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At the end of 2022 and in the following months, an increase in the incidence of Streptococcus pyogenesinfections was observed in many European countries that was simultaneously accompanying to enhance of invasive infections (iGAS). We have showed a risen trend of S. pyogenesinfections among preschoolers after the pandemic event. A thorough epidemiological investigation of both paediatric and adult samples positive for S. pyogenesindicate a more complex scenario leading to need of important improvement in surveillance programs.

The pandemic marked the beginning of an era of dynamic and rapid changes in the diagnosis of respiratory infections. Herein we describe Legionnaires’ disease trend in the years 2016–2023 in a large Italian hospital showing how improvements in diagnostic algorithms impact on its detection.

We consider a class of semilinear equations with an absorption nonlinear zero order term of power type, where elliptic condition is given in terms of Gauss measure. In the case of the superlinear equation we introduce a suitable definition of solutions in order to prove the existence and uniqueness of a solution in ℝNwithout growth restrictions at infinity. A comparison result in terms of the half-space Gaussian symmetrized problem is also proved. As an application, we give some estimates in measure of the growth of the solution near the boundary of its support for sublinear equations. Finally we generalize our results to problems with a nonlinear zero order term not necessary of power type.

ABSTRACTIntroduction: Clostridium difficileinfection (CDI) is the most common healthcare-associated infection worldwide. As standard CDI antibiotic therapies can result in unacceptably high recurrence rates, novel therapeutic strategies for CDI are necessary. A recently emerged immunological therapy is a monoclonal antibody against C. difficiletoxin B.Areas covered: In this review, the authors summarize the available pharmacological, preclinical, and clinical data for the CDI treatment based on anti-toxin A (actoxumab) and anti-toxin B (bezlotoxumab) human monoclonal antibodies (HuMabs), and discuss about the potentiality of a therapy that includes HuMab combined administration for CDI.Expert opinion: Although only bezlotoxumab is indicated to reduce recurrence of CDI, experimental studies using a combination of HuMabs actoxumab and bezlotoxumab have shown that bolstering the host immune response against both the C. difficiletoxins may be effective in primary and secondary CDI prevention. Besides neutralizing both the key virulence factors, combination of two HuMabs could potentially offer an advantage for a yet to emerge C. difficilestrain, which is a steady threat for patients at high risk of CDI. However, as actoxumab development was halted, passive immunotherapy with actoxumab/bezlotoxumab is actually impracticable. Future research will be needed to assess HuMab combination as a therapeutic strategy in clinical and microbiological cure of CDI.

Mixed vaginitis is defined as the simultaneous presence of at least two different vaginal pathogens, both contributing to an abnormal vaginal milieu leading to signs and symptoms. Pathogen coinfection occurs frequently in women with vaginitis, and both coinfection and mixed vaginitis have relevant clinical and therapeutic implications. Fenticonazole, an imidazole derivative with a broad spectrum of antimycotic and antimicrobial activity, appears at least as effective as other topical antifungals in the treatment of vulvovaginal candidiasis and can also have a major role in the treatment of mixed infections or coinfections of the lower genital tract. This paper will address the current role of topical fenticonazole as an empiric treatment of vulvovaginal infections, with a focus on the effectiveness in the treatment of mixed vaginitis and the possible implications of this.

The virulence of isolates among different Candidaspecies causing candidemia may play a role in the prognosis of the patients. Furthermore, the potential relationship between genotype and virulence is still unclear and need to be further studied.

AbstractObjective:The early identification of gastric cancer (GC) represents a major clinical challenge. We conducted a systematic review of studies evaluating the miRNA expression profiling as a diagnostic tool in GC.Methods:We performed a search of PubMed, ISI Web of Science and SCOPUS databases for studies on diagnostic miRNAs and GC, published in English up to October 2017. Eligibility criteria included case-control studies evaluating blood or tissue-based miRNA expression profiles, and incorporating at least two detection phases (screening and validation).Results:We included 27 eligible studies, that reported on 97 deregulated miRNAs either in blood or tissue, out of which 30 were reported in at least two studies. Among 22 studies on tissue-diagnostic miRNAs, 13 consistently upregulated miRNAs (miR-214, miR-21, miR-103, miR-107, miR-196a, miR-196b, miR-7, miR-135b, miR-222, miR-23b, miR-25, miR-92 and miR-93), and six consistently downregulated miRNAs (miR-148a, miR-375, miR-133b, miR-30a, miR-193a and miR-204) were reported. Ten miRNAs with inconsistent direction of expression in tissues were identified. Among the five studies performed on blood samples, only one miRNA was consistently upregulated (miR-20a).Conclusions:This review shows that some tissue or blood miRNAs may be considered as potential biomarkers for GC diagnosis, that urgently needs to be confirmed from large prospective studies.

