Clinical Infectious Diseases

MAJOR ARTICLE

Clinical Impact of Polymerase Chain Reaction–Based

Aspergillus and Azole Resistance Detection in Invasive
Aspergillosis: A Prospective Multicenter Study
Sammy Huygens,1,a, Albert Dunbar,1,a Jochem B. Buil,2 Corné H. W. Klaassen,3 Paul E. Verweij,2 Karin van Dijk,4 Nick de Jonge,5 Jeroen J. W. M. Janssen,5
Walter J. F. M. van der Velden,6 Bart J. Biemond,7 Aldert Bart,8 Anke H. W. Bruns,9 Pieter-Jan A. Haas,10 Astrid M. P. Demandt,11 Guy Oudhuis,12

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Peter von dem Borne,13 Martha T. van der Beek,14 Saskia K. Klein,15,16 Peggy Godschalk,17 Lambert F. R. Span,16 Douwe F. Postma,18 Greetje A. Kampinga,19
Johan Maertens,20,21 Katrien Lagrou,21,22 Toine Mercier,20,21 Ine Moors,23 Jerina Boelens,24 Dominik Selleslag,25 Marijke Reynders,26 Willemien Zandijk,3
Jeanette K. Doorduijn,27 Jan J. Cornelissen,27 Alexander F. A. D. Schauwvlieghe,25 and Bart J. A. Rijnders1,
1
Department of Internal Medicine, Section of Infectious Diseases and Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center, Rotterdam, The
Netherlands; 2Department of Medical Microbiology, Radboud University Center, Nijmegen, The Netherlands; 3Department of Medical Microbiology & Infectious Diseases, Erasmus MC, University
Medical Center, Rotterdam, The Netherlands; 4Department of Medical Microbiology, Amsterdam University Medical Centers, Amsterdam, The Netherlands; 5Department of Hematology, Amsterdam
University Medical Centers, Amsterdam, The Netherlands; 6Department of Hematology, Radboud University Center, Nijmegen, The Netherlands; 7Department of Hematology, Amsterdam University
Medical Centers, Amsterdam, The Netherlands; 8Department of Medical Microbiology, Amsterdam University Medical Centers, Amsterdam, The Netherlands; 9Department of Internal Medicine,
Infectious Diseases, University Medical Center Utrecht, The Netherlands; 10Department of Medical Microbiology, University Medical Center Utrecht, The Netherlands; 11Department of Hematology,
Maastricht University Medical Center, The Netherlands; 12Department of Medical Microbiology, Maastricht University Medical Center, The Netherlands; 13Department of Medical Microbiology,
Leiden University Medical Center, The Netherlands; 14Department of Hematology, Leiden University Medical Center, The Netherlands; 15Department of Hematology, Meander Medical Center,
Amersfoort, The Netherlands; 16Department of Hematology, University Medical Center Groningen, The Netherlands; 17Department of Medical Microbiology, Meander Medical Center, Amersfoort,
The Netherlands; 18Department of Internal Medicine and Infectious Diseases, University Medical Center Groningen, The Netherlands; 19Department of Medical Microbiology, University of Groningen,
University Medical Center Groningen, The Netherlands; 20Department of Hematology, University Hospitals Leuven, Leuven, Belgium; 21Department of Microbiology, Immunology and Transplantation,
KU Leuven, Leuven, Belgium; 22Department of Laboratory Medicine and National Reference Centre for Mycosis, University Hospitals Leuven, Leuven, Belgium; 23Department of Hematology, Ghent
University Hospital, Ghent, Belgium; 24Department of Medical Microbiology, Ghent University Hospital, Ghent, Belgium; 25Department of Hematology, AZ St-Jan Brugge-Oostende Hospital, Bruges,
Belgium; 26Department of Laboratory Medicine, Medical Microbiology, AZ St-Jan Brugge-Oostende Hospital, Bruges, Belgium; and 27Department of Hematology, Erasmus University Medical Center,
Rotterdam, The Netherlands

Background. Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time
resistance detection will result in earlier initiation of appropriate therapy.
Methods. In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in
hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole
resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid
(BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA.
Results. Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable
IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in
116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in
8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed
in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83).
Conclusions. Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the
clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR
criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).
Keywords. invasive aspergillosis; azole resistance; Aspergillus PCR; clinical impact.

