Compliance of clinical microbiology laboratories with recommendations for the diagnosis of bloodstream infections: Data from a nationwide survey in Italy.

In 2014, the Italian Working Group for Infections in Critically Ill Patient of the Italian Association of Clinical Microbiologists updated the recommendations for the diagnostic workflow for bloodstream infections (BSI). Two years after publication, a nationwide survey was conducted to assess the compliance with the updated recommendations by clinical microbiology laboratories. A total of 168 microbiologists from 168 laboratories, serving 204 acute care hospitals and postacute care facilities, were interviewed during the period January-October 2016 using a questionnaire consisting of nineteen questions which assessed the level of adherence to various recommendations. The most critical issues were as follows: (a) The number of sets of blood cultures (BC) per 1,000 hospitalization days was acceptable in only 11% of laboratories; (b) the minority of laboratories (42%) was able to monitor whether BCs were over or under-inoculated; (c) among the laboratories monitoring BC contamination (80%), the rate of contaminated samples was acceptable in only 12% of cases;(d) the Gram-staining results were reported within 1 hr since BC positivity in less than 50% of laboratories. By contrast, most laboratories received vials within 2-4 hr from withdrawal (65%) and incubated vials as soon as they were received in the laboratory (95%). The study revealed that compliance with the recommendations is still partial. Further surveys will be needed to monitor the situation in the future.

Synergistic effect of photodynamic therapy at 400 nm and doxycycline against Helicobacter pylori.

Aim: The objective of this study was to investigate the possible synergy between doxycycline and photodynamic therapy against Helicobacter pylori and to evaluate the possible side effects on adenocarcinoma gastric cells with and without protoporphyrin IX. Materials & methods: Three H. pylori strains (ATCC 700392, 43504 and 49503) were grown on solid medium either with, or without, doxycycline at subinhibitory concentrations, and irradiated for 10, 20 and 30 minutes with a 400 nm-peaked light source. The phototoxicity tests on AGS cells were evaluated by MTT assay. Results: The photodynamic therapy and doxycycline combination showed an antibacterial synergistic effect with no significant toxicities. Conclusion: The synergistic treatment could be considered as an interesting therapeutic option.

Role of place of acquisition and inappropriate empirical antibiotic therapy on the outcome of extended-spectrum ß-lactamase-producing Enterobacteriaceae infections.

The impact of inappropriate empirical antibiotic therapy (IEAT) on the outcome of severe infections due to extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-Ent) remains unclear. Current evidence is limited by study design and lack of confounder control. The main objective of this study was to define the outcome of severe infections due to ESBL-Ent according to clinical parameters and place of infection acquisition. Adult hospitalised patients with ESBL-Ent infections were included in a 3-year multicentre prospective study. Primary outcomes were IEAT rates and crude mortality of severe infections, adjusted by place of acquisition [community-acquired infection (CAI), healthcare-associated infection (HCAI) and hospital-acquired infection (HAI)]. Among 729 patients, 519 (71.2%) were diagnosed with HAI, 176 (24.1%) with HCAI and 34 (4.7%) with CAI. Moreover, 32.9% of patients received IEAT; higher rates of IEAT were observed in pneumonia (23%) and deep surgical site infections (19%). HCAIs were more frequently associated with IEAT than HAIs (48.3% vs. 27.9%; OR = 1.7, 95% CI 1.2-2.4). The overall mortality rate for severe infections (n = 264) was 12.1% and was significantly higher in HCAIs (20%) than HAIs (10%) (RR = 2.3, 95% CI 1.01-5.3). IEAT significantly increased the risk of mortality in bloodstream infections (RR = 8.3, 95% CI 2-46.3). Rates of IEAT and overall mortality of ESBL-Ent severe infections were higher in HCAIs than HAIs. Prompt diagnosis of patients with severe HCAIs due to ESBL-Ent is essential since these infections receive high rates of IEAT and significantly higher mortality than HAIs [ClinicalTrials.gov Identifier: NCT00404625].