There is strong evidence that alcoholism leads to dysbiosis in both humans and animals. However, it is unclear how changes in the intestinal microbiota (IM) relate to ethanol (EtOH)‐induced disruption of gut–liver homeostasis. We investigated this issue using selectively bred Sardinian alcohol‐preferring (sP) rats, a validated animal model of excessive EtOH consumption. Independent groups of male adult sP rats were exposed to the standard, home‐cage 2‐bottle “EtOH (10% v/v) versus water” choice regimen with unlimited access for 24 h/d (Group Et) for 3 (T1), 6 (T2), and 12 (T3) consecutive months. Control groups (Group Ct) were composed of matched‐age EtOH‐naïve sP rats. We obtained samples from each rat at the end of each experimental time, and we used blood and colon tissues for intestinal barrier integrity and/or liver pathology assessments and used stool samples for IM analysis with 16S ribosomal RNA gene sequencing. Rats in Group Et developed hepatic steatosis and elevated serum transaminases and endotoxin/lipopolysaccharide (LPS) levels but no other liver pathological changes (i.e., necrosis/inflammation) or systemic inflammation. While we did not find any apparent alteration of the intestinal colonic mucosa, we found that rats in Group Et exhibited significant changes in IM composition compared to the rats in Group Ct. These changes were sustained throughout T1, T2, and T3. In particular, Ruminococcus,Coprococcus, and Streptococcuswere the differentially abundant microbial genera at T3. The KEGG Ortholog profile revealed that IM functional modules, such as biosynthesis, transport, and export of LPS, were also enriched in Group Et rats at T3. We showed that chronic, voluntary EtOH consumption induced liver injury and endotoxemia together with dysbiotic changes in sP rats. This work sets the stage for improving our knowledge of the prevention and treatment of EtOH‐related diseases.

ABSTRACTAccurate identification of pathogenic species is important for early appropriate patient management, but growing diversity of infectious species/strains makes the identification of clinical yeasts increasingly difficult. Among conventional methods that are commercially available, the API ID32C, AuxaColor, and Vitek 2 systems are currently the most used systems in routine clinical microbiology. We performed a systematic review and meta-analysis to estimate and to compare the accuracy of the three systems, in order to assess whether they are still of value for the species-level identification of medically relevant yeasts. After adopting rigorous selection criteria, we included 26 published studies involving Candidaand non-Candidayeasts that were tested with the API ID32C (674 isolates), AuxaColor (1,740 isolates), and Vitek 2 (2,853 isolates) systems. The random-effects pooled identification ratios at the species level were 0.89 (95% confidence interval [CI], 0.80 to 0.95) for the API ID32C system, 0.89 (95% CI, 0.83 to 0.93) for the AuxaColor system, and 0.93 (95% CI, 0.89 to 0.96) for the Vitek 2 system (Pfor heterogeneity, 0.255). Overall, the accuracy of studies using phenotypic analysis-based comparison methods was comparable to that of studies using molecular analysis-based comparison methods. Subanalysis of studies conducted on Candidayeasts showed that the Vitek 2 system was significantly more accurate (pooled ratio, 0.94 [95% CI, 0.85 to 0.99]) than the API ID32C system (pooled ratio, 0.84 [95% CI, 0.61 to 0.99]) and the AuxaColor system (pooled ratio, 0.76 [95% CI, 0.67 to 0.84]) with respect to uncommon species (Pfor heterogeneity, <0.05). Subanalysis of studies conducted on non-Candidayeasts (i.e., Cryptococcus, Rhodotorula, Saccharomyces, and Trichosporon) revealed pooled identification accuracies of =98% for the Vitek 2, API ID32C (excluding Cryptococcus), and AuxaColor (only Rhodotorula) systems, with significant low or null levels of heterogeneity (P> 0.05). Nonetheless, clinical microbiologists should reconsider the usefulness of these systems, particularly in light of new diagnostic tools such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, which allow for considerably shortened turnaround times and/or avoid the requirement for additional tests for species identity confirmation.

