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Background Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. Aims We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. Methods We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. Results CAGTA ≥50 μg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥80 μg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90 min. Conclusions This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections.

Oropharyngeal candidiasis is a common opportunistic mucosal infection of the oral cavity, mainly caused by an overgrowth of Candida albicans. This infection can inhibit nutritional intakes and strongly affect quality of life. To date, standard therapeutic strategies involving the administration of antifungal drugs can bring several side effects, not least the emergence of drug-resistant strains. The purpose of this study is to investigate the effectiveness of Saccharomyces cerevisiae CNCM I-3856 (live or inactivated cells) against oropharyngeal candidiasis. Our results show that administration of S. cerevisiae CNCM I-3856 (live or inactivated cells) in the oral cavity of C57BL/6J mice resulted in a protective effect against oropharyngeal candidiasis. The strongest effect was obtained with live S. cerevisiae CNCM I-3856. This was related to: (1) a decrease in C. albicans load in the oral cavity, esophagus, stomach, and duodenum; (2) an early resolution of inflammatory process in the tongue; (3) a marked reduction in C. albicans virulence factors; and (4) a consistent increase in neutrophil antimicrobial capacity. These findings suggest that S. cerevisiae products are potentially beneficial in the treatment of oropharyngeal candidiasis.

Background/Aims: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins. Methods: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays. Results: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial. Conclusion: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs.

Endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are found pathogens, like bacteria and opportunistic fungi responsible for various endodontic pathologies. As clinical importance is the fact, that biofilm is extremely resistant to common intracanal irrigants, antimicrobial drugs and host immune responses. The aim of this study was to evaluate the in vitro efficacy of a Cu/CaOH

Peri-implantitis&mdash, an infection caused by bacterial deposition of biofilm&mdash, is a common complication in dentistry which may lead to implant loss. Several decontamination procedures have been investigated to identify the optimal approach being capable to remove the bacterial biofilm without modifying the implant surface properties. Our study evaluated whether two different systems&mdash, Ni-Ti Brushes (Brush) and Air-Polishing with 40 &micro, m bicarbonate powder (Bic40)&mdash, might alter the physical/chemical features of two different titanium surfaces&mdash, machined (MCH) and Ca++ nanostructured (NCA)&mdash, and whether these decontamination systems may affect the biological properties of human STRO-1+/c-Kit+ dental pulp stem cells (hDPSCs) as well as the bacterial ability to produce biofilm. Cell morphology, proliferation and stemness markers were analysed in hDPSCs grown on both surfaces, before and after the decontamination treatments. Our findings highlighted that Bic40 treatment either maintained the surface characteristics of both implants and allowed hDPSCs to proliferate and preserve their stemness properties. Moreover, Bic40 treatment proved effective in removing bacterial biofilm from both titanium surfaces and consistently limited the biofilm re-growth. In conclusion, our data suggest that Bic40 treatment may operatively clean smooth and rough surfaces without altering their properties and, consequently, offer favourable conditions for reparative cells to hold their biological properties.

Invasive candidiasis (IC) plays an important role as severe infection. Elder population, immunocompromised individuals, and intensive care unit (ICU) patients, especially when exposed to major surgery, are the most affected. IC diagnosis and treatment are difficult because of the absence of pathognomonic signs and symptoms. In addition, culture-based examination (gold standard) is known to have low sensitivity and long time to report. All these often lead to unnecessary and costly empirical antifungal therapies, burdened also by the onset of drug resistance and serious side effects for the patient. To partially overcome these problems, in recent years, novel noncultural markers have been investigated with the aim of easily and rapidly achieving an early diagnosis of IC. Such novel markers include the pan-fungal antigen (1 → 3)-β-D-glucan (BDG) and the anti–Candida albicans germ tube antibodies (CAGTAs). We retrospectively analyzed the presence of CAGTA on −80 °C stored serum samples, where the level of BDG had been previously assessed in a prospective study conducted in the Azienda Ospedaliero–Universitaria Policlinic of Modena (Pini et al. Infection 44:223–233, 2016). In particular, we selected 29 samples from proven IC episodes and 28 from non-IC cases. The 29 IC samples had been diagnosed as infections by C. albicans (n = 16), C. glabrata (n = 8), C. parapsilosis (n = 1), C. pelliculosa (n = 1), and C. tropicalis (n = 1), while 2 samples had intrasurgery biopsies positive for yeast (compatible with Candida spp.). The 28 control samples (non-IC) included 9 sera with positive blood cultures [E. faecium (n = 5), S. pneumoniae (n = 2), P. aeruginosa + A. baumannii (n = 2)] and 19 negative blood cultures. The CAGTA immunofluorescence assay was performed using 1:40, 1:80, 1:160, and 1:320 dilutions (reference dilution, as indicated by the manufacturer). According to the protocol, the samples were evaluated by the operator-dependent optical reading based on immunofluorescence positive/negative samples. In parallel, with the aim of standardizing the reading, the fluorescence images were captured, and the data were expressed as arbitrary fluorescence units (AFU). Finally, the results were interpreted as positive or negative using a cutoff provided by receiver operating characteristic (ROC) curves (Youden index). The traditional operator-dependent optical reading and the AFU measuring protocol provided comparable information with respect to the processed samples since IC and non-IC sera were correctly identified by the 2 CAGTA reading strategies in most of the cases. Interestingly, the AFU reading enabled a semiquantitative evaluation of the samples and an objective interpretation of the results. Based on the cutoff value, the AFU-based CAGTA procedure demonstrated a sensitivity of 52% and a specificity of 89%, while BDG showed a sensitivity of 90% and a specificity of 75%; the overall accuracy was 70% and 83% for CAGTA and BDG, respectively. The association of the 2 markers greatly increased both sensitivity and accuracy to 97% and 84%, respectively. As expected, when excluding non–C. albicans episodes, the sensitivity of CAGTA increased from 52% to 86%; moreover, with the exclusion of the non–deep-seated episodes, the sensitivity of CAGTA increased to 67% and reached 100% for C. albicans deep-seated candidiasis. Finally, when evaluating the influence of colonization, BDG demonstrated the most drastic decrease in specificity that dropped from 88% in noncolonized to 58% in colonized patients. With the exception of non–C. albicans episodes, CAGTA is a good marker of IC, particularly in the presence of deep-seated candidiasis. The performance of CAGTA greatly increases when used in combination with BDG.

