anti fungal susceptibility
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Anti fungal susceptibility
MAULANA AZAD MEDICAL COLLEGE PG SEMINAR
What are fungi?
Site of action
• Cell wall : (1,3)- β glucan synthase Echinocandins
• Cell membrane: ergosterol synthesis Polyene antibiotics (AMB) Azoles Allylamines (Terbinafine)
• Polymerization of microtubules: Grisiofulvin• Disrufts RNA and DNA: 5 FC
• protien synthesis (EF-2) : Sordarins• t-RNA synthase : Icofungipen• manoprotien : Pradimycin • chitin : Nikkomycins, Polyoxins• glucan synthatase : Aculeacin
• Most of these drugs are fungi-static except Amphotericin-B
Allylamines and Benzylamines Naftifine,Terbinafine, Butenafine.
why there is a need for susceptibility testing
• Increasing immuno-supressive states……..• Increasing incidence of invasive mycosis and
life threatening infections……..• Avaibility of newer drugs…….• Avaibility of standard guide lines…..• Emerging resistance……
Resistant to antifungal agentsCandida krusei Fluconazole intrinsic
Candida glabrata Fuconazole acquired
Caspofungin
Candida albicans Fuconazole acquired
Caspofungin
Candida lusitanae Amphoericin-B
Aspergillus terreus Amphotericin-B intrinsic
Pseudallescheria boydii Amphotericin-B
Paecilomyces lilanicus Amphotericin-B
Fusarium species all
Also to …..
• Provide a reliable measure of the relative activities of two or more antifungal agents.
• Correlate with in vivo activity and predict the likely outcome of therapy.
• Provide a mean with which to monitor the development of resistance among a normally susceptible population of organisms.
• Predict the therapeutic potential of newly discovered investigational agents.
Should we report any fungal isolate and put their sensitivity…..
Isolation of established pathogen from any..In case of commensal/opportunistics should
be considered when.. • Pure culture, repeated culture ….multiple
specimens ….• Sterile body sites…..• Febrile neutropenia or immunocompromised..• Not improving on long term antibiotics
Methods
• Macro-dilution method.• Micro-dilution method.• Disk diffusion method.• Agar dilution method.
Is any method i.e. standardized ?
• U.S.A. ---- CLSI (clinical laboratory standard institute)
• Europe----EUCAST (European Committee on Antimicrobial Susceptibility Testing)
• London ---BSAC (British Society for Antimicrobial Chemotherapy)
CLSI- manuals
• M 27-A2 : second edition (1992)• M 38-A : Approved standard (1998)• M 44-A : Approved standard(2004)
CSLI M-27 A2 method for yeast susceptibility testing
1
• Test medium- RPMI 1640 broth Buffer (MOPS) 0.165M Glucose (0.2%) pH – 7 at 25°C• Medium modification : YNB broth with MOPS for C. neoformans RPMI – 1640 with 2% Glucose
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• Inoculum preparation : SDA 24-hr ( candida spp.) 48-hr (Cryptococcus neoformans)• Stock inoculum suspension : 0.5 McFarland standard 1Х106 to 5Х106 CFU/ml spectrophotometer at 530 nm
3
• Test inoculum : 1: 2000 (macrodilution) or 1: 1000 (microdilution) dilutions with medium of stock inoculum suspension; Inoculum size after inoculation : 0.5Х103 to 2.5Х103 CFU/ml for both methods
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• Drug dilution : additive 10Х (macrodilution) or 2Х (microdilution) two fold drug dilutions
with medium : { Fluconazole, Caspofungin, 5-FC}
or 100Хwith solvent { AMB, other-azoles, Anidulafungin, Micafungin}
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• Drug dilution ranges : 5-FC and Flucytosine --- 0.12 to 64 µg/ml other drugs --------------- 0.03 to 16 µg/ml
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• Methods : macrodilution – 0.9 ml of diluted test
inoculum plus 0.1 ml of 10 Х drug concentration
microdilution –100 µl of diluted test inoculum plus 100 µl of 2 Х drug concentration
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• Growth controls : macrodilution – 0.9 ml of diluted inoculum
plus 0.1 ml of drug free medium ( or plus 2% of solvent)
microdilution –100 µl of diluted inoculum plus 100 µl of drug free medium ( or plus 2% of solvent)
Quality control strains
• Candida parapsilosis ATCC 22019.• Candida krusei ATCC 6258.
3
• Incubation temp.- 35°c.• Incubation time- 24-48 hr for candida species. 48-72 hr for cryptococcus sp.
