Unravelling the Molecular Identification and Antifungal Susceptibility Profiles of Aspergillus spp. Isolated from Chronic Pulmonary Aspergillosis Patients in Jakarta, Indonesia: The Emergence of Cryptic Species

Abstract

Cryptic species of Aspergillus have rapidly increased in the last few decades. Chronic pulmonary aspergillosis (CPA) is a debilitating fungal infection frequently affecting patients with previous TB. The identification and antifungal susceptibility profiles of different species of Aspergillus are important to support the management of CPA. The aim of this study was to describe the molecular and susceptibility profiles of Aspergillus isolated from CPA patients. The species identity of isolates was determined by combined DNA analyses of internal transcribed space (ITS), partial β-tubulin genes, and part of the calmodulin gene. We revealed a high (27%) prevalence of cryptic species among previous tuberculosis patients with persistent symptoms. Twenty-nine (49%) patients met the criteria for diagnosis of CPA with 24% containing Aspergillus cryptic species. This is the first report of five cryptic Aspergillus species from clinical isolates in Indonesia: A. aculea tus, A. neoniger, A. brunneoviolacues, A. welwitschiae, and A. tubingensis. Significantly, there was decreased sensitivity against itraconazole in the CPA group (66% susceptible to itraconazole) compared to the non-CPA group (90% susceptible to itraconazole) (p = 0.003). The species-level characterisation of Aspergillus and its antifungal susceptibility tests demands greater attention to better the management of CPA patients.

Full text

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Citation: Rozaliyani, A.; Abdullah,
A.; Setianingrum, F.; Sjamsuridzal,
W.; Wahyuningsih, R.; Bowolaksono,
A.; Fatril, A.E.; Adawiyah, R.;
Tugiran, M.; Syam, R.; et al.
Unravelling the Molecular
Identification and Antifungal
Susceptibility Profiles of Aspergillus
spp. Isolated from Chronic
Pulmonary Aspergillosis Patients in
Jakarta, Indonesia: The Emergence of
Cryptic Species. J. Fungi 2022,8, 411.
https://doi.org/10.3390/jof8040411
Academic Editor: Nathan P
Wiederhold
Received: 2 March 2022
Accepted: 12 April 2022
Published: 16 April 2022
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4.0/).
Fungi
Journal of
Article
Unravelling the Molecular Identification and Antifungal
Susceptibility Profiles of Aspergillus spp. Isolated from Chronic
Pulmonary Aspergillosis Patients in Jakarta, Indonesia: The
Emergence of Cryptic Species
Anna Rozaliyani 1, 2, * , Asriyani Abdullah 3, Findra Setianingrum 1,2, Wellyzar Sjamsuridzal 4,
Retno Wahyuningsih 1,5 , Anom Bowolaksono 4, Ayu Eka Fatril 1, Robiatul Adawiyah 1,2 , Mulyati Tugiran 1,2,
Ridhawati Syam 1,2, Heri Wibowo 1,3 , Chris Kosmidis 6,7 and David W. Denning 6,7
1Department of Parasitology, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia;
findra.s88@gmail.com (F.S.); retnet2002@gmail.com (R.W.); ayufatril34@gmail.com (A.E.F.);
bundaadah@gmail.com (R.A.); dramulyati@yahoo.co.id (M.T.); ridhawatia@yahoo.com (R.S.);
bowoheri04@gmail.com (H.W.)
2Indonesia Pulmonary Mycoses Centre, Jakarta 10430, Indonesia
3Magister Program of Biomedical Sciences, Faculty of Medicine, Universitas Indonesia,
Jakarta 10430, Indonesia; asriyaniabdullah1464@gmail.com
4Department of Biology, Faculty of Mathematics and Natural Sciences (FMIPA), Universitas Indonesia,
Depok 16424, Indonesia; sjwelly@hotmail.com (W.S.); alaksono@sci.ui.ac.id (A.B.)
5Department of Parasitology, Faculty of Medicine, Universitas Kristen, Jakarta 13530, Indonesia
6Manchester Fungal Infection Group, Faculty of Biology, Medicine and Health, University of Manchester,
Manchester M23 9LT, UK; chris.kosmidis@manchester.ac.uk (C.K.); ddenning@manchester.ac.uk (D.W.D.)
7Manchester Academic Health Science Centre, Division of Infection, Immunity and Respiratory Medicine,
Faculty of Biology, Medicine and Health, University of Manchester, Manchester M23 9LT, UK
*Correspondence: annaroza1110@gmail.com; Tel.: +62-21-3102135; Fax: +62-21-3983201
Abstract:
Cryptic species of Aspergillus have rapidly increased in the last few decades. Chronic
pulmonary aspergillosis (CPA) is a debilitating fungal infection frequently affecting patients with
previous TB. The identification and antifungal susceptibility profiles of different species of Aspergillus
are important to support the management of CPA. The aim of this study was to describe the molecular
and susceptibility profiles of Aspergillus isolated from CPA patients. The species identity of isolates
was determined by combined DNA analyses of internal transcribed space (ITS), partial
β
-tubulin
genes, and part of the calmodulin gene. We revealed a high (27%) prevalence of cryptic species
among previous tuberculosis patients with persistent symptoms. Twenty-nine (49%) patients met the
criteria for diagnosis of CPA with 24% containing Aspergillus cryptic species. This is the first report of
five cryptic Aspergillus species from clinical isolates in Indonesia: A. aculea tus,A. neoniger,A. brun-
neoviolacues,A. welwitschiae, and A. tubingensis. Significantly, there was decreased sensitivity against
itraconazole in the CPA group (66% susceptible to itraconazole) compared to the non-CPA group
(90% susceptible to itraconazole) (p= 0.003). The species-level characterisation of Aspergillus and its
antifungal susceptibility tests demands greater attention to better the management of CPA patients.
