Studies on detection of Candida antigen in the sera of mice inoculated orally with Candida albicans

Summary The authors succeeded in establishing a murine model of systemic candidiasis being disseminated from the primary gastrointestinal lesions caused by oral inoculation of Candida albicans. Using this model, an attempt was made for detecting the Candida antigen by enzyme-linked immunosorbent assay using avidin-biotin (AB-ELISA) from the serum of infected mice.Gastrointestinal candidiasis was formed in all of the 20 mice treated with the drugs (antibiotics, antineoplastic agents, hydrocortisone, etc.) and inoculated orally with C. albicans. Fourteen of these mice suffered from submucosal candidiasis, and C. albicans was cultured from the visceral organs in 12 of them. The assay by AB-ELISA was able to detect 1.0 ng/ml Candida mannan in the mouse serum. The Candida antigen was detected in the sera of 11 of the 14 mice with submucosal candidiasis. However, the antigen could not be detected in the sera of the 6 mice with intramucosal candidiasis.The assay by AB-ELISA is more sensitive and specific for the diagnosis of systemic candidiasis than other serological assays.     

Evaluation of the antigen from chlamydospores of Candida albicans in the serodiagnosis of candidiasis

Antigens have been prepared from the chlamydospores and blastospores of Candida albicans and their precipitin patterns were analysed by two-dimensional immunoelectrophoresis using specific antisera.The two antigens were used in routine serological tests of patients suffering from candidiasis. On double-diffusion tests for the detection of circulating antibodies of Candida albicans, the antigen from chlamydospores displays precipitin lines that differ in number and intensity from those obtained with the antigen from blastospores. The results are briefly discussed in the framework of C. albicans antigen standardization.     

Mannan and D-arabinitol concentrations in serum from a patient with Candida albicans endocarditis

In an attempt to clarify the comparative values of serological and microbiological examinations for the early diagnosis of systemic candidiasis, antibodies against Candida albicans, serum mannan, and the D-arabinitol creatinine ratio were investigated in a patient with aortic valve endocarditis associated with carcinoma of the bile duct. Candida precipitins and the antibody titer against Candida cell wall mannan were examined by an immunodiffusion technique and hemagglutination test, respectively. Serum mannan was tested by enzyme-linked immunosorbent assay (ELISA) using the biotin-streptavidin procedure. The upper limit of negativity of the assay was determined by adding 0.06 to the absorbance of pooled serum from healthy laboratory workers. This value ws about 0.8 ng/ml with ELISA. The D-arabinitol concentration in serum was examined by an enzymatic fluorometric method. Rising antibody titers against C. albicans, mannan antigenemia, and an elevated D-arabinitol creatinine ratio were first observed between the 11th and 12th hospital days. Blood cultures obtained on 8th, 9th, and 11th hospital days grew C. albicans after 3 to 4 days of incubation. Of 11 serum samples, 5 were positive for mannan, whereas D-arabinitol creatinine ratio was positive in 7 of 9 samples. Blood cultures was the earliest evidence of Candida infections in our cases. However, because of saprophytic nature of Candida species, tests for antibodies, antigenemia, and the D-arabinitol creatinine ratio in combination with blood cultures are necessary to confirm systemic candidiasis at an early stage of infection.Abbreviations ELISA enzyme-linked immunosorbent assay     

Pathological observations in experimental candida infection of sensitized guinea pigs

Guinea pigs immunized intramuscularly with heat-killed or viable Candida albicans were infected intracutaneously with C. albicans. Animals with negative delayed hypersensitivity against C. albicans antigen showed similar lesions with non-immunized controls. Delayed hypersensitivity-positive guinea pigs, which were detected in the animals immunized with heat-killed C. albicans in CFA and IFA, demonstrated a delay of the resolution of the inflammatory tissue reaction and, in the animals immunized with C. albicans in CFA, developed a granuloma.These results suggest that both humoral and cell-mediated immunities do not play a significant role for protection against candidiasis and at a late stage of infection, cell-mediated immunity may play a secondary role of the enhancement of resistance to candida infection associated with granuloma formation.     