Received 14 November 2022; editorial decision 06 March 2023; published online 11 March Invasive aspergillosis (IA) is the most common mold infection
2023 in immunocompromised patients and is associated with sig­
a
S. H. and A. D. contributed equally to this work.
nificant morbidity and mortality. Over 15 years, azoles have
Correspondence: B. J. A. Rijnders, Department of Internal Medicine, Section of Infectious
Diseases and Department of Medical Microbiology and Infectious Diseases, Erasmus MC, been used as first-line therapy [1, 2]. Azole resistance in
University Medical Center, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands (b. Aspergillus fumigatus is increasingly reported [3]. It is mostly
rijnders@erasmusmc.nl).
caused by resistance-associated mutations (RAMs) in the
Clinical Infectious Diseases® 2023;77(1):38–45
© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases cyp51A gene encoding for the target enzyme of azoles. We
Society of America. previously noted that 6 weeks after diagnosis, the mortality
This is an Open Access article distributed under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), of culture-positive voriconazole-resistant IA was 21% higher
which permits non-commercial reproduction and distribution of the work, in any medium, pro­ compared with voriconazole-susceptible IA [4]. A delay in ap­
vided the original work is not altered or transformed in any way, and that the work is properly
cited. For commercial re-use, please contact journals.permissions@oup.com
propriate antifungal therapy was associated with 23% higher
https://doi.org/10.1093/cid/ciad141 mortality compared with patients who received appropriate

38 • CID 2023:77 (1 July) • Huygens et al

antifungal therapy immediately and median time to switch Netherlands) to screen for possible azole resistance, with con­
to appropriate antifungal therapy was 10 days [4]. This delay firmation at the reference center.
is mainly caused by slow growth of fungal cultures, transpor­
tation time to a mycology reference laboratory, and suscept­ Primary and Secondary End Points
ibility testing on that isolate. The resistance polymerase The primary end point was the proportion of patients with
chain reaction (PCR) assay could result in a faster and probable IA by an azole-resistant A. fumigatus in whom an­
more sensitive diagnosis of azole resistance, enabling early tifungal treatment failure, defined as death or switch to anti­
appropriate antifungal therapy and reducing the need for fungal agent from another class after at least 5 days of
upfront combination therapy [5]. A PCR assay also enables first-line therapy, was observed in the 6 weeks following
azole-resistance detection in culture-negative samples. diagnosis.
AsperGenius is a multiplex, real-time, PCR assay that allows Secondary end points were overall prevalence of azole resis­

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for simultaneous detection of A. fumigatus and other tance and outcome of patients with an isolated, positive
Aspergillus species as well as mutations in the A. fumigatus Aspergillus PCR on BALf. For this, the 6-week overall mortality
cyp51A gene, which confers resistance to azoles [6]. This in patients with a negative test result for all 3 mycological tests
PCR test can be implemented in any molecular diagnostics (culture/GM/PCR) was compared with mortality in patients
laboratory without the need for specific expertise in mycol­ with an isolated positive PCR (single as well as duplicate posi­
ogy. In the Azole Resistance Management study, we evaluat­ tive PCR). Furthermore, in a post hoc analysis, we evaluated the
ed the clinical value of this PCR assay in patients suspected of influence of low versus high fungal loads based on cycle thresh­
having IA. old (Ct) values of the PCR.