Keywords: Aspergillus; cryptic; antifungal susceptibility; tuberculosis; chronic pulmonary aspergillosis
1. Introduction
Chronic pulmonary aspergillosis (CPA) has been one of the most common causes
of persistent pulmonary symptoms found in post-tuberculosis infection patients. About
three million CPA cases occur worldwide [
1
]. Globally, it was estimated that 1.2 million
pulmonary tuberculosis cases developed into CPA [
2
]. In Indonesia, the prevalence of CPA
is estimated at 378,700 cases [
3
]. Previous studies revealed around 8–56.7% patients with a
history of pulmonary tuberculosis (TB) developed CPA [4–6].
J. Fungi 2022,8, 411. https://doi.org/10.3390/jof8040411 https://www.mdpi.com/journal/jof

J. Fungi 2022,8, 411 2 of 14
Aspergillus fumigatus is the cause in the majority of CPA cases. However, other species
have also been implicated, such as A. flavus,A. niger,A. terreus or A. nidulans [
7
]. The
conventional methods to identify Aspergillus species rely on direct microscopic examination
and culture to support the diagnosis of CPA [
8
]. However, some Aspergillus species are
morphologically indistinguishable and molecular identification is required to identify
these cryptic species [
9
–
12
]. Several genes have been used to facilitate the identification
of Aspergillus at the species level, including internal transcribed spacer (ITS), calmodulin
(CaM), and
β
-tubulin (benA) [
13
]. The increasing number of cryptic species reported
worldwide indicates that these species are of concern due to the variable susceptibility
profile [
14
–
16
]. A recent report revealed that cryptic species comprised 37% of Aspergillus
clinical isolates [
17
], but whether these species play a role in the aetiology of CPA is not
known. Molecular profiling of Aspergillus isolates in CPA has shown the presence of cryptic
species in the UK [
18
]. Therefore, this study aims to identify the genetic profile of Aspergillus
spp. isolated from clinical specimens of previous TB patients with suspected CPA.
2. Materials and Methods
2.1. Aspergillus spp. Isolates
Fifty-nine clinical isolates of Aspergillus were included in this study. The clinical isolates
were recovered from the culture collection of the Mycology Laboratory, Department of
Parasitology, Faculty of Medicine Universitas Indonesia. The sources of culture collection
were the sputum of post-tuberculosis patients with suspected CPA between 2019 and 2020
obtained during routine clinical care. The diagnostic criteria of CPA are: (1) at least one of
these chronic (>3 months) symptoms including haemoptysis, cough, dyspnea, chest pain
and/or fatigue, and (2) positive Aspergillus spp. culture from sputum or positive Aspergillus
antibodies, and (3) radiological appearances suggestive of CPA (at least fungal balls and/or
cavitation confirmed by a CT scan). The study was approved by the Ethics Committee of
the Faculty of Medicine, Universitas Indonesia (95/UN2.F1/ETIK/2019).
2.2. Molecular Identification
DNA extraction was prepared using the two-step extraction method with the pre-
cipitation reagent phenol-chloroform-isoamylalcohol as previously described with modi-
fications [
19
]. The species-specific identification of all isolates was examined by amplifi-
cation of the ITS rDNA gene using ITS1 (5
0
-TCCGTAGGTGAACCTGCGG-3
0
) and ITS4
(5
0
-TCCTCCGCTTATTGATATGC-3
0
) primers [
20
], part of the benA gene using Bt2a (5
0
-
GGTAACCAAATCGGTGCTGCTTCT-3
0
) and Bt2b (5
0
-ACCCTCAGTGTAGTGACCCTTGGC-
3
0
) primers [
21
], partial CaM gene using cmd5 (5
0
-CCGAGTACAAGGAGGCCTTC-3
0
) and
cmd6 (5
0
-CCGATAGAGGTCATAACGTGG-3
0
) primers [
22
]. The PCR amplifications were
conducted as described in detail previously with some modifications [
22
,
23
]. The results of
sequencing were aligned using Mega 6.06TM followed by the basic local alignment search tool
(BLAST) at The National Center for Biotechnology Information (NCBI) and the International
Society for Human and Animal Mycology (ISHAM) databases. Calmodulin was used as the
reference gene isolate in the Flavi and Nigri sections since beta tubulin and ITS may produce
PCR biases [24–27].
2.3. Antifungal Susceptibility Tests
Antifungal susceptibility tests were performed using the disk diffusion method. Sus-
pension of fungal colonies using a 0.9% NaCl solution was prepared with a turbidity
equivalent to 0.5 of the McFarland standard. By using a sterile swab, the suspension was
applied to the surface of the Muller Hinton Agar (MHA). Disk diffusion for amphotericin
B (10
µ
g), voriconazole (1
µ
g) and itraconazole (8
µ
g disks) were obtained commercially
(Liofilchem, Roseto degli Abruzzi, Italy). The plates were incubated at 35
◦
C for 48 h
after applying the disks. The measurement of the zone of inhibition relied on a marked
reduction (80%) of microcolonies after 48 h [
28
,
29
]. Candida krusei ATCC 6258 was used
as a control strain [
28
,
30
]. The interpretations of the zone of inhibition were referenced to

J. Fungi 2022,8, 411 3 of 14
Espinel-Ingroff et al. [
28
]; the zones of inhibition of the Candida krusei ATCC 6258 in this
study were within the reference range.
