C. Terulla’s research while affiliated with Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Pisana and other places

During the period March 2003 – May 2004 at the Laboratory of Clinical Microbiology “Redaelli” LTCRF in Milan, Italy, a total of 529 E. coli, obtained from inpatients of 3 different Long Term Care Rehabilitation Facilities (LTCRFs) in Northern Italy, were processed and 77 ESßLs producers (14.5%) were identified by Vitek System. The results were confirmed by double-disk synergy test with tazobactam (TZP). 61/77 isolates were characterized by higher levels of resistance to cefotaxime (CTX) than to ceftazidime (CAZ). (ß-lactamase production was investigated by analytical isoelectric focusing (IEF) coupled with a bioassay and showed multiple (ß-lactamase bands including one enzyme with pI 8.4 that, in a bioassay, was more active on CTX,ATM than on CAZ. The presence of (ß-lactamase genes was investigated by colony blot hybridization and by PCR amplification of blaTEM, blaSHV and blaCTX-M alleles. 43/61 isolates produced both TEM-1 and CTX-M-type enzymes, 14/61 expressed only CTX-M-type while in 4 cases were found blaCTX-M, blaTEM and blaSHV genes.The remainders (16/77), characterized by high levels of resistance to both CTX and CAZ, produced TEM-1 and SHV-5 enzymes (1/16) and TEM type ESßLs (15/16). Conjugation experiments, performed in liquid medium, confermed that the ESßLs determinants were transferable. Pulsed-field gel electrophoresis profiles of genomic DNA, digested with NotI, were analysed and revealed clonal heterogeneity. Our work confirms the emergence of CTX-M-type enzymes and their spread in Northern Italy also in longterm care and rehabilitation facilities that may be an important reservoir of ES?L producing E. coli.

A prospective 2-year analysis including 322 infant patients with acute respiratory disease (ARD) hospitalized in a pediatric department in northern Italy was carried out to evaluate the role as respiratory pathogens or co-pathogens of recently identified viruses. The presence of respiratory syncitial virus (RSV), human Metapneumoviruses (hMPVs), human Bocaviruses (hBoVs), and human Coronaviruses (hCoVs) was assayed by molecular detection and clinical symptoms evaluated. Nasopharyngeal aspirates from 150 of the 322 infants (46.6%) tested positive for at least one pathogen. Ninety samples (28.0%) tested positive for RSV RNA (61.5% genotype A and 38.5% genotype B), 46 (14.3%) for hMPV RNA (71.7% subtype A and 28.3% subtype B), 28 (8.7%) for hCoV RNA (39.3% hCoV-OC43, 35.7% hCoV-NL63, 21.4% hCoV-HKU1, and 3.6% hCoV-229E), and 7 (2.2%) for hBoV DNA (of the 6 typed, 50% subtype 1 and 50% subtype 2); 21/150 samples revealed the presence of 2 or more viruses. Co-infection rates were higher for hMPVs, hCoVs, and hBoV (38.3%, 46.4%, and 57.1%,) and lower for RSV (23.3%). RSV was associated with the presence of complications (P < 0.001) and hypoxia (P < 0.015). When the presence of RSV alone and the RSV-hMPV co-infections were considered, RSV mono-infected patients resulted to have longer hospitalization and higher hypoxia (P < 0.001). The data highlight that (i) RSV has a central role as a respiratory pathogen of infants, (ii) the wide circulation of recently identified viruses does not reduce the clinical and epidemiological importance of RSV, and that (iii) recently identified agents (hMPVs, hBoVs, and hCoVs) act as primary pathogens or co-pathogens.

From October 2004 through October 2006 a study was performed to evaluate the prevalence of human Metapneumovirus (hMPV) infection in adult hematopoietic stem cell transplant (HSCT) recipients. Sequential nasopharyngeal aspirates (NPA) were collected independently from respiratory symptoms and evaluated for hMPV-RNA by polymerase chain reaction (PCR) and sequence analysis. Results indicate epidemiological and molecular differences between the 2004-2005 and 2005-2006 periods and that hMPV seems not to symptomatically affect HSCT patients or cause late respiratory sequelae. In addition, data collected suggest a hospital origin of hMPV infection in most HSCT patients during the 2004-2005 period.

Introduzione. Metapneumovirus umano (hMPV), associato a infezio-ni respiratorie in età pediatrica, è stato isolato anche in soggetti adulti, in particolare immunocompromessi. Per valutarne il ruolo patogeno in questi soggetti è stato condotto uno studio prospettico in pazienti sottoposti a trapianto di cellule staminali emopoietiche (HSCT). Metodi. Da ottobre 2004 a ottobre 2005 si sono esaminati cam-pioni di aspirato nasofaringeo (NPA) ottenuti, indipen-dentemente dalla presenza di sintomatologia respirato-ria, da soggetti HSCT; i campioni, prelevati a differen-ti tempi dal trapianto, sono stati esaminati per la pre-senza di hMPV mediante RT-PCR. Parallelamente si sono esaminati campioni di NPA ottenuti da pazienti pediatrici sintomatici. I campioni sono stati tipizzati mediante sequenziamento delle regioni amplificate. Risultati. Si sono valutati 107 campioni ottenuti da 21 pazienti HSCT e 244 campioni ottenuti da 244 pazienti pediatri-ci. MPV-RNA è stato rilevato in 53 dei 107 NPA (49.5%) ottenuti da 18 pazienti HSCT (85.7%). Dei campioni positivi, 6 sono stati raccolti all'ingresso del paziente in ospedale, 12 durante il regime di condizionamento e 35 da 15 giorni a 3 mesi dopo il trapianto. La presenza di MPV-RNA veniva persistentemente rilevata per periodi da 26 a 94 giorni senza differenze nella distribuzione sta-gionale ed in assenza di segni o sintomi respiratori spe-cifici. Nei pazienti pediatrici 37 dei 244 campioni (15.1%) risultavano positivi, con un picco di prevalenza in gennaio e febbraio. L'analisi di sequenza ha eviden-ziato genotipo A e marcata omologia di sequenza in tutti i campioni dei pazienti HSCT e la presenza di entrambi i genotipi (A e B) nei pazienti pediatrici. Conclusioni. Questo studio documenta un'alta prevalenza e persistenza di infezione asintomatica da hMPV in pazienti HSCT e sot-tolinea l'importanza di ulteriori studi sul ruolo della rispo-sta immunitaria antivirale nella patogenesi della malattia.

Sequential nasopharyngeal aspirates from patients without respiratory symptoms undergoing hematopoietic stem cell transplantation (HSCT) were tested for genomic RNA of human metapneumovirus (hMPV). Persistent hMPV infection was documented in most of them and confirmed by virus isolation. hMPV infection etiology was also evaluated during the same period in samples from pediatric patients with acute respiratory diseases (ARDs). Sequence analysis of hMPV in HSCT recipients documented infection by hMPV genotype A and strong interhost similarity; this pattern differs from that observed in pediatric patients with ARDs. The data indicate that HSCT recipients may frequently develop symptomless hMPV infection