Elisa Borghi

Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy.

Patients with inefficient CD4+ T-cell recovery on virogically suppressive highly active antiretroviral therapy constitute a major clinical hurdle given the threat of HIV/AIDS disease progression. We show heightened circulating lipopolysaccharide associated with plasma enterobacterial DNA and highly activated Ki67+CD4+CD8+ in 24 immunologic-nonresponders (CD4+ T-cell ≤ 200; HIV-RNA ≤ 50) compared with 11 full responders (CD4+ T-cell ≥ 400; HIV-RNA ≤ 50). These data provide novel insight into INRs pathogenesis, since they correlate augmented systemic translocation of microbial bioproducts with T-cell hyperactivation.

Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus

ABSTRACT The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.

Molecular Analysis of JC Virus Genotypes Circulating Among the Italian Healthy Population

JC virus (JCV) is an unique virus, but eight different JCV genotypes and various subtypes have been individuated, with a geographic distribution that has been described in general but still needs to be detailed. To define JCV genotype epidemiology in Italy, the authors collected urine from 211 healthy individuals living in Northern, Central, and Southern Continental Italy, and in the two main Italian islands. As screening, JCV DNA was searched using a polymerase chain reaction (PCR) designed to amplify the highly conserved large T (LT) antigen-coding region. Then, to define JCV genotype and trascriptional control region (TCR) organization, fragments of major capsid protein (VP1) and TCR region were amplified and subjected to nucleotide sequencing. The mean frequency of JCV viruria was of 46%, without differences among the four geographically divided groups and between females and males. JCV types 1 and 4 were the most frequently detected, whereas JCV type 2 was rare, and type 3 was found only in one subject. The low frequency of JCV type 2 contrasts with the reported high frequency of this subtype in progressive multifocal leukoen-cephalopathy (PML), and supports its specific role in PML. The data indicate that JCV genotype epidemiology in Italy is quite different from that of other European countries; moreover, differences between the various Italian regions have been observed. An unexpected high frequency of a new variant of JCV type 4 with a stable point mutation (C → G) at nucleotide 1851 was found. Furthermore, all the urinary strains had a TCR showing an archetypal organization.

A case of a progressive multifocal leukoencephalopathy patient with four different JC virus transcriptional control region rearrangements in cerebrospinal fluid, blood, serum, and urine

JC virus (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS). During the acquired immunodeficiency syndrome (AIDS) epidemic, it was the cause of the death in up to 8% of AIDS patients. The genomic organization of JCV and, in particular, the hypervariability of the transcriptional control region (TCR), a regulatory noncoding region, are well known. Given that the TCR plays a central role in the viral replication of JCV, a crucial role in the determination of the neurotropism and in the pathogenic capabilities of the virus is also suspected. Here the authors describe a case of PML that did not respond to highly active antiretroviral therapy (HAART) therapy. There was a simultaneous presence of JCV strains with four different TCR structures in urine, peripheral blood cells, serum, and cerebrospinal fluid (CSF) samples. These data confirmed that the presence of the archetype TCR is restricted to urine, while also suggesting that the degree of the rearrangement varies and increases from the peripheral blood to CSF.

Pediatric obesity is associated with an altered gut microbiota and discordant shifts in Firmicutes populations

Summary An altered gut microbiota has been linked to obesity in adulthood, although little is known about childhood obesity. The aim of this study was to characterize the composition of the gut microbiota in obese (n = 42) and normal‐weight (n = 36) children aged 6 to 16. Using 16S rRNA gene‐targeted sequencing, we evaluated taxa with differential abundance according to age‐ and sex‐normalized body mass index (BMI z‐score). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Members of the Bacteroidetes were generally better predictors of BMI z‐score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. In accordance with these observations, the main metabolites produced by gut bacteria, short chain fatty acids (SCFAs), were higher in obese children, suggesting elevated substrate utilisation. Multiple taxa were correlated with SCFA levels, reinforcing the tight link between the microbiota, SCFAs and obesity. Our results suggest that gut microbiota dysbiosis and elevated fermentation activity may be involved in the etiology of childhood obesity.

Cell surface hydrophobicity: a predictor of biofilm production in Candida isolates?

Biofilm-forming ability undoubtedly represents one of the major medical concerns in clinical practice, being a relevant pitfall for an efficient therapeutic intervention (Donlan & Costerton, 2002). In this scenario, invasive fungal infections, especially those involving Candida species, become an important problem in terms of early diagnosis and medical treatment. A large proportion of Candida infections are associated with biofilm formation, mainly on the surface of implanted medical devices (Douglas, 2003). Rapid methods for biofilm-forming ability evaluation in clinical isolates could thus improve the clinical outcome of the fungal infection. Biofilm formation is a step-by-step and finely regulated process in which the adhesion phase is a crucial event (Chandra et al., 2001). Hydrophobic interactions play a role in the adherence of micro-organisms to a wide variety of surfaces.