2.4. Statistical Analysis
Values were presented using frequencies (%) for categorical variables and means
±
standard deviations and ranges for normally distributed continuous variables. The different
continuous variables were analysed using an independent t-test for CPA and non-CPA
groups or cryptic and sensu stricto groups. Fisher’s exact tests or X2 tests were used for
categorical variables for CPA and non- CPA groups or cryptic and sensu stricto groups.
Data analysis was performed with the use of IBM SPSS V.25 (IBM Corp., Armonk, NY, USA)
statistic software. The significance level was set to p< 0.05.
3. Results
3.1. Patient Characteristics
Amongst the 59 clinical Aspergillus isolates from 46 patients with suspected CPA, DNA
sequencing showed that 16 (27%) isolates were cryptic/rare species and 43 (73%) isolates
were non-cryptic (sensu stricto) species (Table 1). Twenty-nine (49%) of the patients met the
criteria for CPA, while thirty (51%) patients were diagnosed with other conditions. Seven
(24%) of 29 CPA patients had cryptic Aspergillus isolates from their cultures. Amongst the
A. Fumigati section, all were A. fumigatus sensu stricto. Likewise, most of the Flavi were
A. flavus sensu stricto (88%). In contrast, most (68%) of the A. Nigri section was identified
as cryptic species. The Clavati section consisted of one A. clavatus sensu stricto. The CPA
patients had a higher rate of haemoptysis (79% vs. 43%, p= 0.005) and chronic haemoptysis
(38% vs. 13%, p= 0.039) compared to the non-CPA group, which was unrelated to whether
strains were or were not cryptic.
Table 1.
Aspergillus identification according to the section of the isolates recovered and patient’s
clinical features included in this study.
All
(n= 59)
CPA
(n= 29)
Non-CPA
(n= 30) p-Value Cryptic
(n= 16)
Sensu
Stricto
(n= 43)
p-Value
Section
Fumigati 28 (49%) 15 (54%) 13 (43%) 0.519 0 (0%) 28 (65%) <0.005
Clavati 1 (2%) 1 (3%) 0 (0%) 0.492 0 (0%) 1 (2%) 1
Flavi 8 (14%) 2 (7%) 6 (20%) 0.254 1 (6%) 7 (16%) 0.427
Nigri 22 (37%) 11 (38%) 11 (37%) 0.920 15 (93%) 7 (16%) <0.005
Symptoms
Haemoptysis 36 (61%) 23 (79%) 13 (43%) 0.005 12 (75%) 24 (56%) 0.236
Massive
haemoptysis 19 (32%) 12 (41%) 7 (23%) 0.170 7 (44%) 12 (28%) 0.348
Recurrent
haemoptysis 15 (25%) 11 (38%) 4 (13%) 0.039 3 (19%) 12 (28) 0.738
Abbreviations: CPA: chronic pulmonary aspergillosis. The grey background highlighted the cryptic and sensu
stricto variables and their p-values.
3.2. Isolate Identification
The 59 isolates were morphologically classified as the A. Fumigati section (47%, n= 28),
A. Clavati section (2%, n= 1), A. Flavi section (14%, n= 8), and A. Nigri section (37%,
n= 22). The combination of ITS, beta tubulin, and calmodulin sequences generated in
this study identified ten (four non-cryptic and six cryptic species) different species across
these 59 isolates. In order of decreasing prevalence, A fumigatus (47%, n= 28), A. flavus
(12%, n= 7), A. niger (12%, n= 7), A. brunneoviolaceus (12%, n= 7), A. tubingensis (5%,
n= 3), A. aculeatus (3%, n= 2), A. neoniger (3%, n= 2), A. clavatus (2%, n= 1), A. welwitschiae
(2%, n= 1) and A. tamarii (2%, n= 1) accounted for the identified isolates (Table 2). We
repeated the DNA extraction and sequencing steps for the nine selected available isolates
with discrepancies resulting between three primers.

J. Fungi 2022,8, 411 4 of 14
Table 2. Molecular identification of all isolates.
No Sections Sample Code Final ID Genes Used for ID Diagnosis Amphotericin Itraconazole Voriconazole
1
Fumigati
006-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Resistant Susceptible
2 012-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Susceptible Intermediate
3 013-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Intermediate
4 014-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Susceptible Susceptible
5 015-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Susceptible Susceptible Susceptible
6 018-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Resistant
7 019-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Intermediate
8 020-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Intermediate Resistant
9 022-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Resistant
10 023-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Susceptible Susceptible
11 025-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
12 026-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Intermediate
13 027-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Resistant
14 036-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Intermediate
15 048-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Susceptible Resistant Intermediate
16 069-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
17 080-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Resistant
18 083-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Susceptible Susceptible
19 084-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Susceptible Resistant Resistant
20 085-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Resistant
21 091-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Resistant Resistant
22 092-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Resistant Resistant
23 094-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Susceptible Susceptible Resistant
24 097-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Resistant Resistant
25 101-BT A. fumigatus
sensu stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
26 103-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Resistant Intermediate Susceptible
27 109-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Intermediate Intermediate Susceptible
28 110-BT A. fumigatus
sensu stricto ITS, BenA, CaM CPA Intermediate Susceptible Resistant
29 Clavati 064-BT A. clavatus sensu
stricto ITS, BenA, CaM CPA Susceptible Susceptible Resistant

J. Fungi 2022,8, 411 5 of 14
Table 2. Cont.
