Challenges in the diagnosis and management of atypical fungal keratitis during the COVID-19 pandemic: a case series

Fungal keratitis is a time-sensitive ocular infection that often requires a high index of suspicion followed by intensive medical/surgical interventions to achieve a successful clinical outcome. COVID-19 pandemic-related restrictions, necessitated the modification of conventional protocols and guidelines associated with the treatment of keratomycosis. We report four cases of atypical fungal keratitis with poorly differentiated clinical characteristics. The challenges faced during their management were (1) the dilemma of clinically differentiating fungal (Scedosporium and Purpureocillium lilacinum) and bacterial keratitis; (2) treatment of Scedosporium and Trichosporon keratitis with natamycin monotherapy; (3) mixed infection of Candida albicans and Aureobasidium pullulans and continuing medications before rescraping the corneal ulcer against the recommended treatment guidelines; (4) phenotypic identification and differentiation among morphologically resembling fungi; and (5) decision making arising out of disparities between KOH and fungal culture results. Three patients responded well to conservative treatments. The fourth patient underwent therapeutic keratoplasty but was lost to follow-up due to travel-related pandemic restrictions. This case series seeks to broaden the clinician’s knowledge of rare and emerging moulds as presumptive aetiologies of keratomycosis. It also intends to emphasize the significance of early microbiological investigations, (direct microscopy and culture), in resource-limited settings, for initiating empirical treatment for a better visual prognosis.


INTRODUCTION
Keratomycosis or fungal keratitis (FK) is an invasive infection of the corneal stroma.Corneal opacification secondary to keratitis, is also known to be the second most common cause of blindness after cataract [1].Developing, tropical and subtropical countries have reported almost half of the world's FK [2].In India, corneal ulcers are caused by bacteria and fungi in equal proportions.Cyril et al. [3] inferred that only 66 % of corneal specialists were able to clinically distinguish between fungal and bacterial corneal ulcers.Hence, the rationale for empirical therapy must be reviewed.Early diagnosis and treatment of FK also avoids intraocular involvement and complications [4][5][6].This underlines the importance of diagnostic mycological investigations during the initial examination of the patient.Contemporary investigators have reported Aspergillus spp.and Fusarium spp. as the most common etiological agents for fungal keratitis [7][8][9][10].Rarer fungal pathogens like Acremonium spp., Alternaria spp., Penicillium spp., Bipolaris spp., Curvularia spp., Phialophora spp., Blastomyces spp., Sporothrix spp., Exophiala spp., Scedosporium spp., Cylindrocarpon spp., Purpureocillium spp., Lasiodiplodia theobromae, Metarhizium anisopliae and Pythium insidiosum have also been described [11].Conventional treatment protocols had been bypassed during the COVID-19 pandemic owing to patient's financial and travel constraints and the hospital's logistic constraints for prioritizing in-patient admissions, based on their COVID-19 status.Accordingly, patients preferred to access the nearby healthcare facilities post-lockdown.This provides an opportunity to expand the knowledge of the healthcare providers to the rarer aetiologies of common clinical conditions.In this case series, we share our unusual experiences of diagnosing and treating four atypical cases of fungal keratitis during the COVID-19 pandemic.

CASE PRESENTATION Case 1
A 68-year-old man with type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) on treatment, presented to our outpatient department (OPD) with complaints of swelling of the left eye and difficulty in opening the eyes for 3 days.On examination, his best corrected visual acuity (BCVA) in the right eye was 6/18 and left eye counting fingers ½ m.The right eye was normal following a slit lamp examination.There was a central circular ring-shaped 3×3 mm anterior stromal infiltrate around a pre-existing central scar in the left eye.Patient was administered 0.5 % topical Moxifloxacin and 1 % Amikacin every 2 h, along with 2 % Homatropine twice daily after a provisional diagnosis of bacterial keratitis.However, potassium hydroxide (KOH) mount of the corneal scraping revealed septate hyphae.Hence, the patient was treated with 5 % Natamycin every 2 h.The ulcer healed completely with scarring on day 30.Scedosporium spp. was isolated from the fungal culture (Table 1).BCVA improved to counting fingers (CF) 2 m partly due to central scarring and diabetic retinopathy.

