Diffusion of OXA-48 carbapenemase among urinary isolates of Klebsiella pneumoniae in non-hospitalized elderly patients.

BACKGROUND: Recently, a dramatic increase of Klebsiella pneumoniae positive for OXA-48 ß-lactamases was observed first in the hospital setting and later in the long-term care facilities (LTCFs) and community in the Zagreb County, particularly, in urinary isolates. The aim of the study was to analyse the epidemiology and the mechanisms of antibiotic resistance of OXA-48 carbapenemase producing K. pneumoniae strains isolated from urine of non-hospitalized elderly patients. RESULTS: The isolates were classified into two groups: one originated from the LTCFs and the other from the community. Extended-spectrum ß-lactamases (ESBLs) were detected by double disk-synergy (DDST) and combined disk tests in 55% of the isolates (51/92). The ESBL-positive isolates exhibited resistance to expanded-spectrum cephalosporins (ESC) and in majority of cases to gentamicin. LTCFs isolates showed a significantly lower rate of additional ESBLs and consequential resistance to ESC and a lower gentamicin resistance rate compared to the community isolates, similarly to hospital isolates in Zagreb, pointing out to the possible transmission from hospitals.ESBL production was associated with group 1 of CTX-M or SHV-12 ß-lactamases. Ertapenem resistance was transferable from only 12 isolates. blaOXA-48 genes were carried by IncL plasmid in 42 isolates. In addition IncFII and IncFIB were identified in 18 and 2 isolates, respectively. Two new sequence types were reported: ST4870 and ST4781. CONCLUSIONS: This study showed eruptive and extensive diffusion of OXA-48 carbapenemase to LTCFs and community population in Zagreb County, particularly affecting patients with UTIs and urinary catheters. On the basis of susceptibility testing, ß-lactamase production, conjugation experiments, MLST and plasmid characterization it can be concluded that there was horizontal gene transfer between unrelated isolates, responsible for epidemic spread of OXA-48 carbapenemase in the LTCFs and the community The rapid spread of OXA-48 producing K. pneumoniae points out to the shortcomings in the infection control measures.

Klebsiella pneumoniae carbapenemase (KPC) in urinary infection isolates.

Recently, emergence of carbapenem-resistance, in particular due to Klebsiella pneumoniae carbapenemase (KPC), was observed among K. pneumoniae causing urinary tract infections in Croatia. The aim of the study was to characterize, antimicrobial susceptibility, carbapenem resistance, virulence traits and plasmid types of the urinary KPC positive isolates of K. pneumoniae. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method. The transferability of meropenem resistance was determined by conjugation (broth mating method) employing Escherichia coli J63 strain resistant to sodium azide. Genes encoding broad and extended-spectrum ß-lactamases, plasmid-mediated AmpC ß-lactamases, group A and B carbapenemases, and carbapenem hydrolyzing oxacillinases (blaOXA-48like), respectively, were determined by Polymerase chain reaction (PCR). In total 30 KPC-positive K. pneumoniae urinary isolates collected from different regions of Croatia were analysed. The isolates were uniformly resistant to all tested antibiotics except for variable susceptibility to gentamicin, sulphamethoxazole/trimethoprim, and colistin, respectively. Four isolates were resistant to colistin with MICs values ranging from 4 to 16 mg/L. All tested isolates were susceptible to ceftazidime/avibactam. Sixteen isolates transferred meropenem resistance to E. coli recipient strain by conjugation. Other resistance markers were not co-transferred. PCR was positive for blaKPC and blaSHV genes in all isolates whereas 13 isolates tested positive also for blaTEM genes. PCR based replicon typing (PBRT) revealed the presence of FIIs in 13 and FIA plasmid in two strains. The study showed dissemination of KPC-producing K. pneumoniae in urinary isolates, posing a new epidemiological and treatment challenge. Sulphamethoxazole/trimethoprim, colistin, and ceftazidime/avibactam remain so far, as the therapeutic options.

