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OPINION Update on fungal keratitis
Prashant Garg a, Aravind Roy b, and Sanhita Roy c

Purpose of review
The aim of this review article is to present an overview of some of the seminal work published in the last
18 months (July 2014 to December 2015).
Recent findings
The published literature highlights the need for the identification of fungal isolates to species and
subspecies level using molecular methods. Molecular methods helped us identify some of the unknown
fungi such as Pythium – fungi that causes keratitis unresponsive to conventional antifungal therapy.
Although not popular fungal in-vitro susceptibility tests are showing better correlation between resistance
and clinical outcomes. Several groups are trying to understand host responses controlling disease
production as well as inflammation. On therapy front researchers are working to develop drug formulations
and delivery systems that will provide superior pharmacokinetics and bioavailability. Collagen cross-linking
and injections of antifungal agents into the corneal stroma and anterior chamber continue to be attractive
to clinicians and more and more researchers are publishing their experiences.
It is an interesting time in the history of mycotic keratitis with a lot of positive developments. Molecular
methods will help improved diagnosis and clinico-therapeutic correlation. Drug delivery devices with
superior pharmacokinetics are on horizon.
collagen cross-linking, fungal keratitis, in-vitro susceptibility, mycotic ulcer treatment trial, Pythium

INTRODUCTION variation. The variability is influenced by local

Fungi are important causes of keratitis especially in environment; predominant occupation, availability
countries with tropical climate and account for nearly of antimicrobial drugs and even gross national
income [1,4 ]. The epidemiology of fungal keratitis

40% of all isolates from corneal ulcer cases [1,2 ]. As


many of the nations with preponderance of fungal also varies widely throughout the world and even
cause are also most populated and have a higher between different regions of the same country. An
prevalence of corneal ulceration, the contribution Indo-Hungarian Fungal Keratitis (IHFK) Working
of fungi, as causes of corneal blindness cannot be group published global overview of epidemiology
underestimated. Further, it is now established that wherein they identified that more than 70 different
monocular vision loss from corneal opacity adversely fungal species have been incriminated to be causa-
affects vision-related quality of life [3]. Therefore, it tive agents of keratitis. Fusarium, Aspergillus, and
will be prudent to carry out and popularize high- Curvularia were the major fungal genera among
quality research on this topic of public health priority. molds whereas Candida constituted majority among
The outbreak of Fusarium keratitis provided yeast group. Molds are predominant pathogens of
stimulus in generating interest and financial support keratitis in tropical and subtropical areas whereas
for studying fungal infections. In the last decade we
have witnessed a good quality research addressing a
Tej Kohli Cornea Institute, KAR campus, L. V. Prasad Eye Institute,
several seminal aspects of this disorder. In this article
Hyderabad, bTej Kohli Cornea Institute, KVC campus, L. V. Prasad Eye
we will present an update of the research work on Institute, Vijayawada and cBrien Holden Eye Research Centre, L. V.
fungal corneal ulcer published over past 18 months. Prasad Eye Institute, Hyderabad, Andhra Pradesh, India
Correspondence to Prashant Garg, L. V. Prasad Eye Institute, L. V.
Prasad Marg, Banjara Hills, Hyderabad 500034, AP, India. Tel: +91
EPIDEMIOLOGY 40 30612345; fax: +91 40 23548271; e-mail:
The epidemiology of corneal ulceration, including Curr Opin Ophthalmol 2016, 27:333–339
causative microorganisms shows wide geographic DOI:10.1097/ICU.0000000000000272

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Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

Corneal and external disorders

fungal agents many of which were previously

KEY POINTS unreported from corneal ulcer cases. One such
 Identification of fungal isolates to species level using uncommon pathogen belongs to Pythium genus of
molecular methods will help us better understand the Oomycota division. Pythium was first reported from
epidemiology, clinical picture, and treatment outcomes. Thailand in 1985. The human infections caused by
this uncommon pathogen are classified into four
 Pythium, a relatively unknown cause of keratitis
groups – cutaneous, vascular, ocular, and dissemi-
produces clinical picture mimicking other commonly
identified fungi but does not respond to nated [6]. In the year 2015, two reports of ocular
medical treatment. infections by this pathogen were published in
English literature [7,8 ]. The reports identified

 In-vitro susceptibility tests for antifungal agents are corneal trauma and exposure to water as the most
better standardized. There are species level differences
common predisposing factors. The clinical picture is
in susceptibility among fungal isolates and the
resistance to few antifungal drugs correlates well with characterized by tentacle-like or dot-like infiltrates
poor outcomes. surrounding the main infiltrate, hyphate edges, and
reticular subepithelial as well as superficial infiltra-
 Newer formulations and drug delivery systems such as tion. Overall 10% potassium hydroxide mount dem-
liposomes and nanoparticles hold promise for better
onstrates broad flat ribbon-like aseptate or sparsely
treatment of fungal infections.
septate filaments with right-angled branching. The
organism grows on blood or chocolate agar as a flat,
feathery, glabrous, and colorless or light-brown
yeasts are reported in higher numbers from colony. The typical sporangium with bursting out
developed nations and temperate areas [2 ].

