High Detection Rates of Human Bocavirus in Infants and Small Children with Lower Respiratory Tract Infection from Croatia.

BACKGROUND: Human bocavirus (HBoV) is known to cause lower respiratory tract infections (LRTI) in children and may result in substantial morbidity and mortality. The aim of this study was to determine HBoV prevalence among hospitalized infants and small children with acute LRTI in Zagreb, Croatia, as well as to evaluate HBoV DNA quantity in samples in relation to the patients' age and co-infection with other respiratory viruses. METHODS: During winter season 2016/2017, a total of 295 children younger than three years of age who were admitted to hospitals with LRTI were tested for the presence of HBoV, respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) types 1 to 3, and human metapneumovirus (HMPV). HBoV was detected with a real-time PCR method, and the other viruses were diagnosed using monoclonal antibodies in direct fluorescence assay. RESULTS: Viral etiology was proven in 225/295 (76.3%) of patients. The most commonly diagnosed virus was RSV (59.3%), followed by HBoV (23.1%), PIVs (4.4%), ADV (3.1%), and HMPV (1.4%). HBoV-infected children were older than RSV-infected children; likewise, detection rates of HBoV infection increased with age, while RSV infection rates decreased with age. In 51% of HBoV-positive samples an additional respiratory virus was also detected. There was no difference in HBoV DNA quantity between samples with single virus detection and those with multiple virus detection (p = 0.056), although samples positive only for HBoV showed higher cycle threshold values. There was no difference in HBoV DNA quantity in samples of different age groups (p > 0.05). CONCLUSIONS: Frequent detection of HBoV in small children with LRTI, even in combination with other viruses, highlights its role in the pathogenesis of respiratory disease.

Comparative genomics of human rubulavirus 2.

Although human rubulavirus 2 (HPIV2) is an important respiratory pathogen, little is known about its molecular epidemiology. We performed a comparative analysis of the full-length genomes of fourteen HPIV2 isolates belonging to different genotypes. Additionally, evolutionary analyses (phylogenetic reconstruction, sequence identity, detection of recombination and adaptive evolution) were conducted. Our study presents a systematic comparative genetic analysis that complements prior analyses and utilizes full-length HPIV2 genomes to provide a basis for future work on the clinical significance, molecular variation and conservation, and evolution of HPIV2.

Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene.

BACKGROUND: Small hydrophobic (SH) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (HRSV). Its coding region constitutes less than 50% of the complete gene length, enabling SH gene to be highly variable and the SH protein highly conserved. In standard HRSV molecular epidemiology studies, solely sequences of the second hypervariable region of the glycoprotein gene (HVR2) are analyzed. To what extent do the strains identical in HVR2 differ elsewhere in genomes is rarely investigated. Our goal was to investigate whether diversity and inter-genotype differences observed for HVR2 are also present in the SH gene. METHODS: We sequenced 198 clinical samples collected within a limited area and time frame. In this HRSV collection, rapid and significant changes in HVR2 occurred. RESULTS: Over 20% of strains from this pool (containing HRSV genotypes NA1, ON1, GA5, BA9 and BA10) would be incorrectly assumed to be identical to another strain if only the HVR2 region was analysed. The majority of differences found in SH gene were located in the 5' untranslated region (UTR). Seven indels were detected, one was genotype GA5 specific. An in-frame deletion of 9 nucleotides (coding for amino acids 49-51) was observed in one of group A strains. Fifteen different SH protein sequences were detected; 68% of strains possessed the consensus sequence and most of others differed from the consensus in only one amino acid (only 4 strains differed in 2 amino acids). The majority of differing amino acids in group A viruses had the same identity as the corresponding amino acids in group B strains. When analysis was restricted to strains with identical HVR2 nucleotide sequences and differing SH protein sequences, 75% of differences observed in the SH ectodomain were located within region coding for amino acids 49-51. CONCLUSIONS: Basing HRSV molecular epidemiology studies solely on HVR2 largely underestimates the complexity of circulating virus populations. In strain identification, broadening of the genomic target sequence to SH gene would provide a more comprehensive insight into viral pool versatility and its evolutionary processes.

Genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in Croatia.

Human metapneumovirus (HMPV) is recognized as a global and frequent cause of acute respiratory tract infections among people of all ages. The objectives of this study were molecular epidemiology and evolutionary analysis of HMPV strains which produced moderate and severe acute respiratory tract infections in children in Croatia during four consecutive seasons (2011-2014). A total of 117 HMPV-positive samples collected from hospitalized pediatric patients presenting with acute respiratory tract infections and tested by direct immunofluorescence assay were first analyzed by amplifying a part of the F gene. Sixteen samples were further analyzed based on complete F, G, and SH gene sequences. HMPV genome was identified in 92 of 117 samples (78%) and the circulation of multiple lineages of HMPV was confirmed. In 2011, 2012, and 2014, subgroups A2 and B2 co-circulated, while B1 gained prevalence in 2013 and 2014. The study established the presence of a novel subcluster A2c in Croatia. This subcluster has only recently been detected in East and Southeast Asia. This study provides new insights into epidemiology and genetic diversity of HMPV in this part of Europe.

Genetic analysis of human parainfluenza virus type 3 obtained in Croatia, 2011-2015.

PURPOSE: This study investigated the HPIV3 circulating strains in Croatia and whether the other parts of HPIV3 genome (F gene and HN 582 nucleotides fragment) could be equally suitable for genetic and phylogenetic analysis. METHODOLOGY: Clinical materials were collected in period 2011-2015 from children suffering from respiratory illnesses. In positive HPIV3 samples viral genome was partially amplified and sequenced for HN and F genes. Obtained sequences were analysed by phylogenetic analysis and genetic characterization was performed. RESULTS: All samples from this study belonged to subcluster C and over a short period of time, genetic lineage C3a gained prevalence over the other C genetic lineages, from 39 % in 2011 to more than 90 % in 2013 and 2014. Phylogenetic classifications of HPIV3 based on the entire HN gene, HN 582 nt fragment and entire fusion (F) gene showed identical classification results for Croatian strains and the reference strains. Molecular analysis of the F and HN glycoproteins, showed their similar nucleotide diversity (Fcds P=0.0244 and HNcds P=0.0231) and similar Ka/Ks ratios (F Ka/Ks=0.0553 and HN Ka/Ks=0.0428). Potential N-glycosylation sites, cysteine residues and antigenic sites are generally strongly conserved in HPIV3 glycoproteins from both our and the reference samples. CONCLUSION: The HPIV3 subclaster C3 (genetic lineage C3a) became the most detected circulating HPIV3 strain in Croatia. The results indicated that the HN 582 nt and the entire F gene sequences were as good for phylogenetic analysis as the entire HN gene sequence.

Clinical and virological characteristics of hantavirus infections in a 2014 Croatian outbreak.

INTRODUCTION: Croatia is endemic for hemorrhagic fever with renal syndrome (HFRS), with both Puumala (PUUV) and Dobrava virus (DOBV) documented. Several large outbreaks were recorded in 1995, 2002, and 2012. We analyzed demographic, clinical, laboratory, and virological characteristics of HFRS cases detected in three geographically close natural foci (Ogulin, Slunj, and the Plitvice Lakes surroundings) during the 2014 outbreak. METHODOLOGY: From January to December 2014, 122 patients with suspected HFRS were tested for hantavirus IgM/IgG antibodies using an indirect immunofluorescence assay (IFA). Cross-reactive samples were further tested using a western blot (WB). For hospitalized patients from Ogulin area, clinical and laboratory data were analyzed. RESULTS: Acute infection was documented in 57 (46.7%) patients, of whom 75.4% were hospitalized. Ten (8.2%) patients were found to be IgG seropositive. Patients were 15-69 years of age and predominantly male (74.5%). The outbreak started in winter months, with most cases recorded from May to July (80.7%). The most frequently reported symptoms were fever (96.3%), chills/shivering (62.9%), and lumbar pain (48.1%). Mild clinical form was found in 66.7% patients, moderate in 18.5%, and severe in 14.8% patients (all but one infected with PUUV). One patient died. Using IFA, 48.8% patients showed monotypic antibody response, while in 51.2%, cross-reactive antibodies were found. PUUV was confirmed in 94.7% and DOBV in 5.3% HFRS cases by WB. CONCLUSIONS: Central mountainous Croatian regions are still highly endemic areas for HFRS. A higher percentage of severe PUUV infections could be at least partly associated with a patient's immune status.

