Phylogenetic Analysis of Multi-Drug Resistant Klebsiella pneumoniae Strains From Duodenoscope Biofilm: Microbiological Surveillance and Reprocessing Improvements for Infection Prevention.

Duodenoscopes have been described as potential vehicles of patient-to-patient transmission of multi-drug resistant organisms. Carbapenem-resistant Enterobacteriaceae duodenoscope related infections have been described by the Center for Disease Control and the US Food and Drug Administration consequently to outbreaks occurring in the United States. These evidences suggested that improved microbiological surveillance and endoscope design optimization could represent valid tools to improve infection control. At this aim, in this study an example of duodenoscope microbiological surveillance and reprocessing improvement analyzing strains component of bacterial biofilm by phylogenetic analysis has been proposed. From September 2016 to December 2017, duodenoscope instruments were subjected to microbial surveillance by post-reprocessing cultures of liquid collected by internal channels of instruments after injection and aspiration cycles and membrane filtration. During surveillance seventeen Klebsiella pneumoniae, of which 10/17 (58.8%) MDR and KPC strains were collected from duodenoscope instruments plus one MDR Klebsiella pneumoniae strain from the rectal swab performed before ERCP procedure in an inpatient. The surveillance allowed evidencing potential failure of reprocessing procedure and performing consequent reprocessing improvements including the contaminated instruments quarantine until their negativity. Phylogenetic analysis of whole genome sequence of duodenoscope strains plus inpatients MDR strains, showed intermixing between duodenoscopes and inpatients, as evidenced by minimum spanning tree and time-scale Maximum Clade Credibility tree. In minimum spanning tree, three groups have been evidenced. Group I including Klebsiella pneumoniae strains, isolated from inpatients before microbiological surveillance adoption; group II including intermixed Klebsiella pneumoniae strains isolated from inpatients and Klebsiella pneumoniae strains isolated from duedonoscopes and group III including Klebsiella pneumoniae strains exclusively from duedonoscope instruments. In the Maximum Credibility Tree, a statistically supported cluster including two Klebsiella pneumoniae strains from duedonoscope instruments and one strains isolated from an inpatient was showed. From the first microbiologic surveillance performed on September 2016 and after the reprocessing improvement adoption, none MDR or susceptible Klebsiella pneumoniae strain was isolated in the following surveillance periods. In conclusion, these results should encourage hospital board to perform microbiological surveillance of duodenoscopes as well as of patients, by rectal swabs culture, and rapid molecular testing for antimicrobial resistance before any endoscopic invasive procedure.

Multi-drug resistant Pseudomonas aeruginosa nosocomial strains: Molecular epidemiology and evolution.

Pseudomonas aeruginosa causes a wide variety of nosocomial infections. In the study, phylogenetic, selective pressure analysis and homology modelling were applied to oprD efflux pump gene with the aim to characterize multi-drug resistant strains circulating in the nosocomial setting, their transmission dynamics and ongoing evolution. One hundred ninety-three consecutive inpatients with Pseudomonas aeruginosa infection were enrolled at the University Campus Bio-Medico of Rome, between January 2015 and December 2016. oprD gene was sequenced in 20 nosocomial multi-drug resistant P. aeruginosa strains. Phylogeographic, selective pressure, residue conservation analysis and homology modelling were performed. Clinical epidemiological data were extracted from patient medical records. Multi-drug resistant strains accounted for the 36% of total strains and were responsible of 20 cases of nosocomial infections. P. aeruginosa infections occurred prevalently in the West area, especially at the location IIIW and in the Geriatric ward. The time of the most recent common ancestor indicated that strains could have been introduced in the hospital since the end of the year 2009 with the most probable location in general surgery ward. By selective pressure analysis, 29 positions under diversifying selection have been identified and mapped onto the OprD model. Most of the observed residue substitutions are predicted to be destabilizing and some of them occurred in the Loops 2 and 3 that are involved in solute selection and carbapenem susceptibility. The molecular and evolutionary analysis of Multi-drug resistant strains circulating in the nosocomial setting may provide useful insights into the epidemiology and the mechanisms leading to resistance, contributing to infection control improvement.

