Mucormycosis: an update

Livio Pagano

Istituto di Ematologia

Roma

Fungal agents

Yeasts Moulds

Candida

Cryptococcus

Trichosporon spp

Rhodotorula

Modified from de Pauw et al, Med J Hematol Infect Dis 2011

SEPTATE

HYPHAE

Aspergillus spp

Scedosporium

Fusarium

ASEPTATE

HYPHAE

Zygomycetes

Dermatophytes

Trichophyton

Microsporon

Dimorphics

Histoplasma

Blastomyces

Coccidioides

Penicillium

Chamilos et al, Haematologica 2006 91: 986-9

Spectrum of Invasive Mold Infections at

autopsy

MD Anderson Hospital

Lehrnbecher et al. J Infect 2010

Mucorales are characterized by wide hyphaes (10-20 mm), irregularly shaped, with acute angle

ramifications.

They are ubiquitarian, saprophytic, aerobic agents, that find an otpimal substratum for their growth in meat and sugar at a temperature of about 25°C-35°C

Virchows Arch [Pathol Anat] 102: 543-564 , 1885

direct inoculationinhalation

Fungus acquisition

Pathogenesis and classes of “high risk” patients for Mucormycosis

Substratum with high acidity (Ketoacidosis)

Macrophage inhibition in controlling spores germination (steroids = Autoimmune diseases)

Altered neutrophils chemotaxis (Diabetes)

Ferrioxamine transformation in ferrirhizoferrin (IRCand Trasfusion overload recipients)

Immunodepressant therapy (graft = HSCT and SOT)

NEUTROPENIA (hematological malignancy= ↑↑AML)

GR SPA FRA FRA BEL CH ITA EU

Mono Multi Multi Multi Mono Mono Multi Multi

24 6 531 101 31 19 60 230

Petrikkos et al. CMI 2014

Case-series collected

between 1993-2007

Proportion of HMs in IM case-series:

developed vs developing countries

Years All cases % HMs

cases

Roden et al, 2005 Global 1940-2003 929 21%

Bitar et al, 2009 France 1997-2006 531 17.3%

Ruping et al, 2010 Global 2006-2009 41 63.4%

Saegeman et al, 2010 Belgium 2000-2009 31 77%

Kara et al, 2009 Turkey 2001-2005 20 60%

Skiada et al, 2011 Europe 2004-2007 212 54%

Chakrabarti et al, 2009 India 2006-07 178 1.1%

Invasive Non-Aspergillus Mold Infections in

HSCT Recipients, United States, 2001-200615,820 HSCT 77 with Mucorales/983 IFDs

(0.08%)

Overall 12-month cumulative

incidence: 0.3%

0.85%

Park et al, Emerg Infect Dis 2011

Over a 10-year period

31 proven/probable cases

Strasfeld et al. Clin Lymph Myeloma & Leuk 2013

The most frequent

infection form is the

rino-cerebral

one, linked to the capability of fungi to

penetrate in the body through nasal and

nosepharyngeal mucosa

(typical of diabetic patient)

In neutropenic patients

can be instead more

easily observed the

picture of mycotic

pneumonia with a

penetration mechanism

similar to the previous

one

Petrikkos et al. CMI 2014

Laboratory diagnostics

Colonization by Zygomycetes

seems definitely rare (it’s easier

to isolate a Rhizopus as a

contaminating agent than a

Mucor)

Culture studies show a low

sensitivity (more than 75% of

cases with histologically

diagnosed zygomycetes

pneumonia don’t develop fungi

at cultures)

Reduced growth in culture

Galactomannan

negative in case of mucormycosis (as well as glucan)

Galactomannan

mold active prophylaxis decrease the predictive value (low

incidence of IA)

serum GM assay

o not reliable as surveillance in asymptomatic pts on

effective prophylaxis (results negative or FP)

o useful to diagnose pts with a clinical suspicion

Duarte CID 2014;

Evidence of Zygomycetes in the

BAL fluid

Fungiflora Y staining clearly showed fungal walls because the

fluorescence specifically attached to polysaccharides, such as

cellulose or chitin, which are present in fungal walls. Under

fluorescent microscopy, typical hyphae displayed brilliant green

fluorescence, which readily differentiated them from exfoliated

cells

Radiological Pictures

Halo-Sign

Air-Crescent-Sign

Mucormycete

s

Aspergillus

No radiological differences

But “reversed halo sign” may be suggestiveLegouge et al, CID 2014

Focal rounded area of ground-glass opacity surrounded

by a crescent or complete ring of consolidation

Histologically RHS represents infarcted lung tissue with a

greater ammount of hemorrage at the perifery of the lesion

“reversed halo sign”(RHS)

