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لطلبة الدراسات العليا- مراجعة Clinlcal Mycology
Antifungal Susceptibility Testing
It is now possible for the clinical microbiology laboratory to perform reliable in vitro antifungal susceptibility tests on a wide range of yeasts and moulds. The aim of this article is to a provide review of what is currently available to the clinical laboratory along with some practical comments on antifungal susceptibility testing. Major advances with the standardisation and clinical interpretation of in vitro antifungal susceptibility testing have been made in recent years. These include the introduction of standard reference methods for both yeasts and moulds by the NCCLS1,2,3 and publication of interpretive breakpoints, especially for Fluconazole and Itraconazole against Candida infections4. In the antifungal susceptibility testing world, the NCCLS have set the benchmark methodology by providing laboratory tested, reproducible, consensus peer review standards that are updated on a regular basis.
NCCLS M27-A2 standard for yeasts:
See NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Second Edition. NCCLS document M27-A2 [ISBN 1-56238-469-4]. NCCLS, Pennsylvania, USA .
The M27-A2 standard is intended for testing yeasts including Candida species and Cryptococcus species (C. neoformans and C. gattii) by using either a macro or micro broth dilution test system. It recommends the use of RPMI-1640 medium (with glutamine and phenol red, without bicarbonate)(Sigma #R-7755 St. Louis, USA) supplemented with 0.2% glucose and buffered to a pH of 7.0 with 0.165 mol/L MOPS (3-[N-morpholino] propanesulfonic acid)(Sigma #M-6270), inoculum standardized to 0.5 McFarland using a densitometer and incubation at 35oC. It is essential to use the exact reagents as listed from Sigma. Plates are read at 24 hours for non-fastidious yeasts like Candida or at 48 to 72 hours for slower growing yeasts like Cryptococcus.
The microdilution wells should be visualised with the aid of a reading mirror and the growth in each well should be compared with that of the growth control. A numerical score from 0 to 4 is given to each well using the following scale: 0 = optically clear, 1 = slightly hazy, 2 = prominent reduction in turbidity compared with that of the drug-free growth control, 3 = slight reduction in turbidity compared with that of the drug-free growth control, 4 = no reduction in turbidity compared with that of the drug-free growth control. The MIC for amphotericin B is the lowest concentration with a score of 0 (optically clear). The MICs for the azoles and 5FC are the lowest concentrations with a score of 2 (prominent decrease in turbidity).
Interpretation of results: For the most part Amphotericin B MICs for Candida species cluster between 0.25 and 1.0 ug/ml. However it must be stressed that the M27 method does not consistently permit detection of resistant strains and isolates with MICs of > 1.0 ug/ml should be considered likely to be resistant. Antibiotic Medium 3 supplemented with 2 % glucose may permit more reliable detection of resistance but this medium is not standardized and substantial lot-to-lot variability is possible. On a brighter note, interpretative breakpoints for Candida against Fluconazole, Itraconazole and 5-Fluorocytosine have been established (table 1). These breakpoints have also been used for isolates of Cryptococcus where there is also some correlation between elevated MIC and treatment failure1,5.
Trailing end points. Some azoles, particularly fluconazole, exhibit a phenomenon known as trailing. Trailing occurs when the turbidity continually decreases as the drug concentration increases but the suspension fails to become optically clear (partial inhibition of growth over an extended range of antifungal concentrations). For most isolates, the difference between reading at 24 hours versus 48 hours is minimal and will not alter the interpretative category (i.e. does not change whether the isolate would be read as “susceptible” or “resistant”). However some isolates show a dramatic rise in MIC over time (e.g. for fluconazole from 0.5 ug/ml at 24 hours to 256 ug/ml at 48 hours). This trailing phenomenon has been reported as occurring in about 5% of isolates,6 however some studies have reported that up to 20% of C. albicans isolates read at 48 hours may show trailing to fluconazole that would alter the interpretation from “susceptible” to “resistant” . To help resolve this issue the M27-A2 methodology for Candida has provided both 24 hour and 48 hour microdilution MIC ranges for the two QC strains and eight systemic antifungal agents . Ideally plates should be read at 24 hours whenever there is sufficient growth.
Etest showing a trailing end point.
The NCCLS M27-A21 method is now the benchmark to validate all other methods against. As a result several commercial systems are now available to the clinical laboratory for antifungal susceptibility testing.
NCCLS M44-P standard for yeasts by disk diffusion:
See NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Proposed Guideline. NCCLS document M44-P [ISBN 1-56238-488-0]. NCCLS, Pennsylvania, USA 2003.
The M44-P standard is a newly established methodology for disk diffusion testing of Candida species. Other yeast genera and moulds have yet to be validated using this method. The standard includes zone interpretive criteria for fluconazole and recommended quality control ranges for fluconazole and voriconazole. It recommends the use of Mueller-Hinton agar supplemented with 0.2% glucose and 0.5 ug/ml methylene blue dye medium. Mueller-Hinton agar is readily available and shows acceptable batch-to-batch reproducibility, the glucose provides a suitable growth for most yeasts and the addition of methylene blue enhances the zone edge definition. The pH of the medium needs to be between 7.2 and 7.4 at room temperature after gelling. The inoculum is standardized to 0.5 McFarland using a densitometer and plates should be incubated at 35oC for 24 hours. Some strains where insufficient growth has occurred after 24 hours may need to be read after 48 hours incubation. Commercially prepared paper disks for fluconazole (25 ug) and voriconazole (1 ug) are available from Oxoid and Becton Dickinson.