Case 2
A 48-year-old man with T2DM, presented with a post-traumatic pupil associated with a third nerve palsy involving the right eye.Limitation of elevation of the right eye with the right upper eye lid was noted.Corneal sensation was intact without any other cranial nerve involvement.After 3 weeks of follow up, he developed pain and redness in the same eye.His BCVA was CF 3 m, and cornea showed a 2.5×1.5 mm linear anterior stromal infiltrate with an overlying epithelial defect.KOH mount done on the corneal scraping revealed fungal elements.Therefore, he was treated with topical 5 % Natamycin for every 2 h, 0.5 % Moxifloxacin thrice daily and 1 % Cyclopentolate thrice daily.Ulcer healed completely by day 28.Trichosporon asahii was reported from fungal culture (Table 1).BCVA improved to 6/18.(Fig. 1a, b)

Case 3
A 76-year-old woman with a history of plant trauma, presented with pain, redness and watering of right eye for 3 days.She was previously being treated for dyslipidaemia and systemic hypertension.Her BCVA in right eye was 6/36 and that in her left eye was 6/12.Cornea of the right eye showed a 4×4 mm paracentral mid stromal infiltrate with surrounding corneal oedema.Empirical treatment was initiated with topical 5 % Natamycin every 2 h,0.3 % Moxifloxacin thrice daily and 2 % Homatropine drops twice daily.On day 20, she developed a 2.5 mm hypopyon after a brief symptomatic improvement (Fig. 2a).KOH mount Greyish-white cottony colonies on the obverse with reverse black pigmentation (Fig. 3a) Hyaline septate hyphae with percurrent, lateral and terminal conidiogenous cells [21].Graphium conidiation (septate hyphae with long conidiophores cemented to form synnemata) (Fig. 3b)

Yeast-like cells with hyphae
White coloured creamy to pasty colonies at 37 °C (Fig. 5c) Gram positive oval budding yeast cells with hyphae and pseudo hyphae (Fig. 5a)

4
No fungal elements Obverse powdery, cottony, velvety pink to lilac-coloured colonies with reverse off-white pigmentation (Fig. 6a) Septate hyaline hyphae.Branched conidiophores bearing metulae with tapering phialides bending away from the axis of the conidiophore.Oval to fusiform catenulate phialoconidia (Fig. 6a) Purpureocillium lilacinum from the corneal scraping showed sparsely septate hyphae with yeast-like cells.On day 25, treatment was changed to fortified 0.15 % Amphotericin B hourly along with Natamycin.On day 30, the ulcer healed but the hypopyon persisted.Oral Itraconazole 200 mg bd was added to her treatment regimen.As contaminant fungi were isolated from fungal culture, rescraping was done for a second culture on day 35, which grew Aureobasidium pullulans and Candida albicans (Table 1).The ulcer healed completely on day 60, with paracentral thinning and a leucomatous opacity (Fig. 2b, c, d).Her topical medications were tapered gradually.

Case 4
A 46-year-old man migrant farmer presented with a history of on-field foreign body injury with pain and redness in the right eye.This was treated with 5 % topical Natamycin and oral Fluconazole 150 mg at another hospital.Unsatisfied with his recovery, he consulted our OPD with an 8×7 mm large non-healing corneal ulcer and a 3.5 mm hypopyon.His BCVA was counting fingers close to face (CFCF) in the right eye and 6/9 in the left eye.Due to the intraocular spread and poor response to parenteral and topical antibiotics and antifungals, he underwent therapeutic keratoplasty.KOH mount done from the patient's corneal button was negative for fungal elements.Hence, the patient was empirically started on fortified Ceftazidime every 2 h, 5 % Natamycin six times a day, Tobramycin eye drops every 2 h and oral Ciprofloxacin 500 mg.He was advised to review after 3 days.However, we lost our patient to follow up as he returned to his hometown post-lockdown.So, his treatment response also could not be assessed.Purpureocillium lilacinum was isolated from the fungal culture (Table 1).

