Molecular Detection of Aspergillus Fumigatus in A Sample of Iraqi Patients

Aspergillus is a filamentous fungus, it is disseminated in different environments like air, soil, water and food, and cause many problems when it spores inhaled by the host especially in those whom immunocompromised persons. the lung is the most organ effect by Aspergillus spores [1]. Aspergillus fumigates is the most pathogenic among other species of Aspergillus, it characterized with wide distribution through the release of spores and it optimal grows between 25°C - 30°C, A. fumigates associated with high mortality rate (90%) among aspergillosis infections. In spite of its high mortality, invasive aspergillosis still understudied and underdiagnosed when it compared with other disorders [2,3].

Allergic bronchopulmonary aspergillosis (ABPA) is a lung fungal infection, it caused because the hypersensitivity reaction of Aspergillus antigens that colonized into the airways of humans. This disease is commonly among patients with bronchial asthma and those having cystic fibrosis [4]. Aspergillosis is not common in skin caused by Aspergillus species; it occurs by contagious or by hyphal transmission from blood [5].

Aspergillus sinusitis accounts around 9% of all rhinosinusitis cases. Polyps and thick secretions, patients with Aspergillus sinusitis are characterized with neutropenia, diabetes mellitus, excessive use of antibiotics, and immunosuppressive drugs, all these factors may be predisposing for Aspergillus sinusitis [6]. Diabetic patients have more than 25% a higher risk of developing to infect with pulmonary aspergillosis, A. fumigates is the most causative agent of this infection due to it could invade the pulmonary vasculature and cause thrombosis, pulmonary infarctions among diabetic patients [7]. In immunocompetent humans, they can remove A. fumigatus spores and prevent Aspergillosis problems. However, in immunocompromised patients especially whom use immunosuppressive drugs, A. fumigatus colonize the target organ and may develop Aspergillosis ([8].

Azole derivatives represent the first choice for aspergillosis treatment. itraconazole is widely used among other azoles in treat of invasive aspergillosis [9]. Azoles inhibit of the Cyp51 proteins which act with lanosterol-14 alpha demethylase in synthesis of A. fumigatus' ergosterol. The presence of mutations could affect the inhibition azoles and lead to decrease of these antifungals [10].

Samples Collection

One hundred fifty samples (swabs and scraps) were collected from patients with (asthma, cutaneous and sinusitis) problems who attended respiratory, dermatology and ENT consulting clinics at Baquba Teaching Hospital for a period extended from 2^nd of January 2024 to the 30^th of June 2024. The samples were collected from diabetic and non diabetic patients (88 and 62 samples), respectively. The clinical samples were diagnosed by consultant physicians.

Conventional Detection of Aspergillus Fumigatus

The conventional detection of Aspergillus fumigatus was done by culturing the isolates on Sabouraud Dextrose Agar (SDA) medium incubating at 37C for 1-2 weeks. Next, the isolates were stained using Lactophenol cotton blue onto glassed slide. For additional identification, a single hypha of isolates was sub-cultured on and C-Zapek Dox agar (CZA) medium with incubating at 37°C for 48-72 hrs. [11].

Antibiotics Susceptibility Test

The susceptibility of A. fumigatus isolates was done toward commonly antibiotics used in treatment of fungal infections using disk diffusion method. Aspergillus fumigatus isolates cultured on Mueller Hinton agar medium were tested toward Amphotericin B (20 µg) and itraconazole (10 µg) as described by researchers [12].

Detection of Virulence Genes of Aspergillus Fumigates Isolates Using PCR Method

Extraction of Aspergillus Fumigates DNA: Genomic DNA of Aspergillus fumigatus isolates was extracted according to the protocol of BIO-pure Extraction kit for fungal DNA extraction.

