Rifaximin use favoured micafungin-resistant Candida spp. infections in recipients of allogeneic hematopoietic cell transplantation.

Damage to gut mucosa following conditioning regimens may favour bacterial infections that can trigger graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Rifaximin, an oral and non-absorbable antibiotic, has been recently proposed as effective prophylaxis to reduce bacterial infections in the gut and consequently acute GvHD in this setting. The present study evaluated safety and outcomes of HSCT patients that were treated with rifaximin prophylaxis at Perugia University Hospital. Rifaximin prophylaxis was introduced as standard of care in HSCT patients in May 2018. We retrieved data from 118 consecutive transplants, and we compared the outcomes of rifaximin-treated patients with historical controls that did not receive antibiotic prophylaxis. While incidences of neutropenic fever, documented bacterial infections, and aGvHD were similar in the two groups, we found an increased frequency of invasive candidiasis and clinically relevant Candida spp. infections in rifaximin-treated patients (5 patients vs 1 patient, 25% [± 0.99%] vs 1% [± 0.01%], p < .0001). Three rifaximin-treated patients experienced life-threating candidemia (2 C. krusei, 1 C. orthopsilosis). Rifaximin was the only factor that increased the risk of Candida spp. infections. Rifaximin could have contributed to microbiome disruption which favoured an outbreak of life-threatening Candida infections. This important complication forced us to halt its use. Larger, prospective studies are needed to assess the impact of rifaximin prophylaxis on incidence of bacterial infections, aGvHD, and survival of HSCT patients.

IL-22 and IDO1 affect immunity and tolerance to murine and human vaginal candidiasis.

The ability to tolerate Candida albicans, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in IL22 and IDO1 genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to Candida, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.

An immunomodulatory activity of micafungin in preclinical aspergillosis.

OBJECTIVES: Micafungin inhibits 1,3-ß-D-glucan synthase and interferes with fungal cell wall synthesis. Clinically, micafungin has been shown to be efficacious for the treatment of invasive fungal infections. However, little is known about the immunomodulatory activity of micafungin in these infections. METHODS: We evaluated the immunomodulatory activity of escalating doses of micafungin in murine and human polymorphonuclear neutrophils (PMNs) in vitro and in vivo in different preclinical models of invasive aspergillosis, including mice deficient for selected innate immune receptors. RESULTS: Micafungin was able to regulate PMN cytokine response to Aspergillus fumigatus conidia by decreasing the expression of tumour necrosis factor-α and increasing that of interleukin-10 (IL-10). In vivo, the therapeutic efficacy of micafungin was strictly dose-dependent, with the maximum activity observed at the highest dose, concomitant with reduced inflammatory pathology. The anti-inflammatory activity of micafungin required IL-10 and occurred through signalling via the TLR2/dectin-1 and TLR3/TRIF pathways. CONCLUSION: Together, these findings suggest that the therapeutic efficacy of micafungin in aspergillosis is orchestrated by the activation of innate immune receptors affecting the inflammatory/anti-inflammatory balance during infection.

Acquired echinocandin resistance in a Candida krusei blood isolate confirmed by mutations in the fks1 gene.

We describe a case of bloodstream infection caused by a Candida krusei strain that developed echinocandin resistance during caspofungin therapy. Three mutations were found in the HS1 region of the fks1 gene, two of them have never been reported either in C. krusei nor in C. albicans.

An atypical, pigment-producing Metschnikowia strain from a leukaemia patient.

A yeast strain was isolated from the sputum sample of a leukaemia patient in the Spirito Santo Hospital of Pescara, Italy. The fungus produced a pigment that formed a reddish halo around colonies, and was identified and deposited as a Metschnikowia spp. (accession number IHEM 25107-GenBank accession number JQ921016) in the BCCM/IHEM collection of biomedical fungi and yeasts (Bruxelles, Belgium). Although the physiology of the strain was close to that of Metschnikowia sinensis, the D1/D2 sequence did not correspond to any previously described Metschnikowia species. Phylogeny of the genus Metschnikowia is complex and requires far more analysis. We present the first non-M. pulcherrima Metschnikowia spp. isolate recovered from a human, and emphasize the role of man as a transient carrier of environmental yeasts, the pathogenicity of which still needs to be defined.

Role of innate immune receptors in paradoxical caspofungin activity in vivo in preclinical aspergillosis.

