Colonization and infection due to carbapenemase-producing Enterobacteriaceae in liver and lung transplant recipients and donor-derived transmission: a prospective cohort study conducted in Italy.

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-ß-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.

Automatic environmental disinfection with hydrogen peroxide and silver ions versus manual environmental disinfection with sodium hypochlorite: a multicentre randomized before-and-after trial.

BACKGROUND: New technologies for automated disinfection have been developed, including the use of hydrogen peroxide atomized by specific equipment, with associated silver compounds. AIMS: To compare the effectiveness of an automated disinfection system with hydrogen peroxide <8% and silver ion versus a manual method with 0.5% sodium hypochlorite solution when evaluating the reduction of microbial mesophilic contamination and Clostridium difficile presence; and to evaluate the time required for both of these processes. METHODS: This was a randomized multicentre trial performed in different hospital wards that had been occupied previously by patients with Clostridium difficile infection. When patients were discharged their rooms were randomized to one of two decontamination arms. The surfaces where sampled using swabs, before and after disinfection. Swab samples were cultured for quantitative detection of microbial mesophilic contamination and qualitative detection of C. difficile. FINDINGS: Before disinfection, 13% of surfaces decontaminated with hydrogen peroxide and silver ions and 20% of surfaces decontaminated with sodium hypochlorite showed presence of C. difficile spores. After disinfection, the samples containing C. difficile were 0% (P < 0.001) in the group decontaminated with hydrogen peroxide and silver ions, and were 3% (P < 0.001) in the group decontaminated with sodium hypochlorite. This difference was not statistically significant; nor was the difference in the reduction of the microbial mesophilic contamination. CONCLUSION: The differences between the groups were not statistically significant; however, the disinfection with hydrogen peroxide and silver ions is preferable due to less dependence on operators.

New evidence of cutaneous leishmaniasis in north-eastern Italy.

BACKGROUND: Human leishmaniasis is on increase in the Mediterranean Europe. However, the exact prevalence of cutaneous leishmaniasis (CL) is largely unknown as underdiagnosis and under reporting are common. OBJECTIVE: To evaluate epidemiological, clinicopathological and microbiological aspects of CL cases occurring in the Bologna Province, north-eastern Italy. METHODS: We performed a retrospective, observational study on CL cases diagnosed in the Bologna Province between January 2013 and December 2015. RESULTS: During 2013-2015, 30 cases of CL were identified in the Bologna Province with an average incidence of 1.00/100 000, with an increase of fourfold to 12-fold as compared to previous years. 16 of 30 (53%) CL cases presented as single, typical lesions. CL diagnosis was carried out by histological and molecular techniques, although in 7 of 29 (24%) PCR-positive cases, amastigotes were not visible on histology. CONCLUSIONS: We report new evidence of CL cases in a focal area of north-eastern Italy in 2013-2015. Our study highlights the importance of CL surveillance in the Mediterranean basin and emphasizes the need for the molecular laboratory surveillance of CL in endemic areas.

Characterization of antibody response in neuroinvasive infection caused by Toscana virus.

OBJECTIVES: Among sandfly-borne pathogens, Toscana virus (TOSV) is a prominent cause of summer meningitis in Mediterranean Europe. Here, we assessed the kinetics of anti-TOSV antibodies over time in 41 patients diagnosed with TOSV meningitis or meningoencephalitis in northeastern Italy. METHODS: Acute and follow-up serum samples were collected up to 20 months after diagnosis of TOSV infection and tested for the presence of specific antibody using immunoenzymatic and indirect immunofluorescence assays. In addition, maturation of anti-TOSV IgG over time was evaluated as well as production of neutralizing antibodies. RESULTS: Specific IgM and IgG response was present at diagnosis in 100% of patients; TOSV-specific IgM and IgG were detected in patients' sera up to 6 and 20 months after diagnosis, respectively. The avidity index (AI) increased over the first month after infection in 100% of patients and most cases exceeded 60% by Day 30 post infection. The AI subsequently plateaued then declined at 20 months after diagnosis. Finally, neutralization assay to TOSV was performed in 217 sera collected from 41 patients; 69.6% of tested samples resulted in reactive and moderate levels of neutralizing antibodies observed during all phases of infection despite high titres of total anti-TOSV IgG. CONCLUSIONS: Specific antibody response develops rapidly and is long-lasting for neuroinvasive TOSV infection. Serodiagnosis of neuroinvasive TOSV requires simultaneous detection of specific IgM and IgG. Moderate levels of neutralizing antibodies were maintained over the study period, while the protective role of antibodies lacking neutralizing activity is unclear and requires further evaluation.

Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories.

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

Ease-of-use protocol for the rapid detection of third-generation cephalosporin resistance in Enterobacteriaceae isolated from blood cultures using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

An ease-of-use protocol for the identification of resistance against third-generation cephalosporins in Enterobacteriaceae isolated from blood culture bottles was evaluated using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. A cefotaxime hydrolysis assay from chocolate agar subcultures using antibiotic discs and without inoculum standardization was developed for routine work flow, with minimal hands-on time. This assay showed good performance in distinguishing between cefotaxime-susceptible and cefotaxime-resistant strains, with excellent results for Escherichia coli (sensitivity 94.7%, specificity 100%). However, cefotaxime resistance was not detected reliably in Enterobacteriaceae expressing AmpC genes or carbapenemase-producing Klebsiella pneumoniae.

Increased number of cases of Chikungunya virus (CHIKV) infection imported from the Caribbean and Central America to northern Italy, 2014.

This report describes an increased number of cases of Chikungunya virus (CHIKV) infection imported in northern Italy (Emilia-Romagna region) during the period May-September 2014, indicating that the recent spread of CHIKV and its establishment in the Caribbean and in central America, resulted in a high number of imported cases in Europe, thus representing a threat to public health. From May to September 2014, 14 imported cases of CHIKV infection were diagnosed; the patients were returning to Italy from Dominican Republic (n = 6), Haiti (n = 3), Guadeloupe (n = 2), Martinique (n = 1), Puerto Rico (n = 1) and Venezuela (n = 1). Phylogenetic analysis performed on the envelope protein (E1) gene sequences, obtained from plasma samples from two patients, indicated that the virus strain belongs to the Caribbean clade of the Asian genotype currently circulating in the Caribbean and Americas. The rise in the number of imported cases of CHIKV infection should increase healthcare professionals' awareness of the epidemiological situation and clinical presentation of CHIKV infection in order to enhance surveillance and early diagnosis in the forthcoming season of vector activity in Europe and North America.

Persistence of anti-chikungunya virus-specific antibodies in a cohort of patients followed from the acute phase of infection after the 2007 outbreak in Italy.

Chikungunya is a mosquito-borne infection of humans, and its diffusion has increased worldwide. In 2007 an outbreak occurred in Italy. In this study, the antibody response of 133 patients followed up starting from the acute phase of infection was investigated. Antibody titres were periodically scored up to 1 year since the infection: 82.7% of the IgM antibody disappeared within 12 months, and the IgG response lasted longer than 12 months. Nevertheless, the IgG mean titre was lower in 95.5% of patients at the end of follow-up, thus suggesting a decrease within a relatively short period.

Comparative evaluation of the new xTAG GPP multiplex assay in the laboratory diagnosis of acute gastroenteritis. Clinical assessment and potential application from a multicentre Italian study.