Carriage rates and risk factors during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C ST-11 (cc11) in Tuscany, Italy: a cross-sectional study.

BACKGROUND: During 2015-2016 an outbreak of invasive meningococcal disease due to N. meningitidis serogroup C ST-11 (cc11) occurred in Tuscany, Italy. The outbreak affected mainly the age group 20-30 years, men who have sex with men, and the area located between the cities of Firenze, Prato and Empoli, with discos and gay-venues associated-clusters. A cross-sectional-survey was conducted to assess the prevalence and risk factors for meningococcal-carriage, in order to address public health interventions. METHODS: A convenience sample of people aged 11-45 years provided oropharyngeal swab specimens and completed questionnaires on risk factors for meningococcal carriage during a 3 months study-period, conducted either in the outbreak-area and in a control-area not affected by the outbreak (cities of Grosseto and Siena). Isolates were tested by culture plus polymerase chain reaction. Serogroup C meningococcal isolates were further characterized using multilocus sequence typing. Univariate and multivariate analyses were performed to estimate adjusted odds ratios (AORs) for meningococcal carriage. RESULTS: A total of 2285 oropharyngeal samples were collected. Overall, meningococcal carriage prevalence was 4.8% (n = 110), with nonencapsulated meningococci most prevalent (2.3%; n = 52). Among encapsulated meningococci, serogroup B was the most prevalent (1.8%; n = 41), followed by serogroup Y (0.5%; n = 11) and serogroup C (0.2%; n = 4); one carrier of serogroup E and one of serogroup Z, were also found (0.04%). Three individuals from the city of Empoli were found to carry the outbreak strain, C:ST-11 (cc11); this city also had the highest serogroup C carriage prevalence (0.5%). At the multivariate analyses, risk factors for meningococcal carriage were: illicit-drugs consumption (AOR 6.30; p < 0.01), active smoking (AOR 2.78; p = 0.01), disco/clubs/parties attendance (AOR 2.06; p = 0.04), being aged 20-30 years (AOR 3.08; p < 0.01), and have had same-sex intercourses (AOR 6.69; p < 0.01). CONCLUSIONS: A low prevalence of meningococcal serogroup C carriage in an area affected by an outbreak due to the hypervirulent N. meningitidis serogroup C ST-11 (cc11) strain was found. The city of Empoli had the highest attack-rate during the outbreak and also the highest meningococcal serogroup C carriage-prevalence due to the outbreak-strain. Multivariate analyses underlined a convergence of risk factors, which partially confirmed those observed among meningococcal outbreak-cases, and that should be considered in targeted immunization campaigns.

Evaluation of candidemia and antifungal consumption in a large tertiary care Italian hospital over a 12-year period.

PURPOSE: An early adequate antifungal therapy based on the knowledge of local epidemiology can reduce the candidemia-attributable mortality and the length of hospitalization. We performed a retrospective study to analyze the epidemiology of candidemia and the antifungal consumption in our hospital. METHODS: We analyzed Candida spp. isolated from the blood, and their susceptibility profile from 2005 to 2016 in Careggi University Hospital, Florence, Italy. We also performed a stratified analysis by clinical setting where Candida spp. were isolated (Medical Wards, Surgery, Intensive Care Unit-ICU). Then, we retrospectively reviewed the annual consumption of antifungal agents and calculated the defined daily dosing for 10,000 hospital days. RESULTS: The rate of candidemia was higher in ICU than other settings and Candida albicans was the first cause of candidemia (61.2%). After adjustment for hospital days, the rate of C. albicans showed a statistically significant parabolic trend (p < 0.001), with a peak of incidence in 2010. After 2010, we observed a reduction of candidemia due to both C. albicans and non-albicans species. Between 2005 and 2015, we reported an increasing increased use of echinocandins. As far as resistance profile is concerned, only one Candida glabrata isolate was resistant to caspofungin (1.9%) and 30% of C. glabrata were resistant to fluconazole. CONCLUSIONS: Our data describe C. albicans as the first cause of candidemia in all the studied settings and the low rate of echinocandin resistance, despite their increased use over the study period. ICU was confirmed as the setting with the highest incidence of candidemia.