A case of methicillin-resistant Staphylococcus aureus wound infection: phylogenetic analysis to establish if nosocomial or community acquired.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is rapidly increasing in both hospital and community settings. A 71-year-old man admitted at the Department of Orthopaedics and Trauma Surgery, University Campus Bio-Medico of Rome, with MRSA wound infection consequent to orthopedic surgery was studied and the MRSA transmission evaluated by phylogenetic analysis.

The role of procalcitonin in the diagnosis of bacterial infection after major abdominal surgery: Advantage from daily measurement.

Postsurgical infections represent an important cause of morbidity after abdominal surgery. The microbiological diagnosis is not achieved in at least 30% of culture with consequent worsening of patient outcome. In this study, procalcitonin measurement, during the first 3 days after abdominal surgery, has been evaluated for the early diagnosis of postsurgical infection.Ninety consecutive patients subjected to major abdominal surgery at the University Campus Bio-Medico of Rome, have been included. PCT concentrations were measured by time-resolved amplified cryptate emission (TRACE) assay at admission and at the first, second, and third day after surgery. PCT levels were compared using the Mann-Whitney test and by ANOVA test for variance analysis. Receiver operating characteristic (ROC) analysis was performed to define the diagnostic ability of PCT in case of postsurgical infections.PCT values resulted significantly different between patients developing or not developing postsurgical infections. PCT >1.0 ng/mL at first or second day after surgery and >0.5 ng/mL at third day resulted diagnostic for infectious complication, whereas a value <0.5 ng/mL at the fifth day after surgery was useful for early and safety discharge of patients.In conclusion, PCT daily measurement could represent a useful diagnostic tool improving health care in the postsurgical period following major abdominal surgery and should be recommended.

Susceptibility to measles in migrant population: implication for policy makers.

BACKGROUND: Despite a large measles outbreak is taking place in WHO European region, currently no data are available on measles immunization coverage in the asylum seeker and migrants hosted in this area. METHODS: Two hundred and fifty-six migrants upon their arrival in Italy on March, April and May 2016 were screened for measles virus IgG antibodies by chemiluminescence immunoassay (Liaison XL analyzer, Diasorin, Italy). The virus susceptibility in this cohort, the differences between the official country reported and the observed measles immunization coverage and the impact of current measles outbreak on the asylum seekers hosted in the largest Asylum Seeker centres of Italy, were evaluated. RESULTS: The prevalence of subjects with positive result for measles IgG antibodies ranged between 79.9% and 100%. In Senegal, Mali, Nigeria, Pakistan and Bangladesh, the measles IgG seroprevalence observed was greater than the vaccinal coverage reported by WHO after I dose of vaccine. Based on data regarding the II dose coverage, the ASs population presented a seroprevalence greater to that expected. CONCLUSION: On the basis of the results obtained, extraordinary screening and vaccination campaigns in the migrant population, especially in the course of large outbreaks, could represent a resource to reach an adequate measles immunization coverage and to control this infectious disease.

Sentinel surveillance data from Eritrean migrants in Italy: The theory of "Healthy Migrants".

BACKGROUND: Eritrean migrants accounted for the majority of people who drowned in the Mediterranean this year. Recently, data deficit about international migration has been reported. METHODS: One hundred and thirty-three Eritrean migrants, upon their arrival in Italy, were accommodated at the asylum seekers center of Castelnuovo di Porto, together with 25 sanitary workers, and received microbiological surveillance at the Clinical Pathology and Microbiology Unit of the University Campus Bio-Medico of Rome. RESULTS: Gram-negative bacteria, mostly Klebsiella pneumoniae and Klebsiella oxytoca, were frequently recovered in surveillance swabs. Gram-positive bacteria, represented by Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus dysgalactiae and Staphylococcus aureus, were also isolated in surveillance swabs. In migrants, polymicrobial swabs were recovered at high frequency, and unusual microorganisms, rarely found in our country, were isolated. CONCLUSION: The polymicrobial colonization and the high prevalence of gram-negative bacteria represent the baseline characteristics of this migrant population from Eritrea. The presence of unusual microorganisms can be potentially pathogenic to asymptomatic carriers at risk of developing clinical disease if immunosuppressed or subjected to invasive procedures. In conclusion, active microbiological surveillance can represent an advantage for the host country in terms of data collection and by tracing unusual or resistant microorganisms by monitoring migrants' health status.

