mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Review article

Filamentous fungal infections of the cornea: a global overview of

epidemiology and drug sensitivity

szlo
La
Kredics,1 Venkatapathy Narendran,2 Coimbatore Subramanian Shobana,3 Csaba

gvo
Va € lgyi,1,4 Palanisamy Manikandan2,5 and Indo-Hungarian Fungal Keratitis Working Groupa
1
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary, 2Aravind Eye Hospital and Postgraduate Institute
of Ophthalmology, Coimbatore, Tamil Nadu, India, 3Department of Microbiology, PSG College of Arts & Science, Coimbatore, Tamil Nadu, India, 4Botany
and Microbiology Department, King Saud University, Riyadh, Kingdom of Saudi Arabia and 5Department of Medical Laboratory Sciences, College of
Applied Medical Sciences, Majmaah University, Kingdom of Saudi Arabia

Summary Fungal keratitis is a serious suppurative, usually ulcerative corneal infection which
may result in blindness or reduced vision. Epidemiological studies indicate that the
occurrence of fungal keratitis is higher in warm, humid regions with agricultural
economy. The most frequent filamentous fungal genera among the causal agents are
Fusarium, Aspergillus and Curvularia. A more successful therapy of fungal keratitis
relies on precise identification of the pathogen to the species level using molecular
tools. As the sequence analysis of the internal transcribed spacer (ITS) region of the
ribosomal RNA gene cluster (rDNA) is not discriminative enough to reveal a species-
level diagnosis for several filamentous fungal species highly relevant in keratitis
infections, analysis of other loci is also required for an exact diagnosis. Molecular
identifications may also reveal the involvement of fungal species which were not
previously reported from corneal infections. The routinely applied chemotherapy of
fungal keratitis is based on the topical and systemic administration of polyenes and
azole compounds. Antifungal susceptibility testing of the causal agents is of special
importance due to the emergence and spread of resistance. Testing the applicability
of further available antifungals and screening for new, potential compounds for the
therapy of fungal keratitis are of highlighted interest.

Key words: Eye infections, keratitis, Aspergillus, Fusarium, Curvularia, epidemiology, molecular identification,
antifungal susceptibility.

usually ulcerative corneal infection which may result

Introduction
Fungal keratitis – or mycotic keratitis, keratomycosis –
is becoming a frequent and serious suppurative,
Correspondence: L. Kredics, Department of Microbiology, Faculty of
€ z

Science and Informatics, University of Szeged, Ko ep fasor 52, H-6726,
Szeged, Hungary.
Tel.: +36 62 544 516. Fax: +36 62 544 823.
E-mail: kredics@bio.u-szeged.hu
a
Indo-Hungarian Fungal Keratitis (IHFK) Working Group authors are listed
in the Appendix.
Submitted for publication 21 December 2014
Revised 27 January 2015
Accepted for publication 13 February 2015
in reduced vision or blindness.1 Especially during the
past few decades, the incidence of fungal keratitis has
increased by several times, and hundreds of reports
covering epidemiology, clinical data, case descriptions
and research results appeared in the literature – par-
ticularly from developing countries. Although yeast
infections occur more frequently in mild climates, fila-
mentous fungal aetiology is more common in tropical
regions where it accounts for more than 50% of all
corneal ulcers.2,3 The epidemiological pattern of fungal
keratitis varies widely throughout the world and even
between regions of the same country.4,5 Since the first
report of fungal keratitis diagnosed in a farmer,6 peo-
ple involved in soil-related work such as agriculture

