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Evaluation of CHROMagar Candida for rapid screening of clinical specimens for Candida species.

CHROMagar Candida is a new differential culture medium that allows selective isolation of yeasts and simultaneously identifies colonies of Candida albicans, Candida glabrata, Candida tropicalis and Candida krusei. We evaluated this medium and compared it with a reference medium, Sabouraud glucose agar, for the presumptive identification of yeast species isolated directly on the medium from 1150 clinical specimens. A total of 731 specimens showed no growth, 299 isolates (70.2%) showed growth to the same extent on both media. Forty mixed cultures were detected on both media. More than one isolate was detected in 30 of the tested specimens on either CHROMagar (26 specimens) or Sabouraud glucose agar (four specimens). We found a sensitivity of 98.8% and a specificity of 100% for C. albicans, 66.7% and 99.8% for C. tropicalis, 100% and 100% for C. krusei, and 98% and 95.7% for C. glabrata. Regarding these results, CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of yeasts and better detection of mixed cultures in clinical specimens.

Candida↗

Candida africana sp. nov., a new human pathogen or a variant of Candida albicans?

Atypical Candida strains were isolated from patients in Madagascar, Angola and Germany. These isolates were slow growing and were unable to produce chlamydospores. They had atypical carbohydrate assimilation profiles. All strains were unable to assimilate the amino sugars N-acteylglucosamine and glucosamine as well as the disaccharide trehalose and the organic acid DL-lactate. They were germ-tube-positive in serum, but only some of these organisms produced pseudohyphae after a long incubation. As shown by Fourier transform infrared spectroscopy the atypical Candida isolates clustered as a monophyletic group different from C. albicans and C. dubliniensis. All strains belonged to C. albicans serotype B. Considering all data presented here, this group of Candida strains differs from any other known member of the genus Candida. Therefore, it is suggested to represent a new species within the genus Candida for which the name Candida africana is proposed.

Angola↗

Amphotericin B tolerance: a characteristic of Candida parapsilosis not shared by other Candida species.

Thirty yeast isolates from clinical specimens were tested for their susceptibility to amphotericin B at 30 C, 37 C, and 39 C. Of the six Candida albicans, five Candida tropicalis, one Candida guilliermondii, one Candida krusei, one Candida pseudotropicalis, two Torulopsis glabrata, and four Cryptococcus neoformans isolates tested, all were inhibited at amphotericin B concentrations of less than or equal to 0.4 micrograms/ml and killed by concentrations of amphotericin B that were less than or equal to 16-fold higher than the minimal inhibitory concentration (MIC). Although growth of Candida parapsilosis was also inhibited by concentrations of amphotericin B of less than or equal to 0.4 micrograms/ml, minimal fungicidal concentrations of amphotericin B were greater than or equal to 32-fold higher than MICs for each of the 10 isolates examined. This unique susceptibility pattern of C. parapsilosis resembles the antibiotic tolerance observed with Staphylococcus aureus. Variations in temperature within the experimental range did not affect the amphotericin B susceptibility for any of the yeasts examined.

Amphotericin B↗

Persistent oral candidosis by non-albicans Candida strains including Candida glabrata in a human immunodeficiency virus-infected patient observed over a period of 6 years.

A 38-year-old woman infected with human immunodeficiency virus (HIV) presented with persistent oral candidosis in which non-albicans Candida strains were the predominant yeasts in most of the examinations performed over a period of 6 years. Oral treatment with fluconazole had no effect on clinical signs of oral candidosis. In 8 of a total of 11 specimens, Candida glabrata, Candida parapsilosis and Candida tropicalis were at least suspected as the causative pathogens of oral candidosis. The non-response to fluconazole in our patient could be explained by in vitro resistance to fluconazole of detected Candida glabrata and Candida tropicalis isolates.

AIDS-Related Opportunistic Infections↗

Candida albicans killing by RAW 264.7 mouse macrophage cells: effects of Candida genotype, infection ratios, and gamma interferon treatment.

