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Molecular Identification of Fusarium Species Complex Isolated from Clinical Samples and Its Antifungal Susceptibility Patterns

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Molecular Identification of Fusarium Species Complex Isolated from Clinical Samples and Its Antifungal Susceptibility Patterns Current Medical Mycology 2019, 5(4): 43-49 Molecular identification of Fusarium species complex isolated from clinical samples and its antifungal susceptibility patterns Yashik Bansal1, Nidhi Singla1*, Neelam Kaistha1, Sunandan Sood2, Jagdish Chander1 1 Department of Microbiology, Government Medical College Hospital, Chandigarh, India 2 Department of Ophthalmology, Government Medical College Hospital, Chandigarh, India Article Info A B S T R A C T Article type: Background and Purpose: More than 300 Fusarium species are grouped into Original article approximately 23 species complexes out of which around 70 are involved in human infections. The nomenclature of these species has undergone considerable changes in recent years. These species cause localized infections in individuals while inducing systemic infections mainly in immunocompromised patients. The present study was Article History: conducted to identify Fusarium species in clinical isolates by molecular methods and Received: 17 August 2019 determine their in vitro minimum inhibitory concentration (MIC) patterns to address the Revised: 15 October 2019 lack of data in this domain in Northern India. Accepted: 25 November 2019 Materials and Methods: For the purpose of the study, Fusarium isolates obtained from various clinical samples were sent to the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands, for molecular identification. The MIC testing was performed using the microbroth dilution method as per the Clinical and Laboratory Standards * Corresponding author: Institute reference method (M38-A2). Nidhi Singla Results: Fusarium was isolated from 33 patients (i.e., 1, 1, 2, 14, and 15 cases with Department of Microbiology, endophthalmitis, sinusitis, pulmonary involvement, onychomycosis, and keratitis, Government Medical College Hospital, respectively). These 33 isolates belonged to three species complexes, namely F. solani Chandigarh, India. species complex (FSSC; n=13), F. fujikuroi species complex (FFSC; n=13), and F. Email: [email protected] incarnatum equiseti species complex (FIESC; n=7). The species identified within FSSC, FFSC, and FIESC included F. keratoplasticum (n=6)/F. falciforme (n=6)/F. solani (n=1), F. proliferatum (n=7)/F. sacchari (n=5)/F. anthophilum (n=1), and F. incarnatum SC species (n=6)/F. equiseti SC species (n=1), respectively. The MIC results showed that all isolates had a lower MIC against amphotericin B than against the other antifungal agents. Conclusion: Timely diagnosis and appropriate treatment will facilitate the improvement of patient outcomes. Keywords: Fusarium, Molecular identification, Keratitis, Onychomycosis, Taxonomy How to cite this paper Bansal Y, Singla N, Kaistha N, Sood S, Chander J. Molecular identification of Fusarium species complex isolated from clinical samples and its antifungal susceptibility patterns. Curr Med Mycol. 2019; 5(4): 43-49. DOI: 10.18502/cmm.5.4.2149 Introduction he fungi belonging to the genus Fusarium Geejayessia, and Albonectria) [11, 12]. In January possess such attributes as the capability to grow 2013, the International Code of Nomenclature for T on a wide range of substrates and present as algae, fungi, and plants prohibited the dual biofilms [1] on water and in plumbing systems, nomenclature system. Therefore, the genus Fusarium setting the ground for the widespread distribution of was recognized as the standard name because it was this fungus [2, 3]. These species are well-known plant the most commonly used name in the literature [13]. pathogens [4] that account for the contamination and However, during the 2017 event at Shenzhen, China, spoilage of food [5]. More than 300 Fusarium species the 19th International Botanical Congress reversed its [6] have been identified, out of which around 70 Article 57.2 and allowed to use the competing asexual species are involved in human infections, causing and sexual names in the literature [14]. Still, the fusariosis [7]. The incidence of fungal infections in nomenclature has undergone substantial changes in humans is increasing with the rising population of recent years [7]. Fusarium species are now grouped immunocompromised individuals [8, 9]. Accordingly, into 23 grossly phylogenetic species complexes [6]. there is a proportional increase in the morbidity and The species reported to be most commonly mortality caused by fusariosis [10]. associated with human infections are F. solani (60%) Fusarium used to have a dual nomenclature based on and