Detection of anti-Candida antibodies by the indirect immunofluorescence assay in patients with cancer in the orofacial region

An indirect immunofluorescence assay was performed to detect antibodies toCandida albicans blastospores and germ tubes. Serum specimens were obtained from 82 patients with neoplastic diseases in the orofacial region and thrush of the oral mucosa.C. albicans was identified in the oral cavity of 63 patients investigated but serum anti-Candida antibodies were detected in only 23 of them. Serological examination showed that titers of antibodies toC. albicans blastospores ranged from 1∶20 to 1∶1280. High titers from 1∶640 to 1∶1280 were detected in patients without antibiotic, cytostatic, or radiotherapeutic treatment. The titers of antibodies toC. albicans germ tubes ranged from 1∶20 to 1∶640. Our results indicate that titers of antibodies to theC. albicans germ tubes were lower and were detected in a smaller number of patients.     

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores

The effect of germ tube induction on the antigenic variability in C. albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45–47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.Abbreviations GYE glucose-yeast extract agar     

Detection of invasive Candida albicans infection using a specific 99mTc-labeled monoclonal antibody for the C. albicans germ tube

Accurate diagnosis is critical for effective treatment of the invasive infection by Candida albicans. Here, we investigated whether a ^99m technetium (Tc)-labeled Fab’ fragment of the monoclonal antibody specific for the C. albicans germ tube could specifically identify an invasive C. albicans infection. The germ tube of C. albicans was used as an immunogen to obtain monoclonal antibodies and the Fab’ fragment of MAb03.2 C1–C2 with highest affinity and specificity was labeled with ^99mTc. In vitro binding assays showed that the labeled Fab’ preferentially bound to the germ tubes of C. albicans (4.23 ± 0.17 × 10² Bq per 1 × 10⁷ cells). These values were significantly higher than those for blastospores of C. albicans, blastospores of heat-killed C. albicans, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli (P < 0.05). By using in vivo biodistribution and planar imaging with single photon emission computed tomography, we demonstrated a significant specific accumulation of radioactivity in C. albicans-infected tissues. In summary, ^99mTc-MAb03.2 C1–C2 Fab’ is able to specifically accumulate in C. albicans-infected tissues, but not in tissue infected with A. fumigatus or bacteria or in a sterile inflammation. This study provides a new and specific radiopharmaceutical for the diagnosis of invasive C. albicans infections.     

Germ-tube formation by atypical strains of Candida albicans

Atypical isolates of Candida albicans which failed to produce germ tubes in routine diagnostic procedures were examined for their ability to produce germ tubes in various media. Bovine serum was more effective than defined media for induction of germ tubes in the majority of isolates. A few strains formed appreciable germ tubes only in bovine serum with added thioglycollate or cysteine. One strain did not produce germ tubes in any medium. Germ-tube maturation appeared to be dependent upon mitochondrial RNA polymerase activity. The failure by an isolate to produce germ tubes, particularly in tests without strictly controlled conditions, does not preclude the possibility that the organism is C. albicans.     

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.     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody     

Prevalence, Antigenic Specificity, and Bactericidal Activity of Poultry Anti-Campylobacter Maternal Antibodies

Poultry are considered the major reservoir for Campylobacter jejuni, a leading bacterial cause of human food-borne diarrhea. To understand the ecology of C. jejuni and develop strategies to control C. jejuni infection in the animal reservoir, we initiated studies to examine the potential role of anti-Campylobacter maternal antibodies in protecting young broiler chickens from infection by C. jejuni. Using an enzyme-linked immunosorbent assay (ELISA), the prevalence of anti-C. jejuni antibodies in breeder chickens, egg yolks, and broilers from multiple flocks of different farms were examined. High levels of antibodies to the organism were detected in serum samples of breeder chickens and in egg yolk contents. To determine the dynamics of anti-Campylobacter maternal antibody transferred from yolks to hatchlings, serum samples collected from five broiler flocks at weekly intervals from 1 to 28 or 42 days of age were also examined by ELISA. Sera from the 1-day and 7-day-old chicks showed high titers of antibodies to C. jejuni. Thereafter, antibody titers decreased substantially and were not detected during the third and fourth weeks of age. The disappearance of anti-Campylobacter maternal antibodies during 3 to 4 weeks of age coincides with the appearance of C. jejuni infections observed in many broiler chicken flocks. As shown by immunoblotting, the maternally derived antibodies recognized multiple membrane proteins of C. jejuni ranging from 19 to 107 kDa. Moreover, in vitro serum bactericidal assays showed that anti-Campylobacter maternal antibodies were active in antibody-dependent complement-mediated killing of C. jejuni. Together, these results highlight the widespread presence of functional anti-Campylobacter antibodies in the poultry production system and provide a strong rationale for further investigation of the potential role of anti-C. jejuni maternal antibodies in protecting young chickens from infection by C. jejuni.     

