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BackgroundPatients colonized with carbapenem-resistant Enterobacteriaceae (CRE) are at higher risk of developing CRE infection after liver transplantation (LT), with associated high morbidity and mortality. Prediction model for CRE infection after LT among carriers could be useful to target preventive strategies.MethodsMultinational multicenter cohort study of consecutive adult patients underwent LT and colonized with CRE before or after LT, from January 2010 to December 2017. Risk factors for CRE infection were analyzed by univariate analysis and by Fine-Gray subdistribution hazard model, with death as competing event. A nomogram to predict 30- and 60-day CRE infection risk was created.ResultsA total of 840 LT recipients found to be colonized with CRE before (n = 203) or after (n = 637) LT were enrolled. CRE infection was diagnosed in 250 (29.7%) patients within 19 (interquartile range [IQR], 9–42) days after LT. Pre- and post-LT colonization, multisite post-LT colonization, prolonged mechanical ventilation, acute renal injury, and surgical reintervention were retained in the prediction model. Median 30- and 60-day predicted risk was 15% (IQR, 11–24) and 21% (IQR, 15–33), respectively. Discrimination and prediction accuracy for CRE infection was acceptable on derivation (area under the curve [AUC], 74.6; Brier index, 16.3) and bootstrapped validation dataset (AUC, 73.9; Brier index, 16.6). Decision-curve analysis suggested net benefit of model-directed intervention over default strategies (treat all, treat none) when CRE infection probability exceeded 10%. The risk prediction model is freely available as mobile application at https://idbologna.shinyapps.io/CREPostOLTPredictionModel/.ConclusionsOur clinical prediction tool could enable better targeting interventions for CRE infection after transplant.

This study compared standard of care testing (SOC) to BioFire® FilmArray® Pneumonia plus Panel (PNplus). PNplus detects 15 bacteria with semiquantitative log bin values, 7 antibiotic resistance markers, three atypical bacteria (AB), and eight viral classes directly from bronchoalveolar lavage-like specimens (BLS) and sputum-like specimens (SLS). Fifty-two laboratories from 13 European countries and Israel tested 1234 BLS and 1242 SLS with PNplus and SOC. Detection rates and number of pathogens/samples were compared for PNplus pathogens. PNplus bin values and SOC quantities were compared. Three thousand two hundred sixty-two bacteria in PNplus were detected by PNplus and/or SOC. SOC detected 57.1% compared to 95.8% for PNplus (p ≤ 0.0001). PNplus semiquantitative bin values were less than SOC, equal to SOC, or greater than SOC in 5.1%, 25.4%, and 69.6% of results, respectively. PNplus bin values were on average ≥ 1 log than SOC values (58.5% 1–2 logs; 11.0% 3–4 logs). PNplus identified 98.2% of MRSA and SOC 55.6%. SOC detected 73/103 AB (70.9%) and 134/631 viruses (21.2%). PNplus detected 93/103 AB (90.3%) and 618/631 viruses (97.9%) (p ≤ 0.0001). PNplus and SOC mean number of pathogens/samples were 1.99 and 1.44, respectively. All gram-negative resistance markers were detected. PNplus and SOC results were fully or partially concordant for 49.1% and 26.4% of specimens, respectively. PNplus was highly sensitive and detected more potential pneumonia pathogens than SOC. Semiquantification may assist in understanding pathogen significance. As PNplus generates results in approximately 1 h, PNplus has potential to direct antimicrobial therapy in near real time and improve antimicrobial stewardship and patient outcomes.

Infections are a major complication of solid organ transplants (SOTs). This study aimed to describe recipients’ characteristics, and the frequency and etiology of infections and transplant outcome in liver and lung SOTs, and to investigate exposures associated to infection and death in liver transplant recipients. The study population included recipients of SOTs performed in Italy during a 1-year period in ten Italian lung transplant units and eight liver transplant units. Data on comorbidities, infections, retransplantation, and death were prospectively collected using a web-based system, with a 6-month follow-up. The cumulative incidence of infection was 31.7% and 47.8% in liver and lung transplants, respectively, with most infections occurring within the first month after transplantation. Gram-negatives, which were primarily multidrug-resistant, were the most frequent cause of infection. Death rates were 0.42 per 1000 recipient-days in liver transplants and 1.41 per 1000 recipient-days in lung transplants. Infection after SOT in adult liver recipients is associated to an increased risk of death (OR = 13.25; p-value < 0.001). Given the frequency of infection caused by multidrug-resistant microorganisms in SOT recipients in Italy and the heavy impact of infections on the transplant outcome, the reinforcement of surveillance and control activities to prevent the transmission of multidrug-resistant microorganisms in SOT recipients represents a priority. The implementation of the study protocol in liver and lung transplant units and the sharing of results have increased the awareness about the threat due to antimicrobial resistance in the country.

Objective: To provide the first Italian data on pathogens causing early-onset sepsis (EOS) and their antimicrobial susceptibility, after the successfully prevention of Group B streptococcus (GBS) EOS. Methods: Retrospective area-based cohort study from Emilia-Romagna (Italy). Cases of EOS registered (from 2009 to 2012) in all gestational age neonates were reviewed. Results: Live births (LB) numbered 146 682. Ninety neonates had EOS and 12 died (incidence rates of 0.61 and 0.08/1000 LB, respectively). EOS and mortality were the highest among neonates with a birth weight n = 27, 0.18/1000 LB) and Escherichia coli (n = 19, 0.13/1000 LB). Most infants affected by E. coli EOS were born preterm (n = 13), had complications (n = 4) or died (n = 7). Among 90 isolates tested, only 3 were resistant to both first line empirical antibiotics. Multivariate logistic regression analysis showed that low gestational age, caesarean section and low platelet count at presentation were significantly associated with death or brain lesions (area under ROC curve = 0.939, H-L = 0.944, sensitivity 76.0%, specificity 90.7%). Conclusions: GBS slightly exceeds E. coli as a cause of EOS. However, E. coli is the prominent cause of death, complications and in most cases affects preterm neonates. Empirical antimicrobial therapy of EOS seems appropriate.