F. oxysporum (20%) [2]. However, these two different ascomycete teleomorphs (i.e., Gibberella, species have been elevated to species complex status, Nectria, Neocosmospora, Haematonectria, Cyanonectria, and newer species have been identified and described Copyright© 2019, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY) License (http://creativecommons.org/) which permits unrestricted use, distribution and reproduction in any medium, provided appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fusarium molecular identification Bansal Y et al. recently [7]. Some of these species complexes that are molecular identification at species level. To this end, commonly implicated in human infections include F. the DNAs of the clinical isolates were extracted and solani species complex (FSSC), F. fujikuroi species amplified by polymerase chain reaction (PCR) complex (FFSC), F. incarnatum-equiseti species and sequenced as described before [21]. Genus complex (FIESC), F. oxysporum species complex identification and amplification were performed using (FOSC), F. dimerum species complex (FDSC), F. ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’)/ITS4 (5’- chlamydosporum species complex (FCSC), and F. TCCTCCGCTTATTGATATGC-3’) or ITS5 (5’- tricinctum species complex (FTSC) [7]. GAAGTAAAAGTCGTAACAAGG-3’) and NL4 (5’- The majority of Fusarium infections in GGTCCGTGTTTCAAGACGG-3’) under standard immunocompetent individuals are superficial and conditions. subcutaneous, while systemic or disseminated Species level identification was carried out via the infections are seen in immunocompromised individuals multilocus sequence analysis using elongation factor 1- [4, 15]. Although Fusarium can be identified fairly alpha and the DNA-directed RNA polymerase second well morphologically, molecular tests facilitate the largest subunit [21]. The PCR condition included [22] accurate identification of the species and their predenaturation for 3 min at 95°C, five cycles of 45 sec classification in proper species complex [16]. Most of at 95°C, 45 sec at 58°C, and 2 min at 72°C, five cycles Fusarium species invariably show very high resistance of 45 sec at 95°C, 45 sec at 56°C, and 2 min at 72°C, to various antifungal agents. Accordingly, there is a 30 cycles of 45 sec at 95°C, 45 sec at 52°C, and 2 min species-specific resistance pattern to a particular at 72°C, and a postelongation step of 8 min at 72°C. antifungal agent [17, 18]. Amphotericin B is the only Amplicon purification was accomplished using the antifungal drug found to be consistently active during Sephadex G-50 fine (HE Healthcare, Uppsala, in vitro studies on various Fusarium species causing Sweden). The amplicons were subjected to direct human infections [19]. sequencing with the ABI Prism BIGDYE terminator In such a background of increasing fusariosis, cycle sequence kit (Applied Biosystems, Foster City, susceptible host (immunocompromised individuals), CA, USA). The ABI Prism 3730XL Sequencer was changing nomenclature, taxonomy, and high in vitro adopted to perform the final analysis. For antifungal resistance, the present study was conducted identification purposes, the isolates were compared to in a tertiary care center in North India to address the the sequences in the FUSARIUM MLST database lack of data in this domain in this region. (http://www.cbs.knaw.nl/fusarium/) and Gen-Bank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Materials and Methods Antifungal susceptibility testing was carried out The present study was conducted on all the samples by broth microdilution method as per the Clinical received in the Mycology Laboratory of the Laboratory Standards Institute (CLSI) Reference Department of Microbiology wherein Fusarium Method for Broth Dilution Antifungal Susceptibility species had been isolated over a period of 24 months Testing of Filamentous Fungi, Approved Standard- (i.e., October 2012 till October 2014). The obtained Second Edition (M38-A2) [23]. The tested antifungals samples included corneal scrapings, donor sclera, (i.e. amphotericin-B, voriconazole, itraconazole, recipient cornea, vitreous and aqueous tap, nail fluconazole, caspofungin, and anidulafungin) were clippings, nasal polyps, sputum, and pleural or other commercially sourced as powders from the Sigma- body fluids. Aldrich. The clinical samples were processed in the Non-germinated conidial suspensions prepared by a Department of Microbiology using the direct spectrophotometric procedure and conidial test inocula KOH/Calcofluor white mount examination, fungal in a range of approximately 0.4×104 to 5×104 produced culture, and antifungal susceptibility testing. The reproducible minimum inhibitory concentration (MIC). KOH and Calcofluor white mount (fluorescent For inoculum preparation, conidia formation
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