© 2015 Blackwell Verlag GmbH doi:10.1111/myc.12306

L. Kredics et al.
and construction have been frequently reported to be
more prone to corneal infection.4 In addition, the
development and widespread use of broad-spectrum
antibiotics and steroids, the frequent – and sometimes
prolonged – use of contact lenses, and the growing
number of corneal surgeries performed (especially
penetrating keratoplasty) have also been referred to be
among the predisposing factors.
Corneal ulceration is the second most common
cause of blindness after cataract.2 Vision loss is fre-
quently attributed to misdiagnosis or the lack of
advanced investigative tools to accurately identify the
casual agents, the mimicking clinical picture of the
disease, which potentially lead the ophthalmologists/
clinicians to initiate an inappropriate antifungal ther-
apy.4 In this context, although most cases of fungal
keratitis exhibit uniform symptoms and clinical fea-
tures, the importance of an accurate identification of
the aetiological agent has been stressed as it impacts
the therapeutic outcome.1,4,5 Furthermore, the limited
availability of antifungal drugs as well as the therapy-
refractive nature and the diversity of fungal causative
agents continue to retain the disease as a significant
public health problem.
The aim of this review was to provide a global over-
view about the epidemiology of filamentous fungal
keratitis, to evaluate the possibilities available for
molecular identification of the causal agents, as well
as to discuss the therapeutic efficiency of different anti-
fungal compounds and the antifungal susceptibility
data available in the literature.
Studies on the epidemiology of filamentous
fungal keratitis
A comprehensive evaluation of the literature
(Table 1)2,3,5,7–106 reveals that more than 70 different
fungal species were reported to be pathogenic to human
cornea with a variation in the predominant genus
depending on the geographical area studied. A warm,
humid climate and/or regions with an agricultural
economy highly favour fungal keratitis. Mycotic kerati-
tis may account for more than 50% of all cases of
culture-proven microbial keratitis and of ophthalmic
mycoses, especially in tropical and subtropical areas.18
In contrast, Candida spp. were reported in more numbers
from developed countries.3 The majority of the large-
scale studies were reported from India,2,5,9,11,12,14,16,20–
23,27,29,30,32,35,38,39,45–48,50,54,59–61,63,65,66,70,76–80,83,
88–94,96–99,101,102,104,105
followed by the USA,7,8,17,24,
34,41,43,44,49,73,100 25,31,42,52,67,75
China and Brazil,26,37,
68,71,72,82
whereas there is a lack of large
2 © 2015 Blackwell Verlag GmbH
epidemiological studies from European countries, which
is mainly due to the low number of cases in Europe. The
six largest numbers of fungal isolates involved in kerati-
tis epidemiological studies were 1648,65 1458,67
1360,29 1226,47 122494 and 110032 from Hyderabad
(India), Zhengzhou (China), Hyderabad, Tirunelveli,
Pondicherry and Tirunelveli (India), respectively; five of
these studies were carried out in South Indian states.
Although, culture techniques remained the corner-
stone of the diagnosis of most cases of fungal kerati-
tis,107 the isolation rates varied among the studies
(Table 1). This varying culture positivity could be
attributed to prior treatments with antifungals, the
refractive nature of fungi and the application of inap-
propriate culture methods.95 In many cases the fungal
nature of the infection was determined only in patho-
logical sections.
Earlier, isolation of the causative fungus was the
only tool available for the confirmation and the find-
ings were applied as a key for therapy8 without identi-
fying the pathogen at the species level. In several early
studies on the epidemiology of fungal keratitis, the
clinical profile of the patient and the examination of
microscopic and cultural morphologies were applied
for the identification of the causative agent. As a
result, only a few of the investigated isolates were
reported at the species level, most of them were identi-
fied at the genus level only.22–24 In some cases the
genus-level identification was also difficult, therefore,
the isolates were frequently reported as unidentified
pigmented (dematiaceous) or non-pigmented (hyaline)
fungal species.2,19–21 Even if a species-level identity is
provided, it has to be noted that the morphology-based
identification of the isolates may lead to a delayed or
false diagnosis in a significant number of cases.4
The individual, larger scale studies – especially those
published at the end of the last and the beginning of
the recent century – have not provided detailed species
identifications for isolates of Fusarium, Aspergillus and
Curvularia, the major fungal genera involved in kerato-
mycosis (Table 1). This is distressing, as the antifungal
susceptibility patterns may vary between different spe-
cies of the same genus,4,61 and the species pattern of
the predominant genera can be diverse within a given
geographical area. Therefore, the lack of species-level
data is becoming a real-time concern.4
Most of the studies on fungal keratitis across the
globe have identified and reported both fusaria and
aspergilli, or one of these two genera as the predomi-
nant fungal taxa causing human keratitis.38,47,65
Fusarium and Aspergillus were commonly identified as
the causative agents: even studies that dealt with low
 Epidemiology of filamentous fungal keratitis

Table 1 Incidence of filamentous fungi among culture-proven cases of fungal keratitis.

Number Other
of fungal Fusarium Aspergillus Curvularia fungi
Country Period of study isolates (%) (%) (%) (%) Reference Year

