Next Article in Journal
Antifungal Activities of Sulfur and Copper Nanoparticles against Cucumber Postharvest Diseases Caused by Botrytis cinerea and Sclerotinia sclerotiorum
Next Article in Special Issue
Marine Compounds with Anti-Candida sp. Activity: A Promised “Land” for New Antifungals
Previous Article in Journal
Culturable Yeast Diversity of Grape Berries from Vitis vinifera ssp. sylvestris (Gmelin) Hegi
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoF
Volume 8
Issue 4
10.3390/jof8040411
jof-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
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

by
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
1
Department of Parasitology, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia
2
Indonesia Pulmonary Mycoses Centre, Jakarta 10430, Indonesia
3
Magister Program of Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia
4
Department of Biology, Faculty of Mathematics and Natural Sciences (FMIPA), Universitas Indonesia, Depok 16424, Indonesia
5
Department of Parasitology, Faculty of Medicine, Universitas Kristen, Jakarta 13530, Indonesia
6
Manchester Fungal Infection Group, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M23 9LT, UK
7
Manchester Academic Health Science Centre, Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M23 9LT, UK
*
Author to whom correspondence should be addressed.
J. Fungi 2022, 8(4), 411; https://doi.org/10.3390/jof8040411
Submission received: 2 March 2022 / Revised: 8 April 2022 / Accepted: 12 April 2022 / Published: 16 April 2022
(This article belongs to the Special Issue Antifungal Drugs 2022)
Browse Figures
Versions Notes

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.

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 [4,5,6].
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 [9,10,11,12]. 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 [14,15,16]. 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 precipitation reagent phenol-chloroform-isoamylalcohol as previously described with modifications [19]. The species-specific identification of all isolates was examined by amplification of the ITS rDNA gene using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers [20], part of the benA gene using Bt2a (5′-GGTAACCAAATCGGTGCTGCTTCT-3′) and Bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) primers [21], partial CaM gene using cmd5 (5′-CCGAGTACAAGGAGGCCTTC-3′) and cmd6 (5′-CCGATAGAGGTCATAACGTGG-3′) 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 [24,25,26,27].

2.3. Antifungal Susceptibility Tests

Antifungal susceptibility tests were performed using the disk diffusion method. Suspension 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 µg), voriconazole (1 µg) and itraconazole (8 µg 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 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 Χ2 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.

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.

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.
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 excluded 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).
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. brunneoviolaceus (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. aculeatus isolates from clinical specimens with many of them susceptible to antifungals [9,33,34,35]. 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 molecular 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 different 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 discordance 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 different 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 calmodulin 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 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 [43,44,45]. 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 methods. 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 Indonesia. 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 [47,48,49]. 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 [51,52,53].
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 previous 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 antifungal 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 [57,58,59]. It is likely that patients in this study never had antifungal therapy because they were suspected to have post-tuberculosis lung disease. Azole resistance can be acquired without exposure to antifungal during azole therapy but also from the environment, for example, after exposure to triazole fungicides [60,61,62]. 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.

