Joseph M. Bliss

Susceptibility of Candida Species to Photodynamic Effects of Photofrin

ABSTRACT The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of ≥3 μg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.

Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1

Capsules are well‐studied components of the bacterial surface that modulate interactions between the cell and its environment. Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals. Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1. In this review, the current understanding of the functions of the kps gene products is summarized. Further, a proposed mechanistic model for capsule expression is presented and discussed. The model is based on the premise that the numerous components of the kps cluster form a hetero‐oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion. We view the ATP‐binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co‐ordinate synthesis and translocation. The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram‐ negative bacteria.

Candida parapsilosis is a significant neonatal pathogen a systematic review and meta-analysis

Background: Candida is the third most common cause of late-onset neonatal sepsis in infants born at <1500 g. Candida parapsilosis infections are increasingly reported in preterm neonates in association with indwelling catheters. Methods: We systematically reviewed neonatal literature and synthesized data pertaining to percentage of C. parapsilosis infections and mortality by meta-analyses. We also reviewed risk factors, virulence determinants, antimicrobial susceptibility patterns and outlined clinical management strategies. Results: C. parapsilosis infections comprised 33.47% (95% confidence interval [CI]: 30.02, 37.31) of all neonatal Candida infections. C. parapsilosis rates were similar in studies performed before the year 2000, 33.53% (95% CI: 30.06, 37.40) (28 studies), to those after 2000, 27.00% (95% CI: 8.25, 88.37) (8 studies). The mortality due to neonatal C. parapsilosis infections was 10.02% (95% CI: 7.66, 13.12). Geographical variations in C. parapsilosis infections included a low incidence in Europe and higher incidence in North America and Australia. Biofilm formation was a significant virulence determinant and predominant risk factors for C. parapsilosis infections were prematurity, prior colonization and catheterization. Amphotericin B remains the antifungal drug of choice and combination therapy with caspofungin or other echinocandins may be considered in resistant cases. Conclusion: C. parapsilosis is a significant neonatal pathogen, comprises a third of all Candida infections and is associated with 10% mortality. Availability of tools for genetic manipulation of this organism will identify virulence determinants and organism characteristics that may explain predilection for preterm neonates. Strategies to prevent horizontal transmission in the neonatal unit are paramount in decreasing infection rates.

Vertical and horizontal transmission of Candida albicans in very low birth weight infants using DNA fingerprinting techniques.

Background: Very low birth weight infants (VLBW, ≤1500 g) are at increased risk for invasive disease caused by fungi, and colonization is an important risk factor. This study was designed to examine the effect of maternal flora on Candida colonization of VLBW infants. Methods: Body site samples were collected within 24 hours of delivery from mothers who gave birth to VLBW infants, from their infants at birth, and then weekly for 12 weeks or until death or discharge. Yeast isolates were identified as Candida albicans by standard methods and typed by DNA fingerprinting using a C. albicans strain-specific DNA probe (CARE-2). Results: Sixty-six percent (50/76) of mothers were colonized with yeast and 51% (39/76) of their infants had a Candida species isolated at least once. Of 46 infants born to C. albicans-colonized mothers, 18 (39%) became colonized with C. albicans. Twenty-two percent (17/76) of the infants in the study were colonized with C. albicans by 1 week of age; 76% of these infants (13/17) were born to C. albicans-colonized mothers suggesting vertical transmission. DNA fingerprinting was performed on these 13 mother–infant pairs and 11 pairs demonstrated identical band patterns, confirming vertical transmission. However, of all infants colonized with C. albicans by the first week of age, just 65% (11/17) had a maternal source, and among all infants colonized at any time point, only 41% (11/27) became colonized by vertical transmission. Conclusions: Both vertical and horizontal transmission contribute to Candida colonization of VLBW infants in the neonatal intensive care unit.

Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens.

A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.