ABSTRACTIn recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of =2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies.

ABSTRACTThe BD Phoenix system was evaluated for species-level identification of yeasts (250 clinical isolates) and compared with the Vitek 2 system, using ribosomal internal transcribed spacer (ITS) sequence analysis as the gold standard. Considering only the species included in each system's database, 96.3% (236/245) and 91.4% (224/245) of the isolates were correctly identified by BD Phoenix and Vitek 2, respectively.

ABSTRACTThe widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of Candida albicansafter a 3-h incubation in the presence of “breakpoint” level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) µg/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65 C. albicansisolates, including 13 fks1mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 µg/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 µg/ml. In this way, the drug resistance of C. albicansisolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant FKS1genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.

ABSTRACTAspergillus fumigatusbiofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitrointeractions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatusbiofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatusbiofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatusbiofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

ABSTRACTMatrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fksmutant isolates of Candidaand Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candidaisolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

ABSTRACTWe evaluated the usefulness of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcusidentification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcusisolates (72 Cryptococcus neoformansand 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

We report the first documented case of a posttraumatic fungal keratitis caused by Neosartorya udagawae. The patient was empirically treated with fluconazole until a corneal scraping grew an Aspergillus fumigatus-like fungus, and itraconazole therapy was then established. A sequence-based approach assigned the isolate to the species. Five months after completion of antifungal therapy, endophthalmitis occurred and orbital exenteration was necessary.

ABSTRACTWe report the first documented case of a posttraumatic fungal keratitis caused by Neosartorya udagawae. The patient was empirically treated with fluconazole until a corneal scraping grew an Aspergillus fumigatus-like fungus, and itraconazole therapy was then established. A sequence-based approach assigned the isolate to the species. Five months after completion of antifungal therapy, endophthalmitis occurred and orbital exenteration was necessary.

Objectives: We evaluated a new approach for the rapid detection of clarithromycin resistance in Helicobacter pylori, based on PCR and denaturing HPLC (DHPLC).Methods: A 180 bp fragment of the 23S rRNA gene was amplified using DNA from 81 clinical H. pylori isolates (51 isolates were shown to be resistant to clarithromycin by Etest), and, directly, from 101 gastric biopsies from patients with digestive diseases, who were infected with H. pylori as assessed by a 13C-urea breath test, histology and/or culture. DHPLC was used to detect mutations in all the PCR products.Results: DHPLC profiles for the 30 susceptible isolates all showed homoduplex peaks; the resistant isolates consistently generated heteroduplex peaks that were easily distinguishable from the wild-type H. pylori reference strain. Sequencing revealed point mutations in all the resistant isolates. Overall, five different mutations were detected. Four of these mutations (A2142G, A2142C, A2143G and T2182C) are known to be associated with clarithromycin resistance; the remaining mutation (C2195T) has not been previously described. This novel single-base substitution was found in combination with the common mutation A2143G. Of the biopsies tested, 25 specimens generated heteroduplexes due to sequence alterations (mutation A2142G, A2142C or A2143G). In one of these specimens, A2143G was found together with the novel mutation T2221C; in another, a mixture of wild-type and mutant (A2143G) sequences was detected. For 20 culture-positive out of the 25 biopsies DHPLC results confirmed the presence of clarithromycin resistance.Conclusions: Our results suggest that the PCR–DHPLC assay is a valid tool for rapid assessment of clarithromycin resistance in H. pylori and that in the future it could be used directly on biopsy specimens, avoiding the need for culture-based methods.

ABSTRACTAlthough to a lesser extent than diagnostic bacteriology, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently revolutionized the diagnostic mycology workflow. With regard to filamentous fungi (or molds), the precise recognition of pathogenic species is important for rapid diagnosis and appropriate treatment, especially for invasive diseases. This review summarizes the current experience with MALDI-TOF MS-based identification of common and uncommon mold species of Aspergillus, Fusarium, Mucorales, dimorphic fungi, and dermatophytes. This experience clearly shows that MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly with common species or typical strains of filamentous fungi.

