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Phenotypic and genotypic characterization

آبان ۵, ۱۳۹۷

Drug susceptibility profile of Candida glabrata clinical isolates from Iran and genetic resistant mechanisms to caspofungin

شهریور ۲۶, ۱۳۹۷

Genotyping of clinical isolates of Candida glabrata from Iran by multilocus sequence typing and determination of population structure and drug resistance profile

شهریور ۲۶, ۱۳۹۷

Case Report

Clinical and mycological characteristics of keratitis caused by Colletotrichum gloeosporioides: A case report and review of literature

Alireza Izadi¹, Mohammad Soleimani², Roshanak Daie Ghazvini¹, Seyed Jamal Hashemi¹, Mohssen Gramishoar¹, Kazem Ahmadikia¹, Mehdi Aminizadeh², Mohammad Ghahri³, Zohre Abedinifar³, Aleksandra Barac⁴, Sadegh Khodavaisy¹

^۱ Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

^۲ Department of Ocular Trauma and Emergency, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran

^۳ Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran

^۴ Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Abstract

Introduction: Colletotrichum species are well-known plant pathogens, which have been increasingly reported as the cause of keratitis or subcutaneous lesions in humans. In this study we reported a rare case of fungal keratitis from Iran and reviewed the literature.

Case Presentation: A 69-year-old man whose right eye was injured by herbal material was examined by slit-lamp biomicroscopy and mycology investigation of corneal scrapings was done. The grown filamentous fungal was identified as Colletotrichum gloeosporioides based on morphological characteristics and DNA sequence of the internal transcribed spacer region. The isolated strain was sensitive to amphotericin B, caspofungin, anidolafungin, micafungin, voriconazole, and relatively resistant to fluconazole, and itraconazole. Patient was successfully treated with voriconazole.

Conclusions: This report highlights that the early and accurate identification and therapy can helpful to management keratitis caused by rare pathogen C. gloeosporioides.

Key words: Colletotrichum gloeosporioides; Keratitis; Molecular identification.

J Infect Dev Ctries 0000; 0(00):000-000. doi:10.3855/jidc.14492

(Received 15 December 2020 – Accepted 14 February 2021)

Copyright © ۲۰۲۱ Izadi et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Fungal keratitis (FK) is one of the main causes of ocular morbidity [1]. Delay or inappropriate treatment can be potentially leading cause of blindness [2]. In recent years, the genus Colletotrichum has been recognized as an infrequent cause of FK [2-4]. Colletotrichum species are common plant pathogens in tropical and subtropical regions worldwide [3-5]. Trauma with plant materials is one of the main predisposing factors in keratitis caused by these fungi [6]. Among Colletotrichum genus, only a few species are known to be pathogenic for humans. The Colletotrichum spp. have been associated with human ophthalmic infections including C. dematium, C. coccodes, C. gloeosporioides, C. graminicola, C. crassipes, C. capsicii [7]. Based on the literature, C. gloeosporioides considered as the second most common cause of FK after Colletotrichum dematium [8,9]. Though Colletotrichum infection in humans is rare, to avoid ophthalmic complications an early identification of pathogenic fungus and their antibiotic susceptibility can be helpful for management of the infection. Molecular techniques may help in accurate diagnosis of the agent [7]. Close resemblance of the curved conidia of Fusarium and Colletotrichum spp. may mislead inexperienced laboratory personnel [3]. Here, we review the literature and report a case of FK. To our knowledge, this the first case report documenting the clinical and mycological characteristics of FK caused by C. gloeosporioides from Iran.

Case presentation

A 69-year-old male farmer presented to Farabi eye hospital in Tehran with a sudden loss of visual acuity. He mentioned a history of ocular trauma in right eye by herbal material 10 days earlier. On initial examination of the patient, visual acuity (VA) was counting fingers in the right eye and 20/25 in the left eye. Slit lamp examination of the affected eye revealed superficial corneal infiltration with a size of 4×۳ mm and epithelial defect without hypopyon (Figure 1). Corneal scrapings are obtained using sterile surgical blades for mycological examination. One sample was used to perform KOH mount and routine Gram’s staining,

Figure 1. Slit lamp photographs of right eye.