Kinetics of Anti-Mannan Antibodies Useful in Confirming Invasive Candidiasis in Immunocompromised Patients

In studying the anti-mannan antibodies longitudinally in serial serum samples of three immunocompromised patients, it was observed that anti-mannan antibodies started to increase shortly after the moment that cultures of deep-tissue sites became positive with Candida albicans. The mean anti-mannan antibody titers determined in a group of 36 immunocompromised patients with invasive candidiasis increased within two weeks after the probable onset of invasive candidiasis. In contrast, anti-mannan antibody levels in serial serum samples of 14 immunocompromised patients who were only colonized with C. albicans remained stable or decreased over time. The HA test measuring the anti-mannan antibodies was 64% sensitive and 89% specific in determining invasive candidiasis. In contrast, antibodies specific for candidal cytoplasmic antigens or enolase alone were of little value in confirming invasive candidiasis in these immunocompromised patients.     

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.     

Detection of Candida sp. mannan antigen by indirect ELISA-inhibition

Summary We have obtained mannans from four Candida species: C. albicans A, C. albicans B and C. tropicalis; antimannan sera against C. albicans A, C. albicans B and C. tropicalis were obtained by immunizing rabbits sub-cutaneously with the respective yeast extract. The efficacy of these sera in reacting with mannans obtained from three Candida sp. has been proven by indirect ELISA-inhibition.Any of three immune sera can be used to detect mannan antigen from the three Candida sp. tested. This confirms the existence of crossed reactivity and the possibility of detecting mannan antigen in serum from patients infected by different Candida sp., although we had only one immune serum and one Candida mannan.     

Purification of cytoplasmic antigens from the mycelial phase of Candida albicans: Possible advantages of its use in Candida serology

The serology of candidiasis is complicated by the use of poorly defined antigens. Total extracts of the yeast phase have been commonly used as cytoplasmic antigen, without regard to the significant amounts of carbohydrate that may contaminate such preparations. This is particularly true in the case of commercially available antigens that have been used as cytoplasmic antigens but actually are richer in carbohydrate than in protein. Affinity chromatography in concanavalin A — Sepharose provides a simple procedure to separate carbohydrates, mainly mannan, from protein antigens in whole Candida extracts. By using mannan-poor antigens, the specificity of serological reactions can be increased considerably, since both the positive reactions seen in asymptomatic donors and the cross-reactions seen in patients infected with other fungi are due to anti-mannan antibodies. In contrast, both anti-mannan and anti-cytoplasmic antigen antibodies can be detected in patients suspected of systemic candidiasis. On the other hand, absolute specificity may never be achieved for systemic candidiasis. We have found antibodies against cytoplasmic antigen in a patient allergic to C. albicans, in whom the microorganism was isolated from fecal material. It appears that, under favorable conditions, mucosal sensitization may also trigger a systemic reaction directed against both mannan and cytoplasmic antigens.Publication no. 341 from The Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.     

Puesta al día en la candidiasis invasora en el paciente crítico no neutropénico

Invasive candidiasis in non-neutropenic critically ill patients remains a challenge for clinicians due to its association with high morbidity and mortality rates, increased incidence, and health-care costs. It is well known that early diagnosis and treatment are associated with a better prognosis. For these reasons a thorough update has been performed in this setting focused on recent Spanish epidemiology, new predictive scores and microbiological tests such as mannan antigen, mannan antibodies, Candida albicans germ-tube antibodies or (1→3)-β-D-glucan detection, molecular techniques for the detection of fungal-specific DNA, advances in antifungal treatment and educational programs in Spain. An early diagnostic and therapeutic algorithm is proposed based on the combination of scores and microbiological test. The aim of this review is to provide physicians with the best information available in order to improve the prognosis of these patients.     

