1. Identification, Susceptibility & Resistance
Dr Caroline B. Moore
Mycology Reference Centre
University Hospital of South Manchester
The University of Manchester

2. Yeasts

3. Why bother with identification & susceptibility testing?
Significant number of invasive fungal infections
Increase in ‘at-risk’ patient population

4. Why bother with identification & susceptibility testing?
Significant number of invasive fungal infections
Increase in ‘at-risk’ patient population
Increase in non-C. albicans species
Increase in ‘rarer’ species
The literature provides many examples of an increase in species other than Candida albicans.

5. Proportion of nosocomial Candida infections caused by different species in a teaching hospital
Berrouane et al. J Clin Micro 1999; 37:531

6. Why bother with identification & susceptibility testing?
Significant number of invasive fungal infections
Increase in ‘at-risk’ patient population
Increase in non-C. albicans species
Increase in ‘rarer’ species
Increase in drug resistance

7. Increasing fluconazole resistance in Intensive Care Unit
Moore et al. ECMM 1998

8. Why bother with identification & susceptibility testing?
Significant number of invasive fungal infections
Increase in ‘at-risk’ patient population
Increase in non-C. albicans species
Increase in ‘rarer’ species
Increase in drug resistance
More drug choices available
Informed therapeutic choice

9. How do we identify yeasts?
Culture morphology
Chromagar

10. How do we identify yeasts?
Germ tube test
Microscopic morphology
Terminal chlamydospores
Arthrospores
Other tests

11. How do we identify yeasts?
Traditional methods
Wickerham tests
Auxacolor
Commercial kits
numerous!
Vitek-2
API ID 32C
Molecular methods

12. MICs He uses statistics like a drunken man uses lamp-posts ...
For support rather than illumination

13. Do we have a good test of susceptibility for yeasts?
CLSI (NCCLS) M27-A3 method
USA standard
EUCAST Document E-Def 7.1 method
European standard
Extensive clinical correlation work

14. Do we have a good test of susceptibility for yeasts?
Disc diffusion
no MIC obtained
screening method
E-test strips
MIC value obtained
problematic endpoints

15. Some are more equal than others!
Do we have a good test of susceptibility for yeasts?
An array of commercial formats
Vitek-2
YeastOne
Fungitest
Others…
Some are more equal than others!
Molecular methods

16. Candida albicans Most common cause of candidosis (50-70%)
Can cause a wide range of diseases
May cause vaginal and oral/oesophageal thrush
Produces germ tubes
Attributable mortality (18-25%)
differences in patient age and underlying illness

17. Candida albicans - + ++ + ++ ++ ++
Azole susceptibility can depend on HIV status
FLU ITR VOR POS AMB 5FC CASP
C.albicans
C.albicans -
FLU-resistant
Rates of resistance seen in our laboratory
flucytosine 6%
fluconazole 1.2%
5% of C. albicans with reduced susceptibility

18. Candida parapsilosis complex
Candida parapsilosis, Candida orthopsilosis, Candida metapsilosis
2nd most common species in blood, related to catheters and glucose solutions including TPN
Often seen in neonates
Generally do not cause thrush
Less pathogenic than C. albicans

19. Candida parapsilosis complex
FLU ITR VOR POS AMB 5FC CASP
C.parapsilosis
Rates of resistance seen in our laboratory
fluconazole 0.4%

20. Candida glabrata 3rd or 4th most common species in blood
Increasing in many institutions
Common cause of thrush, particularly vaginal
Generally thought as unable to produce pseudohyphae in vitro
Evidence of ability to grow as filamentous organism

21. Rates of resistance seen in our laboratory 1992-2006
Candida glabrata
Usually reduced susceptibility to fluconazole
Responds poorly to amphotericin B
FLU ITR VOR POS AMB 5FC CASP
C.glabrata
Rates of resistance seen in our laboratory
fluconazole 53%

22. Candida tropicalis
Usually 4th most common species in blood, much commoner in some institutions
Generally does not cause thrush
More invasive than other Candida species
High attributable mortality (33-50%)
High frequency (~80-100%) of infection if coloniser during neutropenia

23. Rates of resistance seen in our laboratory 1992-2006
Candida tropicalis
Usually fluconazole susceptible, but may develop resistance quickly
FLU ITR VOR POS AMB 5FC CASP
C.tropicalis
Rates of resistance seen in our laboratory
flucytosine 20%
fluconazole 46%

24. Candida krusei 5th most common species in blood
Does not cause thrush, except in late-stage AIDS
High mortality in leukaemic patients

25. Candida krusei May be a breakthrough species
Always fluconazole resistant and amphotericin intermediate
FLU ITR VOR POS AMB 5FC CASP
C.krusei

26. Cryptococcus neoformans
Neurotropic fungus
Most common predisposing factor is AIDS
Also organ transplant recipients or cancer patients receiving chemotherapeutics
Produces a polysaccharide capsule - major virulence factor

27. Cryptococcus neoformans
Combination therapy generally used
FLU ITR VOR POS AMB 5FC CASP
Crypto.
neoformans
Cryptococcus would generally be susceptible to some degree to most of the drugs – the exception is caspofungin which is intrinsically resistant.
The most commonly used agents for treatment of cryptococcal infections are amphotericin B and flucytosine in combination.

28. High degree of mortality
Rhodotorula mucilaginosa
Image Courtesy of Carsten Kettner
Saccharomyces species
Malassezia species
High degree of mortality

29. Can be intrinsically resistant to some antifungal drugs
Species of…. Trichosporon Geotrichum Blastoschizomyces
Can be intrinsically resistant to some antifungal drugs

30. Moulds - the Aspergilli

31. Increasing incidence of invasive aspergillosis in hematopoietic stem cell transplant recipients
Marr et al. Clin Infect Dis 2002; 34:909

32. What tests do we have to identify moulds?
morphology
microscopy
additional tests
molecular methods
Experience!

33. Can be a secondary pathogen in cases of carcinoma or TB
Aspergillus
Wide spectrum of disease – dependent on immune status
Allergic bronchopulmonary aspergillosis
Pulmonary aspergilloma
Invasive aspergillosis
Can be a secondary pathogen in cases of carcinoma or TB

34. Aspergillus fumigatus complex
most common cause of aspergillosis ~90% of cases

35. Aspergillus niger complex
Common cause of ear infections

36. Aspergillus terreus complex
Cause of superficial, eye, ear and systemic infections

37. Aspergillus flavus complex
Cause of pulmonary and sinus infections

38. In vitro susceptibility of filamentous fungi
ITR VOR POS AMB 5FC CASP FLU
Asp. fumigatus /
Asp. niger
Asp. terreus
Asp. flavus

39. Azole resistance in A. fumigatus complex
Itraconazole
recent literature 0 - 5%
7% RMLM 1992 – 2007 (n = 519)
significant increase in resistance since 2004 – now 13%
Itraconazole resistant isolates
58% cross-resistant to voriconazole
66% cross-resistant to posaconazole
Data from Dr Susan Howard

40. Azole cross resistance of Asp. fumigatus complex
MIC mg/L
Mosquera & Denning.
Antimicrob Agents Chemother
2002; 46:556

41. Other Aspergilli
Klich MA. Identification of common Aspergillus species (2002). CBS.

42. Non-Aspergillus moulds

43. Increasing frequency of non-Aspergillus mould infections in hematopoietic stem cell transplant recipients
Marr et al. Clin Infect Dis 2002; 34:909

44. Range of disease from cutaneous through to disseminated infection
Fusarium spp.
Range of disease from cutaneous through to disseminated infection

45. Scedosporium spp.
Can cause subcutaneous infections but dissemination may occur in immunocompromised host

46. Cunninghamella Rhizomucor Rhizopus Absidia Mucor etc…
Mucorales
Cunninghamella Rhizomucor Rhizopus Absidia Mucor etc…

47. In vitro susceptibility of filamentous fungi
ITR VOR POS AMB 5FC CASP FLU
Asp. fumigatus /
Asp. niger
Asp. terreus
Asp. flavus
Fusarium /- +/
Scedosporium +/- + +/ /- -
Mucorales

48. Others… Alternaria Plus many more… Acremonium Paecilomyces Penicillium
Wangiella
Acremonium
Penicillium
Cladophialophora
Phialophora
Alternaria
Plus many more…

49. Take time to ‘know’ the fungus in your hospital….
Better the devil….