(A) Corneal ulcer with infiltration and the centrifugal, linear, circular spread of fungal infection through the corneal channels, (B) Corneal scarring after treatment with voriconazole.

while the second was inoculated into Sabouraud dextrose agar (SDA) supplemented with 0.5% chloramphenicol and sheep blood agar plates, which incubated at 28°C and 37°C, respectively. Microscopic examinations by KOH mount and gram’s staining of sample showed septate filamentous fungal fragments and did not show any bacterial cells. Based on clinical findings and the result of microscopic examination of corneal scraping, treatment was initiated with topical 0.1% voriconazole, vancomycin and ceftazidime hourly. Culture of corneal scrapings revealed brownish-black filamentous fungal colonies, consisting of numerous sclerotia on SDA (Figure 2). The

Figure 2. Fungal colonies growth on Sabouraud dextrose agar.

(A) The growing fungus produced brown-gray concentrically ringed colonies. Colletotrichum gloeosporioides produced orange mucus beads centrically after 10 days at 30°C. (B) Reverse side showing brownish-black pigmentation.

microscopic examination of fungal colonies showed abundant cylindrical non-septate conidia with blunt ends and darkly pigmented with white aerial hyphae (Figure 3), which are features characteristic of the genus Colletotrichum. DNA was extracted using a DNA isolation kit (Gene All DNAextraction kit; Gene All, Germany) according to the manufacturer’s instructions and stored at -20°C prior to use. To confirm the Colletotrichum species identification, the Internal Transcribed Spacer (ITS) region was sequenced, as previously described [10]. Yielded sequence was subjected to Basic Local Alignment Search Tool (BLAST) program (http://www.blast.ncbi.nlm.nih.gov/Blast). The DNA

Figure 3. (A) Lactophenol cotton blue tease mount from the colony. Hyphae with abundant cylindrical conidia with blunt ends (magnification X400); (B) brownish appressoria, which are characteristic features of Colletotrichum gloeosporioides (magnification X400).

sequence of the ITS gene matched that of Colletotrichum gloeosporioides species (GenBank MT130718) by showing 99% similarity with the ex-type strain of the species (GenBank MT065696). Additionally, in vitro antifungal testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 for filamentous fungi [11]. The minimal inhibitory concentrations (MICs) of amphotericin B, fluconazole, voriconazole, itraconazole, caspofungin, anidulafungin and micafungin were 0.5, >64, 0.5, 8, 0.5, 0.5 and 0.25 respectively. The patient was treated with voriconazole every hour, levofloxacin 4 hours and passing 42 days, the lesion turned into corneal scar and there were no signs of a recurrence after 2 months of treatment with voriconazole.

Discussion

Colletotrichum species are coelomycetous soil fungi that are an uncommon cause of keratitis worldwide [5, 6]. Traumatic insemination is important for the initiation of Colletotrichum infection [2]. Thorough search of literature using “Keratitis” and “Colletotrichum” showed Colletotrichum spp. keratitis is increasingly reported (Table 1). Fernandez et al. reported, FK due to Colletotrichum was in 10 of 360