References

  1. Bongomin, F.; Gago, S.; Oladele, R.O.; Denning, D.W. Global and multi-national prevalence of fungal diseases—Estimate precision. J. Fungi 2017, 3, 57. [Google Scholar] [CrossRef] [PubMed]
  2. Denning, D.W.; Pleuvry, A.; Cole, D.C. Global burden of chronic pulmonary aspergillosis as a sequel to pulmonary tuberculosis. Bull. World Health Organ. 2011, 89, 864–872. [Google Scholar] [CrossRef] [PubMed]
  3. Wahyuningsih, R.; Adawiyah, R.; Sjam, R.; Prihartono, J.; Ayu, E.; Wulandari, T.; Rozaliyani, A.; Ronny, R.; Imran, D.; Tugiran, M.; et al. Serious fungal disease incidence and prevalence in Indonesia. Mycoses 2021, 64, 1203–1212. [Google Scholar] [CrossRef]
  4. Setianingrum, F.; Rozaliyani, A.; Syam, R.; Adawiyah, R.; Tugiran, M.; Sari, C.Y.I.; Burhan, E.; Wahyuningsih, R.; Rautemaa-Richardson, R.; Denning, D.W. Evaluation and comparison of automated and manual ELISA for diagnosis of chronic pulmonary aspergillosis (CPA) in Indonesia. Diagn. Microbiol. Infect. Dis. 2020, 98, 15124. [Google Scholar] [CrossRef] [PubMed]
  5. Setianingrum, F.; Rozaliyani, A.; Adawiyah, R.; Syam, R.; Tugiran, M.; Sari, C.Y.I.; Nandipinto, F.; Ramnath, J.; Arifin, A.R.; Handayani, D.; et al. A prospective longitudinal study of chronic pulmonary aspergillosis in pulmonary tuberculosis in Indonesia (APICAL). Thorax 2021. [Google Scholar] [CrossRef]
  6. Nguyen, N.T.B.; Le Ngoc, H.; Nguyen, N.V.; Van Dinh, L.; Van Nguyen, H.; Nguyen, H.T.; Denning, D.W. Chronic pulmonary aspergillosis situation among post tuberculosis patients in vietnam: An observational study. J. Fungi 2021, 7, 532. [Google Scholar] [CrossRef] [PubMed]
  7. Tone, K.; Suzuki, J.; Alshahni, M.M.; Kuwano, K.; Makimura, K. Species-specific detection of medically important aspergilli by a loop-mediated isothermal amplification method in chronic pulmonary aspergillosis. Med. Mycol. 2019, 57, 703–709. [Google Scholar] [CrossRef] [PubMed]
  8. Denning, D.W.; Page, I.D.; Chakaya, J.; Jabeen, K.; Jude, C.M.; Cornet, M.; Alastruey-izquierdo, A.; Bongomin, F.; Bowyer, P.; Chakrabarti, A.; et al. Case Definition of Chronic Pulmonary Aspergillosis in Resource-Constrained Settings. Emerg. Infect. Dis. 2018, 24, e171312. [Google Scholar] [CrossRef]
  9. Gautier, M.; Normand, A.C.; Ranque, S. Previously unknown species of Aspergillus. Clin. Microbiol. Infect. 2016, 22, 662–669. [Google Scholar] [CrossRef] [Green Version]
  10. Balajee, S.A.; Nickle, D.; Varga, J.; Marr, K.A. Molecular Studies Reveal Frequent Misidentification of Aspergillus fumigatus by Morphotyping. Eukaryot. Cell 2006, 5, 1705–1712. [Google Scholar] [CrossRef] [Green Version]
  11. Tam, E.W.T.; Chen, J.H.K.; Lau, E.C.L.; Ngan, A.H.Y.; Fung, K.S.C.; Lee, K.C.; Lam, C.W.; Yuen, K.Y.; Lau, S.K.P.; Woo, P.C.Y. Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: Characterization by internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-ti. J. Clin. Microbiol. 2014, 52, 1153–1160. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Rozaliyani, A.; Sedono, R.; Sjam, R.; Tugiran, M.; Adawiyah, R.; Setianingrum, F.; Jusuf, A.; Sungkar, S.; Hagen, F.; Meis, J.F.; et al. Molecular typing and antifungal susceptibility study of Aspergillus spp. in intensive care unit (ICU) patients in Indonesia. J. Infect. Dev. Ctries. 2021, 15, 1014–1020. [Google Scholar] [CrossRef] [PubMed]
  13. Houbraken, J.; Kocsub, S.; Visagie, C.M.; Yilmaz, N.; Meijer, M.; Kraak, B.; Hubka, V.; Bensch, K.; Samson, R.A.; Frisvad, J.C. Classification of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales): An overview of families, genera, subgenera, sections, series and species. Stud. Mycol. 2020, 95, 5–169. [Google Scholar] [CrossRef] [PubMed]