This study investigated the possible mechanisms underlying the paradoxical caspofungin activity in vivo in preclinical aspergillosis. We evaluated the activity of escalating doses of caspofungin in vivo in different preclinical models of invasive aspergillosis, including mice deficient for selected innate immune receptors. The therapeutic efficacy of caspofungin in experimental invasive aspergillosis was strictly dose dependent, being observed at doses of 0.1 and 1 mg/kg of body weight depending on the experimental models. Paradoxical increase in pulmonary fungal burden as well as inflammatory pathology was observed at the highest dose of caspofungin (5 mg/kg), occurred independently of the so-called Eagle effect and susceptibility to caspofungin in vitro, and was contingent upon the presence of TLR2, Dectin-1, and TLR9. Increased expression of Dectin-1 and TLR9 were observed upon exposure to caspofungin in vitro and in vivo. Together, these findings suggest that the net activity of caspofungin in vivo is orchestrated by the activation, directly or indirectly, of multiple innate immune receptors.

Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient- and donor-dependent mechanisms of antifungal immunity.

The C-type lectin receptor Dectin-1 plays a pivotal role in antifungal immunity. In this study, the recently characterized human DECTIN1 Y238X early stop codon polymorphism leading to diminished Dectin-1 receptor activity was studied in relation to invasive aspergillosis susceptibility and severity in patients receiving hematopoietic stem cell transplantation. We found that the presence of the DECTIN1 Y238X polymorphism in either donors or recipients of hematopoietic stem cell transplantation increased susceptibility to aspergillosis, with the risk being highest when the polymorphism was present simultaneously in both donors and recipients (adjusted hazard ratio = 3.9; P = .005). Functionally, the Y238X polymorphism impaired the production of interferon-γ and interleukin-10 (IL-10), in addition to IL-1ß, IL-6, and IL-17A, by human peripheral mononuclear cells and Dectin-1 on human epithelial cells contributed to fungal recognition. Mechanistically, studies on preclinical models of infection in intact or bone marrow-transplanted Dectin-1 knockout mice revealed that protection from infection requires a distinct, yet complementary, role of both donor and recipient Dectin-1. This study discloses Dectin-1 deficiency as a novel susceptibility factor for aspergillosis in high-risk patients and identifies a previously unsuspected role for Dectin-1 in antifungal immunity that is the ability to control both resistance and tolerance to the fungus contingent on hematopoietic/nonhematopoietic compartmentalization.

Immunodetection of proteins in ancient paint media.

Diagnostic immunology is a powerful tool, widely used in clinical and biochemical laboratories for detecting molecules. In recent years, the technique has been adapted to materials sciences as a result of the extensive advances achieved in immunology. Today, many companies supply custom antibodies as well as new high-performance bioprobes for virtually any use. The idea of using immunodetection in the field of conservation science is not new. This analytical methodology is, in fact, particularly attractive for investigating biopolymers in painting materials; it is highly sensitive and selective with respect to the biological source of the target molecules. Among biopolymers, proteins have been widely used in the past as painting binders, adhesives, and additives in coating layers. An accurate assessment of these materials is necessary to obtain deeper insights into an artist's technique as well as to design proper restoration and conservation methods. In spite of the diagnostic potential offered by immunodetection-based techniques, some analytical drawbacks had, until recently, limited their use in routine applications in conservation science. In this Account, we highlight the most important results achieved in our research on the development of analytical methodologies based on the use of enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM) techniques for the highly sensitive and specific identification of proteins in artistic and archeological materials. ELISA and IFM offer two alternative analytical routes to this final goal: ELISA provides a fast, cost-effective, quantitative analysis of microsamples put in solution, whereas IFM combines the immunodetection of the targeted molecules with the characterization of their spatial distribution. The latter approach is of great value in the stratigraphic investigation of paintings. We discuss the limits and strengths of these methodologies in the context of the complex matrixes usually found in the investigated materials and the prolonged aging that they have undergone. Immunology is a relatively new technique in conservation science, providing a rich new field for innovation. We see two areas that are particularly ripe for future contributions. The commercial manufacture of antibodies specifically tailored for use in cultural heritage studies holds enormous potential. Moreover, the need for further refinement of detection systems in immuno-fluorescence techniques, especially the suppression of the autofluorescence background in painting materials, offers an abundance of opportunities for researchers. Immunology is a relatively new technique in conservation science, providing a rich new field for innovation.

An Italian Case of Disseminated Histoplasmosis Associated with HIV.