USA, Texas 1962 10 3 (30.0) 2 (20.0) – 5 (50.0) 7 1962

USA, Florida January 1968–July 1970 33 15 (45.5) 11 (33.3) 1 (3.0) 6 (18.2) 8 1971
India, Jamnagar 1985 37 12 (32.5) 10 (27.0) 2 (5.4) 13 (35.1) 9 1985
Bangladesh, Chittagong 1987 7 1 (14.3) 2 (28.6) – 4 (57.1) 10 1987
India, Madras (south) 1980–1982 68 8 (11.8) 36 (52.9) 4 (5.9) 20 (29.4) 11 1989
India, Madras NA 322 44 (13.7) 55 (17.1) – 223 (69.2) 12 1989
Paraguay April 1988–April 1989 26 11 (42.3) 5 (19.2) 1 (3.9) 9 (34.6) 13 1991
India, Karnataka October 67 – 23 (34.3) 2 (3.0) 42 (62.7) 14 1992
1985–September 1988
Bangladesh, 11 months 51 10 (19.6) 19 (37.3) 6 (11.8) 16 (31.3) 15 1994
Chittagong
India, Chandigarh 6 years 61 10 (16.4) 25 (41.0) 5 (8.2) 21 (34.4) 16 1994
(north)
USA, Miami January 1982–January 1992 127 79 (62.2) 5 (4.0) 11 (8.7) 32 (25.1) 17 1994
Ghana, Accra NA 65 34 (52.3) 10 (15.4) 2 (3.1) 19 (29.2) 18 1995
Singapore January 1991–December 1995 29 15 (51.7) 4 (13.8) 1 (3.5) 9 (31.0) 19 1997
India, Madurai (south) January 1994–March 1994 155 73 (47.1) 25 (16.1) 6 (3.9) 51 (32.9) 2 1997
India, Madurai NA 63 22 (34.9) 22 (34.9) 5 (8.0) 14 (22.2) 20 1998
India, Mumbai (west) 1988–1996 387 33 (8.5) 219 (56.6) 10 (2.6) 125 (32.3) 21 1999
India, Hyderabad February 1991–June 1995 21 3 (14.3) 7 (33.3) 3 (14.3) 8 (38.1) 22 2000
India, New Delhi NA 13 4 (30.8) 6 (46.1) 2 (15.4) 1 (7.7) 23 2000
USA, Pennsylvania, January 1991–March 1999 24 6 (25) 1 (4.2) – 17 (70.8) 24 2000
China January 1996 and 97 63 (65.0) 14 (14.4) – 20 (20.6) 25 2001
December 1999
Brazil 1983–1997 25 8 (32.0) 4 (16.0) – 13 (52.0) 26 2001
Ghana June 1999–May 2001 109 46 (42.2) 38 (34.9) 1 (0.9) 24 (22.0) 27 2002
Thailand (central) January 1988–December 2000 34 12 (35.3) 7 (20.6) 7 (20.6) 8 (23.5) 28 2002
India, Hyderabad January 1991–December 2000 1360 506 (37.2) 417 (30.7) 54 (4.0) 383 (28.1) 29 2002
(south)
India, Tiruchirapalli June 1999–May 2001 353 141 (40.0) 76 (21.5) 36 (10.2) 100 (28.3) 27 2002
(south)
India, Tirunelveli September 1999–March 2001 554 254 (45.8) 135 (24.4) 35 (6.3) 130 (23.5) 30 2002
(south)
China, Hong Kong April 1997–August 1998 5 3 (60.0) – – 2 (40.0) 31 2002
India, Tirunelveli September 1999–August 2002 1100 471 (42.8) 286 (26.0) 55 (5.0) 288 (26.2) 32 2003
Paraguay 1988–2001 209 41 (19.6) 37 (17.7) 15 (7.2) 116 (55.5) 33 2004
USA, Florida January 1980–January 2002 421 208 (49.4) 30 (7.1) 34 (8.1) 149 (35.4) 34 2004
India, Madurai December 2002–June 2003 100 63 (63.0) 26 (26.0) 3 (3.0) 8 (8.0) 35 2004
Taiwan, Taipei January 1992–December 2001 34 10 (29.4) 5 (14.7) – 19 (55.9) 36 2004
Brazil 1975–2003 233 137 (58.8) 28 (12.0) 1 (0.4) 67 (28.8) 37 2005
India, New Delhi January 1999–June 2001 191 24 (12.6) 78 (40.8) 63 (33.0) 26 (13.6) 38 2005
India, West Bengal January 2001–December 2003 710 132 (18.6) 373 (52.5) – 205 (28.9) 39 2005
Nepal, Dharan August 1998–July 2001 200 45 (22.5) 75 (37.5) 27 (13.5) 53 (26.5) 40 2005
USA, Kentucky January 1981–December 2004. 89 18 (20.2) 9 (10.1) 9 (10.1) 53 (59.6) 41 2006
China January 1999–December 2004 596 437 (73.3) 72 (12.1) – 87 (14.6) 42 2006
USA, Florida January 2004–December 2005 122 66 (54.1) 19 (15.6) 29 (23.8) 8 (6.5) 43 2006
USA, Florida January 1999–June 2006. 59 24 (40.7) 7 (11.9) 9 (15.2) 19 (32.2) 44 2006
India, Coimbatore February 1997–January 2004 37 17 (46.0) 11 (29.7) 2 (5.4) 7 (18.9) 45 2006
(south, children)
India, New Delhi January 2000–December 2004 77 6 (7.8) 43 (55.8) 2 (2.6) 26 (33.8) 46 2006
India, Tirunelveli September 1226 511 (41.7) 305 (24.9) 81 (6.6) 329 (26.8) 47 2006
1999–September 2002
India, Tiruchirappalli January 1–December 31, 2003 93 32 (34.4) 29 (31.2) 9 (9.7) 23 (24.7) 48 2006
USA, New York January 1987–June 2003 61 6 (9.8) 7 (11.5) – 48 (78.7) 49 2006

(continued)

© 2015 Blackwell Verlag GmbH 3

L. Kredics et al.

Table 1 (continued)

Number Other
of fungal Fusarium Aspergillus Curvularia fungi
Country Period of study isolates (%) (%) (%) (%) Reference Year

