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Vol. 42, No. 1 Azole-Resistant Aspergillus fumigatus:

January 1, 2020

www.cmnewsletter.com What You Need To Know

In ThIS ISSue
1 Azole-Resistant Aspergillus
fumigatus: What You Need
To Know
6 Salmonella enterica
Serotype Typhi Soft Tissue
Infection Following Direct
Inoculation: a Rare Route of
Transmission
A case report
Shawn R. Lockhart, Karlyn Beer, and Mitsuru Toda, Mycotic Diseases Branch, Centers for Disease
Control and Prevention, Atlanta, Georgia
Abstract
Aspergillosis is one of the most common fungal infections. The predominant cause of aspergillosis is
the species Aspergillus fumigatus. There have been increasing reports of A. fumigatus isolates that are
resistant to azole antifungals. The predominant causes of this resistance are environmentally acquired
mutations in the target gene, CYP51A, known as TR 34/L98H and TR 46/Y121F/T289A. They consist
of a tandem repeat in the promoter region (TR) and one or two amino acid changes in the protein
sequence, respectively. Unfortunately, the capacity in the United States for mold antifungal suscepti-
bility testing is limited, so the extent of azole resistance in clinical practice is largely unknown. This
review discusses the causes and implications of azole-resistant A. fumigatus and the role that antifungal
susceptibility testing might play in its identification.

Note to Authors Introduction patients with hematologic malignancy. The mor-

Please note that CMN will
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reports for publication in
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Corresponding author:
Shawn R. Lockhart,
Ph.D.,
D(ABMM), F(AAM), Mycotic
Diseases Branch, Centers for
Disease Control and Prevention,
1600 Clifton Rd., Mailstop G-
11, Atlanta, GA 30333. Tel.:
404-639-2569. E-mail:
gyi2@cdc.gov
0196-4399/©2020 Elsevier Inc.
All rights reserved
Anyone who has processed respiratory specimens
in a clinical microbiology laboratory is familiar
with the white-bordered, felt-like mold colonies
of various colors that are typical of Aspergillus
(Fig. 1 and 2). Aspergillus species are ubiquitous
in the environment, and airborne conidia can be
found both indoors and outdoors. Perhaps due to
their ubiquitous nature, Aspergillus species are the
most frequently encountered molds from respi-
ratory specimens and one of the most frequent
causes of invasive mold infections [1]. Although
there are more than 250 species of Aspergillus,
four species cause almost 90% of all human
Aspergillus infections: A. fumigatus, A. flavus, A.
terreus, and A. niger. The most common species
encountered in clinical practice is A. fumigatus,
responsible for over half of aspergillosis cases in
the United States [2]. It can cause several types
of disease, including asthma, allergic broncho-
pulmonary aspergillosis, chronic obstructive
pulmonary aspergillosis, and invasive pulmo-
nary aspergillosis, of which the last can lead to
disseminated disease. Aspergillosis is the most
common cause of invasive mold infections among
tality rate associated with invasive aspergillosis is
dependent upon the patient population, but in a
recent study of 960 patients with various under-
lying conditions, the 12-week mortality was 35%
[3]. Aspergillus isolates, especially A. fumigatus,
that are resistant to medical azole treatment are
associated with treatment failure and increased
mortality. In a study comparing 90-day mortal-
ity among 196 patients with voriconazole-sus-
ceptible and voriconazole-resistant A. fumigatus
infections, 90-day mortality was 25% higher for
those with a resistant infection [4].
Susceptibility Testing of Aspergillus
Susceptibility testing of molds is not common in
the United States, even though the Clinical and
Laboratory Standards Institute (CLSI) has devel-
oped a standard for mold antifungal susceptibil-
ity testing (AFST) [5]. No automated systems or
custom kits are available for susceptibility testing
of molds, so many laboratories develop AFST
individually. Most laboratories that have validated
mold AFST use either standard broth microdi-
lution or gradient diffusion strips (such as Etest)

Clinical Microbiology Newsletter 42:1,2020 | ©2020 Elsevier 1

Figure 1. Colonies of A. fumigatus growing on Sabouraud dextrose Figure 2. Micrograph of A. fumigatus stained with lactophenol cotton
agar. Colonies are typically green with a white border, turning slate blue. Conidial heads are columnar and occur only on the upper portion
grey with age. of the vesicle; uniseriate with a phialide that produces chains of
smooth globose conidia.