Table 1. Summary of previously reported cases reports of Colletotrichum gloeosporioides keratitis. Authors (Reference) Country, Year No. of case Age, Sex Predisposing factor and risk factor Eye direction Initial visual activity Hypopyon Medical therapy Fungal identification method Outcome María Borrás-Mánez et.al. (5) Spain, 2015 ۱ ۷۵ M trauma with plant material (orange tree branch) NM NM Yes Voriconazole, Keratoplasty microscopic, culture and molecular (sequnsing) Evisceration S.T. Pote et al. (19) India, 2017 ۱ ۴۵ M trauma with with stone chip Right ۶/۶۰ Yes Amphotericin-B, Voriconazole, Natamycin microscopic, culture and molecular (DNA sequencing of its region) CI Lipeng Wang et al. (15) China, 2019 ۱ ۵۲ M trauma with plant material (apple tree branch) Right NM NM Natamycin, Levofloxacin, Voriconazole microscopic, culture, MALDI-TOF, DNA sequencing of ITS, CI Atsushi Shiraishi et al. (4) Japan, 2011 ۳ ۸۰ F trauma with dust particles right ۲۰/۲,۰۰۰ Yes Voriconazole, Pimaricin microscope, culture, DNA sequencing of ITS CI ۷۱ F trauma with plant material Left ۲۰/۸۰ NM Voriconazole, Natamycin, Moxifloxacin microscope, culture, DNA sequencing of ITS CI ۶۰ M trauma with plant material (orange tree branch) Right ۲۰/۳۰۰ NM Voriconazole, Miconazole, Natamycin, Levofloxacin microscope, culture, DNA sequencing of ITS CI Noriaki Yamamoto et al. (9) Japan, 2001 ۱ ۸۲ M Injured by insect, cataract surgery 6 months previously, myelodysplasic syndrome Left ۲۰/۳۰ NM Natamycin, Fluconazole microscope, culture CI Ning Hung et al. (3) Taiwan, 2020 ۵ ۷۲ M Farming, dirty water contact NM ۲۰/۲۰۰ NM Natamycin microscope, culture, DNA sequencing of ITS CI ۲۲ M trauma with metallic body NM ۲۰/۳۰ NM No antifungal medication microscope, culture, DNA sequencing of ITS CI ۷۹ F Farming, soil contact NM ۲۰/۱۲۵ NM Amphotericin B, Voriconazole microscope, culture, DNA sequencing of ITS CI ۶۳ F trauma with plant material NM ۲۰/۱۰۰ NM No antifungal medication microscope, culture, DNA sequencing of ITS CI ۷۷ F Unknown NM ۲۰/۸۰۰ NM Natamycin, Amphotericin B microscope, culture, DNA sequencing of ITS CI J. Lamarca et al. (20) Spain, 2015 ۱ ۵۶ F trauma with plant material (orange tree branch) Left NM Yes Natamycin, Voriconazole, Keratoplasty microscope, culture, PCR CI Ismail Zakariya-Yousef Breval et al.(21) Spain, 2019 ۱ ۴۵ M trauma with plant material (orange tree branch) Right NM Yes Voriconazole microscope, culture CI Our Case Iran, 2020 ۱ ۶۹ M trauma with plant material Right counting fingers No Voriconazole microscope, culture, DNA sequencing of ITS CI M: male; F: female; NM: not mentioned; CI: clinical improvement.