Objectives A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. Methods Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. Results Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor–recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-β-lactamase (VIM)-producing Enterobacter aerogenes. Conclusions This study showed a low risk of donor–recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018–2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018–2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Staphylococcus aureus infection led to a case of goat abortion, and four veterinarians contracted S.aureus infection from the goat during and after the abortion. Three veterinarians assisted a doe during the dystocic delivery of a dead foetus. Seventy-two hours after the dystocia, which ended with the goat's death, the veterinarians who assisted during the kidding and the veterinarian who performed the necropsy showed the presence of multiple, isolated, painful pustules 1–5mm in diameter located along their forearms and knees. S.aureus was isolated from the pustules of the veterinarians, the placenta and uterus of the goat, the organs (brain, thymus gland, abomasum, liver and spleen) of the foetus, the scrotum and eye swabs of the buck, and mammary pustules of another goat from the same herd. Histological analysis revealed purulent metritis and inflammation of the placental cotyledons. Additional investigations eliminated the chances of other infections. S.aureus isolates recovered from the veterinarians, goats, foetus and buck were sensitive to the tested anti-microbials and did not encode staphylococcal enterotoxin genes (sea, ser, sep, see, seg and sei). The isolates were closely related, as indicated by the results of Fourier-transform infrared spectroscopy and comparative whole-genome sequencing analysis. The results of this study clearly support the hypothesis that an episode of professional zoonosis was caused by S.aureus infection during the abortion and also highlight the need for bacterial subtyping in epidemiological surveys.

Objectives We examined factors associated with follow-up blood cultures (FUBCs) in patients with monomicrobial Gram-negative (GN) bloodstream infection (BSI) and investigated the impact of FUBCs on therapeutic management and patient outcome. Methods A retrospective cohort analysis was conducted of adult patients diagnosed with GN-BSI at a tertiary-care university hospital during 2013–2016. FUBCs performed between 24 hours and 7 days after index BCs was the exposure variable. Risk factors for 30-day mortality were analysed by multivariate Cox analysis on the overall cohort, including FUBCs as a time-varying covariate and on 1:1 matched patients according to Sequential Organ Failure Assessment (SOFA) score and time to FUBC. Results In 278 (17.6%) of 1576 patients, FUBCs were performed within a median of 3 and 2 days after index BCs and active antibiotic therapy initiation. Persistent BSI was found in 107 (38.5%) of 278 patients. FUBCs were performed in more severely ill patients, with nonurinary sources, difficult-to-treat pathogens and receipt of initial inappropriate therapy. Source control and infectious disease consultation rates were higher among patients with preceding FUBCs and was associated with longer treatment duration. Thirty-day mortality was 10.4%. Independent risk factors for mortality were Charlson comorbidity index (hazard ratio (HR) 1.12) SOFA (HR 1.11), septic shock (HR 2.64), urinary source (HR 0.60), central venous catheter source (HR 2.30), complicated BSI (HR 2.10), carbapenem resistance (HR 2.34), active empiric therapy (HR 0.68), source control (HR 0.34) and FUBCs (HR 0.48). Association between FUBCs and lower mortality was confirmed in the 274 matched pairs. Conclusions FUBCs were performed in more severe GN-BSIs, yielding a high rate of persistent BSI. In this context, FUBCs were associated with lower mortality.

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Urinary tract infections (UTIs) are among the most common bacterial infections in children, and Escherichia coli is the main pathogen responsible. Several guidelines, including the recently updated Italian guidelines, recommend amoxicillin-clavulanic acid (AMC) as a first-line antibiotic therapy in children with febrile UTIs. Given the current increasing rates of antibiotic resistance worldwide, this study aimed to investigate the three-year trend in the resistance rate of E. coli isolated from pediatric urine cultures (UCs) in a metropolitan area of northern Italy. We conducted a retrospective review of E. coli-positive, non-repetitive UCs collected in children aged from 1 month to 14 years, regardless of a diagnosis of UTI, catheter colonization, urine contamination, or asymptomatic bacteriuria. During the study period, the rate of resistance to AMC significantly increased from 17.6% to 40.2% (p < 0.001). Ciprofloxacin doubled its resistance rate from 9.1% to 16.3% (p = 0.007). The prevalence of multidrug-resistant E. coli rose from 3.9% to 9.2% (p = 0.015). The rate of resistance to other considered antibiotics remained stable, as did the prevalence of extended spectrum beta-lactamases and extensively resistant E. coli among isolates. These findings call into question the use of AMC as a first-line therapy for pediatric UTIs in our population, despite the indications of recent Italian guidelines.

Meropenem/Vaborbactam (MEM-VAB) is a novel carbapenem- β-lactamase inhibitor active against KPC-producing Enterobacteria. Herein, we evaluate the incidence of meropenem/vaborbactam-resistance among KPC-producing K. pneumoniae (KPC-Kp) bloodstream infection in a large Italian hospital. Meropenem/vaborbactam-resistance was found in 8% (n = 5) KPC-Kp, while 5% (n = 3) strains exhibited cross-resistance to ceftazidime/avibactam (CAZ-AVI). Genomic analysis revealed that meropenem/vaborbactam-resistance was associated with truncated OmpK35 and insertion of glycine and aspartic acid within OmpK36 at position 134–135 (GD134–135). Notably, no specific mutation was associated to cross-resistance. No specific antimicrobial treatment was related to favorable clinical outcomes, while cross-resistance was not associated to higher clinical and/or microbiological failures. Our study indicated that resistance to meropenem/vaborbactam was due to porins mutations and is associated with reduced susceptibility to both ceftazidime/avibactam and carbapenems.

Objective: To investigate the rate of and the risk factors for breakthrough-IFI (b-IFI) after orthotopic liver transplantation (OLT) according to the new definition proposed by Mycoses-Study-Group-Education-and-Research-Consortium (MSG-ERC) and the European-Confederation-of-Medical-Mycology (ECMM). Methods: Multicenter prospective study of adult patients who underwent OLT at three Italian hospitals, from January 2015 to December 2018. Targeted antifungal prophylaxis (TAP) protocol was developed and shared among participating centers. Follow-up was 1-year after OLT. B-IFI was defined as infection occurring during exposure to antifungal prophylaxis. Risk factors for b-IFI were analyzed among patients exposed to prophylaxis by univariable analysis. Results: We enrolled 485 OLT patients. Overall compliance to TAP protocol was 64.3%, 220 patients received antifungal prophylaxis, 172 according to TAP protocol. Twenty-nine patients were diagnosed of IFI within 1year after OLT. Of them, 11 presented with b-IFI within 17 (IQR 11-33) and 16 (IQR 4-30) days from OLT and from antifungal onset, respectively. Then out of 11 patients with b-IFI were classified as having high risk of IFI and were receiving anti-mould prophylaxis, nine with echinocandins and one with polyenes. Comparison of patients with and without b-IFI showed significant differences for prior Candida colonization, need of renal replacement therapy after OLT, re-operation, and CMV infection (whole blood CMV-DNA >100000 copies/mL). Although non-significant, a higher rate of b-IFI in patients on echinocandins was observed (8.2% vs 1.8%, P=.06). Conclusions: We observed 5% of b-IFI among OLT patients exposed to antifungal prophylaxis. The impact of echinocandins on b-IFI risk in this setting should be further explored.