No Sections Sample Code Final ID Genes Used for ID Diagnosis Amphotericin Itraconazole Voriconazole
30
Flavi
052-BT A. tamarii CaM Non-CPA Resistant Susceptible Intermediate
31 066-BT A. flavus sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
32 069-BT A. flavus sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
33 071-BT A. flavus sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Intermediate
34 080-BT A. flavus sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
35 086-BT A. flavus sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Resistant
36 092-BT A. flavus sensu
stricto ITS, BenA, CaM CPA Resistant Susceptible Susceptible
37 103-BT A. flavus sensu
stricto ITS, BenA, CaM CPA Resistant Susceptible Intermediate
38
Nigri
057-BT A. niger sensu
stricto ITS, BenA, CaM Non-CPA Resistant Resistant Resistant
39 083-BT A. niger sensu
stricto ITS, BenA, CaM CPA Resistant Susceptible Susceptible
40 064-BT A. niger sensu
stricto ITS, BenA, CaM CPA Resistant Resistant Intermediate
41 074-BT A. niger sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
42 079-BT A. niger sensu
stricto ITS, BenA, CaM CPA Intermediate Susceptible Susceptible
43 085-BT A. niger sensu
stricto ITS, BenA, CaM Non-CPA Resistant Susceptible Susceptible
44 103-BT A. niger sensu
stricto ITS, BenA, CaM CPA Susceptible Susceptible Susceptible
45 076-BT A. welwitschiae BenA, CaM Non-CPA Susceptible Susceptible Resistant
46 099-BT A. tubingensis BenA, CaM Non-CPA Susceptible Susceptible Susceptible
47 101-BT A. tubingensis BenA, CaM CPA Resistant Susceptible Susceptible
48 068-BT A.
brunneoviolaceus CaM Non-CPA Intermediate Susceptible Susceptible
49 073-BT A. aculeatus ITS, BenA, CaM CPA Intermediate Susceptible Susceptible
50 100-BT A. aculeatus ITS, BenA, CaM CPA Resistant Susceptible Susceptible
51 060-BT A.
brunneoviolaceus CaM CPA Susceptible Susceptible Susceptible
52 006-BT A.
brunneoviolaceus CaM CPA Intermediate Susceptible Susceptible
53 061-BT A.
brunneoviolaceus CaM Non-CPA Susceptible Susceptible Susceptible
54 062-BT A.
brunneoviolaceus CaM Non-CPA Intermediate Susceptible Susceptible
55 069-BT A.
brunneoviolaceus CaM Non-CPA Susceptible Susceptible Susceptible
56 098-BT A.
brunneoviolaceus CaM Non-CPA Resistant Susceptible Susceptible
57 086-BT A. tubingensis ITS, BenA, CaM Non-CPA Resistant Resistant Intermediate
58 089-BT A. neoniger CaM CPA Intermediate Susceptible Susceptible
59 097-BT A. neoniger CaM CPA Resistant Intermediate Intermediate
Abbreviations: ID: identification; CPA: chronic pulmonary aspergillosis; ITS: internal transcribed spacer; CaM:
calmodulin; benA: and β-tubulin (benA).
3.3. Antifungal Susceptibility Profiles
Of the 59 isolates tested, 19% (n= 11) were susceptible to amphotericin B, 53% (n= 31)
were susceptible to voriconazole, and 78% (n= 46) were susceptible to itraconazole based on
disk diffusion tests (Table 3). Cryptic species had higher mean values of zones of inhibition
to all three antifungals used in this study compared to the non-cryptic species.

J. Fungi 2022,8, 411 6 of 14
Table 3. Antifungal susceptibility profiles of Aspergillus isolates using disk diffusion method.
All
(n= 59) CPA
(n= 29)
Non-CPA
(n= 30) p-Value Cryptic
(n= 16)
Sensu Stricto
(n= 43) p-Value
Amphotericin B
Zone of inhibition (range) 0–22 0–22 2–21.1 2–21.1 0–22
Mean of inhibition zone ±SD 10.8 ±4.8 11.4 ±4.9 10.3 ±4.8 0.381 13.9 ±4.6 9.7 ±4.5 0.002
Susceptible 11 (19%) 6 (21%) 5 (17%) 0.748 5 (31%) 6 (14%) 0.149
Intermediate 8 (14%) 6 (21%) 2 (7%) 0.145 5 (31%) 3 (7%) 0.028
Resistant 40 (68%) 17 (59%) 23 (77%) 0.170 6 (38%) 34 (79%) 0.002
Voriconazole
Zone of inhibition (range) 2–44 2–44 7–41.3 11.8–41.3 2–44
Mean of inhibition zone ±SD 18.9 ±9.2 19.6 ±10.7 18.1 ±7.7 0.541 26.4 ±10.4 16.1 ±7.1 <0.005
Susceptible 31 (53%) 17 (59%) 14 (47%) 0.358 12 (75%) 19 (44%) 0.035
Intermediate 12 (20%) 4 (14%) 8 (27%) 0.333 3 (19%) 9 (21%) 1
Resistant 16 (27%) 8 (28%) 8 (27%) 11 (6%) 15 (35%) 0.045
Itraconazole
Zone of inhibition (range) 2–37 2–37 11–32 11–37 2–30
Mean of inhibition zone ±SD 19.3 ±6.2 19.3 ±7.5 19.2 ±4.8 0.939 22.7 ±7.3 18 ±5.3 0.009
Susceptible 46 (78%) 19 (66%) 27 (90%) 0.030 14 (88%) 32 (74%) 0.481
Intermediate 4 (7%) 4 (14%) 0 (0%) 0.052 1 (6%) 3 (7%) 1
Resistant 9 (15%) 6 (21%) 3 (10%) 0.299 1 (6%) 8 (19%) 0.421
Abbreviations: CPA: chronic pulmonary aspergillosis; SD: standard deviations. The grey background highlighted
the cryptic and sensu stricto variables and their p-values.
