Abstract
Seventy-two A. fumigatus clinical isolates from China were investigated for azole resistance based on mutations of cyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.
TEXT
Invasive aspergillosis, caused by Aspergillus fumigatus, is often rapidly progressive and can be life-threatening (1, 2). Successful treatment relies almost exclusively on triazoles, such as itraconazole (ITR), posaconazole (POS), and voriconazole (VOR) (1, 3). However, prolonged use of triazoles increases the likelihood of the development of drug resistance (4–8). Since the first report of itraconazole-resistant A. fumigatus in 1997, (6) azole-resistant A. fumigatus has been widely identified in clinical isolates from all over the world (4, 8–16). Meanwhile, azole-resistant A. fumigatus has also been discovered in the wild (16–19). The percentages of azole-resistant A. fumigatus reported from 2000 to 2014 in different study cohorts with various populations and geographic locations range from 2.1% to 8% (15, 20, 21). An extremely high percentage was noted in one study carried out in Britain, where resistant A. fumigatus were found to have a subpopulation of >20% (12).
The mechanism for azole action is 14α-demethylase, a product of the gene cyp51A, that figures in fungal ergosterol biosynthesis. So far, the majority of A. fumigatus azole-resistant strains have been associated with mutations of cyp51A, in which the point mutation alone leads to amino acid substitutions at positions G54, G138, M220, G432, and G448 (13, 22–25) or combines with the tandem repeat sequence in the cyp51A promoter. The latter has been widely reported as the TR34/L98H and TR46/Y121F/T289A mutations (26–28). In a global epidemiological survey (ARTEMIS) (29), eight strains of itraconazole-resistant A. fumigatus with a TR34/L98H mutation were isolated from Hangzhou (China). At the same time, Chen et al. identified the M220I mutation in an itraconazole-resistant A. fumigatus from China (24). In the present study, we continue our investigations of azole-resistant A. fumigatus isolated from clinical settings. Seventy-two A. fumigatus cultures were obtained from patients with symptoms of lung or respiratory infections during the 2011-to-2014 period from four cities in South Central China: Zhengzhou, Nanjing, Shanghai, and Fuzhou. Isolates were identified as A. fumigatus by both microscopic and culture morphology, by temperature tolerance, and by β-tubulin sequencing (22, 23, 30–32). Susceptibility to triazole drugs was assayed using a broth microdilution method according to the EUCAST protocol E. DEF 9.1 (33). The interpretive criteria used were as follows: for itraconazole, susceptible was ≤1 mg/liter, intermediate was 2 mg/liter, and resistant was >2 mg/liter; for posaconazole, susceptible was ≤0.125 mg/liter, intermediate was 0.25 mg/liter, and resistant was >0.25 mg/liter; and for voriconazole, susceptible was ≤1 mg/liter, intermediate was 2 mg/liter, and resistant was >2 mg/liter (34). We choose here an ITR- and VOR-centered strategy for preliminary screening for azole resistance in our A. fumigatus isolates because most POS-resistant strains also showed reduced susceptibility to ITR (10, 12, 35). Therefore, the MIC values for POS were tested only when an isolate had an ITR MIC greater than the breakpoint. The entire cyp51A gene and its promoter region were amplified and sequenced using the primers listed in Table 1.
TABLE 1.
Primers used in cyp51A PCR amplification and sequencing
| Primer name | Primer sequence |
|---|---|
| P450-A1a | 5′-ATGGTGCCGATGCTATGG |
| P450-A2a | 5′-CTGTCTCACTTGGATGTG |
| CYP-ad1 | 5′-AATCCGTAAGGTTTCACGAATA |
| CYP-ad2 | 5′-AGTTCATCAAGTACGGCTTG |
| CYP-S-REV | 5′-CACGCAAAGAAGAACTTGTAG |
| CYP-X-FW | 5′-TATGTCAACCTTGGTGTGATTCTG |
| CYP51A-5b | 5′-ATAATCGCAGCACCACTTCAGA |
| CYP51A-7b | 5′-CCTTGTCACCGTCAAGACGG |
| CYP51A-6b | 5′-TGGATGTGTTTTTCGACCGCTT |
| CYP51A-8b | 5′-CGGATCGGACGTGGTGTATG |
| 1-1R | 5′-AAGACCATTGGCGGTTCTGT |
This study project was reviewed and approved by the medical research ethics committee of Peking Union Medical College, in accordance with the principles of the Declaration of Helsinki.