of motile zoospores through the sporangiophore

may be demonstrated from the isolates. The infec-
tion does not respond to conventional antifungal
IDENTIFICATION OF FUNGAL SPECIES BY treatment and most cases require surgical interven-
MOLECULAR METHODS tions. These reports have certainly enhanced aware-
Most laboratories use culture characteristics ness about this pathogen and we can expect more
and morphology of spores for the identification of publications on this topic in near future.
fungal isolates. But unfortunately, not all samples
despite being positive for fungus on microscopic
examination reveal growth on culture. Even IN-VITRO SUSCEPTIBILITY OF FUNGAL
sporulation is a challenge. The outcomes are: high ISOLATES
proportion of unidentified fungi and lack of identi- Unlike bacterial infections, in-vitro susceptibility
fication of pathogens to species level. These can tests for antifungal agents are not used in the routine
be real concerns if we consider that the clinical clinical practice. It is believed that the test results
features, antifungal susceptibility and treatment do not correlate well with in-vivo clinical response.
outcome of fungal keratitis are variable and might However, in the past 2 decades there has been a lot
be related to differences at species level within the of progress in standardization of antifungal suscepti-
same genus. Further, there is a possibility of geo- bility tests and we now have methods approved by
graphical variation in species pattern of predomi- American and European standards (CLSI and
nant genera, a challenge for epidemiological studies. EUCAST). Both institutions have developed break-
This highlights the need for molecular identifi- points of some antifungal agents against Candida
cation of pathogens. IHFK working group performed and Aspergillus species [9]. Sun et al. [10 ] found that

multilocus DNA sequencing on 65 Fusarium keratitis in mycotic ulcer treatment trial a two-fold increase
isolates from India. The identified species showed in minimum inhibitory concentration (MIC) for
wide variability and could be clubbed into four natamycin was associated with a larger 3-month
clades. The largest included Fusarium solani species infiltrate or scar size and increased odds of corneal
complex followed by Fusarium dimerum, Fusarium perforation. This and previously published trials
fujikuroi, and Fusarium oxysporum [5 ]. This study

[11–14] point that in-vitro susceptibility test may

reaffirmed the genetic diversity of clinical strains of have a prognostic value and help us making crucial
fungi and the need for molecular identification. decisions in the management of fungal keratitis.
Investigators have also looked at species vari-
ations in susceptibility. In 2013–2014 several stud-
PYTHIUM KERATITIS ies evaluated this variability among Fusarium and
Application of DNA-based methods resulted in the Aspergillus isolates [15–18]. In the year 2015, Motu-
identification of several uncommon filamentous kupally et al. [19] and Spierer et al. [20] published

334 Volume 27  Number 4  July 2016

Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

Update on fungal keratitis Garg et al.

in-vitro susceptibility of ocular isolates of Candida further. Dectin-1 is upregulated in HCEC in

and compared susceptibility of C. albicans versus response to C. albicans that in turn mediates
non-albicans, respectively. These studies highlight induction of peptidoglycan recognition proteins
the value of antifungal susceptibility tests, not (PGLYRP) 2, 3, and 4 in an NFkB-dependent manner.
only as a guide to treatment but also as an epide- PGLYRP are conserved innate immunity proteins
miological and surveillance tool for estimating known mainly for their antibacterial properties
antifungal resistance. and the study by Hua et al. [23 ] showed

their production even in fungal infection. Indole-

amine-2,3-dioxygenase (IDO), an enzyme involved
PATHOGENESIS in tryptophan catabolism, also imparts protective
Immunopathogenesis of fungal keratitis is summar- immunity to corneal epithelial cells against A. fumi-
ized in Fig. 1. Several investigators are making gatus in a Dectin-1-mediated pathway [24 ]. Inhi-

attempts to better understand host responses bition of IDO in the mouse model of A. fumigatus
of fungal keratitis. Once hyphae invade corneal resulted in increased disease severity and expression
stroma, innate immune cells like neutrophils, of inflammatory cytokines like IL-6 and IL-1b. LOX-
macrophages, and dendritic cells mediate the 1 is a membrane receptor for oxidized low-density
host defense. These cells recognize conserved lipoprotein, and is upregulated during A. fumigatus
structure of fungal cell wall primarily through infection of HCEC. Li et al. [25 ] in their experiments