[6]. While broth microdilution is the standard, good correlation is though few Aspergillus breakpoints exist, this does not mean there
achieved between gradient diffusion strips and broth microdilution are no other interpretive criteria. Both EUCAST and CLSI have
for itraconazole, voriconazole, and amphotericin B (Fig. 3) [7,8]. developed epidemiological cutoff values (ECVs or ECOFFs) for
Gradient diffusion strips are generally easier to implement in labo- a number of Aspergillus species and antifungal combinations [14]
ratories without experience performing susceptibility testing. In (http://www.eucast.org/mic_distributions_and_ecoffs/). While
addition to these two methodologies, another recently developed an ECV is not a breakpoint, it does help to determine whether
assay uses a 4-well azole agar plate with RPMI agar in wells con- an isolate is wild type (has an MIC value that is consistent with a
taining itraconazole (4 µg/ml), voriconazole (2 µg/ml), posacon- normal distribution and no resistance mutations) or non-wild type
azole (0.5 µg/ml), and no drug [9,10]. The plate is inoculated with
a known concentration of Aspergillus conidia and then monitored
for growth. Growth in any of the wells containing azoles indi-
cates the possibility of resistance and that the isolate should be
investigated further. The European Committee on Antimicrobial
Susceptibility Testing (EUCAST) recently adopted a standard for
the use of these plates for Aspergillus susceptibility testing [11].

One of the commonly cited reasons for not validating mold

susceptibility testing is that interpretive criteria have not been
established to qualify the results. Although 0.008 µg/ml is usually
interpreted as susceptible and 16 µg/ml is usually interpreted as
resistant, what about MIC values in the middle, such as 0.5, 1, or
2 µg/ml? EUCAST established breakpoints for some of the azole
antifungals and amphotericin B against Aspergillus species in 2012
[12, 13]. However, these breakpoints are valid only when using
the EUCAST method of AFST. Fortunately, CLSI approved its
first breakpoint for a mold at the 26 January 2019 meeting of the
Antifungal Susceptibility Testing Subcommittee. The breakpoint
for susceptibility of A. fumigatus against voriconazole is ≤0.5 µg/
ml, and the breakpoint for resistance is ≥2 µg/ml. CLSI does not Figure 3. Etests showing a voriconazole-susceptible (left) and an
have breakpoints for other Aspergillus species or other molds. Even itraconazole-resistant (right) A. fumigatus isolate.