The effect of standard-dose wintertime vitamin D supplementation on influenza infection in immunized nursing home elderly residents.

AIM: To investigate whether three-month oral vitamin D supplementation (800 IU in drops) reduces the risk of influenza infection in elderly nursing home residents vaccinated against influenza. METHODS: This cross-sectional observational study enrolled 97 participants (73.2% women) who received one dose of seasonal trivalent 2016-2017 influenza vaccine. The patients were randomized into an experimental group, which received vitamin D supplementation for three months starting on the day of vaccination, and a control group, which did not receive vitamin D supplementation. The primary outcome was the number of influenza infections laboratory-confirmed using a rapid point-of-care test based on nasal swabs collected during vitamin D supplementation. The secondary outcome was serum 25-hydroxyvitamin D level at the end of the study. RESULTS: The mean age ±standard deviation was 78.5± 8.8 years. All participants had vitamin D deficiency at baseline. Twenty-three participants who developed signs of respiratory infections during the study were tested for influenza virus. Although the number of influenza-positive participants was lower in the group receiving vitamin D supplementation as compared with the control group (5 vs 12), this difference was not significant. Vitamin D supplementation failed to increase 25(OH)D levels after three months of supplementation. CONCLUSION: Elderly nursing home residents in Zagreb County have a significant vitamin D deficiency. The recommended national supplementation of 800 IU daily failed to lead to vitamin D sufficiency and did not reduce the risk of influenza infection among the vaccinated elderly.

Human Papillomavirus (HPV) Prevalence, Temporal Dynamics and Association with Abnormal Cervical Cytology Findings in Women from Croatia: Is there a Compounding Effect of Low-Risk/High-Risk HPV Co-Infection?

BACKGROUND: Human papillomavirus (HPV) is a major risk factor for cervical dysplasia and invasive cervical cancer; therefore, regular screening by cervical smear cytology or HPV testing is recommended. We aimed to determine the overall and risk group-specific HPV prevalence, age distribution, and temporal trends and to appraise the correlation of HPV positivity with abnormal cervical cytological findings. METHODS: This retrospective, single-center study involved a total of 751 women (aged 18 - 67) concurrently subjected to HPV DNA testing and cervical cytology evaluation over a 10-year period in Zagreb, Croatia. Digene HC2 HPV DNA test (Qiagen Corporation, USA) was employed in screening specimens for both low-risk and high-risk HPV risk groups. The cytology was reported using the Bethesda system and in accordance with uniform classification of uterine cervix cytological findings in Croatia "Zagreb 2002". Statistical significance was set at p < 0.05. RESULTS: The overall HPV prevalence in our study population was 48.6%, and the 18 - 30 age group presented with the highest infection burden (p = 0.046). A decrease in low-risk and high-risk mono-positivity has been observed over the 10-year period; conversely, there was a significant increase in low-risk/high-risk co-positivity (p = 0.007). Low-risk/high-risk HPV co-infection resulted in a compounding effect which increased the occurrence of abnormal cells, HPV-associated changes and low grade squamous intraepithelial lesions (LSIL/cervical intraepithelial neoplasia grade I) in cervical cytology when compared to mono-infection with either low-risk or high-risk HPV. On the other hand, such effect has not been demonstrated for high grade squamous intraepithelial lesions (HSIL/ cervical intraepithelial neoplasia grades II and III). CONCLUSIONS: The overall HPV prevalence in female outpatients was high, underscored with rising co-positivity rates. Such co-infection with both low-risk and high-risk HPV (predominantly seen in women younger than 30) can exhibit a compounding effect in the occurrence of cytological abnormalities and low grade squamous intra-epithelial lesions (LSIL), which has to be considered in future diagnostic and screening algorithms.

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.

An Outbreak of Human Parainfluenza Virus 3 (Phylogenetic Subcluster C5) Infection among Adults at a Residential Care Facility for the Disabled in Croatia, 2018.