The overall MICs of ITR and VOR for the 72 isolates are presented in Table 2. Four isolates were identified as azole resistant from a total of 72 A. fumigatus isolates, of which three are highly resistant to ITR and harbor the TR34/L98H mutation that was described previously by Lockhart et al. (29) In their ARTEMIS study, a total of eight azole-resistant isolates (itraconazole MIC of ≥16 mg/liter) were recovered from Hangzhou, and all exhibited the TR34/L98H mutation together with S297T and F495I. As did Lockhart et al., we found that the TR34/L98H mutation is still a predominant mutation which is quite common in Europe and some other Asian countries (10, 15, 17, 18, 20, 36, 37). Some evidence suggests that this resistant genotype is likely to have arisen as a side effect of widespread use of agricultural fungicides. The TR34/L98H mutation may also take hold and flourish in the wild and may spread throughout the world over time.
TABLE 2.
MICs for triazoles and corresponding mutation type for cyp51Aa
| Strain identification | MIC (mg/liter) |
Genotype of cyp51A | Specimen type | Geographical origin | ||
|---|---|---|---|---|---|---|
| ITR | VOR | POS | ||||
| Control strains | ||||||
| MYA3626 | 0.5 | 0.5 | 0.0625 | |||
| AF293 | 0.25 | 0.0625 | 0.0313 | |||
| Resistant isolates | ||||||
| Shhs18 | 4 | 2 | 0.5 | G432A | Sputum | Shanghai |
| Shjt40 | 16 | 1 | 0.5 | TR34/L98H | Sputum | Shanghai |
| Shjt42b | 16 | 2 | 0.5 | TR34/L98H/S297T/F495I | Sputum | Fuzhou |
| Nj21-76 | 16 | 0.25 | 0.25 | TR34/L98H/S297T/F495I | Sputum | Nanjing |
| Susceptible isolates | ||||||
| Shjt1 | 0.25 | 1 | ND | Sputum | Shanghai | |
| Shjt2 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Shjt3 | 0.5 | 1 | ND | Sputum | Shanghai | |
| Shjt4 | 0.5 | 1 | ND | Sputum | Shanghai | |
| Shjt5 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Shjt6 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Shjt7 | 0.25 | 0.25 | ND | N248K | Sputum | Shanghai |
| Shjt8 | 0.5 | 0.25 | ND | Sputum | Shanghai | |
| Shjt9 | 0.5 | 0.5 | ND | D343N | BALF | Shanghai |
| Shjt11 | 0.25 | 0.5 | ND | Throat swabs | Shanghai | |
| Shjt12 | 0.5 | 0.25 | ND | Sputum | Shanghai | |
| Shjt13 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Shjt14 | 1 | 0.25 | ND | Sputum | Shanghai | |
| Shjt15 | 0.5 | 0.125 | ND | N248K | Sputum | Fuzhou |
| Shjt16 | 0.5 | 0.25 | ND | Sputum | Fuzhou | |
| Shjt17 | 1 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt18 | 0.125 | 1 | ND | Sputum | Fuzhou | |
| Shjt19 | 0.5 | 0.25 | ND | Sputum | Fuzhou | |
| Shjt21 | 0.5 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt22 | 1 | 0.25 | ND | Sputum | Fuzhou | |
| Shjt23 | 0.5 | 0.25 | ND | Sputum | Fuzhou | |
| Shjt24 | 0.5 | 0.25 | ND | D343N | Sputum | Fuzhou |
| Shjt25 | 1 | 1 | ND | Throat swabs | Fuzhou | |
| Shjt26 | 0.5 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt27 | 0.5 | 0.5 | ND | N248K | Sputum | Shanghai |