Georgiadou S – Clin Infect Dis 2011;52:1144

23%

18%

15%9%

8%

8%

6%4% 4% 2%1%1% 1%

organizing pneumonia

paracoccidioido

TBC

plm embolism

aspergillosis

zygomycosis

sarcoidosis

adeno-k

“RHS should be considered a relatively non specific sign”

Only 8% of reversed halo sign are in Mucormycosis

Stanzani et al. Clin Infect Dis 2012

Clin Nucl Med 2012

• Optimal for PCR-based diagnosis• Complications <1%. • NO false positivity

(NO CONTAMINATION)• Sensibility 80%; PPV 100% • Diagnosis in 53% of not diagnostic BAL

Int J Hematol. 2009 Jun; 89(5):624-7. Epub 2009 May 27.

Role of CT-guided percutaneous lung biopsyin diagnosis of pulmonary fungal infection

in patients with hematologic diseases.Shi JM, Cai Z, Huang H, Ye XJ, He JS, Xie WZ, Zhang J, Zhou XY, Luo Y, Lin Y, Li L, Zheng WY, Wei GQ, Lin MF.

Nosari A et al, Haematologica 2003Carrafiello G et al Radiol Med 2006

Laas-Florl et al Clin Infect Dis 2007

Gupta S. Hematologic Oncology 2009

The role of PCR

Hammond et al, JCM 2011

This is a useful tool that can improve tissue diagnosis of mucormycosis and

characterization of culture-negative IMD, thus facilitating targeted antifungal

therapy, but performed on formalin-fixed paraffin-embedded tissue samples

Early diagnosis and monitoring of mucormycosis by detection of

circulating DNA in serum: retrospective analysis of 44 cases

collected through the French Surveillance Network of Invasive

Fungal Infections (RESSIF)

Mucorales qPCR not only

could confirm the IM

diagnosis when other

63 mycological arguments

were present but also could

anticipate this diagnosis.

Quantification of 64 DNA

loads may also be a useful

adjunct to treatment

monitoring

Millon et al, CMI 2016

Good for follow-up !!

Mucorales-specific T cells as surrogate

diagnostic marker in high-risk patients

3 proven Mucormycosis

Mucorales-specific T cells, produced predominantly IL-4, IFN-γ, IL-10, may be

detected only in patients with IM

These cells specific were able to directly induce damage of Mucorales

Specific T cells contribute to human immune responses against Mucorales and

could be evaluated as a surrogate diagnostic marker of IM

Potenza et al, Blood 2011

ALGORITMO NEL PAZIENTE EMATOLOGICO

Pagano et al., Br J Haematol 2009; 146(6):597-606

Jeong et al. Mycoses 2015

2005-2014

30 pts with

proven/probable IM

Amphotericin B, posaconazole, and isavuconazole have activity against Mucorales

References: 1. Sabatelli F, et al. Antimicrob Agents Chemother. 2006;50(6):2009-2015. 2. Yamazaki T, et al. Int J Antimicrob Agents. 2010;36(4):324-31. 3. González GM.

Med Mycol. 2009;47(1):71-76. 4. The European Committee on Antimicrobial Susceptibility Testing. www.eucast.org. Accessed 31 March 2016. 5. Pfaller MA, et al. Diagn

Microbiol Infect Dis. 2015;82(4):303-313. 6. Thompson GR 3rd, et al. J Antimicrob Chemother. 2009;64(1):79-83. 7. Al-Hatmi AM, et al. J Antimicrob Chemother.

2015;70(4):1068-1071. 8. Deng S, et al. Antimicrob Agents Chemother. 2013;57(4):1974-1977. 9. Chowdhary A, et al. Clin Microbiol Infect. 2014;20(suppl 3):47-75. 10.

Najafzadeh MJ, et al. Antimicrob Agents Chemother. 2014;58(9):5629-5631. 11. Guinea J, et al. Antimicrob Agents Chemother. 2008;52(4):1396-1400. 12. Lackner M, et al.

Antimicrob Agents Chemother. 2012;56(5):2635-2642. 13. Arendrup MC, et al. Antimicrob Agents Chemother. 2015;59(12):7735-7742. 14. Cresemba, SPC.

