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Objectives: To assess the incidence of colonization and infection with carbapenemase producing Enterobacteriaceae (CPE) and carbapenem resistant Acinetobacter baumannii (CR-Ab) in the ICUs of our city hospitals before and during COVID-19 pandemic. Methods: Multicentre before-after cross sectional study to compare the rates of colonization and infection with CPE and/or CR-Ab in two study periods, period 1 (Jan-Apr 2019) and period 2 (Jan-Apr 2020). Incidence rate ratios (IRR) and 95% CI of weekly colonization and infection rates for each period were compared for the two study periods with Poisson regression. Weekly trends in the incidence of colonization or infection for each study period were summarized using local weighted (Loess) regression. Results: There was no significant change in either IRR and weekly trend in CPE colonization and infection during the two study periods. A shift from KPC to other CPE mechanisms (OXA-48 and VIM) was observed during period 2. Compared to period 1, during period 2 the IRR of colonization and infection with CR-Ab increased of 7.5 and 5.5-fold, respectively. Genome sequencing showed that all CR-Ab strains belonged to the CC92/IC2 clonal lineage. Clinical strains clustered closely into a single monophyletic group in one of the three centres, whereas segregated in two different clusters in the other two centres, strongly appoints for the occurrence of horizontal transmission. Conclusion: Our findings remark the need of pursuing infection control activities targeted against the spread of antimicrobial resistance intra and inter hospitals during COVID-19 pandemic, and if necessary re-modulating them according to the new organizational structures imposed by the pandemic.

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.

The burden of anaerobic bacteremia ranged from 2 to 14% of patients with positive BCs among centers, reflecting different hospital sizes and medical activities. Cutibacterium acnes was the most frequently recovered anaerobe in some centers, suggesting different medical activities / BCs laboratory procedures. Antimicrobial resistance rate in the B. fragilis group is worrisome. It may be associated with treatment failures and requires continuous surveillance.

The burden and epidemiology of anaerobic bacteremia is poorly described. The aim of the study was to characterize the incidence and evolution trends of anaerobic bacteremia in Europe and neighbouring countries in 2012 and 2018 both in terms of microbial epidemiology and antimicrobial resistance. Retrospective multicenter study: 17 laboratory hospitals from 12 countries Results data: total number of adult patients with anaerobes, patients’ age, gender and hospitalization ward, laboratory methods, identification of isolates and antimicrobial susceptibility results were analysed. Burden of anaerobic bacteremia • The mean frequency of patients with anaerobes in BCs was 5% of patients, with important variations according to centers (2% to 14%), probably reflecting their medical activity. The reported incidence moderately increased between 2012 and 2018. • These patients were frequently hospitalized in emergency wards and intensive care units (25% and 22% of isolates), followed by medicine, digestive surgery and oncology (6.7%) Epidemiology of anaerobes in blood cultures • 50% of anaerobic BCs were obtained from emergency and Intensive care units, demonstrating their clinical importance • 4 genera represented 75% of isolates : Bacteroides, Cutibacterium, Clostridium and Fusobacterium • Cutibacterium acnes frequency significantly varied among centers, suggesting variations in Laboratory management of BCs (incubation time). Antimicrobial resistance reported rates • Antimicrobial resistance rates varied according to genera and remained stable between 2012 and 2018. • Clindamycin resistance was the most frequently observed phenotype, predominating in Bacteroides spp, Clostridium spp and anaerobic Gram positive rods. • 10% of Bacteroides spp were resistant to piperacillin- tazobactam. Carbapenems and metronidazole resistance rate were below 5%. • Metronidazole resistance was frequently reported in anaerobic Gram Positive cocci

