Emergence of Echinocandin Resistance in a Patient with Chronic Pulmonary

AspergillosisCristina Jiménez-Ortigosa1, Caroline Moore2, David W. Denning3 and David S. Perlin1

1Public Health Research Institute-New Jersey Medical School, Newark, NJ, USA2Mycology Reference Centre, Manchester, University Hospital of South Manchester, Manchester UK

3University of Manchester, Manchester, UK

POSTER 5647 / ePOSTER P1754

ABSTRACT RESULTS

Background: Echinocandin drugs inhibit the β-(1,3)-glucan synthase (GS), which is responsible for the synthesis of β-(1,3)-glucan, an essential

component of the fungal cell wall. The echinocandin micafungin exhibits high antifungal activity against Aspergillus spp. in vitro and is effective in clinical

studies against aspergillosis. Micafungin is expected to increase the efficacy rate of treatment in patients with severe aspergillosis when used in

combination with another antifungal drugs such as azoles. Mutations in FKS genes encoding GS enzymes have been linked to echinocandin resistance

(ER) in Candida spp. To date, no ER A. fumigatus isolates showing a characteristic fks mutation have been recovered from patients after exposure to an

echinocandin. The aim of this study was to analyse strains isolated from a patient with an aspergilloma and chronic pulmonary aspergillosis who first

failed azole therapy and then was treated with micafungin. We assessed the presence of an fks1 mutation and its affect on inhibition of GS by different

echinocandin drugs.

Material/methods: Susceptibility testing was performed on twelve A. fumigatus isolates according to the recommendations of CLSI document M38-A2

for two echinocandins drugs (micafungin and caspofungin). fks1 gene was amplified and sequenced for the clinical isolates that showed high MECs for

the echinocandins tested. GS was isolated by product entrapment and activity was assessed by glucan polymerization assay. IC50 values for GS

inhibition were determined using a sigmoidal response curve. The 66-year-old female patient has had a right upper lobe aspergilloma for many years

and presented with life-threatening haemoptysis. She initially responded well to a sequence of triazoles, but then developed pan-azole resistance and

further haemoptysis. She then received 2 years of 6x weekly micafungin 150 mg daily, with terbinafine 500 mg. Additional symptoms heralded failure of

micafungin.

Results: Out of the twelve isolates analysed, only one (24053B) showed an epidemiological cut off value (ECV) ≥ 0.5 µg/ml for echinocandins. The

MECs of this isolate for micafungin and caspofungin were 2 µg/ml, between 16.6-66.6-fold higher MEC than the sensitive strain (ATCC13073). The fks1

gene was fully sequenced and a mutation leading to a F675S amino acid substitution was found in the hot spot 1. β-(1,3)-glucan synthase (GS)

complexes were isolated from the isolate 24053B and the inhibition kinetic value (50% inhibitory concentration [IC50]) was obtained to confirm the ER

phenotype. GS complexes isolated from the isolate 24053B yielded higher IC50 (>10000-fold) for all echinocandins tested relative to wild-type GS

complexes.

Conclusions: The Fks1 point mutation F675S of the A. fumigatus isolate 24053B yielded a β-(1,3)-glucan synthase enzyme with highly reduced

sensitivity to echinocandin drugs resulting in elevated MECs. To date, this is the first reported case of echinocandin resistance due to a point mutation in

the fks1 gene in an A. fumigatus clinical isolate.

INTRODUCTION

Invasive aspergillosis (IA) is a life-threatening mycosis mainly caused by the pathogenic fungus Aspergillus fumigatus. Triazole antifungal drugs

represent first-line therapy for all forms of aspergillosis. Voriconazole is used as the primary azole for treatment of IA, while itraconazole is the choice to

treat chronic pulmonary aspergillosis and posaconazole is more often used for prophylaxis (1, 2). Even though acquired resistance during therapy is

rare, epidemiological studies have shown that the number of clinical isolates showing an azole-resistant phenotype have increased over the past decade

in patients with prolonged exposure to azoles (3). Alterations in the cyp51A gene leading to amino acid substitutions in the target enzyme 14-α lanosterol

demethylase are the primary cause for azole resistance (1, 4, 5) although non-cyp51A-mediated resistance has been recently reported (6).