INTRODUCTION: Although highly pertinent for children, outbreaks of human parainfluenza virus (HPIV) may cause up to 15% of all respiratory illnesses in adults and predispose them to serious adverse outcomes, with HPIV serotype 3 (HPIV3) being the most common. This study represents the first report of an HPIV3 outbreak among adults at a long-term health-care facility in Croatia. METHODS: A retrospective study was conducted to investigate an outbreak of acute respiratory infection (ARI) at a single residential care facility for the disabled in Croatia. Demographic, epidemiological, and clinical data were collected for all residents, while hospitalized patients were appraised in detail by laboratory/radiological methods. Multiplex PCR for respiratory viruses and sequencing was performed. Partial HPIV3 HN 581 nt sequences were aligned with HPIV3 sequences from the GenBank database to conduct a phylogenetic analysis, where different bioinformatic approaches were employed. RESULTS: In late June 2018, 5 of the 10 units at the facility were affected by the outbreak. Among the 106 residents, 23 (21.7%) developed ARI, and 6 (26.1%) of them were hospitalized. HPIV3 was identified in 18 (73%) of the residents and 5 (83%) of the hospitalized individuals. Isolated HPIV3 strains were classified within the phylogenetic subcluster C5 but grouped on 2 separate branches of the phylogenetic tree. During the entire outbreak period, none of the institution's employees reported symptoms of ARI. CONCLUSIONS: Our study has shown that this health care-associated outbreak of HPIV3 infection could have been linked to multiple importation events. Preventive measures in curbing such incidents should be enforced vigorously.

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.

Antimicrobial Sensitivity Profile of Chlamydia trachomatis isolates from Croatia in McCoy Cell Culture System and Comparison with the Literature.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is the most common bacterial agent of sexually transmitted infections around the world, but susceptibility testing of this pathogen is rarely pursued due to its intracellular niche. The principal aims of this research were to determine in vitro sensitivity profile of urogenital chlamydial strains isolated from Croatian patients and to compare obtained concentration values of different antimicrobial drugs mutually and with the literature. METHODS: Forty strains of C. trachomatis isolated during 2010-2012 at the National Reference Laboratory for Chlamydia and two reference strains were subjected to susceptibility testing in 96-well microtiter plates containing McCoy cell monolayers. Minimal inhibitory concentration (MIC) and minimal chlamydicidal concentration (MCC) were determined for azithromycin, doxycycline, and levofloxacin. Briefly, strains were inoculated on McCoy cells, followed by addition of serially diluted antimicrobial drugs. Upon incubation, growth of C. trachomatis was detected using fluorescein-conjugated antibody to the lipopolysaccharide genus antigen under the inverted fluorescent microscope. RESULTS: All chlamydial strains were susceptible to the antibiotics tested (MIC < 4 pg/mL), thus the pattern of homotypic or heterotypic resistance has not been found. MCC values were equal or 1-5 dilutions higher than MIC values. Statistically significant differences in the effectiveness of antimicrobial agents in vitro have been proven. Significant correlation has been found for MCCs in the case of two antimicrobial pairs: azithromycin and levofloxacin, and doxycycline and levofloxacin. Comparison of medians for different clinical samples did not reveal any significant difference. CONCLUSIONS: Although resistant strains have not been found in this study, several literature reports of unsuccessfully treated genitourinary infections caused by C. trachomatis require our alertness for possible discovery of resistant strains. Considering the overall antibiotic burden worldwide, pursuing this kind of research is crucial in order to detect possible decreased susceptibility (or even resistance) of chlamydial strains, despite the laborious and time-consuming methodology.

Detection of Macrolide and/or Fluoroquinolone Resistance Genes in Mycoplasma genitalium Strains Isolated from Men in the Northwest Region of Croatia in 2018-2023.