Using amphotericin B, the mean values for the zone of
≥
inhibition for non-cryptic and
cryptic isolates were 9.7
±
4.5 mm (range 0–22 mm) and 13.9
±
4.6 mm (range
2–21 mm
)
(p= 0.002), respectively, indicative of cryptic species being more susceptible. In line with
this, the proportion of resistant isolates in the non-cryptic group (79%) is higher (p= 0.002)
than in the cryptic group (34%) for amphotericin B. However, the non-cryptic group
(7%) showed a lower (p= 0.028) number of intermediate isolates against amphotericin B
compared to the cryptic group (31%).
Voriconazole revealed higher (p< 0.005) mean values for the zone of inhibition in the
cryptic group (26.4
±
10.4) compared to the non-cryptic group (16.1
±
7.1). Itraconazole
showed higher (p= 0.009) mean values for the zone of inhibition in the cryptic group
(22.7
±
7.3) compared to the non-cryptic group (18.9
±
5.3). There are no differences in
antifungal susceptibility profiles based on disease classification (CPA and non-CPA), except
there were a significantly lower number (p= 0.003) of susceptible isolates in the CPA group
(66%) compared to the non-CPA group (90%) against itraconazole. The scatter plots are
shown in Figure 1A–C. The zone of inhibitions of the quality control strain was within the
diameter ranges of the reference.
Amongst the four sections of Aspergillus (Fumigati,Clavati,Flavi, and Nigri), the highest
rate of resistance against amphotericin B was observed in the Flavi section (100%, n= 8).
Meanwhile, the highest rate of resistance against itraconazole and voriconazole was seen
in the Fumigati section (itraconazole: 21%, n= 6; voriconazole: 43%, n= 12). We ex-
cluded the Clavati section from these comparisons because this section only had one isolate.
A. clavatus sensu stricto was susceptible against amphotericin and itraconazole but resistant
to voriconazole (Figure 2). The Fumigati section showed the highest rates of resistance
for azoles with 6 and 12 isolates showing resistance to itraconazole and voriconazole,
respectively (Figure 3).
There were 22 isolates in the Nigri section, consisting of 7 (32%) isolates of A. niger sensu
stricto and 15 (68%) isolates belonging to cryptic species (
Supplementary Tables S1–S4
).
The proportion of CPA and the non-CPA group from cryptic isolates is nearly the same. Of
these 15 cryptic species isolates, there were 7 (47%) isolates from CPA patients. Meanwhile,
A. niger sensu stricto classified as CPA was 57% (4/7) (Figure 4). Seven isolates of cryptic
Aspergillus from the Nigri section classified as CPA were from A. aculeatus (n= 2), A. neoniger
(n= 2), A. tubingensis (n= 1), and A. brunneoviolaceus (n= 2). Meanwhile, eight isolates
were classified as the non-CPA consisting of A. welwitschiae (n= 1), A. tubi (n= 2) and
A. brunneoviolaceus (n= 5).

J. Fungi 2022,8, 411 7 of 14
J. Fungi 2022, 8, x FOR PEER REVIEW 7 of 15
Figure 1. Scatter plot diagrams of zone inhibition diameters of disk diffusion against amphotericin
B (A), voriconazole (B) and itraconazole (C) in the CPA and non-CPA groups. Zone diameter cate-
gories (dash lines): amphotericin B (susceptible ≥ 15 mm; intermediate 13 to 14 mm; resistant ≤ 12
mm), itraconazole and voriconazole (susceptible ≥ 17 mm; intermediate 14 to 16 mm; resistant ≤ 13
mm) (15).
Amongst the four sections of Aspergillus (Fumigati, Clavati, Flavi, and Nigri), the high-
est rate of resistance against amphotericin B was observed in the Flavi section (100%, n =
8). Meanwhile, the highest rate of resistance against itraconazole and voriconazole was
seen in the Fumigati section (itraconazole: 21%, n = 6; voriconazole: 43%, n = 12). We ex-
cluded the Clavati section from these comparisons because this section only had one iso-
late. A. clavatus sensu stricto was susceptible against amphotericin and itraconazole but
resistant to voriconazole (Figure 2). The Fumigati section showed the highest rates of re-
sistance for azoles with 6 and 12 isolates showing resistance to itraconazole and voricon-
azole, respectively (Figure 3).
Figure 1.