Diagnostic mycology findings
Corneal epithelium was scraped off from the ulcer, with aseptic precautions, using a sterile no.11 blade, under direct vision through slit lamp, after instilling 0.5 % Proparacaine anaesthetic eye drops.Direct microscopy of the patient samples was done with 10 % KOH.Corneal scrapings and the resected corneal button (case 4), were streaked in a superficial 'C' pattern, on Saboraud Dextrose Agar (SDA), without Chloramphenicol or Cycloheximide, to recover those saprobic fungi, which are not only inhibited by these antimicrobials, but are also implicated in opportunistic infections.The culture plates were incubated, at 37 and 28 °C for 4 weeks, to enable the isolation of dimorphic fungi.Cultures were reviewed every day for the first 2 weeks, and every alternate day, during the third and fourth weeks.Fungal growth observed on the "C streaks'' on SDA, with or without a positive KOH mount from clinical samples, were considered significant.Growth outside the "C streaks'' were regarded as contaminants [9].Bacterial growth was ruled out by negative cultures done concurrently on HI Media Sheep Blood Agar.Moulds growing on SDA, were identified phenotypically, as per standard laboratory protocols -colony morphology, growth rate, Lactophenol Cotton Blue (LPCB) staining and Riedles Slide Culture Method.Gram stain was done for the identification of isolated yeast colonies on SDA.

DISCUSSION
The World Health Organization (WHO) has included infectious keratitis as one of the many neglected tropical diseases [12].Among the several risk factors reviewed by Shahram et al. [13], corneal trauma accounted for 40-60 % of cases of FK.Notable among others are long-term steroid use, diabetes, use of contact lens, farming, past penetrating keratoplasty and exposure keratitis.In this case series, T2DM (cases 1 and 2), trauma and foreign body exposure during farming (cases 3 and 4) were identified as co-existent and independent risk factors of microbial keratitis.Moreover, alterations in the tear components, commensals and the enzyme activities in a long-term hyperglycaemic ocular microenvironment facilitate fungal adherence, proliferation and deep-layer penetration [14].Xin et al. [15] observed that delayed medical attention-seeking by farmers, especially during harvest season, resulted in a poorer response to antifungal drugs and higher rates of surgery.

Therapeutic and diagnostic challenges in fungal keratitis
Good communication between the ophthalmologist and the mycologist ensured the isolation of all the aetiological fungi from culture.After initial slit-lamp examination, corneal scraping is done for 10 % KOH mount and fungal culture.Other samples include corneal biopsies (if clinical suspicion persisted despite negative KOH and culture results) and anterior chamber aspirates (if there was intraocular involvement) [13].
The diagnostic utility of a KOH mount had been calculated by Bharati et al. [16] on 3298 eye samples with resultant sensitivity, specificity positive and negative predictive values of 99.3, 99.1, 98.5 and 99.6 %, respectively.In the same study, insufficient samples, inexperienced observers and small size of the corneal ulcer yielded negative KOH results.
Fungal cultures remain the gold standard test, especially in resource-limited laboratories.However, the samples have to be incubated for up to 4 weeks.It can also turn out to be false positive (due to the isolation of environmental contaminants) or false negative (due to scanty specimens, poor sampling, previous medications and deep corneal involvement) [13].In our routine clinical practice, the overlying epithelium along with necrotic material and mucus are debrided, after corneal scraping, for better drug penetration.The antifungal agents are also started hourly for 48 h till the signs of healing are visible.Candida infections are treated with 0.15 % Amphotericin B or 1 % Econazole; alternatives are 5 % Natamycin, 2 % Fluconazole and 1 % Voriconazole.Filamentous fungi are treated with 5 % Natamycin or 1 % Econazole or alternatively 0.15 % Amphotericin B 1 % Miconazole or 1 % Voriconazole.Broad spectrum antibiotics prevent bacterial co-infection.Mydriatics or cycloplegics (1 % Cyclopentolate, 2 % Homatropine or 1 % Atropine) prevent posterior synechiae.If there is no treatment response despite patient compliance to the antifungal regimen, there is a possibility of the drug not acting against the implicated fungus.In such situations, the treatment is temporarily suspended for the next 24 h and a repeat corneal scraping is sent for culture.This ensures the recovery of resistant variants of the same or a new fungus and helps the ophthalmologist to suitably modify the treatment regimen [17].
The examination findings in advanced fungal keratitis are indistinguishable from that of bacterial keratitis [7].Drug-related concerns include limited spectrum of action of available antifungals, poor ocular drug penetration of systemic antifungals, and limited commercial availability of topical antifungals.Drug toxicity and associated ocular complications occur due to the prolonged clinical course causing delayed corneal healing.The fungistatic action of the current antifungals are likely to cause recurrence of FK [3].Patients' refractory to medical therapy, undergo an expensive keratoplasty with a high risk of transplant rejection, and a donor tissue is hardly ever available [1].

