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In Slovenia, primary acellular pertussis vaccines (ACVs) containing pertactin (PRN) were mostly used during 1999-2016; ACVs without PRN were introduced in 2017. Among 123 Bordetella pertussis strains collected during 2002-2020, a total of 48 were PRN-deficient; 44 were collected after 2017. Changes to ACVs could increase PRN-deficient B. pertussis and infections.

Molecular testing for human papillomaviruses (HPV) is gradually replacing cytology in cervical cancer screening. In this longitudinal population-based cohort study, 4140 women 20 to 64 years old attending organized screening were tested at baseline by five different screening methods and followed for 9 years. To assess long-term safety, the cumulative risks of CIN2+/CIN3+ were estimated after a negative baseline result obtained by conventional cytology and four clinically validated HPV assays: Hybrid Capture 2 (hc2), RealTime High Risk HPV assay (RealTime), cobas 4800 HPV Test (cobas&#95;4800), and Alinity m HR HPV (Alinity). HPV-negative women at baseline had a substantially lower risk for CIN2+ compared to those with normal baseline cytology: 0.84% (95% CI, 0.46-1.22), 0.90% (95% CI, 0.51-1.29), 0.78% (95% CI, 0.42-1.15), and 0.75% (95% CI, 0.39-1.11) for hc2, RealTime, cobas&#95;4800, and Alinity, respectively, compared to 2.46% (95% CI, 1.88-3.03) for cytology. No differences were observed between HPV assays in longitudinal sensitivity (range: 86.21%-90.36%) and negative predictive values (range: 99.54%-99.70%) for CIN2+ in women ≥30 years, but were significantly different from cytology (p < .05). The 9-year cumulative risk of CIN2+ differed significantly between HPV genotypes, reaching 32.1% (95% CI, 14.5-46.1) for HPV16, 24.9% (95% CI, 4.7-40.8) for HPV18/45, 27.2% (95% CI, 14.6-37.8) for HPV31/33/35/52/58, and 8.1% (95% CI, 0.0-16.7) for HPV39/51/56/59. Four clinically validated HPV assays showed comparable safety and better assurance against precancerous lesions than cytology, but some important differences were identified in the performance characteristics of HPV assays impacting the referral rate. Information about the HPV genotype is valuable for guiding further clinical action in HPV-based screening programs., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Lower respiratory tract infections (LRTIs) are the fourth leading cause of death worldwide, among which Escherichia coli ( E. coli ) pneumonia is considered a rare phenomenon. Treatment options for LRTIs have become limited, especially for extended-spectrum β-lactamase-producing E. coli (ESBL-EC), which are usually resistant to other groups of antimicrobials as well. The aim of our study was to compare the phenotypic resistance profiles and genotypes of ESBL-EC isolates associated with LRTIs before (pre-COVID-19) and during (COVID-19) the COVID-19 pandemic. All isolates were screened for antimicrobial resistance genes (ARGs) and virulence-associated genes (VAGs) and assigned to phylogenetic groups, sequence types and clonal groups by PCR. During the pandemic, a significantly lower proportion of ciprofloxacin-, levofloxacin- and trimethoprim-sulfamethoxazole-resistant ESBL-EC isolates was retrieved from lower respiratory tract (LRT) samples. PCR-based genotypization revealed greater clonal diversity and a significantly lower proportion of isolates with bla TEM , aac(6')-Ib-cr and qacEΔ1 genes. In addition, a higher proportion of isolates with the integrase gene int1 and virulence genes sat and tsh was confirmed. The lower prevalence of fluoroquinolone resistance and greater genetic diversity of ESBL-EC isolated during the COVID-19 period may have been due to the introduction of new bacterial strains into the hospital environment, along with changes in clinical establishment guidelines and practices.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae , Mycoplasma pneumoniae and Legionella pneumophila/ spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

To better understand the natural history of anogenital warts (AGWs) and the dynamics of HPV6/11 infection in regional hairs, 32 newly diagnosed male patients with AGWs and 32 age-matched healthy controls were closely followed. During enrollment and six follow-up visits (every 2.6 months), 43 AGW tissues and 1232 anogenital and eyebrow hair samples were collected. This is the closest longitudinal monitoring of AGW patients to date. Patients were treated according to standards of care. The HPV6/11 prevalence was 19.9% in the patients' hair samples (HPV6 B1 in 53.1%) and 0% in the controls. The highest HPV6/11 prevalence was found in pubic hairs (29.0%) and the lowest in eyebrows (7.1%). The odds of having HPV6/11-positive hairs increased with smoking, shaving the anogenital region, and age. A close association between HPV6/11 presence in hairs and clinically visible AGWs was observed. The proportion of patients with visible AGWs and HPV6/11-positive hairs declined during follow-up with similar trends. No particular HPV6/11 variant was linked with an increased AGW recurrence, but the sublineage HPV6 B1 showed significantly higher clearance from hairs. Despite treatment, 78.1% and 62.5% of the AGW patients experienced one and two or more post-initial AGW episodes, respectively. The patients with HPV6/11-positive hairs or visible AGWs at a preceding visit demonstrated substantially higher odds of presenting with visible AGWs at a subsequent visit.

The COVID-19 pandemic has strained healthcare systems globally. Shortages of hospital beds, reassignment of healthcare workers to COVID-19-dedicated wards, an increased workload, and evolving infection prevention and control measures have potentially contributed to the spread of multidrug-resistant bacteria (MDRB). To determine the impact of the COVID-19 pandemic at the University Medical Center Ljubljana, a tertiary teaching hospital, we analyzed the monthly incidence of select bacterial species per patient from 2018 to 2022. The analysis was performed for all isolates and for MDRB isolates. The data were analyzed separately for isolates from all clinical samples, from blood culture only, and from clinical and surveillance samples. Our findings revealed an increased incidence density of patients with Enterococcus faecium , Staphylococcus aureus , Escherichia coli , and Pseudomonas aeruginosa isolates from clinical samples during the COVID-19 period in the studied hospital. Notably, the incidence density of MDRB isolates-vancomycin-resistant E. faecium , extended-spectrum betalactamase-producing K. pneumoniae , and betalactam-resistant P. aeruginosa -from clinical samples increased during the COVID-19 period. There were no statistically significant differences in the incidence density of patients with blood culture MDRB isolates. We observed an increase in the overall MDRB burden (patients with MDRB isolates from both clinical and surveillance samples per 1000 patient days) in the COVID-19 period in the studied hospital for vancomycin-resistant E. faecium , carbapenem-resistant K. pneumoniae, and betalactam-resistant P. aeruginosa and a decrease in the methicillin-resistant S. aureus burden., Competing Interests: The authors declare no conflicts of interest.

Background: Antibiotic resistance is one of the most serious global health problems and threatens the effective treatment of bacterial infections. Of greatest concern are infections caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC). The aim of our study was to evaluate the prevalence and molecular characteristics of ESBL-EC isolated over an 18-year pre-COVID period from lower respiratory tract (LRT) samples collected from selected Slovenian hospitals., Objectives and Methods: All isolates were identified by MALDI-TOF and phenotypically confirmed as ESBLs by a disk diffusion assay. Using a PCR approach, 487 non-repetitive isolates were assigned to phylogroups, sequence type groups, and clonal groups. Isolates were also screened for virulence-associated genes (VAGs) and antimicrobial resistance genes., Results: The prevalence of ESBL-EC isolates from LRT in a large university hospital was low (1.4%) in 2005 and increased to 10.8% by 2019. The resistance profile of 487 non-repetitive isolates included in the study showed a high frequency of group 1 bla CTX-M (77.4%; n = 377), bla TEM (54.4%; n = 265) and aac(6')-Ib-cr (52%; n = 253) genes and a low proportion of bla SHV and qnr genes. Isolates were predominantly assigned to phylogroup B2 (73.1%; n = 356), which was significantly associated with clonal group ST131. The ST131 group accounted for 67.6% (n = 329) of all isolates and had a higher number of virulence factor genes than the non-ST131 group. The virulence gene profile of ST131 was consistent with that of other extraintestinal pathogenic E. coli (ExPEC) strains and was significantly associated with ten of sixteen virulence factor genes tested. Using ERIC-PCR fingerprinting, isolates with the same ERIC-profile in samples from different patients, and at different locations and sampling dates were confirmed, indicating the presence of "hospital-adapted" strains., Conclusion: Our results suggest that the ESBL-EC isolates from LRT do not represent a specific pathotype, but rather resemble other ExPEC isolates, and may be adapted to the hospital environment. To our knowledge, this is the first study of ESBL-EC isolated from LRT samples collected over a long period of time., (© 2024. The Author(s).)