Histoplasma capsulatum is a dimorphic fungus, endemic in the Americas, Africa (var. duboisii), India, and Southeast Asia. H. capsulatum infection is rarely diagnosed in Italy, while in Latin America, progressive disseminated histoplasmosis (PDH) is one of the most frequent AIDS-defining illnesses and causes of AIDS-related deaths. We report a case of PDH and new HIV infection diagnosis in a Cuban patient, who has been living in Italy for the past 10 years. Bone marrow aspirate and peripheral blood smear microscopy suggested H. capsulatum infection. The diagnosis was confirmed with the culture method identifying its thermal dimorphism. Liposomal amphotericin B was administered alone for 10 days and then for another 2 days, accompanied with voriconazole; the former was stopped for probable side effects (persistent fever and worsening thrombocytopenia), and voriconazole was continued to complete 4 weeks. PDH maintenance treatment consisted of itraconazole for one year. Antiretroviral therapy (ART) was started on the third week of antifungal treatment. At the 3-year follow-up, the patient is adherent on ART, the virus was suppressed, and she has an optimal immune recovery. This case highlights the need to suspect histoplasmosis in the differential diagnosis of opportunistic infections in immunocompromised persons, native to or who have traveled to endemic countries.

Immunity and tolerance to Aspergillus fumigatus.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defence without necessarily eliminating the fungus or causing unacceptable levels of host damage. Efficient responses to the fungus require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) and regulatory (Treg) cell responses. A division of labour occurred between functionally distinct Treg that were coordinately activated by a CD28/B.7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation was controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of interleukin (IL10) and cytotoxic T lymphocyte antigen 4 (CTLA4) on indoleamine 2,3-dioxygenase (IDO). The levels of IFNgamma produced in this early phase set the subsequent adaptive stage by conditioning the IDO-dependent tolerogenic program of DCs and the subsequent activation and expansion of tolerogenic Treg, which produced IL10 and transforming growth factor (TGF)beta, inhibited Th2 cells, and prevented allergy to the fungus. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

Immunochemical Methods Applied to Art-Historical Materials: Identification and Localization of Proteins by ELISA and IFM.

Despite the large diffusion of natural organic substances in art-historical materials, their characterization presents many challenges due to the chemical complexity and instability with respect to degradation processes. Among natural products, proteins have been largely used in the past as binders but also as adhesives or additives in coating layers. Nevertheless, biological identification of proteins in art-historical objects is one of the most recent achievements obtained in heritage science thanks to the development of specifically tailored bio-analytical strategies. In the context of this active emerging discipline, immunological methods stand out for sensitivity, specificity and versatility for both protein recognition and localization in micro-samples. Furthermore, the growing use of immunological techniques for advanced diagnostics and clinical applications ensures continuous improvement in their analytical performance. Considering such, this review provides an overview of the most recent applications of enzyme linked immunosorbent assay and immunofluorescence microscopy techniques in the field of heritage materials. Specifically, the main strengths and potentials of the two techniques as well as their limits and drawbacks are presented and discussed herein.

Pulsed field gel electrophoresis and random amplified polymorphic DNA molecular characterization of Ralstonia pickettii isolates from patients with nosocomial central venous catheter related bacteremia.

Over a period of 18 months 3 clusters of central venous catheter-related Ralstonia pickettii bacteremia occurred in 3 different units of the same hospital. In order to investigate the relatedness of the clinical isolates we studied 15 strains using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques. The combined analysis of the results obtained by these two methods led us to conclude that all the patients except one were infected by a single clone comprising two variants circulating in the units. Only one case was due to a different strain, probably originating outside the ward. PFGE and RAPD appear to be discriminatory techniques to study the clonal relationship among the isolates and can represent a good tool to perform the epidemiological investigation of an outbreak.

Fungal colonization on stoneworks. Interaction fungi-powdered stone samples.

The microbial activity plays an important role in the biodegradative processes implied in stonework decay. In natural environments it is not possible to separate the damage produced by microorganisms from damage caused by physical and chemical agents. In vitro assays carried out with microbes isolated from weathered stones are required in order to understand the biological mechanisms involved in stone deterioration. We have described, as commented in the text, how fungal colonization observed on scaglia may be the result of the fine grain size of rock, whereas inhibition of growth on marble may depend on the surface characteristics of calcite grains after grinding. The extent of microbial growth clearly depends on the quantity of cations released in solution. However, fungal growth may, in turn, induce a decrease in pH, thus promoting mineral chemical attack. These observation points to selective action of fungal species in promoting weathering well evidenced by the presence of different extents of cations released in suspension from the same sample. Detailed studies are in progress in order to go into this question.

A case of Candida guilliermondii abortion in an Arab mare.

Ascending infections of equine uterus frequently result in placentitis and abortions; most of these infections are bacterial and are less commonly due to fungi. This report describes an abortion case in an Arab mare due to Candida guilliermondii that was diagnosed via cytological, histological, cultural and biomolecular assays. The histological lesions found were severe necrotizing placentitis associated with fetal pneumonia. To our knowledge this is the first case of C. guilliermondii abortion reported in equine species.