Seroepidemiology of cytomegalovirus infections in Croatia.

BACKGROUND: Cytomegalovirus (CMV) is endemic worldwide, with marked differences in the seroprevalence rates between countries. The aim of this study was to analyze the seroprevalence of CMV infections in Croatia. METHODS: During a 3-year period (2013-2015) 2438 consecutive serum samples collected from Croatian residents were tested for the presence of CMV IgM and IgG antibodies using enzyme-linked immunoassay. The IgM/IgG positive samples were further tested for IgG avidity. RESULTS: The overall seroprevalence rates for CMV IgG and IgM antibodies were 74.4 % and 4.3 %, respectively. The IgG seroprevalence showed significant differences between population groups: children/adolescents 54.6 %, general adult population 77.2 %, hemodialysis patients 91.4 % (p < 0.001). Seropositivity of CMV was strongly age-dependent with prevalences ranging from 53.0 % in children less than 10 years old to 93.8 % in persons above 60 years (p < 0.001). There was no difference in the prevalence rate between women with normal pregnancy and women with poor obstetric history. Gender and place of residence were not associated with CMV seropositivity. Using IgG avidity, current/recent primary CMV infection was confirmed by a low/borderline avidity index (AI) in 46.7 % participants, while in 53.3 % a high AI indicated CMV reactivation or reinfection. Primary infections were detected mainly in children and adolescents (83.2 % and 70.5 %, respectively), while reactivation/reinfection was common in persons older than 40 (77.0-100 %). Reactivation/reinfection was most commonly detected in hemodialysis patients (92.3 %). Logistic regression showed that older age and being on hemodialysis were significant predictors of CMV seropositivity. CONCLUSION: Infections with CMV are widespread in the Croatian population. Older age and being on hemodialysis appear to be the main risk factors for CMV infection.

Genetic Variability and Sequence Relatedness of Matrix Protein in Viruses of the Families Paramyxoviridae and Pneumoviridae.

BACKGROUND: The families Paramyxoviridae and Pneumoviridae comprise a broad spectrum of viral pathogens that affect human health. The matrix (M) protein of these viruses has a central role in their life cycle. In line with this, molecular characteristics of the M proteins from variable viruses that circulated in Croatia were investigated. METHODS: Sequences of the M proteins of human parainfluenza virus (HPIV) 1-3 within the family Paramyxoviridae, human metapneumovirus (HMPV), and human respiratory syncytial virus from the family Pneumoviridae were obtained and analyzed. RESULTS: M proteins were very diverse among HPIVs, but highly conserved within each virus. More variability was seen in nucleotide sequences of M proteins from the Pneumoviridae family. An insertion of 8 nucleotides in the 3' untranslated region in 1 HMPV M gene sequence was discovered (HR347-12). As there are no samples with such an insertion in the database, this insertion is of interest and requires further research. CONCLUSION: While we have confirmed that M proteins were conserved among individual viruses, any changes that are observed should be given attention and further researched. Of special interest is inclusion of HPIV2 M proteins in this analysis, as these proteins have not been studied to the same extent as other paramyxoviruses.

A study of genetic variability of human parainfluenza virus type 1 in Croatia, 2011-2014.