India, New Delhi NA 9 2 (22.2) 5 (55.6) 1 (11.1) 1 (11.1) 50 2006

Paraguay January 1997–December 2000 25 6 (24.0) 2 (8.0) 4 (16.0) 13 (52.0) 51 2006
China 2001–2004 681 395 (58.0) 116 (17.0) – 170 (25.0) 52 2007
Australia, Melbourne July 1996–May 2004 49 6 (12.2) 7 (14.3) – 36 (73.5) 53 2007
India, New Delhi December 2005 486 71 (14.6) 268 (55.1) 15 (3.1) 132 (27.2) 54 2007
Turkey, West Anatolia January 1990–December 2005 50 25 (50.0) 10 (20.0) – 15 (30.0) 55 2007
Japan, Tokyo January 1999–December 2003 6 – – – 6 (100.0) 56 2007
UK, London December 1993–January 2007 66 12 (18.2) 7 (10.6) – 47 (71.2) 3 2007
Malaysia January 2004–April 2005 4 2 (50.0) 1 (25.0) – 1 (25.0) 57 2008
Thailand January 2001–December 2004 49 13 (26.5) 9 (18.4) 2 (4.1) 25 (51.0) 58 2008
India, Chandigarh January 1999–December 2003 34 8 (23.5) 14 (41.2) 4 (11.8) 8 (23.5) 59 2008
(north)
India, New Delhi NA 39 2 (5.1) 22 (56.4) 7 (18.0) 8 (20.5) 60 2008
India, Ujjain April 2006–November 2007 37 2 (5.4) 20 (54.1) 6 (16.2) 9 (24.3) 61 2008
Australia, 1998–2008 16 8 (50.0) 2 (12.5) 2 (12.5) 4 (25.0) 62 2008
Queensland
India, Chennai July 2006–May 2008 20 7 (35.0) 8 (40.0) 2 (10.0) 3 (15.0) 63 2009
(south)
Nepal, Dharan January 2007–July 2008 12 8 (66.6) 2 (16.7) 2 (16.7) 0 64 2009
India, Hyderabad February 1991–June 2001 1648 588 (35.7) 478 (29.0) 106 (6.4) 476 (28.9) 65 2009
(south)
India, Mumbai NA 40 6 (15.0) 20 (50.0) 4 (10.0) 10 (25.0) 66 2009
China, Zhengzhou January 2000–March 2009 1458 1076 (73.8) 195 (13.4) – 187 (12.8) 67 2009
Brazil, Southeastern 2000–2004 66 44 (66.7) 7 (10.6) – 15 (22.7) 68 2009
UK, London December 39 2 (5.1) 6 (15.4) – 31 (79.5) 69 2009
2003–November 2005
India, Varanasi January 2004–December 2008 36 7 (19.5) 17 (47.2) 3 (8.3) 9 (25.0) 70 2010
Brazil, S~
ao Paulo 1975–2007 94 28 (29.8) 9 (9.6) – 57 (60.6) 71 2010
Brazil, Uberlandia July 2001–August 2004 18 11 (61.1) 3 (16.7) 1 (5.6) 3 16.6) 72 2010
USA, Philadelphia April 1999–December 2008 66 29 (44.0) 2 (3.0) 2 (3.0) 33 (50.0) 73 2010
Sierra Leone January 2005–January 2006 26 1 (3.8) 4 (15.4) – 21 (80.8) 74 2010
China October 139 67 (48.2) 26 (18.7) 1 (0.7) 45 (32.4) 75 2011
2004–September 2009
India, Amristar NA 86 11 (12.8) 36 (41.9) 7 (8.1) 32 (37.2) 76 2011
India, Assam April 2007–March 2009 184 46 (25.0) 27 (14.7) 13 (7.1) 98 (53.2) 77 2011
India, Bhubaneswar July 2006–December 2009 215 50 (23.3) 60 (27.9) 10 (4.7) 95 (44.1) 78 2011
India, Gujarat September 2003–June 2005 57 17 (29.8) 12 (21.1) 6 (10.5) 22 (38.6) 79 2011
India, Pondychery January 2009–August 2008 373 134 (35.9) 97 (26.0) 84 (22.5) 58 (15.6) 80 2011
South Korea, January 2000 and 34 16 (47.0) 2 (6.0) – 16 (47.0) 81 2011
Jeonbuk December 2007
Brazil, S~
ao Paulo July 1975–September 2007 364 189 (51.9) 33 (9.1) – 142 (39.0) 82 2011
India, Maharashtra January 2005–December 2005 25 4 (16.0) 14 (56.0) 2 (8.0) 5 (20.0) 83 2011
Saudi Arabia, Riyadh January 1984–December 2004 106 12 (11.3) 28 (26.4) – 66 (62.3) 84 2011
Nepal January 2004–December 2008 150 19 (12.7) 50 (33.3) 15 (10.0) 66 (44.0) 85 2012
Saudi Arabia January 2006 and 87 24 (27.6) 15 (17.2) 1 (1.2) 47 (54.0) 86 2012
December 2009
Vietnam 2008 351 143 (40.7) 91 (25.9) 9 (2.6) 108 (30.8) 87 2012
India, Ahmedabad July 2007–June 2008 31 7 (22.6) 11 (35.5) 5 (16.1) 8 (25.8) 88 2012
India, Maharashtra December 311 109 (35.0) 56 (18.0) 10 (3.2) 136 (43.8) 89 2012
2004–December 2009
India, West Bengal February 2007–January 2011 399 81 (20.3) 151 (37.8) 41 (10.3) 126 (31.6) 90 2012
India, Karnataka September 2006–August 2007 28 16 (57.2) 9 (32.1) 1 (3.6) 2 (7.1) 91 2012
India, Delhi April 2009–April 2010 22 – 6 (27.2) 8 (36.4) 8 (36.4) 92 2012
India, West Bengal NA 15 4 (26.7) 4 (26.7) – 7 (46.6) 93 2012

(continued)

4 © 2015 Blackwell Verlag GmbH

 Epidemiology of filamentous fungal keratitis

Table 1 (continued)

Number Other
of fungal Fusarium Aspergillus Curvularia fungi
Country Period of study isolates (%) (%) (%) (%) Reference Year

India, Pondicherry 2003–2009 1224 431 (35.2) 353 (28.9) 123 (10.0) 317 (25.9) 94 2012
India, Coimbatore 2005–2008 1087 554 (50.9) 200 (18.3) 79 (7.2) 254 (23.3) 5 2012
Thailand July 2007–May 2009 12 5 (41.7) 1 (8.3) 1 (8.3) 5 (41.7) 95 2012
India, Baroda June 2009–May 2012 73 26 (35.6) 14 (19.2) 4 (5.5) 29 (39.7) 96 2013
India, Gujarat September 26 3 (11.5) 20 (76.9) 1 (3.9) 2 (7.7) 97 2013
(west) 2006–February 2008
India, Karnataka January–June 2012 36 7 (19.4) 16 (44.5) 4 (11.1) 9 (25.0) 98 2013
India, Karnataka December 38 16 (42.1) 11 (28.9) 2 (5.3) 9 (23.7) 99 2013
2009–February 2011
USA, Kansas July 2001–June 30, 2011 50 19 (38.0) 10 (20.0) – 21 (42.0) 100 2013
India, January–June, 2011 62 15 (24.2) 18 (29.0) 4 (6.5) 25 (40.3) 101 2013
Bhubaneswar
India, Karnataka July 2009–June 2010 23 4 (17.5) 17 (73.9) 1 (4.3) 1 (4.3) 102 2013
Tunisia, Sfax January 60 29 (48.3) 13 (21.7) – 18 (30.0) 103 2014
1995–December 2012
India, Madurai 2006–2011 103 32 (31.1) 26 (25.2) 11 (10.7) 34 (33.0) 104 2014
India, New Delhi January 63 10 (15.9) 32 (50.8) 1 (1.6) 20 (31.7) 105 2014
2004–January 2012
Egypt, Assiut NA 25 3 (12.0) 13 (52.0) – 9 (36.0) 106 2014