Biocidal products prevent the spread of pathogenic microorganisms, including extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), which is one of the most alarming health problems worldwide. Quaternary ammonium compounds (QACs) are surface-active agents that interact with the cytoplasmic membrane and are widely used in hospitals and food processing environments. A collection of 577 ESBL-EC, isolated from lower respiratory tract (LRT) samples, was screened for QAC resistance genes oqxA ; oqxB ; qacEΔ1 ; qacE ; qacF/H/I ; qacG ; sugE (p); emrE; mdfA; sugE (c) ; ydgE; ydgF ; and for class 1, 2, and 3 integrons. The prevalence of chromosome-encoded genes ranged from 77 to 100%, while the prevalence of QAC resistance genes encoded on mobile genetic elements (MGEs) was relatively low (0-0.9%), with the exception of qacEΔ1 (54.6%). PCR screening detected the presence of class 1 integrons in 36.3% (n = 210) of isolates, which were positively correlated with qacEΔ1 . More correlations between QAC resistance genes, integrons, sequence type group ST131, and β-lactamase genes were presented. The results of our study confirm the presence of QAC resistance genes and also class 1 integrons commonly found in multidrug-resistant clinical isolates and highlight the potential role of QAC resistance genes in the selection of ESBL-producing E. coli in hospitals., Competing Interests: The authors declare no conflict of interest.

Periodontal disease is a chronic oral inflammatory disorder initiated by pathobiontic bacteria found in dental plaques-complex biofilms on the tooth surface. The disease begins as an acute local inflammation of the gingival tissue (gingivitis) and can progress to periodontitis, which eventually leads to the formation of periodontal pockets and ultimately results in tooth loss. The main problem in periodontology is that the diagnosis is based on the assessment of the already obvious tissue damage. Therefore, it is necessary to improve the current diagnostics used to assess periodontal disease. Using lipidomic analyses, we show that both crucial periodontal pathogens, Porphyromonas gingivalis and Tannerella forsythia , synthesize ceramide phosphoethanolamine (CPE) species, membrane sphingolipids not typically found in vertebrates. Previously, it was shown that this particular lipid can be specifically detected by an aegerolysin protein, erylysin A (EryA). Here, we show that EryA can specifically bind to CPE species from the total lipid extract from P. gingivalis . Furthermore, using a fluorescently labelled EryA-mCherry, we were able to detect CPE species in clinical samples of dental plaque from periodontal patients. These results demonstrate the potential of specific periodontal pathogen-derived lipids as biomarkers for periodontal disease and other chronic inflammatory diseases.

Background: Repeated rotation of empiric antibiotic treatment strategies is hypothesized to reduce antibiotic resistance. Clinical rotation studies failed to change unit-wide prevalence of antibiotic resistant bacteria (ARB) carriage, including an international cluster-randomized crossover study. Unit-wide effects may differ from individual effects due to "ecological fallacy". This post-hoc analysis of a cluster-randomized crossover study assesses differences between cycling and mixing rotation strategies in acquisition of carriage with Gram-negative ARB in individual patients., Methods: This was a controlled cluster-randomized crossover study in 7 ICUs in 5 European countries. Clinical cultures taken as routine care were used for endpoint assessment. Patients with a first negative culture and at least one culture collected in total were included. Community acquisitions (2 days of admission or less) were excluded. Primary outcome was ICU-acquisition of Enterobacterales species with reduced susceptibility to: third- or fourth generation cephalosporins or piperacillin-tazobactam, and Acinetobacter species and Pseudomonas aeruginosa with reduced susceptibility for piperacillin-tazobactam or carbapenems. Cycling (altering first-line empiric therapy for Gram-negative bacteria, every other 6-weeks), to mixing (changing antibiotic type every empiric antibiotic course). Rotated antibiotics were third- or fourth generation cephalosporins, piperacillin-tazobactam and carbapenems., Results: For this analysis 1,613 admissions were eligible (855 and 758 during cycling and mixing, respectively), with 16,437 microbiological cultures obtained. Incidences of acquisition with ARB during ICU-stay were 7.3% (n = 62) and 5.1% (n = 39) during cycling and mixing, respectively (p-value 0.13), after a mean of 17.7 (median 15) and 20.8 (median 13) days. Adjusted odds ratio for acquisition of ARB carriage during mixing was 0.62 (95% CI 0.38 to 1.00). Acquired carriage with ARB were Enterobacterales species (n = 61), Pseudomonas aeruginosa (n = 38) and Acinetobacter species (n = 20), with no statistically significant differences between interventions., Conclusions: There was no statistically significant difference in individual patients' risk of acquiring carriage with Gram-negative ARB during cycling and mixing. These findings substantiate the absence of difference between cycling and mixing on the epidemiology of Gram-negative ARB in ICU., Trial Registration: This trial is registered with ClinicalTrials.gov, registered 10 January 2011, NCT01293071., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SH has received consulting fees from Sandoz. All other authors have declared that no competing interests exist

This study focuses on the feasibility of collaborative robot implementation in a medical microbiology laboratory by demonstrating fine tasks using kinesthetic teaching. Fine tasks require sub-millimetre positioning accuracy. Bacterial colony picking and identification was used as a case study. Colonies were picked from Petri dishes and identified using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. We picked and identified 56 colonies (36 colonies of Gram-negative Acinetobacter baumannii and 20 colonies of Gram-positive Staphylococcus epidermidis ). The overall identification error rate was around 11%, although it was significantly lower for Gram-positive bacteria (5%) than Gram-negative bacteria (13.9%). Based on the identification scores, it was concluded that the system works similarly well as a manual operator. It was determined that tasks were successfully demonstrated using kinesthetic teaching and generalized using dynamic movement primitives (DMP). Further improvement of the identification error rate is possible by choosing a different deposited sample treatment method (e.g., semi-extraction, wet deposition).

Scope: These ESCMID guidelines address the targeted antibiotic treatment of third-generation cephalosporin-resistant Enterobacterales (3GCephRE) and carbapenem-resistant Gram-negative bacteria, focusing on the effectiveness of individual antibiotics and on combination versus monotherapy., Methods: An expert panel was convened by ESCMID. A systematic review was performed including randomized controlled trials and observational studies, examining different antibiotic treatment regimens for the targeted treatment of infections caused by the 3GCephRE, carbapenem-resistant Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii. Treatments were classified as head-to-head comparisons between individual antibiotics and between monotherapy and combination therapy regimens, including defined monotherapy and combination regimens only. The primary outcome was all-cause mortality, preferably at 30 days and secondary outcomes included clinical failure, microbiological failure, development of resistance, relapse/recurrence, adverse events and length of hospital stay. The last search of all databases was conducted in December 2019, followed by a focused search for relevant studies up until ECCMID 2021. Data were summarized narratively. The certainty of the evidence for each comparison between antibiotics and between monotherapy and combination therapy regimens was classified by the GRADE recommendations. The strength of the recommendations for or against treatments was classified as strong or conditional (weak)., Recommendations: The guideline panel reviewed the evidence per pathogen, preferably per site of infection, critically appraising the existing studies. Many of the comparisons were addressed in small observational studies at high risk of bias only. Notably, there was very little evidence on the effects of the new, recently approved, β-lactam/β-lactamase inhibitors on infections caused by carbapenem-resistant Gram-negative bacteria. Most recommendations are based on very-low- and low-certainty evidence. A high value was placed on antibiotic stewardship considerations in all recommendations, searching for carbapenem-sparing options for 3GCephRE and limiting the recommendations of the new antibiotics for severe infections, as defined by the sepsis-3 criteria. Research needs are addressed., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: To determine whether azithromycin (AZI) as an adjunct to scaling and root planing (SRP), when compared to placebo, decreases the number of sites demonstrating pocket depth (PD) ≥ 5 mm and bleeding on probing (BOP) 12 months post-treatment in stage III/IV periodontitis patients., Materials and Methods: In a double-blind randomized parallel-arm placebo-controlled trial, 40 stage III/IV periodontitis patients received steps 1 and 2 of periodontal treatment in two sessions within 7 days. Patients then received systemic antibiotic therapy (n = 20; AZI 500 mg/day, 3 days) or placebo (n = 20). Additional instrumentation of residual diseased sites (DS) - sites with PD ≥ 5 mm and BOP - was performed at the 3-, 6- and 9-month follow-ups. The primary outcome variable was the number of DS at the 12-month re-evaluation. Using a multivariate multilevel logistic regression model, the effects of gender, age, antibiotic therapy, presence of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans, smoking, tooth being a molar and interdental location were evaluated., Results: The number of DS after 12 months was similar in the test (median (Me) = 4, interquartile range (IQR) = 0-6) and control (Me = 3, IQR = 1-6.5) groups. Both groups showed substantial but equivalent improvements in periodontal parameters, with no intergroup differences at initially shallow or deep sites. The logistic regression showed a lower odds ratio (OR) for the healing of DS on molars (OR = 0.29; p < 0.001) and in smokers (OR = 0.36; p = 0.048)., Conclusion: Stage III/IV periodontitis patients showed significant but comparable improvements in periodontal parameters and the number of residual DS at the 12-month revaluation regardless of treatment type. This may have been the result of the additional instrumentation received by patients at residual DS in both treatment groups., Clinical Relevance: Treatment with AZI + SRP provided no additional benefits after 12 months in terms of periodontal parameters or the number of persisting sites with PD ≥ 5 mm + BOP as compared to SRP plus placebo., Trial Registration: EUDRA-CT: 2015-004306-42; https://www.clinicaltrialsregister.eu/ctr-search/trial/2015-004306-42/SI , registered 17. 12. 2015., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Due to the paucity of recent literature on perianal streptococcal disease (PSD), we performed a comprehensive analysis of clinical characteristics of PSD and its management., Methods: We conducted a retrospective search in the laboratory information system of the Institute of Microbiology and Immunology, Ljubljana, Slovenia, between January 2006 and December 2016 and identified patients with suspected PSD. We reviewed patients' medical records and obtained data on patient age and sex, concomitant illnesses, duration of complaints, signs and symptoms of PSD, epidemiological history, date of diagnosis, microbiological characteristics of beta-hemolytic streptococcal isolates, additional laboratory findings, duration and type of systemic and/or topical therapy, and recurrence of PSD., Results: We identified 64 pediatric and eight adult PSD cases in total. The most common signs and symptoms were perianal erythema (67/72; 93.1%), anal fissures (28/72; 38.8%), itching (22/72; 30.6%), and blood-streaked stools (19/72; 26.4%). The duration of symptoms varied from < 1 week to > 1 year, with 58.3% of patients experiencing symptoms between 1 week and 6 months. The majority of patients received systemic (63/72; 87.5%) and topical (56/72; 77.8%) treatment., Conclusions: Although the signs and symptoms of PSD are non-specific, clinicians should be highly suspicious of the disease in adults and especially in preschool children with perianal complaints. Despite being a common disease, there is still considerable delay in correct diagnosis and treatment, prolonging the discomfort of PSD patients.

Human papillomavirus type 159 (HPV159) was identified in an anal swab sample and preliminarily genetically characterized by our group in 2012. Here we present a detailed molecular in silico analysis that showed that the HPV159 viral genome is 7443 bp in length and divided into five early and two late genes, with conserved functional domains and motifs, and a non-coding long control region (LCR) with significant regulatory sequences that allow the virus to complete its life cycle and infect novel host cells. HPV159, clustering into the cutaneotropic Betapapillomavirus ( Beta -PV) genus, is phylogenetically most similar to HPV9, forming an individual phylogenetic group in the viral species Beta -2. After testing a large representative collection of clinical samples with HPV159 type-specific RT-PCR, in addition to the anal canal from which the first HPV159 isolate was obtained, HPV159 was further detected in other muco-cutaneous (4/181, 2.2%), mucosal (22/764, 2.9%), and cutaneous (14/554, 2.5%) clinical samples, suggesting its extensive tissue tropism. However, because very low HPV159 viral loads were estimated in the majority of positive samples, it seemed that HPV159 mainly caused clinically insignificant infections of the skin and mucosa. Using newly developed, highly sensitive HPV159-specific nested PCRs, two additional HPV159 LCR viral variants were identified. Nevertheless, all HPV159 mutations were demonstrated outside important functional domains of the LCR, suggesting that the HPV159 viral variants were most probably not pathogenically different. This complete molecular characterization of HPV159 enhances our knowledge of the genome characteristics, tissue tropism, and phylogenetic diversity of Beta -PVs that infect humans.

Our study evaluates the performance of two rapid phenotypical tests to detect colistin resistance in Enterobacterales: Alifax rapid AST colistin test using the HB&L system and Rapid Polymyxin NP test prepared in-house. A collection of well-characterized 53 colistin-susceptible and 66 colistin-resistantEnterobacterales isolates was used. The results obtained using both rapid tests were compared to the reference broth microdilution. Overall categorical agreement was 81.5% for Alifax test and 98.3% for Rapid Polymyxin NP test. Based on our results, the Rapid Polymyxin NP test is superior to the Alifax test that performed inadequate for Enterobacter spp., (© 2021. The Author(s).)

The Alinity m (Abbott Molecular, Des Plaines, IL) automated molecular analyzer allows continuous loading of samples and sample-to-result molecular detection of several microorganisms. The detection of SARS-CoV-2 by the Alinity m was compared with that of the cobas 6800 (Roche Molecular Systems, Branchburg, NJ; standard comparator) in a manufacturer-independent clinical evaluation on 2157 consecutive nasopharyngeal swab samples. Valid initial results on Alinity m and cobas 6800 were obtained from 2129 (98.7%) and 2157 (100%) samples, respectively. The overall percent agreement (95% CI) was 98.3% (2092/2129 [97.6%-98.7%]); positive percent agreement, 100% (961/961 [99.6%-100%]); negative percent agreement, 96.8% (1131/1168 [95.7%-97.7%]); and high κ value, 0.965 (0.954-0.976). There were 37 discordant results on Alinity m and, based on discordant analyses, including previous and/or follow-up PCR results, 22 could be considered analytically true positive with high probability. Due to a lack of additional information and an inability to perform repeated/further testing, the status of the remaining 15 discordant results remained unresolved. The throughput of the two analyzers was compared using testing on 564 samples in parallel across two 8-hour shifts in clinical practice. The turnaround times were compared using processing of 94 routine samples in parallel on each working day for 5 consecutive days. The two analyzers showed similar performance, with certain differences that have potential importance in some laboratory settings., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: Seroprevalence surveys provide crucial information on cumulative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure. This Slovenian nationwide population study is the first longitudinal 6-month serosurvey using probability-based samples across all age categories., Methods: Each participant supplied two blood samples: 1316 samples in April 2020 (first round) and 1211 in October/November 2020 (second round). The first-round sera were tested using Euroimmun Anti-SARS-CoV-2 ELISA IgG (ELISA) and, because of uncertain estimates, were retested using Elecsys Anti-SARS-CoV-2 (Elecsys-N) and Elecsys Anti-SARS-CoV-2 S (Elecsys-S). The second-round sera were concomitantly tested using Elecsys-N/Elecsys-S., Results: The populations of both rounds matched the overall population (n = 3000), with minor settlement type and age differences. The first-round seroprevalence corrected for the ELISA manufacturer's specificity was 2.78% (95% highest density interval [HDI] 1.81%-3.80%), corrected using pooled ELISA specificity calculated from published data 0.93% (95% CI 0.00%-2.65%), and based on Elecsys-N/Elecsys-S results 0.87% (95% HDI 0.40%-1.38%). The second-round unadjusted lower limit of seroprevalence on 11 November 2020 was 4.06% (95% HDI 2.97%-5.16%) and on 3 October 2020, unadjusted upper limit was 4.29% (95% HDI 3.18%-5.47%)., Conclusions: SARS-CoV-2 seroprevalence in Slovenia increased four-fold from late April to October/November 2020, mainly due to a devastating second wave. Significant logistic/methodological challenges accompanied both rounds. The main lessons learned were a need for caution when relying on manufacturer-generated assay evaluation data, the importance of multiple manufacturer-independent assay performance assessments, the need for concomitant use of highly-specific serological assays targeting different SARS-CoV-2 proteins in serosurveys conducted in low-prevalence settings or during epidemic exponential growth and the usefulness of a Bayesian approach for overcoming complex methodological challenges., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in humans, but its importance in small animal practice is increasing. Here, we present a case of feline otitis externa (OE) caused by MRSA; both hemolytic and nonhemolytic variants with a stable phenotype were recovered from the external auditory canal after infection was detected by routine otoscopy. One isolate per variant underwent antimicrobial susceptibility testing (AST) by broth microdilution method, conventional spa typing and whole-genome sequencing (WGS). The results showed that both variants were genetically related and were of sequence type (ST) 1327, SCC mec type IV and spa type t005. AST and WGS showed that both isolates were resistant to β-lactams and sensitive to all tested non-β-lactam antibiotics. Both isolates were pvl -negative, but encoded several other virulence genes ( aur , hlgABC , sak , scn , seg , sei , sem , sen , seo and seu ). Genetic background of the mixed hemolytic phenotype was not identified; no differences in the agr locus or other regulatory regions were detected. Three single-nucleotide polymorphisms were identified but could not be associated with hemolysis. This well-documented case of MRSA infection in companion animals adds to the reports of MRSA infections with a mixed hemolytic phenotype.

Objectives: To determine the prevalence, viral load, tissue tropism, and genetic variability of novel human papillomavirus (HPV) type 179, which is etiologically associated with sporadic cases of common warts in immunocompromised patients, and phylogenetically related HPV types 135 and 146., Methods: The representative collection of 850 HPV-associated clinical samples (oral/nasopharyngeal/anal, archival specimens of oral/oropharyngeal/conjunctival/cervical/skin cancer, benign lesions of the larynx/conjunctiva/skin, and eyebrows), obtained from immunocompetent individuals, was tested for the presence of HPV179, HPV135, and HPV146 using type-specific real-time PCRs. To assess the genetic diversity of the HPVs investigated in the non-coding long control region (LCR), several highly sensitive nested PCR protocols were developed for each HPV type. The genetic diversity of HPV179 was additionally determined in 12 HPV179 isolates from different anatomical sites of an only immunocompromised patient included in the study., Results: HPV179, HPV135, and HPV146 were detected in 1.4, 2.0, and 1.5% of the samples tested, respectively, with no preference for cutaneous or mucosal epithelial cells. One (with five single nucleotide polymorphisms; SNPs), four (with one to six SNPs), and four (with one to eight SNPs) genetic variants of HPV179, HPV135, and HPV146, respectively, were identified among eligible samples. HPV179 isolates from the immunocompromised patient exhibited the identical LCR nucleotide sequence, suggesting that HPV179 can cause generalized HPV infections., Conclusions: HPV179, HPV135, and HPV146 have a mucocutaneous tissue tropism and are associated with sporadic infections in immunocompromised and immunocompetent individuals. Because the majority of mutations were found outside the major functional domains of the respective LCRs, we assume that HPV179, HPV135, and HPV146 genetic variants pathogenically do not differ from their prototypes. In addition, no association was found between specific HPV179, HPV135, and HPV146 genetic variants and anatomical sites of infection and/or specific neoplasms., Competing Interests: Our co-author Branko Pirš. M.D. is an owner of the medical center Dermacenter that provides publicly funded medical services in the field of dermatovenerology (see details below). Nevertheless that one of our co-authors (BP) is an owner of the Dermacenter, this does not alter our adherence to PLOS ONE policies on sharing data and materials. All other co-authors have no conflicts of interest to declare.

Background: Accurate anti-SARS-CoV-2 assays are needed to inform diagnostic, therapeutic, and public health decisions. The first manufacturer-independent head-to-head comparison of two rapid high-throughput automated electrochemiluminescence double-antigen sandwich immunoassays targeting total anti-SARS-CoV-2 antibodies against two different viral proteins, Elecsys Anti-SARS-CoV-2 (Elecsys-N) and Elecsys Anti-SARS-CoV-2 S (Elecsys-S) (Roche Diagnostics), was performed in a routine setting during the exponential growth phase of the epidemic's second wave., Methods: The diagnostic specificity of Elecsys-N and Elecsys-S was initially evaluated on a panel of 572 pre-COVID-19 samples, showing 100 % specificity of both assays. Elecsys-N/Elecsys-S head-to-head comparison used 3,416 consecutive blood samples from individuals that were tested for the presence of anti-SARS-CoV-2 within commercial out-of-pocket serologic testing., Results: Elecsys-N/Elecsys-S head-to-head comparison showed overall agreement of 98.68 % (3,371/3,416; 95 % CI, 98.23-99.03 %), positive agreement of 95.16 % (884/929; 95 % CI, 93.52-96.41 %), and a high kappa value of 0.996 (95 % CI, 0.956-0.976). Previous SARS-CoV-2 PCR positivity was identified in 14/24 (58.3 %) Elecsys-N negative/Elecsys-S positive individuals and in 4/21 (19.0 %) Elecsys-N positive/Elecsys-S negative individuals., Conclusion: The first Elecsys-N/Elecsys-S head-to-head comparison showed excellent agreement of two highly specific and rapid high-throughput automated anti-SARS-CoV-2 assays. An important question is whether laboratories offering two different antibody assays could benefit from combining the assays; if so, should use be concomitant or sequential-and, in the latter case, in which order? Based on our results, we favor concomitant over sequential Elecsys-N/Elecsys-S use when testing individuals for anti-SARS-CoV-2 antibodies in high-incidence settings; for example, during the exponential or stationary growth phase of the COVID-19 epidemic., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Urinary tract infections (UTIs) represent a serious global health issue, especially due to emerging multidrug-resistant UTI-causing bacteria. Recently, we showed that the human amniotic membrane (hAM) could be a candidate for treatments and prevention of UPEC and Staphylococcus aureus infections. However, its role against multidrug-resistant bacteria, namely methicillin-resistant S. aureus (MRSA), extended-spectrum beta-lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae , vancomycin-resistant Enterococci (VRE), carbapenem-resistant Acinetobacter baumannii , and Pseudomonas aeruginosa has not yet been thoroughly explored. Here, we demonstrate for the first time that the hAM homogenate had antibacterial activity against 7 out of 11 tested multidrug-resistant strains, the greatest effect was on MRSA. Using novel approaches, its activity against MRSA was further evaluated in a complex microenvironment of normal and cancerous urinary bladder urothelia. Even short-term incubation in hAM homogenate significantly decreased the number of bacteria in MRSA-infected urothelial models, while it did not affect the viability, number, and ultrastructure of urothelial cells. The hAM patches had no antibacterial activity against any of the tested strains, which further exposes the importance of the hAM preparation. Our study substantially contributes to basic knowledge on the antibacterial activity of hAM and reveals its potential to be used as an antibacterial agent against multidrug-resistant bacteria.

Purpose: To evaluate the effect of a full-mouth disinfection protocol (FMD) on periodontal parameters, glycaemic control and subgingival microbiota of periodontal patients with type 1 and type 2 diabetes, as well as those without diabetes., Materials and Methods: This study included 33 patients with periodontitis. Eleven of them were type 1 diabetes patients, 11 were type 2 diabetes patients, and 11 were non-diabetics. At baseline and 3 months after the FMD, the periodontal parameters of each patient were recorded, samples of capillary blood for the chairside assessment of HbA1c were taken, and plaque samples from the two deepest periodontal pockets were collected to test for the presence of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf) and Treponema denticola (Td)., Results: Bleeding on probing (BOP), probing pocket depth (PPD), clinical attachment level (CAL) and glycated haemoglobin (HbA1c) decreased statistically significantly (p < 0.05) in all three groups 3 months after FMD. Only the proportion of Pg in the control group decreased statistically significantly (p < 0.05), while the proportion of other bacteria decreased or remained the same, whereby the differences were not statistically significant. Moreover, the proportion of Aa in type 1 diabetics increased statistically significantly (p < 0.05)., Conclusion: The FMD protocol improves periodontal parameters and glycaemic control of type 1 and type 2 diabetes patients with periodontitis.

Purpose: To investigate the subgingival microbiological profiles of patients with periodontitis, to determine their stage and grade scores and to evaluate the differences in the microbiota among different stages and grades. Materials and Methods: Sixty-seven (n = 67) periodontitis patients were selected. Periodontitis staging and grading, following the 2018 classification system, were defined. Following a clinical examination, subgingival samples were taken from the deepest periodontal pocket of each quadrant for cultivation, identification and quantification. The prevalence, proportion and counts of nine selected periodontal pathogens were determined, and differences between periodontitis stages III and IV and grades B and C were assessed. Results: All nine cultivable periodontal bacteria were detected, of which the most prevalent was P. intermedia (91.0%) and the least prevalent were E. corrodens (9.0%) and C. ochracea (9.0%). The frequency of detection of the two main target pathogens, A. actinomycetemcomitans and P. gingivalis, was 41.8% and 76.1%, respectively. The prevalence (grade B: 80.6%, grade C: 55.6%, p = 0.035) and total counts (grade B: 19.8 colony forming units - CFU/ml-4 (1.9-52.8); grade C: 4.0 CFU/ml-4 (0.0-26.4); p = 0.022) of F. nucleatum were statistically significantly higher in grade B than in grade C periodontitis patients, whereas the counts of P. gingivalis and A. actinomycetemcomitans were similar between grades and stages. Conclusion: Our study suggests that relevant differences between the various grades of periodontitis exist only in the numbers of F. nucleatum. Prevalence and quantities of other cultivable species between different stages and grades of periodontitis seem to be similar.

Background: Our aim was to determine if azithromycin therapy, as an adjunct to scaling and root planing (SRP), decreases the number of pathobiontic subgingival plaque species and sites demonstrating pocket depth (PD) ≥ 5 mm and bleeding on probing (BOP) 6 months post-treatment., Methods: In a double-blind randomized parallel-arm placebo-controlled trial, 40 patients received nonsurgical periodontal treatment in two sessions within 7 days. Patients then received systemic antibiotic therapy (n = 20, azithromycin 500 mg/day for 3 days) or placebo (n = 20). Pooled microbiologic samples were taken before and 6 months after therapy and analysed by established culture methods. The primary outcome variable was the number of sites with PD ≥ 5 mm and BOP at the 6-month re-evaluation. Using multivariate multilevel logistic regression, the effects of gender, age, antibiotic therapy, presence of P. gingivalis or A. actinomycetemcomitans, smoking, tooth being a molar and interdental location were evaluated., Results: The number of sites with PD ≥ 5 mm and BOP after 6 months was similar in the test (Me = 4, IQR = 0-11) and control (Me = 5, IQR = 1-22) group. Adjunctive azithromycin treatment, compared to SRP alone, resulted in more frequent eradication of A. actinomycetemcomitans (p = 0.013) and C. rectus (p = 0.029), decreased proportion (p = 0.006) and total counts (p = 0.003) of P. gingivalis, and decreased proportion of C. rectus (p = 0.012). Both groups showed substantial but equivalent improvements in periodontal parameters, with no intergroups differences at initially shallow or deep sites. The logistic regression showed a lower odds ratio for healing of diseased sites on molars (OR = 0.51; p <  0,001)., Conclusion: Despite significant changes in numbers of A. actinomycetemcomitans, P. gingivalis and C. rectus, patients with periodontitis do not benefit from adjunctive systemic azithromycin in terms of number of persisting sites with PD ≥ 5 mm and BOP., Trial Registration: EUDRA-CT: 2015-004306-42; https://www.clinicaltrialsregister.eu/ctr-search/trial/2015-004306-42/SI , registered 17. 12. 2015.

Objectives: The first hospital outbreak of carbapenemase-producing Enterobacteriaceae in Slovenia occurred in 2014-2016. Whole genome sequencing was used to analyse the population of carbapenem-resistant Klebsiella pneumoniae collected in Slovenia in 2014-2017, including OXA-48 and/or NDM-1 producing strains from the outbreak., Methods: A total of 32 K. pneumoniae isolates were analysed using short-read sequencing. Multi-locus sequence typing and core genome multi-locus sequence typing were used to infer genetic relatedness. Antimicrobial resistance markers, virulence factors, plasmid content and wzi types were determined. Long-read sequencing was used for six isolates for detailed analysis of plasmids and their possible transmission., Results: Overall, we detected 10 different sequence types (STs), the most common being ST437 (40.6%). Isolates from the initial outbreak belonged to ST437 (12/16) and ST147 (4/16). A second outbreak of four ST15 isolates was discovered. A new ST (ST3390) and two new wzi types (wzi-556, wzi-559) were identified. blaOXA-48 was found in 17 (53.1%) isolates, blaNDM-1 in five (15.6%), and a combination of blaOXA-48/NDM-1 in seven (21.9%) isolates. Identical plasmids carrying blaOXA-48 were found in outbreak isolates sequenced with long-read sequencing technology., Conclusions: Whole genome sequencing of Slovenian carbapenem-resistant K. pneumoniae isolates revealed multiple clusters of STs, two of which were involved in the first hospital outbreak of carbapenem producing K. pneumoniae in Slovenia. Transmission of the plasmid carrying blaOXA-48 between two STs was likely to have occurred. A previously unidentified second outbreak was also discovered, highlighting the importance of whole genome sequencing in detection and/or characterization of hospital outbreaks and surveillance of drug-resistant bacterial clones., Competing Interests: The authors have declared that no competing interests exist.

The usefulness of the ATP bioluminescence method for monitoring surface hygiene was evaluated in a hospital pharmacy cleanroom. The sensitivity of the method was found to be appropriate for assessing the efficiency of cleaning and disinfection. ATP bioluminescence was superior to the traditional microbiological culture-based method for detecting unclean surfaces (p < .05)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Alinity m HR HPV assay (Alinity) is a novel human papillomavirus (HPV) assay that individually identifies genotypes HPV16, HPV18, and HPV45 while reporting on 11 other high-risk HPV (hrHPV) genotypes in two aggregates: HPV31/33/52/58 and HPV35/39/51/56/59/66/68. The clinical performance of Alinity for screening for cervical cancer was evaluated in population-based settings. For women aged ≥30 years, the clinical sensitivity ( n  = 68) and specificity ( n  = 3,077) for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) of Alinity were 100.0% and 92.4%, respectively, and were not inferior to those of the Qiagen Digene Hybrid Capture 2 high-risk HPV DNA assay (hc2) ( P  = 0.0006 and P  < 0.0001, respectively). The intralaboratory reproducibility and interlaboratory agreement of Alinity were 96.7% (kappa, 0.92) and 98.7% (kappa, 0.97), respectively. In the group ≥30 years of age, women who were baseline hrHPV negative had a lower risk for CIN2+ at 3 years using Alinity (0.04%) than those with a normal baseline cytology (0.65%) and had a risk comparable to that determined by the Abbott RealTime High Risk HPV assay (0.04%), hc2 (0.08%), or the Roche Cobas 4800 HPV assay (0.04%). High-risk HPV16/18 infection was associated with a significantly higher baseline and 3-year CIN2+ and CIN3+ risk than the absence of HPV16/18 or the presence of hrHPVs at the baseline (all P values were <0.05). The baseline CIN2+ risk was 8.8% for those with HPV31/33/52/58 infection and 2.5% for those with HPV35/39/51/56/59/66/68 infection, while the 3-year CIN2+ risk was 17.0% and 4.9%, respectively (relative risk, 3.4 [ P  = 0.03] and 3.5 [ P  = 0.003], respectively), suggesting that extended genotyping by Alinity may be valuable in improving patient risk stratification. Alinity fulfills international consensus guideline criteria for primary cervical cancer screening and can be considered clinically validated, demonstrating safety comparable to that of other clinically validated HPV tests., (Copyright © 2019 American Society for Microbiology.)

Acinetobacter baumannii primarily causes colonization, yet it can be an opportunistic pathogen associated with hospital-acquired infections. Many countries report rapid spread of carbapenem-resistant Acinetobacter baumannii (CRAb) which limits treatment options, with colistin frequently being the last line treatment option. The aim of our study was to evaluate a recently developed rapid method, namely the Rapid ResaPolymyxin test, for detection of colistin resistance (ColR) in Acinetobacter baumannii. This test was used for rapid screening of colistin resistance in a clinical setting where there is endemicity of CRAb isolates. A total of 82 A. baumannii clinical isolates were included in the evaluation. The majority of them were resistant to carbapenems (75/82, 91.5%). A total of 37 isolates (45.1%) were resistant to colistin, all being resistant to carbapenems. None of the ColR isolates carried the plasmid-mediated mcr-1 to -5 genes. The Rapid ResaPolymyxin NP test reached a 95.1% categorical agreement with results of reference broth microdilution method, with 93.3% sensitivity and specificity, and positive and negative predictive values being respectively at 92.3% and 97.7%. The Rapid ResaPolymyxin NP test performed well on our collection of clinical and surveillance CRAb isolates from the Central Slovenia region. The test is inexpensive and easy to integrate into laboratory workflow. The main value of the test is rapid categorization of susceptibility and resistance which has important implications with respect to the treatment strategy as well as the infection control measures.

Purpose. Increasing consumption of colistin as treatment for infections with multidrug-resistant (MDR) Gram-negative bacilli (GNB) has been accompanied by increasingly frequent reports of colistin-resistant (ColR) MDR GNB. Higher selective pressure creates a favourable environment that can facilitate the spread of ColR isolates. Monitoring of asymptomatic ColR GNB carriage can give us a better understanding of this emerging healthcare problem, particularly in wards with higher polymyxin selective pressure and prevalence of carbapenem-resistant GNB. Our aim was to assess the ColR GNB colonization rate in intensive care unit (ICU) patients and evaluate the performance of two surveillance protocols using a selective medium. Methodology. A prospective study included 739 surveillance samples (rectal swabs and tracheal aspirates) from 330 patients that were screened for ColR GNB carriage using SuperPolymyxin medium. Two approaches were used: direct sample plating and overnight pre-enrichment of samples followed by plating. Colistin resistance was confirmed with broth microdilution. ColR isolates were molecularly screened for plasmid-mediated mcr genes. Results. A total of 44/739 samples (45 ColR GNB isolates) were positive for ColR GNB, which included 31/330 (9.4 %) colonized patients; mcr genes were not detected. The direct plating method only identified 17/45 (37.8 %) isolates correctly, whereas the pre-enrichment protocol identified all 45 ColR GNB. Conclusion. The colonization rate among our ICU patients was 9.4  %. Based on our findings, the pre-enrichment step is necessary for the determination of ColR GNB carriage - even though the time to result takes an additional day, fewer than half of ColR GNB carriers were detected using the direct plating protocol.

Knowing the normal cleanroom microbiota is the basis for ensuring microbiological quality; assess changes and the introduction of new sampling methods. During our study, we prepared a catalogue of cleanroom microorganisms located in four different cleanrooms in University Clinical Centre Ljubljana Pharmacy. Catalogue is prepared as a basis for assessing the suitability of the new rapid microbiological method and subsequent correlation of the results of both methods. The results of our study showed that 78% of isolated bacteria are Gram-positive. However, in more than 70% isolated bacteria were the part of the normal human microbiota, 10-15% of the microorganisms originated from the air, mainly spore-forming bacteria of the genus Bacillus and fungi, and 5-10% of the Gram-negative microorganisms that originated from the water and represent the potential endotoxins (pyrogens).

Molluscum contagiosum (MC) manifests as small, umbilicated papules caused by the molluscum contagiosum virus (MCV). The extent of clinical misdiagnosis of MC is unknown. Combined clinical, histopathological, and virological evaluation of 203 consecutive patients with clinical diagnosis of MC treated at a university hospital during a 5-year period showed the correct clinical diagnosis in 188 of 203 (92.6%) patients. All 15 clinically misdiagnosed MC lesions were histopathologically and virologically confirmed as either common or anogenital warts caused by different human papillomaviruses. The MCV1/MCV2 subtypes ratio was 1.54:1, and the distribution of MCV subtypes differed across patients' age and anatomical location of lesions.

Background: Patients on hemodialysis are at high risk for hepatitis C virus (HCV) infection if measures for effective control of HCV infection in the hemodialysis environment are not implemented. Whereas in developed countries isolated small-scale outbreaks of HCV in hemodialysis units are occasionally reported, HCV transmission in the hemodialysis environment still represents a substantial problem in low-resource countries. This study systematically assessed the prevalence of HCV infection among all patients at all hemodialysis centers in Kosovo, determined the HCV genotype distribution, and reviewed the main risk factors associated with HCV infection in this group of patients., Methods: From January to March 2013, blood samples from all patients undergoing hemodialysis at all seven hemodialysis centers in Kosovo were collected. The samples were screened for the presence of anti-HCV antibodies, and seropositive samples were also tested for HCV RNA. Genotyping was performed by sequencing the core region of the HCV genome. Subsequently, face-to-face interviews were conducted with consented patients attending hemodialysis in December 2015 and with the management of all hemodialysis centers in Kosovo., Results: The overall seroprevalence of HCV infection among hemodialysis patients in Kosovo was 53.0% (354/668), ranging from 22.3 to 91.1% at different centers. HCV RNA was detected in 323/354 (91.2%) seropositive patients. The most frequent HCV genotype was genotype 1a (62.2%), followed by genotypes 4d (33.1%), 1b (4.0%), and 2c (0.7%). The duration of hemodialysis and receiving dialysis at more than one center were identified as independent significant predictors of anti-HCV positivity. Shortage of staff, lack of resources, and inconsistent use of hygienic precautions and/or isolation strategies were observed., Conclusions: The prevalence of HCV infection among hemodialysis patients in Kosovo is extremely high. The relatively low prevalence of HCV infection in the general population, predominance of two otherwise rare HCV genotypes among hemodialysis patients, and longer history of hemodialysis as a predictor of HCV infection all indicate nosocomial transmission due to inappropriate infection control practices as the main transmission route.

Background: Whether antibiotic rotation strategies reduce prevalence of antibiotic-resistant, Gram-negative bacteria in intensive care units (ICUs) has not been accurately established. We aimed to assess whether cycling of antibiotics compared with a mixing strategy (changing antibiotic to an alternative class for each consecutive patient) would reduce the prevalence of antibiotic-resistant, Gram-negative bacteria in European intensive care units (ICUs)., Methods: In a cluster-randomised crossover study, we randomly assigned ICUs to use one of three antibiotic groups (third-generation or fourth-generation cephalosporins, piperacillin-tazobactam, and carbapenems) as preferred empirical treatment during 6-week periods (cycling) or to change preference after every consecutively treated patient (mixing). Computer-based randomisation of intervention and rotated antibiotic sequence was done centrally. Cycling or mixing was applied for 9 months; then, following a washout period, the alternative strategy was implemented. We defined antibiotic-resistant, Gram-negative bacteria as Enterobacteriaceae with extended-spectrum β-lactamase production or piperacillin-tazobactam resistance, and Acinetobacter spp and Pseudomonas aeruginosa with piperacillin-tazobactam or carbapenem resistance. Data were collected for all admissions during the study. The primary endpoint was average, unit-wide, monthly point prevalence of antibiotic-resistant, Gram-negative bacteria in respiratory and perineal swabs with adjustment for potential confounders. This trial is registered with ClinicalTrials.gov, number NCT01293071., Findings: Eight ICUs (from Belgium, France, Germany, Portugal, and Slovenia) were randomly assigned and patients enrolled from June 27, 2011, to Feb 16, 2014. 4069 patients were admitted during the cycling periods in total and 4707 were admitted during the mixing periods. Of these, 745 patients during cycling and 853 patients during mixing were present during the monthly point-prevalence surveys, and were included in the main analysis. Mean prevalence of the composite primary endpoint was 23% (168/745) during cycling and 22% (184/853) during mixing (p=0·64), yielding an adjusted incidence rate ratio during mixing of 1·039 (95% CI 0·837-1·291; p=0·73). There was no difference in all-cause in-ICU mortality between intervention periods., Interpretation: Antibiotic cycling does not reduce the prevalence of carriage of antibiotic-resistant, Gram-negative bacteria in patients admitted to the ICU., Funding: European Union Seventh Framework Programme., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10-7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1.

Beta-hemolytic streptococci (BHS) are the most common causative agents of perianal streptococcal dermatitis (PSD). This study evaluates the distribution of BHS isolates in perianal bacterial cultures. We retrospectively reviewed microbiological results for perianal BHS that were isolated in our laboratory between 2006 and 2015. We identified a total of 105 BHS isolates from rectal swabs and swabs of clinically intact perianal skin. The majority of BHS were of group A (GABHS) (73/105; 69.5%), followed by group B BHS (GBBHS) (27/105; 25.7%), and non-group A or B BHS (5/105; 4.8%). The distribution of GABHS was age-specific, with the majority of GABHS obtained from young children. All BHS isolates were susceptible to penicillin. GABHS were universally susceptible to clindamycin, whereas 1.4% were resistant to erythromycin. GBBHS were resistant to erythromycin and clindamycin in 14.8% and 7.4% of cases. In addition, we wanted to emphasize the importance of correct diagnosis of PSD. Hence, we provide a review of protocols that can decrease the time to diagnosis and treatment of PSD, reduce patients' discomfort, and prevent unnecessary diagnostic procedures.

Introduction: The present study describes the development and evaluation of the first multiplex type-specific quantitative real-time PCR (RT-PCR), enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of human papillomavirus (HPV) types HPV2, 27, and 57 in a single PCR reaction., Results: The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 108 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 108, 107, 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children's warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional Low-risk Alpha-PV PCR., Conclusion: The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.

Background/purpose: Providing a tight coronal seal is key for the success of endodontic treatment, therefore the study aimed to assess bacterial microleakage of materials used for short- and long-term temporization., Materials and Methods: One hundred and twenty-eight human upper-third molars were divided into six experimental groups ( n  = 20) and two control groups: negative ( n  = 4) and positive ( n  = 4). The standardized access cavities were prepared and filled with: (1) Cavit; (2) Fuji II LC; (3) Fuji IX; (4) Voco Clip; (5) AdheSE and Tetric EvoCeram; (6) Excite and Tetric EvoCeram. The crown of each tooth was sectioned to obtain 5.5-mm-high disks, which were assembled in a standard setup for bacterial microleakage studies using Streptococcus mutans . The monitoring lasted 90 days. Kaplan-Meier survival analysis was performed., Results: The lowest amount of leaking samples was found in AdheSE and Tetric EvoCeram (31.3%), Cavit (33.3%), and Excite and Tetric EvoCeram groups (35.3%), followed by Fuji II LC (66.7%), Voco Clip (83.3%). and Fuji IX (88.2%) groups. According to the day of microleakage, materials could be classified in three groups with statistically significant differences (P < 0.05). In the first group were Cavit (70 days), AdheSE and Tetric EvoCeram (68 days), and Excite and Tetric EvoCeram (65 days), in the second group were Voco Clip (44 days) and Fuji II LC (43 days), and in the third group was Fuji IX (21 days)., Conclusion: None of the tested materials were able to completely prevent bacterial microleakage. Adhesively bonded composites and Cavit offer better sealing compared with glass ionomer cements, resin modified glass ionomer cements, and composites without the use of an adhesive system.

Background: The aim of this study was to assess whether serology and spirometry and the combination of both can complement culture-based detection for earlier recognition of Pseudomonas aeruginosa infection in children with cystic fibrosis., Methods: A 4 year longitudinal prospective study that included 67 Slovenian children with cystic fibrosis with a mean age of 10.5 years was conducted. Serology, spirometry and a scoring system combining serology and spirometry were assessed and compared. Infection was confirmed with isolation of Pseudomonas aeruginosa from respiratory samples., Results: There was a significantly positive correlation between serology and the combination of serology and spirometry and Pseudomonas aeruginosa isolation (P < 0.01 for both) and a significantly negative correlation between spirometry and Pseudomonas aeruginosa isolation (P < 0.05). An increase in serology for 1 ELISA unit increased the possibility of Pseudomonas aeruginosa isolation 1.6 times. A fall in FEV1% predicted for 10% increased the possibility of Pseudomonas aeruginosa isolation 9.8 times. Binary logistic regression analysis was used to determine the odds ratios and 95% confidence intervals for all three approaches. Serology had the highest specificity (0.80) and the combination of serology and spirometry the highest sensitivity (0.90). Both had a high negative predictive value (0.93 and 0.79 respectively)., Conclusion: Using serology and the combination of serology and lung function measurement can be beneficial for earlier detection of infection with Pseudomonas aeruginosa in children with cystic fibrosis when done simultaneously with standard culture-based detection from respiratory samples.

Introduction: Of the 350 million individuals chronically infected with hepatitis B virus (HBV) worldwide, approximately 15 to 20 million have been exposed to hepatitis D virus (HDV). This study determined for the first time the HDV prevalence in Slovenian patients with chronic HBV infection. In addition, a literature search was performed to identify all HDV prevalence studies from European countries., Methods: A total of 1,305 HBsAg-positive serum samples, obtained from the same number of patients, were randomly selected from 2,337 patients referred to the Slovenian national reference laboratory for viral hepatitis between 1998 and 2015. All samples were retrospectively tested for the presence of total anti-HDV antibodies. Anti-HDV-positive patients were additionally tested for the presence of anti-HDV IgM antibodies, HDV antigen, and HDV RNA., Results: Total anti-HDV antibodies were detected in three of the 1,305 patients tested (0.23%; 95% CI: 0.08-0.67%), of whom one patient had recovered from the past HDV infection and two patients had an ongoing chronic HDV infection. The literature search identified 36 peer-reviewed HDV prevalence studies published between 1983 and 2016 and originating from 21 European countries., Conclusions: The observed prevalence of HDV infection in Slovenia was among the lowest reported in Europe and worldwide. Due to the observed low prevalence of HDV infection, routine diagnostic testing for HDV should not be considered in differential diagnosis of exacerbation of liver disease in Slovenian patients with chronic HBV infection.

The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116-188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116-188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained.