Anti-Candida albicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans

Background

Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis.

Neonatal Intensive Care Unit Candidemia: Epidemiology,Risk Factors,Outcome, and Critical Review of Published Case Series

Evaluation of epidemiological trends, risk factors, and clinical outcome associated with candidemia at a neonatal intensive care unit is reported. From January 2005 to December 2009, forty candidemia cases were identified. C. albicans and C. parapsilosis were the most common species recovered (69 and 24%, respectively). All C. parapsilosis strains were susceptible to antifungals, whereas, C. albicans exhibited higher resistance rates to azoles. Low birth weight, low gestational age, presence of central lines, endotracheal intubation, total parenteral nutrition, previous use of antibiotics, steroids, previous episode(s) of bacteremia and prolonged stay in intensive care unit were common features associated with candidemia. C. albicans was most often isolated from extremely low birth weight neonates as compared to non-albicans Candida (P < 0.01). Mortality rate was 35.7% and was associated with low gestational age (P < 0.01), low birth weight (P < 0.01), and presence of renal failure (P < 0.05). Furthermore, a critical review of recent published case series is presented.     

FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950–1,185 cm⁻¹), protein (1,480–1,720 cm⁻¹), and the fatty acids (2,840–3,000 cm⁻¹) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as β-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.     

Development and evaluation of a rapid identification test for candida albicans

Summary A new technique for the rapid identification ofC. albicans has been developed and evaluated. This yeast can be identified in one hour by the formation of germ tubes after inoculation in 1/2 ml of human or animal plasma, and commercial plasma substitutes.C. albicans also forms germ tubes within 2 to 4 hours after inoculation in human serum and incubation at 37° C.Filamentation ofC. albicans in these blood derivatives is a reliable method for the identification of this yeast. It is more rapid than the assimilation and fermentation sugar tests and chlamydospore formation.Assimilation and fermentation sugar tests are used to identify those isolates ofCandida that fail to produce filaments in plasma or serum.     

Separation and characterization of morphological elements of Candida albicans by Urovison density gradient

Results presented in this paper show that Urovison may be used as a medium for the separation of morphological elements ofC. albicans by means of the density gradient.Blastospores ofC. albicans present variable density, however, those with higher pseudogerminative capacity are situated in the smaller range of densities (1,090: 1,142 g/ml).Germinated blastospores are formed predominantly in the densities close to 1,120 g/ml.Chlamydospores occupy large scale of densities, however, their highest number has been detected in the vicinity of the band corresponding to density of 1,110 g/ml.     

Development of the entomopathogenic fungus Beauveria bassiana in liquid cultures

Growth and development of B. bassiana was followed in four liquid media: peptone, peptone-glucose, glucose and glucose-peptone-yeast extract. Six developmental stages were defined: (I) the unswollen conidium, (II) the swollen conidium, (III) emergence of the germ tube, (IV) elongation of the germ tube and formation of the first septum, (V) polar and bipolar elongation (growth) of the resulting mycelium and initiation of a blastospore and, (VI) seccession of that blastospore. Conidia of B. bassiana produced germ tubes in all liquid media. Blastospores were produced in all liquid media except glucose. In peptone-glucose, the yield of blastospores was four-fold higher than in glucose-peptone-yeast extract. However, biomass production was highest in peptone-glucose-yeast extract.     

An Evaluation of Serodiagnostic Tests in Patients with Candidemia: Beta-Glucan,Mannan, Candida Antigen by Cand-Tec and D-Arabinitol

The serodiagnostic tests, beta-glucan, mannan, candida antigen by Cand-Tec, and D -arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta-glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test-D® and Endospecy®), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand-Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D -Arabinitol was the least sensitive, because a D -arabinitol/creatinine ratio greater than 2.0 μmol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti-fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.     