We assessed the performance of PhenoMatrix digital imaging software in detection of Group B Streptococcus from recto-vaginal swabs plated on a specific chromogenic medium, using the WASP automated processor. PhenoMatrix algorithm showed a sensitivity of 100% and a specificity of 64.5%. False-positive results were mainly due to commensal viridans streptococci.

Objectives Current evidence suggests that early diagnosis of sepsis and timely detection of antimicrobial resistance are crucial to improve mortality rates among patients. The aim of this study was to evaluate a rapid method for the identification of Gram-negative bacteria from positive blood cultures (BCs), combined with the detection of extended spectrum β-lactamases (ESβL) and carbapenemases production, by means of MALDI-TOF/MS analysis. Methods During the study, all BCs positive for Gram-negative rods were selected. Starting from bacterial pellets obtained directly from BC broths, species identification and hydrolysis assays were achieved through MALDI-TOF/MS (Bruker). In particular, we performed a hydrolysis assays of cefotaxime (CTX) and ertapenem (ERT) for the rapid detection of resistance via ESβL and carbapenemases, respectively. These results were compared with the routine workflow, including BC subcultures and confirmation phenotypic methods. Finally, a comparison of the turnaround-time (TAT) between the two protocols was conducted. Results Overall, 185 BCs positive for Enterobacteriaceae were collected. In terms of species identification, we observed a concordance of 95.9% comparing MALDI-TOF/MS results to the subculture-based method. The sensitivity and specificity for CTX hydrolysis assay were 91.1% and 92%, respectively; ERT hydrolysis assay showed a sensitivity of 96.2% and a specificity of 99.2%. The TAT of the proposed MALDI TOF/MS-based protocol was significantly lower compared with the routine workflow (P Conclusions The proposed protocol can provide reliable bacterial identification and data concerning β-lactam resistance in only 3 hours, positively improving management of patients in terms of antimicrobial stewardship.

Objectives: The aim of this study was to evaluate the risk factors for candidaemia in patients with liver cirrhosis. Methods: This was a case–control–control (1:2:2) study performed in four Italian tertiary centres from 2006 to 2015. Cases were patients with liver cirrhosis developing candidaemia. For every case of candidaemia we enrolled two additional patients undergoing blood cultures for suspected infection yielding isolation of a bacterial pathogen (control A) and two additional patients undergoing blood cultures for suspected infection yielding negative results (control B). Patients were matched according to age, sex and model for end stage liver disease at hospital admission. Results: During the study period 90 cases, 180 controls A and 180 controls B were included. At multivariate analysis assessed by means of multinomial conditional regression models, factors independently associated with candidaemia were previous (15 days (RRR 4.63 (95% CI 2.11–10.18), p

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Background Serum bactericidal titres (SBTs) were widely used in the 1970s and 1980s to monitor antimicrobial therapy but are now seldom recommended. It is the only laboratory test that integrates drug pharmacodynamics, host pharmacokinetics and synergistic or antagonistic interactions of antimicrobial combinations into a single index of antimicrobial activity. We hypothesized that SBTs could play a renewed role in monitoring antibiotic treatment of multidrug-resistant Gram-negative infections. However, the last critical appraisal of the test was published over 30 years ago. Objectives This narrative review provides an updated assessment of the SBT test and its methodological limitations. We performed a diagnostic meta-analysis to estimate the value of SBTs for predicting clinical failure or death during antibiotic treatment. Sources A comprehensive literature search of PubMed including all English publications was performed in December 2019 using the Medical Subject Headings (MeSH search terms “serum”, “bactericidal”, “inhibitory”, “titre”, “monitoring”, “anti-infective agents” “antimicrobial therapy” and “therapeutic drug monitoring”). Content Although standardized methods for performing SBTs were approved in 1999, the test remains labour intensive, and results may not be available until 72 hr. However, the use of non-culture-based endpoints (i.e. spectrophotometric or fluorescent) may shorten test time to 24 hr. Despite considerable heterogeneity in published studies, a meta-analysis of 11 evaluable studies published from 1974 to 2007 indicated a critical SBT result (peak SBT ≤1:8 or trough ≤1:2) is associated with a diagnostic odds ratio for clinical failure during antibiotic treatment of 12.27 (95% confidence interval 5.28–28.54) and a 5.32 (95% 1.32–21.42) odds of death. Implications SBTs have prognostic value for identifying patients at high risk for antibiotic treatment failure, but the slow turnaround time of the current test limits its clinical utility. Standardization of a more rapid SBT testing method is needed.

Background To compare the prognostic utility of the new definition of difficult-to-treat resistance (DTR) vs established definitions in a cohort of patients with Gram-negative bloodstream infections (GNBSIs). Methods This was a retrospective single-center study of adult patients with monomicrobial GNBSI, hospitalized from 2013 to 2016. DTR was defined as isolate demonstrating intermediate or resistant phenotype to all reported agents in the carbapenem, beta-lactam, and fluoroquinolone classes. Carbapenem resistance (CR) was defined according to 2015 Centers for Disease Control and Prevention criteria. Each isolate was further classified according to the Magiorakos et al. criteria as non-multidrug-resistant (non-MDR), MDR, extensively drug-resistant (XDR), or pan-drug-resistant (PDR). The primary outcome was all-cause 30-day mortality. Results Overall, 1576 patients were analyzed. Enterobacteriaceae accounted for 88.7% of BSIs, with Escherichia coli (n = 941) and Klebsiella pneumoniae (n = 326) being the most common pathogens. Pseudomonas aeruginosa was the most common nonfermentative bacteria (n = 130, 8.2%). Overall, 11% of strains were defined as DTR and 13% as CR. Episodes were further classified as non-MDR (68.8%), MDR (21.9%), XDR (8.8%), and PDR (0.4%). The prevalence rates of DTR, CR, and XDR were similar among Enterobacteriaceae and Acinetobacter baumannii, whereas they differed in P. aeruginosa. All the analyzed resistance definitions significantly improved prediction of 30-day mortality when introduced into a baseline multivariate model, to a similar degree: 9%, 10%, and 11% for DTR, Magiorakos, and CR definitions, respectively. Conclusions DTR seems a promising tool to identify challenging GNBSIs, mainly those due to P. aeruginosa. With the availability of new agents for CR infections, further multicenter assessments of DTR are needed.

