1. Ana Espinel-Ingroff, MS,PHD VCU Medical Center, Richmond, VA, USA
ISHAM 2015:
Current strategies for preventing invasive fungal infections in patients at risk
Revised 2016
Breakpoints and epidemiological cutoff values (BPs and ECVs): Can they predict clinical response?
Ana Espinel-Ingroff, MS,PHD
VCU Medical Center, Richmond, VA, USA

2. Breakpoints and ECVS
EUCAST and CLSI BPs for Candida versus triazoles and echinocandins are the best predictors of patient outcome.
Until recently, we only had MIC/MEC distributions for mould species.
The focus of the presentation will be:
1. To summarize criteria to define ECVs for CLSI methods as well as provided available CLSI ECVs for Aspergillus spp.
2. To summarize and contrast CLSI cutoffs with other endpoints proposed as either BPs or ECVs based on in vitro/in vivo data, including murine models (infection and PK/PD data models) and clinical data.

3. Triazole Resistance and A. fumigatus
In 1997, Denning et al. reported two isolates with MICs >8 mcg/ml and no clinical response to treatment. (JAC 40: 401)
In 2001, Mellado et al. reported that cyp51A and cyp51B genes coded 14-α sterol demethylase in Aspergillus spp. (JCM 39: 2431)
Subsequently, specific point cyp51A mutations were identified as responsible for elevated triazole MICs:
Codon G54, M220 G138, L98 and others.
Point mutations became associated with patient failure and azole resistance has increased among Apergillus spp. especially in Europe.

4. Role of the ECV ECVs separate WT from non-WT isolates.
Non-WT isolates may harbor resistance mechanisms and usually correlate with high MICs or MECs.
A categorization of an isolate as non-WT indicates decreased susceptibility to the agent evaluated or potential phenotypic resistance.
Non-WT MIC/MEC values are >ECV.

5. Triazole MICs (µg/ml) for
A. fumigatus mutants
Cyp51 mutation
Itra
Isa
Posa
Vori
CLSI ECV WT 1
0.06-1
0.5-2
0.25/0.5
G54
>8
0.25-2
L98
8-16
0.5-1
4-8
M220
1-8
0.5-4
G138,Y431,
G434, G448, F46Y
2->8 16
0.5-16
4-16
Gregson et al ; 57: Lepak et al. AAC. 2013; 57: & 57: Kidd et al doi.1111/myc
Espinel-Ingroff & Turnidge Rev Iberoam Micol. CLSI M57, M

6. Available ECVs for M38 method
ECVs have been defined for some Aspergillus spp. and:
- Amphotericin B, caspofungin, and four triazoles (isavuconazole, itraconazole, posaconazole and voriconazole)
Mucorales and Fusarium spp.:
- Amphotericin B and triazoles
Espinel-Ingroff & Turnidge Rev Iberoam Micol.
CLSI M57 & M

7. Criteria for the calculation of ECVs for CLSI methods

8. Criteria for ECV calculation
1. MICs/MECs generated by specific methodology (CLSI M27, M38, commercial method)
E.g., any method modifications and/or abnormal distributions are not acceptable
2. Distribution must be accompanied by within range QC isolate(s) data during the testing period.
3. MIC/MEC data must be generated by a minimum of 3 different laboratories (to allow interlaboratory variability evaluation) and 100 isolates/species/agent.
4. In the case of CLSI-ECVS, the raw data should be available to the CLSI Subcommittee for review and approval by a majority of voting members.
Espinel-Ingroff & Turnidge Rev Iberoam Micol.

9. Abnormal distributions
Bimodal=“saddle” between two modes
Truncated= mode at highest or lowest drug concentration
Similar bars=Same or similar modes
Samples of abnormal distributions and modal variability in the next slide.
Turnidge & Patterson. Clin. Microbiol. Rev. 2007; 20:

10. Participant Laboratory No.
______________________________________________________________________________________________________________________________________________________________________________________________________________________
Pool of the 14 single caspofungin MIC distributions received
for the calculation of caspofungin ECV for C. albicans:
Espinel-Ingroff & Turnidge Rev Iberoam Micol.
Participant Laboratory No.
Agent
MIC 4 20 3 2 18 19 1 7 11 12 9 14 6 10
Caspofungin
0.0079 92
0.01
136 28
181
114
1181
141
0.03 13 31
755
670
2037 15
343 38
0.06 55
659
544
898 39 59
218
0.12 8 69
357 49 68 24
128 72
345
0.25
212
117
332 58
106
0.5
176 5 16 71
108 97
552 30
The examination of modes (bolded) in each participant laboratory indicated:
Interlaboratory variability (up to five dilutions) which precluded the calculation of caspofungin ECVs and Candida spp. for reference methods.
Abnormal distributions with aberrant modes: (i) truncated, mode at lowest conc. tested: lab 3 distribution (ii) and bimodal: lab 12 distribution. The total number of caspofungin MICs is 11,550. Espinel-Ingroff et al. AAC :

11. Analysis
Distributions from the different labs are screened for abnormal distributions and size, unusual modal variation, and percentage of data provided by each lab.
It is recommended that data should be normalized (weighting) when a lab. contributes >50 to the “pool” used for the analysis, but a decision has not been made regarding the ECV when values are different (see next slide).
The ECV is calculated by the iterative statistical method as described by Turnidge et al. (Microsoft Excel workbook [ECOFFinder]).
The MIC/MEC that captures at least 97.5% of the modeled WT population is the ECV for the particular species and agent.
Turnidge et al. Clin. Microbiol. Infect. 2006; 12: ; Espinel-Ingroff & Turnidge Rev Iberoam Micol.

12. Posaconazole ECV of 0. 25 mcg/ml (97
Posaconazole ECV of 0.25 mcg/ml (97.5% value) using the iterative statistical method (ECOFFinder) before normalizing (weighting [W]) the data, when one lab provided >50% of the data.

13. Posaconazole ECV of 0. 5 mcg/ml (97
Posaconazole ECV of 0.5 mcg/ml (97.5% value) using the iterative statistical method (ECOFFinder) after normalizing (weighting [W]) the data, when one lab provided >50% of the data.

14. ECVs and other susceptibility endpoints for A
ECVs and other susceptibility endpoints for A. fumigatus and the triazoles

15. ECVs for A. fumigatus complex (4 labs)
CLSI
mode
ECV: <1 mcg/mL
Number of isolates
                                                      
EUCAST
mode
Itraconazole
37 isolates >4 mcg/mL
7 mutants
Rodrigez-Tudela et al. AAC. 2008; 52:
Espinel-Ingroff et al JCM ; 48:
Meletiadis et al. AAA :
MIC (mcg/mL)

16. ECVs for A. fumigatus complex (4 labs)
CLSI
mode
Number of isolates
                                                      
ECV: < 1 mcg/mL
Voriconazole
EUCAST
mode
Rodrigez-Tudela et al. AAC. 2008; 52:
Espinel-Ingroff et al JCM ; 48:
Meletiadis et al. AAA :
MIC (mcg/mL)

17. S-BPs: Voriconazole and A. fumigatus Dissem. murine infection
Model
WT/mutants
Target
MIC endpoint
Reference
In vitro IPA model
AUC/MIC= 55:
GA suppression
<0.5 mcg/mL
1Jeans et al.
In vitro PK/PD
Trough/MIC= 2:
Optimal treatment
< 0.25 mcg/mL
2Siopi et al.
Dissem. murine infection
Reduced load
Prolonged surv.
3Salas et al.
Clinical trials
Clinical response
<1 mcg/mL (WT)
4Rodriguez-T
1JID 2012; 206: JAC AAC 2013; 57: Curr Fungal Infect Rep 2012; 5:
Espinel-Ingroff & Turnidge Rev Iberoam Micol.

18. ECVs for A. fumigatus complex (4 labs)
CLSI
mode
EUCAST Mode
*ECV: < mcg/ml
                                                      
CLSI ECV < 0.5 mcg/mL
before normalization of the data
Number of isolates
Posaconazole
*Rodrigez-Tudela et al. AAC. 2008; 52:
Espinel-Ingroff et al. JCM ; 48:
*Meletiadis et al. AAA :
MIC (mcg/mL)

19. ECVs: Posaconazole and A. fumigatus complex
Reference
0.12 mcg/mLt
Meletiadis
AAC. 2012; 56:
<0.25 mcg/mL
Rodriguez-T
AAC 2008; 52: 2649
<0.25/0.5 mcg/ml?
Pending
Espinel-Ingroff et al.
JCM ; 48:
tListed as EUCAST S-BP; Espinel-Ingroff & Turnidge Rev Iberoam Micol.

20. S-BPs: Posaconazole and A. fumigatus complex
Model
Reference
< 0.25 mcg/mL
In vitro data
PK/PD
1Verweij*
<0.06 mcg/mL
WT and mutants
2Meletiadis
mcg/mL
Murine IPA
WT MICs mcg/mL
non-WT MICs 2-8 mcg/mL
(Cyp51 mutations)
Target: Human PK and current static dose PD
3Lepak
1Drug Res Upgrades 2009; 12: AAC. 2012; 56: AAC. 2013; 57:
*Mavridou et al. AAC 2011; ; Espinel-Ingroff & Turnidge Rev Iberoam Micol.

21. ECVs for A. fumigatus complex (8 labs)
100
200
300
400
500
600
0.016
0.032
0.064
0.125
0.25
0.5 1 2 4
>4
Mode
CLSI ECV: < 1 mcg/mL
Isavuconazole
Number of isolates
EUC Mode/ECV:
1/2 mcg/mL
Cyp51 TR/L98H
&/or M220
MICs>4 mcg/ml
Chowdhary et al. JAC 2012; 67: Espinel-Ingroff et al. 2013; AAC 57: Howard et al. 2013; AAC 57:
MIC (mcg/mL)

22. S-BP: Isavuconazole and A. fumigatus complex
Model
Murine neutropenic IA
Predictor of treatment response
Isolates
WT MICs= μg/mL
Mutants= μg/mL (cyp51 mutants)
Endpoint
Efficacy lung burden
14 day survival
<0.5 μg/mL
Conclusions
AUC/MIC: correlation with outcome.
MIC better predictor than mutation.
Lepak et al. AAC. 2013; 57: 6284 ; Espinel-Ingroff & Turnidge
Rev Iberoam Micol.

23. S-BP: Isavuconazole and A. fumigatus complex
Model
Clinical trials
Predictor of treatment response
Isolates
Baseline isolates
28 A. fumigatus
7 A. flavus
6 A. terreus
0.25 to >4 mcg/mL
Endpoint
PK/PD data, Efficacy
Successful outcomes
Conclusions
Susceptible BP
< 1 mcg/mL
Presented by L. Kovanda; CLSI meeting January 2015

24. Azole resistance among A. fumigatus complex in Australia
Between , 13 A. fumigatus complex isolates with elevated MICs of itraconazole, posaconazole and/or voriconazole were collected at the National Mycology Reference Centre, Adelaide.
Molecular analysis confirmed that 4 isolates were Aspergillus lentulus, known to have reduced susceptibility to triazoles.
Among the A. fumigatus, the following cyp51A gene mutations (TR34/L98H, G54R, F46Y, Y431S and G448S) were observed) in 6 of the 9 isolates.
MICs were >ECVs for all isolates with at least one of the agents.
Kidd et al doi.1111/myc.12324

25. ECVs for A. terreus complex (5 labs)
Mode
ECV: < 1 mcg/mL
Number of isolates
                                                      
Voriconazole
Espinel-Ingroff et al. JCM ; 48:
MIC (mcg/mL)

26. A. terreus complex Model Murine neutropenic IA Fungal burden Survival
ECV: Voriconazole and
A. terreus complex
Model
Murine neutropenic IA
Fungal burden
Survival
Galactomannan reduction
Isolates
MICs= μg/mL
Mechanisms of resistance: not provided
Endpoint
MICs < 1 μg/mL
Best survival and tissue burden reduction
Salas et al. AAC. 2013; 57:

27. MIC distributions of amphotericin B against Aspergillus fumigatus complex (7 labs)
CLSI mode
CLSI < 2 mcg/mL
BP: 1 mcg/mL
EUC Mode: 0.25 mcg/mL
(mcg/mL)
Espinel-Ingroff et al 2011; AAC 55: : 27

28. Amphotericin B and Aspergillus spp.
MICs <1 µg/ml: clinical response (6 pts infected with A. fumigatus and A. flavus)
MICs >2 µg/ml: failure (22/23 patients)
Treatment: amphotericin B for invasive aspergillosis
Lass-Florl et al. JAC. 1998; 42:
MICs >1 μg/ml A. terreus : poor response to amphotericin B formulations
Steinbach, et al. AAC. 2004; 38:
The relationship is not significant.
Hachem et al. Cancer 2004; Baddley et al. JCM 2009; 47:

29. ECV for A. fumigatus complex (5 labs)
Mode
Number of isolates
                                                      
< 0.5 mg/L
Caspofungin
Espinel-Ingroff et al. AAC 2011; 55:
MIC (mcg/mL)

30. Caspofungin ECVs
Correlation of high echinocandin MECs with FKS1 gene mutations remains to be determined for Aspergillus spp.
Caspofungin MECs for A. fumigatus of >1 μg/ml from breakthrough infections in patients receiving this agent have been reported, but without genetic information.
Madureira et al. B. Int J Antimicrob Agents. 2007; 30:

31. Conclusions for A. fumigatus
CLSI ECVs based on in vitro
data
S-BPs based on mutants/models/clinical data
Posaconazole < μg/mL
Mutants vs WT strains
Voriconazole <1 μg/mL
Itraconazole <1 μg/mL
Isavuconazole <1 μg/mL
< μg/mL
<0.06 μg/mL
< 1 μg/mL
Preliminary results support the potential clinical utility
of triazole ECVs for A. fumigatus in detecting emerging
resistance (non-WT MICs) due to cyp51A mutations.

32. Conclusions
Similar data are needed for triazoles and other moulds as well for other antifungal agents.
ECVs have only been defined for Aspergillus spp., Fusarium spp. and the Mucorales

33. Thank you