Interpretative zone sizes and equivalent MICs have been set for Candida against Fluconazole Disk tests are inexpensive and easy to set up and provide an ideal screening test . However it is recommended that all strains that appear resistant should be confirmed against the M27-A2 microbroth dilution standard. Disk testing may be adapted for use with other fungi including sporulating moulds but once again the results need to be validated by using the appropriate NCCLS reference method.
NCCLS M38-A standard for moulds:
See NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard. NCCLS document M38-A [ISBN 1-56238-470-8]. NCCLS, Pennsylvania, USA 2002.
The M38-A standard describes a method for testing antifungal susceptibility of filamentous fungi (moulds) that cause invasive infections, including Aspergillus spp., Fusarium spp., Pseudallescheria (Scedosporium) spp., zygomycetes and other pathogenic moulds. It recommends the use RPMI-1640 medium (with glutamine, without bicarbonate, and with phenol red as a pH indicator)(Sigma #R-7755 St. Louis, USA) supplemented with 0.2% glucose and buffered to a pH of 7.0 with 0.165 mol/L MOPS (3-[N-morpholino] propanesulfonic acid)(Sigma #M-6270) as used in the M27-A2 standard for yeasts.
However for the moulds the inoculum preparation of conidial or sporangiospores suspensions must be adjusted using a spectrophotometer with a test inoculum in the range 0.4x104 to 5x104 CFU/ml providing the most reproducible MIC data. The optical density (OD) at 530nm required is dependant on the conidial or sporangiospores size of the mould being tested; i.e. for Aspergillus and Sporothrix species the OD = 0.09 - 0.11; for Fusarium, Pseudallescheria (Scedosporium), and Rhizopus species the OD = 0.15 – 0.17; for Bipolaris and Histoplasma species the OD = 0.2. Note the addition of a very small drop of Tween 20 as a wetting agent will help to facilitate the preparation of Aspergillus inocula.
Microdilution trays are incubated at 35oC; and may be read at 24 hours for Rhizopus species; 48 hours for Aspergillus, Fusarium and Sporothrix species; and 72 hours for slower growing moulds like Pseudallescheria (Scedosporium) species. Most moulds may be read at 48 hours. Once again turbidity in the microdilution wells should be scored with the aid of a reading mirror and compared with that of the growth control. A numerical score from 0 to 4 is given to each well using the following scale: 0 = optically clear or absence of growth, 1 = slight growth (25% of growth control), 2 = prominent reduction in growth (50% of growth control), 3 = slight reduction in growth (75% of growth control), 4 = no reduction in growth. The MIC for amphotericin B, Itraconzole, Voriconazole and Posaconazole is the lowest concentration with a score of 0 (optically clear). The MICs for 5-fluorocytosine, Fluconazole and Ketoconazole are the lowest concentrations with a score of 2 or lower (50% growth reduction).
Interpretation of results: For Amphotericin B end points are typically well defined with most moulds clustering between 0.5 and 2.0 ug/ml. However some species such as Aspergillus terreus, Acremonium strictum, Pseudallescheria boydii and Scedosporium prolificans show higher MICs in the range of 2 to 16 ug/ml (although very little data are available, MICs above 2 ug/ml have been associated with treatment failures). RPMI medium may also be unreliable in detecting resistance to Amphotericin B. For 5-Fluorocytosine most mould MICs are greater than 64 ug/ml, the only exception are some isolates of Aspergillus and the dematiaceous fungi. Similarly for Fluconazole most mould MICs are greater than 64 ug/ml, the only exception are some isolates of the dimorphic fungi and dermatophytes. For Itraconazole, Voriconazole and Posaconazole the end points are typically well defined with MICs ranging from 0.0313 to 16 ug/ml.e
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بتاريخ : 29-Jun-2008 الساعة : 04:40 PM رقم #2
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لطلبة الدراسات العليا مراجعة Clinical Mycology
Mycological Terms
.
Acrogenous: Conidia born at the tip of the conidiophore.
Acropetal: A chain of conidia in which the youngest conidium is at the tip and the oldest is at the base.
Acropleurogenous: Conidia developing at the tip and along the sides of the conidiophore.
Adiaconidia: A large, globose, thick-walled conidium, usually produced by Emmonsia (Chrysosporium) parvum, in the lungs of humans and animals.
Aerial mycelium: Hyphal elements growing above the agar surface.
Aleurioconidium (pl. aleurioconidia): A thallic conidium released by lysis or fracture of the supporting cell.
Ameroconidium (pl. ameroconidium): A one-celled conidium.
Anamorph: An asexual state of a fungus.
Annellide: A specialized conidiogenous cell producing conidia in basipetal succession by a series of short percurrent proliferations (annellations). The tip of an annellide increases in length and becomes narrower as each subsequent conidium is formed.
Annelloconidium (pl. annelloconidia): A conidium produced by an annellide.
Apophysis: A swelling. The term is primarily applied to the funnel-shaped swelling of a sporangiophore, immediately below the columella, seen in some zygomycetes.
Arthric: A type of conidial ontogeny involving the conversion and subsequent disarticulation of a determinant conidiogenous hypha.
Arthroconidium (pl. arthroconidia): A thallic conidium released by either the splitting of a double septum or by the fragmentation or lysis of a disjunctor cell.
Ascocarp: A fruiting body containing asci and ascospores.
Ascomycetes: A group of fungi that reproduce sexually by the endogenous formation of ascospores in an ascus.
Ascomycetous: Referring to the Ascomycetes.
Ascospore: A haploid spore produced within an ascus following karyogamy and meiosis.
Ascus (pl. asci): A sac-like cell containing ascospores. Asci are characteristic of the Ascomycetes.