Background VCN-01 is an oncolytic adenovirus with replication restricted to cells with a nonfunctional retinoblastoma pathway. Upon selective replication VCN-01 expresses the matrix remodeling-enzyme hyaluronidase to enhance virus spreading and tumor uptake of different therapeutics, including immune check-point inhibitors. In a phase I performed in pancreatic carcinoma VCN-01 reached tumors after systemic administration and induced CD8-infiltration, tumor inflammation and PD-1/PD-L1 up-regulation. We hypothesize these intratumor effects may help to overcome the observed resistance to Durvalumab and other PD-(L)-1 checkpoint inhibitors Trial Design NCT03799744 is a multi-center, open-label dose-escalation phase I study evaluating the safety, tolerability and efficacy of the combination of VCN-01 plus durvalumab in R/M HNSCC patients who have progressed on prior PD-(L)-1 checkpoint inhibitors. Patients need to have neutralizing antibodies levels against adenovirus ≤1/350 dilution to be included. The study follows a 3 + 3 design with two dose levels for VCN-01 (3,3.1012 & 1.1013 vp) combined with Durvalumab at a fixed dose of 1500mg intravenous (i.v.). Two treatment arms are explored: I) Concomitant single i.v. dose of VCN-01 with durvalumab on cycle 1 day 1 followed by durvalumab Q4W; II) Sequential single i.v. VCN-01 on cycle 1 day -14 plus durvalumab on cycle 1 day 1 followed by durvalumab Q4W. Patients continue durvalumab Q4W until disease progression, unacceptable toxicity or withdrawal of consent. The primary objective of the study is to evaluate the safety and tolerability of VCN-01 with durvalumab in the two regimens of administration and to establish the recommended phase II dose. Secondary objectives are progression free survival, overall response rate by irRECIST /RECIST, VCN-01 pharmacokinetics and shedding in blood. Other exploratory biomarkers will be analyzed in tumor biopsies (immune markers), serum (hyaluronidase levels), stool (microbiome) and imaging (dynamic contrast-enhanced MRI, diffusion weighted imaging and T2 mapping). The study opened recruitment in April 2019 with 7 out of 18 planned patients enrolled at time of submission. Clinical trial identification NCT03799744. Legal entity responsible for the study Catalan Institute of Oncology (ICO). Funding VCN Biosciences, AstraZeneca. Disclosure M. Jove: Advisory / Consultancy: Boehringer Ingelheim. I. Brana: Advisory / Consultancy, Research grant / Funding (self): Orion Pharma; Speaker Bureau / Expert testimony, Research grant / Funding (self): Bristol-Myers Squibb; Speaker Bureau / Expert testimony, Research grant / Funding (self), Travel / Accommodation / Expenses: AstraZeneca; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Merck Serono; Research grant / Funding (self): Celgene; Research grant / Funding (self): Gliknik; Research grant / Funding (self): GSK; Research grant / Funding (self): Janssen; Research grant / Funding (self): KURA; Research grant / Funding (self): MSD; Research grant / Funding (self): Novartis; Research grant / Funding (self): Pfizer; Research grant / Funding (self): Shattuck; Research grant / Funding (self): Northern Biologics; Research grant / Funding (self): Rakutan Aspirian; Research grant / Funding (self): Naobiotics. M. Taberna: Honoraria (self), Advisory / Consultancy, Non-remunerated activity/ies: Merck; Honoraria (self), Non-remunerated activity/ies: AstraZeneca; Honoraria (self): Bristol-Myers Squibb; Advisory / Consultancy: Nanobiotics. E. Garralda: Leadership role, Research grant / Funding (institution): Novartis; Leadership role: Principia Biopharma Inc; Leadership role: Lilly, S.A; Advisory / Consultancy, Leadership role: Roche / Genentech Inc; Leadership role: Loxo Oncology Inc; Advisory / Consultancy, Leadership role: F.Hoffmann La Roche Ltd; Leadership role: Symphogen A/S; Speaker Bureau / Expert testimony, Leadership role, Travel / Accommodation / Expenses: MSD; Leadership role: Incyte; Leadership role: Pharma Mar; Leadership role: Kura Oncology; Leadership role: Macrogenics; Leadership role, Travel / Accommodation / Expenses: Glycotope; Leadership role: Pierre Fabre; Leadership role: Cellestia Biotech; Leadership role, Travel / Accommodation / Expenses: Menarini Ricerche; Leadership role: Blueprint Medicines; Leadership role: Beigene; Leadership role: Sierra Oncology; Leadership role: Genmab; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Bristol-Myers Squibb; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Janssen Global Services ; Advisory / Consultancy: Ellipses Pharma; Advisory / Consultancy: NeoMed Therapeutics; Advisory / Consultancy: SeaGen; Speaker Bureau / Expert testimony: ThermoFisher. G. Capella: Advisory / Consultancy, Shareholder / Stockholder / Stock options: VCN Biosciences. R. Alemany: Advisory / Consultancy, Shareholder / Stockholder / Stock options: VCN Biosciences; Research grant / Funding (institution): Lokon Pharma; Research grant / Funding (institution): Mologen. E. Blasi: Full / Part-time employment: VCN Biosciences. C. Blasco: Full / Part-time employment: VCN Biosciences. M. Cascallo Piqueras: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: VCN Biosciences. R. Mesia Nin: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck Serono; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Nanobiotix; Advisory / Consultancy: Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche. All other authors have declared no conflicts of interest.

Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule.