number of fungal isolates have not missed these two
genera. In addition, unidentified isolates were also
common.10,31,50
Importance of molecular identification
methods in the diagnosis of fungal keratitis
infections
It is very important to get exact, species-specific infor-
mations about the causal agents of filamentous fungal
keratitis. This, however, is not possible from epidemio-
logical and antifungal susceptibility studies where the
causal agents were identified at the genus level only,
or the identifications were carried out based on mor-
phological characters alone. The generally applied
strategy of sequence-based molecular identification is
the amplification of a fragment from a specific locus
by polymerase chain reaction (PCR), followed by
sequence determination and analysis of the resulting
sequence with a nucleotide–nucleotide BLAST (Basic
Local Alignment Search Tool)108 search in the nucleo-
tide sequence collection of the National Center for Bio-
technology Information (NCBI). The application of this
approach resulted in the diagnosis of several keratitis
cases caused by uncommon filamentous fungal agents,
many of them previously unreported from corneal
infections (Table 2).109–146 These included Lagenidium
and Pythium species from the Oomycota division, a ser-
ies of species from the Botryosphaeriales, Capnodiales,
Diaporthales, Eurotiales, Glomerellales, Hypocreales,
Microascales, Pleosporales, Sordariales and Xilariales
orders of the Ascomycota division, as well as Schizophyl-
lum commune from Basidiomycota. The initial reports
applying sequence-based approaches for the species-
level diagnosis of filamentous fungal keratitis cases
were published during the first years of the 21st cen-
tury and reported the occurrence of Pythium insidio-
sum,110 Alternaria alternata,135 Alt. infectoria136 and
Fusarium polyphialidicum.128 Sequence-based molecular
approaches were becoming more widespread during
the past years: more than 80% of the filamentous
fungal keratitis case reports listed in Table 2 were
published after 2008.
The locus most frequently applied for species identifi-
cation in the case reports was the internal transcribed
spacer (ITS) region of the ribosomal RNA gene cluster
(rDNA). The evaluation of six DNA regions as poten-
tial DNA barcodes for the kingdom Fungi revealed that
the ITS region has the highest probability of successful
identification for the broadest range of fungi147 includ-
ing non-sporulating molds.113 However, there is a
problem with ITS-based strategies in relation with fila-
mentous fungal keratitis pathogens: the sequence of
the ITS region is not discriminative enough to reveal a
species-level diagnosis for several species belonging to
Fusarium, Aspergillus and Curvularia, the three genera
most frequently occurring in filamentous fungal kerati-
tis infections. In the case of these genera, the sequence
analysis of other loci, like fragments of the b-tubulin
(b-tub), calmodulin (cal), RNA polymerase II second

© 2015 Blackwell Verlag GmbH 5

L. Kredics et al.

Table 2 Causal agents of filamentous fungal keratitis recognised in case reports by sequence-based identification.

Taxonomic status
Species (division, order, family) Sequenced locus (GenBank ID) Reference Year