Background: The global spread of carbapenemase-producing Enterobacteriaceae is of great concern to health services worldwide. In particular, multi-drug resistant Klebsiella pneumoniae (KP) harbouring KPC enzymes has been causing epidemics of international proportions. The analysis of the metabolic profiles of KP strains could represent an intriguing approach to obtain useful information to set up new diagnostic methods and to develop new antimicrobial strategies. The aim of this study was to characterize the metabolic profiles of several KP strains, characterized by specific resistance pattern. Materials/methods: A total of 59 KP strains, isolated from clinical samples submitted to the Microbiology Unit of S.Orsola-Malpighi Hospital of Bologna (Italy) for diagnostic procedures, were included in the study. In particular, 27 carbapenemase-negative and 32 carbapenemase-positive strains were analyzed. For each strain, bacteria were grown overnight in Mueller-Hinton (MH) broth. Afterwards, the suspension was standardized (2.8 McF) and centrifuged to separate the cell pellet from the supernatant. The metabolomic analysis was performed by means of 1H-NMR spectroscopy analysis (Avance III Spectrometer; Bruker), starting from 700 μL of filtered supernatants (‘external metabolome’) and 100 μL of cell lysates (‘internal metabolome’). The same protocol was used to study the metabolic profile of 8 KP strains (4 wild-type and 4 KPCproducing) under ‘antibiotic-stress’, allowing the bacteria to grow in MH broth with a meropenem concentration corresponding to 1/8 of the MIC value.Results: A total of 44 and 32 molecules, mainly belonging to organic acids, amino acids and alcohols, were detected in the external and the internal metabolome, respectively. In basal conditions, we found 6 metabolites (acetate, isobutyrate, lysine, phenylacetate, hydroxybutyrate) whose concentration was significantly different between carbapenemase-positive and carbapenemase-negative KP strains. TheTable shows the molecules that differed between carbapenemase-positive and carbapenemase-negative strains under ‘meropenem-stress’.Conclusions: The metabolomic analysis allows to detect molecules that significantly differed between carbapenemase-positive and carbapenemase-negative KP strains, both in basal conditions and under ‘antibiotic stress’.

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.

INTRODUZIONE: Negli ultimi anni si è assistito ad un notevole aumento dei fenomeni di resistenza ai farmaci carbapenemici in ceppi di Klebsiella pneumoniae (KP), per lo più mediati dalla produzione di carbapenemasi di tipo KPC. Lo studio del profilo metabolico di questi microorganismi rappresenta un approccio innovativo in grado di fornire informazioni utili per la messa a punto di nuove metodiche diagnostiche e lo sviluppo di nuovi agenti terapeutici. Lo scopo del presente lavoro è stato quello di valutare il profilo metabolico di diversi ceppi di KP, caratterizzati da specifici fenotipi di resistenza, mediante spettrometria di risonanza magnetica nucleare. METODI: Sono stati inclusi nello studio 59 ceppi di KP, di cui 27 non-produttori di carbapenemasi (16 wild-type e 11 produttori di ESBL) e 32 produttori di carbapenemasi (24 KPC, 4 MBL e 4 OXA-48). I ceppi sono stati isolati da vari campioni clinici provenienti da pazienti ricoverati presso il Policlinico Sant’Orsola-Malpighi di Bologna. Tramite micro-diluizione in brodo secondo linee guida EUCAST, sono state calcolate le minime concentrazioni inibenti (MIC) del meropenem. Dopo una crescita O/N in brodo Mueller-Hinton (MH), la sospensione batterica di ogni ceppo è stata standardizzata a 2.8 McF ed è stata centrifugata al fine di separare il pellet batterico dal surnatante. Il surnatante è stato filtrato e congelato, fino al momento dell’analisi metabolica, mentre i metaboliti intracellulari sono stati estratti processando il pellet con metanolo freddo. L’analisi metabolomica è stata condotta a partire da 700 μL di surnatante e da 100 μL di lisato intracellulare, tramite spettroscopia di risonanza magnetica nucleare (1H-NMR), con spettrometro AVANCE III (Bruker). Lo stesso protocollo è stato inoltre utilizzato per studiare il profilo metabolico di 8 ceppi di KP (4 wild-type e 4 KPC produttori) in condizioni di ‘stress’, facendo crescere i ceppi in presenza di meropenem ad una concentrazione pari a 1/8 della MIC. RISULTATI: I risultati preliminari evidenziano la presenza di differenze nel profilo metabolico fra ceppi produttori e non produttori di carbapenemasi. Per i ceppi non-carbapenemasi produttori le MIC del meropenem erano comprese tra 0.008 e 0.25 mg/L, mentre per i ceppi produttori variavano tra 0.5 e 512 mg/L. In caso di ‘stress’ indotto da concentrazioni subletali di meropenem, emerge una maggior ‘perturbazione metabolica’ nei ceppi KPC-produttori. CONCLUSIONI: Lo studio metabolomico permetterà di ampliare la conoscenza delle basi fisiologiche della cellula batterica e dei meccanismi di virulenza associati. Inoltre, l’identificazione di biomarker metabolici specifici potrebbe aprire nuovi scenari per l’approccio diagnostico e terapeutico delle infezioni da ceppi di KP produttori di carbapenemasi