Statistical Analyses
METHODS
For the primary end point, the incidence of treatment failure
Study Design and Population was compared with a fixed hypothetical 75% incidence that
A prospective multicenter study was performed in 12 centers in can be expected when patients with an azole-resistant A. fumi­
the Netherlands and Belgium. The study was approved by the gatus are treated with azoles and switch to a non-azole therapy
institutional review boards at all sites, and patients provided when treatment failure is clinically diagnosed [9]. Patients
written informed consent. with IA in whom a mixed azole-susceptible/azole-resistant
We included adult patients with a hematological malignancy Aspergillus infection was demonstrated were excluded because
and a new pulmonary infiltrate on a computed tomography it could not be excluded that only the azole-susceptible strain
(CT) scan for which bronchoalveolar fluid (BALf) sampling was causing IA while the resistant strain was a colonizer.
was planned or performed within 48 hours. Invasive fungal dis­ Because the use of real-time detection of azole resistance in pa­
ease (IFD) was classified according to the updated Consensus tients allows for a proactive change from the first-line therapy
Definitions of Invasive Fungal Disease From the European with voriconazole to other agents as soon as resistance is detect­
Organization for Research and Treatment of Cancer and the ed, a lower incidence of treatment failure can be expected. The
Mycoses Study group Education and Research Consortium goal of the study therefore was to demonstrate that this
(2020 EORTC/MSGERC) (Supplementary Methods 1) [7]. A PCR-based approach reduces the incidence of treatment failure
diagnostic and therapeutic protocol was agreed on by those compared with the presumed 75%. We anticipated that failure
at the study sites. This incorporated the AsperGenius PCR on would be reduced to 35% when the antifungal therapy was
BALf. Operational information on this PCR assay is available changed to liposomal amphotericin B (L-AmB) as soon as resis­
in the manufacturer’s instructions and Supplementary tance was documented. Using these percentages, at least 15 cas­
Methods 2. This consensus protocol also provided a guideline es of azole resistance would have to be enrolled to have 90%
on antifungal treatment (Supplementary Methods 3, power to show that treatment failure is significantly less than
Supplementary Figure 1) [8]. 75%. To compare the observed treatment failure with this
Baseline characteristics, serum and BALf galactomannan 75%, the exact Clopper–Pearson 95% confidence interval (CI)
(GM), culture results, antifungal treatment, and mortality up for the observed proportion of treatment failure was calculated,
to week 12 were registered. GM testing on serum and BALf and the P value of the observed proportion vs 75% was calcu­
with the Bio-Rad Platelia Aspergillus Ag assay and fungal cul­ lated using a general z test.
ture was performed at the study site. Phenotypic resistance test­ Secondary end points were analyzed using Mann–Whitney U
ing with the European Committee on Antimicrobial and Pearson χ2 tests as appropriate. All tests were 2-tailed with a
Susceptibility Testing (EUCAST) standard was performed at significance level of 0.05. A receiver operating
the Dutch and Belgian Mycology Reference Center. Some of characteristic (ROC) curve was generated to evaluate the dis­
the centers also used the VIPcheck (Mediaproducts BV, the criminative power of the fungal load (reported as Ct value) to

Impact of Resistance PCR in Aspergillus • CID 2023:77 (1 July) • 39

Table 1. Baseline Patient Characteristics include a wedge-shaped, lobar, or segmental infiltrate) were
present in 291, while 11 patients had atypical findings (eg,
Characteristic Total Patients, N = 323
ground glass opacities). Results of the chest CT scan in combi­
Age, median (interquartile range), y 63 (53–69) nation with complete mycological data (ie, PCR, GM, and cul­
Male sex (%) 219/320 (68)
ture result) were available for 276. Of these patients, 99 (36%)
Allogeneic stem cell transplant (%) 102/322 (32)
Autologous stem cell recipient (%) 13/322 (4)
were classified as probable IA based on the radiological findings
Underlying hematological disease (%) in combination with a positive GM, culture, or duplicate PCR
Acute myeloid leukemia 163/321 (51) test. Possible IFD was diagnosed in 169 of 276 (61%). Eight of
Myelodysplastic syndrome 40/321 (13) 276 patients showed atypical radiological findings, of whom 2
Acute lymphoblastic leukemia 20/321 (6) had a positive mycological criterion. Supplementary Data 4
Other 98/321 (30)
and Supplementary Data 6 (Supplementary Tables 1 and 2, re­