Lagenidium sp. Oomycota, Lagenidiales, Lagenidiaceae ITS (JX646749) 109 2013

Pythium insidiosum Oomycota, Pythiales, Pythiaceae ITS (NA) 110 2001
Pythium insidiosum Oomycota, Pythiales, Pythiaceae ITS (GU584093) 111 2011
Auerswaldia lignicola Ascomycota, Botryosphaeriales, 18S rDNA gene (KC866317.1) 112 2013
Botryosphaeriaceae
Botryosphaeria rhodina Ascomycota, Botryosphaeriales, ITS (EF446281) 113 2008
Botryosphaeriaceae
Cladosporium cladosporioides Ascomycota, Capnodiales, Cladosporiaceae ITS (NA) 114 2009
Phomopsis phoenicicola Ascomycota, Diaporthales, Diaporthaceae ITS (100% identity with FJ 889452) 115 2011
Aspergillus viridinutans Ascomycota, Eurotiales, Aspergillaceae b-tub (NA) 116 2012
Aspergillus tamarii Ascomycota, Eurotiales, Aspergillaceae ITS (EF525554) b-tubulin (EF525555) 117 2007
calmodulin (EF525556)
Aspergillus pseudotamarii Ascomycota, Eurotiales, Aspergillaceae Calmodulin (KC202290) 118 2013
Aspergillus nomius Ascomycota, Eurotiales, Aspergillaceae ITS (GQ221261) b-tubulin (GQ221262) 119 2009
calmodulin (GQ221263)
Aspergillus tubingensis Ascomycota, Eurotiales, Aspergillaceae b-tubulin (EU600389 and EU600388) 120 2009
Aspergillus brasiliensis Ascomycota, Eurotiales, Aspergillaceae b-tubulin (EU600386 and EU600387) 121 2010
Neosartorya udagawae Ascomycota, Eurotiales, Aspergillaceae ITS (100% identity with AB250781) 122 2011
Colletotrichum gloeosporioides Ascomycota, Glomerellales, Glomerellaceae ITS1 (NA) 123 2009
Colletotrichum truncatum Ascomycota, Glomerellales, Glomerellaceae ITS (100% identity with GU227878) 124 2011
Plectosporium tabacinum Ascomycota, Glomerellales, ITS (100% identity with AM408781) 125 2012
Plectosphaerellaceae
Beauveria bassiana Ascomycota, Hypocreales, Cordycipitaceae ITS (HF675188), 28S rRNA (HF675189) 126 2014
Fusarium equiseti Ascomycota, Hypocreales, Nectriaceae ITS (100% identity with GQ505694) 127 2012
Fusarium polyphialidicum Ascomycota, Hypocreales, Nectriaceae ITS (99% identity with X94172) 128 2003
Fusarium proliferatum Ascomycota, Hypocreales, Nectriaceae ITS (EF446290) 113 2008
Fusarium verticillioides Ascomycota, Hypocreales, Nectriaceae ITS (100% identity with AY533376) 127 2012
Neocosmospora vasinfecta Ascomycota, Hypocreales, Nectriaceae ITS (EF373539) 129 2008
Fusarium temperatum Ascomycota, Hypocreales, Nectriaceae tef1 (KF956084) b-tub (KF956080) 130 2014
Purpureocillium lilacinum Ascomycota, Hypocreales, ITS (AB808481 and AB909369) 131 2014
Ophiocordycipitaceae
Scedosporium apiospermum Ascomycota, Microascales, Microascaceae ITS (99% identity with AY213680) 132 2005
Sporothrix pallida Ascomycota, Ophiostomatales, ITS (100% identity with EF127880) 133 2013
Ophiostomataceae LSU (100% identity with EF139121)
b-tub (100% identity with EF139110)
Corynespora cassiicola Ascomycota, Pleosporales, ITS (100% identity with AY238606) 134 2013
Corynesporascaceae
Alternaria alternata Ascomycota, Pleosporales, Pleosporaceae ITS (NA) 135 2002
Alternaria infectoria Ascomycota, Pleosporales, Pleosporaceae ITS (AY168773) 136 2003
Edenia gomezpompae Ascomycota, Pleosporales, Pleosporaceae ITS (KC193601) 137 2013
Pyrenochaeta keratinophila1 Ascomycota, Pleosporales, Pleosporaceae ITS (EU885415) 138 2010
Ulocladium sp.2 Ascomycota, Pleosporales, Pleosporaceae ITS (AY943384) 139 2006
Chaetomium atrobrunneum Ascomycota, Sordariales, Chaetomiaceae ITS (HQ222986) 140 2012
Chaetomium sp. Ascomycota, Sordariales, Chaetomiaceae ITS (HQ906667) 141 2012
Thielavia subthermophila Ascomycota, Sordariales, Chaetomiaceae ITS (99% identity with AJ271575) 142 2009
Cladorrhinum bulbillosum Ascomycota, Sordariales, Lasiosphaeriaceae ITS (98% identity with FM955448) 143 2011
Pestalotiopsis clavispora Ascomycota, Xilariales, Amphisphaeriaceae ITS (100% identity with EF119336) 144 2013
Phaeoisaria sp.3 Ascomycota, Xilariales, Diatrypaceae 28S rDNA (97% identity 145 2010
with JQ429231)
Schizophyllum commune Basidiomycota, Agaricales, Schizophyllaceae 18S rDNA (JQ695912) 146 2013

NA, not available.