Scatter plot diagrams of zone inhibition diameters of disk diffusion against amphotericin B (
A
),
voriconazole (
B
) and itraconazole (
C
) in the CPA and non-CPA groups. Zone diameter categories (dash
lines): amphotericin B (susceptible
≥
15 mm; intermediate 13 to 14 mm; resistant
≤
12 mm), itraconazole
and voriconazole (susceptible ≥17 mm; intermediate 14 to 16 mm; resistant ≤13 mm) (15).
J. Fungi 2022, 8, x FOR PEER REVIEW 8 of 15
Figure 2. Resistance profiles of Aspergillus in each section. Fumigati and Nigri sections showed re-
sistance in all three classes of antifungals (amphotericin B, voriconazole and itraconazole). There is
no itraconazole resistance detected from the Flavi section.
Figure 3. Antifungal resistances of Aspergillus in each section (A) Flavi section, (B) Nigri section, (C)
Fumigati section and its correlation with CPA diagnosis.
There were 22 isolates in the Nigri section, consisting of 7 (32%) isolates of A. niger
sensu stricto and 15 (68%) isolates belonging to cryptic species (Supplementary Tables S1–
Figure 2.
Resistance profiles of Aspergillus in each section. Fumigati and Nigri sections showed
resistance in all three classes of antifungals (amphotericin B, voriconazole and itraconazole). There is
no itraconazole resistance detected from the Flavi section.

J. Fungi 2022,8, 411 8 of 14
J. Fungi 2022, 8, x FOR PEER REVIEW 8 of 15
Figure 2. Resistance profiles of Aspergillus in each section. Fumigati and Nigri sections showed re-
sistance in all three classes of antifungals (amphotericin B, voriconazole and itraconazole). There is
no itraconazole resistance detected from the Flavi section.
Figure 3. Antifungal resistances of Aspergillus in each section (A) Flavi section, (B) Nigri section, (C)
Fumigati section and its correlation with CPA diagnosis.
There were 22 isolates in the Nigri section, consisting of 7 (32%) isolates of A. niger
sensu stricto and 15 (68%) isolates belonging to cryptic species (Supplementary Tables S1–
Figure 3.
Antifungal resistances of Aspergillus in each section (
A
)Flavi section, (
B
)Nigri section,
(C)Fumigati section and its correlation with CPA diagnosis.
J. Fungi 2022, 8, x FOR PEER REVIEW 9 of 15
S4). The proportion of CPA and the non-CPA group from cryptic isolates is nearly the
same. Of these 15 cryptic species isolates, there were 7 (47%) isolates from CPA patients.
Meanwhile, A. niger sensu stricto classified as CPA was 57% (4/7) (Figure 4). Seven isolates
of cryptic Aspergillus from the Nigri section classified as CPA were from A. aculeatus (n =
2), A. neoniger (n = 2), A. tubingensis (n = 1), and A. brunneoviolaceus (n = 2). Meanwhile,
eight isolates were classified as the non-CPA consisting of A. welwitschiae (n = 1), A. tubi
(n = 2) and A. brunneoviolaceus (n = 5).
There was no azole resistance detected from CPA from cryptic isolates compared
with two isolates (A. tubingensis and A. welwitschiae) detected as resistance from the non-
CPA cryptic group. Amphotericin B resistance was observed in three isolates (A. tubingen-
sis, A. aculeatus, A. neoniger) from CPA cryptic isolates compared to two isolates from the
non-CPA cryptic group (A. brunneoviolaceus and A. tubingensis).
Figure 4. Aspergillus spp. isolates distribution based on chronic pulmonary aspergillosis (CPA) di-
agnosis.
4. Discussion
This is the first report of the clinical isolation of several cryptic species including A.
aculeatus, A. neoniger, A. brunneoviolaceus, A. welwitschiae, A. tubingensis and A. clavatus
from Indonesia. Several papers identified some of these cryptic isolates such as A. brunne-
oviolaceus (previously A. fijiensis), A. japonicus, A. tubingensis, A. carbonarius from the envi-
ronment in Indonesia [31,32]. Twenty-seven percent of Aspergillus isolates in this study
were classified as cryptic species. This rate is nearly the same as a multicenter study from
China which revealed that 21.3% of clinical isolates of Aspergillus belong to cryptic species
[16]. At 5-years follow-up, the mortality rate was 27% with two patients dying because of
CPA related to A. tubingiensis and Aspergillus sydowii [16]. There is no previous study
about cryptic species from CPA patients in Indonesia. However, a recent report showed
that 15.6% of invasive aspergillosis patients were infected by cryptic isolates [12].
Aspergillus aculeatus is mostly found in plants; however, previous studies recovered
A. aculeatus isolates from clinical specimens with many of them susceptible to antifungals
[9,33–35]. Two patients with CPA and A. aculeatus in our study had amphotericin B re-
sistant isolates. A. brunneoviolaceus has been previously described as an etiological cause
of CPA [16] and we found one CPA isolate in our study. The occurrence of these cryptic
species in our study revealed the diversity of fungal etiology of CPA in Indonesia.
Figure 4.
Aspergillus spp. isolates distribution based on chronic pulmonary aspergillosis (CPA) diagnosis.