A new selective broth enrichment automated method for detection of carbapenem-resistant Enterobacteriaceae from rectal swabs.

We evaluated the performance of an automated, rapid (TAT 4-8 h), liquid-culture method for carbapenem-resistant Enterobacteriaceae (CRE) detection from rectal swabs, in a setting of KPC-producing Klebsiella pneumoniae endemicity. With 600 samples (22 positive for CRE, 3.7%), the system sensitivity and specificity, at 8 h, were 100% and 99.2%, respectively.

The Role of DNA Amplification and Cultural Growth in Complicated Acute Appendicitis.

Bacterial growth of peritoneal fluid specimens obtained during surgical procedures for acute appendicitis may be useful to optimize further antibiotic therapy in complicated cases. DNA amplification represents a fast technique to detect microbial sequences. We aimed to compare the potential of DNA amplification versus traditional bacterial growth culture highlighting advantages and drawbacks in a surgical setting. Peritoneal fluid specimens were collected during surgery from 36 children who underwent appendectomy between May and December 2012. Real-time polymerase chain reaction (RT-PCR) and cultures were performed on each sample. RT-PCR showed an amplification of 16S in 18/36 samples, Escherichia coli (in 7 cases), Pseudomonas aeruginosa (3), Fusobacterium necrophorum (3), Adenovirus (2), E.coli (1), Klebsiella pneumoniae (1), Serratia marcescens/Enterobacter cloacae (1). Bacterial growth was instead observed only in four patients (3 E.coli and 1 P.aeruginosa and Bacteroides ovatus). Preoperative C-reactive protein and inflammation degree, the most reliable indicators of bacterial translocation, were elevated as expected. DNA amplification was a quick and useful method to detect pathogens and it was even more valuable in detecting aggressive pathogens such as anaerobes, difficult to preserve in biological cultures; its drawbacks were the lack of biological growths and of antibiograms. In our pilot study RT-PCR and cultures did not influence the way patients were treated.

Proposal for a New Score-Based Approach To Improve Efficiency of Diagnostic Laboratory Workflow for Acute Bacterial Meningitis in Adults.

Microbiological tests on cerebrospinal fluid (CSF) utilize a common urgent-care procedure that does not take into account the chemical and cytological characteristics of the CSF, resulting sometimes in an unnecessary use of human and diagnostic resources. The aim of this study was to retrospectively validate a simple scoring system (bacterial meningitis-Careggi score [BM-CASCO]) based on blood and CSF sample chemical/cytological parameters for evaluating the probability of acute bacterial meningitis (ABM) in adults. BM-CASCO (range, 0 to 6) was defined by the following parameters: CSF cell count, CSF protein levels, CSF lactate levels, CSF glucose-to-serum glucose ratio, and peripheral neutrophil count. BM-CASCO was retrospectively calculated for 784 cases of suspected ABM in adult subjects observed during a four-and-a-half-year-period (2010 to 2014) at the emergency department (ED) of a large tertiary-care teaching hospital in Italy. Among the 28 confirmed ABM cases (3.5%), Streptococcus pneumoniae was the most frequent cause (16 cases). All ABM cases showed a BM-CASCO value of ≥3. Most negative cases (591/756) exhibited a BM-CASCO value of ≤1, which was adopted in our laboratory as a cutoff to not proceed with urgent microbiological analysis of CSF in cases of suspected ABM in adults. During a subsequent 1-year follow-up, the introduction of the BM-CASCO in the diagnostic workflow of ABM in adults resulted in a significant decrease in unnecessary microbiological analysis, with no false negatives. In conclusion, BM-CASCO appears to be an accurate and simple scoring system for optimization of the microbiological diagnostic workflow of ABM in adults.