Micafungin (MCF) is a member of the echinocandin class of antifungal agents (alongside with caspofungin and anidulafungin) that targets the fungal cell

wall by the inhibition of the β-(1,3)-glucan synthase, an enzyme unique to fungi and responsible for the synthesis of β-(1,3)-glucan. MCF has

demonstrated in vitro and in vivo activity against A. fumigatus and shown synergistic activity when used in combination therapy with azoles

(isavuconazole, itraconazole, voriconazole) or with amphotericin B (7, 8). Echinocandin resistance has been linked to mutations in the FKS genes –

which encode the β-(1,3)-glucan synthase enzymes- in Candida spp (9-11). In A. fumigatus an in vitro engineeried point mutation in the fks1 gene

showed to be sufficient to confer resistance to echinocandin drugs (12).

To date, no echinocandin resistant A. fumigatus isolates showing a characteristic fks mutation have been recovered from patients after exposure to an

echinocandin. In this study, we describe the first documented case of echinocandin resistance in A. fumigatus due to a point mutation in the hot spot 1 of

the fks1 gene in a female 66-year-old patient with an aspergilloma and chronic pulmonary aspergillosis that failed azole therapy.

Antifungal susceptibility testing was performed in triplicate in accordance with the guidelines described in CLSI document M38-A2 (13). The drugs

used were isavuconazole (ISA) (Astellas Pharma USA, Inc., Northbrook, IL), itraconazole (ITR) (Sigma-Aldrich, St. Louis, MO), voriconazole (VRC)

(Pfizer Inc., New York, NY), posaconazole (POS) (Merck Sharp & Dohme Corp., Rahway, NJ), amphotericin B (AMB) (Sigma-Aldrich, St. Louis, MO),

caspofungin (CSF) (Merck Sharp & Dohme Corp., Rahway, NJ), micafungin (MCF) (Astellas Pharma USA, Inc., Northbrook, IL) and terbinafine (TERB)

(Novartis Pharmaceuticals, East Hanover, NJ) and were obtained as standard powders from their manufacturers. All drugs were dissolved in 100%

dimethyl sulfoxide (DMSO) with the exception of CSF and MCF that were dissolved in water. Stock solutions of the drugs were kept at −86°C. Microtiter

plates were read visually and the MIC was determined using 100% inhibition of growth at 48h for azoles, AMB and TERB. In the case of the

echinocandins (CSF and MCF) plates were read under the microscope after 24h of growth to determine the MEC [C. krusei ATCC 6258 and C.

parapsilosis ATCC 22019 were used as quality control strains through all experiments].

fks1 and cyp51A sequencing. DNA from spores was extracted following the protocol published by Hervás-Aguilar et al, 2007 (14). PCR and

sequencing primers were designed based on the sequences of the genes Affks1 (GenBank accession no. U79728.1), Afcyp51A (GenBank accession

no. JX283445.1) and Aflvcyp51A (Gene ID: 7913518) and are listed in Table 1. PCR amplifications were carried out in a BIORAD iCycler (Biorad) in a

50 μl reaction volume containing 50 ng of genomic DNA, 0.2 μM of each primer and 25 μl of EmeraldAmp Master MIX (Takara Bio Inc). PCR products

were purified by QIAquick PCR Purification Kit (Qiagen). Automated fluorescent sequencing was performed in both 5’ and 3’ by Genewiz NJ lab (South

Plainfield, NJ). Sequences were assembled and edited using SeqMan II and EditSeq software packages (Lasergene 13.0; DNAStar, Inc., Madison, WI).

Table 1. Oligonucleotides used for fks1 and cyp51A PCR and sequencing.

GS isolation and assay. 1,3-β-D-glucan synthase (GS) complexes were isolated from the ATCC13073 and MD24053B strains. Cell growth and

disruption, membrane protein extraction, partial GS purification by product entrapment, and polymerization assays were performed as described

previously (10, 11). Inhibition curves and 50% inhibitory concentrations (IC50s) were determined using a sigmoidal response (variable-slope) curve and a

two-site competition fitting algorithm with GraphPad Prism software, version 4.0 (10).