Combination therapies are frequently used in the treatment of multidrug-resistant Klebsiella pneumoniae infection without consensus regarding which combination is the most effective. We compared bactericidal titres from sera collected from critically ill patients receiving meropenem plus tigecycline (n= 5), meropenem plus colistin (n= 5), or meropenem, colistin and tigecycline (n= 5) against K. pneumoniae isolates that included ESBL-producing (n= 7) and KPC-producing strains (n= 14) with varying sensitivity patterns to colistin and tigecycline. Meropenem concentrations (Cmin) were measured in all samples by LC-MS/MS, and indexed to respective pathogen MICs to explore differences in patterns of bactericidal activity for two versus three drug combination regimens. All combination regimens achieved higher SBTs against ESBL (median reciprocal titre 128, IQR 32–256) versus KPC (4, IQR 2–32) strains. Sera from patients treated with meropenem-colistin yielded higher median SBTs (256, IQR 64–512) than either meropenem-tigecycline (32, IQR 8–256; P' 0.001). The addition of tigecycline was associated with a lower probability of achieving a reciprocal SBT above 8 when meropenem concentrations were below the MIC (P= 0.04). Although the clinical significance is unknown, sera from patients receiving tigecycline-based combination regimens produce lower serum bactericidal titres against ESBL or KPC-producing K. pneumoniae. SBTs may represent a useful complimentary endpoint for comparing pharmacodynamics of combinations regimens for MDR Enterobacteriaceae.

Objective To investigate the impact of colonization with carbapenemase-producing Enterobacteriaceae (CPE) on the CPE infection risk after liver transplantation (LT). Methods Prospective cohort study of all adult patients undergoing LT at our centre over an 8-year period (2010–2017). Individuals were screened for CPE colonization by rectal swabs at inclusion onto the waiting list, immediately before LT and weekly after LT until hospital discharge. Asymptomatic carriers did not receive decolonization, anti-CPE prophylaxis or pre-emptive antibiotic therapy. Participants were followed up for 1 year after LT. Results We analysed 553 individuals who underwent a first LT, 38 were colonized with CPE at LT and 104 acquired colonization after LT. CPE colonization rates at LT and acquired after LT increased significantly over the study period: incidence rate ratios (IRR) 1.21 (95% CI 1.05–1.39) and 1.17 (95% CI 1.07–1.27), respectively. Overall, 57 patients developed CPE infection within a median of 31 (interquartile range 11–115) days after LT, with an incidence of 3.05 cases per 10 000 LT-recipient-days and a non-significant increase over the study period (IRR 1.11, 95% CI 0.98–1.26). In multivariable analysis, CPE colonization at LT (hazard ratio (HR) 18.50, 95% CI 6.76–50.54) and CPE colonization acquired after LT (HR 16.89, 95% CI 6.95–41.00) were the strongest risk factors for CPE infection, along with combined transplant (HR 2.60, 95% CI 1.20–5.59), higher Model for End-Stage Liver Disease at the time of LT (HR 1.03, 95% CI 1.00–1.07), prolonged mechanical ventilation (HR 2.63, 95% CI 1.48–4.67), re-intervention (HR 2.16, 95% CI 1.21–3.84) and rejection (HR 2.81, 95% CI 1.52–5.21). Conclusions CPE colonization at LT or acquired after LT were the strongest predictors of CPE infection. Prevention strategies focused on LT candidates and recipients colonized with CPE should be investigated.

The prevalence and microbiology of concomitant respiratory bacterial infections in patients with SARS-CoV-2 infection are not yet fully understood. In this retrospective study, we assessed respiratory bacterial co-infections in lower respiratory tract samples taken from intensive care unit-hospitalized COVID-19 patients, by comparing the conventional culture approach to an innovative molecular diagnostic technology. A total of 230 lower respiratory tract samples (i.e., bronchial aspirates or bronchoalveolar lavages) were taken from 178 critically ill COVID-19 patients. Each sample was processed by a semi-quantitative culture and by a multiplex PCR panel (FilmArray Pneumonia Plus panel), allowing rapid detection of a wide range of clinically relevant pathogens and a limited number of antimicrobial resistance markers. More than 30% of samples showed a positive bacterial culture, with Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus the most detected pathogens. FilmArray showed an overall sensitivity and specificity of 89.6% and 98.3%, respectively, with a negative predictive value of 99.7%. The molecular test significantly reduced the turn-around-time (TAT) and increased the rates of microbial detection. Most cases missed by culture were characterized by low bacterial loads (104–105 copies/mL). FilmArray missed a list of pathogens not included in the molecular panel, especially Stenotrophomonas maltophilia (8 cases). FilmArray can be useful to detect bacterial pathogens in lower respiratory tract specimens of COVID-19 patients, with a significant decrease of TAT. The test is particularly useful to rule out bacterial co-infections and avoid the inappropriate prescription of antibiotics.

Infections continue to be one of the leading causes of morbidity and mortality in liver transplant recipients. We retrospectively reviewed the symptomatic infectious episodes that occurred during the first year post-transplant to determine time of onset, causative pathogens and cell-mediated immunity response patterns. Ninety-eight of the 202 (48.5%) recipients enrolled developed at least one infectious episode. The total number of infectious episodes was 135: 77 (57.1%) bacterial, 45 (33.3%) viral and 13 (9.6%) fungal. The most frequently isolated bacteria were Escherichia coli (21 isolates) and Klebsiella pneumoniae (19 isolates). Overall, extended-spectrum beta lactamase-producing and methicillin-resistant organisms were responsible for 29 (29/77; 37.7%) infectious episodes. Members of the herpes virus group, in particular cytomegalovirus (34/45 viral infections, 75.5%), were detected. Candida species (9 isolates) followed by Aspergillus species (4 isolates) were isolated. The majority of infections (63%) occurred during the early post-transplant phase (

Objectives The aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection. Methods We collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing. Results During the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime–avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime–avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of blaKPC gene. Conclusions Our results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the blaKPC gene or porin deficiency associated with higher blaKPC copy number and is also related to the meropenem/vaborbactam resistance.

