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Anti fungal susceptibility

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Anti fungal susceptibility

  1. 1. Anti fungal susceptibilityMAULANA AZAD MEDICAL COLLEGE PG SEMINAR
  2. 2. What are fungi?
  3. 3. Do we have any anti fungal available?
  4. 4. • Drugs with their site of action
  5. 5. Site of action• Cell wall : (1,3)- β glucan synthase Echinocandins• Cell membrane: ergosterol synthesis Polyene antibiotics (AMB) Azoles Allylamines (Terbinafine)
  6. 6. • 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
  7. 7. • Most of these drugs are fungi-static except Amphotericin-B Allylamines and Benzylamines Naftifine,Terbinafine, Butenafine.
  8. 8. why there is a need for susceptibility testing
  9. 9. • Increasing immuno-supressive states……..• Increasing incidence of invasive mycosis and life threatening infections……..• Avaibility of newer drugs…….• Avaibility of standard guide lines…..• Emerging resistance……
  10. 10. Resistant to antifungal agentsCandida krusei Fluconazole intrinsicCandida glabrata Fuconazole acquired CaspofunginCandida albicans Fuconazole acquired CaspofunginCandida lusitanae Amphoericin-BAspergillus terreus Amphotericin-B intrinsicPseudallescheria boydii Amphotericin-BPaecilomyces lilanicus Amphotericin-BFusarium species all
  11. 11. 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.
  12. 12. 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
  13. 13. Do we have any methods ?
  14. 14. Methods• Macro-dilution method.• Micro-dilution method.• Disk diffusion method.• Agar dilution method.
  15. 15. 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)
  16. 16. CLSI- manuals• M 27-A2 : second edition (1992)• M 38-A : Approved standard (1998)• M 44-A : Approved standard(2004)
  17. 17. CSLI M-27 A2 method for yeast susceptibility testing
  18. 18. 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
  19. 19. 2• 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
  20. 20. 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
  21. 21. 4• 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}
  22. 22. 5• Drug dilution ranges : 5-FC and Flucytosine --- 0.12 to 64 µg/ml other drugs --------------- 0.03 to 16 µg/ml
  23. 23. 6• 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
  24. 24. 7• 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)
  25. 25. Quality control strains• Candida parapsilosis ATCC 22019.• Candida krusei ATCC 6258.
  26. 26. 3• Incubation temp.- 35°c.• Incubation time- 24-48 hr for candida species. 48-72 hr for cryptococcus sp.
  27. 27. Quality ANTIFUNGAL MIC AT 48 HR MIC AT 24 HR MIC AT 48 HRcontrol strains AGENTS MACRODILUTN MICRODILUTN MICRODILUTNC.Parapsilosis AMB 0.25-1 0.25-2 0.5-4ATCC 22019 FLUCONAZOLE 2-8 0.5-4 1-4 ITRACONAZOLE 0.06-0.25 0.12-0.5 0.12-0.5 VORICONAZOL NA 0.016-0.12 0.03-0.25 KETOCONAZOL 0.06-0.25 0.03-0.25 0.06-0.5 5-FC 0.12-0.5 0.06-0.25 0.12-0.5C. Krusei ATCC AMB 0.5-2 0.5-0.2 1-46258 FLUCONAZOLE 16-64 8-64 16-128 ITRACONAZOLE 0.12-0.5 0.12-1 0.25-1 VORICONAZOL NA 0.06-0.5 0.12-1 KETOCONAZOL 0.12-0.5 0.12-1 0.25-1 5-FC 4-16 4-16 8-32
  28. 28. 8• 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 standardo microdilution—shows 50% growth inhibition
  29. 29. Microtitre plate with in stand with reading mirror
  30. 30. Susceptibility cut-off for yeast (µg/ml) S SDD ID RFLUCONAZOLE ≤8 16-32 ≥ 64ITRACONAZOLE ≤ 0.12 0.25-0.5 ≥1VORICONAZOL ≤1 2 ≥4FLUCYTISINE ≤4 ---- 8-16 ≥ 32
  31. 31. EUCAST Antifungal Susceptibility Testing Subcommittee (AFST)• EUCAST DEFINITIVE DOCUMENT• E Def 7.1• MIC• Yeast (fermentative)• Broth dilution
  32. 32. Differences of CLSI and EUCAST conditions for antifungal susceptibility testing for yeasts Difference between CLSI (USA) and EUCAST (Europe) CLSI EUCASTSuitability Yeasts Fermentative yeastsTest medium RPMI 0.2% glucose RPMI 2% glucoseMicrotitration plates U-shaped wells Flat-bottom wellsTemperature 35°C 35-37°CLength of incubation 24-48 h 24 hReading Visually PhotometricallyEndpoint 100% AMB , 50% 5FC, azoles, 90% AMB , 50% 5FC, azoles, candins candins