  14. Nedel, W.L.; Pasqualotto, A.C. Treatment of Infections by Cryptic Aspergillus Species. Mycopathologia 2014, 178, 441–445. [Google Scholar] [CrossRef]
  15. Alastruey-izquierdo, A.; Alcazar-Fuoli, L.; Rivero-Menéndez, O.; Ayats, J.; Castro, C.; García-Rodríguez, J.; Goterris-Bonet, L.; Ibáñez-Martínez, E.; Linares-Sicilia, M.J.; Martin-Gomez, M.T.; et al. Molecular Identification and Susceptibility Testing of Molds Isolated in a Prospective Surveillance of Triazole Resistance in Spain (FILPOP2 Study). Antimicrob. Agents Chemother. 2018, 62, e00358-18. [Google Scholar] [CrossRef] [Green Version]
  16. Tsang, C.C.; Tang, J.Y.M.; Ye, H.; Xing, F.; Lo, S.K.F.; Xiao, C.; Han, L.; Wu, A.K.L.; Ngan, A.H.Y.; Law, K.C.; et al. Rare/cryptic Aspergillus species infections and importance of antifungal susceptibility testing. Mycoses 2020, 63, 1283–1298. [Google Scholar] [CrossRef]
  17. Sabino, R.; Gonçalves, P.; Melo, A.M.; Sim, D.; Oliveira, M.; Francisco, M.; Viegas, C.; Carvalho, D.; Martins, C.; Ferreira, T.; et al. Trends on Aspergillus Epidemiology—Perspectives from a National Reference Laboratory Surveillance Program. J. Fungi 2021, 7, 28. [Google Scholar] [CrossRef]
  18. Bongomin, F.; Moore, C.B.; Masania, R.; Rowbotham, E.; Alastruey-Izquierdo, A.; Novak-Frazer, L.; Richardson, M.D. Sequence analysis of isolates of Aspergillus from patients with chronic and allergic aspergillosis reveals a spectrum of cryptic species. Future Microbiol. 2018, 13, 1557–1563. [Google Scholar] [CrossRef] [Green Version]
  19. Gontia-Mishra, I.; Tripathi, N.; Tiwari, S. A simple and rapid DNA extraction protocol for filamentous fungi efficient for molecular studies. Indian J. Biotechnol. 2014, 13, 536–539. [Google Scholar]
  20. White, T.; Bruns, T.; Lee, S.; Taylor, J. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Academic Press: New York, NY, USA, 1990; pp. 315–322. [Google Scholar]
  21. Glass, N.L.; Donaldson, G.C. Development of Primer Sets Designed for Use with the PCR To Amplify Conserved Genes from Filamentous Ascomycetes. Appl. Environ. Microbiol. 1995, 61, 1323–1330. [Google Scholar] [CrossRef] [Green Version]
  22. Hong, S.-B.; Go, S.-J.; Shin, H.-D.; Frisvad, J.C.; Samson, R.A. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia 2005, 97, 1316–1329. [Google Scholar] [CrossRef] [PubMed]
  23. Singh, B.K.; Nazaries, L.; Munro, S.; Anderson, I.C.; Campbell, C.D. Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community. Appl. Environ. Microbiol. 2006, 72, 7278–7285. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Samson, R.A.; Visagie, C.M.; Houbraken, J.; Hong, S.B.; Hubka, V.; Klaassen, C.H.W.; Perrone, G.; Seifert, K.A.; Susca, A.; Tanney, J.B.; et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud. Mycol. 2014, 78, 141–173. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Ashtiani, N.M.; Kachuei, R.; Yalfani, R.; Harchegani, A.B.; Nosratabadi, M. Identification of Aspergillus sections Flavi, Nigri, and Fumigati and their differentiation using specific primers. Le Infez. Med. 2017, 25, 127–132. [Google Scholar]
  26. Yang, R.H.; Su, J.H.; Shang, J.J.; Wu, Y.Y.; Li, Y.; Bao, D.P.; Yao, Y.J. Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing. PLoS ONE 2018, 13, e0206428. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Bellemain, E.; Carlsen, T.; Brochmann, C.; Coissac, E.; Taberlet, P.; Kauserud, H. ITS as an environmental DNA barcode for fungi: An in silico approach reveals potential PCR biases. World J. Microbiol. Biotechnol. 2010, 31, 189–999. [Google Scholar] [CrossRef] [Green Version]
  28. Espinel-Ingroff, A.; Arthington-Skaggs, B.; Iqbal, N.; Ellis, D.; Pfaller, M.A.; Messer, S.; Rinaldi, M.; Fothergill, A.; Gibbs, D.L.; Wang, A. Multicenter evaluation of a new disk agar diffusion method for susceptibility testing of filamentous fungi with voriconazole, posaconazole, itraconazole, amphotericin B, and caspofungin. J. Clin. Microbiol. 2007, 45, 1811–1820. [Google Scholar] [CrossRef] [Green Version]