Members of the AP-1 family, c-Jun and c-Fos, functionally interact with JC virus early regulatory protein large T antigen

ABSTRACT The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early inducible transcription factors which were shown to be activated by a variety of stress-related stimuli and to be involved in numerous biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. We have recently demonstrated the involvement of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the human polyomavirus, JC virus (JCV), genome. Here, we further examined their role in JCV gene regulation and replication through their physical and functional interaction with JCV early regulatory protein large T antigen (T-Ag). Transfection and replication studies indicated that c-Jun and c-Fos can significantly diminish T-Ag-mediated JCV gene transcription and replication. Affinity chromatography and coimmunoprecipitation assays demonstrated that c-Jun and T-Ag physically interact with each other. Results from band shift assays showed that the binding efficiency of c-Jun to the AP-1 site was reduced in the presence of T-Ag. In addition, we have mapped, through the use of a series of deletion mutants, the regions of these proteins which are important for their interaction. While the c-Jun interaction domain of T-Ag is localized to the middle portion of the protein, the T-Ag interacting domain of c-Jun maps to its basic-DNA binding region. Results of transient-transfection assays with various c-Jun mutants and T-Ag expression constructs further confirm the specificity of the functional interaction between c-Jun and T-Ag. Taken together, these data demonstrate that immediate-early inducible transcription factors c-Jun and c-Fos physically and functionally interact with JCV major early regulatory protein large T-Ag and that this interaction modulates JCV transcription and replication in glial cells.

Rare mycoses of the oral cavity: a literature epidemiologic review.

Stomatologic fungal infections display different etiologies, pathogenesis, and clinical presentations. The incidence of rare mycoses of oral cavity is very low. These infections can involve both immunocompromised and immmunocompetent patients with common predisposing factors, such as diabetes or suffering from diseases causing immune system impairment. Oral mycoses can cause acute, chronic, and mucocutaneous lesions. Candidiasis is the most common mouth mycosis. Although occasionally primary mouth pathogens, Cryptococcus spp. or filamentous fungi (Aspergillus spp. and zygomycetes) can cause oral mycoses, with the oral localization more commonly secondary to a more serious systemic infection. The diagnosis of oral mycoses is based on clinical examination; for yeasts, culture is necessary to identify the etiologic agents; for filamentous fungi, in particular for zygomycetes and dimorphic, a definitive diagnosis can be made by histologic examination and pertinent stains with or without isolation of the fungus from the same site.

Antifungal drug resistance in Candida species

Invasive Candida infections are well established infectious entities of immunocompromised or critically ill patients and are characterised by high morbidity and mortality. Owing to the common eukaryotic structure of fungi and humans, a limited number of antifungal drugs is available for therapeutic purposes. In this unsatisfactory scenario, the emergence of drug resistance represents an important health problem. Failure of antifungal treatment can be related to host factors, to the pharmacokinetic and pharmacodynamic parameters of the drug, or to morphological, reproductive modalities and biofilm production of the fungus itself. Innate or acquired antifungal resistance derives from the presence or onset of molecular mechanisms related to the toxic activity of the drug itself. The resulting resistance can thus be extended to different molecules of the same class according to a greater or lesser affinity of the molecules for the target. In addition, non-specific cellular mechanisms of extrusion of toxic substances, such as overexpression of efflux pumps, can play a role involving different antifungal classes. Here we briefly review the current antifungal susceptibility testing methods and their usefulness as predictors of antifungal resistance in Candida spp., focusing on assessment of the involved molecular mechanisms.

Comparative Evaluation of the Vitek 2 Yeast Susceptibility Test and CLSI Broth Microdilution Reference Method for Testing Antifungal Susceptibility of Invasive Fungal Isolates in Italy: the GISIA3 Study

ABSTRACT The newly available AST-YS01 Vitek 2 cards were evaluated, and the results were compared with those obtained by the CLSI M27-A2 microdilution reference method. Clinical fungal isolates, including 614 isolates of Candida spp., 10 Cryptococcus neoformans isolates, 1 Geotrichum capitatum isolate, and 2 quality control strains, were tested for their susceptibilities to amphotericin B, fluconazole, and voriconazole using both methods. The majority of fungal isolates were susceptible to all antifungal agents tested: the MIC90 values determined by the Vitek 2 and CLSI methods were 0.5 and 1 μg/ml, respectively, for amphotericin B; 8 and 16 μg/ml, respectively, for fluconazole; and <0.12 and 0.25 μg/ml, respectively, for voriconazole. Overall there was excellent categorical agreement (CA) between the methods (99.5% for amphotericin B, 92% for fluconazole, 98.2% for voriconazole), but discrepancies were observed within species. The CAs for fluconazole were low for Candida glabrata and Candida krusei when the results of the CLSI method at 48 h were considered. Moreover, the fully automated commercial system did not detect the susceptibility of Cryptococcus neoformans to voriconazole. The Vitek 2 system can be considered a valid support for antifungal susceptibility testing of fungi, but testing of susceptibility to agents not included in the system (e.g., echinocandins and posaconazole) should be performed with other methods.