Background Expression of hyaluronidase from the selective oncolytic adenovirus VCN-01 modifies tumor matrix in pancreatic tumours. Such effect enhances gemcitabine uptake and induces potent immunomodulatory signals that could help recruit CD8+-T cells. Methods VCN-01 was administered as a single IV injection to 40 patients with ECOG 0-1 at doses ranging from 1E11 to 1E13 viral particles per patient (vp) as a single agent in patients with advanced solid tumors or in combination with gemcitabine/nab-paclitaxel in patients with advanced pancreatic adenocarcinoma using two independent administration regimens (concomitant or sequential). Blood levels of VCN-01 DNA and hyaluronidase together with several immunological markers and VCN-01 presence in paired tumor biopsies were measured. Response was assessed using RECIST v1.1 criteria. Results The most commonly reported VCN-01-associated adverse events of grade 3 or higher included transaminase increase, thrombocytopenia and neutropenia, accounting for 27% and 10% of patients respectively for the combination of VCN-01 with gemcitabine/nab-paclitaxel in the concomitant and the sequential regimen. Recommended Phase 2 Dose (RP2D) was 1E+13vp/patient both in monotherapy and in combination with chemotherapy in a sequential regimen whereas 3.3E+12vp was the RP2D for the concomitant regimen. VCN-01 reached and replicated in tumors when administered systemically. VCN-01 dose correlated with hyaluronidase positivity in sera and sustained viremia for over 3 weeks indicating replication. VCN-01 administration changed tumour environment into a more pro-inflammatory state, inducing CD8 T-cells infiltration and upregulation of IDO in 64% patients. RECIST evaluation resulted in a response rate between 40-45% ORR depending on regimen including a complete response and several partial responses. Conclusions VCN-01 shows a good safety profile when administered systemically in combination with the standard of care for pancreatic cancer. Its ability to reach systemically the tumours and modify tumour matrix to favour immune infiltration results in evidences of clinical activity. Clinical trial identification 2012-005555-16. Legal entity responsible for the study VCN Biosciences. Funding VCN Biosciences. Disclosure R. Garcia-Carbonero: Advisory / Consultancy: AAA; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy, Research grant / Funding (institution): Ipsen; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): MSD; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Pharma Mar; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Sanofi; Advisory / Consultancy, Research grant / Funding (institution): Servier; Research grant / Funding (institution): ARMO Biosciences; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Pharmacyclics; Research grant / Funding (institution): Boston Medicals; Research grant / Funding (institution): Boehringer; Research grant / Funding (institution): VCN Biosciences. M. Gil Martin: Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony: PharmaMar; Speaker Bureau / Expert testimony: AstraZeneca. R. Alvarez Gallego: Advisory / Consultancy: Celgene; Advisory / Consultancy, Research grant / Funding (institution): Shire. T. Macarulla Mercade: Advisory / Consultancy: Servier; Advisory / Consultancy: Shire; Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Baxter; Advisory / Consultancy, Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Agios; Research grant / Funding (institution): Aslan; Advisory / Consultancy: AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Halozyme; Research grant / Funding (institution): Immunomedics; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Merimarck; Research grant / Funding (institution): Millenium; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Pharmacyclics. M.C. Riesco Martinez: Speaker Bureau / Expert testimony: Merck. C. Guillen-Ponce: Research grant / Funding (institution): BeiGene; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): PH Research; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Erytech Pharma; Research grant / Funding (institution): VCN Biosciences; Research grant / Funding (institution): Halozyme. N. Vidal: Research grant / Funding (institution): VCN Biosciences. F.X. Real: Speaker Bureau / Expert testimony: Roche; Research grant / Funding (institution): VCN Biosciences. V. Maliandi: Full / Part-time employment: VCN Biosciences. A. Mato-Berciano: Full / Part-time employment: VCN Biosciences. M. Bazan-Peregrino: Full / Part-time employment: VCN Biosciences. G. Capella: Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: VCN Biosciences. R. Alemany: Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: VCN Biosciences; Research grant / Funding (institution): Lokon Pharma; Research grant / Funding (institution): Mologen. E. Blasi: Full / Part-time employment: VCN Biosciences. C. Blasco: Full / Part-time employment: VCN Biosciences. M. Cascallo: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: VCN Biosciences. R. Salazar: Advisory / Consultancy, Research grant / Funding (institution): VCN Biosciences; Advisory / Consultancy: Agendia; Advisory / Consultancy: Guardiant Health; Advisory / Consultancy, Research grant / Funding (institution): Roche Diagnostics; Advisory / Consultancy: Ferrer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Ipsen; Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): Roche Pharma; Advisory / Consultancy: Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Speaker Bureau / Expert testimony: AZD; Speaker Bureau / Expert testimony: Celgene; Leadership role, Shareholder / Stockholder / Stock options: Sace MedHealth; Research grant / Funding (institution): PsiOxus; Research grant / Funding (institution): Mologen. All other authors have declared no conflicts of interest.

Background Dense stroma is a hallmark of hard-to-treat cancers such as pancreatic adenocarcinoma. VCN-01 oncolytic adenovirus is designed to replicate in cancer cells with dysfunctional RB1 pathway, improve tumor targeting and express hyaluronidase to enhance virus intratumor spreading and facilitate drug extravasation into the tumor. Methods The mechanisms of action of VCN-01 were tested in pancreatic xenograft models after both intravenous and intratumor administration. In a proof of concept phase I clinical trial study (8 patients), VCN-01 was escalated up to 1E+11vp per Endoscopic Ultrasound (EUS)-guided intratumor injection (3x) combined with the standard of care, SoC (gemcitabine or nab-paclitaxel/gemcitabine) in patients with advanced pancreatic adenocarcinoma. Results VCN-01 alone showed antitumor efficacy in preclinical models and further increased its efficacy when combined with SoC chemotherapy. VCN-01 replicated within injected tumors regardless of chemotherapy. Hyaluronidase expression by VCN-01 facilitated tumor extravasation and increased uptake of chemotherapy agents and antibodies. Data obtained from a phase 1 clinical trial of VCN-01 administered by repeated intratumor injection showed that the combination treatment was generally well-tolerated although dose limiting toxicity was found at highest dose. When combined with chemotherapy, VCN-01 stabilized the injected lesion in all patients, with a patient showing long progression free survival of 134 weeks. VCN-01 was detected as secondary peaks in blood several days after injection and in tumors at day 20-28, indicating virus replication. Hyaluronidase PH20 presence in sera was confirmed for all patients injected at 1E+11vp. Elastography of VCN-01-treated tumors evidenced reduction in tumor stiffness regardless of nab-paclitaxel co-administration. Conclusions VCN-01 replicates and expresses hyaluronidase after intratumor injection both in preclinical models and in patients with pancreatic cancer. Enhanced chemotherapy access and reduction of tumor stiffness favours local control of pancreatic tumours treated with VCN-01 + standard of care. Clinical trial identification EudraCT: 2012-005556-42. Legal entity responsible for the study VCN Biosciences. Funding VCN Biosciences. Disclosure M. Hidalgo: Advisory / Consultancy, Speaker Bureau / Expert testimony: Celgene; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy: Novartis; Advisory / Consultancy: MSD; Advisory / Consultancy, Research grant / Funding (institution): EMD; Speaker Bureau / Expert testimony: Ipsem; Speaker Bureau / Expert testimony: Shire; Advisory / Consultancy: SOBI; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Champions Oncology; Advisory / Consultancy: Agenus; Advisory / Consultancy: Erytech; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Pharmacyte; Advisory / Consultancy: Bioline; Advisory / Consultancy, Shareholder / Stockholder / Stock options: BioOncotech; Advisory / Consultancy, Research grant / Funding (institution): Oncomatrix; Advisory / Consultancy: VCN Biosciences; Advisory / Consultancy: Bayer; Advisory / Consultancy: BMS; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Nelum; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Eng T Cells. M. Bazan-Peregrino: Full / Part-time employment: VCN Biosciences. B. Laquente: Advisory / Consultancy, Research grant / Funding (institution): Celgene; Advisory / Consultancy: Servier; Research grant / Funding (institution): Mylan. R. Alvarez Gallego: Advisory / Consultancy: Celgene; Advisory / Consultancy, Research grant / Funding (institution): Shire. A. Mato-Berciano: Full / Part-time employment: VCN Biosciences. M. Gimenez-Alejandre: Full / Part-time employment: VCN Biosciences. V. Maliandi: Full / Part-time employment: VCN Biosciences. M.C. Riesco Martinez: Speaker Bureau / Expert testimony: Merck. M. Perez-Carreras: Advisory / Consultancy: Intercept; Research grant / Funding (institution): GENFIT. G. Capella: Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: VCN Biosciences. R. Alemany: Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: VCN Biosciences; Research grant / Funding (institution): Lokon Pharma; Research grant / Funding (institution): Mologen. R. Salazar: Advisory / Consultancy, Research grant / Funding (institution): VCN Biosciences; Advisory / Consultancy: Agendia; Advisory / Consultancy: Guardant Health; Advisory / Consultancy, Research grant / Funding (institution): Roche Diagnostics; Advisory / Consultancy: Ferrer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Ipsen; Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): Roche Farma; Advisory / Consultancy: Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Speaker Bureau / Expert testimony: AZD; Speaker Bureau / Expert testimony: Celgene; Leadership role, Shareholder / Stockholder / Stock options: Sace MedHealth; Research grant / Funding (institution): PsiOxus; Research grant / Funding (institution): Mologen. E. Blasi: Full / Part-time employment: VCN Biosciences. C. Blasco: Full / Part-time employment: VCN Biosciences. M. Cascallo: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: VCN Biosciences. R. Garcia-Carbonero: Advisory / Consultancy: AAA; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy, Research grant / Funding (institution): Ipsen; Advisory / Consultancy: Lilly; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): MSD; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: PharmaMar; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Sanofi; Advisory / Consultancy: Servier; Research grant / Funding (institution): ARMO; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Pharmacyclics; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Boehringer; Research grant / Funding (institution): VCN Biosciences. All other authors have declared no conflicts of interest.