Procalcitonin and MR-Proadrenomedullin Combination with SOFA and qSOFA Scores for Sepsis Diagnosis and Prognosis: A Diagnostic Algorithm.

PURPOSE: The third Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defined sepsis as an organ dysfunction consequent to infection. A Sequential Organ Failure Assessment (SOFA) score at least 2 identifies sepsis. In this study, procalcitonin (PCT) and midregional pro-adrenomedullin (MR-proADM) were evaluated along with SOFA and quick SOFA (qSOFA) scores in patients with sepsis or septic shock. METHODS: A total of 109 septic patients and 50 patients with noninfectious disease admitted at the Department of Internal Medicine and General Surgery of the University Hospital Campus Bio-Medico of Rome were enrolled. PCT and MR-proADM were measured with immunoluminometric assays (Brahms, Hennigsdorf, Germany). Data were analyzed with receiver-operating characteristic (ROC) curve analysis, likelihood ratios, and Mann-Whitney U test using MedCalc 11.6.1.0 package. RESULTS: At ROC curve analysis, PCT showed the highest area under the curve and positive likelihood ratio values of 27.42 in sepsis and 43.62 in septic shock. MR-proADM and SOFA score showed a comparable performance. In septic shock, lactate showed the most accurate diagnostic ability. In sepsis, the best combination was PCT with MR-proADM with a posttest probability of 0.988. Based upon these results, an algorithm for sepsis and septic shock diagnosis has been developed. MR-proADM, SOFA, and qSOFA scores significantly discriminated survivors from nonsurvivors. CONCLUSIONS: PCT and MR-proADM test combination represent a good tool in sepsis diagnosis and prognosis suggesting their inclusion in the diagnostic algorithm besides SOFA and qSOFA scores. Furthermore, MR-proADM as marker of organ dysfunction, with a turn around time of about 30 min, has the advantage to be more objective and rapid than SOFA score.

Visceral leishmaniasis triggering an adult-onset Still's disease: a unique case.

Adult-onset Still's disease (AOSD) due to visceral leishmaniasis (VL) has not been previously reported. This case report analyzes a single episode of AOSD probably due to a visceral leishmaniasis successfully treated with pentamidine isethionate and prednisone.

Multi-drug resistant Klebsiella pneumoniae strains circulating in hospital setting: whole-genome sequencing and Bayesian phylogenetic analysis for outbreak investigations.

Carbapenems resistant Enterobacteriaceae infections are increasing worldwide representing an emerging public health problem. The application of phylogenetic and phylodynamic analyses to bacterial whole genome sequencing (WGS) data have become essential in the epidemiological surveillance of multi-drug resistant nosocomial pathogens. Between January 2012 and February 2013, twenty-one multi-drug resistant K. pneumoniae strains, were collected from patients hospitalized among different wards of the University Hospital Campus Bio-Medico. Epidemiological contact tracing of patients and Bayesian phylogenetic analysis of bacterial WGS data were used to investigate the evolution and spatial dispersion of K. pneumoniae in support of hospital infection control. The epidemic curve of incident K. pneumoniae cases showed a bimodal distribution of cases with two peaks separated by 46 days between November 2012 and January 2013. The time-scaled phylogeny suggested that K. pneumoniae strains isolated during the study period may have been introduced into the hospital setting as early as 2007. Moreover, the phylogeny showed two different epidemic introductions in 2008 and 2009. Bayesian genomic epidemiology is a powerful tool that promises to improve the surveillance and control of multi-drug resistant pathogens in an effort to develop effective infection prevention in healthcare settings or constant strains reintroduction.

The Liver as Another Possible Target Organ for Bacillus cereus Infection.