Objectives. Acute rheumatic fever (ARF) is a post-infectious non-suppurative condition resulting from an autoimmune response to a Group A streptococcus (GAS) pharyngeal infection. In the last decades, the incidence of ARF showed a significant decrease in industrialized Countries. However, sporadic outbreaks have been reported in combination with the reappearance of certains emm-types of GAS. Here, we report a case of ARF occurred in a child from a primary school in Bologna, North of Italy. In addition, we describe the epidemiological investigation of GAS strains isolated from pharyngeal swabs collected during a surveillance program conducted in his classroom. Methods. In November 2012, a 11-year-old previously healthy white boy suffered for a GAS pharyngitis and was treated with clarithromycin for 10-days. In February 2013, he complained about left knee joint pain and retrosternal pain and was admitted to the hospital since a diagnosis of ARF was suspected. The patient underwent a complete clinical examination, an echocardiography and an electrocardiography (ECG). Several blood samples were taken to perform white blood cell count (WBC), antistreptolysine O titer (ASO) and inflammation markers. A pharyngeal swab was collected for GAS investigation. A surveillance program, based on pharyngeal swabs screening, was started in the classroom since case definition of ARF. Pharyngeal swabs were seeded on blood agar plates and β-haemolytic colonies were identified with mass-spectrometry (MALDI-TOF). In case of GAS isolation, antibiogram was performed using MICroSTREP plus1 panel (Microscan). Susceptibility was determined according to EUCAST criteria. Emm-type of each GAS isolates was determined by PCR and sequencing. Streptococcal pyrogenic exotoxin (spe) profile of each isolate, were performed by multiplex PCR Results. During hospitalization, echocardiography showed a mild mitral regurgitation. ECG revealed a first-degree atrioventricular block (PR=200-440 ms). The cardiac auscultation highlighted a faint systolic murmur. Blood tests showed an elevated ASO (2264 U/L) and inflammation markers and a mild leucocytosis (18.070/mmc). According to Jones criteria a diagnosis of ARF was established. A GAS strain showing a mucoid phenotype was isolated from pharyngeal swab of ARF case and the typing analysis conducted on it identified the presence of emm18 gene. Superantigen profile showed the presence of speA, speC, speG, speL, speM genes. Seven out of 24 pharyngeal swabs collected for surveillance resulted positive for GAS. Detailed microbiological characteristics are shown in table 1. Patient was given a therapy with corticosteroids for 21 days and benzilpenicillin (1.200.000 U im), with a gradual resolution of symptoms. Pharyngeal swab culture were negative after 2 weeks of hospitalization. Conclusion. Our case highlights that ARF has not disappeared in developed Countries. Moreover, as observed in our experience, a high circulation of certain mucoid GAS strains, like emm-type 18, could be a risk factor for ARF development.

