Distribution, molecular characterization, and treatment options for dermatophyte and non-dermatophyte fungi isolated from human and animal samples: An integrated computational and experimental approach
Main Article Content
Keywords
Arthroderma multifidum, Aspergillus sublatus, Aspergillus sydowii, Azole antifungals, Cutaneous infections, ITS, Molecular docking, Thymoquinone
Abstract
Background: Cutaneous infections caused by dermatophytes and non-dermatophytes fungi have become significant public health and veterinary concern.
Objective: The current study deals with the distribution, characterization, and antifungal susceptibility profile of these fungi.
Materials and Methods: A total of 40 sample were screened by KOH mount; positive samples were cultured on SDA. Purified isolates were identified by macroscopic and microscopic characters. Molecular characterization by PCR and sanger sequencing of ITS region confirmed the fungi. Evolutionary trees were constructed using MEGA 11. Different treatment options were evaluated, including amphotericin B, nystatin, fluconazole, itraconazole, and essential oil of Nigella sativa. Initially molecular docking was performed against selected fungal targets (5eqb and 5v5z) using molecular-operating environment (MOE v.2018.01) and Maestro modeling interface 12.3. In vitro antimycotic activity was performed by agar disc diffusion, followed by micro-broth dilution method.
Results: KOH mount revealed that 65% (13/20) samples positive for fungal components. Culture positivity for samples was 23.07% for dermatophytes and 76.92% for non-dermatophytes. Dermatophytes were detected in skin samples. However, non-dermatophytes were isolated from all types of samples. Sequence analysis confirmed the distribution of Arthroderma multifidum, Aspergillus sydowii, and Aspergillus sublatus. In-silico analysis revealed itraconazole as the most effective antimycotic agent with -7.93 kcal/mol (5eqb) and -10.89 (5v5z) kcal/mol. In vitro analysis strengthens docking analysis, itraconazole was the most effective agent with the highest mean ZOI against A. multifidum (72.00±3.46 mm), A. sydowii (62.00±3.00 mm), and A. sublatus (64.00±1.73 mm). Moreover, essential oil of N. sativa was the most effective antifungal against A. sydowii (ZOI; 31.00±3.46 mm, MIC; 3.125±0.00 mg/mL).
Conclusion: The current study is the first report of A. sydowii and A. sublatus in onychomycosis and hair infections in Pakistan. This study provides awareness about the existence of new fungal pathogens and highlights the availability of effective treatment options for emerging fungal infections.
References
2 Chanyachailert P, Leeyaphan C, Bunyaratavej S. Cutaneous fungal infections caused by dermatophytes and non-dermatophytes: An updated comprehensive review of epidemiology, clinical presentations, and diagnostic testing. J Fungi. 2023 Jun 20;9(6):669–88.
3 Shah A, Mirza R, Sattar A, Khan Y, Khan SA. Unveiling onychomycosis: Pathogenesis, diagnosis, and innovative treatment strategies. Microb Pathog. 2025 Jan;198(1):107111.
4 Gupta AK, Summerbell RC, Venkataraman M, Quinlan EM. Nondermatophyte mould onychomycosis. J Eur Acad Dermatol Venereol. 2021 Sep;35(9):1628–41.
5 Farwa U, Abbasi SA, Mirza IA, Amjad A, Ikram A, et al. Non-dermatophyte moulds as pathogens of onychomycosis. J Coll Physicians Surg Pak. 2011 Sep;21(9):597–600.
6 Menu E, Filori Q, Dufour JC, Ranque S, L’ollivier C. A repertoire of clinical non-dermatophyte moulds. J Fungi. 2023 Apr 10;9(4):433–502.
7 Smith J. Cutaneous fungal infections. US Pharm. 2015; 40(12):35–9.
8 Soomra H, Shar H, Soomra M. Fungal biota of the domestic animals in a city in Pakistan. Pak J Med Sci. 2010;26(4):964–7.
9 Chang C, Wechtaisong W, Chen Y, Cheng C, Chung S, et al. Prevalence and risk factors of zoonotic dermatophyte infection in pet rabbits in northern Taiwan. J Fungi. 2022 Jun 18;8(6):627–38.
10 Chen Q, Yang Q, Chen H, Yao Y, Shen Y, et al. Zoonotic fungus Arthroderma multifidum causing chronic pulmonary infection. Int J Infect Dis. 2023 Mar 1;130(3):17–9.
11 Puumala E, Fallah S, Robbins N, Cowen LE. Advancements and challenges in antifungal therapeutic development. Clin Microbiol Rev. 2024 Jan 5;37(1):e00142–23.
12 Agu C, Afiukwa A, Orji OU, Ezeh EM, Ofoke IH, Ogbu CO, et al. Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in disease management. Sci Rep. 2023;13(1):13398.