Phagocytic cells such as neutrophils and macrophages are potential components of the immune defense that protects mammals against Candida albicans infection. We have tested the interaction between the mouse macrophage cell line RAW 264.7 and a variety of mutant strains of C. albicans. We used an end point dilution assay to monitor the killing of C. albicans at low multiplicities of infection (MOIs). Several mutants that show reduced virulence in mouse systemic-infection models show reduced colony formation in the presence of macrophage cells. To permit analysis of the macrophage-Candida interaction at higher MOIs, we introduced a luciferase reporter gene into wild-type and mutant Candida cells and used loss of the luminescence signal to quantify proliferation. This assay gave results similar to those for the end point dilution assay. Activation of the macrophages with mouse gamma interferon did not enhance anti-Candida activity. Continued coculture of the Candida and macrophage cells eventually led to death of the macrophages, but for the RAW 264.7 cell line this was not due to apoptotic pathways involving caspase-8 or -9 activation. In general Candida cells defective in the formation of hyphae were both less virulent in animal models and more sensitive to macrophage engulfment and growth inhibition. However the nonvirulent, hypha-defective cla4 mutant line was considerably more resistant to macrophage-mediated inhibition than the wild-type strain. Thus although mutants sensitive to engulfment are typically less virulent in systemic-infection models, sensitivity to phagocytic macrophage cells is not the unique determinant of C. albicans virulence.

Animals↗

Candida detection system (CAND-TEC) to differentiate between Candida albicans colonization and disease.

Eighty-three serum specimens from 24 patients infected with Candida albicans were examined for circulating Candida protein antigens with the Candida Detection System (CAND-TEC; Ramco Laboratories, Inc., Houston, Tex.). The medical records of each patient were reviewed for clinical evidence of Candida colonization or disease, predisposing factors for infection, underlying illness, the presence of a contaminated indwelling venous catheter, intravenous amphotericin B therapy, and outcome. Forty-nine serum specimens with antigen titers of 1:2 or less were obtained either from colonized patients or at a time when disseminated disease was not yet clinically suspected. Except for five specimens from two colonized patients, one with a contaminated arterial line, the other specimens with titers of 1:8 or greater (n = 14) were obtained from patients who had been clinically diagnosed and treated for disseminated candidiasis. Serum specimens with titers of 1:4 were often from patients with deep-seated candidal infection but were not uniformly diagnostic; in this situation additional specimens should be tested for Candida antigen titers. Only 1 of 24 serum specimens from patients with no evidence of C. albicans infection had a Candida protein antigen titer of 1:8. With a 1:8 or greater titer as a criterion for dissemination, the sensitivity of the CAND-TEC system was 71%, with a specificity of 98%. If the 1:8 titer for the colonized patient with a contaminated arterial line is not considered a false-positive result, the CAND-TEC sensitivity was 83%. The latex agglutination assay appears to be a useful, rapid, and noninvasive means of laboratory diagnosis of systemic candidiasis. The recovery of C. albicans from at least three body sites may also be a useful predictor of disseminated disease.

Antigens, Fungal↗

Oligonucleotide fingerprinting of isolates of Candida species other than C. albicans and of atypical Candida species from human immunodeficiency virus-positive and AIDS patients.