Polymerase Chain Reactions Method

The PCR constitute of 25 μLs as total volumes which were made from Go Taq Green Master Mix (12.5 μLs) containing (GoTaq DNA polymerase supplied in 2X Green GoTaq reaction buffer (pH 8.5), 400 μM each of dATP, dTTP dGTP, and dCTP, 3 mM MgCl2, bluish loading dye to analyze PCR product using agarose gel electrophoresis), forward primers (1 μLs), reverse primers (1 μLs), DNA (2 μLs), and nuclease free water (8.5 μLs). Sequences of primers each of CYB51 and Calmodulin genes for A. fumigates isolates and the program was done using PCR thermo cycler with condition (30 cycles) are mentioned in Table 1 and 2.

Agarose gel electrophoreses

After PCR. carrying out, agarose gel electrophoreses was done to detect the existence and integrity for the PCR. product. A gram of the powder (agarose) was resolved in fifty mls of buffered Tres Borate EDTA. (T.B.E.) to be agarose gel at pH 8. Next, mixture was dissolved using microwave. one microliter of ethidium bromide (10 mg/ ml) was added to agarose solution, with stirred to mix and mixture was cooled at 45°C. After comb fixed in one cm away from the margin, the agarose solution was put in tray of gel. After solidifying of gel, the comb was sided and the gel tray was put in the tank which filled with 0.5X buffered TBE. Five microliters of A. fumigatus DNA were disorder with 2 μl of bromophenol blue dye (loading buffer). Samples were put onto the gel wells, the electrical power was turn on 100 volt/mAmp for 1 hr. DNA mobile from (-) cathode pole to (+) anode. Stained bands visible by UV transiluminator at 350 nm.

Molecular Detection of Virulence Genes for Aspergillus fumigatus Isolates using Gene Sequencing Method

For gene sequencing, twelve isolates of A. fumigatus in both directions were sent to Microgen Inc., South Korea to detect of substitution mutations for both genes. The sent isolates were two isolates for each studied groups among diabetic and non-diabetic patients. The sequencing data of targeted gene that received from Microgen Inc. were assembled and translated to contig format and text document using Contig Express module of Vector NTI 9.0 program. All the reference nucleotide sequences of targeted genes of identified microbial vaginitis from (www.ncbi) and aligned using Clustal W method of MEGA4 program.

Table 1: The primer of Virulence Genes for Aspergillus Fumigatus Isolates

Table 2: PCR Program Virulence Genes for Aspergillus Fumigatus Isolates

Ethical Approval

The protocol, the study information form was confirmed by local ethic board based on the decision 1674 in 4\1\2024.

Among the diabetic patients, the percentages of A. fumigatus isolated from asthma, cutaneous and sinusitis aspergillosis were 19.2% (5 out of 26), 13.8% (4 out of 29) and 27.3% (9 out of 33) respectively. Whereas, among the nondiabetic patients, the percentages of A. fumigatus isolated from asthma, cutaneous and sinusitis aspergillosis were 8.3% (2 out of 24), 19.1% (4 out of 21) and 17.6% (3 out of 17), respectively as shown in Table 3. No statistically significant difference was revealed in aspergillosis among diabetic and non-diabetic patients (p>0.05). The macroscopic examination of A. fumigates isolates grow as a velvety, suede like surface with greenish blue colored mycelia. the microscopic appearance of A. fumigatus showed smoothed wall conidiophore with swollen vesicle with phialids coating on the upper half of its surface.

Antibiotics Susceptibility Test for Aspergillus Fumigatus Isolates

Disc diffusion method (Kirby-Bauer) was used to perform susceptibility test of two antibiotics (Amphotericin B and itraconazole) against all A. fumigates (18 isolates for diabetic patients and 9 isolates for non-diabetic patients). The results were compared with CLSI stander (CLSI, 2017). Among diabetic patients of aspergillosis, five isolates of A. fumigatus (27.8%) were resistant toward both antibiotics. Whereas, among non-diabetic patients of aspergillosis, three isolates of A. fumigatus (33.3%) were resistant toward both antibiotics. No statistically significant difference was revealed in resistance of A. fumigatus isolates toward both studied antifungals (p>0.05) (Table 4).

Molecular Identification of Aspergillus Fumigatus Isolates

Detection the Virulence Genes of A. Fumigatus Isolates Using Singleplex PCR: The molecular weight of CYP51 and Calmodulin genes for A. fumigatus isolates were 140 bp and 461 bp, respectively. This was indicated sign for successes reaction. Figure 1 A and B.