  33. 33. Breakpoints (µg/ml) according to CLSI and EUCAST for Candida species* only for C. albicans, C. parapsilopsis, C. tropicalis** tenative break points; NS: Non susceptiblesDrug CLSI EUCASTAmphotericin 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 >0.125Posaconazole - -Caspofungin NS >2** -Anidulafungin NS >2** -
  34. 34. • Breakpoints from one method cannot be extrapolated to another method
  35. 35. Broth-based alternative approaches foryeasts modifications of reference method• Colorimetric methods• Spectrophotometric method• Flow cytometry methodTo improve interlaboratory reproducibilityBetter serve clinical laboratory needs
  36. 36. Sensititre yeast one & fungitest
  37. 37. Spectrophotometric methods
  38. 38. CLSI 44-A
  39. 39. • Disc diffusion susceptibility testing.• Candida species.• Good correlation with microdilution method.• Antifungal agents: Fluconazole Itraconazole Voriconazole
  40. 40. • 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
  41. 41. • 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.
  42. 42. Recommended quqlity control zone- diameter (mm) rangesAnti fungal Disk content C. albicans C.parapsilosis C.tropicalis C. krusei ATCC 90028 ATCC 22019 ATCC 750 ATCC 6258Fluconazole 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
  43. 43. Interpretative guidelines for zone diametersANTIFUNGAL susceptible (S) susceptible-dose resistant (R)DRUGS dependent (SDD)Fluconazole ≥ 19 mm 15-18 mm ≤ 14 mmItraconazole* ≥ 23 mm 14-22 mm ≤ 13 mmVoriconazole* ≥ 17 mm 14-16 mm ≤ 13 mm
  44. 44. 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
  45. 45. • E Test (AB Biodisk-Sweden) Amphotericin-B, Fluconazole, 5-FC, Ketoconazole, Itraconazole, VoriconazoleFDA : Fluconazole,Itraconazole & 5-FC solidified RPMI medium supplemented with 2% Glucose
  46. 46. MHA- C. albicans (flu: MIC-0.38 µg/ml)C. glabrata (flu: MIC >256 µg/ml) & C. lusitanea (AMB)
  47. 47. Candida species clinical isolates to Caspofungin by Etest in RPMI
  48. 48. CSLI M-38 AStandard broth dilution methods for moulds
  49. 49. 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
  50. 50. Recommended OD ranges and mean inoculum sizesFungus OD ranges Mean Inoculum size( 106CFU/ml)Aspergillus species 0.09-0.11 1.6Bipolaris species 0.2-0.4 0.6Cladophialaphora bantiana 0.15-0.17 1.1Dactylaria constricta 0.15-0.17 1.1Fusarium species 0.15-0.17 3Paecilomyces lilanicus 0.09-0.13 2.1Rhizophus arrhizus 0.15-0.17 1.3Scedosporium apisospermum 0.15-0.17 1Scedosporium prolificans 0.15-0.17 0.8Sporothrix schenckii 0.09-0.11 2
  51. 51. 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)
  52. 52. 4• 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
  53. 53. 5 INCUBATION CONDITIONSMold TimeRhizopus arrhizus 24-hrAspergillus species, Bipolaris species, Fusarium species,Paecilomyces 48-hrlilanicus, Sporothrix schenckii, Tricoderma longobrachiatum, wangielladermatidisPseudallescheria boydii (Scedosporium apiospermum), 72-hrCladophialaphora bantiana, Dactylaria constricta, Scedosporiumproliferans
  54. 54. 6• End point determination visual : absence of growth with respect to GC
  55. 55. MEC caspofungin to Aspergillus
  56. 56. Broth based alternative approach for moulds• Colorimetric method• Spectrophotometric method
  57. 57. Agar based alternative approaches• E test ( AMB, Azoles)• Disk diffusion ( under investigation)• Agar dilution method (not standardised)
  58. 58. Commercial kits• VITEK 2 (BioMerieux) fully automated system
  59. 59. 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
  60. 60. Problems of concern …..• Narrow range of MICs (amphotericin B) Use other media (i.e. AM3) Use E-test
  61. 61. 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 immunity inadequate concentration of the drug at theinfection site infections associated with catheters/prostheticdevices acting as substrates for biofilm growth
  62. 62. 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.
  63. 63. • 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.

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