Oligonucleotide fingerprinting of genomic DNA from oral isolates of four different Candida species other than C. albicans and atypical chlamydospore-positive isolates from human immunodeficiency virus (HIV)-positive individuals and AIDS patients was investigated as a means for differentiating between isolates within individual species. Oligonucleotides composed of simple repetitive sequence motifs, including (GACA)4, (GATA)4, (GGAT)4, (GTG)5, and (GT)8, all yielded fingerprints suitable for strain segregation of 8 C. tropicalis isolates, 12 Torulopsis (Candida) glabrata isolates, 8 atypical Candida isolates, and, except for (GATA)4, 2 C. krusei probe in turn and so generate several distinct DNA fingerprints of the same DNA sample. However, none of the probes yielded fingerprints suitable for strain segregation with three C. parapsilosis isolates. The (GATA)4 probe was also used to detect restriction fragment length polymorphisms among a genetically closely related group of atypical Candida isolates on primary isolation from an additional HIV-infected patient. These chlamydospore-positive atypical Candida isolates were sucrose positive, were of C. albicans serotype A, hybridized weakly with the C. albicans-specific mid-repeat sequence probe 27A, and yielded fingerprint profiles by random polymorphic DNA analysis that were distinct from those derived from C. albicans isolates. The C. stellatoidea ex-type strain NCPF 3108 was indistinguishable from the atypical Candida isolates in all these tests and also yielded an identical carbohydrate and nitrogen source assimilation profile by using the ID 32C yeast identification system.

AIDS-Related Opportunistic Infections↗

Comparison of the MUREX C. albicans, Albicans-Sure, and BactiCard Candida test kits with the germ tube test for presumptive identification of Candida albicans.

The MUREX C. albicans (MC)(Murex Diagnostics), Albicans-Sure (AS) (Clinical Standards Laboratories), and BactiCard Candida (BC) (Remel) test kits were compared with the germ tube (GT) test for the rapid, presumptive identification of Candida albicans. All three test kits detect the enzymes L-proline aminopeptidase and beta-galactosaminidase in yeast cells grown on culture media and are based on the principle that C. albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C system and conventional methods) and included 303 C. albicans isolates, 153 Candida glabrata isolates, 70 Candida tropicalis isolates, 36 Candida parapsilosis isolates, 13 isolates of other Candida spp., 5 Cryptococcus neoformans isolates, and 3 Saccharomyces cerevisiae isolates. The MC, AS, BC, and GT tests detected 299 (98.7%), 300 (99.0%), 301 (99.3%), and 287 (94.7%) C. albicans isolates, respectively. There was one false-positive result with both the MC and BC kits and two false-positive results with the GT test. The enzymatic methods evaluated in this study provide rapid and accurate alternatives to the GT test for the presumptive identification of C. albicans.

Candida albicans↗

Optimizing voriconazole susceptibility testing of Candida: effects of incubation time, endpoint rule, species of Candida, and level of fluconazole susceptibility.

Voriconazole is a new triazole antifungal agent that has potent activity against many isolates of Candida, including Candida krusei and Candida glabrata. In this work, we studied the impact of glucose supplementation, incubation time, agitation of the plates prior to reading, endpoint determination rule, visual versus spectrophotometric reading, Candida species, and fluconazole MIC on the MIC of voriconazole for Candida isolates tested by using the microdilution format assay of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A antifungal susceptibility testing methodology. For both voriconazole and fluconazole, a spectrophotometric endpoint of 50% reduction in turbidity relative to the growth control correlated most closely with the NCCLS-defined visual endpoint of "prominent decrease in turbidity." Correlation was generally better after 24 h of incubation than after 48 h. Supplementation of the medium to contain 20 g of glucose/liter did not alter the MIC significantly but did enhance growth and simplify visual readings. All Candida species appeared potentially susceptible to voriconazole, including isolates of C. krusei. For some isolates for which fluconazole MICs were markedly elevated voriconazole MICs were also elevated, but the clinical significance of these observations remains to be determined.

Amphotericin B↗

Rapid identification of the Candida species from direct blood cultures by CHROMagar Candida.