Aseptate: Lacking septa, often pertaining to the hyphae seen in zygomycetes (also see coenocytic).
Ballistoconidium (pl. ballistoconidia): A conidium that is forcible discharged.
Basidiomycetes: A group of fungi that reproduce sexually by the exogenous formation of basidiospores from a basidium.
Basidiospore: A haploid spore produced on a basidium following karyogamy and meiosis.
Basidium (pl. basidia): A cell that gives rise to a basidiospore. Basidia are characteristic of the Basidiomycetes.
Basipetal: A chain of conidia, the oldest conidium is at the apex and the youngest is at the base.
Basocatenulate: A chain of conidia having the youngest cell at the base.
Bipolar budding: Blastoconidia developing at the opposite poles of a parent cell.
Biseriate: Phialides arising from metulae as in the genus Aspergillus.
Biverticillate: Two or rarely three levels of branching directly below the phialides as in the genus Penicillium.
Blastic: A form of conidial development where there is a recognizable enlargement or "blowing out" of a conidial initial before being delimited by a septum.
Blastocatenate: A chain of conidia having the youngest cell at the tip.
Blastoconidium (pl. blastoconidia): An asexual conidium that forms by a blowing out or budding process.
Bud: A young conidium. Usually used to denote the young blastoconidia of yeasts.
Budding: Asexual multiplication by the production of a small outgrowth or bud from a parent cell.
Capsule: A hyaline mucopolysaccharide sheath around the cell wall of certain yeasts e.g. Cryptococcus and Rhodotorula.
Catenulate: Conidia arranged in chains.
Chlamydoconidium (pl. Chlamydoconidia): A thick-walled, thallic conidium formed within the vegetative hyphae. Chlamydoconidia function as organs of perennation rather than dissemination.
Clamp connection: A specialized hyphal bridge over a septum in the Basidiomycetes.
Clavate: Club-shaped.
Cleistothecium (pl. cleistothecia): An enclosed ascocarp containing randomly dispersed asci.
Coenocytic: Infrequently septate, multi-nucleate hyphae as in the Zygomycetes.
Collarette: A small collar. Usually, a remnant of a cell wall present at the tip of a phialide, or around a sporangiophore.
Columella (pl. columellae): A sterile dome-like structure at the tip of a sporangiophore or within a sporangium.
Columnar: Forming a column.
Conidiogenous cell: A cell that forms conidia.
Conidiophore: A specialized hypha upon which conidia develop.
Conidium (pl. conidia): An asexual reproductive propagule formed in any manner that does not involve cytoplasmic cleavage. Conidia function as organs of dissemination.
Cottony: Having a loose and coarse texture.
Cylindrical: Cylindric, having parallel walls and circular cross-section.
Dematiaceous: A dark brown, greenish gray or black colour.
Dermatophyte: A fungus belonging to the genera Epidermophyton, Microsporum, or Trichophyton with the ability to utilize keratin to infect hair, nail and skin.
Denticle: A small projection or peg on which conidia are produced.
Determinate conidiophore: The conidiophore does not alter in length after the formation of conidia.
Deuteromycetes: An artificial subdivision to accommodate those fungi where only the asexual state is known.
Dichotomous: A type of hyphal branching into two equal forks.
Dictyoconidium (pl. dictyoconidia): A conidium with both longitudinal and transverse septa; a muriform conidium.
Didmoconidium (pl. didymoconidia): A two celled conidium.
Dimorphic: Having two different morphological forms.
Disjunctor cell: An empty cell that fragments and/or undergoes lysis to release a conidium.
Dolipore septum: A characteristic septum found in the Basidiomycetes that flares out near the pore to form an elongate channel.
Double septum: A two layered septum that may undergo centripetal separation (schizolysis) to release a conidium.
Downy: Having a short and dense mycelial texture.
Dysgonic: A slow growing variant.
Echinulate: Covered with delicate spines.
Ectothrix: Natural hair invasion by a dermatophyte characterized by arthroconidia on the outside of the hair shaft.
Effuse: Spread out, radiate.
Elliptical: Oval, with a symmetric curve.
Elongate: Lengthened.
Endospore: A spore produced within a spherule.
Endothrix: Natural hair invasion by a dermatophyte characterized by the development of arthroconidia within the hair shaft only.
Erect: Upright.
Evanescent: Disappearing.
Exudate: Droplets of fluid formed on the surface of a colony.
Falcate: Curved like a sickle.
Flexuous: Wavy.
Floccose: Fluffy or cottony.
Foot cell: A basal cell of a conidiophore as seen in Aspergillus and Fusarium.
Fusiform: Spindle-shaped, tapering toward the end.
Geniculate: Bent like a knee.
Germ tube: The initial hypha that develops from a conidium or spore.
Glabrous: Smooth.
Gloiospora: Conidia aggregated in slimy heads at the tip of an annellide or phialide.
Guttulate: Containing one or more oil droplets.
Gymnothecium (pl. gymnothecia): A non-ostiolate ascocarp composed of loosely interwoven hyphae and containing randomly dispersed asci.
Heterothallic: A fungus that requires mating between two compatible strains for sexual reproduction to occur.
Hilum: A scar at the base of a conidium.
Holoblastic: A mode of blastic conidium ontogeny in which all the cell wall layers of the conidiogenous cell are involved in conidium development.
Holothallic: A mode of thallic conidium ontogeny in which all the cell wall layers of the conidiogenous cell are involved in conidium development.
Homothallic: A fungus capable of sexual reproduction on a single thallus.