The transition from commensalism to pathogenicity of Candida albicans reflects both the host inability to mount specific immune responses and the microorganism’s dimorphic switch efficiency. In this study, we used whole genome sequencing and microarray analysis to investigate the genomic determinants of the phenotypic changes observed in two C. albicans clinical isolates (YL1 and YQ2). In vitro experiments employing epithelial, microglial, and peripheral blood mononuclear cells were thus used to evaluate C. albicans isolates interaction with first line host defenses, measuring adhesion, susceptibility to phagocytosis, and induction of secretory responses. Moreover, a murine model of peritoneal infection was used to compare the in vivo pathogenic potential of the two isolates. Genome sequence and gene expression analysis of C. albicans YL1 and YQ2 showed significant changes in cellular pathways involved in environmental stress response, adhesion, filamentous growth, invasiveness, and dimorphic transition. This was in accordance with the observed marked phenotypic differences in biofilm production, dimorphic switch efficiency, cell adhesion, invasion, and survival to phagocyte-mediated host defenses. The mutations in key regulators of the hyphal growth pathway in the more virulent strain corresponded to an overall greater number of budding yeast cells released. Compared to YQ2, YL1 consistently showed enhanced pathogenic potential, since in vitro, it was less susceptible to ingestion by phagocytic cells and more efficient in invading epithelial cells, while in vivo YL1 was more effective than YQ2 in recruiting inflammatory cells, eliciting IL-1β response and eluding phagocytic cells. Overall, these results indicate an unexpected isolate-specific variation in pathways important for host invasion and colonization, showing how the genetic background of C. albicans may greatly affect its behavior both in vitro and in vivo. Based on this approach, we propose that the co-occurrence of changes in sequence and expression in genes and pathways driving dimorphic transition and pathogenicity reflects a selective balance between traits favoring dissemination of the pathogen and traits involved in host defense evasion. This study highlights the importance of investigating strain-level, rather than species level, differences, when determining fungal–host interactions and defining commensal or pathogen behavior.

Respiratory samples from Pneumocystis jirovecii pneumonia (PJP) cases collected at a tertiary-care university hospital in Modena were analyzed for the presence of specific polymorphisms in the mitochondrial large subunit ribosomal RNA (mtLSU-rRNA). Retrospectively, 57 cases were selected in a six-year period and 34 out of the 57 processed BAL samples returned PCR positive results, thus allowing further molecular analysis. The following P.jirovecii genotype distribution was observed: genotype 3 (50%), genotype 2 (23%), genotype 1 (18%), genotypes 1 or 4 (9%). These data add novel insights on P.jirovecii epidemiology, investigating a previously unstudied area of Northern Italy. A peculiar local distribution is highlighted with respect to other areas within the national panorama, thus encouraging further in-depth studies in an attempt to better understand the overall situation concerning P.jirovecii genotype circulation.

Background Recently, we published data suggesting a mutualistic relationship between HSV-1 and Candida. albicans; in particular: (a) HSV-1 infected macrophages are inhibited in their anti-Candida effector function and (b) Candida biofilm protects HSV-1 from inactivation. The present in vitro study is aimed at testing the effects of Candida biofilm on HSV-1 sensitivity to pharmacological and physical stress, such as antiviral drugs (acyclovir and foscarnet) and laser UVA1 irradiation. We also investigated whether fungus growth pattern, either sessile or planktonic, influences HSV-1 sensitivity to antivirals. Methods Mature Candida biofilms were exposed to HSV-1 and then irradiated with laser light (UVA1, 355 λ). In another set of experiments, mature Candida biofilm were co-cultured with HSV-1 infected VERO cells in the presence of different concentrations of acyclovir or foscarnet. In both protocols, controls unexposed to laser or drugs were included. The viral yield of treated and untreated samples was evaluated by end-point titration. To evaluate whether this protective effect might occur in relation with a different growth pattern, HSV-1 infected cells were co-cultured with either sessile or planktonic forms of Candida and then assessed for susceptibility to antiviral drugs. Results UVA1 irradiation caused a 2 Log reduction of virus yield in the control cultures whereas the reduction was only 1 Log with Candida biofilm, regardless to the laser dose applied to the experimental samples (50 or 100 J/cm2). The presence of biofilm increased the IC90 from 18.4–25.6 J/cm2. Acyclovir caused a 2.3 Log reduction of virus yield in the control cultures whereas with Candida biofilm the reduction was only 0.5 Log; foscarnet determined a reduction of 1.4 Log in the controls and 0.2 Log in biofilm cultures. Consequently, the ICs50 for acyclovir and foscarnet increased by 4- and 12-folds, respectively, compared to controls. When HSV-1 was exposed to either sessile or planktonic fungal cells, the antiviral treatments caused approximately the same weak reduction of virus yield. Conclusions These data demonstrate that: (1) HSV-1 encompassed in Candida biofilm is protected from inactivation by physical (laser) and pharmacological (acyclovir or foscarnet) treatments; (2) the drug antiviral activity is reduced at a similar extent for both sessile or planktonic Candida.