We evaluated the ability of CHROMagar Candida to identify Candida species isolated directly from blood cultures. A total of 50 clinical isolates of Candida were incubated at 35 degrees C, and once growth was established, an aliquot of each was plated onto CHROMagar Candida medium. A control specimen was plated directly from Sabouraud's dextrose agar. Following incubation at 30 degrees C, all yeast isolates were identified by colony morphology and colour. We were able to identify all isolates of C. albicans (n = 20), C. tropicalis (n = 14), C. glabrata (n = 6), and C. krusei (n = 5), which were isolated from blood or from control cultures. This study demonstrated that CHROMagar Candida reliably isolated and identified yeast taken directly from blood cultures. We conclude that this rapid and easy method of identifying Candida species will enable clinicians to quickly choose the appropriate antifungal agent. This should decrease patient morbidity and mortality.

Blood↗

Studies on stimulation of cell-mediated cytotoxicity by skin test antigens. I. Candida antigen stimulation of cell-mediated cytotoxicity in vitro correlated with the skin test response to candida antigen in vivo.

The magnitude of the skin test response in a group of normal volunteers to intradermal Candida antigen correlated closely with the level of cytolytic activity stimulated by Candida antigen in vitro in the peripheral blood lymphocytes from those same individuals. The cytolytic activity stimulated by Candida antigen was cell mediated, and cold target inhibition studies demonstrated that Candida antigen-stimulated effector cells were capable of cross-species (i.e., nonspecific) killing. Analysis of the Candida antigen-induced effector cell population for various cell markers did not enable absolute identification of the cell responsible for the killing. The findings in this study indicate that the stimulation of cell-mediated cytolysis by Candida antigen may be related to the delayed-type hypersensitivity response produced by the same antigen.

Animals↗

[Acute Candida arthritis. Isolation of Candida krusei in a heroin addict].

Candida arthritis in men is very rare. In most cases Candida albicans is isolated from joints (commonly the knee) of immature infants or of immunodeficient subjects without underlying joint disease; Candida non albicans is isolated from joints of immunodeficient patients with underlying joint disease. A case of Candida arthritis in a heroin addict is described. This is the second report of Candida arthritis in a heroin addict, but it is the first in which Candida krusei has been isolated.

Adult↗

An update on clinically relevant, rare, and emerging Candida and Saccharomycotina yeasts that have been recently reclassified from Candida.

SUMMARYMany yeast species causing life-threatening invasive infections that were formerly classified in the genus Candida have been reclassified due to their evolutionary and phylogenetic relationships elucidated by DNA sequencing methods that are increasingly using whole genomes. This review explores the evolving taxonomy, epidemiology, and clinical implications of clinically relevant, rare, emerging Candida and Saccharomycotina yeasts that have recently been reclassified from Candida. This article highlights the urgent need for intensified research efforts to enhance knowledge and improve outcomes in the management of infections caused by these yeasts. Communicating results from molecular phylogenetic studies of yeasts, which lead to their reclassification, is of great importance to the medical mycology community to implement such results in clinical practice.

Humans↗

Immunosuppression and recovery of drug-impaired host resistance against Candida albicans infection by oxoglaucine.

The immunosuppressive action of aporphinoid alkaloid oxoglaucine was studied in experimental Candida albicans (C. albicans) infection in mice. The alkaloid augmented host resistance to pathogen applied to mice (6-8 weeks of age) at a low dose of 2 mg kg(-1) in 3 days and impaired it at a high dose of 10 mg kg(-1). The suppressive activity observed under the latter schedule correlated with the inhibited proliferative response of splenic cells to mitogens and with decreased popliteal lymph node (PLN) reaction to C. albicans. Treatment of mice with oxoglaucine (at the age of 5 days) at a dose of 5 mg kg(-1) in 3 consecutive days increased the susceptibility to Candida inoculation at the age of 6 weeks. Delayed type hypersensitivity (DTH) response to C. albicans was enhanced after pretreatment of adult mice and was suppressed after administration to newborn mice. Long-time treatment (10 days) with oxoglaucine, cyclophoshamide or prednisolone at a dose of 10 mg kg(-1) increased the rate of mortality of Candida-infected mice. Combined pretreatment of mice with cyclophosphamide or prednisolone (5 days at a dose of 5 mg kg(-1)) followed by oxoglaucine (5 days at a dose of 5 mg kg(-1)), prolonged the survival of infected mice.

