A 5-year study of the performance of the Verigene Gram-positive blood culture panel in a pediatric hospital.

High accuracy of direct from positive blood culture molecular panels is imperative, particularly for the detection of resistance determinants as it allows for antimicrobial optimization prior to conventional susceptibility testing. In this study, we provide extensive data since implementation of the Verigene Gram-positive blood culture panel (BC-GP) in 2013. Within 5 years, 1636 blood culture bottles positive for a Gram-positive organism were tested on the BC-GP panel. The BC-GP panel identified 1520 Gram-positive organisms in 1636 (92.9%) blood cultures tested. For positive blood cultures, we observed 96.4% (806/834) concordance to the species level. Compared with conventional antimicrobial susceptibility testing, the positive percent agreement (PPA) of methicillin-resistant SA (MRSA) (50) and methicillin-resistant SE (MRSE) (365) was 100%. The mecA gene was detected in two methicillin-susceptible Staphylococcus aureus (MSSA) and one methicillin-susceptible S. epidermidis (MSSE) with a negative percent agreement (NPA) of 99.1% (221/223) and 99.2% (120/121), respectively. The PPA and NPA for vancomycin-resistant Enterococcus faecium (VRE) was 100%. The BC-GP panel demonstrated excellent performance and clinicians can confidently de-escalate antimicrobial therapy in the absence of mecA and vanA/B gene.

Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection.

Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared with wild-type. Collectively, these studies demonstrate a novel stage-specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin.

Polymeric Nanofiber/Antifungal Formulations Using a Novel Co-extrusion Approach.

We report the successful implementation of a novel melt co-extrusion process to fabricate ca. 1 µm diameter fibers of poly(caprolactone) (PCL) containing the antifungal compound clotrimazole in concentrations between 4 and 8 wt%. The process involves co-extrusion of a clotrimazole-loaded PCL along with poly(ethylene oxide) (PEO) as a co-feed, with subsequent removal of PEO to isolate PCL-clotrimazole fibers. In vitro tests of the clotrimazole-containing fibers against the fungus Aspergillus fumigatus, Candida albicans, and Trichophyton mentagrophytes strains demonstrated good antifungal activity which was maintained for more than 3 weeks. An in vivo study using a mouse model showed the lowest tissue fungal burden for PCL-clotrimazole when compared to a PCL-only patch and untreated controls. Comparative studies were conducted with clotrimazole-containing PCL fibers fabricated by electrospinning. Our data showed that the co-extruded, clotrimazole-containing fibers maintain activity for longer times vs. electrospun samples. This, coupled with the much higher throughput of the co-extrusion process vs. electrospinning, renders this new approach very attractive for the fabrication of drug-releasing polymer fibers.

Interleukin 17 expression in peripheral blood neutrophils from fungal keratitis patients and healthy cohorts in southern India.

Interleukin 17A (IL-17) production by peripheral blood neutrophils was examined in patients with fungal keratitis and in uninfected individuals in southern India, which has high levels of airborne Aspergillus and Fusarium conidia. Il17a gene expression and intracellular IL-17 were detected in all groups, although levels were significantly elevated in neutrophils from patients with keratitis. There were no significant differences in plasma IL-17 and IL-23 between patients with keratitis and uninfected individuals; however, combined data from all groups showed a correlation between the percentage IL-17 producing neutrophils and plasma IL-23, and between plasma IL-17 and IL-6 and IL-23.

Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum.

Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections.

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease.

Act1 is a negative regulator of B-cell activation factor of the TNF family (BAFF) and CD40L-induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti-nuclear IgG autoantibody (ANA-IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T-cell dependent or T-cell independent manner, we investigated the role of T-cell help during Act1-mediated autoimmunity. Act1-deficiency was bred onto C57Bl/6 (B6.Act1(-/-) ) mice and B6.TCRß(-/-) TCRδ(-/-) Act1(-/-) (TKO) mice were generated. While TCRß/δ-sufficient B6.Act1(-/-) mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA-IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1(-/-) nor TKO mice developed SjS-like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1(-/-) mice. Interestingly, BAFF-driven transitional B-cell abnormalities, previously reported in BALB/C.Act1(-/-) mice, were intact in B6.Act1(-/-) mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE-like disease in B6.Act1(-/-) mice, but not BAFF-driven transitional B-cell differentiation.

Cladophialophora bantiana brain abscess and concurrent pulmonary cryptococcus neoformans infection in a patient twenty years after renal transplantation.