Biochemical and Immunochemical Studies of Fungi

Crude mannans extracted from Candida albicans and Saccharomyces cerevisiae by autoclaving yeast cells in citrate buffer (pH 7.0) according to Peat's method, were fractionated repeatedly by column chromatography on DEAE-Sephadex, acetate form, yielding neutral and acidic mannans. The former fraction showed a single peak by boundary electrophoresis and ultracentrifugal analysis, while the latter contained small amounts of phosphorus and protein. Using purified mannans as controls, various serological experiments were carried out with mannan antigens extracted from C. albicans with 45% phenol water and with 3% NaOH. No remarkable differences were observed in the antigenic activity of 4 mannan antigens from C. albicans, and the purified mannan exhibited very high antigenic activity. It was found that the mannan of S. cerevisiae was antigenically less specific than that of C. albicans mannan. The difference in serological specificity between mannans of both species may reflect not only differences in mannopyranose linkages but differences in the structure of the macromolecules.     

Rapid enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to Candida albicans

A rapid enzyme-linked immunosorbent assay (ELISA) where the performance time was shortened to 4h was compared with counter-immunoelectrophoresis (CIE) and a standard ELISA procedure for the detection of IgG antibodies to Candida albicans in 61 patients with suspected invasive candidosis. Using a C. albicans cytoplasmic antigen the rapid ELISA compared well with CIE and the standard ELISA. Seventeen sera that reacted with two concentrations of C. albicans antigen in CIE were also positive in both forms of ELISA. Four sera that were CIE-negative were positive in the standard ELISA and three were also positive in the rapid ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of different forms of candidosis.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

On the Immunochemical and Biochemical Studies of Fungi

The nature of the cross reaction of the mycelial mannan of Trichophyton rubrum and galactomannan isolated from the culture medium of Aspergillus fumigatus with antisera of Saccharomyces cerevisiae and Candida albicans is described. Cross-reactivity of polysaccharides of both T. rubrum and A. fumigatus was weak with antisera of C. albicans and S. cerevisiae, but the galactomannan of A. fumigatus showed slightly stronger activity than the mannan of T. rubrum which possesses more closely related chemical structure of the mannans of S. cerevisiae and C. albicans.     

Detection of anti-<Emphasis Type="Italic">Candida</Emphasis> antibodies in neonates from a neonatal intensive care unit

Anti-Candida antibodies were determined in a group of preterm neonates from a neonatal intensive care unit with serious diseases including candidemia. Antibodies toC. albicans blastospores,i.e. antibodies toC. albicans surface mannan and toC. albicans germ tubes were detected. Higher titers of antibodies to blastospores (1:320) occurred in all patients examined while antibodies toC. albicans germ tubes (with the highest titer of 1:160) were present in 32 out of 66 neonates examined. The highest titers of both anti-C. albicans blastospore antibodies and anti-C. albicans germ tube antibodies were detected in neonates with candidemia and disorders of saccharide metabolism.     

Response to Oxidative Stress in Eight Pathogenic Yeast Species of the Genus <Emphasis Type="Italic">Candida</Emphasis>

In the course of an infection, the formation of reactive oxygen species by phagocytes and the antioxidant defense mechanisms of microorganisms play a crucial role in pathogenesis. In this study, isolates representing 8 pathogenic Candida species—Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida tropicalis—were compared with regard to their resistance to oxidative stress in vitro. We evaluated degree of resistance, induction of oxidative damage, capacity to adapt, and induction of antioxidant enzymes. The species showed variable sensitivity to oxidative attack. C. albicans, C. glabrata, and C. krusei were more resistant to oxidative stress under the conditions tested; C. parapsilosis and C. tropicalis presented medium resistance; and C. dubliniensis, C. famata, and C. guilliermondii were more sensitive. The overall greater resistance to oxidative stress of C. albicans and C. glabrata may provide an advantage to these species, which are the major causative agents of candidiasis.     

In vitro and in vivo evaluation of the antifungal activity of fluoxetine combined with antifungals against Candida albicans biofilms and oral candidiasis

Abstract

Candida albicans biofilms are responsible for oral candidiasis. Fluoxetine is a widely used antidepressant, with certain anti-Candida activities. The antifungal activity of fluoxetine combined with various antifungals against C. albicans biofilms and oral candidiasis was evaluated in this study. The morphological change in the inhibition of fluoxetine on C. albicans biofilms was observed using SEM. The interactions between fluoxetine and antifungals against C. albicans biofilms were evaluated using microdilution checkerboard methods, FICI and the ΔE model. The synergistic combination was tested in vivo on the mice model of oral candidiasis. SEM imaging showed fluoxetine inhibited hyphal growth and biofilm formation. Fluoxetine combined with caspofungin exhibited synergistic effects against C. albicans biofilms. Antagonistic effects occurred when fluoxetine was combined with amphotericin B or terbinafine. Further, the fluoxetine combined with caspofungin significantly reduced the lesion score and CFU of C. albicans on the murine tongue (p?<?0.05), and relieved oral candidiasis of the infected mice.     

