A study on fungal speciation and drug susceptibility testing

2020 ◽  
Vol 10 (2) ◽  
pp. 57-62
Author(s):  
Shaik Mujafar ◽  
Boddu Ganesh ◽  
Kesani Meghana ◽  
Nuthalapati Amani ◽  
Shaik Faizan Ali
Keyword(s):  
Candida Albicans ◽  
Bacterial Infections ◽  
Susceptibility Testing ◽  
Clinical Presentation ◽  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Candida Rugosa ◽  
Clinical Manifestations ◽  
Drug Susceptibility Testing ◽  
Important Species

Fungal infections affect many people but most of these do not come to light as they are mild in clinical presentation. Candidiasis is the frequent fungal infection involving mucosa, skin, nails and internal organs caused by different species of Candida and Candida albicans being the prototype. The clinical manifestations vary with duration and severity. It occurs mostly as a comorbid disease with a primary disease or disorder. Candida comes under the phylum Fungi Imperfecti, order Moniliales and family Cryptococcaceae. Genus Candida comprises of 20 important species recognised as pathogenic in humans, of which 7 are renowned opportunistic pathogens. The following are some of the known species: Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Candida guilliermondii, Candida parapsilosis, Candida lusitaniae, Candida kefyr, Candida rugosa, Candida dubliniensis and Candida viswanathii. Among all the fungi, 600 species are identified to be causing infections in human. Candida albicans being one of the normal human commensals may cause infections from mild to severe forms, which is influenced by molecules that helps in adhesion and invasion, hydrolases, yeast to hyphal transition, biofilms etc. In general, equal importance has not been give to fungal infections as is being given for bacterial infections. Nowadays newly emerging species are on the tract as to cause infections and their identification profile being indeterminate, which could be confirmed only by molecular methods. Overall C.tropicalis was the frequent species causing infections clinically and resistance was demonstrated against azoles and caspofungin by C.albicans and C.krusei. This may be due to extensive use of echinocandins as empirical therapy without susceptibility testing which can increase the development of resistance and may deduct treatment options. Hence, routine antifungal susceptibility testing has to be done. Out of all these things accurate epidemiological analysis and data can be provided regarding the burden of fungal infections around the globe. As tip of the iceberg, most of the fungal infections are not being reported as it is unnoticed without prominent clinical presentation and due to lack of complete documentation.

scholarly journals Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

2000 ◽  
Vol 44 (10) ◽  
pp. 2752-2758 ◽  
Author(s):  
Rama Ramani ◽  
Vishnu Chaturvedi
Keyword(s):  
Flow Cytometry ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Broth Microdilution ◽  
Nitrogen Base ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACT Candida species other than Candida albicansfrequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing ofCandida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired ttest values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

scholarly journals Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

2017 ◽  
Vol 55 (6) ◽  
pp. 1883-1893 ◽  
Author(s):  
Cheryl Leong ◽  
Antonino Buttafuoco ◽  
Martin Glatz ◽  
Philipp P. Bosshard
Keyword(s):  
Susceptibility Testing ◽  
Skin Diseases ◽  
Antifungal Susceptibility ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Broth Microdilution ◽  
Content Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Colorimetric Indicator

ABSTRACTMalasseziais a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays forMalasseziaspp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing ofMalasseziathat is based on the CLSI and EUCAST assays forCandidaand other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of allMalasseziaspp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13Malasseziaspecies to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC90values of 0.03 to 1.0 μg/ml, 0.06 to 0.5 μg/ml, and 0.03 to 2.0 μg/ml, respectively. AllMalasseziaspp. were resistant to echinocandins and griseofulvin. SomeMalasseziaspp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treatMalasseziaskin infections. In summary, our assay enables the fast and reliable susceptibility testing ofMalasseziaspp. with a large panel of different antifungals.

scholarly journals Mitochondrial Cochaperone Mge1 Is Involved in Regulating Susceptibility to Fluconazole in Saccharomyces cerevisiae and Candida Species

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Liesbeth Demuyser ◽  
Erwin Swinnen ◽  
Alessandro Fiori ◽  
Beatriz Herrera-Malaver ◽  
Kevin Verstrepen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Iron Metabolism ◽  
Candida Glabrata ◽  
Human Population ◽  
Bacterial Infections ◽  
Fungal Infections ◽  
Pathogenic Fungi ◽  
Recurrent Infections ◽  
Iron Sulfur Clusters ◽  
Iron Sulfur

ABSTRACT MGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae. We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicans. IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker’s yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans. IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker’s yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans.

scholarly journals Colistin and Isavuconazole Interact Synergistically In Vitro against Aspergillus nidulans and Aspergillus niger