The aim of this study was to investigate risk factors for treatment failure in patients receiving in vitro-active therapy with β-lactam/β-lactamase inhibitor (BL/BLI) for Enterobacteriaceae bloodstream infection (E-BSI). This was a retrospective, single-centre study of patients diagnosed with E-BSI at an Italian centre over a 4-year period. Exclusion criteria were age

Background Data about the optimal management of immunosuppressive therapy in liver transplant (LT) recipients with bloodstream infection (BSI) are missing. We aimed to describe the management of immunosuppressive therapy at diagnosis of BSI in LT recipients and to assess its impact on 28-day mortality. Methods We performed a single-center retrospective study of all LT recipients diagnosed with BSI, over 10-year period. Multivariate Cox regression analysis of risk factors for all cause 28-day mortality was adjusted for the propensity score of being managed with "any reduction" in immunosuppressive therapy at the diagnosis of BSI. Results We identified 209 episodes of BSI in 157 LT recipients: 107 (68%) male, median age 54 (IQR 48-63) years. "Any reduction" was made in 90 (43%) cases including: dosage reduction of ≥1 immunosuppressive drug in 31 (15%), discontinuation of ≥1 immunosuppressive drug in 28 (13%), both dosage reduction and discontinuation in 13 (6%), complete withdrawal of immunosuppressive therapy in 18 (9%) cases. All-cause 28-day mortality rate was 13.4%, varying from 22% to 7% (P = .002) in cases with and without "any reduction". Cox regression showed septic shock (aHR 3.15, P = .007) and "any reduction" (aHR 2.50, P = .02) as independent risk factors for all-cause 28-day mortality, while Escherichia coli (aHR 0.38, P = .03) and source control (aHR 0.43, P = .04) were protective factors. The final model did not change after the introduction of the propensity score for "any reduction". Conclusions Any reduction in the immunosuppressive therapy was common and was associated with worse outcome in LT recipients developing BSI.

none 11 no Objectives: To investigate the impact of treatment duration on mortality and on relapse in patients with Escherichia coli bloodstream infection (BSI). Methods: Retrospective single-centre study of patients diagnosed with E. coli BSI at our centre over a 4-year period. Exclusion criteria: age 10 days) therapy. Primary end point was all-cause mortality within 90 days after index BSI. Secondary end point was relapse, defined as repeat isolation of E. coli from blood cultures within 90 days after index BSI, in patients with documented clinical cure and completion of therapy for the initial episode. Results: Of the 856 analysed patients: 426 received short and 430 received long therapy. All-cause mortality at day 90 occurred in 47 patients; on multivariate analysis, short therapy was not associated with a higher risk of mortality, also after adjusting the model for the propensity score of receiving short therapy. Relapse occurred in 42 patients. Independent risk factors for relapse using death as competing risk were immunosuppression (subhazard ratio 4.67, p < 0.001), and end-stage liver disease (subhazard ratio 2.58, p 0.013). The propensity-weighted estimation of the average treatment effect for relapse reduction with long therapy (>10 days) was -1.6% (p 0.26) in the total population, and -7.1% (p 0.18) in immunocompromised patients. Conclusions: We could not identify shorter treatment duration as a risk factor for mortality and for relapse in patients with E. coli BSI. open Giannella, M.*; Pascale, R.; Toschi, A.; Ferraro, G.; Graziano, E.; Furii, F.; Bartoletti, M.; Tedeschi, S.; Ambretti, S.; Lewis, R.E.; Viale, P. Giannella, M.*; Pascale, R.; Toschi, A.; Ferraro, G.; Graziano, E.; Furii, F.; Bartoletti, M.; Tedeschi, S.; Ambretti, S.; Lewis, R.E.; Viale, P.

BACKGROUND & AIMS: Bloodstream infections (BSIs) in cirrhotic patients are 10-fold more common than in non-cirrhotic patients and increasingly caused by resistant pathogens. We examined 162 BSI episodes in cirrhotic patients to describe the etiology and risk factors for 30-day mortality. METHODS: We retrospectively analyzed all consecutive BSIs in patients with liver cirrhosis at our 1350-bed teaching hospital (January 2008 to June 2012). Cox-proportional hazard regression was used to analyze the impact of disease and treatment-related variables on the crude 30-day mortality. RESULTS: BSI episodes were identified in 162 patients, including 29 mixed infections. Most of episodes were classified as hospital acquired or healthcare associated (93%). Gram-negative bacteria (GNB), Gram-positive bacteria and Candida spp. caused 64%, 38%, and 10% of episodes, respectively. GNB were classified as multi-drug resistant (MDR) and extensively drug resistant (XDR) in 25% and 21% of cases, respectively. The overall crude 30-day mortality rate was 29%. Four risk factors were independently associated with 30-day crude mortality: worsening of MELD score from baseline (the last MELD score available in the 2weeks prior BSI) to that at BSI onset (HR 1.11 per point increase, 95% CI 1.07-1.15, p

Summary Objective The identification of patients colonized or infected with carbapenemase-producing Enterobacteriaceae (CPE), in order to control and prevent the global spread of multidrug-resistant (MDR) pathogens. Methods From June 1 to June 15, 2012, eight Citrobacter freundii strains with reduced susceptibility to carbapenems were isolated from rectal swabs of hospitalized patients during active screening following the detection of a Klebsiella pneumoniae carbapenemase (KPC) -positive patient on the ward. All isolates were analyzed phenotypically and molecularly by PCR and sequencing. Genotype clustering was performed by multilocus sequence typing (MLST) analysis. Results The isolates showed high rates of multidrug resistance profile. A phenotypic assay for carbapenemase production suggested the presence of metallo-β-lactamase (MBL). The blaVIM-1 gene was detected in all imipenem-resistant C. freundii isolates. MLST showed that the C. freundii isolates shared the same sequence type (ST). Phylogenetic analysis revealed a strict relationship with an ST5 C. freundii isolate from a diarrhea patient in China. Conclusions Our findings showed that the active surveillance program for CPE was useful, not only for the detection of KPC-producers, but also to identify and control the spread of other MDR pathogens that could expand the spectrum of circulating MDR pathogens.

Introduction: the purpose of this retrospective multicenter study was to assess whether the risk of developing bloodstream infections (BSI) due to carbapenem-resistant Klebsiella pneumoniae (CRKP) in colonized patients is influenced by the occurrence of BSI due to other pathogens. Methods: from January 2012 to March 2014, all patients with at least one rectal swab positive for CRKP and at least 30 days of previous hospital stay were included in the study. The primary outcome measure was CRKP BSI, defined as a time-to-event endpoint. The role of potential predictors was evaluated through univariable and multivariable Cox regression analyses, considering previous BSI as a time-dependent variable. Results: during the study period, 353 patients met the inclusion criteria. Thirty-seven developed a CRKP BSI (11%). A higher incidence of CRKP BSI was observed in presence rather than in absence of previous BSI. In the final multivariable model of risk factors for CRKP BSI, multisite colonization (hazard ratio [HR] 13.73, 95% confidence intervals [CI] 3.29-57.32, p < 0.001), ICU stay (HR 3.14, 95% CI 1.19-8.31, p = 0.021), and previous BSI (p = 0.026, with the overall effect being mainly due to Enterococcus spp. BSI vs absence of BSI, HR 6.62, 95% CI 2.11-20.79) were associated with the development of CRKP BSI, while an inverse association was observed for age (HR 0.98, 95% CI 0.95-1.00, p = 0.027). Conclusions: previous BSI due to other pathogens were associated with an increased risk of CRKP BSI that was independent of other factors in colonized patients with prolonged hospital exposure.