Toll-like receptors, C-type lectin receptors, and showed that LOX-1 induces production of CXCL1
NOD-like receptors, including inflammasomes. and TNFa, causes infiltration of neutrophils via
C-type lectin receptors like Dectin-1 and Dectin-2 activation of p38. Pearlman et al. investigated the
mediate secretion of chemokines (CXCL1 and role of IL-17 in the pathogenesis of fungal keratitis.
CXCL2) and proinflammatory cytokines (IL-1b Using a murine model of fungal keratitis the group
and TNFa) [21]. Liu et al. [22] studied the role of showed that IL-17 produced from both neutrophils
Syk signaling in antifungal innate immunity of and TH17 cells aids in fungal killing by contributing
human corneal epithelial cells (HCEC) and found to increased generation of reactive oxygen species
that A. fumigatus stimulates expression of IL-6, IL-8, [26 ,27 ]. Bhoomiraj et al. [28] using deep sequenc-
&& &

and CXCL1 in HCEC in Syk-dependent manner. ing determined a complete profile of microRNAs
The role of Dectin-1 and -2 is also being explored expressed in fungal keratitis in humans.


Dectin 1/2
TLRs Dectin 1/2
Macrophages Syk TLRs
IL-17 receptor
Stroma Dectin 1/2


Dendritic cells

CXC chemokines, IL-1β, IL-17 ROS Fungal killing


FIGURE 1. The cartoon depicting the sequence of events in fungal keratitis. Once fungi invade a breached corneal
epithelium, it germinates into hyphae and initiates an immune response via different pathogen recognition receptors present on
resident macrophages and dendritic cells. This results in Dectin-1 and Dectin-2-mediated activation of NFkB through CARD9
leading to production of CXC chemokines and IL-1b that in turn mediates recruitment of neutrophils.

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Corneal and external disorders

As fungal infection causes serious inflammation explored the role of intrastromal and intracameral
of the cornea, better approaches to control inflam- injections in conjunction with topical therapy. Both
mation needs to be studied also. Triggering receptor these approaches involve off label use of drugs.
expressed on myeloid cells-1 (TREM-1), a regulator Sharma et al. [36] in a randomized comparative
of inflammation, has recently been shown to be evaluation concluded that as an adjunct to natamy-
upregulated in rat corneal epithelium during A. cin therapy intrastromal injections of voriconazole
fumigatus infection implicating its possible role as do not offer any beneficial effect over topical vor-
a therapeutic target to control inflammation during iconazole in recalcitrant fungal keratitis. Mimouni
fungal invasion [29 ]. et al. [37] evaluated safety and efficacy of intrastro-

Gao et al. [30 ] showed the protective role mal injection of natamycin 5% in experimental

of chitinase 3-like 1 that stimulates the expression Fusarium keratitis and found that the combined
of antimicrobial peptides and anti-inflammatory treatment offers no advantage over standard
factors in C. albicans-infected C57BL/6 corneas. treatment with topical therapy alone. Contrary to
The same group also showed that natural killer cells these findings Kalaiselvi et al. [38 ] and Tu and Hou

are recruited by CXCL10-CXCR3-mediated signal- [39] found that intrastromal injection of voricona-
ing in response to C. albicans keratitis [31 ]. zole is well tolerated and effective way of treating

All these studies have enhanced our understand- patients with deep recalcitrant fungal keratitis
ing of the pathogenesis of this blinding disease who do not respond to combined topical voricona-
and will facilitate identification of potential targets zole and natamycin treatment. Similar conflicting
for therapeutic intervention that will also inhibit results have been published on the role of intra-
tissue damage. cameral injections of antifungal agents [40,41 ].

From all these studies we can infer that

injections of antifungal drugs have if at all a limited
TREATMENT role and can be tried in patients not responding to
Management of mycotic keratitis poses several maximal medical therapy while awaiting surgery.
challenges to clinicians. The newer azoles (vorico-
nazole and posaconazole) and echinocandine
(micafungin and caspofungin) suffered a setback DRUG FORMULATIONS
after the mycotic ulcer treatment trial showed that In addition to exploring newer molecules and
voriconazole is inferior to natamycin both on effi- routes of administration investigators are also trying
cacy and safety parameters [32]. Sharma et al. [33] modified drug formulations and drug delivery sys-
reevaluated the comparative safety and efficacy of tems. In past 18 months several such modifications
voriconazole and natamycin in a double-masked were tested and are described in this section.
randomized multicenter clinical trial and found Liposomal formulations of drugs: liposomes
similar results. are small, spherical, and enclosed compartments
Realizing the need for better evidence on the separating an aqueous medium from another by a
management of fungal keratitis FlorCruz and Evans phospholipid bilayer. Investigators have attempted
performed a systematic review and published it to develop liposomal formulations of antifungal
under Cochrane Database of Systematic Review of drugs, including amphotericin B, voriconazole,
the Cochrane Library. The review further confirmed and itraconazole. The basic goal of liposomes is
that pharmacological advantages of voriconazole do to provide superior pharmacokinetics and biodistri-
not translate into clinical superiority in the manage- bution while minimizing toxicity. A group from
ment of filamentous fungal keratitis. Apart from Brazil was able to show that the liposomal formu-
this the review also demonstrated that the level of lation of voriconazole was able to deliver about
evidence for most other comparisons is weak and 47.85  5.72 mg/cm2 of the drug into porcine cornea
highlighted the need for more randomized clinical after 30 min of permeation test; drug levels much
trials to assess the role of different antifungal agents higher than the MIC of several keratitis fungal
[34 ]. Qiu et al. [35] also published a similar isolates [42 ]. Leal et al. [43] assessed the antifungal
&& &