Detection of Substitution Mutations of Virulence Gene for A. Fumigatus Isolates by Gene Sequencing

After performing of the alignment between the amino acid reference sequences of CYP51 and Calmodulin genes for A. fumigatus isolates and amino acids sequences of ten isolates of this study. For diabetic patients, all isolates of A. fumigatus for sinusitis and asthma samples have substitution mutations for both genes. For non diabetic patients, one isolate of sinusitis samples has substitution mutation for CYP51 gene only. All isolates of cutaneous samples for both genes did not have any substitution mutations among the studied groups.

Table 3: Percentage of Aspergillus Fumigatus Isolates Concerning Study Groups

Table 4: Antibiotics susceptibility Test for Aspergillus Fumigatus Isolates

Table 5: Percentage of Aspergillus Fumigatus Isolates Concerning Their Ages

Figure 1: Agarose Gel Electrophoreses of PCR Products of: (A)Cyp51 And (B) Calmodulin Genes for A. Fumigatus Using 1% Agarose Gel At 100 Volt/Mamp For 1 Hr. Lane M 100 Bp Dna Ladder, (1-4) Pcr Products

Relation of Study Groups, Percentage of Isolates with Age Groups

Among diabetic patients, the age group (40-49) years old was the most infected with A. fumigatus that isolated from cutaneous and sinusitis infections with a percentage (50.0%). Whereas, among non diabetic patients, the age groups (40-49) and (30-39) years old were the most infected with A. fumigatus that isolated from cutaneous and sinusitis infections with the percentages (100.05) and (50.0%), respectively. No statistically significant difference was revealed between aspergillosis and age groups in both studied groups (p>0.05) (Table 5).

Invasive aspergillosis especially that caused A. fumigatus affect immunocompromised patients include those with hematologic malignancies, patients who underwent lung or liver cirrhosis, diabetic patients, using of antibiotics for prolong periods and those with organ transplant [15,16]. The truly and soon diagnosis of aspergillosis and rapidly determination of suitable antifungal drug led to improve survival significant [17]. Molecular detection methods so important in identification of pathogenic fungi in special methods that detect about the substitution mutation of some virulence genes like gene sequencing method. For example, CYP51 gene in Aspergillosis caused by A. fumigatus play an important role in mechanism of triazole actions due to the mutations that encoded triazoles targeted enzymes which necessary for ergosterol biosynthesis in fungal plasma membranes [18]. This result disagreed with [19,20] whom isolated A. fumigatus with sputum more than sinusitis and cutaneous samples with a percentage (68.75%). This genus is widespread airborne mold pathogens, which led to the increasing cases of Aspergillosis during past decades [21]. Patient populations may be played a role in the incidence of invasive Aspergillosis and explanation the varies in these results. Also, Calmodulin gene was used for identifying A. fumigatus from other species in cases of Aspergillosis [22]. Azoles are widely used in treatment of human and animal infections which caused by A. fumigatus, in protection of crops against diseases caused by fungi or in wood preservation as biocides. Therefore, azoles present as residues from these materials in many closet areas such as surfaced water, groundwater and sediments), these may be an opportunity for development of resistant Aspergillus isolates toward antifungals [23]. Studies indicated that many azoles act an inhibit CYP51 and calmodulin genes which mean that these antifungals can interacted with iron atoms of the heme group of CYP51 and the closely amino acids become in reverse and competitive manners, that way challenge the usual substrates for binding site [24]. These results were closed with [25] which revealed that CYP51 gene mutations were associate with A. fumigatus resistant to azoles, and made of possible roles in maintain some levels of resistance when CYP51 gene functions are weak. Also, these results were closed with results of [26] which find that the percentage of azoles resistance was 3.2% among isolates of A. fumigatus and out of these resistant isolates, 78% were A. fumigatus had mutation of Cyp51 gene. Whereas, these results disagreed with [22] which revealed that no mutation with calmodulin gene for A. fumigatus clinical isolates. Aspergillosis was invasive in immunocompromised patients that used corticosteroid for prolong periods or patients with neutropenia [27]. Aspergillus fumigatus has ability to biosynthesize different from secondary metabolites like fumagillins, fumitoxin, gliotoxin, these metabolites may cause serious health hazard and involved in impairing the host immune system [28].