Louse-Borne Relapsing Fever with Meningeal Involvement in an Immigrant from Somalia to Italy, October 2015.

INTRODUCTION: Borrelia recurrentis, transmitted by Pediculus humanus humanus, is the etiological agent of louse-borne relapsing fever (LBRF). Currently the main focus of endemicity of LBRF is localized in East African countries. From July 2015 to October 2015, 36 cases of LBRF have been diagnosed in Europe in immigrants from the Horn of Africa. Here we report a case of LBRF with meningitis diagnosed in Florence, Italy, in an immigrant arrived from Somalia. CASE STUDY: In October 2015, a 19-year-old Somali male presented to the emergency department of the Azienda Ospedaliero Universitaria Careggi, Florence, Italy, with a 3-day history of high fever. The patient had disembarked in Sicily 10 days before admission after a long migration trip from his country of origin. On clinical examination, neck stiffness was found. Main laboratory findings were thrombocytopenia, increased procalcitonin, and increased polymorphonucleates in the cerebrospinal fluid. Suspecting a possible meningitis, the patient was treated with ceftriaxone, pending results of laboratory testing for malaria, and developed severe hypotension that was treated with fluid resuscitation and hydrocortisone. Hemoscopic testing revealed the presence of spirochetes and no malaria parasites. The patient rapidly improved with doxycycline for 7 days and ceftriaxone for 11 days, then was lost to follow-up. Total DNA from blood was extracted, and amplification and sequencing with universal 16S rDNA primers D88 and E94 revealed a 100% identity with B. recurrentis A1. CONCLUSIONS: LBRF is a rare but emerging infectious disease among vulnerable displaced immigrants from the Horn of Africa. Since immigrants from endemic areas can carry the vector with them, the infection should be suspected even in subjects with compatible clinical features living in the same place where new arrival immigrants are hosted. Healthcare providers should be aware of this condition to implement adequate diagnostic, therapeutic, and public health measures.

Large Nosocomial Outbreak of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Traced to Clonal Expansion of an mgrB Deletion Mutant.

We describe a large hospital outbreak (93 bloodstream infections) of colistin-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates which was mirrored by increased colistin consumption. The outbreak was mostly traced to the clonal expansion of an mgrB deletion mutant of an ST512 strain that produced KPC-3.

First Human Case of Meningitis and Sepsis in a Child Caused by Actinobacillus suis or Actinobacillus equuli.

We report the first human case of meningitis and sepsis caused in a child by Actinobacillus suis or A. equuli, a common opportunistic pathogen of swine or horses, respectively. Identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and real-time PCR assay. A previous visit to a farm was suspected as the source of infection.

Intestinal carriage of Shewanella xiamenensis simulating carriage of OXA-48-producing Enterobacteriaceae.

Positivity for bla(OXA-48)-like carbapenemase genes was revealed by molecular testing of a surveillance rectal swab from a patient who had previously been colonized and infected by an OXA-48-producing Klebsiella pneumoniae. Positivity was due to a coincidental carriage of Shewanella xiamenensis harboring a new bla(OXA-48)-like gene, while the K. pneumoniae was no longer present.

Serratia marcescens outbreak in a neonatal intensive care unit: crucial role of implementing hand hygiene among external consultants.