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.

The detection of Aspergillus antigen (galactomannan) is considered a reliable marker for the diagnosis of invasive aspergillosis (IA), yet the sensibility and specificity of the assays commonly employed in routine are not optimal. The aim of the present study was to investigate whether the detection of another panfungal antigen, the (1,3)-b-D-glucan could have an auxiliary role in the identification of patients with IA. The study was carried out on 63 sera belonging to patients who had been screened for galactomannan, according to the clinical suspect of IA. Our data show that the positive galactomannan results were not confirmed by positive (1,3)-b-D-glucan results in patients receiving therapy with beta-lactam antibiotics associated with tazobactam, whereas in all the other cases, with the exception of four, the results of the (1,3)-b-D-glucan test were confirmatory of the galactomannan results.

Purpose: We investigated the clinical performance of (1 → 3)-β-d-glucan (BG), as an early marker of invasive fungal infections (IFI), in different clinical settings. Methods: BG serum levels were assessed by Fungitell (Associates of Cape Cod, Inc), in parallel with galactomannan (GM) when requested by clinicians. By a prospective monocentric study, 270 episodes at risk or with suspect of IFI were enrolled, namely 58 proven-probable invasive aspergillosis (IA), 27 proven invasive candidiasis (IC), 11 possible IC, 16 P.jirovecii pneumonia (PJP), 4 episodes of other IFI and 154 non-IFI controls. Results: We found that (a) the BG overall sensitivity, specificity, positive predictive value and negative predictive value (NPV) were 87.9, 80.5, 76.7 and 89.9 %, respectively; (b) the highest sensitivity was found in the IC groups, followed by PJP, IA and other IFI groups; (c) an association was observed between BG kinetics and patients outcome; (d) in the IA episodes, the combination of BG or GM vs GM alone increased sensitivity from 60.0 to 83.3 % in the haematological patients; (e) false-positive BG results were related to Gram-negative infections or infusion of polyclonal IgM-enriched immunoglobulins, where high levels of BG were indeed detected. Conclusion: Besides strengthening its overall good clinical performance, we provide evidence that serum BG correlates with clinical outcome and that, once used in combination with GM, BG allows to enhance IFI diagnosis rate. The high sensitivity and NPV, observed in the Intensive Care Unit setting, open to BG validation as a marker for assessment of antifungal treatment.

Hyaluronic acid (HA) has several clinical applications (aesthetic surgery, dermatology, orthopaedics and ophtalmology). Following recent evidence, suggesting antimicrobial and antiviral properties for HA, we investigated its effects on 15 ATCC strains, representative of clinically relevant bacterial and fungal species. The in vitro system employed allowed to assess optical density of broth cultures as a measure of microbial load in a time-dependent manner. The results showed that different microbial species and, sometimes, different strains belonging to the same species, are differently affected by HA. In particular, staphylococci, enterococci, Streptococcus mutans, two Escherichia coli strains, Pseudomonas aeruginosa, Candida glabrata and C. parapsilosis displayed a HA dose-dependent growth inhibition; no HA effects were detected in E. coli ATCC 13768 and C. albicans; S. sanguinis was favoured by the highest HA dose. Therefore, the influence of HA on bacteria and fungi warrants further studies aimed at better establishing its relevance in clinical applications.

A protein microarray serological assay was used to assess the antibody profile of 102 women subjected to in vitro fertilization treatment. The studies were conducted on pairs of serum and follicular fluid samples, collected from each woman on the same day at the time of oocyte recovery. The samples, stored as frozen aliquotes, were assessed by both microarray and ELISA. Follicular fluids and sera were screened to detect the presence of specific IgG and IgM antibodies against seven vertically transmitted pathogens. The IgG reactivity of follicular fluids closely mirrored that of serum in all the patients and for all the antigens, with an agreement of more than 85%. IgM antibodies were undetectable in follicular fluids. The antibody patterns were subsequently related to the biological and clinical outcomes of in vitro fertilization cycles. The results showed that varicella zoster virus (VZV) IgG positive women and cytomegalovirus (CMV) IgG negative women had on average a higher number of inseminated, good quality oocytes compared to VZV IgG negative and CMV IgG positive women. In addition, the rate of successful embryo transfers was significantly higher in Toxoplasma gondii IgG negative women than in their positive counterparts. Overall, the microarray was proven to be a suitable tool for detecting analytes in follicular fluids, therefore supporting its application in a wide spectrum of investigations.

Results of recent molecular studies have provided evidence of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. While there are initial data pertaining to the virulence of these Candida species with respect to reconstituted epidermal and oral epithelial tissues, there have been no studies, as of yet, on their interaction with immune cells. Employing an in vitro infection model using microglial cells, we investigated the pathogenetic potential of different isolates of each of these three species. We show that C. metapsilosis isolates are more susceptible to microglia-mediated antifungal activity, as compared with those of C. parapsilosis and C. orthopsilosis. Interestingly, C. metapsilosis isolates are also phagocytosed to a lower extent, but the yeast-containing phagosomes exhibit the highest degree of acidification in comparison with the phagosomes containing C. parapsilosis or C. orthopsilosis. Furthermore, when assessing microglia secretory response to infection, comparable high levels of MIP-1α and little or no TNF-α production are observed with all of these Candida species. Finally, unlike C. metapsilosis infected cells, microglial cells infected with C. parapsilosis and C. orthopsilosis release high and time-dependent levels of lactate dehydrogenase (LDH). Overall, these findings point to C. metapsilosis as the least virulent member of the 'C. parapsilosis' complex.