OBJECTIVE: Gastroenteritis caused by a single pathogen or multiple pathogens remains a major diagnostic challenge for the laboratory. The treatment of diarrhoea is based on microbiological results. Diagnosis is achieved using different laboratory techniques that have variable sensitivity and specificity. xTAG GPP is a new multiplex PCR assay that simultaneously detects 15 different pathogens responsible for diarrhoea. The results of the first multicentre study in Italy to evaluate the potential clinical application of the GPP assay in the laboratory diagnosis of diarrhoea are reported here. METHODS: Faeces specimens (N=664) from hospitalized patients were tested with the GPP assay using a Luminex 200 instrument. All specimens were run using comparator methods following a routine algorithm: culture for bacteria, enzyme immunoassay and PCR for viruses, and microscopy for parasites. RESULTS: Of the samples tested with the GPP, 53.61% (356/664) gave positive results, as compared to 45.33% by routine testing. Of the positive specimens, 34.55% showed the presence of genomic DNA from multiple pathogens. The Luminex method showed an increase in the percentage of positivity of 8.28%. CONCLUSIONS: The GPP assay can be considered a helpful tool for the detection of gastrointestinal pathogens, with a hands-on time of 5h; it provides accurate data for the clinical management of hospitalized patients and for epidemiological surveillance.

Human and entomological surveillance of Toscana virus in the Emilia-Romagna region, Italy, 2010 to 2012.

Toscana virus (TOSV), transmitted by phlebotomine sandflies, is recognised as one of the most important causes of viral meningitis in summer in Mediterranean countries. A surveillance plan based on both human and entomological surveys was started in 2010 in the Emilia-Romagna region, Italy. Clinical samples from patients with neurological manifestations were collected during 2010 to 2012. The surveillance protocol was improved during these years, allowing the detection of 65 human infections. Most of these infections were recorded in hilly areas, where sandflies reach the highest density. Entomological sampling around the homes of the patients resulted in a low number of captured sandflies, while later sampling in a hilly area with high number of human cases (n=21) resulted in a larger number of captured sandflies. Using this approach, 25,653 sandflies were sampled, of which there were 21,157 females, which were sorted into 287 pools. TOSV RNA was detected by real-time PCR in 33 of the pools. The results highlighted the role of Phlebotomus perfiliewi as the main vector of TOSV and a potential link between vector density and virus circulation. This integrated system shows that an interdisciplinary approach improves the sensitiveness and effectiveness of health surveillance.

The experience of West Nile virus integrated surveillance system in the Emilia-Romagna region: five years of implementation, Italy, 2009 to 2013.

Predicting West Nile virus (WNV) circulation and the risk of WNV epidemics is difficult due to complex interactions of multiple factors involved. Surveillance systems that timely detect virus activity in targeted areas, and allow evidence-based risk assessments may therefore be necessary. Since 2009, a system integrating environmental (mosquitoes and birds) and human surveillance has been implemented and progressively improved in the Emilia-Romagna region, Italy. The objective is to increase knowledge of WNV circulation and to reduce the probability of virus transmission via blood, tissue and organ donation. As of 2013, the system has shown highly satisfactory results in terms of early detection capacity (the environmental surveillance component allowed detection of WNV circulation 3­4 weeks before human cases of West Nile neuroinvasive disease (WNND) occurred), sensitivity (capacity to detect virus circulation even at the enzootic level) and area specificity (capacity to indicate the spatial distribution of the risk for WNND). Strong correlations were observed between the vector index values and the number of human WNND cases registered at the province level. Taking into consideration two scenarios of surveillance, the first with environmental surveillance and the second without, the total costs for the period from 2009 to 2013 were reduced when environmental surveillance was considered (EUR 2.093 million for the first scenario vs EUR 2.560 million for the second). Environmental surveillance helped to reduce costs by enabling a more targeted blood unit testing strategy. The inclusion of environmental surveillance also increased the efficiency of detecting infected blood units and further allowed evidence-based adoption of preventative public health measures.

Comparison between MALDI-TOF MS and FilmArray Blood Culture Identification panel for rapid identification of yeast from positive blood culture.

In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast from positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures.

A model of laboratory surveillance for neuro-arbovirosis applied during 2012 in the Emilia-Romagna region, Italy.