INTRODUZIONE Le resistenza ai carbapenemi negli enterobatteri rappresenta ad oggi la principale problematica di antibiotico-resistenza. Negli ultimi anni nel contesto epidemiologico italiano si è osservata una rapida diffusione di microrganismi con queste caratteristiche, legata principalmente a Klebsiella pneumoniae produttrice di carbapenemasi KPC. Una rapida e corretta definizione della produzione di carbapenemasi riveste notevoli implicazioni sia da un punto di vista clinico che epidemiologico per la necessità di mettere in atto il più precocemente possibile una terapia antibiotica appropriata e le idonee misure preventive. In questo lavoro abbiamo valutato la performance analitica di un saggio di LC-MS (Liquid Chromatography Mass Spectrometry) nel determinare la produzione di carbapenemasi in isolati di enterobatteri. MATERIALI E METODI Sono stati selezionati per lo studio 48 isolati clinici (40 K.pneumoniae, 4 E.coli, 4 C.freundii) precedentemente caratterizzati: 20 ceppi produttori di carbapenemasi KPC, 10 di metallo-βlattamasi (MBL), 6 di OXA-48, 12 non produttori di carbapenemasi (6 con ridotta sensibilità ai carbapenemi da produzione di ESBL con deficit di porine e 6 wild-type). La metodica di valutazione della produzione di carbapenemasi prevede l’incubazione per 1 ora a 37°c dei ceppi in esame in presenza di meropenem ad una concentrazione di 0.05 mg/ml. In seguito si procede ad una centrifugazione ed il sovranatante viene utilizzato per il dosaggio del meropenem mediante LC-MS, metodica che coniuga la cromatografia liquida alla spettrometria di massa. L’analisi è stata eseguita su sistema HPLC Prominance UFLC-XR (Shimadzu) accoppiato a spettrometro di massa triplo quadrupolo API 4000 QTRAP (AB Sciex). Una curva di calibrazione permette di ottenere una quantificazione assoluta, mentre l’attività carbapenemasica è stata valutata in base al rapporto tra la concentrazione di meropenem nei campioni incubati con i ceppi e quella dell’antibiotico incubato in assenza di batteri (ratio). RISULTATI Tutti i ceppi produttori di KPC hanno evidenziato una rapida attività di degradazione del meropenem, determinando valori di ratio inferiori a 0.2 in 19 dei 20 campioni. Per gli isolati produttori di MBL i valori di ratio sono risultati tutti inferiori a 0.6, mentre per quelli positivi per OXA-48 in 2 casi i valori di ratio sono risultati sovrapponibili a quelli ottenuti per i ceppi produttori di ESBL. Complessivamente, utilizzando un cut-off di ratio ottimale identificato ad un valore di 0.75, la sensibilità del metodo nel determinare la produzione di carbapenemasi è risultata pari al 94.4%, la specificità del 100%. CONCLUSIONI La metodica di LC-MS valutata in questo studio ha dimostrato una elevata affidabilità diagnostica nella valutazione dell’attività carbapenemasica da parte di ceppi di enterobatteri. La sensibilità non ottimale ottenuta su isolati produttori di OXA-48 è probabilmente legata ad una più lenta attività enzimatica di questa specifica βlattamasi

INTRODUZIONE Le sepsi da gram-negativi multiresistenti rappresentano una delle più gravi minacce per la gestione dei pazienti ospedalizzati. Nei casi di sepsi grave o shock settico una precoce terapia antibiotica appropriata è correlata con una riduzione della mortalità e dei costi sanitari. La recente diffusione di gram-negativi resistenti ai β-lattamici per la produzione di ESBL e carbapenemasi, a cui spesso si associa la resistenza a diverse altre classi di farmaci, ha ulteriormente aumentato il rischio di inappropriatezza. Scopo di questo lavoro è quello di valutare il saggio molecolare Verigene Gram-negative Blood culture test (Nanosphere-Moss) che consente di identificare rapidamente da flacone di emocoltura positivo la specie responsabile della batteriemia e i determinanti di resistenza ai β-lattamici. METODI Sono stati inclusi nello studio 39 casi di batteriemia diagnosticati mediante emocoltura (BactecFX, BD) tra ottobre 2013 e maggio 2014 in pazienti ricoverati in reparti di area critica del Policlinico S.Orsola- Malpighi. Una volta identificata la presenza di bacilli gram-negativi mediante la colorazione di Gram, unitamente alle procedure di routine (subcoltura, identificazione con MALDI-TOF, antibiogramma con VITEK2, Biomerieux), si è proceduto all’esecuzione del saggio Verigene Gram-negative Blood culture test (BC-GN). Il metodo, della durata complessiva di 2 ore, prevede, a partire da 700 ml di brodo, l’estrazione degli acidi nucleici e la successiva ibridazione dei bersagli con sonde che permettono di identificare K.pneumoniae, K.oxytoca, E.coli, S.marcescens, Proteus spp., Citrobacter spp., Enterobacter spp., P.aeruginosa e Acinetobacter spp. e di rilevare la presenza dei principali marker di resistenza ai β-lattamici (CTX-M, KPC, VIM, NDM, IMP e OXA). I risultati ottenuti sono stati confrontati con quelli della routine diagnostica colturale. RISULTATI Il test BC-GN ha identificato 1 o più specie di gram-negativi in 36 campioni, con prevalenza di K.pneumoniae (17) ed E.coli (14). La concordanza con la metodica colturale è risultata del 100%. Per i 3 casi negativi, la coltura ha evidenziato la crescita di specie non incluse tra quelle previste dal metodo (2 M.morganii e 1 C.jejuni). Per quel che riguarda i determinanti di resistenza, il test ha identificato 13 CTX-M, 7 KPC e 2 OXA, e i risultati sono stati del tutto compatibili con il quadro fenotipico dei relativi antibiogrammi. CONCLUSIONI Il saggio molecolare Verigene Gram-negative Blood culture test ha evidenziato nel nostro studio elevata sensibilità e specificità nella identificazione delle specie di gram-negativi responsabili di batteriemia e dei meccanismi di resistenza ai β-lattamici. La metodica, molto rapida e di semplice esecuzione, è di facile implementazione all’interno della routine diagnostica. Peraltro altri studi sono necessari per valutare quale possa essere l’impatto clinico dell’introduzione del test sulla gestione del paziente settico in termini di appropriatezza delle modifiche terapeutiche indotte e dei relativi esiti.