Vaginal candidiasis is a common disorder in women of childbearing age, caused primarily by the dimorphic fungus Candida albicans. Since C. albicans is a normal commensal of the vaginal mucosa, a long-standing question is how the fungus switches from being a harmless commensal to a virulent pathogen. Work with human subjects and in mouse disease models suggests that host inflammatory processes drive the onset of symptomatic infection. Fungal cell wall molecules can induce inflammation through activation of epithelial and immune receptors that trigger pro-inflammatory cytokines and chemokines, but pathogenic fungi can evade recognition by masking these molecules. Knowledge about which cell wall epitopes are available for immune recognition during human infection could implicate specific ligands and receptors in the symptoms of vaginal candidiasis. To address this important gap, we directly probed the surface of fungi present in fresh vaginal samples obtained both from women with symptomatic Candida vaginitis and from women that are colonized but asymptomatic. We find that the pro-inflammatory cell wall polysaccharide β-glucan is largely masked from immune recognition, especially on yeast. It is only exposed on a small percentage of hyphal cells, where it tends to co-localize with enhanced levels of chitin. Enhanced β-glucan availability is only found in symptomatic patients with strong neutrophil infiltration, implicating neutrophils as a possible driver of these cell wall changes. This is especially interesting because neutrophils were recently shown to be necessary and sufficient to provoke enhanced β-glucan exposure in C. albicans, accompanied by elevated immune responses. Taken together, our data suggest that the architecture of C. albicans cell wall can be altered by environmental stress during vaginal candidiasis.

Recently, we showed that herpes simplex virus 1 (HSV-1)-infected monocytes have altered antifungal defences, in particular they show augmented phagocytosis of Candida albicans followed by a failure of the intracellular killing of the ingested fungi. On the basis of these functional data, comparative studies were carried out on the gene expression profile of cells infected with HSV-1 and/or C. albicans in order to investigate the molecular mechanisms underlying such virus-induced dysfunction. Affymetrix GeneChip technology was used to evaluate the cell transcription pattern, focusing on genes involved in phagocytosis, fungal adhesion, antimicrobial activity and apoptosis. The results indicated there was: (a) prevalent inhibition of opsonin-mediated phagocytosis, (b) upregulation of several pathways of antibody- and complement-independent phagocytosis, (c) inhibition of macrophage activation, (d) marked dysregulation of oxidative burst, (e) induction of apoptosis.

We investigated the interplay occurring between pathogens in the course of dual infections, using an in vitro model in which the THP-1 monocytic cell line is first infected with HSV-1 and then exposed to Ca or Cn. These three pathogens share some pathogenic features: they cause opportunistic infections, target macrophages and are neurotropic. Here, we show that HSV-1-infected THP-1 cells exhibited augmented phagocytosis against the two opportunistic fungi but reduced capability to counteract fungal infection: the better ingestion by monocytes was followed by facilitated fungal survival and replication. Reduced IL-12 production was also observed. Cytofluorimetric analysis showed that HSV-1-infected monocytes exhibit: (i) downregulated TLR-2 and TLR-4, critical structures in fungal recognition; (ii) reduced expression of CD38 and CD69, known to be important markers of monocyte activation; and (iii) enhanced expression of apoptosis and necrosis markers, in the absence of altered cell proliferation. Overall, these findings imply that HSV-1 infection prevents monocyte activation, thus leading to a significant dysfunction of the monocyte-mediated anti-Candida response; HSV-1 induced apoptosis and necrosis of monocytes further contribute to this impairment.

We describe an imported case of histoplasmosis, whose serological profile was established by means of a protein-based microarray platform, the recently described mycoarray. Because of its peculiarities, such a novel tool greatly facilitates the rapid and multiparametric assessment of patients' serological status and lends itself to be employed as an aid in the diagnosis of primary mycoses, especially in nonendemic countries.

It has been recently demonstrated that Candida albicans isolates with distinct genomic backgrounds (namely, b and c genotypes) express different susceptibility to antifungal activity by human monocytes in vitro. We show here that, although comparable in their ability to undergo dimorphic transition and in susceptibility to phagocytosis by microglial cells, the b and c isolates show striking differences in terms of intracellular survival. Only the c genotype resists indeed to intracellular killing and eventually replicates inside microglial cells, that in turn respond to fungal infection, preferentially towards the c genotype, with nuclear factor-κB (NF-κB) activation and increased Mip1 α production. These data indicate that C. albicans -microglial cell interaction is strictly dependent upon fungal genotype, strengthening the potential significance of genotyping as prognostic parameter in clinical infections by C. albicans .

Significant changes in the management of fungaemia have occurred in the last decade with increased use of fluconazole prophylaxis, of empirical treatment and of echinocandins as first-line agents for documented disease. The emergence of drug resistance in fungal pathogens has a profound impact on human health given limited number of antifungal drugs. Antifungal resistance in Candida may be either intrinsic or acquired and may be encountered in the antifungal drug exposed but also the antifungal drug naïve patient The variation in resistance rates between centers emphasizes that it is essential to have knowledge of the local Candida species distribution and antifungal resistance rates to guide initial therapy for Candida BSI. Moreover, all Candida isolates from blood and normally sterile sites should be identified to the species level. The Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing have developed breakpoints and epidemiological cutoff values that are now established for Candida spp. Clinical microbiology laboratories will be employed commercial susceptibility assays, rather than reference broth microdilution methods and comparative studies are particularly important. Vitek 2®, Etest® and Sensititre YeastOne® provided a high degree of essential agreement and comparable sensitivity and specificity to BMD-RPMI for identifying resistance to azole and echinocandins in Candida spp.

The emergence of drug resistance in fungal pathogens has a profound impact on human health given limited number of antifungal drugs. Antifungal resistance in Aspergillus spp. infection can be encountered in the antifungal drug-exposed patient due to selection of intrinsically resistant species or isolates with acquired resistance belonging to species that are normally susceptible. Resistance to triazoles is not common in Aspergillus spp., however, triazole resistance in A. fumigatus appears to be increasing in several European countries in recent years and can be clinically relevant. The Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing have developed breakpoints and epidemiological cutoff values that are now established for Aspergillus spp. Clinical microbiology laboratories will be employed commercial susceptibility assays, rather than reference broth microdilution methods and comparative studies are particularly important.