  29. Al-Wathiqi, F.; Ahmad, S.; Khan, Z. Molecular identification and antifungal susceptibility profile of Aspergillus flavus isolates recovered from clinical specimens in Kuwait. BMC Infect. Dis. 2013, 13, 126. [Google Scholar] [CrossRef] [Green Version]
  30. Espinel-Ingroff, A.; Canton, E.; Fothergill, A.; Ghannoum, M.; Johnson, E.; Jones, R.N.; Ostrosky-Zeichner, L.; Schell, W.; Gibbs, D.L.; Wang, A.; et al. Quality control guidelines for amphotericin B, itraconazole, posaconazole, and voriconazole disk diffusion susceptibility tests with nonsupplemented Mueller-Hinton agar (CLSI M51-A document) for nondermatophyte filamentous fungi. J. Clin. Microbiol. 2011, 49, 2568–2571. [Google Scholar] [CrossRef] [Green Version]
  31. Varga, J.; Frisvad, J.C.; Kocsubé, S.; Brankovics, B.; Tóth, B.; Szigeti, G.; Samson, R.A.; Varga, A.; Frisvad, A. New and revisited species in Aspergillus section Nigri Extrolite analysis. Stud. Mycol. 2011, 69, 1–17. [Google Scholar] [CrossRef] [Green Version]
  32. Varga, J.; Kevei, F.; Vagvogyi, C.; Vriesema, A.; Croft, H. Double-stranded RNA mycoviruses in section Nigri of the Aspergillus genus. Can. J. Microbiol. 1994, 40, 325–329. [Google Scholar] [CrossRef] [PubMed]
  33. Treviño-Rangel, R.D.J.; Villanueva-Lozano, H.; Bonifaz, A.; Castañón-Olivares, L.R.; Andrade, A.; Becerril-García, M.A.; Martínez-Reséndez, M.F.; Ayala-Gaytán, J.; Montoya, A.M.; González, G.M. Species distribution and antifungal susceptibility patterns of Aspergillus isolates from clinical specimens and soil samples in Mexico. Med. Mycol. 2021, 59, 1006–1014. [Google Scholar] [CrossRef] [PubMed]
  34. Arabatzis, M.; Kambouris, M.; Kyprianou, M.; Chrysaki, A.; Foustoukou, M.; Kanellopoulou, M.; Kondyli, L.; Kouppari, G.; Koutsia-Karouzou, C.; Lebessi, E.; et al. Polyphasic identification and susceptibility to seven antifungals of 102 Aspergillus isolates recovered from immunocompromised hosts in Greece. Antimicrob. Agents Chemother. 2011, 55, 3025–3030. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Hendrickx, M.; Beguin, H.; Detandt, M. Genetic re-identification and antifungal susceptibility testing of Aspergillus section Nigri strains of the BCCM/IHEM collection. Mycoses 2012, 55, 148–155. [Google Scholar] [CrossRef] [PubMed]
  36. D’hooge, E.; Becker, P.; Stubbe, D.; Normand, A.C.; Piarroux, R.; Hendrickx, M. Black aspergilli: A remaining challenge in fungal taxonomy? Med. Mycol. 2019, 57, 773–780. [Google Scholar] [CrossRef]
  37. Alshehri, B.; Palanisamy, M. Evaluation of molecular identification of Aspergillus species causing fungal keratitis. Saudi J. Biol. Sci. 2020, 27, 751–756. [Google Scholar] [CrossRef]
  38. Peterson, S.W. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia 2008, 100, 205–226. [Google Scholar] [CrossRef]
  39. Hubka, V.; Kolarik, M. β-tubulin paralogue tubC is frequently misidentified as the benA gene in Aspergillus section Nigri taxonomy: Primer specificity testing and taxonomic consequences. Persoonia 2012, 29, 1–10. [Google Scholar] [CrossRef] [Green Version]
  40. Jurjevic, Ž.; Peterson, S.W.; Stea, G.; Solfrizzo, M.; Varga, J.; Hubka, V.; Perrone, G. Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus 2012, 3, 159–173. [Google Scholar] [CrossRef]
  41. Masih, A.; Singh, P.K.; Kathuria, S.; Agarwal, K.; Meis, J.F.; Chowdhary, A. Identification by molecular methods and matrix-assisted laser desorption ionization-time of flight mass spectrometry and antifungal susceptibility profiles of clinically significant rare aspergillus species in a referral chest hospital in Delhi, India. J. Clin. Microbiol. 2016, 54, 2354–2364. [Google Scholar] [CrossRef] [Green Version]