Mycoplasma genitalium (M. genitalium) poses a significant public health challenge due to its association with non-gonococcal urethritis (particularly in men) and antimicrobial resistance. However, despite the prevalence of M. genitalium infections and the rise in resistance rates, routine testing and surveillance remain limited. This is the first study from Croatia that aimed to assess the prevalence and trends of resistance in M. genitalium strains isolated from male individuals by detecting macrolide and fluoroquinolone resistance genes. The study also aimed to explore the factors associated with resistance and changes in resistance patterns over time. Urine samples collected from male individuals in the Zagreb County and northwest region of Croatia between 2018 and 2023 were tested for M. genitalium with the use of molecular methods. Positive samples were subjected to DNA extraction and multiplex tandem polymerase chain reaction (MT-PCR) targeting genetic mutations associated with macrolide (23S rRNA gene) and fluoroquinolone (parC gene) resistance. Of the 8073 urine samples tested from 6480 male individuals (and following the exclusion of repeated specimens), we found that the prevalence of M. genitalium infection was 2.2%. Macrolide resistance was observed in 60.4% of strains, while fluoroquinolone resistance was found in 19.2%. Co-resistance to both antibiotics was present in 18.2% of cases. A statistically significant increase in fluoroquinolone resistance was noted over the study period (p = 0.010), but this was not evident for azithromycin resistance (p = 0.165). There were no statistically significant differences in resistance patterns between age groups, whereas re-testing of patients revealed dynamic changes in resistance profiles over time. The high burden of macrolide resistance and increasing fluoroquinolone resistance underscore the urgent need for comprehensive resistance testing and surveillance programs. The implementation of resistance-guided treatment strategies, along with enhanced access to molecular diagnostics, is pivotal for effectively managing M. genitalium infections.

Comparison of MT-PCR with Quantitative PCR for Human Bocavirus in Respiratory Samples with Multiple Respiratory Viruses Detection.

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

[Comparative urinary bactericidal activity of oral antibiotics against gram-positive pathogens]. / Usporedna urinarna baktericidna aktivnost oralnih antibiotika prema gram-pozitivnim urinarnim patogenima.

In routine bacteriological laboratories the antibacterial activity of antibiotics is determined by in vitro testing, usually by disk-diffusion test. However, in vitro testing does not always reflect antibacterial efficiency of antibiotics in vivo. In this investigation, the urine samples obtained in a single oral dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant Gram-positive urinary tract pathogens. Urinary bactericidal activity of linezolid had been previously compared with ciprofloxacin but not with other oral antibiotics such as beta-lactams. Linezolid showed satisfactory urinary bactericidal titres throughout the whole testing period against all Gram-positive cocci. Fluoroquinolones displayed high and persisting levels of urinary bactericidal activity against staphylococci, but their activity against enterococci was weaker. According to the results of ex-vivo testing amoxycillin could be recommended only for infections caused by E. faecalis. Amoxycillin combined with clavulanic acid can be considered as a therapeutic option for infections caused by S. saprophyticus and E. faecalis. Older cephalosporins had high titres only against S. saprophyticus. Their drawback is a short elimination half-time in urine resulting in rapid decrease of urinary bactericidal titers during dosing interval. Furthermore, they do not show activity against enterococci due to their intrinsic resistance to cephalosporins.

[P16NIK4a expression in premalignant cervical lesions]. / Ekspresija stanicnog proteina p16INK4a kod premalignih promjena vrata maternice.

Increased expression of viral E6 and E7 oncoproteins within the host cells results in an increase in cellular protein p16INK4a expression. That increase may serve as a marker for dysplastic and neoplastic cells of the uterine cervical epithelium. The aim of this study is to assess the p16INK4a protein expression in different stages of cytological abnormality in correlation with the proven high oncogenic risk human papillomavirus infection in order to demonstrate its value as the diagnostic marker. The study included cervical smear samples of 371 patient in whom the viral typization was done. In 171 patient, during their regular gynaecological examination, along with conventional Pap smear sampling an additional smear was taken. Two hundred cervix brush (Rovers Medical DevicesOss, the Netherlands) samples were obtained and analyzed by the LBC method and the ThinPrep2000 machine. All samples were analyzed cytologically, classified according to the Bethesda system, and immunostained with the p16INK4a-specific monoclonal antibody E6H4 (MTM Laboratories, Heidelberg, Germany). A significant difference is seen in p16 positivity between the cytological diagnosis of a high grade cervical squamous intraepithelial lesion and the group with mild dysplasia (chi2=146,48; D.F.=4; p<0,01) while most of the mild dysplastic lesions were p16 negative. In cases of p16 positive mild dysplastic lesions, that positivity is the result of viral integration into the host genome, which would imply lesions at high risk of further progression. The potential and value of p16INK4a protein not only as a prognostic, but also as a diagnostic factor is proved in this way. Reproducibility of the p16 staining technique renders it suitable for follow-up monitoring as well as for comparison of the cytological results.