| Shjt28 | 0.5 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt29 | 1 | 0.25 | ND | BALF | Fuzhou | |
| Shjt30 | 0.25 | 1 | ND | Sputum | Fuzhou | |
| Shjt31 | 1 | 0.5 | ND | N248K | Sputum | Fuzhou |
| Shjt32 | 0.125 | 0.5 | ND | Sputum | Shanghai | |
| Shjt33 | 1 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt34 | 0.5 | 0.25 | ND | Sputum | Shanghai | |
| Shjt35 | 0.5 | 1 | ND | Throat swabs | Shanghai | |
| Shjt36 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Shjt37 | 0.5 | 0.5 | ND | N248K | Sputum | Shanghai |
| Shjt38 | 1 | 0.25 | ND | Sputum | Shanghai | |
| Shjt41 | 1 | 0.5 | ND | Sputum | Shanghai | |
| Shjt42a | 1 | ND | ND | Sputum | Fuzhou | |
| Shjt43 | 1 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt44 | 1 | 0.5 | ND | Sputum | Fuzhou | |
| Shjt45 | 0.5 | 0.5 | ND | N248K | Sputum | Shanghai |
| Shjt47 | 1 | 0.25 | ND | Sputum | Shanghai | |
| Shjt48 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Shjt49 | 1 | 0.25 | ND | N248K | Sputum | Shanghai |
| Shjt50 | 1 | 0.5 | ND | Sputum | Shanghai | |
| Shjt51 | 1 | 0.25 | ND | Sputum | Shanghai | |
| Shhs1 | 0.25 | 1 | ND | Sputum | Shanghai | |
| Shhs2 | 0.5 | 1 | ND | Sputum | Shanghai | |
| Shhs5 | 1 | 0.25 | ND | Throat swabs | Shanghai | |
| Shhs8 | 1 | 1 | ND | Sputum | Shanghai | |
| Shhs9 | 0.25 | 0.25 | ND | Sputum | Shanghai | |
| Shhs11 | 0.5 | 0.25 | ND | Sputum | Shanghai | |
| Shhs12 | 0.125 | 0.5 | ND | Sputum | Shanghai | |
| Shhs13 | 0.5 | 1 | ND | Sputum | Shanghai | |
| Shhs14 | 0.25 | 1 | ND | Sputum | Shanghai | |
| Shhs15 | 1 | 0.5 | ND | Sputum | Shanghai | |
| Shhs16 | 0.5 | 0.25 | ND | Sputum | Shanghai | |
| Shhs17 | 0.5 | 0.5 | ND | Sputum | Shanghai | |
| Nj7-20 | 1 | 0.25 | ND | Sputum | Nanjing | |
| Nj9-72 | 1 | 0.25 | ND | BALF | Nanjing | |
| Nj10-106 | 0.5 | 0.5 | ND | Sputum | Nanjing | |
| Nj10-119 | 1 | 0.25 | ND | BALF | Nanjing | |
| Nj11-87 | 1 | 0.25 | ND | Sputum | Nanjing | |
| Nj12-10 | 1 | 0.5 | ND | Sputum | Nanjing | |
| Hn1 | 0.0625 | 0.25 | ND | BALF | Henan | |
| Hn2 | 0.5 | 0.25 | ND | Sputum | Henan | |
| Hn3 | 0.125 | 0.125 | ND | Sputum | Henan | |
| Hn4 | 0.5 | 0.25 | ND | Sputum | Henan | |
ND, not determined; BALF, bronchoalveolar lavage fluid.
Interestingly, the combined TR34/L98H and S297T/F495I genotype of cyp51A in azole-resistant A. fumigatus from Hangzhou described above is also observed in this current study. We found S297T and F495I mutations appearing in two of the three azole-resistant isolates along with the TR34/L98H mutation. However, one isolate with a high resistance to triazole drugs (ITR MIC of 16 mg/liter, VOR MIC of 1 mg/liter, and POS MIC of 0.5 mg/liter) has the TR34/L98H mutation exclusively without either an accompanying S297T or F495I mutation. Furthermore, neither S297T nor F495I was found after we sequenced the remaining 68 A. fumigatus ITR-susceptible isolates.