1
Described as new species.
2
Reported as Ulocladium atrum.
3
Reported as Carpoligna sp.

6 © 2015 Blackwell Verlag GmbH


largest subunit (rpb2) or translation elongation factor
1a (tef1) genes is required for an exact diagnosis. For
example, the loci b-tub and/or cal were applied for the
identification of different Aspergillus species,116–121
whereas b-tub and tef1 were applied for the identifica-
tion of the novel opportunist Fus. temperatum as kera-
titis pathogens.130
Studies are also available in the literature, where
sequence-based identification was performed for larger
sets of fungal cultures deriving from keratitis cases.
Oechsler et al. [148] examined 58 archived Fusarium
isolates from ocular sources (cornea, contact lens, vit-
reous and anterior chamber) of 52 patients by
sequence analysis of the ITS region, and identified
them as members of the species complexes Fus. solani
(FSSC, 75%), Fus. oxysporum (FOSC, 16%), Fus. incarn-
atum-equiseti (FIESC 5%) and Fus. dimerum (FDSC,
2%). A multilocus sequence typing strategy based on
the combined analysis of a portion of tef1, the ITS
region and D1 and D2 domains of the 28S rDNA as
well as two contiguous regions of rpb2 was applied for
the taxonomic investigation of Fusarium isolates from
the multistate contact lens-associated US keratitis out-
breaks of 2005 and 2006.149–152 The method identi-
fied members of the FSSC (including Fus. falciforme
and the recently described species Fus. keratoplasticum
and Fus. petroliphilum152), FOSC, FFSC (Fus. fujikuroi
species complex; Fus. proliferatum, Fus. thapsinum and
Fus. verticillioides), FIESC and FDSC among the causal
agents.150,151 Azor et al. [153] used b-tub sequences
along with morphological examinations for the identi-
fication of 48 Fusarium isolates deriving from various
clinical sources. The strains were identified as Fus.
dimerum (4), Fus. incarnatum (6) and Fus. sacchari (4).
In a retrospective study, Homa et al. [154] identified
the causal agents of Fusarium keratitis that occurred
at an eye hospital in South India during the period of
2010–2011. Sequences of the ITS region as well as
partial sequences of the b-tub and tef1 genes were used
for molecular identification at the species complex –
and where possible, at the species – level. Representa-
tives of five species complexes of the genus Fusarium
could be detected, with members of the FSSC being the
most frequent (75.71%), followed by FDSC (8.57%),
FFSC (8.57%), FOSC (4.29%) and FIESC (2.86%). Spe-
cies-level identification could be performed for the
members of the FDSC and FFSC by the simultaneous
analysis of ITS, tef1 and b-tub sequences as well as
macro- and microscopic morphology.155 The isolates
from FDSC proved to be F. delphinoides strains. The
FFSC isolates were identified as F. verticillioides, as well
as F. napiforme – a species firstly detected from
© 2015 Blackwell Verlag GmbH
Epidemiology of filamentous fungal keratitis
keratitis.156 In another study from the same hospital
focusing on the genus Aspergillus, fragments of the ITS
region, b-tub and cal genes were involved in the
molecular identification of keratitis isolates.5 Asp.
flavus proved to be the predominant species (75%),
followed by Asp. fumigatus and Asp. terreus. A set of
isolates represented Aspergillus species which have not
been reported from keratitis cases before: Asp. tamarii,
Asp. pseudotamarii and Asp. nomius from section Flavi,
as well as Asp. tubingensis and Asp. brasiliensis from
section Nigri of the genus were firstly recognised as
potential causal agents of fungal keratitis (Table 2).
The firstly detected cases with the involvement of
these species were described in detailed case
reports.117–121 Besides the above-mentioned species,
further representatives of the genus, including Asp.
amstelodami from section Aspergillus, Asp. melleus from
section Circumdati, Asp. lentulus from section Fumig-
ati,157 Asp. sydowii and Asp. variecolor from section
Nidulantes, as well as Asp. neoniger 157 and Asp. wel-
witschiae (syn. Asp. awamori) from section Nigri5,157
were also detected as rarely occurring keratitis patho-
gens. Among the newly identified agents of keratitis,
Asp. tamarii proved later to be more frequent than ini-
tially suspected: since the publication of the original
case report a series of further 17 cases were diagnosed
as Asp. tamarii keratitis, suggesting that the lack of
reports in the literature is rather due to the lack of
recognition (identification of Asp. tamarii as Asp. flavus)
than to the lack of occurrence.4 During the molecular
identification of Curvularia isolates from kerati-
tis,158,159 sequences of the ITS and intergenic spacer
(IGS) regions of the nuclear ribosomal RNA gene clus-
ter, as well as of cal, b-tub and tef1 gene fragments
were analysed. The latter three loci and ITS did not
prove variable enough to distinguish among the three
species, however, clearly distinctive species motifs
could be found in the IGS region, the use of which
can therefore be recommended as sequence-based
identification procedure of Curvularia keratitis iso-
lates.159 The isolates from South Indian keratitis cases
proved to belong to the species Cur. spicifera and Cur.
hawaiiensis.158,159
When a sequence-based method is applied for the
routine diagnosis of fungal keratitis, the time needed
for identification can be substantially reduced by per-
forming the amplification of the targeted fragment
directly from the corneal samples. Ferrer et al. [160]
applied seminested PCR amplification of the ITS2/5.8S
region and subsequent sequence analysis to success-
fully detect various fungal pathogens (Can. parapsilosis,
Alt. alternata, Scedosporium apiospermum, Asp. niger and
7
L. Kredics et al.
Asp. fumigatus) in ocular samples. In a study per-
formed 10 years later by Ferrer and Ali
o [161] with
27 corneal samples deriving from 20 keratitis patients,
besides the five species detected in the initial study,
four further Candida species (Can. albicans, Can. famata,
Can. sake, Can. dubliniensis), and six further filamentous
fungi, Alt. infectoria, Asp. oryzae, Fus. oxysporum
(FOSC), Fus. solani (FSSC), Pyrenochaeta keratinophila
and Paecilomyces sp. could be diagnosed by sequence
analysis of the amplified ITS2/5.8S fragments. The
same identification strategy was also applied by other
authors.95,162,163 Ghosh et al. [162] diagnosed 27
cases of fungal keratitis from corneal samples, the
causal agents reported were Candida spp. (including
Can. parapsilosis and Can. rugosa), Aspergillus spp.
(including Asp. flavus and Asp. fumigatus), Fusarium
spp. (including members of the FSSC) Colletotrichum
spp. (including Col. truncatum), Curvularia spp. (includ-
ing Cur. lunata), Cladosporium spp. (including Cla. oxy-
sporum), Penicillium brocae and Cochliobolus sp. Embong
et al. [163] also applied the same method for the diag-
nosis of 11 fungal keratitis cases from corneal scraping
samples, and reported the occurrence of Fusarium spp.
(including members of the FSSC), Cladosporium sp.,
Asp. flavus, Trichosporon asahii and Glomerella cingulata.
The authors suggested that ITS PCR-based molecular
identification should be applied as the gold standard
for the identification of corneal fungal pathogens dur-
ing screening diagnosis tests when an early mycotic
keratitis is suspected. In a study from 2012, Tananu-
vat et al. [95] identified the causal agents of 30 fungal
keratitis cases by the same seminested strategy and
reported the occurrence of Candida spp. (Can. albicans,
Can. parapsilosis, Can. etchellsii), Fusarium spp. (includ-
ing Fus. proliferatum, other members of the FFSC, as
well as members of the FOSC), Botryosphaeria sp.,
Erysiphe guercicola, Cladosporium spp. including Cla.
colocasiae and Cla. oxysporum, Curvularia spp. including
Cur. affinis, Alt. alternata, Botryosphaeria rhodina,
Acremonium sp., Asp. fumigatus as well as the basidio-
mycetes Cryptococcus pseudolongus, Exidiopsis calcea,
Phanerochaete sordida and Hyphodontia sp., however,
the respective sequences were not submitted to the
GenBank database. Can. albicans, Alternaria sp., Curvu-
laria sp., Scedosporium apiospermum and Asp. flavus
could also be rapidly detected in corneal samples by
amplification and sequencing of the entire ITS
region.164 Kuo et al. [165] reported the detection of
Cla. cladosporioides, Col. gloeosporioides, Hypocreales sp.
including members of the FSSC, Acremonium sp.,
Phomopsis sp., Malassezia sp. including Mal. restricta,
Ramularia coleosporii, Phaeoacremonium parasiticum and
8 © 2015 Blackwell Verlag GmbH
Corynespora spp. including Cor. cassiicola (the latter
three for the first time) as keratitis pathogens after
sequencing of cloned ITS fragments that were ampli-
fied from corneal scrapings.
During the BLAST searches performed with
sequences of the ITS region, fungi with sequences
showing 99–100% identity are generally identified at
the species level, whereas a genus level identification
is made in the cases when the homology is between
95% and 99%. However, the results of NCBI BLAST
searches have to be treated carefully. There are many
sequences in the GenBank database which were depos-
ited under an incorrect species name, therefore, the
sequence records, and if available, the related publica-
tions should be thoroughly reviewed before the accep-
tance of a diagnosis. Possible pitfalls of BLAST-based
identifications were demonstrated by Badenoch et al.
[166] in a letter reflecting to the article of Bag-
yalakshmi et al. [113]: the identifications reported for
eight fungal keratitis cases (Thielavia tortuosa, Botryosp-
haeria dothidea, Glo. cingulata, Asp. terreus, Bipolaris sp.,
Rhizoctonia bataticola, Macrophomina phaseolina, Pythi-
um insidiosum) were found to be uncertain. It was em-
phasised that the degree of confidence for an
identification depends not just on the quality of the
sequence (good identity is needed with high-quality
sequences from expert laboratories) but also on the
level of coverage.166 Another example is the article of
Kumar et al. [167] who amplified and sequenced the
ITS1 region from four corneal samples and reported
the identification of the causal agents of keratitis based
on alignments of ITS1 sequences as Nectria haematoc-
occa, Can. albicans and Cur. papendorfii (two cases).
However, the ‘Nectria haematococca’ ITS1 sequence
recovered from the sequence alignment published by
the authors showed only 94% sequence identity with
ITS1 of the reference strain N. haematococca ATCC
MYA-4622 (Fusarium solani subsp. pisi), and a BLAST
search performed in November 2014 allows the identi-
fication of the isolate only at the species complex level,
as a member of the FSSC. Furthermore, the diagnosis
of Cur. papendorfii keratitis for two cases based on only
the presented ITS1 sequences is also highly uncertain.
The interpretation of BLAST search results is especially
difficult in cases when at the time of the analysis there
are no hits in the NCBI database with sufficient level
of sequence identity to the query sequence. This was
the situation in the case report of Chew et al. [145]
who identified a fungal keratitis isolate as Carpoligna
sp. (Chaetosphaeriales) based on an NCBI BLAST search
performed on April 2008, which revealed 91% identity
of the 28S rDNA query sequence with the