Case 1
The fungus could have possibly entered as a result of trivial trauma caused by rubbing of the eye and diabetic corneal erosion [18,19].The four clinically encountered species belonging to the Scedosporium species complex are S. apiospermum, S. boydii, S. auranticum and S. deboogi [20].As all the above-mentioned species appear morphologically similar in culture, we could only identify the synanamorph, Graphium, of the fungus along with the conidiogenous cells.(Fig. 3a, b).The latest ECMM-ISHAM-ASM guidelines recommend Voriconazole as the first-line therapy across all patterns of organ involvement [21].Mycotic ulcer treatment trial ll demonstrated no therapeutic benefit of adding oral Voriconazole to topical antifungal therapy [22].However, two cases of Scedosporium keratitis were treated with topical and oral Voriconazole [18,23].Ramakrishnan et al. [24] treated S. keratitis among seven out of the ten patients with a combination of Natamycin and Fluconazole.Natamycin monotherapy was employed by Rathi et al. [25] in five out of eight cases.Our patient also recovered with topical 5 % Natamycin monotherapy alone.Thus, it can be inferred that the treatment must be tailored according to clinical response.

Case 2
The patient with a history of trauma had no epithelial defects during the preliminary examination.But, with an associated elevation deficit of the right eye with the involvement of the right eyelid, exposure of the cornea during sleep could have predisposed to FK after 3 weeks.Geotrichum spp., morphologically resembles Trichosporon spp.due to the predominant arthroconidia and hence, it was excluded by urease and assimilation tests.Trichosporon spp.(Fig. 4a, b) are known to rarely cause FK in Indian case reports [26,27].They are intrinsically resistant to Echinocandins with low MICs to both Voriconazole and Posaconazole.This patient also responded well to empirical 5 % Natamycin monotherapy due to the early initiation of treatment.His ulcer also resolved completely with minimal scarring (Fig. 1a, b).

Case 3
Clinical findings were consistent with FK in this dual infection with Aureobasidium pullulans and C. albicans.Candida keratitis have been reported more in the west than in Asia [28].Few published case reports also highlight topical steroid use, previous ocular surface diseases or keratorefractive surgeries as risk factors for Candida keratitis [29][30][31].As this patient had none of these factors, we presume that C. albicans may have been acquired from his own flora [32].The clinical findings of the patient must be corroborated to determine if Aureobasidium pullulans, a phaeohyphomycete, is a contaminant in clinical samples or a pathogen.The thin and thick-walled septate dematiaceous hyphae of A. pullulans (Fig. 5a, b) can resemble another non-pathogenic phaeohyphomycete, Hormonema dematioides [20].Augustin et al. [33] noted good recovery with Fluconazole, Natamycin and repeated scraping.Empirical treatment with topical 5 % Natamycin was started in this case as the patient refused consent for corneal scraping.She consented to the procedure only when her recovery was unsatisfactory.Since contaminants were isolated from the initial fungal culture and hypopyon persisted, rescraping had to be done.Her travel constraints made us deviate from the conventional practice of stopping her medication before rescraping.Itraconazole and 0.15 % fortified Amphotericin B were added.Studies report higher in vitro antifungal resistance to Fluconazole, Voriconazole and Echinocandins for A. pullulans than Itraconazole, Posaconazole and Amphotericin B [34,35].Intravenous preparation of Amphotericin B was reconstituted for topical administration in this patient due to its commercial non-availability.The short shelf life (5-7 days) of the formulations increased the treatment cost and OPD visits of our patient.Even though the patient was non-compliant to 5 % Natamycin, she improved with Amphotericin B and Itraconazole.Nevertheless, delayed treatment and healing caused a dense and larger scar (Fig. 2a-d).