2020 ◽  
Vol 8 (9) ◽  
pp. 1447
Author(s):  
Patrick Schwarz ◽  
Elie Djenontin ◽  
Eric Dannaoui
Keyword(s):  
Aspergillus Niger ◽  
Aspergillus Nidulans ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Reference Method ◽  
Important Species ◽  
Agar Diffusion ◽  
Agar Diffusion Assay ◽  
Diffusion Assay

The in vitro interactions of isavuconazole in combination with colistin were evaluated against 55 clinical Aspergillus species isolates belonging to the five most important species (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) responsible for human aspergillosis by a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing. Selected isolates (A. nidulans, n = 10; A. niger, n = 15) were additionally evaluated by an agar diffusion assay using isavuconazole gradient concentration strips with or without colistin incorporated Roswell Parc Memorial Institute (RPMI) agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index. Using the checkerboard method, combination isavuconazole–colistin was synergistic for 100% of the 15 A. nidulans isolates and for 60% of the 20 A. niger isolates. No interactions were found for any of the other isolates. By agar diffusion assay, minimal inhibitory concentrations (MICs) in combination decreased compared to isavuconazole alone for 92% of the isolates. No interactions were found for any A. nidulans isolates, but synergy was observed for 40% of the A. niger isolates. A poor essential agreement of EUCAST and gradient concentration strip MICs at ± 2 log2 dilutions with 0% was obtained. Antagonistic interactions were never observed regardless of the technique used.

scholarly journals Mutations in the fks1 Gene in Candida albicans, C. tropicalis, and C. krusei Correlate with Elevated Caspofungin MICs Uncovered in AM3 Medium Using the Method of the European Committee on Antibiotic Susceptibility Testing

2008 ◽  
Vol 52 (9) ◽  
pp. 3092-3098 ◽  
Author(s):  
Marie Desnos-Ollivier ◽  
Stéphane Bretagne ◽  
Dorothée Raoux ◽  
Damien Hoinard ◽  
Françoise Dromer ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Clinical Isolates ◽  
Antibiotic Susceptibility Testing ◽  
Wild Type ◽  
European Committee ◽  
Rpmi Medium

ABSTRACT Mutations in two specific regions of the Fks1 subunit of 1,3-β-d-glucan synthase are known to confer decreased caspofungin susceptibility on Candida spp. Clinical isolates of Candida spp. (404 Candida albicans, 62 C. tropicalis, and 21 C. krusei isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans, 2 C. tropicalis, and 1 C. krusei isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC90) were subjected to molecular analysis in order to identify mutations in the fks1 gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six C. albicans isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For C. tropicalis, one isolate showed an L644W mutation, and for one C. krusei isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 μg/ml for wild-type isolates and from 1 to 8 μg/ml for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 μg/ml of caspofungin, while caspofungin MICs for all mutant isolates were ≥0.5 μg/ml. These data demonstrate that clinical isolates of C. albicans, C. tropicalis, and C. krusei with decreased susceptibility to caspofungin in vitro have diverse mutations in the fks1 gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.

scholarly journals Diutina (Candida) rugosa complex: The biofilm ultrastructure, extracellular matrix, cell wall component and antifungal susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole

2020 ◽  
Author(s):  
Sri Raja Rajeswari Mahalingam ◽  
Priya Madhavan ◽  
Chong Pei Pei
Keyword(s):  
Extracellular Matrix ◽  
Amphotericin B ◽  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Candida Rugosa ◽  
Yeast Cells ◽  
Confocal Scanning Laser Microscopy ◽  
Prosthetic Heart Valves ◽  
Total Carbohydrate ◽  
Surgical Interventions