Hulle cells: Thick-walled cells with characteristic thin-walled pores, usually associated with cleistothecia of Aspergillus.
Hyaline: Colourless.
Hyalo-: A prefix meaning hyaline to lightly coloured.
Hypha (pl. hyphae): A single filament of a fungus.
Hyphomycetes: A class of mycelial moulds which reproduce asexually by conidia on hyphae or aggregations of hyphae.
Intercalary: Within a hyphal element.
Lageniform: Flask-shaped.
Lanceolate: Lance-shaped.
Lanose: Woolly.
Lateral: On the side.
Lenticular: Shaped like a double convex lens.
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بتاريخ : 01-Jul-2008 الساعة : 01:41 PM رقم #3
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لطلبة الدراسات العليا - مراجعة Clinical Mycology
Mycological Terms
Macroconidium (pl. macroconidia): The larger of two different types of conidia produced by a fungus in the same manner.
Macronematous: Having a conidiophore that is morphologically different from the vegetative hyphae.
Merosporangium (pl. merosporangia): A small cylindrical sporangium with the sporangiospores aligned in a row.
Metula (pl. Metulae): A sterile cell below the phialides of some Aspergillus and Penicillium species.
Microconidium (pl. microconidia): The smaller of two different types of conidia produced by a fungus in the same manner.
Micronematous: Having a conidiophore that is not morphologically different from the vegetative hyphae.
Mucoid: Sticky or slimy
Multiseptate: Having several septa.
Multipolar budding: Blastoconidia developing at different sites on the surface of a parent cell.
Muriform: A conidium with both longitudinal and transverse septa.
Mycelium (pl. mycelia): The mass of hyphae making up the thallus of a fungus.
Niger: Black.
Nonseptate: Without septa.
Obclavate: Club-shaped in reverse; the distal region is smaller.
Obpyriform: Pear-shaped in reverse; the distal region is larger.
Olivaceous: Olive-grey colour.
Ostiole: An opening or pore in an ascocarp or a pycnidium.
Ovoid: Egg-shaped.
Pectinate: Like the teeth of a comb.
Pedicel: A slender stalk.
Pellicle: A film-like or skin-like surface growth.
Penicillus (pl. penicilli): The brush-like conidiophore of Penicillium.
Percurrent: Conidiogenous cell growth where a new axis grows through the previous apex.
Peridium: The outer wall of an ascocarp.
Perithecium (pl. perithecia): An enclosed ascocarp characterized an apical ostiole and by asci arranged in a basal tuft or hymenium layer.
Phaeo-: A prefix meaning darkly pigmented.
Phialide: A specialized conidiogenous cell that produces conidia in basipetal succession without increasing in length.
Phialoconidium (pl. phialoconidia): A conidium produced from a phialide.
Phragmoconidium (pl. phragmoconidia): A conidium having two or more transverse septa.
Pleomorphic: Having more than one form.
Pleurogenous: Born on the sides of a conidiophore or hyphae.
Poroconidium (pl. poroconidia): A conidium produced through a small pore in a conidiogenous cell.
Pseudohyphae: A string of elongated blastoconidia formed in some yeasts that resemble a hypha-like filament.
Pycnidium (pl. pycnidia): An asexual fruiting body containing conidia.
Pyriform: Pear-shaped.
Rachis: An extension of a sympodial proliferating conidiogenous cell bearing conidia.
Racquet hyphae: A hypha composed of a number of cells swollen at one end resembling a tennis racquet.
Retrogressive conidial development: The conidiogenesis cell becomes shorter during the successive development of conidia.
Rhizoids: A short branching root-like hyphae seen in some Zygomycetes.
Sclerotium (sclerotia): A mass of thick-walled cells formed by the vegetative hyphae that function as an organ of perennation.
Semimacronematous: Having a conidiophore that is only slightly morphologically different from the vegetative hyphae.
Septum (pl. septa): A cross wall in a hypha.
Spinulose: Covered in small spines.
Solitary: Alone.
Sporangiolum (pl. ): A small sporangium producing a small number of sporangiospores.
Sporangiophore: A specialized hypha that bears a sporangium.
Sporangiospore: An asexual spore produced within a sporangium.
Sporangium (pl. sporangia): A sac-like structure producing asexual spores endogenously by cytoplasmic cleavage.
Spore: a reproductive propagule formed by either meiosis or mitosis. However, if by asexual means, cleavage of cytoplasm is usually involved.
Sporodochium (pl. sporodochia): A cushion-shaped mass of hyphae bearing conidiophores.
Stellate: Star-shaped
Sterigma (pl. sterigmata): A small pointed structure upon which a basidiospore forms.
Stolon: A running hypha from which rhizoids and sporangiospores arise.
Striate: Having lines or minute furrows.
Subglobose: Not quite round or spherical.
Sympodial: A mode of conidiogenous cell growth which results in the development of conidia on a geniculate or zig-zag rachis.
Synnema (pl. synnemata): A group of erect conidiophores that are cemented together producing conidia at the apex and/or along the sides.
Teleomorph: The sexual state of a fungus.
Thallic: A mode of conidial ontogeny where a conidium is formed from a pre-existing hyphal segment or cell.
Toruloid: Having swellings at intervals.
Truncate: Cut off sharply.
Tuberculate: Having small wart-like structures.
Uniserate: Phialides arising directly from a vesicle as in Aspergillus.
Verrucose: Having many warts.
Verticillate: Having branches arranged in verticils or whorls.
Vesicle: A swollen cell.