Activity Variable activity Little or no activity

TRIAZOLES

AMB

FIRST GENERATION SECOND GENERATION

Fluconazole Itraconazole Voriconazole Posaconazole Isavuconazole

Aspergillus spp1-4

Fusarium spp.1,7

Chromoblastomycosis1,8

Phaeohyphomycoses1,9,10

Scedosporium apiospermum1,11,12

Scedosporium prolificans1,11,12

Mucorales1,13

Number of

isolates

MIC50 VALUES

Voriconazole Posaconazole Isavuconazole AMB

Lichtheimia corymbiferaa 12 16 0.125 1 ≤0.03

Rhizopus oryzaea 6 8 0.5 1 0.25

Rhizopus microsporusa 26 8 0.5 1 0.125

Rhizomucor pusillusa 9 8 0.06 0.5 ≤0.03

Lichtheimia ramosaa 5 8 ≤0.03 0.25 ≤0.03

Mucor circinelloides G-Ia 5 >16 0.5 8 ≤0.03

Mucor circinelloides G-IIa 9 >16 2 8 0.06

Ability to grow under varying conditions

Production of several virulence factors

(proteases, subtilase)

Ability to accelerated synthesis and remodeling

of membrane and cell wall –

Assimilative capacity of the iron from Hb’ host

Angio-invasive growth in humans

Adaptability in hostile conditions

Resistance to systemic antifungal therapies

Factors influencing the course of ZygomycosisPagano et al, Haematologica 2004

Univariate analysis

Age

Sex (M vs F)

Hematological disease

Steroids use

Hematological disease phase

PMN recovery (yes vs no)

Infection site (lung vs other)

AmB compounds vs other

L-AmB vs other treatments

P-value

ns

0.033

ns

ns

ns

0.01

ns

0.01

0.001

At a multivariate analysis only the treatment with L-AmB[RR=0.5 (CI 0.3-0.8; p=0.0001)] is significant

How is the better dose of L-AmB ?

3 mg/kg ? (Pagano et al, Haematologica 2004)

3 → 5 mg/kg ? (Petrikkos et al, Eur J Clin Micr Infect Dis 2003)

7.5 →15 mg/kg ? (Walsh et al, AAC 2001)

Probably 6-7 mg/kg must be considered the better

dosage

Lanternier et al. JAC 2015

Fail to demonstrate a superior efficacy !!

Delaying > 6 days AMB-based therapy was

associated with a doubling in mortality

≤6, n=35

>6, n=35

48,6%82,9%

0%

20%

40%

60%

80%

100%

% m

ort

ality

at 12

weeks p=0.029, ≤6 days vs. >6 days

Chamilos et al. Clin Infect Dis 2008

0%

20%

40%

60%

80%

100%

0 5 10 15 20 25 30 35 40 45

Days from symptoms to initiation of appropriate therapy

Mort

ality

at 12 w

eeks

CART identified breakpoint

Posaconazole in the treatment of

proven/probable Mucormycosis

Van Burik et al, CID 2006Greenberg et al, AAC 2006

Patients: 24 Recovered: 19

79%

All refractory or resistant

11 rhino-CNS infection, 4 pneumonia

POS 800 mg/d (200 mg x4 o 400 mg x2)

Duration therapy ranged from 8 to 1004 d

Patients: 91 Recovered: 55

60%

All refractory or resistant

22 rhino-CNS infection, 37 pneumonia

POS 800 mg/d (200 mg x4 o 400 mg x2)

Duration therapy ranged from 6 to 1005 d

Two separate, but overlapping series of patients receiving posaconazole within compassionate use

protocols of the manufacturer have been published

Vehreschild et al, CMI 2011

Different underlying disease Different phase of treatment Frequently combinations (!?)

Mucormycosis Infection Only

N = 38

MucormycosisN = 46

DRC Assessed Proven / Probable

N = 37

IntolerantN = 5

RefractoryN = 11

PrimaryN = 21

Marty et al, Lancet Infect Dis 2016

Overall Response for Mucormycosis

Overall Response at EOT

Primary N = 21

%

RefractoryN = 11

%

IntolerantN = 5

%

Total N = 37

%

Success 31.6 36.4 20.0 31.4

Complete 15.8 18.2 0 14.3

Partial 15.8 18.2 20.0 17.1

Failure 68.4 63.6 80.0 68.6

Stable 31.6 18.2 40.0 28.6

Progression 36.8 45.5 40.0 40.0

WHY?

WHAT?

The mortality rate of mucormycosis with

monotherapy remains unacceptable

The proposed strategy should have shown:

1) Markedly improved survival in relevant animal

models of IM.