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METHODS

CASE REPORT

A 66-year-old life-long non-smoker female patient with an aspergilloma and chronic pulmonary

aspergillosis, diagnosed after coughing up blood who later developed azole resistance on

voriconazole. The patient (MD) complained of weight loss, fatigue and severe breathlessness. She

had suffered recurrent chest infections for many years. MD had severe kyphoscoliosis as a child

with insertion of spinal rods in early adulthood. She first presented in 2001 with an irritating cough

and several treatments with antibiotics that failed to alleviate it. After 2 years, the cough worsened

and the patient developed a fever. MD then coughed up large amounts of blood (haemoptysis)

and had a very severe bleed which was treated with embolization and oral tranexamic acid. MD

continued to cough and produced green sputum and lose weight. Aspergillus precipitin titre was

high and the CT scan demonstrated chronic cavitary pulmonary aspergillosis with fungal ball

(aspergilloma). She was started on itraconazole in 2005 but failed to respond despite satisfactory

blood drug levels, and was switched to voriconazole in 2006. Considerable improvement was seen

initially and the patient gained some weight. Voriconazole therapy continued for 2 years. However,

the Aspergillus titre remained high and the cough continued. Further tests showed the trough

plasma levels of voriconazole to be more than 0.5 mg/L, however Aspergillus fumigatus isolates

recovered were resistant to itraconazole, voriconazole and posaconazole. Thus, a portocath was

inserted for IV amphotericin B but it was felt that her Aspergillus was probably amphotericin B-

resistant as she continued to deteriorate, which was supported by in vitro data. In 2010, the patient

started micafungin IV 150mg 6x weekly with oral terbinafine daily. She improved substantially and

continued to take micafungin, with terbinafine to minimise the risk of resistance. She remained on

this combination, until she developed more haemoptysis and a micafungin resistant A. fumigatus

was grown and therapy was discontinued in 2013. She remained off therapy for over 2 years.

Haemoptysis recurred and she was trialed on isavuconazole (2015/6).

Effect of Fks1 F675S substitution on 1,3-

β-D-glucan synthase inhibition. Product-

entrapped 1,3-β-D-glucan synthase (GS)

complexes were extracted from a prototype

wild type strain (ATCC13073) and the

MD24053B clinical isolate (Fig 3A), and the

echinocandin inhibition parameter IC50 (half

maximal inhibitory concentration) was

determined. The fks1 mutant enzyme

showed significantly higher IC50 values than

corresponding enzyme isolated from the

wild-type (>106-fold change for both CSF

and MCF) (Fig. 3B).

REFERENCES

DNA sequencing of the drug target gene fks1 was performed on the isolate MD24053B. A

point mutation in the hot-spot 1 (HS1) at nucleotide position 2072 (T to C), leading to an

F675S amino acid substitution, was found. The HS1 region of fks1 gene correspond to

nt2071-3003 (aa675-684) of the published A. fumigatus Af293 fks1 gene (Afu6g12400).

The equivalent mutation in the highly conserved HS1 region of Fksp is well known to

confer echinocandin resistance in Candida albicans (F641S) and other Candida spp. and

has been linked with echinocandin clinical failure (9-11) (Fig. 2 and Table 3).

Fig. 2. Sequencing of the A. fumigatus Hot Spot 1 (HS1) region of the fks1 gene. The HS1 sequence

is shown in black and the mutation found in the clinical isolate MD24053B is shown in a purple box

(TTC x TCC in the first codon of the HS1 sequence). The wild-type strain ATCC13073 is also shown.Fig. 3. A. Strains used in the assay grown for 2 days at 37º C on PDA plates. B. Echinocandin inhibition profiles for

product-entrapped 1,3-β-D-glucan synthase enzyme complexes assessed by the incorporation of [3H]-glucose into

radiolabeled product and using a sigmoidal-response (variable-slope) curve. Echinocandin inhibition kinetics yielding 50%

inhibitory concentrations (IC50s) were obtained and are expressed in nanograms per milliliter. Titration curves for CSF and

MCF for wild-type (ATCC13073) and clinical isolate MD24053B of A. fumigatus are shown.

9. Garcia-Effron G, Katiyar SK, Park S, Edlind TD, Perlin DS. 2008. A naturally occurring proline-to-alanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis,

and Candida metapsilosis accounts for reduced echinocandin susceptibility. Antimicrob. Agents Chemother. 52:2305–2312.