Case 4
This patient presented with an advanced ulcer due to Purpureocillium lilacinum (previously Paecilomyces lilacinus) [36].The slender tapering whorls of phialides of Purpureocillium lilacinum were differentiated from the 'Penicillus'' or 'brush-like' phialoconidia of Penicillium spp.[20] (Fig. 6a, b).The fungi invades the injured or diseased ocular surface [37] and elicits an innate immune response due to the production of hydrolytic enzymes and the release of mycotoxic peptides such as leucinostatins [38].Corneal inflammation alters its transparency due to the associated oedema, and changes in the refractive index affect vision [39].Several case series [40][41][42] mention that the ocular tropism of this fungi occurs due to tropical climate, contact lens use and exposure to vegetable matter.Though the probability of FK was higher, it was still clinically challenging to rule out bacterial keratitis.The fungus is resistant to Amphotericin B, Fluconazole and Echinocandins and sensitive to second-generation triazoles [37,43,44].KOH was negative (possibly due to the absence of viable fungi in the scanty sample) and no response to empirical antifungal therapy was noted.Medical line of treatment has been successful only in a few cases [43][44][45][46].Foreseeing the possibility of intraocular spread, the patient underwent penetrating keratoplasty.But, the outcome of postoperative management could not be assessed as the patient could not be followed up.
In this case series, the central ulcers (noted in cases 1 and 2) and hypopyon (noted in case 3) could be successfully managed without admitting the patients, despite COVID-19-related constraints.Butt et al. [47] also concur that there were no significant differences in the treatment outcomes of admitted and self-medicating microbial keratitis patients.
There were a few limitations in this case series.The use of confocal microscopy could have avoided repeated scraping of the cornea and decreased the patient visits in case 3. We were unable to follow up on case 4 due to lockdown-related constraints.Due to the ongoing standardization of testing protocols in our laboratory, antifungal susceptibility testing, that could have influenced the treatment outcomes of all our patients was not done.It is in this context that interactions with the diagnostician can help prioritize culture over KOH testing in resource-limited settings for scanty samples, depending on the stage of presentation of the corneal ulcer.Fungi may also be directly identified from scanty samples by molecular methods like Conventional PCR followed by post-amplification procedures [restriction enzyme digestion and analysis, single-base extension, hybridization probes or capillary-based molecular sequencing, and single-strand conformational polymorphism (SSCP)] [13].Molecular testing is not always feasible due to logistic constraints in developing countries.Therefore, the importance of phenotypic identification up to the genus level, which was done for all our patients in this case series, cannot be overlooked.
Tawde et al. [48] have tempero spatially correlated the source of fungi causing keratitis by environmental sampling.The state of Kerala receives an annual rainfall of 300 cm and provides a moist environment for the thriving fungi [49].Furthermore, the devastating floods in 2018 and 2019, as well as the excessive rainfall in the following years, have expanded the ecological niche of the fungi to newer areas.This could have possibly triggered regional spikes in the occurrence of atypical keratomycosis.So, we believe that conventional treatment guidelines need to be individualized to the stage of the presentation of fungal keratitis.In this regard, a trend analysis tracing the atypical fungi causing keratomycosis to their environmental habitats, may be undertaken to guide further modifications in the treatment protocols.

CONCLUSION
Early diagnosis and treatment, backed by good communication with the microbiologist, more so during contingencies, help in prevention of the ocular complications and improvement of the visual outcome.Disparities between the findings of KOH smears and fungal cultures must be discussed with the clinician so that the plan of care may be revised according to the patient's clinical profiles.Hence, microbiological cultures are mandatory to identify the etiological agents of microbial keratitis because the regional climatic changes may dictate the ecological niche of the microbes.