A case of liver abscess due to Bacillus cereus infection in an immunocompetent 59-year-old man is reported. Percutaneous drainage and antimicrobial therapy, with vancomycin and levofloxacin afterwards, have been demonstrated to be an appropriate treatment, leading to clinical and radiological cure.

Klebsiella pneumoniae blaKPC-3 nosocomial epidemic: Bayesian and evolutionary analysis.

K. pneumoniae isolates carrying blaKPC-3 gene were collected to perform Bayesian phylogenetic and selective pressure analysis and to apply homology modeling to the KPC-3 protein. A dataset of 44 blakpc-3 gene sequences from clinical isolates of K. pneumoniae was used for Bayesian phylogenetic, selective pressure analysis and homology modeling. The mean evolutionary rate for blakpc-3 gene was 2.67×10-3 substitution/site/year (95% HPD: 3.4×10-4-5.59×10-3). The root of the Bayesian tree dated back to the year 2011 (95% HPD: 2007-2012). Two main clades (I and II) were identified. The population dynamics analysis showed an exponential growth from 2011 to 2013 and the reaching of a plateau. The phylogeographic reconstruction showed that the root of the tree had a probable common ancestor in the general surgery ward. Selective pressure analysis revealed twelve positively selected sites. Structural analysis of KPC-3 protein predicted that the amino acid mutations are destabilizing for the protein and could alter the substrate specificity. Phylogenetic analysis and homology modeling of blaKPC-3 gene could represent a useful tool to follow KPC spread in nosocomial setting and to evidence amino acid substitutions altering the substrate specificity.

Phylogenetic Analysis of Human Immunodeficiency Virus Type 2 Group B.

CONTEXT: Human immunodeficiency virus type 2 (HIV-2) infections are mainly restricted to West Africa; however, in the recent years, the prevalence of HIV-2 is a growing concern in some European countries and the Southwestern region of India. Despite the presence of different HIV-2 groups, only A and B Groups have established human-to-human transmission chains. AIMS: This work aimed to evaluate the phylogeographic inference of HIV-2 Group B worldwide to estimate their data of origin and the population dynamics. MATERIALS AND METHODS: The evolutionary rates, the demographic history for HIV-2 Group B dataset, and the phylogeographic analysis were estimated using a Bayesian approach. The viral gene flow analysis was used to count viral gene out/in flow among different locations. RESULTS: The root of the Bayesian maximum clade credibility tree of HIV-2 Group B dated back to 1957. The demographic history of HIV-2 Group B showed that the epidemic remained constant up to 1970 when started an exponential growth. From 1985 to early 2000s, the epidemic reached a plateau, and then it was characterized by two bottlenecks and a new plateau at the end of 2000s. Phylogeographic reconstruction showed that the most probable location for the root of the tree was Ghana. Regarding the viral gene flow of HIV-2 Group B, the only observed viral gene flow was from Africa to France, Belgium, and Luxembourg. CONCLUSIONS: The study gives insights into the origin, history, and phylogeography of HIV-2 Group B epidemic. The growing number of infections of HIV-2 worldwide indicates the need for strengthening surveillance.

Phylogeny of Murray Valley encephalitis virus in Australia and Papua New Guinea.

OBJECTIVE: To study the genetic diversity of Murray Valley encephalitis virus (MVEV) in Australia and Papua New Guinea. METHODS: MVEV envelope gene sequences were aligned using Clustal X and manual editing was performed with Bioedit. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Maximum likelihood analysis was performed using PhyML. The phylogenetic signal of the dataset was investigated by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST. RESULTS: The phylogenetic trees showed two main clades. The clade Ⅰ including eight strains isolated from West Australia. The clade Ⅱ was characterized by at least four epidemic entries, three of which localized in Northern West Australia and one in Papua New Guinea. The estimated mean evolutionary rate value of the MVEV envelope gene was 0.407 × 10(-3) substitution/site/year (95% HPD: 0.623 × 10(-4)-0.780 × 10(-3)). Population dynamics defines a relative constant population until the year 2000, when a reduction occurred, probably due to a bottleneck. CONCLUSIONS: This study has been useful in supporting the probable connection between climate changes and viral evolution also by the vector point of view; multidisciplinary monitoring studies are important to prevent new viral epidemics inside and outside new endemic areas.