BACKGROUND: Serratia marcescens represents an important pathogen involved in hospital acquired infections. Outbreaks are frequently reported and are difficult to eradicate. The aim of this study is to describe an outbreak of Serratia marcescens occurred from May to November 2012 in a neonatal intensive care unit, to discuss the control measures adopted, addressing the role of molecular biology in routine investigations during the outbreak. METHODS: After an outbreak of Serratia marcescens involving 14 neonates, all admitted patients were screened for rectal and ocular carriage every two weeks. Extensive environmental sampling procedure and hand sampling of the staff were performed. Antimicrobial susceptibility pattern and molecular analysis of isolates were carried out. Effective hand hygiene measures involving all the external consultants has been implemented. Colonized and infected babies were cohorted. Dedicated staff was established to care for the colonized or infected babies. RESULTS: During the surveillance, 65 newborns were sampled obtaining 297 ocular and rectal swabs in five times. Thirty-four Serratia marcescens isolates were collected: 11 out of 34 strains were isolated from eyes, being the remaining 23 isolated from rectal swabs. Two patients presented symptomatic conjunctivitis. Environmental and hand sampling resulted negative. During the fifth sampling procedure no colonized or infected patients have been identified. Two different clones have been identified. CONCLUSIONS: Ocular and rectal colonization played an important role in spread of infections. Implementation of infection control measures, involving also external specialists, allowed to control a serious Serratia marcescens outbreak in a neonatal intensive care unit.

Accuracy of different methods for susceptibility testing of gentamicin with KPC carbapenemase-producing Klebsiella pneumoniae.

Performance of Vitek2, Etest, and TREK broth microdilution (BMD) panels was evaluated versus reference CLSI BMD for gentamicin susceptibility testing with 57 bloodstream isolates of KPC-producing Klebsiella pneumoniae. Compared with reference BMD, the Essential Agreement and Categorical Agreement for TREK panels, Vitek2, and Etest were 91.2%, 31.6%, and 61.4%, respectively, and 86%, 21%, and 52.6%, respectively. Four very major discrepancies occurred with Vitek2. In these 4 strains, gentamicin resistance was associated with the presence of an armA aminoglycoside resistance determinant.

Update on the antibiotic resistance crisis.

Antibiotics tend to lose their efficacy over time due to the emergence and dissemination of resistance among bacterial pathogens. Strains with resistance to multiple antibiotic classes have emerged among major Gram-positive and Gram-negative species including Staphylococcus aureus, Enterococcus spp., Pseudomonas aeruginosa, Acinetobacter spp. Enterobacteriaceae, and Neisseria gonorrhoeae. With some Gram-negatives, resistance may involve most or even all the available antimicrobial options, resulting in extremely drug-resistant or totally drug-resistant phenotypes. This so-called 'antibiotic resistance crisis' has been compounded by the lagging in antibiotic discovery and development programs occurred in recent years, and is jeopardizing the essential role played by antibiotics in current medical practices.

Refractory disseminated fusariosis by Fusarium verticillioides in a patient with acute myeloid leukaemia relapsed after allogeneic hematopoietic stem cell transplantation: a case report and literature review.

BACKGROUND: Fusarium species are among the leading fungal pathogens to cause invasive mould infections in patients with hematopoietic malignancy. The Fusarium species most frequently involved in human infections are Fusarium solani, Fusarium oxysporum and Fusarium verticillioides. However, identification is a cumbersome and time-consuming task. Fusarium is resistant in vitro to many of the antifungal agents and the management of fusariosis is not well defined. OBJECTIVES: To emphasise the difficulty of identifying Fusarium spp. by conventional methods and the need of new rapid molecular tests to achieve earlier diagnosis and appropriate therapy. METHODS: A disseminated Fusarium infection due to F. verticillioides was documented in a neutropenic refractory patient with acute myeloid leukaemia, relapsed after allogeneic hematopoietic stem cell transplantation. RESULTS: The patient died despite liposomal amphotericin B and voriconazole combination and "in vitro" susceptibility of agents employed. Morphological and molecular identification of F. verticillioides was obtained only after the death of the patient. CONCLUSIONS: This case highlights the poor outcome of an invasive fungal disease caused by Fusarium in aplastic patients. Identification of members of Fusarium genus remains restricted to selected laboratories and should be introduced into routine mycological diagnostics. In immunocompromised patients, diagnosis of fusariosis is directly related to prompt diagnosis and to patient's status. Current diagnosis methods and therapeutic options are discussed.