2 Clinical Microbiology Newsletter 42:1,2020 | ©2020 Elsevier

(likely to have a mutation that confers resistance). For a bug-drug
combination where it is generally known that the drug is effica-
cious for treatment, like Aspergillus and the newer azoles, ECVs
may be used to determine whether an isolate has acquired muta-
tions and is therefore not wild type. This designation is challeng-
ing, though, because there are few clinical data to inform as to the
interpretive value of these MICs. Thus, it is unknown whether an
MIC result that falls outside the ECV range would be predictive
of treatment failure [15].
Azole Resistance in A. fumigatus
The prevalence of azole resistance in A. fumigatus has been
increasing for more than a decade [16-18]. The most frequently
encountered resistance mutations are those that occur in the
14-α-demethylase gene, CYP51A, a component of the ergosterol
synthesis pathway and the target of the azole antifungals. This
gene is orthologous to ERG11, the gene target of azoles in Candida
species. Multiple amino acid substitutions that lead to azole resis-
tance have been identified in the Cyp51A protein. These mutations
generally occur in isolates from patients that have received long-
term azole therapy or prophylaxis. Depending on the mutation,
they can confer resistance to either a single azole, multiple azoles,
or all the azoles (Table 1). To confound matters, approximately
30% of isolates that show in vitro azole resistance do not have an
identifiable resistance mechanism [17]. It is thought that efflux
pumps play a role in resistance in these isolates [19]. While the
incidence of azole-resistant A. fumigatus is increasing globally, it
is not yet common in patients in the United States [20].
Resistance can develop under selection pressure from azoles, and
this can happen inside the human body when a patient is on long-
term azole therapy or in the environment, where A. fumigatus is
exposed by chance to azoles used for other purposes. A study by
Snelders and colleagues in 2008 initially identified a trend toward
increasing azole resistance in A. fumigatus isolates in the Neth-
erlands [17]. The most frequent cause of resistance was a pair of
mutations found in the 14-α-demethylase gene (CYP51A) called
TR 34 /L98H (TR 34 ). The mutations consist of a tandem 34-bp
repeat in the promoter region of the gene that leads to increased
gene expression (TR 34) and an amino acid change from lysine to
histidine at amino acid 98 (L98H) that hinders the binding of
azoles to the target. A worrisome aspect of the TR 34 mutation is
that it is believed to be derived from the use of azole fungicides
Table 1. The most common mutations in CYP51A that lead to azole resistance in A. fumigatus
Mutation Found in patients
TR34/L98H Yes
TR46/Y121F/T289A Yes
G54E/R/V Yes
G138C/S Yes
M220I/R/K/V Yes
G448S No
a Some isolates with these mutations are pan-azole resistant, but further work needs to be done to decipher other contributing mutations within the same isolate.
that are used in agriculture rather than from exposure to azoles
during treatment or prophylaxis in patients [21,22].
Since the initial discovery in the Netherlands in 1998, A. fumiga-
tus isolates with TR 34/L98H have been identified in Asia, Africa,
Australia, South America, and North America [23]. In addition,
a second set of mutations, TR 46/Y121F/T289A (TR 46), has been
identified. Like TR34, TR46 is associated with the use of azole fun-
gicides in agriculture [13,14,23,24].
To determine whether and to what extent these fungicide-associ-
ated resistant isolates are present in the United States, the Cen-
ters for Disease Control and Prevention (CDC) conducted passive
surveillance for azole-resistant A. fumigatus in the United States.
Of the >1,000 A. fumigatus isolates collected from 22 states during
2011 to 2013, none contained the TR 34 or TR 46 mutation [25].
The first U.S. isolates with either TR 34 or TR 46 were identified
from a national reference laboratory that retrospectively exam-
ined all isolates collected during 2001 to 2014. In that collec-
tion, they found that 26 isolates had elevated MIC values to more
than one azole. One isolate from 2008 and one from 2014, both
from Pennsylvania, contained the TR 34 mutation [26]. In addi-
tion, two isolates with the TR 46 mutation, one from Arizona in
2008 and one from an unknown location in 2012, were identified
[26]. Another report, published in 2016, identified a patient from
Michigan who had an isolate containing the TR 46 mutation [27].
In 2015, CDC reinitiated passive surveillance for azole-resistant
A. fumigatus. During 2015 to 2017, five additional isolates with the
TR34 mutation were identified, two from Pennsylvania, two from
Virginia, and one from California, although these positive results
represented only 0.3% of all of the isolates tested [20]. These iso-
lates and two others with TR 34 mutations were identified among
7 patients, of whom 3 died and 4 had not been previously treated
with azole medication [28]. Patients had no epidemiologic links,
suggesting a variety of different sources contributed to their A.
fumigatus specimens.
Role of Agricultural Azoles in A. fumigatus Resistance
Certain agricultural azole fungicides, including difenoconazole,
tebuconazole, and propiconazole, are structurally similar to medi-
cal azoles, such as itraconazole, voriconazole, and posaconazole
[29]. Azole fungicides are used in agriculture (crop protection
from fungal diseases and promotion of increased crop yields) and
in other applications, such as preservation of wood and other
Found in nature Primary resistancea
Yes Itraconazole/posaconazole
Yes Voriconazole
Yes Itraconazole/posaconazole
No Itraconazole/posaconazole
Yes Itraconazole/posaconazole/voriconazole
No Voriconazole