13 Shalaby M, Din N, Hamd A. Isolation, identification, and in vitro antifungal susceptibility testing of dermatophytes from clinical samples at Sohag University Hospital in Egypt. Electron Physician. 2016;8(3):2557–67.
14 Gupta C, Tripathi K, Tiwari S, Rathore Y, Nema S, et al. Current trends of clinicomycological profile of dermatophytosis in Central India. J Am Podiatr Med Assoc. 2014;13(10):23–6.
15 Poluri V, Indugula P, Kondapaneni L. Clinicomycological study of dermatophytosis in South India. J Lab Physicians. 2015;7(2):84–9.
16 Gnat S, Lagowski D, Nowakiewicz A, Dyląg M, Osińska M, et al. Detection and identification of dermatophytes based on currently available methods—A comparative study. J Appl Microbiol. 2021;130(1):278–91.
17 Dalis J, Kazeem H, Kwaga J, Kwanashie C, Yakubu B, et al. Molecular characterization of dermatophytes isolated from cattle in Plateau State, Nigeria. Vet Microbiol. 2018;219(1):212–8.
18 Halgren TA. MMFF VI. MMFF94s option for energy minimization studies. J Comput Chem. 1999;20(7):720–9.
19 Monk BC, Tomasiak M, Keniya V, Huschmann FU, Tyndall JD, O’Connell JD, et al. Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer. Proc Natl Acad Sci U S A. 2014;111(10):3865–70.
20 Phienluphon A, Kondo K, Mikami B, Nagata T, Katahira M. Structural insights into the molecular mechanisms of substrate recognition and hydrolysis by feruloyl esterase from Aspergillus sydowii. Int J Biol Macromol. 2023;253(1):127188.
21 Aala F, Yusuf UM, Khodavandi A, Jamal F. In vitro antifungal activity of allicin alone and in combination with two medications against six dermatophytic fungi. Afr J Microbiol Res. 2010;4(4):380–5.
22 Prajapati S, Sharma M, Kumar A, Gupta P, Dwivedi B, et al. Antimicrobial activity of different homoeopathic drugs and their potencies against Aspergillus niger in vitro. Indian J Res Homoeopathy. 2019;13(3):150–8.
23 Pfaller A, Castanheira M, Diekema J, Messer A, Jones N. Triazole and echinocandin MIC distributions with epidemiological cutoff values for differentiation of wild-type strains from non-wild-type strains of six uncommon species of Candida. J Clin Microbiol. 2011;49(10):3800–4.
24 Taxonomy. Taxonomy (Arthrodermataceae) [Internet]. National Center for Biotechnology Information. [cited n.d.]. Available from: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=34384
25 Yamaguchi S, Sano A, Hiruma M, Murata M, Kaneshima T, Murata Y, et al. Isolation of dermatophytes and related species from domestic fowl (Gallus gallus domesticus). Mycopathologia. 2014;178(1–2):135–43.
26 Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–25.
27 Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;39(4):783–91.
28 Kumar S, Tamura K, Nei M. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004;101(30):11030–5.
29 Tamura K, Stecher G, Kumar S. MEGA 11: Molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021 Jun 25;38(7):3022–7.
30 Martinez-Rossi NM, Peres NTA, Bitencourt TA, Martins MP, Rossi A. State-of-the-art dermatophyte infections: Epidemiological aspects, pathophysiology, and resistance mechanisms. J Fungi. 2021;7(8):629–46.
31 Araya S, Tesfaye B, Fente D. Epidemiology of dermatophyte and non-dermatophyte fungi infection in Ethiopia. Clin Cosmet Investig Dermatol. 2020;13(1):291–7.
32 Teklebirhan G, Bitew A. Prevalence of dermatophytic infection and the spectrum of dermatophytes in patients attending a tertiary hospital in Addis Ababa, Ethiopia. Int J Microbiol. 2015;2015(1):1–5.
33 Woldeamanuel Y, Leekassa R, Chryssanthou E. Clinico-mycological profile of dermatophytosis in a reference centre for leprosy and dermatological diseases in Addis Ababa. Mycopathologia. 2006;161(3):167–72.
34 Fentaw S, Gentachew T, Assefa M. A five-year retrospective study of dermatophytosis and dermatomycoses at the Mycology Referral Laboratory of EHNRI, Addis Ababa, Ethiopia. Cienc Biol. 2010;41(1):1–7.
35 Bitew A. Dermatophytosis: Prevalence of dermatophytes and non-dermatophyte fungi from patients attending Arsho Advanced Medical Laboratory, Addis Ababa, Ethiopia. Dermatol Res Pract. 2018;2018:1–6.
36 Ridzuan M, Ruth M, Arffah K. Isolation of dermatophytes from infected stray cats in Selangor. J Berkala Epidemiol. 2021;9:231–8.
37 Smagulova AM, Kukhar YV, Glotova TI, Glotov AG, Kim AS. First record of Trichophyton benhamiae isolated from domestic cats in Russia. Med Mycol Case Rep. 2023;40:16–21.