Molecular epidemiology of human parainfluenza viruses type 1 (HPIV1) was investigated. Samples were collected from patients hospitalized in Croatia during the three consecutive epidemic seasons (2011-2014). Results indicated co-circulation of two major genetic clusters of HPIV1. Samples from the current study refer to clades II and III in a phylogenetic tree of haemagglutinin-neuraminidase (HN) gene. Additional phylogenetic trees of fusion (F) and phosphoprotein (P) genes confirmed the topology. Analysis of nucleotide diversity of entire P, F and HN genes demonstrated similar values: 0.0255, 0.0236 and 0.0237, respectively. However, amino acid diversity showed F protein to be the most conserved, while P protein was the most tolerant to mutations. Potential N- and O-glycosylation sites suggested that HPIV1 HN protein is abundantly glycosylated, and a specific N-glycosylation pattern could distinguish between clades II and III. Analysis of potential O-glycosylation sites in F protein indicated that samples from this study have two potential O-glycosylation sites, while publicly available sequences have five potential sites. This study provides data on the molecular characterization and epidemic pattern of HPIV1 in Croatia.

IgG Avidity: an Important Serologic Marker for the Diagnosis of Tick-Borne Encephalitis Virus Infection.

A total of 52 serum samples from patients with symptoms suggestive of tick-borne encephalitis virus (TBEV) infection and positive IgM and/or IgG antibodies were tested for IgG avidity. Acute/recent TBEV infection was confirmed by low/borderline avidity index (AI) in 94.8% IgM positive/IgG positive samples, while in 5.2% high AI was found indicating persisting IgM antibodies. Majority of IgM negative/IgG positive samples (78.6%) showed high AI consistent with past TBEV infection. However, in 21.3% patients without measurable IgM antibodies current/recent infection was confirmed by AI. IgG avidity represents an additional serologic marker that improves diagnosis of TBEV, especially in cases of atypical antibody response.

Genetic diversity among human parainfluenza virus type 2 isolated in Croatia between 2011 and 2014.

The dynamics and evolution of the human parainfluenza virus type 2 (HPIV2) in Croatia, and also globally, are largely unknown. Most HPIV2 infections are treated symptomatically outside the hospital setting. Thus, the diagnosis is missing making it difficult to follow the genetic variation and evolution of the HPIV2. This study explores hospitalized HPIV2 cases in Croatia during 4-year period (2011-2014). Most cases in this period were reported in October or November (68.75%) and most of patients were under 2 years of age (81.25%). For molecular analyses, we used the F and HN gene sequences and showed that although both regions are equally suitable for phylogenetic analyses it would be advantageous to use regions longer than 2 kb for HPIV2 analyses of isolates which are spatially and temporally closely related. We show here that the dominant cluster in this area was cluster G3 while only one strain isolated in this period was positioned in the distant cluster G1a. Further monitoring of the HPIV2 will determine whether cluster G3 will remain dominant or it will be overruled by cluster G1a. This will be important for the surveillance of virus circulation in population and significance of the viral infection. J. Med. Virol. 88:1733-1741, 2016. © 2016 Wiley Periodicals, Inc.

Early Evolution of Human Respiratory Syncytial Virus ON1 Strains: Analysis of the Diversity in the C-Terminal Hypervariable Region of Glycoprotein Gene within the First 3.5 Years since Their Detection.

