1. Anti fungal susceptibility
MAULANA AZAD MEDICAL COLLEGE
PG SEMINAR

2. What are fungi?

5. Do we have any anti fungal available?

6. • Drugs with their site of action

7. Site of action
• Cell wall : (1,3)- β glucan synthase
Echinocandins
• Cell membrane: ergosterol synthesis
Polyene antibiotics (AMB)
Azoles
Allylamines (Terbinafine)

8. • 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

9. • Most of these drugs are fungi-static except
Amphotericin-B
Allylamines and Benzylamines
Naftifine,Terbinafine, Butenafine.

10. why there is a need for susceptibility
testing

11. • Increasing immuno-supressive states……..
• Increasing incidence of invasive mycosis and
life threatening infections……..
• Avaibility of newer drugs…….
• Avaibility of standard guide lines…..
• Emerging resistance……

12. Resistant to antifungal agents
Candida 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

13. 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.

14. 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

15. Do we have any methods ?

16. Methods
• Macro-dilution method.
• Micro-dilution method.
• Disk diffusion method.
• Agar dilution method.

18. 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)

19. CLSI- manuals
• M 27-A2 : second edition (1992)
• M 38-A : Approved standard (1998)
• M 44-A : Approved standard(2004)

20. CSLI M-27 A2 method for yeast
susceptibility testing

23. 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

24. 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

25. 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

26. 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}

27. 5
• Drug dilution ranges :
5-FC and Flucytosine --- 0.12 to 64 µg/ml
other drugs --------------- 0.03 to 16 µg/ml

28. 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

29. 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)

30. Quality control strains
• Candida parapsilosis ATCC 22019.
• Candida krusei ATCC 6258.

31. 3
• Incubation temp.- 35°c.
• Incubation time- 24-48 hr for candida species.
48-72 hr for cryptococcus sp.

32. Quality ANTIFUNGAL MIC AT 48 HR MIC AT 24 HR MIC AT 48 HR
control strains AGENTS MACRODILUTN MICRODILUTN MICRODILUTN
C.Parapsilosis AMB 0.25-1 0.25-2 0.5-4
ATCC 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 0.12-0.5
C. Krusei ATCC AMB 0.5-2 0.5-0.2 1-4
6258 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

33. 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 standard
o microdilution—shows 50% growth inhibition

34. Microtitre plate with in stand with reading
mirror

37. 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

38. EUCAST Antifungal Susceptibility Testing
Subcommittee (AFST)
• EUCAST DEFINITIVE DOCUMENT
• E Def 7.1
• MIC
• Yeast (fermentative)
• Broth dilution

39. Differences of CLSI and EUCAST conditions for
antifungal 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, 90% AMB , 50% 5FC, azoles, candins
candins

40. 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** -

41. • Breakpoints from one method
cannot be extrapolated to another method

42. Broth-based alternative approaches for
yeasts modifications of reference method
• Colorimetric methods
• Spectrophotometric method
• Flow cytometry method
To improve interlaboratory reproducibility
Better serve clinical laboratory needs

43. Sensititre yeast one & fungitest

44. Spectrophotometric methods

45. CLSI 44-A

46. • Disc diffusion susceptibility testing.
• Candida species.
• Good correlation with microdilution method.
• Antifungal agents:
Fluconazole
Itraconazole
Voriconazole

47. • 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

48. • 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.

49. Recommended quqlity control zone-
diameter (mm) ranges
Anti fungal Disk content C. albicans C.parapsilosis C.tropicalis C. krusei
ATCC 90028 ATCC 22019 ATCC 750 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

50. Interpretative guidelines for zone
diameters
ANTIFUNGAL susceptible (S) susceptible-dose resistant (R)
DRUGS dependent (SDD)
Fluconazole ≥ 19 mm 15-18 mm ≤ 14 mm
Itraconazole* ≥ 23 mm 14-22 mm ≤ 13 mm
Voriconazole* ≥ 17 mm 14-16 mm ≤ 13 mm

51. 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

52. • 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

53. MHA- C. albicans (flu: MIC-0.38 µg/ml)
C. glabrata (flu: MIC >256 µg/ml) & C. lusitanea (AMB)

54. Candida species clinical isolates to Caspofungin
by Etest in RPMI

55. CSLI M-38 A
Standard broth dilution
methods for moulds

56. 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

57. 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

58. 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)

59. 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

60. 5
INCUBATION CONDITIONS
Mold Time
Rhizopus arrhizus 24-hr
Aspergillus species, Bipolaris species, Fusarium species,Paecilomyces 48-hr
lilanicus, Sporothrix schenckii, Tricoderma longobrachiatum, wangiella
dermatidis
Pseudallescheria boydii (Scedosporium apiospermum), 72-hr
Cladophialaphora bantiana, Dactylaria constricta, Scedosporium
proliferans

61. 6
• End point determination visual :
absence of growth with respect to GC

62. MEC caspofungin to Aspergillus

63. Broth based alternative approach for
moulds
• Colorimetric method
• Spectrophotometric method

64. Agar based alternative approaches
• E test ( AMB, Azoles)
• Disk diffusion ( under investigation)
• Agar dilution method (not standardised)

65. Commercial kits
• VITEK 2 (BioMerieux)
fully automated system

66. 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

67. Problems of concern …..
• Narrow range of MICs (amphotericin B)
Use other media (i.e. AM3)
Use E-test

68. 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 the
infection site
 infections associated with catheters/prosthetic
devices acting as substrates for biofilm growth

69. 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.

70. • 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.