Seasonal Coronaviruses and Other Neglected Respiratory Viruses: A Global Perspective and a Local Snapshot.

Respiratory viral infections are the leading cause of morbidity and mortality in the world; however, there are several groups of viruses that are insufficiently routinely sought for, and can thus be considered neglected from a diagnostic and clinical standpoint. Timely detection of seasonality of certain respiratory viruses (e.g., enveloped viruses such as seasonal coronaviruses) in the local context can aid substantially in targeted and cost-effective utilization of viral diagnostic approaches. For the other, non-enveloped and year-round viruses (i.e., rhinovirus, adenovirus, and bocavirus), a continuous virological diagnosis needs to be implemented in clinical laboratories to more effectively address the aetiology of respiratory infections, and assess the overall impact of these viruses on disease burden. While the coronavirus disease 2019 (COVID-19) pandemic is still actively unfolding, we aimed to emphasize the persistent role of seasonal coronaviruses, rhinoviruses, adenoviruses and bocaviruses in the aetiology of respiratory infections. Consequently, this paper concentrates on the burden and epidemiological trends of aforementioned viral groups on a global level, but also provides a snapshot of their prevalence patterns in Croatia in order to underscore the potential implications of viral seasonality. An overall global prevalence in respiratory tract infections was found to be between 0.5 and 18.4% for seasonal coronaviruses, between 13 and 59% for rhinoviruses, between 1 and 36% for human adenoviruses, and between 1 and 56.8% for human bocaviruses. A Croatian dataset on patients with respiratory tract infection and younger than 18 years of age has revealed a fairly high prevalence of rhinoviruses (33.4%), with much lower prevalence of adenoviruses (15.6%), seasonal coronaviruses (7.1%), and bocaviruses (5.3%). These insights represent a relevant discussion point in the context of the COVID-19 pandemic where the testing of non-SARS-CoV-2 viruses has been limited in many settings, making the monitoring of disease burden associated with other respiratory viruses rather difficult.

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.

Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium linked to ocular and urogenital infections with potentially serious sequelae, including blindness and infertility. First-line antibiotics, such as azithromycin (AZT) and doxycycline, are effective, but treatment failures have also been reported. Encapsulation of antibiotics in liposomes is considered an effective approach for improving their local effects, bioavailability, biocompatibility and antimicrobial activity. To test whether liposomes could enhance the antichlamydial action of AZT, we encapsulated AZT in different surface-charged elastic liposomes (neutral, cationic and anionic elastic liposomes) and assessed their antibacterial potential against the C. trachomatis serovar D laboratory strain as well as the clinical isolate C. trachomatis serovar F. A direct quantitative polymerase chain reaction (qPCR) method was used to measure chlamydial genome content 48 h post infection and to determine the recoverable chlamydial growth. All the liposomes efficiently delivered AZT to HeLa 229 cells infected with the laboratory Chlamydia strain, exhibiting the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of AZT even 4-8-fold lower than those achieved with the free AZT. The tested AZT-liposomes were also effective against the clinical Chlamydia strain by decreasing MIC values by 2-fold relative to the free AZT. Interestingly, the neutral AZT-liposomes had no effect on the MBC against the clinical strain, while cationic and anionic AZT-liposomes decreased the MBC 2-fold, hence proving the potential of the surface-charged elastic liposomes to improve the effectiveness of AZT against C. trachomatis.