38 Long S, Carveth H, Chang YM, O’Neill D, Bond R. Isolation of dermatophytes from dogs and cats in the south of England between 1991 and 2017. Vet Rec. 2020;187:e87.
39 Moskaluk A, Woude SV. Two novel species of Arthroderma isolated from domestic cats with dermatophytosis in the United States. Med Mycol. 2022;60:1–10.
40 Dawson CO. Two new species of Arthroderma isolated from soil from rabbit burrows. Sabouraudia. 1963;3:185–91.
41 Ali-Shtayeh MS, Salameh AA, Abu-Ghdeib SI, Jamous RM, Khraim H. Prevalence of tinea capitis as well as of asymptomatic carriers in school children in Nablus area (Palestine). Mycoses. 2002;45:188–94.
42 Shakir S, Saleem S, Rizvi W, Waheed A, Iqbal J. Isolation, identification and antifungal susceptibility of dermatophytes isolated from clinically suspected cases of tinea infections in Pakistan. Microbiol Res J Int. 2019;29:1–11.
43 Arora T, Oberoi L, Malhotra A, Kauri R. Mycological pattern of dermatophytes and non-dermatophytes in a tertiary care hospital. Int J Health Sci Res. 2020;10:37–41.
44 Nouripour-Sisakht S, Mirhendi H, Shidfar MR, Ahmadi B, Rezaei-Matehkolaei A, et al. Aspergillus species as emerging causative agents of onychomycosis. J Mycol Med. 2015;25:101–7.
45 Van Straten MR, Balkis MM, Ghannoun MA. The role of non-dermatophyte molds in onychomycosis: diagnosis and treatment. Dermatol Ther. 2002;15:89–95.
46 Hubka V, Nováková A, Peterson SW, Frisvad JC, Sklenář F, Matsuzawa T, et al. A reappraisal of Aspergillus section Nidulantes with descriptions of two new sterigmatocystin-producing species. Plant Syst Evol. 2016;302(9):1267–99.
47 Henriet SS, Verweij PE, Warris A. Aspergillus nidulans and chronic granulomatous disease: a unique host–pathogen interaction. J Infect Dis. 2012;206(7):1128–37.
48 Abdel-Azeem A, Salem F, Abdel-Azeem M, Nafady N, Mohesien M, Soliman E. Biodiversity of the genus Aspergillus in different habitats. In: Gupta VK, editor. New and Future Developments in Microbial Biotechnology and Bioengineering. Amsterdam: Elsevier; 2016. p. 3–28.
49 Schoch CL, Ciufo S, Domrachev M, Hotton CL, Kannan S, Khovanskaya R, et al. NCBI taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford). 2020;2020:baaa062.
50 Mousavi B, Hedayati MT, Hedayati N, Ilkit M, Syedmousavi S. Aspergillus species in indoor environments and their possible occupational and public health hazards. Curr Med Mycol. 2016;2(1):36–42.
51 Rex JH, Pfaller MA, Walsh TJ, Chaturvedi V, Espinel-Ingroff A, Ghannoum MA, et al. Antifungal susceptibility testing: practical aspects and current challenges. Clin Microbiol Rev. 2001;14:643–58.
52 Rudramurthy S, Dogra S, Shaw D. Antifungal drug susceptibility testing of dermatophytes: laboratory findings to clinical implications. Indian Dermatol Online J. 2019;10:225.
53 Ibrahim SM, Adlan N, Alomair SM, Butaiban I, Alsalman A, Bawazeer A, et al. Evaluation of systemic antifungal prescribing knowledge and practice in the critical care setting among ICU physicians and clinical pharmacists: a cross-sectional study. Antibiotics. 2023;12:238.
54 Mahmoudvand H, Sepahvand A, Jahanbakhsh S, Ezatpour B, Mousavi SA. Evaluation of antifungal activities of the essential oil and various extracts of Nigella sativa and its main component, thymoquinone, against pathogenic dermatophyte strains. J Mycol Med. 2014;24:155–61.
55 Nouri N, Mohammadi SR, Beardsley J, Aslani P, Ghaffarifar F, et al. Thymoquinone antifungal activity against Candida glabrata oral isolates from patients in intensive care units—an in vitro study. Metabolites. 2023;13:580–92.
56 Taha M, Azeiz AZ, Saudi W. Antifungal effect of thymol, thymoquinone and thymohydroquinone against yeasts, dermatophytes and non-dermatophyte molds isolated from skin and nail fungal infections. Egypt J Biochem Mol Biol. 2010;28:109–26.
57 Aljabre SH, Randhawa MA, Akhtar N, Alakloby OM, Alqurashi AM, et al. Antidermatophyte activity of ether extract of Nigella sativa and its active principle, thymoquinone. J Ethnopharmacol. 2005;101:116–19.