We investigated the clinical performance of a polymerase chain reaction (PCR)-based commercial platform, the Myconostica MycAssay™ Aspergillus (MAP), for fungal DNA detection in the serum of patients at risk of invasive aspergillosis (IA). Sixty-four hospitalized patients were prospectively enrolled and a total of 71 different episodes were investigated (30 episodes were clinically/microbiologically classified as IA and 41 as control episodes). When MAP was compared to the galactomannan (GM) assay, no significant differences were found in terms of sensitivity (46.7% vs. 50.0%), specificity (97.6% vs. 95.1%), positive predictive value (PPV) (93.3% vs. 88.2%), and negative predictive value (NPV) (71.4% vs. 72.2%). The corresponding areas under the curve (AUC) of the receiver operating characteristic (ROC) curves were also superimposable. Overall, because of the good agreement between the two assays and considering the high specificity and PPV of the MAP, we suggest the use of this PCR-based platform as a second-level examination for the evaluation of clinically undefined cases where culture or GM have provided positive results.

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. albicans proteins (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.

The aim of this study was to examine three serial isolates of Cryptococcus neoformans from a patient with AIDS for genotypical and phenotypical characteristics. The isolates were obtained during a first episode of cryptococcosis (simultaneous sampling of blood and cerebrospinal fluid) and after a relapse 3 years later (sampling of cerebrospinal fluid). Pulsed-field gel electrophoresis and random amplification of polymorphic DNA revealed that the blood isolate 1525 (first episode) was different from the two cerebrospinal fluid isolates (1526, first episode; 1782, relapse), yet the cerebrospinal fluid isolates were indistinguishable from each other regardless of the analysis performed. Phenotypical studies showed that isolate 1782 had significantly improved resistance to phagocytosis and killing by monocytes and polymorphonuclear cells and an altered efficacy in evoking cytokine response (augmentation of tumour necrosis factor-alpha, interleukin [IL]-1beta, IL-10, and interferon-gamma, decrease of IL-12). Interestingly, capsule size and antifungal drug resistance remained unchanged, while production of phospholipase and protease was consistently enhanced in the 1782 isolate with respect to the 1525 and 1526 isolates. In conclusion, in serial Cryptococcus neoformans isolates from a patient with AIDS, phenotypical changes but not molecular changes were documented, thus supporting the role of microevolution as a pathogenetic mechanism(s) for persistence/reactivation of fungal organisms.

The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences.

Leptospires, the agents of leptospirosis, exert tropism for the central nervous system, in the course of mammal infection. We evaluated the interaction between murine microglial cells and strains of pathogenic L. interrogans leptospires and non-pathogenic L. biflexa leptospires, mainly by flow cytometric assays. In the absence of opsonic conditions microglia are capable of ingesting--even quite slowly--the spirochetes and killing the non-pathogenic strain. The adhesion to microglia, which is quick and relevant for all the strains, does not involve the CR3 integrin receptor. These findings suggest that the murine microglia--in non opsonic conditions in vitro--do not effectively clear the pathogenic leptospires.

Using two isogenic strains of Cryptococcus neoformans, we studied the influence of the capsule in C. neoformans microglial-cell interaction. We demonstrate that the acapsular mutant yeasts (CAP67) are more susceptible to phagocytosis and killing than encapsulated yeasts (B3501) by the murine microglial cells, BV-2. RT-PCR analysis showed that the pattern of gene transcripts for tumour necrosis factor alpha (TNF-a), interleukin (IL)-1beta, IL-6, IL-12p40 and granulocyte macrophage colony stimulating factor remains unchanged following BV-2 cell infection with CAP67 or B3501 yeasts. Moreover, no induction of TNF-alpha secretion occurs in BV-2 cells infected with either B3501 or CAP67 yeasts or exposed to glucuronoxylomannan (GXM) or galactoxylomannan (GalXM). Nevertheless, lipopolysaccharide-induced TNF-alpha secretion is downregulated by cell infection with B3501 or CAP67 yeasts or exposure to GXM or GalXM. Overall, by means of a continuous cell line, it appears that the C. neoformans capsule is detrimental to microglial cell antifungal activity, while no effect can be attributed to the capsule as trend of cytokine gene expression and TNF-alpha secretion.

Background: Pneumococcal meningitis (PM) is a life-threatening disease with a high case-fatality rate and elevated risk for serious neurological sequelae. In this study, we investigated the contribution of three major virulence factors of Streptococcus pneumoniae, the capsule, pneumococcal surface protein A (PspA) and C (PspC), to the pathogenesis of experimental PM. Methods: Mice were challenged by the intracranial route with the serotype 4 TIGR4 strain (wt) and three isogenic mutants devoid of PspA, PspC, and the capsule. Survival, bacterial counts, and brain histology were carried out. To study the interaction between S. pneumoniae mutants and microglia, phagocytosis and survival experiments were performed using the BV2 mouse microglial cell line. Results: Virulence of the PspC mutant was comparable to that of TIGR4. In contrast, survival of animals challenged with the PspA mutant was significantly increased compared with the wt, and the mutant was also impaired at replicating in the brain and blood of infected mice. Brain histology indicated that all strains, except for the unencapsulated mutant, caused PM. Analysis of inflammation and damage in the brain of mice infected with TIGR4 or its unencapsulated mutant demonstrated that the rough strain was unable to induce inflammation and neuronal injury, even at high challenge doses. Results with BV2 cells showed no differences in phagocytic uptake between wt and mutants. In survival assays, however, the PspA mutant showed significantly reduced survival in microglia compared with the wt. Conclusions: PspA contributed to PM pathogenesis possibly by interacting with microglia at early infection stages, while PspC had limited importance in the disease. The rough mutant did not cause brain inflammation, neuronal damage or mouse death, strengthening the key role of the capsule in PM.

Candida albicans is the most prevalent human fungal pathogen associated with biofilm formation on indwelling medical devices. Under this form, Candida represents an infectious reservoir difficult to eradicate and possibly responsible for systemic, often lethal infections. Currently, no information is available on the occurrence and persistence of pathogenic viruses within C. albicans biofilm. Therefore, the aim of this study was to investigate whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CVB5) can be encompassed in Candida biofilm, retain their infectivity and then be released. Thus, cell-free virus inocula or HSV-1-infected cells were added to 24h-old fungal biofilm in tissue culture plates; 48 h later, the biofilm was detached by washing and energetic scratching and the presence of virus in the rescued material was end-point titrated on VERO cells. Planktonic Candida cultures and samples containing only medium were run in parallel as controls. We found that both HSV-1 and CVB5 free virus particles, as well as HSV-1 infected cells remain embedded in the biofilm retaining their infectivity. As a second step, the influence of biofilm on virus sensitivity to sodium hypochlorite and to specific neutralizing antibodies was investigated. The results showed that virus encompassment in fungal biofilm reduces virus sensitivity to chemical inactivation but does not affect antibody neutralization. Overall, these data provide the first in vitro evidence that viruses can be encompassed within Candida biofilm and then be released. Thus, it may be speculated that Candida biofilm can be a reservoir of viruses too, posing a further health risk.