Depending on the findings, our results concluded that diabetic patients were more infected with Aspergillosis than non diabetic patients and the genes of CYP51 and Calmodulin play an important role in the resistance of A. fumigatus towards studied antifungals..

Recommendations

This study is recommended to detect the relationship between virulence genes of A. fumigatus isolated from Aspergillosis patients. Also, we recommended to detect the role of CYP51 and Calmodulin genes of Non-fumigatus Aspergillus species in in the pathogenicity of invasive Aspergillosis or Aspergilloma.

Researchers would like to appear the thankful to the patients and staff of respiratory, dermatology and ENT consulting clinics at Baqubah teaching hospital and the staffs of Biology Department, College of Science, Diyala University for continuous supporting.

1. Grifiths et al. “Role for IL-1 family cytokines in fungal infection.” Front Microbiol, vol. 12, no. 63047, 2021. https://doi.org/10.3389/fmicb.2021.633047.

2. Janssens et al. “Aspergillus and the lung.” Seminars in Respiratory and Critical Care Medicine, vol. 45, no. 1, 2024. https://doi.org/10.1055/s-0043-1777259.

3. Van de Verdonk et al. “Aspergillus fumigatus morphology and dynamic host interaction.” Nat Rev Microbiol, vol. 15, no. 11, 2017, pp. 661–674. https://doi.org/10.1038/nrmicro.2017.90.

4. Sehgal et al. “Prevalence of sensitization to Aspergillus flavus in patients with allergic bronchopulmonary aspergillosis.” Med Mycol, vol. 57, no. 3, 2019, pp. 270–276. https://doi.org/10.1093/mmy/myy012.

5. Nachate et al. “Secondary cutaneous aspergillosis in a child with Behçet disease.” Annals of Pediatric Surgery, vol. 19, no. 13, 2023. https://doi.org/10.1186/s43159-023-00242-2.

6. Rai et al. “Aspergillosis of maxillary sinus diagnosis, management and association with COVID-19: A case report.” Creus, vol. 14, no. 10, 2022, pp. e30191. https://doi.org/10.7759/cureus.30191.

7. Fernández et al. “Invasive aspergillosis in patients with diabetes mellitus as the only risk factor: Case report.” Journal of Investigative Medicine High Impact Case Reports, vol. 11, 2023, pp. 1–8. https://doi.org/10.1177/23247096231175443.

8. Zhang et al. “Primary cutaneous aspergillosis due to Aspergillus fumigatus in immunocompetent patients with diabetes mellitus after tattoo: A case report.” Infection and Drug Resistance, vol. 16, 2023, pp. 791–797. https://doi.org/10.2147/idr.s398858.

9. Nargesi et al. “A whole genome sequencing-based approach to track down genomic variants in itraconazole-resistant species of Aspergillus from Iran.” J. Fungi, vol. 8, no. 1091, 2022. https://doi.org/10.3390/jof8101091.

10. Bader et al. “CYP51A based mechanism of Aspergillus fumigatus azoles drug resistance present in clinical samples from Germany.” Antimicrob. Agents Chemother., vol. 57, no. 8, 2013, pp. 3513–17. https://doi.org/10.1128/AAC.00167-13.

11. Pinto et al. “Aspergillus species and antifungal susceptibility in clinical settings in north of Portugal: Cryptic species and emerging azole resistance in Aspergillus fumigatus.” Front Microbiol, vol. 9, 2018, pp. 46–56. https://doi.org/10.3389/fmicb.2018.01656.

12. Takeda et al. “High detection rates of azole-resistant Aspergillus fumigatus after treatment with azole antifungal drugs among patients with chronic pulmonary aspergillosis in a single hospital setting with low azole resistance.” Med Mycol, vol. 59, 2021, pp. 327–334. https://doi.org/10.1093/mmy/myaa052.