cases (2.8%) [12]. In other series by Kaliamurthy et al. [13] and Hung et al. [3], Colletotrichum was the cause of the keratitis in 7 of 378 cases (1.9%) and in 7 of 65 cases (10.8%), respectively. The ocular trauma and contamination by organic materials were principal risk factors for Colletotrichum keratitis [12, 13]. Our case had a history of ocular injury by herbal material during farm work, similar to other cases reported [8, 9, 14]. Colletotrichum spp. can be identified by nonseptate conidia, presence of appressoria, and (in the later stage) acervuli with setae but the morphological identification is time-consuming and technically challenging [7]. Due to the difficulty of the morphological identification of Colletotrichum spp., molecular approaches based on the nucleic acid sequence of ITS region are the more applicable and accurate method for isolates identification to the species level in the diagnostic laboratory [5, 15]. Most cases of FK caused by Colletotrichum species, particularly C. dematium and C. gloeosporioides respond well to natamycin, possibly due to its easy availability [12, 13]. However, Shiraishi et al. and Fernandez et al. reported that isolates of C. gloeosporioides in their study showed intermediate resistance to natamycin [4, 12]. In our reported case, simultaneous voriconazole regimen leads to complete regression of eye lesion. The excellent results have been reported following voriconazole treatment in cases of FK and endophthalmitis caused by a number of species of fungi [16, 17]. Shiraishi et al. [4] treated 3 cases of C. gloeosporioides keratitis with voriconazole, as well as Mitani et al. reported [2]. However, Mánez et al. [5] reported oral, topical, intravitreal and intrastromal voriconazole showing no improvement of eye lesion. In some case reported combination therapy (natamycin plus amphotericin-B/voriconazole) may be more effective than monotherapy [4]. There is a lack of consensus regarding the optimal therapy against Colletotrichum spp. ophthalmic infection in the literature [15]. Few reports mentioning the drug sensitivity test against C. gloeosporioides [4, 15]. Shiraishi et al. indicated that all three C. gloeosporioides, isolated cases of FK, were sensitive to amphotericin B, itraconazole, miconazole, micafungin, and voriconazole, and relatively resistant to flucytosine, fluconazole, and natamycin [4]. Also, Mitani et al, reported similar results [2]. The MICs of different antifungal drugs on the C. gloeosporioides isolated from case reports are shown in Table 2. Reported studies have indicated that the majority of Colletotrichum isolates have a different degree of resistance to antifungal drugs and therefore combination therapy may be more effective than monotherapy for complete cure of patients having Colletotrichum infections [18, 19].

In conclusion, this is a rare case of FK caused by C. gloeosporioides in Iran. The results suggest that molecular investigations and antifungal susceptibility testing will be required to further understand the epidemiology and optimal management of FK caused by rare fungal pathogens in humans.

Table 2. The MICs of different antifungal drugs on the C. gloeosporioides isolated from reported case. Authors (Reference) Amphotericin B Fluconazole Voriconazole Itraconazole Posaconazole Miconazole Caspofungin Anidolafungin Micafungin ۵-flucytosine Natamycin Lipeng Wang et al. (15) ۰٫۲۵ ۶۴ ۰٫۲۵ ۰٫۲۵ ۰٫۲۵ – ۰٫۲۵ ۰٫۱۲ ۰٫۰۶ ۱۶ – Atsushi Shiraishi et al. (4) ۰٫۱۲۵ >64 ۰٫۵ ۰٫۲۵ – ۰٫۵ – – <0.03 >64 ۴ <0.03 ۶۴ ۰٫۱۲۵ ۰٫۱۲۵ – ۰٫۲۵ – – <0.03 ۲ ۴ <0.03 ۱۶ ۰٫۱۲۵ ۰٫۰۳ – ۰٫۲۵ – – ۰٫۱۲۵ >64 ۲ Presented case ۰٫۵ >64 ۰٫۵ ۸ – – ۰٫۵ ۰٫۵ ۰٫۲۵ – –

Acknowledgements

Authors would like to thank staff of Farabi eye Hospital for their kind help.

 

Authors’ contributions

AI, RDG, SJH and SK drafted the manuscript. AI, MS, MG, KA, MA, MG, ZA and SK conducted the clinical examinations and Laboratory tests. AI, MS, RDG, SJH, AB, and SK reviewed the manuscript and participated in the layout and design of the report. All authors read and approved the final manuscript.

 

Ethics approval and consent to participate

The patient provided a signed written informed consent to undergo the treatment. This report was approved by ethics committee of Tehran University of Medical Sciences (IR.TUMS.SPH.REC.1398.256).

Funding

This study has been funded and supported by Tehran University of Medical Sciences (TUMS); Grant no. 99-1-99-46389. Dr. Aleksandra Barac is funded by the Project of Ministry of Education, Science and Technology of the Republic of Serbia (No. III45005).

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Corresponding author

Sadegh Khodavaisy, PhD

Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Tel : +982188008588

Email : sadegh_7392008@yahoo.com

Conflict of interests: No conflict of interests is declared.