Funding information
This work received no specific grant from any funding agency Comments: No further comment.All comments have been answered by the author(s).

Please rate the quality of the presentation and structure of the manuscript Very good
To what extent are the conclusions supported by the data?Strongly support

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes
Author response to reviewers to Version 1 We have a LABOMED ® LX 500 binocular microscope in our laboratory which does not have an inbuilt camera.Our images had been captured on a mobile phone camera.In compliance to the reviewer's suggestion, we had sent them to a premier research institute, Rajiv Gandhi Centre for Biotechnology, in Thiruvananthapuram, for adding scale bars.As the mobile phone images had a different resolution, this couldn't be done.Adding scale bars from the IMAGE X software was also attempted but to no avail.Therefore, we humbly acknowledge this as our limitation.Kindly oblige.However, Figures 1-4 have been presented together in a single A4 format in the revised manuscript.

Reviewer 6
Reviewer 6 Comments to Author: The manuscript describes the challenges in diagnosing and managing atypical fungal keratitis during the COVID-19 pandemic, intending to broaden the clinician's knowledge of rare but emerging moulds as presumptive aetiologies of keratomycosis.It also intends to reinforce the significance of early microbiological investigations (direct microscopy and culture) in resource-limited settings for initiating empirical treatment towards a better visual prognosis.Overall, the case report is interesting.However, improvements are needed for clarity.
Please rate the quality of the presentation and structure of the manuscript Line 149-The culture plates were incubated at two different temperatures for the isolation of dimorphic fungi from the clinical samples as a part of our Standard Operating Procedure (SOP) manual of our laboratory in order to optimize our laboratory resources.A justification to this effect has been provided in the section on "Diagnostic Mycology Findings" of our manuscript.
Page 6 line 149 -4 weeks should be written in full.Line 149-4 weeks written in full Page 7 line 153 -Why Gram stain was conducted to test fungi?Gram stain is to differentiate between Gram positive and Gram negative bacteria.All fungi should give dark blue colour since the eukaryotic microorganism generally will have cell wall.Please justify the purpose of Gram stain in the study.Similar comment to Table 1 on Candida albicans isolates.
Line 153 and Table 1-Gram stain was done for the morphological identification of the isolated yeast colonies on SDA.The diagnostic protocol has been elaborated and modified by quoting the reference article no-9 in our manuscript-Tilak R, Singh A, Maurya OPS, Chandra A, Tilak V, Gulati AK (2010) Mycotic keratitis in India: a five-year retrospective study.J Infect Dev Ctries 4:171-174.doi: 10.3855/ jidc.309 The heading of the column in Table 1 has also been modified likewise.The authors address the atypical fungal keratitis, reporting an interesting set of cases.This is a hard-totreat clinical entity, whose interest is emphasized by Covid-19 pandemic background in which the cases appeared.In addition, the unusual incidence of the involved fungi and the implicit clinic/laboratory strong interaction are additional interesting points of the manuscript.Moreover, the whole manuscript is well-structured and generally well-written; the good quality of the photographs is noteworthy.
There are a few points that I wish to take to the at-

SUGGESTED REVISIONS AUTHOR RESPONSES
Lines 219-221: the authors refer that when improvement was lacking, the patient's treatment was suspended for 24 h to obtain another sample.Thus, the benefit compensated for the risk.Can the authors argument a little more about this issue?
Line 219-221-The statement noted in these lines has been suitably modified.
Several phrasing or wording shortcomings were noticed: -some full stops are inserted before references or before «