ABSTRACTAn association between atrial myxoma and left ventricular failure is rarely described, is not completely understood, and may have multiple etiologies. We present a 49-year-old man with no history of cardiovascular disease who was admitted to our hospital with pulmonary edema. He was in atrial fibrillation with rapid ventricular response. Echocardiography showed a 10.5-cm left atrial myxoma, which had been asymptomatic until the onset of congestive heart failure in the presence of severe left ventricular systolic dysfunction. Left ventricular inflow obstruction associated with the giant atrial mass could not be the only cause for acute heart failure.

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.

ABSTRACTDespite the current reliance on blood cultures (BCs), the diagnosis of bloodstream infections (BSIs) can be sped up using new technologies performed directly on positive BC bottles. Two methods (the MALDI BioTyper system and FilmArray blood culture identification [BCID] panel) are potentially applicable. In this study, we performed a large-scale clinical evaluation (1,585 microorganisms from 1,394 BSI episodes) on the combined use of the MALDI BioTyper and FilmArray BCID panel compared to a reference (culture-based) method. As a result, the causative organisms of 97.7% (1,362/1,394) of the BSIs were correctly identified by our MALDI BioTyper and FilmArray BCID-based algorithm. Specifically, 65 (5.3%) out of 1,223 monomicrobial BCs that provided incorrect or invalid identifications with the MALDI BioTyper were accurately detected by the FilmArray BCID panel; additionally, 153 (89.5%) out of 171 polymicrobial BCs achieved complete identification with the FilmArray BCID panel. Conversely, full use of the MALDI BioTyper would have resulted in the identification of only 1 causative organism in 97/171 (56.7%) of the polymicrobial cultures. By applying our diagnostic algorithm, the median time to identification was shortened (19.5 h versus 41.7 h with the reference method; P< 0.001), and the minimized use of the FilmArray BCID panel led to a significant cost savings. Twenty-six out of 31 microorganisms that could not be identified were species/genera not designed to be detected with the FilmArray BCID panel, indicating that subculture was not dispensable for a few of our BSI episodes. In summary, the fast and effective testing of BC bottles is realistically adoptable in the clinical microbiology laboratory workflow, although the usefulness of this testing for the management of BSIs remains to be established.

A 51-year-old woman was admitted to the emergency unit with diffuse headache, visus reduction, and paraesthesias of the trigeminal area and the left hand. Three days after admission she showed shaking chills, vomiting, and sudden onset of fever (39·4°C). Blood cultures were performed soon after fever onset. Fever persisted for the whole day, decreasing slowly after 12 hours. No empirical antibiotic treatment was started in order to better define the diagnosis. Fever completely disappeared the day after. Two blood cultures for aerobes were positive for Chryseobacterium indologenes. The patient was discharged with the diagnosis of transient bacteraemia and transferred to the neurology unit for further investigations. C. indologenesinfections are described in 31 studies with a total of 171 cases (pneumonia and bacteraemia being the most frequent). Our case is the first report of transient bacteraemia caused by C. indologenesin an immunocompetent, non-elderly patient without needing medical devices.

ABSTRACTSensititre YeastOne (SYO) is an affordable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method for antifungal susceptibility testing. In this study, the MICs of yeast isolates from 1,214 bloodstream infection episodes, generated by SYO during hospital laboratory activity (January 2005 to December 2013), were reanalyzed using current CLSI clinical breakpoints/epidemiological cutoff values to assign susceptibility (or the wild-type [WT] phenotype) to systemic antifungal agents. Excluding Candida albicans(57.4% of all isolates [n= 1,250]), the most predominant species were Candida parapsilosiscomplex (20.9%), Candida tropicalis(8.2%), Candida glabrata(6.4%), Candida guilliermondii(1.6%), and Candida krusei(1.3%). Among the non-Candidaspecies (1.9%), 7 were Cryptococcus neoformansand 17 were other species, mainly Rhodotorulaspecies. Over 97% of Candidaisolates were susceptible (WT phenotype) to amphotericin B and flucytosine. Rates of susceptibility (WT phenotype) to fluconazole, itraconazole, and voriconazole were 98.7% in C. albicans, 92.3% in the C. parapsilosiscomplex, 96.1% in C. tropicalis, 92.5% in C. glabrata, 100% in C. guilliermondii, and 100% (excluding fluconazole) in C. krusei. The fluconazole-resistant isolates consisted of 6 C. parapsilosiscomplex isolates, 3 C. glabrataisolates, 2 C. albicansisolates, 2 C. tropicalisisolates, and 1 Candidalusitaniaeisolate. Of the non-Candidaisolates, 2 C. neoformansisolates had the non-WT phenotype for susceptibility to fluconazole, whereas Rhodotorulaisolates had elevated azole MICs. Overall, 99.7% to 99.8% of Candidaisolates were susceptible (WT phenotype) to echinocandins, but 3 isolates were nonsusceptible (either intermediate or resistant) to caspofungin (C. albicans, C. guilliermondii, and C. krusei), anidulafungin (C. albicansand C. guilliermondii), and micafungin (C. albicans). However, when the intrinsically resistant non-Candidaisolates were included, the rate of echinocandin nonsusceptibility reached 1.8%. In summary, the SYO method proved to be able to detect yeast species showing antifungal resistance or reduced susceptibility.