Arboviruses with neuroinvasive potential are gaining more attention due to the increased number of cases of autochthonous and imported infections in the human host. Diagnosis of infection caused by these viruses in patients with central nervous system (CNS) diseases is still underestimated and these infections represent an emerging threat to public health. We describe a model suitable for the laboratory surveillance of neuro-arbovirosis that was applied in the Emilia-Romagna region, north-eastern Italy, during the 2012 summer season. One hundred and twenty cases of suspected neuroinvasive infection were tested for arboviral agents on the basis of clinical and laboratory signs and epidemiological data. The most common virus detected was Toscana virus (TOSV): anti-TOSV specific antibodies or viral components were detected in 28.3% of the cases; 79.4% of the TOSV cases were in the acute phase of infection. No cases resulted in acute phase for West Nile (WNV), Usutu (USUV), Chikungunya (CHIKV) or Dengue (DENV) virus infection. Conversely, two patients with a history of staying in a tick-borne encephalitis virus (TBEV) endemic area showed a probable TBEV infection. These results emphasize the importance of a complete and 'ready to act' laboratory diagnostic system to be implemented within the larger frame of a regional integrated surveillance system.

Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013.

An increased number of autochthonous visceral leishmaniasis (VL) cases has recently been reported in Bologna Province in northern Italy. Over six months from November 2012 to May 2013, 14 cases occurred, whereas the average number of cases per year was 2.6 (range: 0-8) in 2008 to 2012. VL was diagnosed in a median of 40 days (range: 15-120) from disease onset. This delay in diagnosis shows the need for heightened awareness of clinicians for autochthonous VL in Europe.

Detection of specific antibodies against West Nile and Usutu viruses in healthy blood donors in northern Italy, 2010-2011.

Neutralizing antibodies against West Nile (WNV) and Usutu (USUV) viruses were measured in 6000 samples collected, between 1 September 2010 and 30 June 2011, from blood donors living in different districts of Emilia-Romagna, northeastern Italy. On the basis of the microneutralization assay (MNTA), 47 (0.78%) subjects were positive for WNV and 14 (0.23%) for USUV. These results were compared with those obtained 2 years ago and suggest an increased circulation of USUV among humans in Emilia-Romagna.

West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention.

West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis antigenic group, has caused sporadic outbreaks in humans, horses and birds throughout many of the warmer regions of Europe for at least 20 years. Occasional cases of West Nile encephalitis have also been associated with infected blood transfusions and organ donations. Currently, WNV appears to be expanding its geographical range in Europe and causing increasing numbers of epidemics/outbreaks associated with human morbidity and mortality. This brief review reports on the current epidemic situation regarding WNV in Europe, highlighting the clinical, diagnostic and preventive measures available for controlling this apparently emerging human pathogen.

Congenital cytomegalovirus infection: patterns of fetal brain damage.

Cytomegalovirus (CMV) is the most prevalent infectious agent causing neurological dysfunction in the developing brain. This study analysed the different patterns of tissue damage, particularly in the brain, of fetuses with documented CMV infection. We studied 45 fetuses at 20-21 weeks of gestation with congenital CMV infection documented by invasive positive prenatal diagnosis. At the time of amniocentesis, abnormal ultrasound findings had been recorded for 13 of the 45 fetuses (29%). Histological and immunohistochemical characterization was performed on the placenta, brain, heart, lung, liver, kidney, and pancreas. The different degrees of brain damage were correlated with tissue viral load, inflammatory response, placental functionality, and extramedullary haematopoiesis. Even though a high CMV load was detected in all amniotic fluids, brain infection occurred in only 62% of the fetuses and with different degrees of severity. Tissues with a low viral load showed a globally weak inflammatory response, and fetuses had only mild brain damage, whereas tissues with a high CMV load showed prominent infiltration of the activated cytotoxic CD8(+) T-lymphocytes responsible for immune-mediated damage. Furthermore, severe placental infection was associated with diffuse villitis and necrosis, consistent with functional impairment and possible consequent hypoxic cerebral damage. Brain injury induced by CMV congenital infection may be the result of uncontrolled viral replication, immune-mediated damage by cytotoxic CD8(+) T-lymphocytes, and, in the presence of placental insufficiency, fetal hypoxia.