Zygospores: A thick-walled sexual spore formed by the fusion of two similar gametangia; characteristic of the Zygomycetes
لطلبة الدراسات العليا- مراجعة Clinlcal Mycology
Antifungal Susceptibility Testing
It is now possible for the clinical microbiology laboratory to perform reliable in vitro antifungal susceptibility tests on a wide range of yeasts and moulds. The aim of this article is to a provide review of what is currently available to the clinical laboratory along with some practical comments on antifungal susceptibility testing. Major advances with the standardisation and clinical interpretation of in vitro antifungal susceptibility testing have been made in recent years. These include the introduction of standard reference methods for both yeasts and moulds by the NCCLS1,2,3 and publication of interpretive breakpoints, especially for Fluconazole and Itraconazole against Candida infections4. In the antifungal susceptibility testing world, the NCCLS have set the benchmark methodology by providing laboratory tested, reproducible, consensus peer review standards that are updated on a regular basis.
NCCLS M27-A2 standard for yeasts:
See NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Second Edition. NCCLS document M27-A2 [ISBN 1-56238-469-4]. NCCLS, Pennsylvania, USA .
The M27-A2 standard is intended for testing yeasts including Candida species and Cryptococcus species (C. neoformans and C. gattii) by using either a macro or micro broth dilution test system. It recommends the use of RPMI-1640 medium (with glutamine and phenol red, without bicarbonate)(Sigma #R-7755 St. Louis, USA) supplemented with 0.2% glucose and buffered to a pH of 7.0 with 0.165 mol/L MOPS (3-[N-morpholino] propanesulfonic acid)(Sigma #M-6270), inoculum standardized to 0.5 McFarland using a densitometer and incubation at 35oC. It is essential to use the exact reagents as listed from Sigma. Plates are read at 24 hours for non-fastidious yeasts like Candida or at 48 to 72 hours for slower growing yeasts like Cryptococcus.
The microdilution wells should be visualised with the aid of a reading mirror and the growth in each well should be compared with that of the growth control. A numerical score from 0 to 4 is given to each well using the following scale: 0 = optically clear, 1 = slightly hazy, 2 = prominent reduction in turbidity compared with that of the drug-free growth control, 3 = slight reduction in turbidity compared with that of the drug-free growth control, 4 = no reduction in turbidity compared with that of the drug-free growth control. The MIC for amphotericin B is the lowest concentration with a score of 0 (optically clear). The MICs for the azoles and 5FC are the lowest concentrations with a score of 2 (prominent decrease in turbidity).
Interpretation of results: For the most part Amphotericin B MICs for Candida species cluster between 0.25 and 1.0 ug/ml. However it must be stressed that the M27 method does not consistently permit detection of resistant strains and isolates with MICs of > 1.0 ug/ml should be considered likely to be resistant. Antibiotic Medium 3 supplemented with 2 % glucose may permit more reliable detection of resistance but this medium is not standardized and substantial lot-to-lot variability is possible. On a brighter note, interpretative breakpoints for Candida against Fluconazole, Itraconazole and 5-Fluorocytosine have been established (table 1). These breakpoints have also been used for isolates of Cryptococcus where there is also some correlation between elevated MIC and treatment failure1,5.
Trailing end points. Some azoles, particularly fluconazole, exhibit a phenomenon known as trailing. Trailing occurs when the turbidity continually decreases as the drug concentration increases but the suspension fails to become optically clear (partial inhibition of growth over an extended range of antifungal concentrations). For most isolates, the difference between reading at 24 hours versus 48 hours is minimal and will not alter the interpretative category (i.e. does not change whether the isolate would be read as “susceptible” or “resistant”). However some isolates show a dramatic rise in MIC over time (e.g. for fluconazole from 0.5 ug/ml at 24 hours to 256 ug/ml at 48 hours). This trailing phenomenon has been reported as occurring in about 5% of isolates,6 however some studies have reported that up to 20% of C. albicans isolates read at 48 hours may show trailing to fluconazole that would alter the interpretation from “susceptible” to “resistant” . To help resolve this issue the M27-A2 methodology for Candida has provided both 24 hour and 48 hour microdilution MIC ranges for the two QC strains and eight systemic antifungal agents . Ideally plates should be read at 24 hours whenever there is sufficient growth.
Etest showing a trailing end point.
The NCCLS M27-A21 method is now the benchmark to validate all other methods against. As a result several commercial systems are now available to the clinical laboratory for antifungal susceptibility testing.
NCCLS M44-P standard for yeasts by disk diffusion:
See NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Proposed Guideline. NCCLS document M44-P [ISBN 1-56238-488-0]. NCCLS, Pennsylvania, USA 2003.
The M44-P standard is a newly established methodology for disk diffusion testing of Candida species. Other yeast genera and moulds have yet to be validated using this method. The standard includes zone interpretive criteria for fluconazole and recommended quality control ranges for fluconazole and voriconazole. It recommends the use of Mueller-Hinton agar supplemented with 0.2% glucose and 0.5 ug/ml methylene blue dye medium. Mueller-Hinton agar is readily available and shows acceptable batch-to-batch reproducibility, the glucose provides a suitable growth for most yeasts and the addition of methylene blue enhances the zone edge definition. The pH of the medium needs to be between 7.2 and 7.4 at room temperature after gelling. The inoculum is standardized to 0.5 McFarland using a densitometer and plates should be incubated at 35oC for 24 hours. Some strains where insufficient growth has occurred after 24 hours may need to be read after 48 hours incubation. Commercially prepared paper disks for fluconazole (25 ug) and voriconazole (1 ug) are available from Oxoid and Becton Dickinson.