Obesity is rapidly increasing in the USA. It is one of the most serious public health problems that significantly increase the risk of many chronic illnesses. Obesity disproportionately affects people with lower incomes and minority groups and imposes a heavy financial burden on the healthcare system in the USA. Thus, there is a critical need for healthcare policies that are designed to stem the growth of obesity. The aims of this paper are to discuss the cost of obesity to the national healthcare expenditure, and to present and discuss various policies and strategies adopted by several states across the country to prevent the growth of obesity. Additionally, the role of interest groups and political will with respect to formulating healthcare policies on preventing obesity are discussed. A multifaceted approach that incorporates market and cultural forces is recommended to prevent obesity and reduce healthcare costs.

ABSTRACTWe previously showed that the mutant strain of Enterococcus faecalislacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyAoperon, ef_3001, renamed pmvE(for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N1-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalisstrain carrying the recombinant plasmid pMSP3535-pmvE(V19/p3535-pmvE), which allows the induction of pmvEby addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalisin the Galleria mellonellainfection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism.

In the last few years, not many studies on the use of robot-assisted therapy to recover hand function in acute stroke patients have been carried out. This randomized-controlled observer trial is aimed at evaluating the effects of intensive robot-assisted hand therapy compared with intensive occupational therapy in the early recovery phases after stroke with a 3-month follow-up. Twenty acute stroke patients at their first-ever stroke were enrolled and randomized into two groups. The experimental treatment was performed using the Amadeo Robotic System. Control treatment, instead, was carried out using occupational therapy executed by a trained physiotherapist. All participants received 20 sessions of treatment for 4 consecutive weeks (5 daysweek). The following clinical scales, Fugl-Meyer Scale (FM), Medical Research Council Scale for Muscle Strength (hand flexor and extensor muscles) (MRC), Motricity Index (MI) and modified Ashworth Scale for wrist and hand muscles (MAS), were performed at baseline (T0), after 20 sessions (end of treatment) (T1) and at the 3-month follow-up (T2). The Barthel Index was assessed only at T0 and T1. Evidence of a significant improvement was shown by the Friedman test for the FM experimental group (EG): P=0.0039, control group (CG): P<0.0001, Box and Block Test (EG: P=0.0185, CG: P=0.0086), MI (EG: P<0.0001, CG: P=0.0303) and MRC (EG: P<0.0001, CG: P=0.001) scales. These results provide further support to the generalized therapeutic impact of intensive robot-assisted treatment on hand recovery functions in individuals with acute stroke. The robotic rehabilitation treatment may contribute toward the recovery of hand motor function in acute stroke patients. The positive results obtained through the safe and reliable robotic rehabilitation treatment reinforce the recommendation to extend it to a larger clinical practice.

The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicansproteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.

ABSTRACTCandidemia has become an important bloodstream infection that is frequently associated with high rates of mortality and morbidity, and its growing incidence is related to complex medical and surgical procedures. We conducted a multicenter study in five tertiary care teaching hospitals in Italy and Spain and evaluated the epidemiology, species distribution, antifungal susceptibilities, and outcomes of candidemia episodes. In the period of 2008 to 2010, 995 episodes of candidemia were identified in these hospitals. The overall incidence of candidemia was 1.55 cases per 1,000 admissions and remained stable during the 3-year analysis. Candida albicanswas the leading agent of infection (58.4%), followed by Candida parapsilosiscomplex (19.5%), Candida tropicalis(9.3%), and Candida glabrata(8.3%). The majority of the candidemia episodes were found in the internal medicine department (49.6%), followed by the surgical ward, the intensive care unit (ICU), and the hemato-oncology ward. Out of 955 patients who were eligible for evaluation, 381 (39.9%) died within 30 days from the onset of candidemia. Important differences in the 30-day mortality rates were noted between institutions: the lowest mortality rate was in the Barcelona hospital, and the highest rate was in the Udine hospital (33.6% versus 51%, respectively; P= 0.0005). Overall, 5.1% of the 955 isolates tested were resistant or susceptible dose dependent (SDD) to fluconazole, with minor differences between the hospitals in Italy and Spain (5.7% versus 3.5%, respectively; P= 0.2). Higher MICs for caspofungin were found, especially with C. parapsilosiscomplex (MIC90, 1 µg/ml). Amphotericin B had the lowest MICs. This report shows that candidemia is a significant source of morbidity in Europe, causing a substantial burden of disease and mortality.