J. Fungi 2022,8, 411 9 of 14
There was no azole resistance detected from CPA from cryptic isolates compared with
two isolates (A. tubingensis and A. welwitschiae) detected as resistance from the non-CPA
cryptic group. Amphotericin B resistance was observed in three isolates (A. tubingensis,
A. aculeatus,A. neoniger) from CPA cryptic isolates compared to two isolates from the
non-CPA cryptic group (A. brunneoviolaceus and A. tubingensis).
4. Discussion
This is the first report of the clinical isolation of several cryptic species including A.
aculeatus,A. neoniger,A. brunneoviolaceus,A. welwitschiae,A. tubingensis and A. clavatus
from Indonesia. Several papers identified some of these cryptic isolates such as A. brun-
neoviolaceus (previously A. fijiensis), A. japonicus,A. tubingensis,A. carbonarius from the
environment in Indonesia [
31
,
32
]. Twenty-seven percent of Aspergillus isolates in this study
were classified as cryptic species. This rate is nearly the same as a multicenter study
from China which revealed that 21.3% of clinical isolates of Aspergillus belong to cryptic
species [
16
]. At 5-years follow-up, the mortality rate was 27% with two patients dying
because of CPA related to A. tubingiensis and Aspergillus sydowii [16]. There is no previous
study about cryptic species from CPA patients in Indonesia. However, a recent report
showed that 15.6% of invasive aspergillosis patients were infected by cryptic isolates [12].
Aspergillus aculeatus is mostly found in plants; however, previous studies recovered A. ac-
uleatus isolates from clinical specimens with many of them susceptible to antifungals [
9
,
33
–
35
].
Two patients with CPA and A. aculeatus in our study had amphotericin B resistant isolates.
A. brunneoviolaceus has been previously described as an etiological cause of CPA [
16
] and
we found one CPA isolate in our study. The occurrence of these cryptic species in our study
revealed the diversity of fungal etiology of CPA in Indonesia.
This study revealed a discordance of molecular identification using three different
primers (ITS, beta tubulin, calmodulin) in the Nigri section. There was a significant number
of medically important strains from the Nigri section [
18
,
36
]. Additionally, the molecu-
lar analysis and genotyping of the Nigri section is difficult [
36
]. The Nigri section was
dominated by the cryptic species in our study (88%) as previously report in recent study
from Portugal (84%) [
17
]. Three isolates were identified as A. aculeatus by ITS and beta
tubulin, while calmodulin grouped the isolates as A. brunneoviolaceus. One isolate was
identified as A. niger by ITS, meanwhile beta tubulin and calmodulin grouped the isolate as
A. welwitschiae. Finally, one isolate was identified as A. flavus by ITS and beta tubulin, while
calmodulin grouped the isolate as A. tamarii. In this study we used calmodulin instead
of beta tubulin and ITS for the reference gene in cases with different results of species
identification in the Flavi and Nigri sections [24,25,37].
The discrepancies between ITS, beta tubulin, and calmodulin in some isolates might
be explained by the existence of a paralogue of the beta tubulin gene named tubC [
36
,
38
,
39
].
The paralogue has different intron numbers in the Nigri section [
38
] and forms two differ-
ent beta tubulin proteins in A. aculeatus and A. japonicus [
38
]. The isolates which contain
two or three beta tubulin genes appeared in different branches of the parsimony tree [
38
].
Ben2f/Bt2b were recommended to be used as primers instead of Bt2a to prevent discor-
dance in the molecular identification of the Nigri section [
39
]. Another explanation was the
presence of the mixed colonies of the Nigri section since it was difficult to distinguish differ-
ent species via microscopy. A. flavus and A. tamarii from the Flavi section are phenotypically
very similar, making it possible to have two different species on one plate [11,12].
Two isolates (068-BT and 069-BT) were identified as A. aculeatus with ITS and beta-
tubulin, while calmodulin showed the result as A. brunneviolaceus. We repeated the calmod-
ulin sequencing after re-examination of the morphology of the fungi microscopically to
exclude mixed culture cases in these two isolates. The second attempt of the calmodulin
sequencing revealed both of the species as A. aculeatus. Recent evidence suggests that
A. brunneviolaceus and A. aculeatus are genetically closely related [
40
,
41
]. Two strains of
A. brunneviolaceus were previously identified as A. aculeatus, all of them coming from the
same highly supported clade [
40
]. In addition, the MSP dendogram from MALDI-TOF MS

J. Fungi 2022,8, 411 10 of 14
clustered A. brunneviolaceus and A. aculeatus together while the phylogenetic tree based on
calmodulin clearly separated these two species. Calmodulin is recommended to distinguish
closely related species of Aspergilli [
24
,
25
]. Therefore, the final identification for 068-BT and
069-BT are A. brunneviolaceus.
One of the gold standards of antifungal susceptibility testing is CLSI broth micro-
dilution [
42
]. This method is labour intensive and not routinely used in our centre. We
used the disk diffusion method as this method is simple and shows excellent correlation
(
93.8–100%
) with the CLSI broth microdilution based on previous studies [
43
–
45
]. However,
the level of agreement between these methods was lower (66.7–87.5%) for amphotericin
B [
43
,
45
], possibly because broth dilution is not generally as accurate as agar-based meth-
ods. The main limitation of the present study is that we did not perform CLSI or EUCAST
methods to confirm the susceptibility profile findings due to resource constraints in Indone-
sia. A previous study showed a higher rate of amphotericin B resistance based on the disk
diffusion test compared to CLSI broth micro-dilution [43].