Interpretative zone sizes and equivalent MICs have been set for Candida against Fluconazole Disk tests are inexpensive and easy to set up and provide an ideal screening test . However it is recommended that all strains that appear resistant should be confirmed against the M27-A2 microbroth dilution standard. Disk testing may be adapted for use with other fungi including sporulating moulds but once again the results need to be validated by using the appropriate NCCLS reference method.
NCCLS M38-A standard for moulds:
See NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard. NCCLS document M38-A [ISBN 1-56238-470-8]. NCCLS, Pennsylvania, USA 2002.
The M38-A standard describes a method for testing antifungal susceptibility of filamentous fungi (moulds) that cause invasive infections, including Aspergillus spp., Fusarium spp., Pseudallescheria (Scedosporium) spp., zygomycetes and other pathogenic moulds. It recommends the use RPMI-1640 medium (with glutamine, without bicarbonate, and with phenol red as a pH indicator)(Sigma #R-7755 St. Louis, USA) supplemented with 0.2% glucose and buffered to a pH of 7.0 with 0.165 mol/L MOPS (3-[N-morpholino] propanesulfonic acid)(Sigma #M-6270) as used in the M27-A2 standard for yeasts.
However for the moulds the inoculum preparation of conidial or sporangiospores suspensions must be adjusted using a spectrophotometer with a test inoculum in the range 0.4x104 to 5x104 CFU/ml providing the most reproducible MIC data. The optical density (OD) at 530nm required is dependant on the conidial or sporangiospores size of the mould being tested; i.e. for Aspergillus and Sporothrix species the OD = 0.09 - 0.11; for Fusarium, Pseudallescheria (Scedosporium), and Rhizopus species the OD = 0.15 – 0.17; for Bipolaris and Histoplasma species the OD = 0.2. Note the addition of a very small drop of Tween 20 as a wetting agent will help to facilitate the preparation of Aspergillus inocula.
Microdilution trays are incubated at 35oC; and may be read at 24 hours for Rhizopus species; 48 hours for Aspergillus, Fusarium and Sporothrix species; and 72 hours for slower growing moulds like Pseudallescheria (Scedosporium) species. Most moulds may be read at 48 hours. Once again turbidity in the microdilution wells should be scored with the aid of a reading mirror and compared with that of the growth control. A numerical score from 0 to 4 is given to each well using the following scale: 0 = optically clear or absence of growth, 1 = slight growth (25% of growth control), 2 = prominent reduction in growth (50% of growth control), 3 = slight reduction in growth (75% of growth control), 4 = no reduction in growth. The MIC for amphotericin B, Itraconzole, Voriconazole and Posaconazole is the lowest concentration with a score of 0 (optically clear). The MICs for 5-fluorocytosine, Fluconazole and Ketoconazole are the lowest concentrations with a score of 2 or lower (50% growth reduction).
Interpretation of results: For Amphotericin B end points are typically well defined with most moulds clustering between 0.5 and 2.0 ug/ml. However some species such as Aspergillus terreus, Acremonium strictum, Pseudallescheria boydii and Scedosporium prolificans show higher MICs in the range of 2 to 16 ug/ml (although very little data are available, MICs above 2 ug/ml have been associated with treatment failures). RPMI medium may also be unreliable in detecting resistance to Amphotericin B. For 5-Fluorocytosine most mould MICs are greater than 64 ug/ml, the only exception are some isolates of Aspergillus and the dematiaceous fungi. Similarly for Fluconazole most mould MICs are greater than 64 ug/ml, the only exception are some isolates of the dimorphic fungi and dermatophytes. For Itraconazole, Voriconazole and Posaconazole the end points are typically well defined with MICs ranging from 0.0313 to 16 ug/ml.e
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بتاريخ : 29-Jun-2008 الساعة : 04:40 PM رقم #2
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لطلبة الدراسات العليا مراجعة Clinical Mycology
Mycological Terms
.
Acrogenous: Conidia born at the tip of the conidiophore.
Acropetal: A chain of conidia in which the youngest conidium is at the tip and the oldest is at the base.
Acropleurogenous: Conidia developing at the tip and along the sides of the conidiophore.
Adiaconidia: A large, globose, thick-walled conidium, usually produced by Emmonsia (Chrysosporium) parvum, in the lungs of humans and animals.
Aerial mycelium: Hyphal elements growing above the agar surface.
Aleurioconidium (pl. aleurioconidia): A thallic conidium released by lysis or fracture of the supporting cell.
Ameroconidium (pl. ameroconidium): A one-celled conidium.
Anamorph: An asexual state of a fungus.
Annellide: A specialized conidiogenous cell producing conidia in basipetal succession by a series of short percurrent proliferations (annellations). The tip of an annellide increases in length and becomes narrower as each subsequent conidium is formed.
Annelloconidium (pl. annelloconidia): A conidium produced by an annellide.
Apophysis: A swelling. The term is primarily applied to the funnel-shaped swelling of a sporangiophore, immediately below the columella, seen in some zygomycetes.
Arthric: A type of conidial ontogeny involving the conversion and subsequent disarticulation of a determinant conidiogenous hypha.
Arthroconidium (pl. arthroconidia): A thallic conidium released by either the splitting of a double septum or by the fragmentation or lysis of a disjunctor cell.
Ascocarp: A fruiting body containing asci and ascospores.
Ascomycetes: A group of fungi that reproduce sexually by the endogenous formation of ascospores in an ascus.
Ascomycetous: Referring to the Ascomycetes.
Ascospore: A haploid spore produced within an ascus following karyogamy and meiosis.
Ascus (pl. asci): A sac-like cell containing ascospores. Asci are characteristic of the Ascomycetes.