Animals↗

Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces.

Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.

Antifungal Agents↗

CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species.

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.

Agar↗

Brief exposure to antimycotics reduces the extracellular phospholipase activity of Candida albicans and Candida tropicalis.

BACKGROUND: Although the phospholipase activity is considered a potential virulence determinant of the pathogenic Candida species, the effect of antimycotics on this attribute is not known. Hence we evaluated the phospholipase activity in 10 isolates each of Candida albicans and Candida tropicalis, after their exposure to antifungals. METHODS: The impact of antimycotics on phospholipase activity was also assessed after exposure of the isolates to sub-minimum inhibitory concentrations of nystatin, amphotericin B and fluconazole. RESULTS: All Candida isolates investigated exhibited phospholipase activity (Pz). In general C. ALBICANS showed relatively higher P(z) activity than C. tropicalis , and exposure of the isolates to antimycotics led to a significant (p < 0.05) reduction in the phospholipase activity. Nystatin and amphotericin B, but not fluconazole, significantly reduced the phospholipase activity of both Candida species. CONCLUSION: These observations, while confirming the higher virulence of C. albicans relative to C. tropicalis, demonstrate for the first time the effect of antifungal agents on extracellular phospholipases of these common opportunistic pathogens.

AIDS-Related Opportunistic Infections↗

Activated lactoferrin and fluconazole synergism against Candida albicans and Candida glabrata vaginal isolates.

OBJECTIVE: To evaluate the fungistatic activity of activated lactoferrin (ALF), fluconazole (FCN) individually and in combination against Candida vaginal isolates as well as to measure the time to recovery from the fungistatic effects after exposure in vitro to threshold minimal inhibitory concentrations (MIC). STUDY DESIGN: Fungistasis patterns for ALF (2.5 mg/mL) and FCN (0.25 mg/mL) were tested at threshold MIC against vaginal isolates of C albicans (n = 5) and C glabrata (n = 5) grown in Sabouraud's dextrose broth against 10(5) yeast inoculum at 37 degrees C for 48 hours by microscale optical density (OD) assay according to the following criteria: "Total stasis" indicates that an agent elicited no change or a change in turbidity <0.1 OD unit for >48 hours (complete growth inhibition), "stasis recovery" (SR) is the time point at which turbidity of a previous stasis system shows an upward growth trend for >0.1 OD unit (recovery from growth inhibition), and "partial stasis" (PS) is proliferation after stasis recovery, measured as a percentage relative to growth control at any time (incomplete growth inhibition). RESULTS: For ALF (2.5 mg/mL), the mean SR time was 15.6 +/- 2 hours for C albicans (n = 5) and 27.5 +/- 2 hours for C glabrata (n = 5). The SR patterns for FCN were strain dependent and showed a wide range of deviation for both Candida species; accordingly, the values were 15.8 +/- 9 hours for C albicans and 25.5 +/- 12 hours for C glabrata. After 48 hours exposure to C albicans, ALF and FCN elicited a mean PS of 27.5 +/- 2% and 24.8 +/- 7%, respectively. The PS values at 48 hours showed a marked variation between C glabrata isolates, 29.1 +/- 24% for ALF and 21.5 +/- 38% for FCN. However, a combination of ALF and FCN at their threshold MIC showed significant drug synergism, causing total stasis of both species of Candida isolates. Thus, no SR for any Candida isolate was detected at or beyond 48 hours. Conversely, native lactoferrin failed to demonstrate such potent synergism with FCN against either Candida species. CONCLUSION: The combination of ALF and FCN at the threshold MIC elicited potent synergism, leading to total fungistasis of C albicans and C glabrata vaginal pathogens. ALF is a new class of fungistatic agent with a mode of action distinct from that of azoles.

Analysis of Variance↗