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Acute GvHD, grade II–IV, n (%) 23/321 (7)
Chronic GvHD, n (%) 19/321 (6)
spectively) provide more information on the classification of
Mild 6/321 (2) these patients and Supplementary Data S8 provides a visual
Moderate 5/321 (2) overview of all positive diagnostics (Supplementary Figure 2).
Severe 8/321 (3) When we used the previous (2008) version of the EORTC/
Use of prednisolonea (%)
MSGERC criteria in which a positive Aspergillus PCR was
<0.3 mg/kg/d 41/310 (13)
>0.3 mg/kg/d 51/310 (17)
not included, only 72 (26%) had a probable IA.
Chemotherapy in last 90 db (%) 195/273 (71)
Neutropenia,c Yes (%) 170/293 (58) Azole Resistance
Abbreviation: GvHD, graft-versus-host disease. The A. fumigatus resistance PCR was performed on the BALf of
a
Median dose of prednisolone in the 21 days preceding bronchoalveolar fluid (BALf)
the 89 patients in whom A. fumigatus DNA was detected. A
sampling.
b
Chemotherapy received in the last 3 months prior to BALf sampling. conclusive resistance PCR for both cyp51A mutation patterns
c
Neutropenia (<500/µL) on day of BALf sampling. (ie, showing wild type or a resistance marker) was obtained
in 58 of 89 (65%) patients. The resistance PCR was more often
conclusive when BALf GM was higher (44% with GM <1.0% vs
predict 6-week mortality or successful resistance testing 92% when ≥1.0). In 8 of 58 patients (14%), resistance markers
(Supplementary Data S9). Analysis was performed with SPSS were detected (Table 2); all of these patients were categorized as
version 28 (IBM, Armonk, NY) probable IA.
Aspergillus was cultured from BALf in 7.5% (24 of 323) of pa­
RESULTS tients. Aspergillus fumigatus was cultured in 21, while Aspergillus
From April 2017 to March 2021, 323 patients who underwent flavus, Aspergillus terreus, and Aspergillus niger were each cul­
bronchoscopy with BALf sampling and fulfilled the host factor tured in 1 patient. Even in patients with GM ≥1.0 on BALf, cul­
criterion of the EORTC/MSGERC definitions were enrolled. tures were positive in only 23% (17 of 74). Supplementary Data 6
Two-thirds were male (68%), and the median age was 63 years. (Supplementary Table 3) provides additional information on the
Seventy percent had an acute leukemia or myelodysplastic syn­ success rate of the resistance PCR and phenotypic resistance
drome, and 32% had received an allogeneic stem cell transplan­ testing.
tation (Table 1). As shown in Table 3, a RAM was detected in 8 patients by PCR.
Six had a positive culture for A. fumigatus. Unfortunately, pheno­
Aspergillus PCR typic resistance testing was also performed in only 4 of them.
Sufficient BALf remained for Aspergillus PCR testing for 293, Resistance was confirmed in 3 of 4. In 1 patient, the BALf culture
and this was used as the denominator for the performance of showed no phenotypic resistance; however, a sputum sample
the PCR. Overall, Aspergillus DNA was detected in BALf of gathered 14 days after inclusion showed phenotypic resistance.
116 (40%) patients and A. fumigatus DNA was detected in 89 In our cohort, there were no patients in whom phenotypic resis­
(30%). Patients were categorized in three groups according to tance was shown that could not be confirmed by the cyp51A resis­
the GM on BALf (<0.5, 0.5–0.99 and ≥1.0) and Aspergillus tance PCR. For details on treatment of these patients, refer to
DNA was more frequently detected with increasing GM Supplementary Data 5.
(Table 2). In patients with a BALf GM <1.0, Aspergillus DNA
could still be detected in 66 of 224 (29%). Primary End Point
After exclusion of patients with the mixed azole-susceptible/
EORTC/MSGERC Classification azole-resistant infection as predefined in the protocol, 6 pa­
Data on radiological findings were available for 302 patients tients with probable azole-resistant IA remained. In 1 of these
and lesions suspect for IFD (which in the 2020 criteria also 6, treatment failure was observed, and an echinocandin was

40 • CID 2023:77 (1 July) • Huygens et al

Table 2. Microbiology Results Including Bronchoalveolar Fluid Galactomannan, AsperGenius Polymerase Chain Reaction, and Culture

BALf GM
<0.5 0.5–0.99 ≥1
Number of patients (n)a 215 32 74
Aspergenius performed 193 31 68
PCR Aspergillus species–positive 50 (26%) 16 (52%) 50 (74%)
PCR Aspergillus species–negative 143 (74%) 15 (48%) 18 (26%)
PCR Aspergillus fumigatus–positive 38 (20%) 12 (39%) 39 (57%)
PCR Aspergillus fumigatus–negative 156 (80%) 19 (61%) 29 (43%)
PCR Aspergillus terreus–positive 1 (0.5%) 0 (0%) 2 (3%)

TR34/L98H PCR successfulb

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19 (50%) 8 (67%) 36 (92%)
TR46/T289A/Y121F PCR successfulb 21 (55%) 5 (42%) 36 (92%)
TR34/L98H and TR46/T289A/Y121F both WT 16 4 32
TR34/L98H and TR46/T289A/Y121F both not successful 15 3 3
TR34/L98H WT and TR46/T289A/Y121F not successful 1 4 0
TR34/L98H not successful and TR46/T289A/Y121F WT 3 1 0
TR34/L98H resistant and TR46/T289A/Y121F WT 1 0 5 (2c)
TR34/L98H WT and TR46/T298A/y121F resistant 1 0 1

Culture positive for Aspergillus species 6 1 17

Culture positive for Aspergillus fumigatus 5 0 16
Culture positive for Aspergillus niger 1 0 0
Culture positive for Aspergillus terreus 0 0 1
Culture positive for Aspergillus flavus 0 1 0
Abbreviations: BALf, bronchoalveolar fluid; GM, galactomannan; PCR, polymerase chain reaction; WT, wild type.
a
Bronchoalveolar fluid (BALf) volume is occasionally too small to perform all tests in all patients. BALf PCR was performed in 293 patients. In 1 patient, galactomannan (GM) was not available;
therefore, total number of patients in this table is 292. In this patient, Aspergillus species PCR was positive and A. fumigatus PCR was negative.
b
The number of patients in the GM subgroup for whom A. fumigatus PCR was positive was used as the denominator (39, 12, and 37 for GM <0.5, GM 0.5–0.99, and GM ≥ 1.0, respectively).
c
In 2 patients, DNA of WT A. fumigatus and the TR34/98H mutation were detected simultaneously.