Monolateral visual loss due to cryptococcal meningitis.

A case of retrobulbar monolateral optic neuritis due to disseminated Cryptococcosis in a 32-year-old man with a new diagnosis of AIDS is described in the era of combination antiretroviral therapy (cART). The patient presented a monolateral rapid visual loss on day 7 of a liposomal amphotericin B treatment. Neuroradiological imaging showed the presence of retrobulbar neuritis. After starting ART and intravenous metilprednisolone 1 g daily for 3 days, we assisted to a progressive improvement of visual acuity. At 3 months of follow-up, complete clinical resolution was obtained. In this case, in the presence of effective antiretroviral and antifungal treatment, a short course of metilprednisolone was a safe therapy.

The C allele of rs5743836 polymorphism in the human TLR9 promoter links IL-6 and TLR9 up-regulation and confers increased B-cell proliferation.

In humans, allelic variants in Toll-like receptors (TLRs) associate with several pathologies. However, the underlying cellular and molecular mechanisms of this association remain largely unknown. Analysis of the human TLR9 promoter revealed that the C allele of the rs5743836 polymorphism generates several regulatory sites, including an IL-6-responding element. Here, we show that, in mononuclear cells carrying the TC genotype of rs5743836, IL-6 up-regulates TLR9 expression, leading to exacerbated cellular responses to CpG, including IL-6 production and B-cell proliferation. Our study uncovers a role for the rs5743836 polymorphism in B-cell biology with implications on TLR9-mediated diseases and on the therapeutic usage of TLR9 agonists/antagonists.

Genetic variability of innate immunity impacts human susceptibility to fungal diseases.

Fungi are a major threat in immunocompromised patients. Despite presenting similar degrees of immunosuppression, not all individuals at-risk ultimately develop fungal diseases. The traditional view of immune suppression as a key risk factor for susceptibility to fungal infections needs to be accommodated within new conceptual advances on host immunity and its relationship to fungal disease. The critical role of the immune system emphasizes the contribution of host genetic polymorphisms to fungal disease susceptibility. This review highlights the present knowledge on innate immunity genetics that associates with susceptibility to fungal diseases.

Polymorphisms in Toll-like receptor genes and susceptibility to infections in allogeneic stem cell transplantation.

OBJECTIVE: Discovery of genetic variations in the genes encoding for Toll-like receptors (TLRs) has highlighted a potential link between genomic variation of the host and susceptibility to infections. MATERIALS AND METHODS: We investigated the association between polymorphisms in the TLR2, TLR4, and TLR9 genes in recipients of allogeneic hematopoietic stem cell transplant and susceptibility to infections caused by cytomegalovirus and filamentous fungi. RESULTS: A significant association was observed between the presence of the T-1237C polymorphism (TLR9) and susceptibility to viral pneumonia (p=0.04; odds ratio [OR]: 1.73). For fungi, a significant association was observed between the presence of the cosegregating Asp299Gly/Thr399Ile polymorphisms (TLR4) and fungal colonization (p=0.003; OR: 10.6). However, susceptibility to fungal infections, predominantly fungal pneumonia, was instead significantly decreased in the presence of the same polymorphisms (p=0.03; OR: 0.23). CONCLUSION: Thus, fungal colonization may not predict susceptibility to infection in the presence of these single nucleotide polymorphisms. The finding that defective viral but not fungal sensing may predict susceptibility to infection highlights the divergent function of TLRs in the pathogenesis of opportunistic infections.

Thymosin alpha1 activates the TLR9/MyD88/IRF7-dependent murine cytomegalovirus sensing for induction of anti-viral responses in vivo.

Reactivation of latent human cytomegalovirus following allogeneic transplantation is a major cause of morbidity and mortality and predisposes to severe complications. Thymosin alpha1 (Talpha1), a naturally occurring thymic peptide, is approved for treatment of some viral infections and as an immune adjuvant. Talpha1 successfully primed dendritic cells (DCs) for anti-microbial T helper type 1 resistance through Toll-like receptor (TLR) 9 signaling. We sought to determine here whether Talpha1 could play a role in murine cytomegalovirus infection (MCMV). To this purpose, susceptible, resistant and TLR-deficient mice were infected with MCMV, treated with Talpha1 and assessed for protection in term of microbiological and immunological parameters. Talpha1 protected susceptible and resistant mice from MCMV infection. The anti-viral effect of Talpha1 occurred through the activation of plasmacytoid DCs via the TLR9/myeloid differentiation primary response gene 88-dependent viral recognition sensing, leading to the activation of IFN regulatory factor 7 and the promotion of the IFN-alpha/IFN-gamma-dependent effector pathway.