Influence of oxidative and osmotic stresses on the structure of the cell wall mannan of Candida albicans serotype A

In this study, we investigated the structural changes in the cell wall mannan of Candida albicans serotype A strain cells cultured under various stress conditions, that is, oxidative stress of 3.5 mM H₂O₂, osmotic stress of 1.5 M NaCl, and heat stress at 37 °C, compared with the normal condition of 30 °C in yeast extract-added Sabouraud liquid medium (YSLM). Based on the ¹H nuclear magnetic resonance (NMR) and fluorophore-assisted carbohydrate electrophoresis (FACE) analyses of the mannans, we showed that the proportion of the terminal β-1,2-linked mannose side chain unit in the mannan increased in the cell proliferation process under both the normal condition and the oxidative stress condition. The osmotic stress induced a slight decrease in the proportion of the β-1,2-linked mannose unit in the acid-labile fraction. The heat stress induced a significant decrease in the proportions of the β-1,2-linked mannose unit in both the acid-labile and acid-stable fractions. Based on these results, we propose that C. albicans significantly changes the mannan structures under various stress conditions and that sufficient attention to the cultural conditions is needed to perform an accurate diagnosis of candidiasis.     

Evaluation of three serological tests for detection of anti-candidal antibodies in diagnosis of invasive candidiasis

Immunoblot detection of antibody against 47 KD cytoplasmic antigen ofCandida albicans was evaluated in diagnosis of invasive candidiasis and compared to whole cell agglutination and gel diffusion tests for detection of anticandidal antibody in 64 patients. The patients included 17 with culture proved candidemia, 34 with significant candiduria (more than 10,000 colony forming units per ml of urine) and 13 with nonsignificant candiduria. Antibody against 47 KD antigen was found to be the best indicator for diagnosis of invasive candidiasis even in patients with malignancy. The sensitivity of this procedure was 82.4%, specificity 86.7%, positive predictive value 77.8%, negative predictive value 89.7% and efficacy 85.1%. The gel diffusion procedure lacked in sensitivity whereas whole cell agglutination lacked in specificity. Detection of antibody against 47 KD antigen proved to be a valuable adjunct in the diagnosis of invasive candidiasis.     

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H₂O₂) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.     

Studies on the Antigenic Activities of Yeasts

In order to provide further information about the immunochemical differences between two mannans of Candida albicans serotype A and serotype B, quantitative precipitation-inhibition tests of anti-C. albicans serotype B serum were carried out in the present study. Oligosaccharides were prepared by acetolysis of a homologous mannan, and a1→2 linked di-, tri-, tetra-, penta- and hexasaccharide were separated in chromatographically homogeneous states. The latter two oligomers contained a small amount of an isomer containing a1→2 and a1→3 linkages. In the precipitation-inhibition tests of anti-C. albicans serotype B serum with its homologous mannan and heterologous mannan of C. albicans serotype A, the inhibitory power of the oligomers was of the following order; hexa-> penta-> tetra-> tri-> disaccharide, and the amounts for 50%-inhibition of the former 4 oligomers were 0.02–0.03, 0.05–0.07, 0.1–0.2 and 0.3–0.4 μmoles respectively, whereas disaccharide was very poor inhibitor. The lower oligomers, a1→2 linked tri- and tetrasaccharide, showed considerably strong inhibitory activities. The results obtained in the present study confirmed that the antigenic determinants of the mannan of C. albicans serotype B is the hexasaccharide moiety corresponding to the longest branched chains of mannan, and moreover, the a1→2 linked tri- and tetrasaccharide moieties play an important factor in dominating immunochemical specificity.