added to L-AmB 42 days after the initiation of antifungal ther­ culture (Table 4, Supplementary Data 7). In our cohort, 240 pa­
apy. This patient eventually died on day 64. Compared with the tients had a negative BALf GM in combination with a negative
predefined historical treatment failure rate of 75%, the treat­ culture. Because the PCR could be performed in 216 of 240, 216
ment failure of 16.7% (95% CI, .5%–64%) we observed was sig­ was used as the denominator. Sixty-two (29%) of them had an
nificantly lower (P = .005) [9]. isolated positive Aspergillus species and/or fumigatus PCR. In
52 of 62 (84%), Aspergillus therapy was initiated in the
Secondary End Points 14 days following BALf sampling. Significantly fewer of those
Overall Prevalence of Azole Resistance with a negative PCR received antifungal therapy (105 of 154,
RAMs associated with azole resistance were found in 8 of 58 68%, P = .019). The median duration of therapy in patients
(14%) with successful resistance PCR results available and in with an isolated positive PCR was 34 days (interquartile range
8 of 293 (2.7%) in whom the PCR was performed. [IQR], 10–123), and this was 18 days (IQR, 7–63) in patients
Phenotypic resistance testing (VIPcheck and/or EUCAST with a negative PCR (P = .045).
method) was available for 18 of 21 (86%) of the culture-positive
cases; in 3, resistance to 1 or more azoles was documented (2 of Mortality According to Subgroup. Of 321 patients with available

3 voriconazole, 1 of 3 posaconazole, 3 of 3 isavuconazole) in data on mortality, 89 (28%) died within 12 weeks. The
BALf. Unfortunately, phenotypic resistance testing was not 6-week as well as the 12-week overall mortality were signifi­
performed on culture for 2 patients in whom a RAM was de­ cantly higher in patients with BALf GM ≥1.0 compared with
tected by PCR. patients with a lower BALf GM (6 weeks: 32% vs 16%,
P = .004; 12 weeks: 40% vs 24%, P = .010). In contrast, the mor­
Outcome of Patients With an Isolated Positive Aspergillus Species tality was not significantly higher in patients with a positive
PCR PCR in duplicate vs a negative PCR (6 weeks: 24% vs 19%,
Characteristics of Patients With an Isolated Positive Aspergillus Species P = .324; 12 weeks: 31% vs 27%, P = .457). Furthermore, the
PCR. We analyzed the clinical impact of a positive PCR result 6-week mortality in patients with an isolated positive PCR re­
in patients with a negative BAL GM and a negative BALf sult was comparable to that for patients who lacked any

Impact of Resistance PCR in Aspergillus • CID 2023:77 (1 July) • 41

 Table 3. Overview of Baseline Characteristics, Treatment, and Outcome on the 8 Azole-Resistant Cases

Resistance AsperGenius Time of

42 • CID 2023:77 (1 July) • Huygens et al

Age, Stem Cell Bronchoalveolar Testing on Resistance Subsequent Antifungal Therapy 6-Week 12-Week
y Sex Disease Transplant Fluid Galactomannan Culture Culture Testing Initial Therapy Therapy Switch, d Failure Mortality Mortality

66 M AML - 1.6 + Azole resistant TR46 Azole L-AmB 1 No No No

53 F Non-Hodgkin + 0.3 + Azole resistant TR34 Azole + L-AmB L-AmB 4 No No No
lymphoma
54 M Hodgkin + 4.8 + Azole resistant TR34 Azole + L-AmB L-AmBa 5 Yes on No No
lymphoma day 42
48 F AML - 5.6 - - TR34 Azole + L-AmB 2 No No Yes
echinocandin
64 F AML + 0.07 - - TR46 Azole NA No No No
57b M Mantle cell - 3.08 + Not tested Mixed pattern: Azole NA No No No
lymphoma WT and TR34
23 M T-cell acute - 8 + Azole TR34 Azole Azole + 15d No No No
lymphocytic susceptiblec L-AmB
leukemia
79 M MW - 5.6 + Not tested Mixed pattern: Azole NA No Yes Yes
WT and TR34
Abbreviations: AML, acute myeloid leukemia; azole, voriconazole or posaconazole; culture, culture of bronchoalveolar fluid; L-AmB, liposomal amphotericin B; WT, wild type; MW, morbus waldenström.
a
Treatment failure: anidulafungin was associated with L-AmB on day 42 due to lack of clinical improvement.
b
Wild-type and TR34/L98H-resistant strains both present in the bronchoalveolar fluid sample.
c
Azole resistance was proven on sputum sample 14 days after start of azole treatment.
d
In this patient, the result of the resistance test became available 14 days after start of initial antifungal therapy with voriconazole. The switch was therefore not due to clinical treatment failure but based on the resistance test which became available at that
time. This patient was not counted for treatment failure in the analysis.