OBJECTIVE: Characterization of the phylogeny and diversity of human respiratory syncytial virus (HRSV) genotype ON1 that occurred during its early evolution (within the first 3.5 years since the detection of the first ON1 strains). ON1 strains have a 72-nucleotide-long in-frame duplication within the second hypervariable domain of the glycoprotein gene (HVR2). METHODS: All available HVR2 sequences of strains belonging to the ON1 genotype published prior to June 20, 2014 were collected. Multiple sequence alignments, phylogeny, phylogeography, sequence clustering and putative protein analyses were performed. RESULTS: The worldwide spread and diversification of ON1 strains are presented. Only in a minority of ON1 strains do the two replicas remain identical, and various ON1 strains possess common differences between the first and the second copy (segments A and B). Mutations of the progenitor sequence were more frequent in segment B, a higher overall diversity on the protein level and more putative glycosylation sites exist in segment B, and, unlike in segment A, positive selection acts on that protein region. CONCLUSIONS: The fast spread of the novel HRSV genotype ON1 has been accompanied by its rapid concurrent diversification. Differences in variability of the two replicas within HVR2 were detected, with C-terminal replica being more variable.

[Usutu virus: a novel flavivirus in Croatia]. / Usutski virus: novi flavivirus u hrvatskoj.

Usutu virus (USUV) belongs to the family Flaviviridae, genus Flavivirus, Japanese encephalitis serocomplex. The virus was discovered in 1959 in South Africa and has emerged since 1996 causing epizootics with high avian mortality in Europe. The importance of USUV in humans is not fully understood. However, several human clinical cases of USUV infection described so far indicate the role of this virus as an antropozoonotic agent. In Croatia, serologic evidence of USUV was first documented in 2011 in two horses from Zagreb and Sisak-Moslavina County. In 2012, USUV neutralizing antibodies were found in one human sample from a resident of a Vukovar-Srijem County. Human clinical cases of USUV infection were detected for the first time during the West Nile virus outbreak from July to September 2013. Three patients with USUV neuroinvasive disease were detected in the City of Zagreb and Zagreb County. Our results indicate USUV circulation in Croatia. Further human cases could be expected in the next transmission seasons.

Seasonal occurrence of human metapneumovirus infections in Croatia.

BACKGROUND: Human metapneumovirus (HMPV) is 1 of the commonest causes of viral ARI especially among pediatric patients. Its incidence varies from year to year in countries belonging to moderate climate zone. The aim of this study was to investigate epidemiologic characteristics of HMPV infections in Croatia. METHODS: During a 4-year period (January 1, 2009, through December 31, 2012), nasopharyngeal aspirates were collected from 2610 children <10 years who were admitted to hospitals with acute respiratory infections. Direct immunofluorescence assay was used to detect the virus from clinical samples. Demographics and clinical data were also analyzed. RESULTS: HMPV was detected in 8.4% of patients. While many of HMPV-infected children were 13-24 months of age (30.9% of all proven HMPV infections), the highest incidence of HMPV infection was recorded in 2- to 5-year-old children (11.4% of all children in this age group). HMPV caused 7.1% of upper respiratory tract infections and 11.7% of lower respiratory tract infections. Annual prevalence rates of HMPV infection varied significantly from year to year (P < 0.001). Peak incidence was detected in spring or winter months, depending on the year. CONCLUSIONS: This study indicates that HMPV infections in Croatia show a biennial outbreak pattern characterized by alternation of winter and spring activity. HMPV outbreaks alternate with respiratory syncytial virus outbreaks.

[POLIOMYELITIS ERADICATION--ONE STEP TO ACHIEVE THE GOAL]. / ERADIKACIJA POLIOMIJELITISA--KORAK DO CILJA.

Poliomyelitis is a very old disease of humans, caused by poliovirus. With appearance of the epidemics in the 20th century, poliomyelitis became a global public health issue. In 1988, the World Health Organization started a campaign for global eradication of poliomyelitis and till now poliomyelitis cases have been reduced by more than 99%. In Croatia, the introduction of vaccination in 1961 resulted in dramatic reduction of paralytic disease. The European region, including Croatia was certified polio free in 2002. However, the final goal of the "polio-free world" has not yet been reached. To reinforce the campaign, the global polio eradication initiative has come up with the Polio Eradication & Endgame Strategic Plan 2013-2018 with detailed program how to resolve the main challenges: (a) continued transmission of wild polioviruses in endemic reservoirs; (b) reinfection of polio-free areas; and (c) outbreaks due to the circulating vaccine-derived polioviruses (cVDPV). Global oral polio vaccine cessation will follow, with the introduction of universal use of inactivated polio vaccine.