Anonymous.
Date report received: 24 May 2023 Recommendation: Minor Amendment

Comments:
The authors address the atypical fungal keratitis, reporting an interesting set of cases.This is a hard-to-treat clinical entity, whose interest is emphasized by Covid-19 pandemic background in which the cases appeared.In addition, the unusual incidence of the involved fungi and the implicit clinic/laboratory strong interaction are additional interesting points of the manuscript.Moreover, the whole manuscript is well-structured and generally well-written; the good quality of the photographs is noteworthy.There are a few points that I wish to take to the attention of the authors: 1. Lines 219-221: the authors refer that when improvement was lacking, the patient's treatment was suspended for 24h to obtain another sample.Thus, the benefit compensated the risk.Can the authors argument a little more about this issue?2. Several phrasing or wording shortcomings were noticed: -some full stops are inserted before references or before «

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Partially support

Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices? No
Is there a potential financial or other conflict of interest between yourself and the author(s)?No If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes

Reviewer 6 :
GoodTo what extent are the conclusions supported by the data?Reviewer 6: Partially support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 6: No: If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Reviewer 6: Yes: 72 and line 76 -spp.should be written if full when first mentioned.Consistency of nomenclature maintained.Spp.mentioned after every genus Page 5 line 91 -3 days should be written as 'three'.All numbers from 1 to 10 should be written in full.Line 91-3 days altered as "three" days Page 5 line 97 -potassium should be small letter.Line 97-potassium written in small letters.Page 5 line 105 -3 weeks should be written as 'three'.Line 105-3 weeks written as "three" Page 5 line 113 -suggest to replace lady with female.Line 113-lady replaced with "woman" in order to maintain uniformity with the other cases presented in this manuscript (as per Reviewer 7 comments) Page 5 line 114 -3 days should be written as 'three'.Line 114-3 days written as "three" Page 5 line 96 and 118-What do you mean by two hourly?Is it twice or two times?Line 96-118-Two hourly means "every two hours".Corrections reflected in the manuscript.Page 6 line 123, Page 12 line 255, Page 12 line 274, Page 13 line 294 -itraconazole should be written in small letters.Please check other names of antifungal used as well in the whole manuscript.Better to standardize it to use capital letters.All the antifungals in this manuscript have been standardized with capital letters in compliance with the reviewer comments.Page 6 line 131 -What is Tab.fluconazole?Line 131-"Tab" Fluconazole replaced with "oral" Fluconazole.Page 6 line 138 -What do you mean by 'two hourly'?Line 96-118-Two hourly means "every two hours".Corrections reflected in the manuscript.Page 6 line 138 -6 should be written in full.Line 138-6 written in full Page 6 line 139 -3 days should be written in full.Line 139-3 days written in full Page 6 line 149 -Why the culture plates were incubated at two different temperatures?Please justify.

7
Page 10 line 204 -..negative smears.Line 204-… negative smears modified as "yield negative KOH results" Page 10 line 206 -4 should be written in full.Line 206-4 weeks written in full Page 11 line 243 -Scedosporium keratitis should be written as S. keratitis when mentioned for the second time.Line 243-Scedosporium keratitis changed to S. keratitis during the second mention.Page 11 line 245 -5 and 8 cases should be written in full.Line 245-5 and 8 cases written in full.Page 12 line 259, 263 -Candida albicans should be written as C. albicans.Line 259,263-Candida albicans modified as "C.albicans"Reviewer Reviewer 7 Comments to Author:

Comments:
The manuscript describes the challenges in diagnosing and managing atypical fungal keratitis during the COVID-19 pandemic, intending to broaden the clinician's knowledge of rare but emerging moulds as presumptive aetiologies of keratomycosis.It also intends to reinforce the significance of early microbiological investigations (direct microscopy and culture) in resource-limited settings for initiating empirical treatment towards a better visual prognosis.Overall, the case report is interesting.However, improvements are needed for clarity.Page 4 line 72 and line 76 -spp.should be written if full when first mentioned.Page 5 line 91 -3 days should be written as 'three'.All numbers from 1 to 10 should be written in full.Page 5 line 97 -potassium should be small letter.Page 5 line 105 -3 weeks should be written as 'three'.Page 5 line 113 -suggest to replace lady with female.Page 5 line 114 -3 days should be written as 'three'.Page 5 line 96 and 118-What do you mean by two hourly?Is it twice or two times?Page 6 line 123, Page 12 line 255, Page 12 line 274, Page 13 line 294 -itraconazole should be written in small letter.Please check other names of antifungal used as well in the whole manuscript.Better to standardised in to use capital letter.Page 6 line 131 -What is Tab.fluconazole?Page 6 line 138 -What do you mean by 'two hourly'?Page 6 line 138 -6 should be written in full.Page 6 line 139 -3 days should be written in full.Page 6 line 149 -Why the culture plates were incubated at two different temperatures?Please justify.Page 6 line 149 -4 weeks should be written in full.Page 7 line 153 -Why Gram stain was conducted to test fungi?Gram stain is to differentiate between Gram positive and Gram negative bacteria.All fungi should give dark blue colour since the eukaryotic microorganism generally will have cell wall.Please justify the purpose of Gram stain in the study.Similar comment to Table 1 on Candida albicans isolates.Page 10 line 204 -..negative smears.Page 10 line 206 -4 should be written in full.Page 11 line 243 -Scedosporium keratitis should be written as S. keratitis when mentioned for the second time.Page 11 line 245 -5 and 8 cases should be written in full.Page 12 line 259, 263 -Candida albicans should be written as C. albicans.

cases Direct microscopy findings -KOH mount Colony morphology of the isolate on SDA at 37 °C/28 °C Smear findings from isolated colonies on SDA (Lactophenol Cotton Blue tease mount preparation/Gram stain) Final Identification of the isolate 1 Septate hyphae
The authors present 4 compelling case studies detailing the difficulties of identifying fungal pathogens implicated in fungal keratitis in developing countries.The author addresses the reviewers comments to a sufficient level.
https://doi.org/10.1099/acmi.0.000570.v2.1 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Anonymous.Date report received: 10 July 2023 Recommendation: Accept Comments: Comments: Authors have fulfilled all minor suggestions I have indicated (reviewer 7).In this version, Line 269, consider «Case 1» instead of «For Case 1».
Lines 58-59 contains Data Summary Statement of the Manuscript.It was initially removed and uploaded on 23 rd June 2023.We got an email from the Editorial Office of the esteemed journal on the same day requesting us to add the Data Summary Statement in the main manuscript folder.

Reviewer 2 recommendation and comments https
Table» or «Figure»; some other are located after those items.Now and then, there are commas that seem to be missing or are misplaced.In addition, spacings and unnecessary capitals (e.g., Potassium, Day, Tab, Itraconazole, Posaconazole, Amphotericin) indicate that an additional review of the English will standardize the manuscript.-Lines89&113:manand woman would be preferred to gentleman and lady -Lines 95-96: regarding moxifloxacin and amikacin administration «two hourly», do the authors mean every 2 hours?-Lines 104 & 249: please verify whether the limitation was of the eye or the eyelid -Line 105: «…any other cranial nerve…», instead of «…any cranial nerve…».-Lines142-143:Thetextrelated to the consent appear to be repeated (in Lines 352-354).I suggest to remove the first appearance.-Line224:«microbialkeratitis»;dothe authors mean «fungal keratitis»?://doi.org/10.1099/acmi.0.000570.v1.3 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.The authors present a thorough and well written case study series demonstrating the use of laboratory culture techniques to identify microbial species associated with fungal keratitis in a resource limited setting.I commend the authors for highlighting limitations of these techniques and improvements required for effective diagnostic testing.I have a handful of minor comments.Line 58 -59: Remove, not required Line 72 -76: Be consistent with nomenclature, include spp.after every genus described Line 153 -156: Please provide references to protocols for species identification used in this study, or alternatively provide additional brief detail.Figures: Please add scale bars to acquired microscopy images.Figures1 -4could be presented as a single figure in A4 format.Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000570.v1.5 © 2023 Arzmi M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.University of Malaya, Oral Biology, 70, JLN SS7/28,, TAMAN KELANA INDAH,, KELANA JAYA, PETALING JAYA, Malaysia https://orcid.org/0000-0002-9470-6412Date report received: 28 April 2023 Recommendation: Minor Amendment rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesPlease rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly support