Molecular characterization of extended-spectrum ß-lactamase-producing Escherichia coli isolated from lower respiratory tract samples between 2002 and 2019 in the Central Slovenia region.

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 ß-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 blaCTX-M (77.4%; n = 377), blaTEM (54.4%; n = 265) and aac(6')-Ib-cr (52%; n = 253) genes and a low proportion of blaSHV 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.

Collaborative Robot Precision Task in Medical Microbiology Laboratory.

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).

Evaluation of two rapid phenotypical tests-Alifax rapid AST colistin test and Rapid Polymyxin NP test-for detection of colistin resistance in Enterobacterales.

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.

Systemic azithromycin as an adjunct to scaling and root planing in patients with stage III/IV periodontitis: 12-month results of a randomized controlled clinical trial.

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.

Influence of adjunctive azithromycin on microbiological and clinical outcomes in periodontitis patients: 6-month results of randomized controlled clinical trial.

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.

Alinity m HR HPV Assay Fulfills Criteria for Human Papillomavirus Test Requirements in Cervical Cancer Screening Settings.

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.

Evaluation of resazurin-based rapid test to detect colistin resistance in Acinetobacter baumannii isolates.

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.

The environmental monitoring in hospital pharmacy cleanroom and microbiota catalogue preparation.

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).

A very high prevalence of hepatitis C virus infection among patients undergoing hemodialysis in Kosovo: a nationwide study.

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.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences.

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.

Assessment of serology and spirometry and the combination of both to complement microbiological isolation for earlier detection of Pseudomonas aeruginosa infection in children with cystic fibrosis.

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.

Global genomic diversity of human papillomavirus 6 based on 724 isolates and 190 complete genome sequences.

UNLABELLED: Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Effect of repeated adjunctive antimicrobial photodynamic therapy on subgingival periodontal pathogens in the treatment of chronic periodontitis.

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).

Genomic diversity of low-risk human papillomavirus genotypes HPV 40, HPV 42, HPV 43, and HPV 44.

In order to investigate the genomic diversity of low-risk human papillomavirus (HPV) genotypes, a total of 108 isolates of HPV 40, HPV 42, HPV 43, or HPV 44, obtained from anal swabs or tissue specimens of patients with anogenital warts, and cervical swabs of women with cervical intraepithelial neoplasia of different grades, were analyzed. The characterization of genomic variants was established by sequencing one third of the viral genome and analysis of three different genomic regions: L1, LCR, and E6. Maximum variant divergence accounted for 0.4-1.1% of the investigated genomic segments. Several novel, potentially important nucleotide substitutions, deletions, and insertions are described. Altogether, among 14 HPV 40 isolates, a total of nine different genomic variants were identified, composed of eight L1, five LCR, and four E6 genomic variants. Among 49 HPV 42 isolates, a total of 30 genomic variants were identified, composed of 20 L1, 18 LCR, and four E6 genomic variants. Among 10 HPV 43 isolates, distributed into two major genomic variant lineages with clearly defined nucleotide signatures, three genomic variants were identified, composed of three L1, two LCR, and two E6 genomic variants. Among 35 HPV 44 isolates, a total of eight HPV 44 and 11 subtype HPV 44 genomic variants were identified, composed of 13 L1, 14 LCR, and 6 E6 genomic variants. A similar level of genomic diversity of HPV 44 and its subtype was identified in our geographic region as has been reported previously on isolates collected worldwide.

SeptiFast real-time PCR for detection of bloodborne pathogens in patients with severe sepsis or septic shock.

Several studies have been performed investigating the role of a real-time multiplex polymerase chain reaction assay LightCycler SeptiFast with inconsistent results. In prospective evaluation of adult patients with severe sepsis or septic shock SeptiFast assay and blood culture results were compared regarding concordance, the impact of SeptiFast assay on antimicrobial therapy adjustment, time to results and the role of SeptiFast assay as a marker of disease severity. 63 blood sample sets were collected from 57 patients. 51 (80.9%) results were concordant negative and 7 (11.1%) concordant posi- tive. In one (1.6%) sample set blood culture was positive and SeptiFast assay negative, in three (4.8%) sample sets with negative blood cultures pathogens were detected by SeptiFast assay and in one (1.6%)patient an additional pathogen was detected by SeptiFast assay. If blood culture is considered as "gold standard", 1 (1.6%) SeptiFast false negative and 4 (6.3%) false positive results were identified (sensitivity 87.5%, specificity 92.6%, negative predictive value 97.8%). Antibiotic treatment was adjusted according to SeptiFast assay in 4 (6.3%) cases. Time to final results was significantly shorter with SeptiFast assay (32 +/- 23 h vs. 97 +/- 28 h, p < 0.0001). Positive SeptiFast assay was not associated with higher mortality, C-reactive protein orprocalcitonin (p = 0.74, p = 0.44 and p = 0.12, respectively). According to our results SeptiFast assay can be used as a valuable add-on to blood culture in diagnostic workup ofpatients with severe sepsis and septic shock but it cannot replace the blood culture.

Identical human papillomavirus (HPV) genomic variants persist in recurrent respiratory papillomatosis for up to 22 years.

Seventy initial and 125 follow-up tissue specimens of laryngeal papillomas, obtained from 70 patients who had had recurrent respiratory papillomatosis for from 1-22 years, were investigated for the presence of human papillomavirus (HPV) DNA and HPV E5a, LCR and/or full-length genomic variants. HPV-6 was found in 130/195, HPV-11 in 63/195, and HPV-6/HPV-11 in 2/195 samples. Within 67/70 (95.7%) patients, all follow-up HPV isolates genetically matched completely initial HPV isolate over the highly variable parts of the genome or over the entire genome. Frequent recurrence of laryngeal papillomas is a consequence of long-term persistence of the identical initial HPV genomic variant.

Prospective Longitudinal Study of Dynamics of Human Papillomavirus 6 and 11 Infection in Anogenital Hairs and Eyebrows of Male Patients with Anogenital Warts and Age-Matched Controls.

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.

Herpesviridae and Atypical Bacteria Co-Detections in Lower Respiratory Tract Samples of SARS-CoV-2-Positive Patients Admitted to an Intensive Care Unit.

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.

The Impact of COVID-19 on Multidrug-Resistant Bacteria at a Slovenian Tertiary Medical Center.

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.

Improvement of pneumococcal pneumonia diagnostics by the use of rt-PCR on plasma and respiratory samples.

BACKGROUND: The aim of the study was to assess the performance of a real-time polymerase chain reaction (rt-PCR) assay on plasma and respiratory samples for the diagnosis of pneumococcal pneumonia. METHODS: Three hundred and forty patients (160 children and 180 adults) with community-acquired pneumonia were included prospectively from January 2011 to May 2012. Blood samples were obtained simultaneously for culture and rt-PCR targeting the lytA gene. Respiratory samples were also obtained: nasopharyngeal swab in nearly all patients and sputum or tracheal aspirate when available. RESULTS: Streptococcus pneumoniae was detected in 222 (65%) of 340 patients: 143 (89%) children and 79 (44%) adults. Pneumonia was assigned as definite pneumococcal in 96 (28.2%) of 340 patients, according to S. pneumoniae detected in blood: in 54 (33.8%) children - by rt-PCR in 51 (31.9%) and by culture in 5 (3.1%); and in 42 (23.3%) adults - by rt-PCR in 41 (22.8%) and by culture in 12 (6.7%). Pneumonia was considered as probably pneumococcal in 19 (10.6%) adults according to S. pneumoniae detected in sputum/tracheal aspirate, by rt-PCR in 19 and by culture in 5. In 18 adults and 89 children with S. pneumoniae detected only in the nasopharynx, pneumonia was considered as possibly pneumococcal; however it should be noted that nasopharyngeal colonization with S. pneumoniae is also common in children with other aetiologies of pneumonia. CONCLUSIONS: rt-PCR on plasma and other samples performed significantly better than culture for the detection of pneumococcal pneumonia (p < 0.0005) in children and adults.