Role of Neutrophil Gelatinase-Associated Lipocalin in the Diagnosis and Early Treatment of Acute Kidney Injury in a Case Series of Patients with Acute Decompensated Heart Failure: A Case Series.

Patients with acute decompensated heart failure (ADHF) frequently develop worsening in renal function until Acute Kidney Injury (AKI). The use of kidney injury biomarkers could be useful in the early diagnosis of AKI. In the present study, the role of the neutrophil gelatinase-associated lipocalin (NGAL), compared to the standard creatinine, in ADHF patients, was analyzed to evaluate if an early treatment could affect the outcome. A case series of 24 ADHF patients was enrolled and patients randomly divided in two groups (Group A and Group B). In Group A, NGAL, creatinine, and eGFR were measured, while in Group B, creatinine and eGFR alone were measured. NGAL was measured by turbidimetric immunoassay and creatinine using an enzymatic spectrophotometric method. In presence of AKI, creatinine increase and eGFR decrease were significantly lower in Group A than in Group B, whereas in absence of AKI the difference between the two groups was not significant. Hospitalization stay was significantly lower in Group A (receiving early treatment based on NGAL) than in Group B. In ADHF patients, plasma NGAL in combination with creatinine was superior to the standard creatinine in the diagnosis and early treatment of AKI with a better outcome and a decreased hospital stay.

Unusual microorganisms and antimicrobial resistances in a group of Syrian migrants: Sentinel surveillance data from an asylum seekers centre in Italy.

BACKGROUND: Three years of civil war in Syria have caused death and increase of communicable diseases. The suffering population has been forced to migrate creating a fertile condition for epidemic spread of infection within the refugee camps. METHODS: Forty-eight Syrian migrants, upon their arrival in Italy, were accommodated at the asylum seekers centre of Castelnuovo di Porto. They received a physical examination and were subjected to microbiological surveillance by blood, rectal, pharyngeal and nasal swabs collection and delivering to the Clinical Pathology and Microbiology Laboratory of the University Campus Bio-Medico of Rome. RESULTS: All refugees resulted negative for HBV, HCV and HIV infections. In swabs a large number of unusual gram-negative bacteria species were isolated, such as Pseudomonas putida, Pseudomonas monteilii, Pseudomonas fulva, Pseudomonas moselii, Aeromonas veronii, Aeromonas caviae, Aeromonas hydrophila, Acinteobacter guilloviae, Acinteobacter lowffii; Acinetobacter johnsonii; Acinteobacter tjernbergae; Pantoea agglomerans; Pantoea calida. Among isolates, strains resistant to carbapenems, ESBL producers and methicillin resistant were found. CONCLUSIONS: The microbiological surveillance performed represents a useful action to understand refugees health status and to trace unusual microorganisms movement even carriers of antimicrobial resistance during migrants traveling.

Procalcitonin, MR-Proadrenomedullin, and Cytokines Measurement in Sepsis Diagnosis: Advantages from Test Combination.

BACKGROUND: Elevated cytokines levels correlate with sepsis severity and mortality but their role in the diagnosis is controversial, whereas Procalcitonin (PCT) has been largely used. Recently, the mid-regional proadrenomedullin (MR-proADM) has been combined with PCT for diagnosis optimization. In this study the combined measurement of PCT, MR-proADM, and cytokines in patients with sepsis was evaluated. METHODS: One hundred and four septic patients and 101 controls were enrolled. Receiver operating characteristic (ROC) analysis and multiple logistic regression were used to evaluate applicant markers for sepsis diagnosis. Markers with best Odds Ratio (OR) were combined, and the posttest probability and a composite score were computed. RESULTS: Based upon ROC curves analysis, PCT, MR-proADM, IL-6, IL-10, TNF-α, and MCP-1 were considered applicant for sepsis diagnosis. Among these PCT, MR-proADM , IL-6, and TNF-α showed the best OR. A better posttest probability was found with the combination of PCT with MR-proADM and PCT with IL-6 or TNF-α compared to the single marker. A composite score of PCT, MR-proADM, and TNF-α showed the best ROC curve in the early diagnosis of sepsis. CONCLUSION: The combination of PCT with other markers should expedite diagnosis and treatment of sepsis optimizing clinical management.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and Bayesian phylogenetic analysis to characterize Candida clinical isolates.