Clinical Microbiology Newsletter 42:1,2020 | ©2020 Elsevier 3
materials [30]. Of the many classes of agricultural fungicides,
azoles represent the largest fungicide class in agricultural and hor-
ticultural use. In the Netherlands, the total volume of fungicides
used has not changed dramatically over time, but the proportion
of azole fungicide use doubled between 1995 and 2007 [22]. In the
United States, azole fungicides are commonly used on wheat, corn,
and soybeans, three of the major crops produced in this country
in metric tons. The estimated usage of azoles in this country also
increased dramatically from 2006 to 2015 [31].
In addition to clinical isolates, A. fumigatus isolates with TR34 and
TR 46 mutations have been found in the environment, including
in patients’ homes and back yards, in flower bulb waste and wood
chips, in hospital gardens, and even in air samples from hospitals
[24,32-35]. They have also been found in sealed tulip bulbs in
Ireland that were imported from the Netherlands, where azole
resistance in A. fumigatus has become common [35]. While all the
environmental A. fumigatus isolates with TR 34 and TR 46 muta-
tions were identified outside the United States, the conundrum
remained that no one in the United States had actually looked in
the environment for these mutations. To see if A. fumigatus isolates
associated with the use of agricultural azoles could be identified
in the United States, isolates were collected from crop debris on
a farm in Georgia that had been treated with tebuconazole and
propiconazole. Of the 200 isolates collected, 20 contained the TR34
mutation and none contained the TR 46 mutation [36]. This single
finding may not be representative of the rest of the United States,
but with the increasing use of agricultural azoles throughout the
country, it indicates potential for increasing incidence, similar to
what has been seen in Europe.
new Antifungals
Medical azoles are the first-line therapy for invasive aspergillo-
sis, and use of azole antifungal medications has improved patient
survival [37]. The Infectious Diseases Society of America rec-
ommends voriconazole as the primary treatment of choice for
most forms of Aspergillus infection; other azoles, as well as for-
mulations of amphotericin B, are also used [38]. Because some
A. fumigatus isolates with the TR 34 mutation are pan-resistant to
the azoles, amphotericin B is left as the only proven treatment
option. However, other classes of mold-active antifungal agents
with activity against Aspergillus are now in phase 2 clinical trials.
Olorofim (formerly F901318) is a mold-active antifungal that tar-
gets dihydroorotate dehydrogenase and inhibits pyrimidine bio-
synthesis [39]. A second mold-active compound in development
is fosmanogepix (formerly APX001A). This compound is a potent
inhibitor of glycophosphatidylinositol biosynthesis [40]. Both com-
pounds have very strong in vitro activity against multiple species
of Aspergillus. The results of phase 3 clinical trials will determine
whether they join the current antifungal armamentarium.
4 Clinical Microbiology Newsletter 42:1,2020 | ©2020 Elsevier
Implications for Clinicians
Although azole-resistant A. fumigatus has been infrequently
reported in the United States to date, these reports likely rep-
resent an underestimate of the number of patients with resistant
isolates. A. fumigatus infection and colonization are not report-
able conditions in any state and are not nationally notifiable to
the CDC, meaning that no formal reporting requirement exists.
Perhaps as a result, clinicians are largely unaware of resistant A.
fumigatus and its added risk of treatment failure and death, espe-
cially among patients with invasive aspergillosis [4]. A retrospec-
tive cohort study in the Netherlands showed 21% higher mortality
in patients with voriconazole-resistant invasive aspergillosis than
in patients infected with voriconazole-susceptible isolates [41].
Unfortunately, the problem of azole-resistant A. fumigatus is still
not widely recognized. In a 2018 survey of 709 infectious disease
physicians, over half were unfamiliar with the concept of azole-
resistant A. fumigatus and only 62% reported having access to
antifungal susceptibility testing [42]. Together, limited awareness
and lack of reporting mechanisms point to a strong need for vigi-
lance among front-line clinicians treating susceptible patients, even
those without prior azole treatment. Clinicians and laboratories
interested in participating in CDC’s voluntary passive surveillance
program are invited to submit all A. fumigatus isolates; more infor-
mation and submission instructions are available online (lhttps://
www.cdc.gov/fungal/aspergillus-resistance.html) .
Summary
Azole-resistant A. fumigatus is present in the United States in
patients and in the environment. Many factors, including increas-
ing use of agricultural azoles, medical prophylaxis, and long-term
treatment of patients, and the growing number of patients sus-
ceptible to fungal infections may contribute to the potential for
increasing azole-resistant A. fumigatus prevalence in the United
States, as it has in parts of Europe. The solution is not a simple
one, but two important steps clinical microbiologists can take are
vigilance and increased AFST capacity. Laboratories can con-
sider implementing susceptibility testing of isolates from patients
thought to have a resistant infection or surveillance for azole-
resistant isolates using one of the protocols mentioned above.
The findings and conclusions in this article are those of the authors
and do not necessarily represent the official position of the Centers
for Disease Control and Prevention.
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6 Clinical Microbiology Newsletter 42:1,2020 | ©2020 Elsevier