  42. Lass-Flörl, C. Susceptibility testing in Aspergillus species complex. Clin. Microbiol. Infect. 2014, 20, 49–53. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Hassan, R.M.; Ismail, D.K.; Elkholy, Y.S. Comparison of e test and disc diffusion methods for susceptibility testing of filamentous fungi; experience of a routine lab. Arch. Clin. Infect. Dis. 2018, 13, e57889. [Google Scholar] [CrossRef] [Green Version]
  44. Serrano, M.C.; Ramírez, M.; Morilla, D.; Valverde, A.; Chávez, M.; Espinel-Ingroff, A.; Claro, R.; Fernández, A.; Almeida, C.; Martín-Mazuelos, E. A comparative study of the disc diffusion method with the broth microdilution and Etest methods for viroconazole susceptibility testing of Aspergillus spp. J. Antimicrob. Chemother. 2004, 53, 739–742. [Google Scholar] [CrossRef] [PubMed]
  45. Gupta, P.; Khare, V.; Kumar, D.; Ahmad, A.; Banerjee, G.; Singh, M. Comparative evaluation of disc diffusion and E-test with broth micro-dilution in Susceptibility testing of amphotericin B, voriconazole and caspofungin against clinical Aspergillus isolates. J. Clin. Diagnostic Res. 2015, 9, DC01–DC04. [Google Scholar] [CrossRef]
  46. Ashu, E.E.; Korfanty, G.A.; Samarasinghe, H.; Pum, N.; You, M.; Yamamura, D.; Xu, J. Widespread amphotericin B-resistant strains of aspergillus fumigatus in Hamilton, Canada. Infect. Drug Resist. 2018, 11, 1549–1555. [Google Scholar] [CrossRef] [Green Version]
  47. Gonçalves, S.S.; Stchigel, A.M.; Cano, J.; Guarro, J.; Colombo, A.L. In vitro antifungal susceptibility of clinically relevant species belonging to Aspergillus section Flavi. Antimicrob. Agents Chemother. 2013, 57, 1944–1947. [Google Scholar] [CrossRef] [Green Version]
  48. Diekema, D.; Messer, S.; Hollis, R.; Jones, R.; Pfaller, M. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi. J. Clin. Microbiol. 2003, 41, 3623–3626. [Google Scholar] [CrossRef] [Green Version]
  49. Guinea, J.; Peláez, T.; Alcalá, L.; Ruiz-Serrano, M.J.; Bouza, E. Antifungal susceptibility of 596 Aspergillus fumigatus strains isolated from outdoor air, hospital air, and clinical samples: Analysis by site of isolation. Antimicrob. Agents Chemother. 2005, 49, 3495–3497. [Google Scholar] [CrossRef] [Green Version]
  50. Bongomin, F.; Harris, C.; Hayes, G.; Kosmidis, C.; Denning, D.W. Twelve-month clinical outcomes of 206 patients with chronic pulmonary aspergillosis. PLoS ONE 2018, 13, e0193732. [Google Scholar] [CrossRef] [Green Version]
  51. van Rhijn, N.; Denning, D.W. Is an azole-resistant Aspergillus hotspot emerging in South-East Asia? Environ. Microbiol. 2021, 23, 7275–7277. [Google Scholar] [CrossRef]
  52. My, D.N.T.; Van, L.T.; Linh, T.H.K.; Tuyen, N.P.; Phuong, N.T.; Thu Anh, N.; Lan, N.P.H.; Ngoc, N.T.B.; Fisher, M.C.; Rhodes, J.; et al. Unprecedented Prevalence of Azole-Resistant Aspergillus fumigatus Identified in the Environment of Vietnam, with Marked Variability by Land Use Type. SSRN Electron. J. 2020. [Google Scholar] [CrossRef]
  53. Duong, T.M.N.; Nguyen, P.T.; Van Le, T.; Nguyen, H.L.P.; Nguyen, B.N.T.; Nguyen, B.P.T.; Nguyen, T.A.; Chen, S.C.A.; Barrs, V.R.; Halliday, C.L.; et al. Drug-resistant aspergillus flavus is highly prevalent in the environment of Vietnam: A new challenge for the management of aspergillosis? J. Fungi 2020, 6, 296. [Google Scholar] [CrossRef] [PubMed]