Amphotericin B showed a higher rate of resistant isolates compared to azoles, and
most of them were non cryptic isolates. All A. flavus isolates and 79% of A. fumigatus isolates
were resistant to amphotericin B. Two out of seven patients with A. flavus resistant isolates
met the criteria of CPA in our study. A previous study from Canada observed that 96.4%
(n= 195) of A. fumigatus isolates developed resistance to amphotericin B [
46
]. The antifungal
susceptibility profiles of A. flavus from our study were in line with a previous study,
which showed that A. flavus was generally less susceptible to amphotericin B compared to
A. fumigatus [
47
–
49
]. Goncalves et al. found 49.4% of A. flavus isolates to be amphotericin B
resistant [47].
The rate of itraconazole resistance in this study is 10% (6/59), slightly higher than
another study in CPA patients which showed 8% resistance after 12 months of itraconazole
therapy [
50
]. Similarly, voriconazole resistance is higher (14%) in this study than another
CPA study, which showed that 4% of patients developed resistance [
50
]. Most of the
azole-resistant isolates were A. fumigatus sensu stricto isolates. A remarkably high number
of resistant strains were detected from environmental isolates of Aspergillus in South East
Asia [51–53].
Amongst 59 isolates, it was found four isolates (7%) showed resistance to all three
antifungals included in this study. Three of them were A. fumigatus sensu stricto from three
CPA patients and one from A. niger sensu stricto from a non-CPA patient. Although it was
implied from our study that the cryptic species are more susceptible than the sensu stricto
species to antifungals, we identified seven resistant isolates from cryptic species. Three
cryptic isolates (A. tubingensis,A. aculeatus, and A. neoniger) from CPA patients showed
amphotericin B resistance. Another four patients with resistant isolates were from the
non-CPA groups: one A. brunneoviolaceus isolate was resistant to amphotericin B, one
A. tubingensis isolate was resistant to itraconazole, one A. welwitschiae isolate was resistant
to voriconazole and one A. tamarii isolate was resistant to amphotericin B. Cryptic species
frequently showed less resistance to antifungals than the sensu stricto species [54,55].
Although the resistance rate of Aspergillus was lower in cryptic species, the clinical
severity of the infections caused by these isolates were not known from our study. A previ-
ous study reported fatal invasive aspergillosis caused by a cryptic Aspergillus species [
56
].
A limitation of our study is the cryptic isolates belonged mostly to the Nigri section. The an-
tifungal susceptibility profiles of other cryptic species from different sections of Aspergillus
other than the Nigri section might indicate different results.
Data on the antifungal susceptibility of any clinical isolates of fungi in Indonesia are
very scarce. This is the first study reporting the antifungal susceptibility profile from CPA
patients in Indonesia. This study showed reduced susceptibility of CPA isolates against
itraconazole. This finding is concerning because itraconazole is a key antifungal agent
for aspergillosis, although some compounds are being investigated for the development
of new antifungal drug options [
57
–
59
]. It is likely that patients in this study never had
antifungal therapy because they were suspected to have post-tuberculosis lung disease.

J. Fungi 2022,8, 411 11 of 14
Azole resistance can be acquired without exposure to antifungal during azole therapy
but also from the environment, for example, after exposure to triazole fungicides [
60
–
62
].
In a large surveillance study from the Netherlands, 64% of patients with itraconazole
resistance never had prior azole treatment [
63
]. Further study is needed to investigate the
environmental Aspergillus isolates in Indonesia, their susceptibility profile and the presence
of resistance mutations. Studies on clinical outcomes of azole treatment in CPA in Indonesia
are urgently needed in order to understand the impact of the reported higher rates of azole
resistance in this population.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jof8040411/s1, Table S1. The antifungal susceptibility profiles
from the Fumigati section. Table S2. The antifungal susceptibility profiles from the Clavati section.
Table S3. The antifungal susceptibility profiles from the Flavi section. Table S4. The antifungal
susceptibility profiles from the Nigri section.
Author Contributions:
Conceptualization, A.R., A.B., R.W., D.W.D. and C.K.; methodology, A.A.,
A.R., W.S., H.W. and F.S.; formal analysis, A.R., A.A., F.S. and A.B.; investigation, A.R., A.A., W.S.,
R.W., A.E.F., R.A., R.S., H.W. and M.T.; data curation, A.R., F.S., A.A. and A.E.F.; writing—original
draft preparation, A.R., A.A. and F.S.; writing—review and editing, W.S., A.E.F., R.W., A.B., H.W.,
R.S., R.A., M.T., C.K. and D.W.D. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was partly funded by United Kingdom-Indonesia Joint Partnership on
Infectious Diseases (Medical Research Council, Newton Fund, Ristekdikti) with a grant number
NKB-282/UN2.RST/HKP.05.00/2020 and MR/S019898/1 and Universitas Indonesia through PUTI
Grant with contract number NKB-1555/UN2.RST/HKP.05.00/2020.
Institutional Review Board Statement:
The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by The Ethics Committee of the Faculty of Medicine,
Universitas Indonesia (ND 071/UN2.F1/ETIK/PPM.00.02/2021).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors gratefully acknowledge the doctors from Persahabatan National
Respiratory Referral Hospital and MH Thamrin hospital for their help with patient’s recruitment, the
staff of Parasitology Laboratory FMUI for laboratory works in Jakarta preparation.
Conflicts of Interest: The authors declared no conflict of interest.
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