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Table 4. Outcome of Patients According to the Mycological Test That Was Positive

GM- and GM- and GM-, Culture-,

Aspergillus Culture-Negative Culture-Negative and Aspergillus
Aspergillus PCR-Positive in but Aspergillus but Aspergillus Species
GM-Positivea Culture-Positivea PCR-Positivea,b Duplicatea PCRb-Positive PCR-Positive in PCR-Negative
(N = 77) (N = 24) (N = 119) (N = 67) (N = 62) Duplicate (N = 28) (N = 154)

Antifungal therapy 72/77 (94%) 23/24 (96%) 105/119 (88%) 62/67 (93%) 52/62 (84%) 24/28 (86%) 105/154 (68%)
started around
bronchoalveolar
lavage (−5, +
14 d) (n/N)
Median duration of 27 (11–73) 38 (17–88) 32 (10–89) 33 (12–89) 34 (10–123) 71 (15–135) 18 (7–63)
antifungal

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treatment (d)—
median
(interquartile
range)
6-week mortality 23/76 (30%) 8/24 (33%) 26/119 (22%) 16/67 (24%) 9/62 (15%) 4/28 (14%) 24/153 (16%)
(n/N)
Abbreviations: GM, galactomannan; PCR, polymerase chain reaction.
a
Irrespective of the other mycological tests. GM in serum and/or bronchoalveolar fluid.
b
Aspergillus species– and/or Aspergillus fumigatus PCR–positive.

mycological evidence (15% when positive vs 16% when nega­ Compared with historical data on the incidence of treatment
tive, P = .829; Table 4). The results were comparable when failure in patients with azole-resistant IA, use of PCR was asso­
we restricted the analysis to patients with an isolated duplicate ciated with a better treatment response. Indeed, treatment fail­
positive PCR (mortality 14% with duplicate positive PCR ure was observed in only 1 of 6 patients [9]. This observation
and 16% when negative, P = .851). Supplementary Data 7 should be interpreted cautiously. First, a randomized trial rath­
(Supplementary Tables 4 and 5) provides additional informa­ er than a comparison with a historical cohort would have been
tion on outcome for subgroups defined by combinations of preferred. However, the required sample size (>1000 patients)
positive diagnostic tests. was deemed unrealistic. Also, current Dutch guidelines recom­
mend combination therapy or L-AmB if real-time resistance
Relevance of the Aspergillus PCR Ct Value. In patients with an iso­ testing is not performed. Consequently, despite its clear limita­
lated positive PCR, the median Ct value was higher (36.4, IQR, tions, a prospective observational study with a historical con­
35.1–37.5) compared with patients with a positive GM or cul­ trol group was considered the only realistic design.
ture (33.8, IQR, 31.8–36.1 and 33.4, IQR, 32.6–36.4, respective­ Despite the inclusion of 323 patients, only 8 patients with
ly). In the entire patient cohort, a cutoff of 33.11 had the best probable IA by an azole-resistant A. fumigatus were identified.
Area Under the Curve (AUC) (65.9%; 95% CI, 53.4–78.3) to Since 2 of the patients had a mixed azole-susceptible/
predict 6-week mortality but with a low sensitivity (52%) and azole-resistant infection, 6 were left for the primary analysis.
moderate specificity (80%). In patients with an isolated positive Therefore, the patient group was smaller than anticipated.
PCR, there was no statistical difference in 6-week mortality Also, in contrast to what was suggested in the flow diagram
based on the cutoff of 33.11, but Ct values <33.11 were present of the protocol (Supplementary Methods 3, Supplementary
in only 6 patients (see Supplemental data S9 for the complete Ct Figure 1), 3 of 8 patients with azole-resistant IA received
value analysis). Finally, a Ct value of 34.6 predicted that the re­ L-AmB or an echinocandin rather than an azole as their initial
sistance PCR would be the most successful (AUC, 79.3%; CI, antifungal therapy before resistance had been detected. So, de­
79.8–94.3; sensitivity, 79.3%; specificity, 90%). spite the agreed-on flow diagram, some clinicians took the risk
for azole resistance into account before it was documented. The
positive outcome of these patients can therefore potentially be
DISCUSSION
explained in part by this initial treatment choice.
Patients with an azole-resistant IA are characterized by an ex­ Of all 72 patients with a probable IA in which A. fumigatus
cess mortality of greater than 20% compared with an azole- was demonstrated, the culture was positive in 20 (28%).
susceptible IA. Early switch to appropriate antifungal therapy However, focusing on the 8 patients with azole-resistant proba­
may reduce the resistance-attributable mortality [4, 10, 11]. ble IA, cultures were positive in 6 of 8 (75%). This means that in 2
We therefore determined if the use of an azole-resistance of 8 (25%) azole resistance would have been missed by culture
PCR improves the outcome of patients with IA. alone. Moreover, as phenotypic resistance testing takes time

Impact of Resistance PCR in Aspergillus • CID 2023:77 (1 July) • 43

and is frequently done at a reference laboratory, PCR-based test­ died of this infection within 6 weeks. This suggests that azoles
ing can decrease the time it takes to demonstrate azole resistance. can continue to be the initial therapy for the large majority of
Historical data on azole resistance are mostly culture based. patients in the Netherlands and Belgium, as long as real-time
Since cultures may be more frequently positive if the IA is caused resistance testing is possible and a change to appropriate anti­
by azole-resistant A. fumigatus, this may lead to an overestima­ fungal therapy is initiated promptly when resistance is detected.
tion of the actual azole resistance frequency in patients with IA. Our observations therefore are in support of the Dutch antifun­
However, the PCR-based resistance prevalence of 14% that we gal treatment guideline in which this approach is considered
observed is comparable to the prevalence based on culture re­ reasonable. This approach also has the advantage of reducing
sults described previously in the Netherlands [12]. overexposure to antifungal drugs that can have side effects
Our study provides insight into the potential value of the sys­ while only few patients will benefit.
tematic use of an Aspergillus PCR on BALf as well as the asso­ In conclusion, real-time PCR-based resistance testing may

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ciation with the quantitative GM result and Ct value of the PCR help to limit the clinical impact of triazole resistance. In con­
[13]. In 116 patients (40%), Aspergillus species DNA was de­ trast, the clinical impact of an isolated positive Aspergillus
tected, and this percentage increased when GM was higher PCR on BALf seems limited. The EORTC/MSGERC PCR crite­
(Table 2). While the A. fumigatus PCR was positive in 89 rion for BALf may need further specification (eg, minimum Ct
(30%) patients, resistance testing was successful in only 58 value and/or PCR positive on >1 BALf sample).
(65%). Also, this resistance PCR was more successful with high­
Supplementary Data
er GM levels. The lower sensitivity of the resistance PCR is in
Supplementary materials are available at Clinical Infectious Diseases online.
line with a previous study and can be explained by the single-
Consisting of data provided by the authors to benefit the reader, the posted
copy nature of the cyp51A gene target in contrast to the fumi­ materials are not copyedited and are the sole responsibility of the authors,
gatus DNA probe that targets a multicopy gene [9, 14]. so questions or comments should be addressed to the corresponding
author.
A positive PCR test result on BALf in a patient with a negative
GM and culture was observed in 1 of every 5 patients (in 1 of ev­
Notes
ery 10 when the duplicate positivity criterion was used). Author contributions. B. R., A. S., and A. D. contributed to study
However, it was not associated with any increase in overall mor­ design. A. S., A. D., and S. H. did the data extraction and analysis. B. R. su­
tality. We cannot conclude whether this lack of clinical impact pervised the statistical analysis. B. R., A. S., A. D., and S. H. wrote the orig­
inal draft. A. D., S. H., J. B. B., C. K., P. V., K. D., N. J., J. J., W. V., B. B., A. B.,
on mortality of a positive PCR was the result of the antifungal
A. H. W. B., P. H., A. D., G. O., P. B., M. B., S. K., P. G., L. S., D. P., G. K.,
therapy that 85% of these patients received or if it reflects colo­ J. M., K. L., T. M., I. M., J. B., D. S., M. R., W. Z., J. D., J. C., A. S., and
nization instead of IPA in most of these patients. The value of B. R. reviewed and edited the manuscript. All authors read and approved
antifungal therapy for these patients remains uncertain. the final version. B. R., A. D., and S. H. had full access to the data in the
study and take full responsibility for the integrity of the data and the accu­
During the conduct of our study, the EORTC/MSGERC defini­ racy of the data analysis.
tions were updated, and the 2020 version now includes a positive Acknowledgments. The authors acknowledge all patients and participat­
Aspergillus PCR as a mycological criterion. For blood samples, ing centers for their efforts during the trial. They thank the Department of
Medical Microbiology and Infectious Diseases of Erasmus Medical Center