10. Garcia-Effron G, Lee S, Park S, Cleary JD, Perlin DS. 2009. Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-beta-d-glucan

synthase: implication for the existing susceptibility breakpoint. Antimicrob. Agents Chemother. 53:3690–3699.

11. Garcia-Effron G, Park S, Perlin DS. 2009. Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive

breakpoints. Antimicrob. Agents Chemother. 53:112–122

12. Rocha EM, Garcia-Effron G, Park S, Perlin DS. 2007. A Ser678Pro substitution in Fks1p confers resistance to echinocandin drugs in Aspergillus fumigatus. Antimicrob. Agents

Chemother 51(11):4174-6.

13. National Committee for Clinical Laboratory Standards. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard.

Document M38-A2, Vol 28, No 16. National Committee for Clinical Laboratory Standards, Wayne, Pa.

14. Hervás-Aguilar A, Rodríguez JM, Tilburn J, Arst HN Jr, Peñalva MA. 2007 Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus

nidulans zinc finger transcription factor PacC. J Biol Chem. 282:34735-47.15. Alanio A, Cabaret O, Sitterle E, Costa JM, Brisse S, Cordonnier C, Bretagne S. 2012. Azole preexposure affects the Aspergillus fumigatus population in patients. Antimicrob

Agents Chemother. 56:4948-50.

Twelve consecutive Aspergillus spp. isolates (11 A. fumigatus and 1 A. flavus) were recovered from sputum cultures and were submitted for susceptibility

testing against several antifungal compounds. According to the epidemiological cut-off values (ECVs), all isolates except one (MD24053B) were classified as

wild-type population for all antifungal drugs tested. Moreover, cyp51A sequencing revealed five mutations not linked to azole resistance in all A. fumigatus

isolates (15). Isolate MD24053B showed an increase in MEC values of 16.6 to 66.6-fold higher for CSF and MCF respectively, compared to the control strain

(ATCC13073, data not shown) and the other 11 isolates collected from the patient (Table 2). Isolate MD24053B showed a diminished growth rate in PDA

plates compared to the other ten A. fumigatus isolates recovered from the patient and lacked the typical dark green color of the colonies formed by A.

fumigatus (Fig. 1B.).

Table 2. MIC/MEC distributions of the antifungal drugs tested in the study for the Aspergillus spp. clinical isolates.

# Oligo Name Sequence 5'→3' Purpose # Oligo Name Sequence 5'→3' Purpose

1 AfFKS1 -751F CCTGAGTTGGTGGTCAAT 13 AfCyp51A F -991 CGTCGATCTGTGTGACAC

2 AfFKS1 R6378 GACTGGCGAAACACGTTG 14 AfCyp51A R 1993 CTAGAAGGAGCAGGACTG

3 AfFKS1F -211 CTGCGACTCGAGATTCAG 15 AfCyp51A F -819 CATGCTGGGAGGAATCTC

4 AfFKS1F +697 GCATGCGCAACATGTATG 16 AfCyp51A F -147 GCTGGTCTCTCATTCGTC

5 AfFKS1F +1562 CGCACAATCGCTTTACAC 17 AfCyp51A F 502 AGAGTCTCATGTGCCACT

6 AfFKS1 1947F CGTCAGTATGTGGCTAGC 18 AfCyp51A F 1151 CACTCCTCTATTCACTCTATC

7 AfFKS1F +2405 GATTTCTCAAGTTTGGAATGC 19 AflvCyp51A_F-511 CAGACTTCGAGGCTTTGG

8 AfFKS1 3043F CGATCAAGCTCCTGTACC 20 AflvCyp51A_R1985 TGGTATTGACGCAGGTAC

9 AfFKS1F +3401 GTCTGACAACCAGAATCAC 21 AflvCyp51A_F-286 CGATGCCAAGTACTCCAA

10 AfFKS1F +4269 GTCCAGGAACTGACAGAG 22 AflvCyp51A_F240 CCACAATGTTGTACGGAG 

11 AfFKS1F +5117 CGAAGTCATGTTCTTCCTTG 23 AflvCyp51A_F886 CTACAAGAATGGACAGCC 

12 AfFKS1R +5931 CTTCGAGGCGCTGGATAC 24 AflvCyp51A_R1811 GGTCATCATACTGTGGAC

Affks1 PCR

amplification

Affks1

sequencing

Afcyp51A PCR

amplification

Afcyp51A

sequencing

A. flavus cyp51A

PCR amplification

A. flavus cyp51A

sequencing

Table 3. fks1 sequencing of the A. fumigatus MD24053B isolate.