Aseptate: Lacking septa, often pertaining to the hyphae seen in zygomycetes (also see coenocytic).
Ballistoconidium (pl. ballistoconidia): A conidium that is forcible discharged.
Basidiomycetes: A group of fungi that reproduce sexually by the exogenous formation of basidiospores from a basidium.
Basidiospore: A haploid spore produced on a basidium following karyogamy and meiosis.
Basidium (pl. basidia): A cell that gives rise to a basidiospore. Basidia are characteristic of the Basidiomycetes.
Basipetal: A chain of conidia, the oldest conidium is at the apex and the youngest is at the base.
Basocatenulate: A chain of conidia having the youngest cell at the base.
Bipolar budding: Blastoconidia developing at the opposite poles of a parent cell.
Biseriate: Phialides arising from metulae as in the genus Aspergillus.
Biverticillate: Two or rarely three levels of branching directly below the phialides as in the genus Penicillium.
Blastic: A form of conidial development where there is a recognizable enlargement or "blowing out" of a conidial initial before being delimited by a septum.
Blastocatenate: A chain of conidia having the youngest cell at the tip.
Blastoconidium (pl. blastoconidia): An asexual conidium that forms by a blowing out or budding process.
Bud: A young conidium. Usually used to denote the young blastoconidia of yeasts.
Budding: Asexual multiplication by the production of a small outgrowth or bud from a parent cell.
Capsule: A hyaline mucopolysaccharide sheath around the cell wall of certain yeasts e.g. Cryptococcus and Rhodotorula.
Catenulate: Conidia arranged in chains.
Chlamydoconidium (pl. Chlamydoconidia): A thick-walled, thallic conidium formed within the vegetative hyphae. Chlamydoconidia function as organs of perennation rather than dissemination.
Clamp connection: A specialized hyphal bridge over a septum in the Basidiomycetes.
Clavate: Club-shaped.
Cleistothecium (pl. cleistothecia): An enclosed ascocarp containing randomly dispersed asci.
Coenocytic: Infrequently septate, multi-nucleate hyphae as in the Zygomycetes.
Collarette: A small collar. Usually, a remnant of a cell wall present at the tip of a phialide, or around a sporangiophore.
Columella (pl. columellae): A sterile dome-like structure at the tip of a sporangiophore or within a sporangium.
Columnar: Forming a column.
Conidiogenous cell: A cell that forms conidia.
Conidiophore: A specialized hypha upon which conidia develop.
Conidium (pl. conidia): An asexual reproductive propagule formed in any manner that does not involve cytoplasmic cleavage. Conidia function as organs of dissemination.
Cottony: Having a loose and coarse texture.
Cylindrical: Cylindric, having parallel walls and circular cross-section.
Dematiaceous: A dark brown, greenish gray or black colour.
Dermatophyte: A fungus belonging to the genera Epidermophyton, Microsporum, or Trichophyton with the ability to utilize keratin to infect hair, nail and skin.
Denticle: A small projection or peg on which conidia are produced.
Determinate conidiophore: The conidiophore does not alter in length after the formation of conidia.
Deuteromycetes: An artificial subdivision to accommodate those fungi where only the asexual state is known.
Dichotomous: A type of hyphal branching into two equal forks.
Dictyoconidium (pl. dictyoconidia): A conidium with both longitudinal and transverse septa; a muriform conidium.
Didmoconidium (pl. didymoconidia): A two celled conidium.
Dimorphic: Having two different morphological forms.
Disjunctor cell: An empty cell that fragments and/or undergoes lysis to release a conidium.
Dolipore septum: A characteristic septum found in the Basidiomycetes that flares out near the pore to form an elongate channel.
Double septum: A two layered septum that may undergo centripetal separation (schizolysis) to release a conidium.
Downy: Having a short and dense mycelial texture.
Dysgonic: A slow growing variant.
Echinulate: Covered with delicate spines.
Ectothrix: Natural hair invasion by a dermatophyte characterized by arthroconidia on the outside of the hair shaft.
Effuse: Spread out, radiate.
Elliptical: Oval, with a symmetric curve.
Elongate: Lengthened.
Endospore: A spore produced within a spherule.
Endothrix: Natural hair invasion by a dermatophyte characterized by the development of arthroconidia within the hair shaft only.
Erect: Upright.
Evanescent: Disappearing.
Exudate: Droplets of fluid formed on the surface of a colony.
Falcate: Curved like a sickle.
Flexuous: Wavy.
Floccose: Fluffy or cottony.
Foot cell: A basal cell of a conidiophore as seen in Aspergillus and Fusarium.
Fusiform: Spindle-shaped, tapering toward the end.
Geniculate: Bent like a knee.
Germ tube: The initial hypha that develops from a conidium or spore.
Glabrous: Smooth.
Gloiospora: Conidia aggregated in slimy heads at the tip of an annellide or phialide.
Guttulate: Containing one or more oil droplets.
Gymnothecium (pl. gymnothecia): A non-ostiolate ascocarp composed of loosely interwoven hyphae and containing randomly dispersed asci.
Heterothallic: A fungus that requires mating between two compatible strains for sexual reproduction to occur.
Hilum: A scar at the base of a conidium.
Holoblastic: A mode of blastic conidium ontogeny in which all the cell wall layers of the conidiogenous cell are involved in conidium development.
Holothallic: A mode of thallic conidium ontogeny in which all the cell wall layers of the conidiogenous cell are involved in conidium development.
Homothallic: A fungus capable of sexual reproduction on a single thallus.