Clinical Candida isolates from two different hospitals in Rome were identified and clustered by MALDI-TOF MS system and their origin and evolution estimated by Bayesian phylogenetic analysis. The different species of Candida were correctly identified and clustered separately, confirming the ability of these techniques to discriminate between different Candida species. Focusing MALDI-TOF analysis on a single Candida species, Candida albicans and Candida parapsilosis strains clustered differently for hospital setting as well as for period of isolation than Candida glabrata and Candida tropicalis isolates. The evolutionary rates of C. albicans and C. parapsilosis (1.93×10(-2) and 1.17×10(-2)substitutions/site/year, respectively) were in agreement with a higher rate of mutation of these species, even in a narrow period, than what was observed in C. glabrata and C. tropicalis strains (6.99×10(-4) and 7.52×10(-3)substitutions/site/year, respectively). C. albicans resulted as the species with the highest between and within clades genetic distance values in agreement with the temporal-related clustering found by MALDI-TOF and the high evolutionary rate 1.93×10(-2)substitutions/site/year.

MALDI-TOF mass spectrometry and blakpc gene phylogenetic analysis of an outbreak of carbapenem-resistant K. pneumoniae strains.

Carbapenem-resistant Klebsiella pneumoniae isolates are an important cause of nosocomial infections. This study evaluated a rapid cost-saving method based on MALDI-TOF technology, was and compared it with phenotypic, genotypic and epidemiological data for characterization of KPC-Kp strains consecutively isolated during a supposed outbreak. Twenty-five consecutive KPC Klebsiella pneumoniae isolates were identified and clustered by the MALDI Biotyper (Bruker, Daltonics). To display and rank the variance within a data set, principal component analysis (PCA) was performed. ClinProTools models were generated to investigate the highest sum of recognition capability and cross-validation. A Class dendrogram of isolates was constructed using ClinproTool. MLST was performed sequencing gapA, infB, mdh, pgi, rpoB, phoE and tonB genes. blakpc and cps genes were typed. Phylogenetic analysis and genetic distance of the KPC gene were performed using the MEGA6 software. PCA analysis defined two clusters, I and II, which were identified in a dendrogram by both temporal split and different antimicrobial susceptibility profiles. These clusters were composed mostly of strains of the same sequence type (ST512), the most prevalent ST in Italy, and the same cps (type 2). In cluster II, blakpc genotype resulted more variable than in cluster I. Phylogenetic analysis confirmed the genetic diversity in both clusters supported by the epidemiological data. Our study confirms that MALDI-TOF can be a rapid and cost-saving method for epidemiological clustering of KPC K. pneumoniae isolates and its association with blakpc genotyping represents a reliable method to recognize possible clonal strains in nosocomial settings.

Turnaround time of positive blood cultures after the introduction of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

A comparative evaluation of the turnaround time (TAT) of positive blood culture before and after matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) introduction in the laboratory routine was performed. A total of 643 positive blood cultures, of which 310 before and 333 after MALDI-TOF technique introduction, were collected. In the post MALDI-TOF period, blood culture median TAT decreased from 73.53 hours to 71.73 for Gram-positive, from 64.09 hours to 63.59 for Gram-negative and from 115.7 hours to 47.62 for anaerobes. MALDI-TOF significantly decreased the TAT of anaerobes, for which antimicrobial susceptibility test is not routinely performed. Furthermore, the major advantage of MALDI-TOF introduction was the decrease of the time for pathogen identification (TID) independently from the species with an improvement of 93% for Gram-positive, 86% for Gram-negative and 95% for anaerobes. In addition, high species-level identification rates and cost savings than conventional methods were achieved after MALDI-TOF introduction.