ESGBIS/BICHROME Study Group: C. Campoli, G. Siccardi, S. Ambretti, A. Stallmach, M. Venditti, C. Lucidi, S. Ludovisi, M. De Cueto, M. D. Navarro, E. Lopez Cortes, E. Bouza, M. Valerio, A. Eworo, R. Losito, M. Senzolo, E. Nadal, A. Ottobrelli, M. Varguvic, C. Badia, G. Borgia, I. Gentile, A. R. Buonomo, E. Boumis, A. Beteta-Lopez, A. Rianda, G. Taliani, S. Grieco., [Objectives] To describe the current epidemiology of bloodstream infection (BSI) in patients with cirrhosis; and to analyse predictors of 30-day mortality and risk factors for antibiotic resistance., [Methods] Cirrhotic patients developing a BSI episode were prospectively included at 19 centres in five countries from September 2014 to December 2015. The discrimination of mortality risk scores for 30-day mortality were compared by area under the receiver operator risk and Cox regression models. Risk factors for multidrug-resistant organisms (MDRO) were assessed with a logistic regression model., [Results] We enrolled 312 patients. Gram-negative bacteria, Gram-positive bacteria and Candida spp. were the cause of BSI episodes in 53%, 47% and 7% of cases, respectively. The 30-day mortality rate was 25% and was best predicted by the Sequential Organ Failure Assessment (SOFA) and Chronic Liver Failure–SOFA (CLIF-SOFA) score. In a Cox regression model, delayed (>24 hours) antibiotic treatment (hazard ratio (HR) 7.58; 95% confidence interval (CI) 3.29–18.67; p < 0.001), inadequate empirical therapy (HR 3.14; 95% CI 1.93–5.12; p < 0.001) and CLIF-SOFA score (HR 1.35; 95% CI 1.28–1.43; p < 0.001) were independently associated with 30-day mortality. Independent risk factors for MDRO (31% of BSIs) were previous antimicrobial exposure (odds ratio (OR) 2.91; 95% CI 1.73–4.88; p < 0.001) and previous invasive procedures (OR 2.51; 95% CI 1.48–4.24; p 0.001), whereas spontaneous bacterial peritonitis as BSI source was associated with a lower odds of MDRO (OR 0.30; 95% CI 0.12–0.73; p 0.008)., [Conclusions] MDRO account for nearly one-third of BSI in cirrhotic patients, often resulting in delayed or inadequate empirical antimicrobial therapy and increased mortality rates. Our data suggest that improved prevention and treatment strategies for MDRO are urgently needed in the liver cirrhosis patients.

Treponema denticola has been identified as an important cause of periodontal disease and hypothesized to be involved in extra-oral infections. The objective of this study was to investigate the role of T. denticola cell length and motility during mouse peritoneal macrophages in vitro uptake. Macrophages, incubated under aerobic and anaerobic conditions, produced a similar amount of TNF-α when stimulated with Escherichia coli LPS. The uptake of FlgE- and CfpA-deficient mutants of T. denticola was significantly increased compared with the wild-type strain, due to cell size or lack of motility. Opsonization with specific antibodies considerably improved the treponemes’ uptake. These results suggest that macrophages, in addition to other phagocytes, could play an important role in the control of T. denticola infection, and that the raising of specific antibodies could improve the efficacy of the immune response toward T. denticola, either at an oral site or during dissemination.

The evidence on genotype-specific risk in women infected with human papillomavirus (HPV) with normal cytology and the importance of the distinction of high-risk (HR)-HPV genotypes in the management of low-grade lesions suggest that the distinction of HR-HPV genotypes has the potential to improve the follow-up of patients treated for high-grade cervical lesions. The aims of this study were to define the persistence of the different HR-HPV in the follow-up of surgical treated women, to detect the changes of genotypes from the pre- to the post-operative status, and to evaluate whether genotype-specific persistence can predict the development of residual or recurrent disease during the follow-up. HR-HPV detection and genotyping was carried out by the Linear Array HPV Genotyping Test on cervical cytological samples from 72 women treated by surgery. The 6-month post-operative HPV status was correlated with the pre-operative HPV genotype and with the residual or recurrent disease within 24 months. It was observed that the residual or recurrent disease in women with persistence of HPV 16 and/or HPV 18 was higher (82.4%) than in women with persistence of at least one HR-HPV type of group 2 (HPV 31, 33, 35, 45, 52, and 58) (66.7%) and at least one type of group 3 (HPV 39, 51, 56, 59, 68, 26, 53, 66, 73, and 82) (14.3%). These data defined HR-HPV groups for the risk of progression of disease and suggested that the identification of persistent infection with different HR-HPV genotypes has the potential to improve the management of these patients.

Background and objectives: High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. Study design: the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. Results: protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance ( K =0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. Conclusions: HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Objectives Infections caused by Klebsiella pneumoniae (Kp) carbapenemase (KPC)-producing strains of Kp have become a significant threat in recent years. To assess their outcomes and identify risk factors for 14 day mortality, we conducted a 4 year (2010–13) retrospective cohort study in five large Italian teaching hospitals. Methods The cohort included 661 adults with bloodstream infections (BSIs; n = 447) or non-bacteraemic infections (lower respiratory tract, intra-abdominal structure, urinary tract or other sites) caused by a KPC-Kp isolate. All had received ≥48 h of therapy (empirical and/or non-empirical) with at least one drug to which the isolate was susceptible. Results Most deaths occurred within 2 weeks of infection onset (14 day mortality: 225/661, 34.1%). Logistic regression analysis identified BSI (OR, 2.09; 95% CI, 1.34–3.29), presentation with septic shock (OR, 2.45; 95% CI, 1.47–4.08), inadequate empirical antimicrobial therapy (OR, 1.48; 95% CI, 1.01–2.18), chronic renal failure (OR, 2.27; 95% CI, 1.44–3.58), high APACHE III score (OR, 1.05; 95% CI, 1.04–1.07) and colistin-resistant isolates (OR, 2.18; 95% CI, 1.37–3.46) as independent predictors of 14 day mortality. Combination therapy with at least two drugs displaying in vitro activity against the isolate was associated with lower mortality (OR, 0.52; 95% CI, 0.35–0.77), in particular in patients with BSIs, lung infections or high APACHE III scores and/or septic shock at infection onset. Combinations that included meropenem were associated with significantly higher survival rates when the KPC-Kp isolate had a meropenem MIC of ≤8 mg/L. Conclusions KPC-Kp infections are associated with high mortality. Treatment with two or more drugs displaying activity against the isolate improves survival, mainly in patients who are critically ill.