the guideline clearly mentions that 2 consecutive samples should for technical support and performing the majority of the AsperGenius po­
be PCR-positive; for BALf, the criterion is “2 or more duplicate lymerase chain reaction assays.
PCR tests positive.” Because BALf sampling is invasive, it is al­ Financial support. This work was supported by Gilead Sciences, Inc, un­
der grant number [IN-NL-131-4187].
most never done twice. We therefore interpreted this criterion
Potential conflicts of interest J. M. reports grants from Gilead Sciences,
as “2 positive PCRs on a single BALf sample.” This EORTC/ Inc; consulting fees, payment for lectures/presentations, and support for
MSGERC criterion will need further specification as we found meetings/travel expenses from Gilead Sciences, Inc, MSD, Pfizer, Takeda,
no association with mortality in patients with a negative GM and F2G; and participation on a data safety monitoring board or advisory
board for Gilead Sciences, Inc, MSD, Pfizer, Takeda, F2G, and Cidara. P. G.
and culture and therefore only fulfilled the PCR BALf criterion. reports partial support for travel to Trends In Medical Mycology confer­
These specifications may consist of a certain minimum Ct value ence. K. L. reports grants from Thermo Fisher Scientific and
threshold because the median Ct value of patients with an isolat­ TECOmedical paid to their institution; consulting fees from Gilead,
MSD, and MRM Health, all paid to their institution; and personal fees
ed positive PCR was much higher (36.4) than in patients with ad­ for lectures/presentations for Pfizer, Gilead, and FUJIFILM Wako. M. R.
ditional mycological evidence (33.1). Also, the guideline could reports support for travel to Trends In Medical Mycology conference.
perhaps state that the PCR should be positive on BALf from 2 J. J. reports grants from Novartis and BMS, both paid to their institution;
payment for lectures from AbbVie, Novartis, Pfizer, and Incyte; and serving
different bronchoscopies or 2 different lobes. In all scenarios,
as president of the Apps for Care and Science, a nonprofit organization,
the PCR criteria that are included should be associated with a supported by AbbVie, Alexion, Amgen, Astellas, BMS, Daiichi-Sankyo,
clinical impact and ideally mortality because the criteria in the Janssen-Cilag, Olympus, Incyte, Sanofi Genzyme, Servier, Jazz, and
guidelines are used for registration trials. Takeda. J. B. reports research grants from Gilead Sciences, Inc, and
F2G. P. V. reports research grants from F2G and Gilead, paid to their insti­
Despite the reasonably large number of patients enrolled tution; honoraria for lectures from F2G, Gilead, and Pfizer, all paid to their
across 12 sites over 4 years, only 1 patient with azole resistance institution; and participation on a data safety monitoring board for F2G,

44 • CID 2023:77 (1 July) • Huygens et al

paid to their institution. S. H. reports support from Gilead for travel to resistance of Aspergillus fumigatus on bronchoalveolar lavage fluid. J Clin
the International Society for Human and Animal Mycology 2022 Microbiol 2015; 53:868–74.
conference. B. R. reports research grants from Gilead Sciences, Inc; support 7. Donnelly JP, Chen SC, Kauffman CA, et al. Revision and update of the consensus
for meetings/travel expenses from Gilead Sciences, Inc, F2G, and Pfizer; definitions of invasive fungal disease from the European Organization for
Research and Treatment of Cancer and the Mycoses Study Group Education
consulting fees from F2G; payment/honoraria for lectures/presentations
and Research Consortium. Clin Infect Dis 2020; 71:1367–76.
from Gilead Sciences, Inc; and participation on a data safety monitoring
8. Schauwvlieghe A, de Jonge N, van Dijk K, et al. The diagnosis and treatment of
board/advisory board for Exevir. All other authors report no potential con­ invasive aspergillosis in Dutch haematology units facing a rapidly increasing prev­
flicts. All authors have submitted the ICMJE Form for Disclosure of alence of azole-resistance. A nationwide survey and rationale for the DB-MSG 002
Potential Conflicts of Interest. Conflicts that the editors consider relevant study protocol. Mycoses 2018; 61:656–64.
to the content of the manuscript have been disclosed. 9. Chong GM, van der Beek MT, von dem Borne PA, et al. PCR-based detection of
Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multi­
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