  54. Pinto, E.; Monteiro, C.; Maia, M.; Faria, M.A.; Lopes, V.; Lameiras, C.; Pinheiro, D. Aspergillus species and antifungals susceptibility in clinical setting in the north of Portugal: Cryptic species and emerging azoles resistance in A. fumigatus. Front. Microbiol. 2018, 9, 656. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Zoran, T.; Sartori, B.; Sappl, L.; Aigner, M.; Sánchez-Reus, F.; Rezusta, A.; Chowdhary, A.; Taj-Aldeen, S.J.; Arendrup, M.C.; Oliveri, S.; et al. Azole-resistance in Aspergillus terreus and related species: An emerging problem or a rare Phenomenon? Front. Microbiol. 2018, 9, 516. [Google Scholar] [CrossRef] [PubMed]
  56. Chrenkova, V.; Hubka, V.; Cetkovsky, P.; Kouba, M.; Weinbergerova, B.; Lyskova, P.; Hornofova, L.; Hubacek, P. Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to Aspergillus sublatus, a Cryptic Species of A. nidulans. Mycopathologia 2018, 183, 423–429. [Google Scholar] [CrossRef]
  57. Kosmidis, C.; Muldoon, E.G. Challenges in the management of chronic pulmonary aspergillosis. Med. Mycol. 2017, 55, 63–68. [Google Scholar] [CrossRef] [Green Version]
  58. Campione, E.; Gaziano, R.; Doldo, E.; Marino, D.; Falconi, M.; Iacovelli, F.; Tagliaferri, D.; Pacello, L.; Bianchi, L.; Lanna, C.; et al. Antifungal effect of all-trans retinoic acid against aspergillus fumigatus in vitro and in a pulmonary aspergillosis in vivo model. Antimicrob. Agents Chemother. 2021, 65, e01874-20. [Google Scholar] [CrossRef]
  59. Cosio, T.; Gaziano, R.; Zuccari, G.; Costanza, G.; Grelli, S.; Di Francesco, P.; Bianchi, L.; Campione, E. Retinoids in fungal infections: From bench to bedside. Pharmaceuticals 2021, 14, 962. [Google Scholar] [CrossRef]
  60. Snelders, E.; Camps, S.M.T.; Karawajczyk, A.; Schaftenaar, G.; Kema, G.H.J.; van der Lee, H.A.; Klaassen, C.H.; Melchers, W.J.G.; Verweij, P.E. Triazole fungicides can induce cross-resistance to medical triazoles in Aspergillus fumigatus. PLoS ONE 2012, 7, e31801. [Google Scholar] [CrossRef] [Green Version]
  61. Denning, D.W.; Park, S.; Lass-Florl, C.; Fraczek, M.G.; Kirwan, M.; Gore, R.; Smith, J.; Bueid, A.; Moore, C.B.; Bowyer, P.; et al. High-frequency triazole resistance found in nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease. Clin. Infect. Dis. 2011, 52, 1123–1129. [Google Scholar] [CrossRef] [Green Version]
  62. Vazquez, J.A.; Manavathu, E.K. Are the TR 46 /Y121F/T289A Mutations in Azole-Resistant Aspergillosis Patient Acquired or Environmental?". Antimicrob. Agents Chemother. 2016, 60, 3259–3260. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. van der Linden, J.W.M.; Snelders, E.; Kampinga, G.A.; Rijnders, B.J.A.; Mattsson, E.; Debets-Ossenkopp, Y.J.; Kuijper, E.J.; van Tiel, F.H.; Melchers, W.J.G.; Verweij, P.E. Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, 2007-2009. Emerg. Infect. Dis. 2011, 17, 1846–1854. [Google Scholar] [CrossRef] [PubMed]
Jof 08 00411 g001 550
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).
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).
Jof 08 00411 g001
Jof 08 00411 g002 550
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.
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.
Jof 08 00411 g002
Jof 08 00411 g003 550
Figure 3. Antifungal resistances of Aspergillus in each section (A) Flavi section, (B) Nigri section, (C) Fumigati section and its correlation with CPA diagnosis.
Figure 3. Antifungal resistances of Aspergillus in each section (A) Flavi section, (B) Nigri section, (C) Fumigati section and its correlation with CPA diagnosis.
Jof 08 00411 g003
Jof 08 00411 g004 550
Figure 4. Aspergillus spp. isolates distribution based on chronic pulmonary aspergillosis (CPA) diagnosis.
Figure 4. Aspergillus spp. isolates distribution based on chronic pulmonary aspergillosis (CPA) diagnosis.
Jof 08 00411 g004
Table
Table 1. Aspergillus identification according to the section of the isolates recovered and patient’s clinical features included in this study.
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-ValueCryptic