Sequence type 258 (ST258) is the most widespread multidrug resistant (MDR) Klebsiella pneumoniae strain worldwide. Here, we report the draft genome sequences of two colistin-resistant MDR K. pneumoniae ST258 clinical strains isolated from hospital patients in Italy. These strains are resistant to β-lactams, cephalosporins, fluoroquinolones, aminoglycosides, macrolides, tetracyclines, carbapenems, and colistin.

BACKGROUND: A risk score for invasive mold disease (IMD) in patients with hematological malignancies could facilitate patient screening and improve the targeted use of antifungal prophylaxis. METHODS: We retrospectively analyzed 1,709 hospital admissions of 840 patients with hematological malignancies (2005-2008) to collect data on 17 epidemiological and treatment-related risk factors for IMD. Multivariate regression was used to develop a weighted risk score based on independent risk factors associated with proven or probable IMD, which was prospectively validated during 1,746 hospital admissions of 855 patients from 2009-2012. RESULTS: Of the 17 candidate variables analyzed, 11 correlated with IMD by univariate analysis, but only 4 risk factors (neutropenia, lymphocytopenia or lymphocyte dysfunction in allogeneic hematopoietic stem cell transplant recipients, malignancy status, and prior IMD) were retained in the final multivariate model, resulting in a weighted risk score 0-13. A risk score of < 6 discriminated patients with low (< 1%) versus higher incidence rates (> 5%) of IMD, with a negative predictive value (NPV) of 0.99, (95% CI 0.98-0.99). During 2009-2012, patients with a calculated risk score at admission of < 6 had significantly lower 90-day incidence rates of IMD compared to patients with scores > 6 (0.9% vs. 10.6%, P

Here, we present the draft genome sequence of Klebsiella pneumoniae subsp. pneumoniae sequence type 512 (ST512) isolated during a KPC-producer outbreak. This strain is resistant to β-lactams, cephalosporins, fluoroquinolones, aminoglycosides, macrolides, tetracyclines, and carbapenems but susceptible to colistin. The ST512-K30BO genome is composed of 289 contigs for 5,392,844 bp with 56.9% G+C content.

Reduced susceptibility to carbapenems in Gram-negative pathogens is an emerging feature of the antibiotic-resistance phenomenom Reports about strains resistant to this class of antibiotics among Enterobacteriaceae, particularly in Klebsiella pneumoniae, are increasing.The aims of this study were to assess the incidence of Klebsiella pneumoniae with reduced susceptibility to carbapenems in Bologna area and to carry out the characterization of these strains.The study included isolates of K. pneumoniae that showed reduced susceptibility to carbapenems, as detected by an automated system (Vitek2, bioMérieux). Between January and May 2009, 26 strains were collected (mainly isolated from urinary samples).These isolates were tested for susceptibility to carbapenems by E-test, to define MIC values for meropenem and ertapenem. Moreover, to detect the production of metallo-beta lactamases (MBL) and carbapenemases (KPC) were respectively performed the Etest with imipenem and imipenem/EDTA (IPM-IPM/EDTA) and the modified Hodge test. Susceptibility assays performed by E-test showed that 25/26 strains were susceptible to meropenem, while for ertapenem 20/26 strains resulted resistant.The modified Hodge test was positive for 1 strain, while all the isolates were negative to the IPM-IPM/EDTA E-test.These results show that, as recently reported, the majority of strains of K. pneumoniae exhibiting reduced susceptibility to carbapenems, especially to ertapenem, are characterized by the production of ESBLs, which likely is associated with the loss of porins. On the other side, one strain was found to produce KPC and this finding confirms that the diffusion of carbapenemases producing K. pneumoniae has also to be considered in this geographic area.

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16(INK4A) (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.

A quantitative evaluation of p16 INK4A overexpression together with its topographical localization in the epithelium of cervical biopsies from non-neoplastic lesions and cervical intraepithelial neoplasias (CIN 1, 2, and 3) was obtained by the development of an objective and sensitive immunohistochemical assay with chemiluminescent detection (CL IHC assay). The cervical biopsy samples were also checked for the presence of human papillomavirus nucleic acids. The quantitative evaluation of p16 INK4A expression was performed by combining 2 parameters: (1) intensity of the chemiluminescent-positive signal in the epithelium and (2) percentage of epithelium interested by the overexpression of p16 INK4A, to obtain a p16 INK4A expression score. A cut-off value was determined by using the receiver-operator characteristic analysis to distinguish between low-grade and high-grade CIN. Quantitative data showed that both p16 INK4A expression parameters increased with worsening grades of CIN and, when combined to obtain the p16 INK4A expression score, they showed a sharp discrimination among different lesions. The differences between the average p16 score of CIN1 versus CIN2 (0.57 versus 1.05), of CIN1 versus CIN3 (0.57 versus 1.31) and of CIN1 versus CIN2/3 (0.57 versus 1.20) were statistically significant. The quantitative evaluation of p16 INK4A expression by CL IHC assay could be therefore an interesting adjuvant method to distinguish different CIN grades and to predict the risk of progression of early CIN lesions.

Objectives.: Cervical intraepithelial neoplasias (CIN) associated with high-risk (HR) human papillomavirus infection, in addition to HR-HPV typing need other viral marker testing to distinguish a subset of lesions with clinical relevant infections. This study has evaluated the significance of viral markers, such as viral load, physical status and E2/E6 ratio, to stratify HPV16 infected women at a single point in time for grade of cervical lesions. Methods.: One hundred sixty-six cytological specimens were selected from women with low (n = 72) and high (n = 94) grade squamous intraepithelial lesions (SIL), and positive to HPV16. All the 72 LSIL were CINI, 83 of the 94 HSIL were CINII/III and 11 SCC (Squamous Cervical Carcinoma). Cytological specimens were analysed by two different SYBR Green Real-time PCR assays (RT-PCR). Specific primers for both E2 and E6 viral genes and GAPDH cellular gene were designed to determine viral load, physical status and E2/E6 ratio. Results.: The viral load was significantly higher in HSIL than in LSIL. In CINI episomal DNA was prevalent (72.2%), mixed forms (episomal and integrated) were 27.8%, suggestive of an early integration of viral DNA into cellular genome, no pure integrated forms were detected. However in CINII/III mixed DNA forms were prevalent (73.5%). In SCC pure integrated DNA was prevalent (81.8%) in absence of episomal forms. E2/E6 ratio decreased significantly from CINI to CINII/III and SCC with a linear trend. The logistic regression analysis showed that viral load higher than 1.38 × 106 genome copies per 300ng of total DNA associated with E2/E6 ratio lower than 0.90 was highly significant in differentiating CINII/III versus CINI, while the only E2/E6 value lower than 0.17 was significant in differentiating SCC from CINI. Conclusions.: Viral load higher than 1.38 × 106 genome copies per 300ng of total DNA and E2/E6 ratio values allow HPV16 infected women with high grade cervical intraepithelial lesions to be recognized. © 2007 Elsevier Inc. All rights reserved.