ABSTRACTWe report the first comparative evaluation between the Bruker Biotyper MS (BMS) and the Vitek MS (VMS) for the identification of yeasts. The rate of correct identifications at the species level was comparable using the commercial databases (89.8% versus 84.3%; P= 0.712), but higher for BMS using an in-house-extended database (100% versus 84.3%; P= 0.245). Importantly, the rate of misidentification was significantly higher for VMS (1% versus 12.1%; P< 0.0001), including the rate of major errors (0% versus 4.5%; P= 0.0036).

Background:Candida mannan (Mn) detection in bronchoalveolar lavage fluid (BALF) was shown to be useful for earlier identification and preemptive therapy targeting in preterm infants at high risk of invasive Candida infection. We investigated whether early detection of Candida Mn in BALF is associated with the presence of some neutrophilic products, as markers of prenatal infection/inflammation.Methods:BALF specimens were collected during the first 48?h of life from mechanically ventilated preterm newborns. Samples were analyzed by high-performance liquid chromatography–electrospray ionization–mass spectrometry. The relative amounts of a-defensins 1–4 and S100A proteins were measured by extracted ion current peak area. Absolute and differential white cell counts in BALF were obtained. Mn antigen concentrations were determined by the Platelia Candida antigen kit.Results:Twenty-five studied neonates were divided into two groups: Mn-positive group and Mn-negative group. Levels of a-defensins 1–4 and S100A12 were significantly higher in the Mn-positive group than in the Mn-negative group. Moreover, positive significant correlations between the absolute number of neutrophils and the levels of a-defensins 1–4 and S100A8 were observed.Conclusion:The detection of Mn antigen in BALF of preterm infants is consistent with evidence of an innate immune response in their lungs as demonstrated by higher levels of a-defensins and S100A proteins.

ABSTRACTEnterococcus faecalisis a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalisto maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published for Staphylococcus aureus(A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672–1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer.

ABSTRACTPeptidylprolyl cis/transisomerases (PPIases) are enzymes involved in protein folding. Analysis of the genome sequence of Enterococcus faecalisV583 allowed for identification of 3 PPIases carrying genes. ef2898encodes an intracellular PPIase which was not shown to be important for the E. faecalisstress response or virulence. The other two PPIases, the parvulin family rotamase EF0685 and the cyclophilin family member EF1534, are expected to be surface-exposed proteins. They were shown to be important for virulence and resistance to NaCl. A ?ef0685?ef1534mutant was also more resistant to oxidative stress, was able to grow under a high manganese concentration, and showed altered resistance to ampicillin and quinolone antibiotics.

Peptidylprolyl cis/trans isomerases (PPIases) are enzymes involved in protein folding. Analysis of the genome sequence of Enterococcus faecalis V583 allowed for identification of 3 PPIases carrying genes. ef2898 encodes an intracellular PPIase which was not shown to be important for the E. faecalis stress response or virulence. The other two PPIases, the parvulin family rotamase EF0685 and the cyclophilin family member EF1534, are expected to be surface-exposed proteins. They were shown to be important for virulence and resistance to NaCl. A Δef0685 Δef1534 mutant was also more resistant to oxidative stress, was able to grow under a high manganese concentration, and showed altered resistance to ampicillin and quinolone antibiotics.