Antifungal pharmacotherapy for neonatal candidiasis

Candida is a leading cause of late onset infection (> 3 days of age) in the premature infant. Therefore, decisions about the diagnosis and management of infections caused by Candida are commonplace in the neonatal intensive care unit. Despite this fact, there are few comparative trials about treatment of neonatal Candida infections to guide the practitioner. New antifungals have been developed in the past decade and some clinical experience has been reported that can be used to guide the treatment of infants with serious Candida infections. This article reviews recent pertinent data with regard to dosing guidelines, efficacy, and toxicities of available systemic antifungal agents in the newborn.

The role of galectin-3 in phagocytosis of Candida albicans and Candida parapsilosis by human neutrophils

Candida albicans causes the majority of invasive candidiasis in immunocompromised adults while Candida parapsilosis is a leading cause of neonatal candidiasis. While much work has focused on how the immune system recognizes and responds to C. albicans, less is known about host interaction with C. parapsilosis. This study investigates the human neutrophil phagocytic response to these species. Neutrophils underwent phagocytosis of C. parapsilosis yeast and C. albicans hyphae much more efficiently than C. albicans yeast. Treatment of neutrophils with a galectin‐3 (gal3) blocking antibody inhibited phagocytosis of C. parapsilosis yeast and C. albicans hyphae, but not C. albicans yeast. The majority of neutrophil gal3 was expressed intracellularly and was secreted from neutrophils after treatment with C. parapsilosis mannan. When neutrophils were treated with exogenous gal3, phagocytosis of both C. albicans and C. parapsilosis yeast increased. Exposure of neutrophils to C. parapsilosis yeast increased phagocytosis of C. albicans yeast and was inhibited by gal3 blocking antibody. Taken together, these data indicate that gal3 secreted from neutrophils may act as a pro‐inflammatory autocrine/paracrine signal in neutrophil phagocytosis and suggest that gal3 has a unique role in neutrophil response to C. parapsilosis yeast and C. albicans hyphae distinct from C. albicans yeast.

Candida parapsilosis and the neonate: epidemiology, virulence and host defense in a unique patient setting

Invasive candidiasis is a common problem in premature infants that leads to high morbidity and mortality. Although Candida albicans has historically been the most prominent species involved in these infections and has therefore been the subject of the most study, Candida parapsilosis is increasing in frequency, and neonates are disproportionately affected. This article reviews unique aspects of the epidemiology of this organism as well as strategies for prophylaxis against invasive candidiasis in general. Additionally, important differences between C.parapsilosis and C.albicans are coming to light related to virulence determinants and interactions with components of host immunity. These developments are reviewed while highlighting the significant gaps in our understanding that remain to be elucidated.

Galectin-3 Plays an Important Role in Protection against Disseminated Candidiasis

Recent in vitro studies have implicated galectin-3 as an important receptor in host recognition and response to specific Candida species; however, its role in protection against disseminated candidiasis in vivo has not been evaluated. This study investigated the importance of galectin-3 in host defense against systemic infection with the highly virulent species Candida albicans, and the less virulent species, C. parapsilosis. Mice deficient in galectin-3 (gal3-/-) were more susceptible to infection than wild-type (WT) mice. When infected with C. albicans, gal3-/- mice died significantly faster and exhibited a trend towards increased fungal burden and increased abscess formation in infected brains compared to WT mice. When infected with C. parapsilosis, gal3-/- mice had significantly higher renal fungal burdens and abscess formation compared to WT mice. To evaluate whether galectin-3 may contribute to susceptibility to candidiasis in human infants, galectin-3 levels in sera of newborn infants, a patient population uniquely susceptible to infections with both C. albicans and C. parapsilosis, were compared to serum galectin-3 levels of adults. Galectin-3 levels were significantly lower in newborn infant sera compared to adult sera. These data indicate that galectin-3 plays an important role in a murine model of disseminated candidiasis and suggest a potential mechanism of neonatal susceptibility to these infections.

Differentiation of Candida albicans and Candida dubliniensis by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae

ABSTRACT To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.