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

Yeasts

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

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.

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

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

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

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

How do we identify yeasts? Culture morphology Chromagar

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

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

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

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

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

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

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

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 1992-2006 flucytosine 6% fluconazole 1.2% 5% of C. albicans with reduced susceptibility

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

Candida parapsilosis complex FLU ITR VOR POS AMB 5FC CASP C.parapsilosis ++ ++ ++ ++ ++ + - Rates of resistance seen in our laboratory 1992-2006 fluconazole 0.4%

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

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 1992-2006 fluconazole 53%

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

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 1992-2006 flucytosine 20% fluconazole 46%

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

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

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

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.

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

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

Moulds - the Aspergilli

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

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

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

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

Aspergillus niger complex Common cause of ear infections

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

Aspergillus flavus complex Cause of pulmonary and sinus infections

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

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

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

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

Non-Aspergillus moulds

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

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

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

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

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

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

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