By means of a double-chamber model, different root canal filling materials and procedures were compared. Briefly, the root canals of single-rooted human teeth, extracted for periodontal reasons, were instrumented and obturated by gutta-percha/Pulp Canal Sealer EWT (PCS) or by Resilon, in association with different sealers (Real Seal, RelyX Unicem or Meta). Obturation was achieved by traditional continuous wave of condensation technique (TCWCT), a modified version of it (MCWCT), or single cone technique (SCT). The obturated roots, inserted in a double-chamber model, were sterilized by gamma irradiation. Next, Enterococcus faecalis was added to the upper chamber and the specimens were incubated at 37 °C for up to 120 days; the development of turbidity in the lower chambers’ broths indicated bacterial leakage through the obturated root canals. The kinetics of leakage were analyzed in different groups by means of Kaplan–Meier statistics and compared by log-rank test. The results showed that root canals obturated with either gutta-percha/PCS using the MCWCT, Resilon/Real Seal SCT or Resilon/RelyX Unicem using the TCWCT displayed significantly better performance than the remaining groups (p

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

Tumour necrosis factor (TNF) secretory activity has been studied in the murine macrophage cell line ANA-1 following in vitro exposure to Candida albicans 200 kDa stress mannoprotein (SMP200). Treatment of ANA-1 murine macrophages with 200 kDa stress mannoprotein results in increased TNF secretion. The phenomenon is (i) dose- and time-dependent, (ii) abrogated by 200 kDa stress mannoprotein preincubation with a specific monoclonal antibody, and (iii) dependent on intact murine macrophage Ca2+/calmodulin-dependent protein kinase function.

We have recently shown that intracerebral (i.c.) administration of heat-killed Cryptococcus neoformans (HCN) enhances mouse resistance to a subsequent local challenge with lethal doses of viable yeast cells. Here we show that i.c. administration of HCN is also effective in significantly delaying brain colonization of mice intravenously infected with viable C. neoformans. PCR analysis revealed that interleukin 6 (IL-6) and IL-1 beta gene expression occurs in brain of HCN-treated mice but not in brains of saline-treated controls. In contrast, no differences are observed in terms of tumor necrosis factor alpha and IL-1 alpha gene transcripts, which are slightly and highly detectable, respectively, in saline-treated mice and which remain such also following HCN treatment. Furthermore, i.c. administration of exogenous IL-6 or IL-1 beta, but not tumor necrosis factor alpha, before local challenge with viable C. neoformans results in significantly reduced microbial counts in the brain and blood and in increased mouse survival. Taken together, these observations provide initial evidence that brain anticryptococcal resistance involves elicitation of a local cytokine response, involving primarily IL-6 and IL-1 beta.

This article investigates the performance of 2 commercial real-time polymerase chain reaction (PCR) assays, MycAssay™ Aspergillus (Myc Asp Assay) and MycAssay™ Pneumocystis (Myc PCP Assay), on the ABI 7300 platform for the detection of Aspergillus (Asp) or Pneumocystis jirovecii (Pj) DNA in bronchoalveolar lavage (BAL) samples from 20 patients. Operationally, patients enrolled were clustered into 3 groups: invasive aspergillosis group (IA, 7 patients), Pj pneumonia group (PCP, 8 patients), and negative control group (5 patients). All the IA patients were Myc Asp Assay positive, whereas 12 non-IA patients returned negative PCR results. Furthermore, 7 of 8 PCP patients were Myc PCP Assay positive, while 9 non-PCP patients were PCR negative. In conclusion, these data provide an early indication of the effectiveness of both the Myc Asp Assay and Myc PCP Assay on the ABI 7300 platform for the detection of either Asp or Pj DNA in BAL from patients with deep fungal infections.

Recently, we demonstrated that intracerebral (i.c.) administration of picolinic acid (PLA) confers protection against a lethal local challenge with the opportunistic pathogen Candida albicans . By histopathological studies, we show here that mice receiving PLA treatment survive challenge and no evidence of fungal invasion is found within the brain compartment. In contrast, PLA-untreated mice succumb to infection within 7–10 days and show massive brain colonization with extensive granulomatous reaction. By PCR analysis, we show that, unlike naive brains, PLA-treated brains show transient activation of TNFα, IL-1β and IL-6 genes. C. albicans infection results in high levels of all cytokine transcripts, the phenomenon being long-lasting in PLA-untreated brains, while gradually declining in PLA-treated brains. The only exception is IL-1β, whose levels remain high at the latest time-points tested, also in PLA-treated brains. Finally, IL-1α, constitutively detectable in naive brains, is slightly enhanced by C. albicans challenge, regardless of prior treatment. These findings, together with the knowledge that PLA is a potent co-stimulus for macrophages, suggest the involvement of cytokine circuits, likely of macrophage origin, in anti- Candida resistance established by PLA at the cerebral level.

We have previously demonstrated that the hyphal form of Candida albicans (H-Candida), but not the yeast form (Y-Candida), acts as a macrophage-stimulating agent. The early response (1 to 3 h) of the macrophage cell line ANA-1 to H-Candida results in enhanced tumor necrosis factor (TNF) transcription and production. Here we show that when coincubation times are prolonged (3 to 24 h), Y-Candida also exhibits stimulatory properties. This phenomenon has been ascribed to the occurrence of the dimorphic transition, as demonstrated by microscopic evaluation of the cultures and by experiments in which both killed Y-Candida and the agerminative strain C. albicans PCA-2 failed to induce cytokine production. TNF produced in response to H-Candida acts as an autocrine and paracrine signal controlling the macrophage secretory response to C. albicans. In fact, addition of anti-TNF polyclonal antibodies to the coculture of ANA-1 macrophages and H-Candida results in a marked and time-dependent decrease of TNF transcript levels. Moreover, pretreatment of macrophages with recombinant TNF for 3 h enhances TNF and induces interleukin-1 production in response to both forms of Candida, while pretreatment for 18 h renders macrophages refractory to any stimuli. Interestingly, the kinetics of interleukin-1 transcription and secretion in response to H-Candida are delayed with respect to those of TNF. Overall, these data indicate that TNF, produced by macrophages in response to H-Candida, regulates its own production as well as that of other soluble factors, thus suggesting that this cytokine plays multiple roles in the immune mechanisms involved in Candida infection.

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo–in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10² CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10⁷ CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.