Recipients of solid organ transplants are at risk for a variety of infections due to their immunocompromised status. The types of infections are often correlated to the timing from their transplant. After about six to twelve months, transplant recipients remain at risk for typical community acquired pathogens, late viral infections, and fungal infections including atypical molds such as Cladophialophora bantiana. C. bantiana is a dematiaceous fungus that has a predilection for infecting the brain and is the most common cause of cerebral phaeohyphomycosis - a term used to describe infections caused by molds that produce dark cell walls. Patients with cerebral abscesses due to C. bantiana infections have an estimated mortality of about 70%. Improved outcomes have been seen in patients who receive both surgical and antifungal therapy. While there are no clear guidelines on antifungal therapy, most cases have been treated with combination amphotericin B, a triazole (itraconazole, voriconazole, or posaconazole) with flucytosine sometimes in conjunction as well. This case describes a patient with C. bantiana brain abscess and concurrent Cryptococcus neoformans pulmonary infection that occurred twenty years after his kidney transplantation. He was treated successfully with two craniotomies for cerebral abscess debridement and liposomal amphotericin B followed by planned lifelong voriconazole.

Eight-Year-Old Male With Primary Amebic Meningoencephalitis.

Naegleria fowleri is a thermophilic free-living amoeba that is found in warm, fresh water and causes primary amebic meningoencephalitis (PAM). The following report demonstrates the rapid and destructive clinical features of PAM in an 8-year-old male who presented with severe headaches approximately 12 days after swimming in a hot spring.

Sustained release of decorin to the surface of the eye enables scarless corneal regeneration.

Disorganization of the transparent collagenous matrix in the cornea, as a consequence of a variety of infections and inflammatory conditions, leads to corneal opacity and sight-loss. Such corneal opacities are a leading cause of blindness, according to the WHO. Public health programs target prevention of corneal scarring, but the only curative treatment of established scarring is through transplantation. Although attempts to minimize corneal scarring through aggressive control of infection and inflammation are made, there has been little progress in the development of anti-scarring therapies. This is owing to eye drop formulations using low viscosity or weak gelling materials having short retention times on the ocular surface. In this study, we report an innovative eye drop formulation that has the ability to provide sustained delivery of decorin, an anti-scarring agent. The novelty of this eye drop lies in the method of structuring during manufacture, which creates a material that can transition between solid and liquid states, allowing retention in a dynamic environment being slowly removed through blinking. In a murine model of Pseudomonas keratitis, applying the eye drop resulted in reductions of corneal opacity within 16 days. More remarkably, the addition of hrDecorin resulted in restoration of corneal epithelial integrity with minimal stromal opacity endorsed by reduced α-smooth muscle actin (αSMA), fibronectin, and laminin levels. We believe that this drug delivery system is an ideal non-invasive anti-fibrotic treatment for patients with microbial keratitis, potentially without recourse to surgery, saving the sight of many in the developing world, where corneal transplantation may not be available.

Pseudomonas aeruginosa Effector ExoS Inhibits ROS Production in Human Neutrophils.

Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.

Fungal antioxidant pathways promote survival against neutrophils during infection.

Filamentous fungi are a common cause of blindness and visual impairment worldwide. Using both murine model systems and in vitro human neutrophils, we found that NADPH oxidase produced by neutrophils was essential to control the growth of Aspergillus and Fusarium fungi in the cornea. We demonstrated that neutrophil oxidant production and antifungal activity are dependent on CD18, but not on the ß-glucan receptor dectin-1. We used mutant A. fumigatus strains to show that the reactive oxygen species-sensing transcription factor Yap1, superoxide dismutases, and the Yap1-regulated thioredoxin antioxidant pathway are each required for protection against neutrophil-mediated oxidation of hyphae as well as optimal survival of fungal hyphae in vivo. We also demonstrated that thioredoxin inhibition using the anticancer drug PX-12 increased the sensitivity of fungal hyphae to both H2O2- and neutrophil-mediated killing in vitro. Additionally, topical application of PX-12 significantly enhanced neutrophil-mediated fungal killing in infected mouse corneas. Cumulatively, our data reveal critical host oxidative and fungal anti-oxidative mediators that regulate hyphal survival during infection. Further, these findings also indicate that targeting fungal anti-oxidative defenses via PX-12 may represent an efficacious strategy for treating fungal infections.