Hulle cells: Thick-walled cells with characteristic thin-walled pores, usually associated with cleistothecia of Aspergillus.
Hyaline: Colourless.
Hyalo-: A prefix meaning hyaline to lightly coloured.
Hypha (pl. hyphae): A single filament of a fungus.
Hyphomycetes: A class of mycelial moulds which reproduce asexually by conidia on hyphae or aggregations of hyphae.
Intercalary: Within a hyphal element.
Lageniform: Flask-shaped.
Lanceolate: Lance-shaped.
Lanose: Woolly.
Lateral: On the side.
Lenticular: Shaped like a double convex lens.
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بتاريخ : 01-Jul-2008 الساعة : 01:41 PM رقم #3
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لطلبة الدراسات العليا - مراجعة Clinical Mycology
Mycological Terms
Macroconidium (pl. macroconidia): The larger of two different types of conidia produced by a fungus in the same manner.
Macronematous: Having a conidiophore that is morphologically different from the vegetative hyphae.
Merosporangium (pl. merosporangia): A small cylindrical sporangium with the sporangiospores aligned in a row.
Metula (pl. Metulae): A sterile cell below the phialides of some Aspergillus and Penicillium species.
Microconidium (pl. microconidia): The smaller of two different types of conidia produced by a fungus in the same manner.
Micronematous: Having a conidiophore that is not morphologically different from the vegetative hyphae.
Mucoid: Sticky or slimy
Multiseptate: Having several septa.
Multipolar budding: Blastoconidia developing at different sites on the surface of a parent cell.
Muriform: A conidium with both longitudinal and transverse septa.
Mycelium (pl. mycelia): The mass of hyphae making up the thallus of a fungus.
Niger: Black.
Nonseptate: Without septa.
Obclavate: Club-shaped in reverse; the distal region is smaller.
Obpyriform: Pear-shaped in reverse; the distal region is larger.
Olivaceous: Olive-grey colour.
Ostiole: An opening or pore in an ascocarp or a pycnidium.
Ovoid: Egg-shaped.
Pectinate: Like the teeth of a comb.
Pedicel: A slender stalk.
Pellicle: A film-like or skin-like surface growth.
Penicillus (pl. penicilli): The brush-like conidiophore of Penicillium.
Percurrent: Conidiogenous cell growth where a new axis grows through the previous apex.
Peridium: The outer wall of an ascocarp.
Perithecium (pl. perithecia): An enclosed ascocarp characterized an apical ostiole and by asci arranged in a basal tuft or hymenium layer.
Phaeo-: A prefix meaning darkly pigmented.
Phialide: A specialized conidiogenous cell that produces conidia in basipetal succession without increasing in length.
Phialoconidium (pl. phialoconidia): A conidium produced from a phialide.
Phragmoconidium (pl. phragmoconidia): A conidium having two or more transverse septa.
Pleomorphic: Having more than one form.
Pleurogenous: Born on the sides of a conidiophore or hyphae.
Poroconidium (pl. poroconidia): A conidium produced through a small pore in a conidiogenous cell.
Pseudohyphae: A string of elongated blastoconidia formed in some yeasts that resemble a hypha-like filament.
Pycnidium (pl. pycnidia): An asexual fruiting body containing conidia.
Pyriform: Pear-shaped.
Rachis: An extension of a sympodial proliferating conidiogenous cell bearing conidia.
Racquet hyphae: A hypha composed of a number of cells swollen at one end resembling a tennis racquet.
Retrogressive conidial development: The conidiogenesis cell becomes shorter during the successive development of conidia.
Rhizoids: A short branching root-like hyphae seen in some Zygomycetes.
Sclerotium (sclerotia): A mass of thick-walled cells formed by the vegetative hyphae that function as an organ of perennation.
Semimacronematous: Having a conidiophore that is only slightly morphologically different from the vegetative hyphae.
Septum (pl. septa): A cross wall in a hypha.
Spinulose: Covered in small spines.
Solitary: Alone.
Sporangiolum (pl. ): A small sporangium producing a small number of sporangiospores.
Sporangiophore: A specialized hypha that bears a sporangium.
Sporangiospore: An asexual spore produced within a sporangium.
Sporangium (pl. sporangia): A sac-like structure producing asexual spores endogenously by cytoplasmic cleavage.
Spore: a reproductive propagule formed by either meiosis or mitosis. However, if by asexual means, cleavage of cytoplasm is usually involved.
Sporodochium (pl. sporodochia): A cushion-shaped mass of hyphae bearing conidiophores.
Stellate: Star-shaped
Sterigma (pl. sterigmata): A small pointed structure upon which a basidiospore forms.
Stolon: A running hypha from which rhizoids and sporangiospores arise.
Striate: Having lines or minute furrows.
Subglobose: Not quite round or spherical.
Sympodial: A mode of conidiogenous cell growth which results in the development of conidia on a geniculate or zig-zag rachis.
Synnema (pl. synnemata): A group of erect conidiophores that are cemented together producing conidia at the apex and/or along the sides.
Teleomorph: The sexual state of a fungus.
Thallic: A mode of conidial ontogeny where a conidium is formed from a pre-existing hyphal segment or cell.
Toruloid: Having swellings at intervals.
Truncate: Cut off sharply.
Tuberculate: Having small wart-like structures.
Uniserate: Phialides arising directly from a vesicle as in Aspergillus.
Verrucose: Having many warts.
Verticillate: Having branches arranged in verticils or whorls.
Vesicle: A swollen cell.
Zygospores: A thick-walled sexual spore formed by the fusion of two similar gametangia; characteristic of the Zygomycetes