CONCLUSION

The Fks1 point mutation F675S of the A. fumigatus isolate 24053B yielded a β-(1,3)-glucan synthase enzyme with highly reduced sensitivity to echinocandin drugs

resulting in elevated MECs, and echinocandin clinical failure. To date, this is the first reported case of echinocandin resistance due to a point mutation in the fks1 gene in

an A. fumigatus clinical isolate.

# Lab no. Date received ID Specimen site ISA ITR POS VOR CSF MCF AMB TERB Cyp51A* Fks1*

1 62194 12/24/2015 Aspergillus fumigatus complex sputum 0.25 0.5 0.25 0.12 0.12 0.03 2 0.5 Y46F, V172M, T248N, E255D, K427E ND$

2 53619 1/28/2015 Aspergillus fumigatus complex sputum 0.25 0.5 0.25 0.25 0.12 0.03 2 0.5 Y46F, V172M, T248N, E255D, K427E ND

3 45755 12/30/2013 Aspergillus fumigatus complex sputum 2 1 1 0.5 0.12 0.03 2 1 Y46F, V172M, T248N, E255D, K427E ND

4 33460 4/10/2012 Aspergillus fumigatus complex sputum 0.25 0.5 0.25 0.12 0.12 0.03 2 1 Y46F, V172M, T248N, E255D, K427E ND

5 30906 10/25/2011 Aspergillus fumigatus complex sputum 0.25 0.5 0.25 0.12 0.12 0.03 2 1 Y46F, V172M, T248N, E255D, K427E ND

6 29576A 7/27/2011 Aspergillus flavus sputum 0.25 0.5 0.25 0.25 0.12 0.03 2 0.06 WT# ND

7 29576B 7/27/2011 Aspergillus fumigatus complex sputum 0.25 0.25 0.25 0.25 0.12 0.03 2 0.5 Y46F, V172M, T248N, E255D, K427E ND

8 24555 7/28/2010 Aspergillus fumigatus complex sputum 0.25 0.25 0.25 0.25 0.12 0.03 2 0.5 Y46F, V172M, T248N, E255D, K427E ND

9 24053A 6/15/2010 Aspergillus fumigatus complex sputum 0.25 0.25 0.25 0.25 0.12 0.03 2 1 Y46F, V172M, T248N, E255D, K427E ND

10 24053B 6/15/2010 Aspergillus fumigatus complex sputum 0.5 0.25 0.25 1 2 2 2 1 Y46F, V172M, T248N, E255D, K427E F675S

11 23525 5/4/2010 Aspergillus fumigatus complex sputum 0.25 0.25 0.12 0.12 0.12 0.03 2 0.5 Y46F, V172M, T248N, E255D, K427E ND

12 22432 2/9/2010 Aspergillus fumigatus complex sputum 0.25 0.25 0.12 0.25 0.12 0.03 2 1 Y46F, V172M, T248N, E255D, K427E ND

*Ref strain used Af293$ND = not determined #Ref strain used A. flavus NRRL3357

MIC/MEC (mg/L)

A.

A. B.

Contact information: Cristina Jimenez-Ortigosa Ph.D. E-mail: jimenecr@njms.rutgers.edu

Strain nt aa

Af293 (Ref) TTCCTGACCCTGTCTTTCAAGGATCCGATCCG FLTLSFKDPI

ATCC13073 TTCCTGACCCTGTCTTTCAAGGATCCGATCCG FLTLSFKDPI

MD24053B TCCCTGACCCTGTCTTTCAAGGATCCGATCCG SLTLSFKDPI

fks1 HS1

Fig. 1. A. Antifungal therapy of the patient. Collection of the Aspergillus spp. isolates for the current microbiological

study is shown (triangles). B. Isolates recovered from the patient grown for 2 days at 37º C on a PDA plate.

B.