13. Melado et al. “Identification of two different 14-a sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species.” J. Clin. Microbiol., vol. 39, no. 7, 2001, pp. 2431–38. https://doi.org/10.1128/jcm.39.7.2431-2438.2001.

14. Dhaban et al. “Molecular identification of Aspergillus fumigatus complex from lung transplant recipient using multilocus sequencing analyses.” J. Assoc. Med. Microbiol. Infect. Dis., vol. 7, no. 1, 2022, pp. 54–63. https://doi.org/10.3138/jammi-2021-0004.

15. Jenk et al. “Point of care diagnosis of invasive aspergillosis in non-neutropenic patients: Aspergillus galactomannan lateral flow assay versus Aspergillus specific lateral flow device test in bronchoalveolar lavage.” Mycoses, vol. 62, no. 3, 2019, pp. 230–236. https://doi.org/10.1111/myc.12881.

16. Hoenigl et al. “Global guidelines and initiatives from the European Confederation of Medical Mycology to improve patient care and research worldwide.” Mycoses, vol. 61, no. 11, 2018, pp. 885–94. https://doi.org/10.1111/myc.12836.

17. Hoenig et al. “Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.” J. Infection, vol. 78, no. 2, 2018, pp. 150–157. https://doi.org/10.1016/j.jinf.2018.09.006.

18. Postina et al. “A comparison of two molecular assays for detection and characterization of A. fumigatus triazoles resistance and Cyp51 mutation.” Front Microbiol, 2018. https://doi.org/10.3389/fmicb.2018.00555.

19. Shrivastava et al. “Isolation of Aspergillus species from various clinical samples in a tertiary care hospital, Ahmedabad.” International Journal of Pharmaceutical and Clinical Research, vol. 14, no. 12, 2022, pp. 452–457.

20. Shrmali. “Isolation of Aspergillus species from sputum sample: Study conducted in a tertiary care hospital, Ahmedabad.” Int J Res Med Sci, vol. 3, no. 3, 2018, pp. 1398–401.

21. P.H. Chandrasekar et al. “Aspergillus: An increasing problem in tertiary care hospitals.” Clin Infect Dis, vol. 30, 2000, pp. 984. https://doi.org/10.1086/313832.

22. Almas et al. “Multilocus sequence analysis of Aspergillus species from clinical samples and the surge of cryptic aspergillosis in southern India.” Journal of Clinical and Diagnostic Research, vol. 18, no. 10, 2024, pp. 04–09. https://doi.org/10.7860/JCDR/2024/72709.20211.

23. H.A. Assress et al. “Antifungal azoles and azole resistance in the environment: Current status and future perspectives.” Rev Environ Sci Biotechnol, vol. 20, 2021, pp. 1011–1041. https://doi.org/10.1007/s11157-021-09594-w.

24. Hagrove et al. “Structure-functional characterization of cytochrome P450 sterol 14α-demethylase (CYP51) from A. fumigatus and molecular basis for antifungal drug development.” J. Biol. Chem., vol. 290, 2015, pp. 23916–23934. https://doi.org/10.1074/jbc.m115.677310.

25. Sanseverino et al. “Activity of azole and non-azole substances against Aspergillus fumigatus in clinical and environmental samples.” Int J Mol Sci, vol. 26, 2025, no. 1033. https://doi.org/10.3390/ijms26031033.

26. J.W. van der et al. “Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.” Emerg Infect Dis, vol. 21, 2015, pp. 1041–1044. https://doi.org/10.3201/eid2106.140717.

27. J.D. Jaffaery et al. “Point-of-care diagnosis of invasive aspergillosis in non-neutropenic patients.” Mycoses, vol. 26, no. 3, 2019, pp. 230–36. https://doi.org/10.1111/myc.12881.

28. J.L. Steenwyk et al. “Variation among biosynthetic gene clusters and virulence across Aspergillus species.” Genetics, vol. 216, 2020, pp. 481–497. https://doi.org/10.1534/gen etics.120.303549.