Quality control strains
ANTIFUNGAL AGENTS
MIC AT 48 HRMACRODILUTN
MIC AT 24 HR MICRODILUTN
MIC AT 48 HR MICRODILUTN
C.Parapsilosis ATCC 22019
AMBFLUCONAZOLEITRACONAZOLEVORICONAZOLKETOCONAZOL5-FC
0.25-12-80.06-0.25NA0.06-0.250.12-0.5
0.25-20.5-40.12-0.50.016-0.120.03-0.250.06-0.25
0.5-41-40.12-0.50.03-0.250.06-0.50.12-0.5
C. Krusei ATCC 6258
AMBFLUCONAZOLEITRACONAZOLEVORICONAZOLKETOCONAZOL5-FC
0.5-216-640.12-0.5NA0.12-0.54-16
0.5-0.28-640.12-10.06-0.50.12-14-16
1-416-1280.25-10.12-10.25-18-32
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• MIC by visual examination : lowest drug conc. AMB : (macro & micro dilution) : no visible growth 5-FC, Azoles, Caspofungin and other
echinocandins : o macrodilution-- that matches an 80%
inhibition standard o microdilution—shows 50% growth inhibition
Microtitre plate with in stand with reading mirror
Susceptibility cut-off for yeast (µg/ml)
S SDD ID R
FLUCONAZOLE ≤ 8 16-32 ≥ 64
ITRACONAZOLE ≤ 0.12 0.25-0.5 ≥ 1
VORICONAZOL ≤ 1 2 ≥ 4
FLUCYTISINE ≤ 4 ---- 8-16 ≥ 32
EUCAST Antifungal Susceptibility TestingSubcommittee (AFST)
• EUCAST DEFINITIVE DOCUMENT • E Def 7.1• MIC• Yeast (fermentative) • Broth dilution
Differences of CLSI and EUCAST conditions forantifungal susceptibility testing for yeasts
Difference between CLSI (USA) and EUCAST (Europe)
CLSI EUCAST
Suitability Yeasts Fermentative yeasts
Test medium RPMI 0.2% glucose RPMI 2% glucose
Microtitration plates U-shaped wells Flat-bottom wells
Temperature 35°C 35-37°C
Length of incubation 24-48 h 24 h
Reading Visually Photometrically
Endpoint 100% AMB , 50% 5FC, azoles, candins
90% AMB , 50% 5FC, azoles, candins
Breakpoints (µg/ml) according to CLSI and EUCAST for Candida species* only for C. albicans, C. parapsilopsis, C. tropicalis** tenative break points; NS: Non susceptibles
Drug CLSI EUCAST
Amphotericin B - -
Flucytosine R ≥32; I 8-16 -
Fluconazole R ≥64; SDD 16-32 R >4*
Itraconazole R ≥1; SDD 0.25-0.5 -
Voriconazole R ≥4 R >0.125
Posaconazole - -
Caspofungin NS >2** -
Anidulafungin NS >2** -
Micafungin NS >2** -
• Breakpoints from one method cannot be extrapolated to another method
Broth-based alternative approaches for yeasts modifications of reference method
• Colorimetric methods• Spectrophotometric method• Flow cytometry methodTo improve interlaboratory reproducibilityBetter serve clinical laboratory needs
Sensititre yeast one & fungitest
Spectrophotometric methods
CLSI 44-A
• Disc diffusion susceptibility testing.• Candida species.• Good correlation with microdilution method.• Antifungal agents: Fluconazole Itraconazole Voriconazole
• Test medium: Muller- Hinton agar Glucose (2%) Methylene blue (0.5µg/ml)• Inoculum preparation : SDA (24-hr old culture)• Test medium : stock inoculum suspension 0.5 McFarland standard 1Х106 to 5Х106 CFU/ml
• Disk contents : Fluconazole (25µg) Itraconazole (10µg) Voriconazole (1µg)• Incubation conditions : 20-24 hr at 35°C• Reading zone diameter : to the nearest whole mm
at the point at which there is prominent reduction in growth.
* Pinpont microcolonies at the zone edge or large colonies within the zone should be ignored.