systematic review in a different journal. activity of a liposomal itraconazole in experimental

Aspergillus flavus keratitis and found it superior
compared with standard topical formulation.
Role of intrastromal and intracameral Drug-loaded polymeric micelles and nanopar-
injections in the management of fungal ticles: They are other forms of drug delivery systems
keratitis tested for fungal keratitis. Jaiswal et al. [44] tested
To overcome the disadvantage of poor corneal zero order delivery of itraconazole via polymeric
penetration of antifungal agents’ investigators have micelles and demonstrated that such controlled

336 Volume 27  Number 4  July 2016

Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

Update on fungal keratitis Garg et al.

release formulations are capable of carrying drug molecular methods for the identification of fungal
to the anterior chamber on topical application. Jain isolates several unanswered questions related to
et al. [45] used cell penetrating peptides (Tat 2) as the epidemiology, drug susceptibility/resistance,
nanocarriers of natamycin and demonstrated and the response to medical treatment are likely
that the conjugated natamycin has enhanced to be answered. In-vitro susceptibility test for fungal
solubility, superior penetration through corneal isolates are going to be used more often. In addition
epithelium, and twice antifungal activity against to newer antifungal agents we will be witnessing
clinical isolates of Fusarium solani. Some other newer formulations and drug delivery systems in
cornea-specific nanoparticle-based formulations near future. Hopefully, all these will translate into
tested are: cornea-targeted nanoformulation of better treatment for our patients suffering from this
natamycin [46], polyethylene glycol-based formu- difficult blinding disease.
lation of ketoconazole [47], and amphotericin-B
entrapped lecithin/chitosan nanoparticles [48]. Acknowledgements
All these formulations have shown a superior We would like to acknowledge the help of Dr Savitri
pharmacokinetics, tissue availability, and biocom- Sharma for her assistance with this manuscript.
patibility. It is time their efficacy be established in
in-vivo animal experiments and human trials. Financial support and sponsorship
Hyderabad Eye Research Foundation, Hyderabad, India,
COLLAGEN CROSS-LINKING FOR supports this work.
Conflicts of interest
Photochemical collagen cross-linking (PCCL)
continues to interest clinicians in exploring its P.G. is currently receiving a grant (099800) from Well-
newer therapeutic applications, including the man- come Trust and the department of biotechnology, Gov-
agement of fungal corneal infections. In past few ernment of India (BT/IN/DBT-MRC(UK)/11/PG/2015–
years several articles, including systematic review 16). He is also member of the advisory board of Nova-
and meta-analysis to assess the potential role of this Bay, Allergan India.
modality in corneal infections have been published S.R. is currently receiving a grant from the Science and
[49–53]. Some of these trials included fungal Engineering Research Board of the Department of Science
keratitis cases as well. However, the results of these and Technology, Government of India.
trials have been contradictory. Bilgihan et al. [54] A.R. has no financial disclosures to make.
and Sun et al. [55] in in-vitro studies found PCCL
with riboflavin to be effective in reducing the via-
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338 Volume 27  Number 4  July 2016

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Update on fungal keratitis Garg et al.

49. Papaioannou L, Miligkos M, Papathanassiou M. Corneal collagen cross- 55. Sun B, Li ZW, Yu HQ, et al. Evaluation of the in vitro antimicrobial properties of
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2016; 35:62–71. ium solani. Int J Ophthalmol 2014; 7:205–210.
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52. Tayapad JB, Viguilla AQ, Reyes JM. Collagen cross-linking and corneal In this study the authors enrolled patients of deep keratomycosis that did
infections. Curr Opin Ophthalmol 2013; 24:288–290. not respond to 2 weeks of medical treatment and randomized into
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activated riboflavin (PACK-CXL) for the treatment of advanced infectious collagen cross linking does not offer any advantage over standard medical
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