(n = 16)		Sensu Stricto
(n = 43)		p-Value
Section
Fumigati28 (49%)15 (54%)13 (43%)0.5190 (0%)28 (65%)<0.005
Clavati1 (2%)1 (3%)0 (0%)0.4920 (0%)1 (2%)1
Flavi8 (14%)2 (7%)6 (20%)0.2541 (6%)7 (16%)0.427
Nigri22 (37%)11 (38%)11 (37%)0.92015 (93%)7 (16%)<0.005
Symptoms
Haemoptysis36 (61%)23 (79%)13 (43%)0.00512 (75%)24 (56%)0.236
Massive haemoptysis19 (32%)12 (41%)7 (23%)0.1707 (44%)12 (28%)0.348
Recurrent haemoptysis15 (25%)11 (38%)4 (13%)0.0393 (19%)12 (28)0.738
Abbreviations: CPA: chronic pulmonary aspergillosis. The grey background highlighted the cryptic and sensu stricto variables and their p-values.
Table
Table 2. Molecular identification of all isolates.
Table 2. Molecular identification of all isolates.
NoSectionsSample CodeFinal IDGenes Used for IDDiagnosisAmphotericin Itraconazole Voriconazole
1Fumigati006-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantResistantSusceptible
2012-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantSusceptibleIntermediate
3013-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleIntermediate
4014-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantSusceptibleSusceptible
5015-BTA. fumigatus sensu strictoITS, BenA, CaMCPASusceptibleSusceptibleSusceptible
6018-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleResistant
7019-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleIntermediate
8020-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantIntermediateResistant
9022-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleResistant
10023-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantSusceptibleSusceptible
11025-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
12026-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleIntermediate
13027-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleResistant
14036-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleIntermediate
15048-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPASusceptibleResistantIntermediate
16069-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
17080-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleResistant
18083-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantSusceptibleSusceptible
19084-BTA. fumigatus sensu strictoITS, BenA, CaMCPASusceptibleResistantResistant
20085-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleResistant
21091-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantResistantResistant
22092-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantResistantResistant
23094-BTA. fumigatus sensu strictoITS, BenA, CaMCPASusceptibleSusceptibleResistant
24097-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantResistantResistant
25101-BTA. fumigatus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
26103-BTA. fumigatus sensu strictoITS, BenA, CaMCPAResistantIntermediateSusceptible
27109-BTA. fumigatus sensu strictoITS, BenA, CaMCPAIntermediateIntermediateSusceptible
28110-BTA. fumigatus sensu strictoITS, BenA, CaMCPAIntermediateSusceptibleResistant
29Clavati064-BTA. clavatus sensu strictoITS, BenA, CaMCPASusceptibleSusceptibleResistant
30Flavi052-BTA. tamariiCaMNon-CPAResistantSusceptibleIntermediate
31066-BTA. flavus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
32069-BTA. flavus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
33071-BTA. flavus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleIntermediate
34080-BTA. flavus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
35086-BTA. flavus sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleResistant
36092-BTA. flavus sensu strictoITS, BenA, CaMCPAResistantSusceptibleSusceptible
37103-BTA. flavus sensu strictoITS, BenA, CaMCPAResistantSusceptibleIntermediate
38Nigri057-BTA. niger sensu strictoITS, BenA, CaMNon-CPAResistantResistantResistant
