7% of patients with influenza had at least one co-morbidity, but only 3% of all patients were vaccinated against influenza. Identification of high risk groups and education of these groups regarding their increased susceptibility to severe forms of influenza, and in particular regarding the importance of influenza vaccination, is essential.

Molecular typing, pathogenicity factor genes and antimicrobial susceptibility of vancomycin resistant enterococci in Belgrade, Serbia

In this study the distribution of species and antimicrobial resistance among vancomycin resistant enterococci (VRE) recovered from clinical specimens obtained from five hospitals in Belgrade was analyzed. Strains were further characterized by pulsed-field gel electrophoresis (PFGE). Polymerase chain reaction (PCR) was used to investigate the presence of vanA and vanB genes and pathogenicity factor genes. Identification of 194 VRE isolates revealed 154 Enterococcus faecium, 21 Enterococcus faecalis, 10 Enterococcus raffinosus and 9 Enterococcus gallinarum. This study revealed existence of 8 major clones of VRE. PCR determined vanA gene to be present in all of the VRE studied. Esp and hyl genes were present in 29.22% and 27.92% of E. faecium, respectively, and in 76.19% and 0 of E. faecalis, respectively. Esp and hyl genes were not found more frequently in members of predominant clones of E. faecium than in single isolates; nor was their presence connected to invasiveness.

Molecular characterization of vancomycin-resistant enterococci in Serbia: intensive care unit as the source.

The purpose of this study was to evaluate the molecular relatedness of clinical isolates of vancomycin-resistant enterococci (VRE) collected from patients of the Clinic for Infectious and Tropical Diseases in Belgrade. Among 40 isolates available for the investigation, 36 were identified as Enterococcus faecium, whereas 2 were Enterococcus faecalis and Enterococcus raffinosus, respectively. Pulsed-field gel electrophoresis (PFGE) typing revealed 21 strain types, comprising 7 clusters which contained at least two isolates and 14 unique PFGE patterns. Although we searched for pathogenicity factor genes (gelE, cylB, asa1, efaAfs, esp, cpd, cob) in representatives of all macro-restriction patterns, they have been confirmed in only one clone of E. faecalis. Genes esp and hyl, commonly found in E. faecium, were yilded in 10 macro-restriction patterns of this species, and their presence could not be ascribed to clonally related strains (p = 0.05). All VRE isolates were multiresistant and positive for vanA gene. Twenty strains of VRE and 6 clusters obtained from Intensive care unit (ICU) are proof of intensive transmission of these microorganisms at this department. The results of this study suggest wide genotypic variability among the clinical VRE isolates, but also intrahospital dissemination of some of them.