Unlabelled: Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B., Importance: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Staphylococcus aureus is among the most important human pathogens. It is associated with different infections and is a major cause of skin and soft tissue infections (SSTIs). The aim of our study was to compare S. aureus isolates associated with SSTIs with isolates obtained from healthy carriers in the Central Slovenia region in terms of antimicrobial susceptibility, genetic diversity by clonal complex (CC)/sequence type, spa type, and by toxin gene profiling. In total, 274 S. aureus isolates were collected prospectively by culturing wound samples from 461 SSTI patients and nasal samples from 451 healthy carriers. We have demonstrated high heterogeneity in terms of CCs and spa type in both groups of isolates. The main clone among SSTI strains was Panton-Valentine leukocidin gene (pvl) positive CC121, whereas the main clone among carrier strains was CC45 carrying a large range of toxin genes. The main spa type in both groups was t091. Pvl was more frequently present in SSTI strains (31.2% SSTI vs 3.6% carrier strains) and staphylococcal enterotoxin C was more frequently present in carrier strains (1.6% SSTI vs 17.0% carrier strains). We have also demonstrated that methicillin-resistant S. aureus was a rare cause (2.8%) of SSTIs in our region., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

Background: Testing cervical smears for the presence of high-risk human papillomaviruses (hrHPV) increases the sensitivity for detecting women with underlying high-grade cervical intraepithelial neoplasia (CIN) and provides better and longer protection against invasive cervical cancer compared to cytology testing alone. The Abbott RealTime High Risk HPV test (RealTime) is a hrHPV DNA test with concurrent partial genotyping for HPV16 and HPV18 and aggregate detection of 12 other hrHPV types that have been extensively analytically and clinically evaluated over the last 6 years., Objectives and Study Design: To provide the first 3-year longitudinal data regarding the clinical performance of RealTime, the risk of CIN2+ according to various negative baseline characteristics, and baseline and future risk for CIN2+ at 3 years for women with baseline infection with various hrHPV types were assessed in a cohort of 3,920 Slovenian women that had hrHPV DNA and/or cytology in 36- to 48-month follow-up results after a baseline screening round in 2009/2010., Results: A total of 36 CIN2+ cases were identified in the second screening round. Of these, 17 CIN2+ cases were identified passively through questionnaires/data registries and 19 cases actively as the result of actions triggered by second-round cytology and/or HPV test results. Accumulation of CIN2+ cases during follow-up occurred predominantly in woman with normal cytology at baseline. Among women >30 years old, significantly better protection against CIN2+ at 3 years was associated with a negative hrHPV DNA result at baseline (risk for CIN2+ 0.04% [95 CI, 0.00-0.22%]) than by normal cytology at baseline (risk for CIN2+ 0.68% [95 CI, 0.40-1.08%]). Women with baseline HPV16 infection had a significantly higher risk of CIN2+ at baseline (21.9% [95 CI, 15.2-30.4%]) and baseline plus future risk at 3 years for CIN2+ (33.3% [95 CI, 24.7-44.0%]) in comparison to women with baseline non-HPV16/18 hrHPV infection (7.0% [95 CI, 4.6-10.2%]) or those that were hrHPV-positive (11.7% [95 CI, 9.1-14.9%])., Conclusions: 3-year longitudinal data reinforce evidence from previous studies that RealTime can be safely used in primary HPV-based cervical cancer screening. Concurrent partial genotyping for HPV16/18 should be strongly considered as a triage method for HPV screen-positive women., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Five patients in a geropsychiatric unit of a psychiatric hospital became abruptly ill with pneumonia caused by Streptococcus pneumoniae serotype 6A. Four other residents were colonized with the same serotype, which has previously not been reported in association with pneumonia outbreaks. Furthermore, serotype 6A is not included in all vaccine types, which may be important for the choice of vaccine in some settings. All isolates showed identical pulsed-field gel electrophoresis restriction patterns.

Background: We wanted to determine the time course of colonization with extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (EPE), sites of colonization and risk factors for prolonged colonization with EPE to obtain information for successful infection control measures., Methods: Rectal swab, urine, throat swab and other clinically relevant samples (wound swab, tracheal aspirate and sputum) were obtained from each participant. Sets of follow-up samples and data about potential risk factors for prolonged colonization with EPE were collected every 3 months for 2 years. Multivariate analysis using a logistic regression model was performed to identify risk factors for prolonged colonization., Results: A total of 114 patients were included in the study, 49 completed the 2-year follow-up. In all, 611 sample sets were collected, 309 (50.6%) of which were positive for ESBL. Of the positive sample sets, 90% had a rectal swab positive for ESBL, the throat swab was positive for ESBL in 17.2% of cases and urine in 36.2% of cases; 10% of positive sample sets had negative rectal swabs with EPE isolated from other sites, most often from urine. Immobility was found to be associated with prolonged carriage (≥ 12 months) of EPE. After 2 years, 15/49 (30.6%) patients were colonized with EPE. In 12/49 (24.5%) patients, transient negativity was observed., Conclusions: We found that prolonged colonization with EPE was common, especially in bedridden patients. Transient negative samples were often observed during the course of colonization. In some patients, urine can be the only positive site from which EPE are isolated.

The aim of this study was to compare the effect of subgingival ultrasonic scaling followed by repeated (three times) antimicrobial photodynamic therapy (PDT), ultrasonic scaling alone (US), and scaling and root planing with hand instruments (SRP) for initial periodontal treatment. Twenty-seven non-smoking systemically healthy chronic periodontitis patients were included. Residual pockets ≥4 mm deep and bleeding on probing were debrided either with SRP, US alone, or US followed by a single episode of PDT during supportive periodontal treatment. Probing pocket depth (PPD), bleeding on probing (BOP), and clinical attachment level (CAL) were monitored over 12 months. The presence of five periodontal pathogens in the pockets was determined by a commercially available micro-IDent test. Intergroup and intragroup statistical analysis was performed. All three treatments resulted in a significant clinical improvement. Additional application of PDT to US failed to result in further improvement in terms of PPD reduction and CAL gain. However, it resulted in a higher reduction of BOP at 3 and 12 months comparing to US alone or SRP (PDT from 25 to 13 and to 9%, US from 23 to 16 and to 12%, and SRP from 17 to 10 and to 9%, respectively). PDT reduced the proportion of positive sites after 6 months for Treponema denticola (TD) significantly more effectively than US or SRP (p < 0.0001). Additionally, PDT resulted in a greater reduction of Aggregatibacter actinomycetemcomitans (AA), Tannerella forsythia (TF), and TD in medium pockets (4-6 mm) (p < 0.02) and of TD in deep pockets (>6 mm) compared to mechanical debridement alone (p < 0.05).

Gammapapillomavirus (Gamma-PV) is a diverse and rapidly expanding PV-genus, currently consisting of 76 fully characterized human papillomavirus (HPV) types. In this study, DNA genomes of two novel HPV types, HPV179 and HPV184, obtained from two distinct facial verrucae vulgares specimens of a 64 year-old renal-transplant recipient, were fully cloned, sequenced and characterized. HPV179 and HPV184 genomes comprise 7,228-bp and 7,324-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2); the non-coding region is typically positioned between L1 and E6 genes. Phylogenetic analysis of the L1 nucleotide sequence placed both novel types within the Gamma-PV genus: HPV179 was classified as a novel member of species Gamma-15, additionally containing HPV135 and HPV146, while HPV184 was classified as a single member of a novel species Gamma-25. HPV179 and HPV184 type-specific quantitative real-time PCRs were further developed and used in combination with human beta-globin gene quantitative real-time PCR to determine the prevalence and viral load of the novel types in the patient's facial warts and several follow-up skin specimens, and in a representative collection, a total of 569 samples, of HPV-associated benign and malignant neoplasms, hair follicles and anal and oral mucosa specimens obtained from immunocompetent individuals. HPV179 and HPV184 viral loads in patients' facial warts were estimated to be 2,463 and 3,200 genome copies per single cell, respectively, suggesting their active role in the development of common warts in organ-transplant recipients. In addition, in this particular patient, both novel types had established a persistent infection of the skin for more than four years. Among immunocompetent individuals, HPV179 was further detected in low-copy numbers in a few skin specimens, indicating its cutaneous tissue tropism, while HPV184 was further detected in low-copy numbers in one mucosal and a few skin specimens, suggesting its dual tissue tropism.