39083-BTA. niger sensu strictoITS, BenA, CaMCPAResistantSusceptibleSusceptible
40064-BTA. niger sensu strictoITS, BenA, CaMCPAResistantResistantIntermediate
41074-BTA. niger sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
42079-BTA. niger sensu strictoITS, BenA, CaMCPAIntermediateSusceptibleSusceptible
43085-BTA. niger sensu strictoITS, BenA, CaMNon-CPAResistantSusceptibleSusceptible
44103-BTA. niger sensu strictoITS, BenA, CaMCPASusceptibleSusceptibleSusceptible
45076-BTA. welwitschiaeBenA, CaMNon-CPASusceptibleSusceptibleResistant
46099-BTA. tubingensisBenA, CaMNon-CPASusceptibleSusceptibleSusceptible
47101-BTA. tubingensisBenA, CaMCPAResistantSusceptibleSusceptible
48068-BTA. brunneoviolaceusCaMNon-CPAIntermediateSusceptibleSusceptible
49073-BTA. aculeatusITS, BenA, CaMCPAIntermediateSusceptibleSusceptible
50100-BTA. aculeatusITS, BenA, CaMCPAResistantSusceptibleSusceptible
51060-BTA. brunneoviolaceusCaMCPASusceptibleSusceptibleSusceptible
52006-BTA. brunneoviolaceusCaMCPAIntermediateSusceptibleSusceptible
53061-BTA. brunneoviolaceusCaMNon-CPASusceptibleSusceptibleSusceptible
54062-BTA. brunneoviolaceusCaMNon-CPAIntermediateSusceptibleSusceptible
55069-BTA. brunneoviolaceusCaMNon-CPASusceptibleSusceptibleSusceptible
56098-BTA. brunneoviolaceusCaMNon-CPAResistantSusceptibleSusceptible
57086-BTA. tubingensisITS, BenA, CaMNon-CPAResistantResistantIntermediate
58089-BTA. neonigerCaMCPAIntermediateSusceptibleSusceptible
59097-BTA. neonigerCaMCPAResistantIntermediateIntermediate
Abbreviations: ID: identification; CPA: chronic pulmonary aspergillosis; ITS: internal transcribed spacer; CaM: calmodulin; benA: and β-tubulin (benA).
Table
Table 3. Antifungal susceptibility profiles of Aspergillus isolates using disk diffusion method.
Table 3. Antifungal susceptibility profiles of Aspergillus isolates using disk diffusion method.
All
(n = 59)		CPA
(n = 29)		Non-CPA
(n = 30)		p-ValueCryptic
(n = 16)		Sensu Stricto
(n = 43)		p-Value
Amphotericin B
Zone of inhibition (range)0–220–222–21.1 2–21.10–22
Mean of inhibition zone ± SD10.8 ± 4.811.4 ± 4.910.3 ± 4.80.38113.9 ± 4.69.7 ± 4.50.002
Susceptible11 (19%)6 (21%)5 (17%)0.7485 (31%)6 (14%)0.149
Intermediate8 (14%)6 (21%)2 (7%)0.1455 (31%)3 (7%)0.028
Resistant40 (68%)17 (59%)23 (77%)0.1706 (38%)34 (79%)0.002
Voriconazole
Zone of inhibition (range)2–442–447–41.3 11.8–41.32–44
Mean of inhibition zone ± SD18.9 ± 9.219.6 ± 10.718.1 ± 7.70.54126.4 ± 10.416.1 ± 7.1<0.005
Susceptible31 (53%)17 (59%)14 (47%)0.35812 (75%)19 (44%)0.035
Intermediate12 (20%)4 (14%)8 (27%)0.3333 (19%)9 (21%)1
Resistant16 (27%)8 (28%)8 (27%)11 (6%)15 (35%)0.045
Itraconazole
Zone of inhibition (range)2–372–3711–32 11–372–30
Mean of inhibition zone ± SD19.3 ± 6.219.3 ± 7.519.2 ± 4.80.93922.7 ± 7.318 ± 5.30.009
Susceptible46 (78%)19 (66%)27 (90%)0.03014 (88%)32 (74%)0.481
Intermediate4 (7%)4 (14%)0 (0%)0.0521 (6%)3 (7%)1
Resistant9 (15%)6 (21%)3 (10%)0.2991 (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.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

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

AMA Style

Rozaliyani A, Abdullah A, Setianingrum F, Sjamsuridzal W, Wahyuningsih R, Bowolaksono A, Fatril AE, 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. Journal of Fungi. 2022; 8(4):411. https://doi.org/10.3390/jof8040411

Chicago/Turabian Style

Rozaliyani, Anna, Asriyani Abdullah, Findra Setianingrum, Wellyzar Sjamsuridzal, Retno Wahyuningsih, Anom Bowolaksono, Ayu Eka Fatril, Robiatul Adawiyah, Mulyati Tugiran, Ridhawati Syam, and et al. 2022. "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" Journal of Fungi 8, no. 4: 411. https://doi.org/10.3390/jof8040411

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop