Prevalence and Molecular Characterization of Human Bocavirus Detected in Croatian Children with Respiratory Infection.

Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs' genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10-4 and 10-5 substitutions per site and year. Recombination was not detected among sequences from this study.

Polyclonal spread of colistin resistant Klebsiella pneumoniae in Croatian hospitals and outpatient setting.

INTRODUCTION: Recently, a marked increase in the rate of colistin resistant Klebsiella pneumoniae was observed in Croatian hospitals and the outpatient setting. This prompted us to analyze the molecular epidemiology of these isolates and the mechanisms of spread. METHODS: In total 46 colistin-resistant K. pneumoniae isolates from five hospitals and the community were analyzed. The presence of genes encoding broad and extended-spectrum ß-lactamases, plasmid-mediated AmpC ß-lactamases and carbapenemases was determined by PCR. Plasmids were characterized by PCR based replicon typing. Isolates were genotyped by pulsed-field gel electrophoresis. Virulence traits such as hemolysins, hyperviscosity and resistance to serum bactericidal activity were determined by phenotypic methods. RESULTS: High resistance rates were observed for cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem, ciprofloxacin and gentamicin. The majority of OXA-48 producing isolates were resistant to ertapenem but susceptible to imipenem and meropenem. Nine strains transferred ertapenem resistance to E. coli recipient strain. Thirty-nine strains were phenotypically positive for ESBLs and harbored group 1 of CTX-M ß-lactamases. OXA-48 was detected in 39 isolates, KPC-2 in four and NDM-1 in one isolate. The isolates belonged to six PFGE clusters. All isolates were found to be resistant to serum bactericidal activity and all except four strains positive for KPC, produced ß-hemolysins. String test indicating hypermucosity was positive in only one KPC producing organism. CONCLUSIONS: The study demonstrated the ability of K. pneumoniae to accumulate different resistance and virulence determinants. We reported dissemination of colistin resistant K. pneumoniae in five hospitals, located in different geographic regions of Croatia and in the outpatients setting. mcr genes responsible for transferable colistin resistance were not found, indicating that resistance was probably due to chromosomal mutations.

The Emerging Role of Rhinoviruses in Lower Respiratory Tract Infections in Children - Clinical and Molecular Epidemiological Study From Croatia, 2017-2019.

Rhinoviruses (RVs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of RVs among the infected children. Therefore, we investigated the rhinovirus (RV) infection prevalence over a 2-year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of RV infections among 590 children hospitalized with acute respiratory infection in north-western and central parts of Croatia. For respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex RT-PCR. To determine the RV species in a subset of positive children, 5'UTR in RV-positive samples has been sequenced. Nucleotide sequences of referent RV strains were retrieved by searching the database with Basic Local Alignment Tool, and used to construct alignments and phylogenetic trees using MAFFT multiple sequence alignment tool and the maximum likelihood method, respectively. In our study population RV was the most frequently detected virus, diagnosed in 197 patients (33.4%), of which 60.4% was detected as a monoinfection. Median age of RV-infected children was 2.25 years, and more than half of children infected with RV (55.8%) presented with lower respiratory tract infections. Most RV cases were detected from September to December, and all three species co-circulated during the analyzed period (2017-2019). Sequence analysis based on 5'UTR region yielded 69 distinct strains; the most prevalent was RV-C (47.4%) followed by RV-A (44.7%) and RV-B (7.9%). Most of RV-A sequences formed a distinct phylogenetic group; only strains RI/HR409-18 (along with a reference strain MF978777) clustered with RV-C strains. Strains belonging to the group C were the most diverse (41.6% identity among strains), while group B was the most conserved (71.5% identity among strains). Despite such differences in strain groups (hitherto undescribed in Croatia), clinical presentation of infected children was rather similar. Our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where RVs should always be considered as potentially serious pathogens.

Viral pathogens associated with acute respiratory illness in hospitalized adults and elderly from Zagreb, Croatia, 2016 to 2018.

AIMS: To investigate the viral etiology of acute respiratory infection (ARI) in hospitalized adults and elderly patients in Croatia, compare the prevalence of detected viruses, and to determine clinical characteristics and seasonal occurrence of investigated infections. METHODS: From January 2016 to June 2018, a total of 182 adult patients presented with symptoms of ARI and admitted to the hospital were tested for 15 respiratory viruses by multiplex reverse-transcription polymerase chain reaction. Clinical data were collected by retrospective analysis of the patient's chart. RESULTS: A virus was identified in 106 (58.5%) of the patients. The most commonly detected virus was influenza virus (41.5%), followed by respiratory syncytial virus (13.8%), human metapneumovirus (13.0%), parainfluenza viruses (12.2%), rhinoviruses (11.4%), adenovirus and coronaviruses with equal frequencies (3.3%), and enterovirus (1.6%). The serum level of C-reactive protein and white blood cell count were significantly lower in patients with respiratory viruses identified when compared with those in whom no virus was detected (P < 0.001 and P = 0.007, respectively). There were no differences in clinical symptoms according to the type of the detected virus, except for more frequent illness exposure recall for influenza infection ( P = 0.010). Influenza, parainfluenza, and pneumoviruses were detected mostly in winter months, while rhinoviruses in autumn and spring. CONCLUSIONS: In addition to influenza, pneumoviruses, rhinoviruses, and parainfluenza viruses play an important role in etiology of ARIs in adults. Fast and accurate laboratory diagnosis for respiratory viruses in routine practice is needed for clinicians optimally manage patients with ARI and potentially avoid the unnecessary use of antimicrobial drugs.

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.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PURPOSE: A dramatic increase in OXA-48 ß-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. METHODS: Carbapenemase and other ß-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 ß-lactamases. In addition to the ß-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, ß-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

COMPARISON OF TWO DIFFERENT METHODS FOR TIGECYCLINE SUSCEPTIBILITY TESTING IN ACINETOBACTER BAUMANNII.

- Tigecycline susceptibility testing (TST) presents a tremendous challenge for clinical microbiologists. Previous studies have shown that the Epsilometer test (E-test) and Vitek 2 automated system significantly overestimate the minimum inhibitory concentrations for tigecycline resistance compared to the broth microdilution method (BMM). This leads to very major errors or false susceptibility (i.e. the isolate is called susceptible when it is actually resistant). The aim of this study was to compare E-test against BMM for TST in carbapenem-resistant and carbapenem-susceptible Acinetobacter (A.) baumannii and to analyze changes in tigecycline susceptibility between two time periods (2009-2012 and 2013-2014), with BMM as the gold standard. Using the EUCAST criteria, the rate of resistance to tigecycline for the OXA-23 MBL-positive, OXA-23 MBL-negative and carbapenemase-negative strains for BMM was 54.5% (6/11), 29.4% (5/17) and 2.7% (1/37), respectively; the OXA-24/40 and OXA-58 producing organisms did not exhibit any resistance. With E-test, all OXA-23 MBL-positive organisms (11/11), 23.5% (4/17) of OXA-23 MBL-negative, and 4.1% of OXA-24/40 (3/74) strains displayed tigecycline resistance; there were no resistant strains among the OXA-58 and carbapenemase-negative isolates. Resistance emerged in the bacterial isolates from 2013 to 2014. Although tigecycline does not display cross-resistance, the highest rates of resistant A. baumannii isolates were observed among those producing VIM MBL, regardless of the testing method. These findings suggest that the commercial E-test does not provide reliable results for TST of A. baumannii. Further confirmation with the dilution method should be recommended, particularly in cases of serious infections.