[TICK-BORNE ENCEPHALITIS VIRUS: EPIDEMIOLOGICAL AND CLINICAL PICTURE, DIAGNOSIS AND PREVENTION]. / VIRUS KRPELJNOG ENCEFALITISA: EPIDEMIOLOSKA I KLINICKA SLIKA, DIJAGNOSTIKA I PREVENCIJA.

Tick-borne encephalitis virus (TBEV) is a small, enveloped virus that belongs to the family Flaviviridae, genus Flavivirus, tick-borne encephalitis serocomplex. There are three subtypes of TBEV: European, Far-Eastern and Siberian subtypes, which differ in geographical distribution, tick vector and clinical manifestation of disease in humans. TBEV is endemic in a wide geographic area ranging from Central Europe and the Scandinavian Peninsula to Japan. The virus is maintained in nature in so-called natural foci in cycles involving ticks and wild vertebrate hosts (mainly small rodents). The principal vector for the European subtype is Ixodes (I.) ricinus tick, whereas for Far-Eastern and Siberian subtypes it is I. persulcatus. In the Baltic States and Finland, co-circulation of two or all three subtypes was documented. Several animals, principally small rodents, serve as virus reservoirs. In the tick population, TBEV is transmitted by feeding/co-feed ing on the same host, transovarially (from infected females to their eggs) and trans-stadially (from one development stage to the next). An infected tick remains infected for life. While most TBE infections in humans occur following a tick bite, alimentary routes of TBEV transmission (consumption of unpasteurized milk/milk products from infected livestock) have also been described. All three tick stages can transmit the infection to humans. In the last decade, an increase of TBE incidence has been observed in some endemic areas. This could be due to a number of interacting factors such as changes in the climatic conditions affecting tick habitats, improvements in the quality of epidemiological surveillance systems and diagnostics, in landscape resources and their utilization and more outdoor recreation activity. In addition, the endemic area of TBEV has expanded to higher altitudes (up to 1500 m), apparently influenced by climatic changes. The typical clinical picture of infection with European subtype TBEV is characterized by a biphasic course (50%-77%). The first phase is characterized by nonspecific, flu-like symptoms followed by an asymptomatic interval of about one week. In 20%-30% of persons who develop symptoms, the second phase occurs with symptoms of central nervous system involvement (meningitis, encephalitis, myelitis, radiculitis). The mortality rate for European subtype is 1%-2%. Diagnosis is usually based on detection of specific antibodies (enzyme immunoassay, indirect immunofluorescent assay, plaque reduction neutralization test). From 1993 to 2013, a total of 777 cases of TBE were reported in Croatia. Endemicity is highest in north-western counties (mean incidence 3.61-6.78/100,000 inhabitants). The majority of patients were older than 20 years (88%). Most cases (73%) were reported from May to July.

[West nile virus infection: re-emergent disease in Croatia]. / Infekcija virusom zapadnog nila: re-emergentna bolest u hrvatskoj.

West Nile virus (WNV) is a small, enveloped, spherical virus that belongs to the family Flaviviridae, genus Flavivirus, Japanese encephalitis serocomplex. Natural reservoirs of WNV are birds, and the main vectors are mosquitoes of the genus Culex. There are seven genetic lineages of WNV. Lineages 1 and 2 are the most widely distributed (Africa, North and South America, Europe, Asia and Australia). About 80% of infections are asymptomatic. In 20% of patients nonspecific febrile disease occurs (West Nile fever). Less than 1% of infected persons will develop neuroinvasive WNV disease (meningitis, encephalitis, and poliomyelitis). In Croatia, antibodies to WNV were demonstrated in humans,bears and horses. In August-September 2012 clinical cases of human WNV neuroinvasive disease and asymptomatic acute infection in horses were reported for the first time in three eastern Croatian counties. The diagnosis was confirmed by serologic tests (enzyme immunoassay, IgG avidity, plaque-reduction neutralization test).