AbstractThe genus Candida is the most common etiological factor of opportunistic fungal infections in humans. The virulence of Candida species is due to a wide repertoire of factors, specifically, the ability to form biofilms. Medical devices such as intravenous catheters, prosthetic heart valves and surgical interventions provide pathogenic microorganisms with a surface to adhere to form biofilm. The objectives of this study were to investigate the biofilm ultrastructure of Diutina (Candida) rugosa (D. rugosa) at different developmental phases using Confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM), quantify β-glucan, total carbohydrate and total protein in the extracellular matrix (ECM) using enzymatic β-glucan kit, phenol-sulfuric acid method and Bradford’s method, respectively, and to identify Sessile Minimum Inhibition Concentrations (SMICs) of amphotericin B, caspofungin, fluconazole, and voriconazole using serial doubling dilution. From the SEM micrographs, D. rugosa biofilms were composed of adherent yeast cells and blastospores with hyphal elements. The ultrastructure of the yeast cells was collapsed and disfigured upon exposure to amphotericin B, fluconazole and voriconazole and the biofilms presented with punctured yeast morphology upon exposure to caspofungin at their respective SMICs. The matrix thickness of embedded yeast cells from CLSM micrographs was 3.9µm at 48h. However, there was reduction in the thickness of the biofilms upon antifungal exposure. The antifungal exposed biofilms exhibit bright, diffuse, green-yellow fluorescence that were not seen in the control. D. rugosa biofilm matrices revealed 172.57µg/mL of carbohydrate, and 27.11µg/mL of protein content. The β-glucan yield in D. rugosa complex planktonic cells were in the range of 2.5 to 4.38%, on the contrary, β-glucan was not detected in the ECM. The SMICs of Diutina biofilm for amphotericin B is 1024μg/mL, caspofungin is 512 μg/mL, whereas fluconazole and voriconazole is 2048 μg/mL, respectively.

scholarly journals Antifungal susceptibility testing of Aspergillus niger on silicon microwells by intensity-based reflectometric interference spectroscopy

2019 ◽  
Author(s):  
Christopher Heuer ◽  
Heidi Leonard ◽  
Nadav Nitzan ◽  
Ariella Lavy-Alperovitch ◽  
Naama Massad-Ivanir ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Susceptibility Testing ◽  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Fungal Growth ◽  
Antifungal Susceptibility Testing ◽  
Label Free ◽  
Broth Microdilution ◽  
Silicon Phase ◽  
Reflectometric Interference Spectroscopy

AbstractThe increasing number of invasive fungal infections among immunocompromised patients and the emergence of antifungal resistant pathogens has resulted in the need for rapid and reliable antifungal susceptibility testing (AFST). Accelerating antifungal susceptibility testing allows for advanced treatment decisions and the reduction in future instances of antifungal resistance. In this work, we demonstrate the application of a silicon phase grating as sensor for the detection of growth of Aspergillus niger (A. niger) by intensity-based reflectometric interference spectroscopy and its use as an antifungal susceptibility test. The silicon gratings provide a solid-liquid interface to capture micron-sized Aspergillus conidia within microwell arrays. Fungal growth is optically tracked and detected by the reduction in the intensity of reflected light from the silicon grating. The growth of A. niger in the presence of various concentrations of the antifungal agents voriconazole and amphotericin B is investigated by intensity-based reflectometric interference spectroscopy and used for the determination of the minimal inhibitory concentrations (MIC), which are compared to standard broth microdilution testing. This assay allows for expedited detection of fungal growth and provides a label-free alternative to standard antifungal susceptibility testing methods, such as broth microdilution and agar diffusion methods.

scholarly journals SUSCEPTIBILITY TEST FOR FUNGI: CLINICAL AND LABORATORIAL CORRELATIONS IN MEDICAL MYCOLOGY

2015 ◽  
Vol 57 (suppl 19) ◽  
pp. 57-64 ◽  
Author(s):  
Ana ALASTRUEY-IZQUIERDO ◽  
Marcia S.C. MELHEM ◽  
Lucas X. BONFIETTI ◽  
Juan L. RODRIGUEZ-TUDELA
Keyword(s):  
Susceptibility Testing ◽  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Fungal Species ◽  
Medical Mycology ◽  
Candida Spp ◽  
Disease Epidemiology ◽  
Resistant Strains ◽  
European Standards

SUMMARYDuring recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

scholarly journals Evaluation of the “Dip Effect” Phenomenon in Antifungal Susceptibility Testing of Candida spp. against Echinocandins by Use of Gradient Concentration Strips

2015 ◽  
Vol 53 (11) ◽  
pp. 3654-3659 ◽  
Author(s):  
Maria Siopi ◽  
Marilena Tsala ◽  
Nikolaos Siafakas ◽  
Loukia Zerva ◽  
Joseph Meletiadis
Keyword(s):  
Candida Albicans ◽  
Candida Tropicalis ◽  
Candida Glabrata ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Practical Approach ◽  
Candida Spp ◽  
Content Type ◽  
Inhibition Zones

The “dip effect” phenomenon complicates antifungal susceptibility testing with gradient concentration strips. Of 60Candidaisolates tested with the three echinocandins, this phenomenon was observed only for caspofungin with most (>90%)Candida albicans,Candida glabrata, andCandida tropicalisisolates and for isolates with CLSI MICs of ≤0.25 mg/liter. In order to facilitate MIC determination, a practical approach was developed using the inhibition zones at 32, 8, 2, and 1 mg/liter, increasing the agreement with the CLSI method >86%.

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