ABSTRACTThe aim of this study was to compare the in vitroand in vivoactivities of micafungin, caspofungin, and anidulafungin against Candida glabrata. The MICs against 28 clinical isolates showed that the overall susceptibilities to caspofungin and to micafungin were not statistically different in the absence of human serum, whereas the isolates were less susceptible to micafungin than to caspofungin in its presence. Minimum fungicidal concentrations, as well as time-kill experiments, showed that caspofungin was more active than anidulafungin, while micafungin was superior to either caspofungin or anidulafungin without serum; its addition rendered caspofungin and micafungin equally effective. A murine model of systemic candidiasis against a C. glabrata-susceptible isolate was performed to study the effects of all three echinocandins, and kidney burden counts showed that caspofungin, micafungin, and anidulafungin were active starting from 0.25, 1, and 5 mg/kg of body weight/day, respectively. Two echinocandin-resistant strains of C. glabratawere selected: C. glabrata30, a laboratory strain harboring the mutation Fks2p-P667T, and C. glabrata51, a clinical isolate harboring the mutation Fks2p-D666G. Micafungin activity was shown to be as effective as or more effective than that of caspofungin or anidulafungin in terms of MICs. In vivostudies against these resistant strains showed that micafungin was active starting from 1 mg/kg/day, while caspofungin was effective only when administrated at higher doses of 5 or 10 mg/kg/day. Although a trend toward colony reduction was observed with the highest doses of anidulafungin, a significant statistical difference was never reached.

ABSTRACTWe evaluated the reliability of the Bruker Daltonik's MALDI Biotyper system in species-level identification of yeasts directly from blood culture bottles. Identification results were concordant with those of the conventional culture-based method for 95.9% of Candida albicans(187/195) and 86.5% of non-albicans Candidaspecies (128/148). Results were available in 30 min (median), suggesting that this approach is a reliable, time-saving tool for routine identification of Candidaspecies causing bloodstream infection.

ABSTRACTCulture-independent molecular techniques such as real-time PCRs offer the potential for early diagnosis of invasive aspergillosis (IA), thereby reducing the disease-associated mortality rate. PCR-based testing is presently excluded from disease-defining consensus criteria due to lack of standardization and clinical validation. A single-center prospective study was conducted to investigate the performance of the commercially available MycAssay Aspergillustest for detecting AspergillusDNA in patients with suspicion of IA. To this end, a total of 158 bronchoalveolar lavage (BAL) fluid specimens that were consecutively collected from hematology (n= 68) and intensive care unit (n= 90) patients were examined. Sixteen of 17 (94.1%) specimens from patients with proven/probable IA were MycAssay positive, and 15 of these 16 patients were also positive by an “in-house” PCR assay. A total of 139 of 141 (98.6%) specimens from patients without proven/probable IA were MycAssay negative. Fifteen of 16 (94.1%) MycAssay-positive patients were also positive for BAL fluid galactomannan (GM) at an index cutoff of =1.0 (index range, 1.1 to 8.3), as were 3 patients without IA but with pulmonary fusariosis. Interestingly, in seven of the PCR-positive BAL specimens that tested culture positive for Aspergillusspecies, cycle threshold values were earlier than those of specimens with a culture-negative result. In conclusion, the MycAssay AspergillusPCR appears to be a sensitive and specific molecular test for the diagnosis of IA, and its performance is comparable to that of the GM assay. However, more large studies are necessary to firmly establish its clinical utility in high-risk settings.

ABSTRACTPhylogenetic analysis of the crystal structure of the Enterococcus faecalisSlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalisbiology, we dissected the genetic organization of the slyA-EF_3001 locus and constructed a slyAdeletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyAmutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyAmutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

Phylogenetic analysis of the crystal structure of the Enterococcus faecalis SlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalis biology, we dissected the genetic organization of the slyA-EF_3001 locus and constructed a slyA deletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyA mutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyA mutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

We tested the activities of anidulafungin and other antifungal agents against clinical isolates of different fungal species. For Candida species, high sessile MIC90s (SMIC90s) were obtained for fluconazole, voriconazole, and amphotericin B, whereas the anidulafungin SMIC90s were very low, as were those for caspofungin. Comparatively, for Aspergillus species, higher SMIC90values were obtained not only for amphotericin B and voriconazole but also for the echinocandins.

ABSTRACTWe tested the activities of anidulafungin and other antifungal agents against clinical isolates of different fungal species. For Candidaspecies, high sessile MIC90s (SMIC90s) were obtained for fluconazole, voriconazole, and amphotericin B, whereas the anidulafungin SMIC90s were very low, as were those for caspofungin. Comparatively, for Aspergillusspecies, higher SMIC90values were obtained not only for amphotericin B and voriconazole but also for the echinocandins.

ABSTRACTMitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called “petite mutants”) upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabratamutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrataisolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1(CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitrogrowth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitrogrowth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivounder azole therapy, even if strongly affecting in vitrogrowth characteristics, can confer a selective advantage under host conditions, allowing the C. glabratamutant to be more virulent than wild-type isolates.

ABSTRACTMethionine sulfoxide reductases A and B are antioxidant repair enzymes that reduce the S- and R-diastereomers of methionine sulfoxides back to methionine, respectively. Enterococcus faecalis, an important nosocomial pathogen, has one msrAgene and one msrBgene situated in different parts of the chromosome. Promoters have been mapped and mutants have been constructed in two E. faecalisstrains (strains JH2-2 and V583) and characterized. For both backgrounds, the mutants are more sensitive than the wild-type parents to exposure to H2O2, and in combination the mutations seem to be additive. The virulence of the mutants has been analyzed in four different models. Survival of the mutants inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide but not in naïve phagocytes is significantly affected. The msrAmutant is attenuated in the Galleria mellonellainsect model. Deficiency in either Msr enzyme reduced the level of virulence in a systemic and urinary tract infection model. Virulence was reconstituted in the complemented strains. The combined results show that Msr repair enzymes are important for the oxidative stress response, macrophage survival, and persistent infection with E. faecalis.

Methionine sulfoxide reductases A and B are antioxidant repair enzymes that reduce the S- and R-diastereomers of methionine sulfoxides back to methionine, respectively. Enterococcus faecalis, an important nosocomial pathogen, has one msrA gene and one msrB gene situated in different parts of the chromosome. Promoters have been mapped and mutants have been constructed in two E. faecalis strains (strains JH2-2 and V583) and characterized. For both backgrounds, the mutants are more sensitive than the wild-type parents to exposure to H2O2, and in combination the mutations seem to be additive. The virulence of the mutants has been analyzed in four different models. Survival of the mutants inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide but not in naïve phagocytes is significantly affected. The msrA mutant is attenuated in the Galleria mellonella insect model. Deficiency in either Msr enzyme reduced the level of virulence in a systemic and urinary tract infection model. Virulence was reconstituted in the complemented strains. The combined results show that Msr repair enzymes are important for the oxidative stress response, macrophage survival, and persistent infection with E. faecalis.

Candidacolonization is an important predictor for development of invasive fungal infection (IFI). We investigated whether early detection of Candidamannan (Mn) in bronchoalveolar lavage fluid (BALF) reduces IFI among preterm infants.

Spinal epidural abscess (SEA) is a rare condition that can be fatal if untreated. Risk factors are immunocompromised states as well as spinal procedures including epidural anesthesia and spinal surgery. The signs and symptoms of SEA are nonspecific and can range from low back pain to sepsis. The treatment of choice is surgical decompression followed by four to six weeks of antibiotic therapy. The most common causative organism in SEA is staphylococcus aureus and spread is usually haematogenous or contiguous from psoas, paraspinal or retropharyngeal abscesses. The exact mechanism by which an epidural abscess causes spinal cord damage is unclear. In fact, the damage is often out of proportion to the degree of compression demonstrated radiologically. There is only a report of a patient with syrinx formation secondary to epidural abscess. We describe the case of a 48-year-old woman with a two-week history of thoracic back pain and evidence of dorsal SEA probably from contiguous psoas abscess. Neurological examination revealed flaccid paraplegia and loss of sphincter control. A spinal MRI scan with Gd-enhancement revealed focal high intensity signal in the T2-weighted and FLAIR images at the level of the vertebral bodies in segments D3-D11. The patient was treated with posterior decompression and drainage of the SEA, but with a poor outcome. Six weeks after the onset of symptoms, an MRI scan showed a newly-formed hydromyelia formation from D4 to D8. The case reported is the second to describe hydromyelia formation secondary to epidural abscess and a poor outcome, experiencing partial improvement without recovery. For this reason, we confirmed that the essential problem of SEA lies in the need for early diagnosis, because the early signs and symptoms may be vague and the “classic” triad of back pain, fever and variable neurological deficits occur in only 13% of patients by the time of diagnosis. Only timely treatment will avoid or reduce permanent neurological deficits before massive neurological symptoms occur. The clear message is that a high index of suspicion and modern imaging techniques are essential.