Complex interaction between different proteinaceous components within the cell-wall structure ofCandida albicans

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface ofCandida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to theC. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluo-rescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.     

Effect of antigen used in the detection of anti -Candida albicans antibodies

Antigens of different origins were used in the investigation of anti-Candida albicans antibodies. This can influence the results obtained.We have assayed three different antigenic preparations weekly for 8 weeks in the study of anti-C. albicans antibodies induced by cutaneous, digestive, and systemic inoculations with C. Albicans ATCC 26555 in rabbits free of specific antibodies, and using indirect immunofluorescence (IIF), direct agglutination (DA) and counterimmunoelectrophoresis (CIE) as serological methods.In IIF and DA, two antigens were used (C. albicans ATCC 26555 and C. albicans NCPF 3153). In CIE we also used a third commercial antigen. All three somatic antigens were used at three different concentrations.Using IIF and DA the titres obtained with both antigens were similar in different inoculations. The IIF was somewhat earlier in the detection of antibodies, and the titre reached was higher when the antigen used was obtained from the inoculated strain.The detection of precipitins by CIE was in most cases only positive with the antigen obtained from the homologous strain, the highest level being reached in the systemic inoculation.     

Inhibition of Germ Tube Formation by Candida albicans by Local Anesthetics: An Effect Related to Ionic Channel Blockade

Formation of germ tubes by Candida albicans has been assumed as a putative virulence factor. Local anesthetics (LAs), e.g., lidocaine and bupivacaine, are known to inhibit germ tube formation. The study confirmed this observation for the novel drug ropivacaine, although it was less potent than the former two drugs. Hypothesizing that the effect is due to blockading ionic channels, we exposed Candida albicans to selective calcium blockers, i.e., nifedipine and verapamil, and to a general blocker of ionic channels, i.e., lanthanum. All blockers inhibited germ tube formation. The effect was dose-dependent and pH-independent. Addition of calcium reverted the effect of the blockers as well as the effect of lidocaine and ropivacaine. The study suggests that the inhibitory effect of LAs on germ tube formation by C. albicans is due to blockade of ionic channels, particularly calcium channels. Therefore, LAs can affect morphology and probably also the pathogenesis of C. albicans. Received: 19 May 1999 / Accepted: 5 October 1999     

Detection of Candida albicans in disseminated candidosis by immunofluorescence staining

An indirect immunofluorescence (IF) method using rabbit anti-Candida albicans was used to detect C. albicans in blood samples of 12 patients with systemic candidosis defined clinically, histologically and by blood cultures. Positive staining of C. albicans could be detected in all of the patients. The findings suggest that IF-method offers a more rapid method in the diagnosis of disseminated candidosis.     

Studies on the Antigenic Activities of Yeasts

In order to identify the antigenic determinant groups of the mannan of C. albicans serotype A, six kinds of manno-oligosaccharides of up to 7 units in chain-length connected by α1→2 linkages were prepared from the partial acetolysate of the parent mannan. In the precipitation-inhibition test of anti-C. albicans serotype A serum with its homologous mannan, inhibitory power of the oligosaccharides was of the following order: heptaose→: hexaose>pentaose>tetraose>triose> biose, and the amounts for 50%-inhibition of the former four oligosaccharides were 0.08, 0.10, 0.50 and 3.0 μmole respectively, and the inhibitory power of the latter two oligomers at 0.5 μmole were 8 and 5% respectively. On the other hand, the cross-inhibition test of anti-C. albicans serotype A serum with the heterologous mannan of C. albicans serotype B afforded the result that the order of inhibitory activities was hexaose>heptaose>pentaose>tetraose>triose> biose, and that the amounts for 50%-inhibition were 0.05, 0.08, 0.1, 0.45, 0.50 and 3.0μmole respectively. Furthermore, the results of inhibition test on the anti-C. albicans serotype A serum absorbed with the mannan of C. albicans serotype B revealed that the biose, triose and tetraose did not show significant inhibitory power in the range employed, whereas the pentaose, hexaose and heptaose did not significantly affect the inhibitory activities. Thus, it was concluded that the antigenic determinants of the mannan of C. albicans serotype A are α1→2 linked hexaose or heptaose moieties. Based on the above facts, the serological differences between two antigenic mannans of C. albicans serotype A and B may reside at least in the length of the antigenic determinants in which the former is longer than the latter considering the length of the α-D-manno-pyranosyl residue.