In this study, we observed a new mutation with an elevated MIC for ITR at residue 432 in a single isolate (Shhs18); we refer to this mutation as G432A (substitution of alanine for glycine). This strain (Shhs18) was isolated from a patient with a severe lung infection who had received fluconazole (FLU) and antibiotics for bacterial and unrelated fungal infections. To better understand whether this FLU treatment introduced any selective pressure for the mutation, we performed amplified fragment length polymorphism (AFLP) analysis and microsatellite analysis to compare the genotype of Shhs18 to that of Shhs15, which was obtained earlier from this same patient before FLU treatment. Given the lack of activity of fluconazole against Aspergillus spp., the results, expectedly, indicated that the azole-resistant G432A isolate is not descended from the earlier azole-susceptible isolate. Our explanation for this genotypic diversity is that either this patient was infected by both azole-susceptible and azole-resistant A. fumigatus or Shhs18 was introduced from the hospital environment later due to the patient's extremely feeble condition (having undergone a tracheotomy procedure). However, such genotypic diversity of A. fumigatus in the same patient has been noted by others (26), lending somewhat greater weight to the former hypothesis.
The significance of the G432 mutation is not well understood at this time. Mutations at residue 432 were observed previously in France (22). Unlike our G432A strain, this strain (G432S) was only resistant to itraconazole (ITR MIC of 16 mg/liter, VOR MIC of 0.38 mg/liter, and POS MIC of 0.25 mg/liter) and had been isolated from a hematological malignancy harvested from a patient who was azole naive. With regard to the G434 neighborhood, another mutation with changes at both Y431 and G434 has been mentioned in the context of azole-resistant A. fumigatus (38). The G432A mutation occurring in Shhs18 is accompanied by moderately elevated MICs for all three triazole drugs (ITR MIC of 4 mg/liter, VOR MIC of 2 mg/liter, and POS MIC of 0.5 mg/liter). These results taken together suggest that the G432 mutation may be related to a reduced susceptibility to triazoles in general. As the G432 mutation was also identified in the cyp51A ortholog gene from azole-resistant Mycosphaerella graminicola (a plant fungal pathogen) (39), we hypothesize that G432 is a hot spot target for selection of azole resistance following pesticide use.
Given the widespread use of itraconazole in clinical settings and of triazole fungicides in agriculture throughout China, the percentage of azole-resistant A. fumigatus isolates in the current study (5.6%) is still not as high as those in most reports from European countries (12, 40). It may be significant that most isolates have been obtained from patients who were naive to azole therapy in our study. This fact may serve to explain the relatively low prevalence of resistance in our study, while the difference in the occurrence of resistance between the azole therapy-free cohort and the azole-treated cohort within the same country has been documented by others (22, 41, 42).
In summary, we observed a genotypic heterogeneity in the azole-resistant A. fumigatus isolates from China. Three genotypes, G432A, TR34/L98H, and TR34/L98H/S297T/F495I, were identified in four azole-resistant isolates. Of these, G432A is a newly reported genotype of azole-resistant A. fumigatus and the TR34/L98H genotype has also been identified in China now for the first time.
Nucleotide sequence accession numbers.
The cyp51A sequences were submitted to GenBank under accession numbers KP270710 to KP270768.
ACKNOWLEDGMENTS
We thank Zhang Zhifeng (the Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China), and Jing Pengwei (the First Affiliated Hospital, Henan University of TCM, Zhengzhou, China) for help with sample collection.
This work was supported by the PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (Grant 33320140189), the Jiangsu Provincial Special Program of Medical Science (Grant BL2012003), the Major National Science and Technology Projects (Grant 2012ZX09301002005001005), and the Technology Foundation for Selected Overseas Chinese Scholar.
We declare no conflicts of interest.
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