2) Available retrospective or observational clinical

data, concordant with preclinical models

3) Involvement of agents already approved for the

use in humans

Echinocandins:

Caspofungin

After the primary damage due to the others drug, penetrates into

the cell and block the glucan synthesis

Azoles:

Itraconazole

Posaconazole

Voriconazole

Antagonist of

ergosterol biosynthesis

Polyenes:

Amphotericin B

Lipid AmB

Nystatin

Act on cell membrane

integrity

Combination Therapy in mucormycosis:

Mechanism of interaction

12 Year Retrospective Review (37cases)

Rhino-orbito-cerebral zygomycosis

Monotherapy with AmB formulation (31 patients) versus a combination of caspofungin

and ABLC or L-AmB (6 patients)

Pts receiving COMBO had

a significantly higher

response rate and survival

LIMITS: LOW NUMBER

most of pts had diabetes

rhinocerebral forms only

all had surgery

Combination AmB-Echinocandin in CNS Mucormycosis Reed et al. Clin Infect Dis 2008

Skiada et al, CMI 2011

34%

32 patients, 8 of them allo-HSCT (25%), not responsive to

monotherapy and with an advanced disease

Pagano et al, Haematologica 2013

Lipid AMB + Posaconazole

ALGORITMO NEL PAZIENTE EMATOLOGICO

Pagano et al., Br J Haematol 2009; 146(6):597-606

Surgery and Mucormycosis

Surgery YesDeath/treated

NoDeath/treated

Morrison et al,

BMT 19931/3 9/10

Tedder et al,

Leuk & Lymph 19944/36 38/56

Pagano et al,

Br J Haematol 19970/5 17/21

Petrikkos et al, Eur J Micr Infect Dis 2003 6/17 7/7

Sims et al, Arch Med Res 2006 2/12 2/4

Total 13/73

(17%)

73/98

(74%)

Mortality in ECMM Study230 cases

Surgical treatment related to decreased mortality (Odds

ratio 0.21, 95% CI 0.09-0.48, p=0.001)

Skiada et al, CMI 2011

ALGORITMO NEL PAZIENTE EMATOLOGICO

Pagano et al., Br J Haematol 2009; 146(6):597-606

Efficacy of growth factors or granulocyte

transfusion in MucormycosesDeaths Improved p-value

Pagano et al, Br J

Haematol 1997

7 2 n.s.

Kontoyiannis et al,

Clin Infect Dis 2000

5 7 n.s.

Roden et al *

CID 2005

3 15 n.r.

Pagano et al,

Haematologica

2004

9 9 n.s.

Kara et al,

Int J Clin Pract

2007

2 3 n.s.

ECMM-Italy 2008 4 4 n.s.

Deaths Improved

Kontoyiannis et al, *

Clin Infect Dis 2000

4 4

Gleisser et al, §Leuk & Lymph 2004

0 1

Mousset et al,**

Ann Hematol 2005

2 4

Roden et al, §CID 2005

5 2

Total 11 11

GTXG-CSF Failure !!

Saoulidis et al, CMI 2014

Lovastatin is able to induce apoptosis-like death of Mucor

racemosus (Roze et al Fungal Genet Biol 1998;25:119-33.)

Control + statin and D-eritro-sfingosin

Statins showed both in vitro and in vivo activity against

zygomycetes (Lukacs et al, J Clin Microbiol 2004;42:5400-2; Chamilos et al, Antimicrob

Agents Chemother 2006; 50:96-103 )

ALGORITMO NEL PAZIENTE EMATOLOGICO

Pagano et al., Br J Haematol 2009; 146(6):597-606

Hyperbaric Oxygen Therapy

Chamilos & Kontojiannis Clin Microbiol Infect 2005

N°28

Mean age 31 (0.5-74)

Underlying diseases

•Diabetes

•Trauma

•Others

17

5

6

Principal site: CNS 21

Post operative 23/25 (92%)

Median sesssion 22 (2-85)

Overall Survival 86%

Better outcome

24 sessions Vs. 6

p= 0.009

Cut-off 9 sessions p=0.003

Proposed mechanisms of iron uptake by Mucorales

Risk

factors

Site of infection Antifungal agents outcome

Diabetes

SOT

Rhino-orbital-cerebral, lung, sinus, gastric

Lipid-AmB+ posaconazole

Lipid-AmB+echinocandin

monotherapy

5/8 cured

8 patients treated with combination therapy including

DFX at the dose of 15-20 mg/Kg

11 pts in L-AmB + deferasirox Vs. 9 L-AmB + placebo

Better results in diabetic patients

characteristics L-AmB+DFX

L-AmB +placebo

No. patients 11 9

Active malignancy 7 (74%) 3 (33%)

Concomitantantifungal Tx

6 (55%) 7 (78%)

2009