Objectives. Currently, the main difficulty in laboratory confirmation of carbapenemases production concerns the identification of class D enzymes (OXA-48, OXA-181 and OXA-181-like), harbored in Enterobacteriaceae. Nevertheless, determination of susceptibility to temocillin provides an important indication of their possible presence. Aims of the study are to evaluate the susceptibility to temocillin of Enterobacteriaceae strains with reduced susceptibility to carbapenems, and to correlate it with the mechanism of resistance defined by genotypic tests, in order to assess the performance of the temocillin-susceptibility test. Methods. 231 strains of Enterobacteriaceae with reduced susceptibility to carbapenems at routine analysis (Vitek2 - BioMérieux), were tested, selected on the basis of the results to disk diffusion synergy test for detection and typing of carbapenemases (DDST, Rosco). These strains, belonging to different genus and species (Klebsiella, Escherichia, Enterobacter, Citrobacter, Proteus), have also been molecularly characterized with Hyplex® PCR (Amplex), a multiplex-PCR for blaKPC, blaVIM, blaIMP, blaNDM and blaOXA-48; their susceptibility to temocillin has been evaluated by disk diffusion test. Results. The 231 tested strains, divided according to DDST, gave the following results: - 33 class A carbapenemase-producers: 33 resulted positive for blaKPC, 31 subsceptible to temocillin, 2 resistant; - 41 class B (MBL) producers: 34 resulted positive for blaVIM (12 susceptible to temocillin, 22 resistant), and 7 for blaNDM (6 susceptible to temocillin, 1 susceptible); - 147 negative to DDST: 2 resulted positive for blaOXA-48, both resistant to temocillin, while the other 145 were negative to multiplex PCR and temocillin-susceptible; - 10 AmpC-producers: all resulted negative to multiplex PCR and susceptible to temocillin. Conclusion. Susceptibility to temocillin proved to be the only phenotypic test that allows the identification of class D carbapenemases-producers strains if combined to DDST, even if this study suggest that temocillin resistance, not performed with DDST, does not provide a result specific for class D carbapenemases. However its introduction in the laboratory routine could allow the clinical microbiologist to provide a reliable result about presence/absence of all main known carbapenemases in strains with reduced susceptibility to carbapenems; particularly, a negative result to DDST, associated with susceptibility totemocillin, allows to exclude with very high probability the presence of any carbapenemases (negative predictive value of 100%).

INTRODUZIONE L’introduzione della spettrometria di massa (MS) MALDI-TOF per l'identificazione (ID) microbica ha migliorato la diagnosi di batteriemia, riducendo turnaround time e costi complessivi. Tuttavia, molti dei protocolli per l’ID diretta da emocoltura positiva tramite MS prevedono procedure complesse comprendenti fasi di lavaggio e di estrazione delle proteine. Anche se tali metodiche hanno mostrato una buona accuratezza diagnostica, i tempi di lavorazione rendono difficile la loro implementazione nella routine diagnostica. In questo lavoro è stato valutato un flusso di lavoro “less time consuming” che prevede una semina concentrata su piastra e una breve incubazione prima dell’analisi dei campioni tramite lo strumento Vitek MS (bioMerieux) METODI Da febbraio ad aprile 2014 sono stati raccolti 351 flaconi positivi. Unitamente all’esecuzione di subcolture e colorazione di Gram, 8ml di sangue da ogni flacone positivo sono stati prelevati e inseriti in una provetta Vacuette Z serum Sepclot activator contenente un gel separatore (Bekton Dickinson). Questa è stata centrifugata a 3500 giri per 10 minuti. Il sovranatante è stato scartato e il pellet risospeso in 100 μl di 0,9% sterile saline solution water. Dopo aver vortexato la provetta per 2 secondi, la soluzione è stata rovesciata su una piastra di agar cioccolato (Meus s.r.l), seminata e fatta asciugare per pochi minuti. In base all'esito della lettura del vetrino Gram: per i bacilli Gram negativi il tempo di incubazione è stato di 1,5h, per tutti gli altri di 3h. Al termine con un'ansa si è raccolta una quantità di patina di crescita e la si è deposta in un target-plate monouso con l'aggiunta di 1μl di matrice HCCA. Gli spot ottenuti sono stati analizzati tramite lo strumento Vitek MS come indicato dalla casa produttrice. I risultati sono stati verificati e confrontati nei giorni successivi ripetendo le ID dalle subcolture (gold standard) tramite MS o se necessario tramite metodi di ID biochimica (Vitek II bioMerieux, Microscan Siemens) e analisi complementari RISULTATI Dei 351 campioni, 214 hanno mostrato la presenza di microrganismi Gram+, 110 di Gram-, 12 di lieviti, 15 hanno mostrato la presenza di flora polimicrobica. Dei 336 campioni con flora monomicrobica, 286 sono risultati identificati correttamente (85,1%), 48 non identificati (14,2%) e solamente 2 con ID erronea (0,5%). I Gram+ e i Gram- hanno avuto una ID corretta rispettivamente nell’83,2% e nel 95,4% dei casi. Tra i microrganismi Gram+ sono stati identificati correttamente 26 su 28 (92,8%) ceppi di S. aureus e 19 su 20 (95%) ceppi di enterococchi. Solo 4 campioni (25%) dei 12 con presenza di lieviti hanno avuto ID corretta. Dei 48 campioni con microrganismi non identificati, 21 sono stati considerati da probabile contaminazione CONCLUSIONI Il protocollo in esame ha dimostrato una buona capacità analitica, in linea con altri protocolli presenti in letteratura che però prevedono passaggi di lavaggio e di estrazione delle proteine. In più c’è da sottolineare come sia molto efficace nell’ID dei microrganismi più rilevanti come Gram-, S. aureus ed enterococchi. Per campioni con presenza di lieviti il protocollo prevede limiti intrinseci alla metodica che non ne suggeriscono l’impiego. In definitiva, essendo la metodica in esame poco laboriosa, pur mantenendo una qualità diagnostica elevata, possiamo concludere come essa possa essere implementabile senza difficoltà nella routine di un laboratorio di microbiologia clinica.

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.

Parvovirus B19 is a frequent contaminant of human blood and plasma derivatives and iatrogenic transmission of B19 infection has been shown to occur through the administration of contaminated products. Manufacturing procedures, generally used for removal or inactivation of enveloped viruses (HIV, HCV and HBV) are not always effective in the elimination of B19 virus. A certain risk of contamination remains for some plasma derivatives due to the high-titer viral load in the starting blood donations and the extreme heat resistance and small size of the virus. This review provides an update on the different approaches currently available to detect, remove or inactivate B19 virus in order to enhance the safety margins of plasma products. Nucleic acid amplification techniques are the methods of choice for the detection of viruses, due to their high specificity and sensitivity. NAT assays are beneficial tools for the identification of contaminated mini-pools or plasma pools and the quantification of B19 contamination. They may also be valuable for testing the removal of B19 virus during manufacturing: since the virus may not be completely inactivated or removed by chemical or physical treatments, residual B19 contamination should always be checked. Solvent-detergent treatments fail to destroy B19 capsids because of the absence of a lipid-envelope, and heat treatments (pasteurization and dry-heat methods) cannot guarantee a complete viral in-activation because of the variable beat sensitivity of the virus.