Recommended quqlity control zone-diameter (mm) ranges
Anti fungal Disk content C. albicans
ATCC 90028
C.parapsilosis
ATCC 22019
C.tropicalis
ATCC 750
C. krusei
ATCC 6258
Fluconazole 25µg 28-39 mm 22-33 mm 26-37 mm -
Itraconazole 10µg - 28-35 mm - -
Voriconazole * 1µg 31-42 mm 28-37 mm - 16-25 mm
Interpretative guidelines for zone diameters
ANTIFUNGAL
DRUGS
susceptible (S) susceptible-dose
dependent (SDD)
resistant (R)
Fluconazole ≥ 19 mm 15-18 mm ≤ 14 mm
Itraconazole* ≥ 23 mm 14-22 mm ≤ 13 mm
Voriconazole* ≥ 17 mm 14-16 mm ≤ 13 mm
Agar based alternative approach for yeast
• NeoSensitabs tablets (A/S rosko-Europe) facility of extra new antifungal drugs: Voriconazole (1µg), Posaconazole (5µg),
Caspofungin (5µg) Muller- Hinton Agar
• E Test (AB Biodisk-Sweden) Amphotericin-B, Fluconazole, 5-FC,
Ketoconazole, Itraconazole, VoriconazoleFDA : Fluconazole,Itraconazole & 5-FC solidified RPMI medium supplemented with 2% Glucose
MHA- C. albicans (flu: MIC-0.38 µg/ml)C. glabrata (flu: MIC >256 µg/ml) & C. lusitanea (AMB)
Candida species clinical isolates to Caspofungin by Etest in RPMI
CSLI M-38 A
Standard broth dilution methods for moulds
1
• Medium for conidial growth : PDA slant at 35°C for 7 days Fusarium spp. may need at 30°C incubation
for the last 4 days.• Inoculum morphology : conidia or sporangiospores
Recommended OD ranges and mean inoculum sizes
Fungus OD ranges Mean Inoculum size( 106CFU/ml)
Aspergillus species 0.09-0.11 1.6
Bipolaris species 0.2-0.4 0.6
Cladophialaphora bantiana 0.15-0.17 1.1
Dactylaria constricta 0.15-0.17 1.1
Fusarium species 0.15-0.17 3
Paecilomyces lilanicus 0.09-0.13 2.1
Rhizophus arrhizus 0.15-0.17 1.3
Scedosporium apisospermum 0.15-0.17 1
Scedosporium prolificans 0.15-0.17 0.8
Sporothrix schenckii 0.09-0.11 2
3
• Stock inoculum suspension : 0.4 Х 106 to 5 Х 106 CFU/ml• Inoculum concentration final : 0.4 Х 106 to 5 Х 106 CFU/ml or 1:50 dilution of stock suspension ( S. apiospermum 2:50)
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• Test medium : RPMI 1640 as in yeast (pH 7)• Format – microdilution assay; total volume /well – 200µl• Drug concentration : 0.01– 8 µg/ml AMB and Itraconazole
5 INCUBATION CONDITIONS
Mold Time
Rhizopus arrhizus 24-hr
Aspergillus species, Bipolaris species, Fusarium species,Paecilomyces
lilanicus, Sporothrix schenckii, Tricoderma longobrachiatum, wangiella
dermatidis
48-hr
Pseudallescheria boydii (Scedosporium apiospermum),
Cladophialaphora bantiana, Dactylaria constricta, Scedosporium
proliferans
72-hr
6
• End point determination visual : absence of growth with respect to GC
MEC caspofungin to Aspergillus
Broth based alternative approach for moulds
• Colorimetric method• Spectrophotometric method
Agar based alternative approaches
• E test ( AMB, Azoles)
• Disk diffusion ( under investigation)• Agar dilution method (not standardised)
Commercial kits
• VITEK 2 (BioMerieux) fully automated system
Problems of concern …..
• Difficulties to determine endpoints/breakpoints in Trailing phenomenon (fluconazole and other azoles, candins) Isolates appear “susceptible” at 24 h and “resistant” at
48 h• Two independent investigations in murine models of
candidiasis demonstrated that isolates should be characterized as “susceptible”
• Trailing can be minimized by reading at 24 h or adding methylene blue
Problems of concern …..
• Narrow range of MICs (amphotericin B) Use other media (i.e. AM3) Use E-test
Conclusion…..
• Despite stardardization of susceptibility testing, MIC values do not always associate with response to antifungal therapy
• Most important factors that make correlation in vitro-in vivo data difficult:disease heterogeneity and bias of host immunityinadequate concentration of the drug at theinfection siteinfections associated with catheters/prostheticdevices acting as substrates for biofilm growth
90-60 rule
• Infections due to susceptible isolates respond to appropriae therapy in 90% of the time.
• Infections due to resistant isolates (or infections due to inapproriate therapy) respond in 60% of the time.
• The local epidemiology of antifungal resistance aids to select empirical treatment
• Despite recent advances, mortality rate from invasive fungal infections remains high and emphasis should be given to :
early diagnosis, rapid restoration of host immunity guided antifungal therapy.