Twenty-five patients with high-grade cervical lesions associated with HPV16 infection were studied at the time of surgical treatment and followed up after conization. Before surgical treatment, the following parameters were analyzed: (1) physical status of HPV16 DNA, (2) viral load, (3) cytological presentation confirmed by histological diagnosis, and (4) colposcopy. At the time of conization, (5) margin presentation, and (6) cone biopsy were evaluated. At each stage of the follow-up (7) physical status of HPV16 DNA, (8) viral load, (9) cytological test, and (10) colposcopy were repeated. The correlation between the different parameters was examined. Significant differences in the viral loads were observed between integrated and episomal forms and between the coexisting integrated/episomal forms and the only episomal form, while no statistically significant differences were observed between integrated and coexisting forms. At the first stage of follow-up, 11 of 25 patients analyzed (44%) were negative to HPV DNA cytology and colposcopy tests, 13 of the 25 (52%) were HPV16 DNA negative, 17 (68%) cytology negative, and 20 (80%) colposcopy negative. At the closing stage, 15 of 25 subjects (60%) were negative to all three tests, 16 of the 25 (64%) were HPV16 DNA negative, 19 (76%) cytology negative, and 20 colposcopy negative (80%). These observations suggest that in surgery treated patients the viral clearance at the closing stage of follow-up was unrelated to the HPV DNA physical status and to the viral load before treatment, but was associated significantly with the effectiveness of surgery treatment, in particular with margin cone presentation. J. Med. Virol. 78:494–500, 2006. © 2006 Wiley-Liss, Inc.

The presence and type of oncogenic papillomavirus (HPV) in classic warty/basaloid vulvar intraepithelial neoplasia and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma was investigated using three techniques, that is, histology, in situ hybridization, and PCR-ELISA. HPV typing was performed using in situ hybridization and PCR-ELISA. Differentiated vulvar intraepithelial neoplasia and invasive keratinizing vulvar squamous cell carcinoma proved completely negative for HPV by PCR-ELISA, in situ hybridization, and histologic examination, while in classic vulvar intraepithelial neoplasia, a HPV positivity of 66.1% was found. HPV 16 was the predominant type, with HPV 35, 33, and 52 types found rarely and sometimes together with HPV 16. PCR-ELISA proved to be the most suitable method to detect and type mucosal oncogenic HPVs. The absolute absence of HPV DNA in differentiated vulvar intraepithelial neoplasias and in keratinizing vulvar squamous cell carcinoma suggests a strong HPV-independent pathway of malignant progression to invasive carcinoma.

Summary Background Some studies have shown that cutaneous and mucosal melanoma biopsy specimens harbour human papillomavirus (HPV), suggesting that this virus may play a role in development and progression of the tumour. Objectives To investigate the presence of HPV DNA and the prevalence of different high-risk mucosal HPV genotypes in primary melanoma (PM) and in acquired dysplastic melanocytic naevi (ADMN). Methods Fifty-one PMs from 18 men and 33 women (median age 55·5 years), 33 ADMN from 15 men and 18 women (median age 35·1 years) and 20 control skin samples from nine men and 11 women (median age 43·5 years) were studied. All diagnoses were made after histological analysis. HPV DNA analysis was made using two different polymerase chain reaction–enzyme-linked immunosorbent assay (PCR–ELISA) methods, namely MY-PCR and GP-PCR. Results Using GP-PCR, mucosal HPVs were detected in 14 PMs (27%; P = 0·0166) and eight ADMN (24%; P = 0·0367), while with MY-PCR, mucosal HPVs were found in 11 PMs (22%; P = 0·04) and five ADMN (15%; P not significant). All control skin samples were negative for mucosal HPVs with both DNA amplification procedures. Conclusions Using our PCR–ELISA methods, the detection of mucosal high-risk HPV genotypes in 24% of precursor lesions (ADMN) and in 27% of PMs adds to the body of evidence indicating a colocalization of mucosal HPV and tumoral melanocytic pathologies.

A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based on a 384-well microtiter plate for detection and typing of oncogenic high- and low-risk human papillomavirus (HPV) in genital lesions is described. The assay relies on PCR consensus amplification, hybridization of the digoxigenin-labeled product by means of type-specific biotin-labeled oligoprobes immobilized on the streptavidin-coated wells of a 384-well microtiter plate, and quantification by means of a horseradish peroxidase-labeled antidigoxigenin antibody and chemiluminescence detection. The method provides semiquantitative information on the viral load, with a limit of detection of 10-50 DNA copies for HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 58, and 59 and high reproducibility (intraassay CV 7.5%, interassay CV 9.5%). Results obtained on 60 clinical samples were concordant with those obtained with a conventional PCR-enzyme-linked immunosorbent assay colorimetric assay. The 384 PCR-CLEIA method, which is amenable to automation, represents a fast and high-throughput method for detecting and typing HPV DNAs in screening programs and evaluating the viral load.

We performed a quasi-experimental study of a multifaceted infection control programme for reducing carbapenem-resistant Enterobacteriaceae (CRE) transmission and bloodstream infections (BSIs) in a 1420-bed university-affiliated teaching hospital during 2010–2014, with 30 months of follow-up. The programme consisted of the following: (a) rectal swab cultures were performed in all patients admitted to high-risk units (intensive-care units, transplantation, and haematology) to screen for CRE carriage, or for any room-mates of CRE-positive patients in other units; (b) cohorting of carriers, managed with strict contact precautions; (c) intensification of education, cleaning and hand-washing programmes; and (d) promotion of an antibiotic stewardship programme carbapenem-sparing regimen. The 30-month incidence rates of CRE-positive rectal cultures and BSIs were analysed with Poisson regression. Following the intervention, the incidence rate of CRE BSI (risk reduction 0.96, 95% CI 0.92–0.99, p 0.03) and CRE colonization (risk reduction 0.96, 95% CI 0.95–0.97, p