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.

Estimating clinical outcome of HPV induced cervical lesions by combination of capsid protein L1 and p16INK4a protein detection.

The aim of this study was to investigate whether is possible to predict clinical outcome of cervical lesion by immunoassaying performed on cervical smears. During the two year study period the cervical smears of 81 patients were collected. All patients were tested for human papillomavirus (HPV) infections using Amplycor HPV test. Sixty-six of them were tested as positive for high risk types (hrHPV) and squamous intraepithelial lesion, and in those patients repeated cervical smears were taken every six months. The rest were hrHPV negative patients with normal smears which were used as a negative control in immunoassays with HPV L1 and p16INK4a antibodies. The results of p16INK4a staining in 66 hrHPV positive patients showed impairment of the cervical lesion in 22 (33.3%) and unchanged cytological finding in 21 (31.9%) p16INK4a positive patients, respectively, while improving of cytological finding was seen only in three (4.5%) p16LNK4a positive patients. On the contrary, impairment of cytological finding was not seen in p16INK4a negative patients, while in 17 out of 20 patients from that group improving or normalisation of cytological finding were detected (p < 0.01). Correlation between L1/p16 pattern and cytological finding showed that only in L1-/p16+ cervical lesions was detected impairment of cytological finding during the study period. In L1 +/p16+ group the cytological finding was the same during the follow up in all 11 patients, while in L1 +/p16- group in most patients (9/11) downgrading or normalisation of Pap test were detected. The usage of p16 and HPV L1 markers can be useful in estimation of biologic potentiality and clinical outcome of cervical lesions.

Extended-spectrum beta-lactamases and plasmid diversity in urinary isolates of Escherichia coli in Croatia: a nation-wide, multicentric, retrospective study.

In recent years, a dramatic increase in the prevalence of Escherichia coli strains producing extended-spectrum ß-lactamases (ESBLs) has been observed - both in the community and in healthcare settings. This multicentric study aimed to characterize ESBLs produced by E. coli isolates causing hospital-onset and community urinary tract infections, as well as to compare their antimicrobial sensitivity patterns, ß-lactamase content and plasmid types. Phenotypic tests for the detection of ESBLs and plasmid-mediated AmpC ß-lactamases were initially pursued, followed by molecular detection of resistance genes, plasmid characterization, genotyping with pulsed-field gel electrophoresis and whole genome sequencing (WGS). The isolates exhibited high level of resistance to expanded-spectrum cephalosporins (ESC) and carried CTX-M (cefotaximase-Munich) or TEM (Temoniera) ß-lactamases. All six representative isolates subjected to WGS belonged to the widespread clone ST131. In conclusion, our study demonstrated dissemination of group 1 CTX-M positive E. coli in different geographic regions of Croatia, but also different components of the health care systems (hospitals, nursing homes and the community) and confirmed the switch from SHV-2 (suphydril variant) and SHV-5 ESBLs to the nation-wide predominance of group 1 CTX-M ß-lactamases. Different plasmids were shown to be associated with the dissemination of blaCTX-M genes in different geographic regions of Croatia.

Antimicrobial Resistance Screening in Chlamydia trachomatis by Optimized McCoy Cell Culture System and Direct qPCR-Based Monitoring of Chlamydial Growth.

Obligate intracellular localization of Chlamydia trachomatis (C. trachomatis) complicates antimicrobial sensitivity testing efforts that we are so accustomed to in routine bacteriology. Cell culture systems with immunofluorescence staining, to identify cellular inclusions in the presence of various concentrations of antimicrobial drugs, are still the most pervasive techniques, but more specific and sensitive nucleic acid concentration measuring methods are increasingly being used. Here we describe how to approach antimicrobial susceptibility/resistance screening in C. trachomatis by using a McCoy cell culture system, optimized by a research group from Croatia, and direct qPCR-based monitoring of chlamydial growth, optimized by a research group from Hungary.

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.