Demonstration of Usutu virus antibodies in horses, Croatia.

We report the first serological evidence of Usutu virus (USUV) infection in horses in Croatia. During 2011, 1380 horse serum samples from healthy animals were collected from six northern Croatian counties. All samples were first screened for West Nile virus (WNV) immunoglobulin G (IgG) antibodies using an enzyme-linked immunosorbent assay (ELISA). Sixty-nine WNV ELISA-reactive samples were further tested for WNV antibodies by a virus neutralization assay (VN assay) and plaque reduction neutralization test (PRNT), and USUV by a VN assay and tick-borne encephalitis virus (TBEV) antibodies by PRNT. During the same period, 306 human serum samples from patients coming for routine testing with no symptoms of acute febrile disease were tested for USUV IgG using ELISA. Reactive samples were tested for both USUV and WNV using a VN assay. USUV-specific neutralizing antibodies were detected in two of 69 WNV ELISA-reactive horse serum samples. Seropositive animals were found in two different regions of Croatia. One additional sample showed specific WNV-neutralizing antibodies that cross-neutralized USUV. Only one human sample (0.3%) was reactive to USUV antibodies in an ELISA test. In a confirmatory test, WNV-neutralizing antibodies were detected, indicating cross-reactive antibodies with USUV in ELISA. The exposure to USUV was documented in two WNV ELISA-reactive horses at distant locations. These results indicate the presence of USUV in northern Croatia.

In vitro susceptibility of urogenital Chlamydia trachomatis strains in a country with high azithromycin consumption rate.

Although Chlamydia trachomatis resistance is not of great concern due to its excellent sensitivity to the currently recommended first-line antibiotics (azithromycin and doxycycline), clinical treatment failures have been reported and some of them were linked to laboratory proved resistance. The aim of this study was to determine in vitro susceptibility to azithromycin and doxycycline for 24 urogenital chlamydial strains isolated in Croatia-a country with the highest consumption of azithromycin in Europe and with very high antibiotic prescription rates. Fourteen isolates from cervical swabs, nine from male urethral swabs, and one isolate from expressed prostatic secretion were tested in McCoy cell culture system. All strains were susceptible to azithromycin and doxycycline with minimal inhibitory concentration for azithromycin and doxycycline ranging from 0.064 to 0.125 µg/mL and 0.016 to 0.064 µg/mL, and minimal chlamydicidal concentration ranging from 0.064 to 2.0 µg/mL and 0.032 to 1.0 µg/mL, respectively. Since we still lack information on whether C. trachomatis is evolving in vivo in response to antibiotic selection pressure, this kind of surveillance for resistance is essential in detecting shifts in antimicrobial susceptibilities.

A study of the genetic variability of human respiratory syncytial virus in Croatia, 2006-2008.

Human respiratory syncytial virus (HRSV) is a common etiological agent of acute lower respiratory tract disease in infants. The molecular epidemiology of HRSV in Croatia over four consecutive seasons (from 2006 to 2008) was investigated. A total of 72 HRSV samples were chosen from 696 screened cases in a pediatric clinic in Zagreb. Molecular characterization of HRSV revealed the predominance of HRSV group B viruses in the first two epidemic seasons and HRSV group A viruses in the next two seasons. According to the phylogenetic analysis, NA1 and BA9 were the predominant circulating HRSV genotypes detected during the study. Overall, 82.9% of all HRSV A strains belonged to the NA1 genotype. The HRSV B genotype BA9, detected in two consecutive seasons (2006 and 2007), was the predominant circulating HRSV B genotype, accounting for 80.6% of all HRSV B strains. This study provides data on the circulation pattern of HRSV genotypes in Croatia and their molecular characterization.