corresponding sequences of Carpoligna pleurothecii
strains, whereas the same BLAST search performed on
November 2014 identified the case isolate as Phaeoisa-
ria sp. (Xilariales) with 97% sequence identity. In
another case, the unique ITS sequence of a keratitis
isolate supported the proposal of a new species: Pyren-
ochaeta keratinophila, the main morphological features
of which were not matching with any other described
species of the genus.138
Sequencing of the fragments with diagnostic value
is generally performed with the aid of external ser-
vices, which is substantially increasing the time and
costs of routine diagnosis. An alternate solution is the
application of diagnostic PCR methods based on spe-
cific primers, which require less time and costs, just
the availability of PCR and gel electrophoresis equip-
ments. The initial effort was the development of a
PCR-based test amplifying a fragment of the cutinase
gene for the diagnosis of Fusarium keratitis,168
whereas the subsequent studies targeted different
regions of the fungal ribosomal RNA gene cluster.
Panfungal primers targeting the ITS region,160,161,169
the 28S rRNA gene170,171 or the 18S rRNA
gene163,172,173 can be applied for the recognition of
fungal involvement in keratitis. Panfungal primers
were designed for 18S rRNA sequences and a nested
PCR method was worked out for the detection of Can.
albicans and two filamentous fungi, Asp. fumigatus and
Fus. solani (FSSC) in ocular samples.172 Another
nested PCR-strategy was used by Gaudio et al. [174]
to detect Can. albicans as well as Asp. fumigatus and
Fus. oxysporum (FOSC) from corneal scrapings of kera-
titis patients. Wang et al. [175] developed a multi-PCR
resulting in different fragment sizes for the differential
diagnosis of aspergilli, fusaria, Penicillium implicatum
and Cur. lunata. Kuo et al. [165] developed a diagnos-
tic test for fungal keratitis based on PCR amplification
of the ITS region and subsequent hybridisation of the
PCR product to a fungus-specific oligonucleotide probe
immobilised on a nylon membrane. Single-stranded
conformation polymorphism (SSCP) analysis of ampli-
cons deriving from different rDNA regions was also
applied for the diagnosis of fungal keratitis on the
basis of size and primary structural differ-
ences,167,176,177 however, size determination is not
considered to be precise enough for a unambigous
identification of the causal agents at the species
level.161 Goldschmidt et al. [178] developed a real-time
PCR high-resolution melting analysis, which is capable
of differentiation between filamentous fungi and yeasts
and discrimination among relevant yeast species in a
single run.
© 2015 Blackwell Verlag GmbH
Epidemiology of filamentous fungal keratitis
Regarding further gemomic regions applied for PCR-
based identification of fungal keratitis isolates, a rapid
method based on a specific EcoRI restriction site in a
fragment amplified from the tef1 gene was developed
for fusaria,154 the parallel application of which with
the method described by He et al. [75] can be sug-
gested for the fast and correct identification of FSSC
members. Variations in the rpb2 nucleotide sequence
among 72 Fusarium isolates enabled the design of 34
allele-specific probes for the identification of all medi-
cally important Fusarium species complexes and 10
human pathogenic Fusarium species including the
most important keratitis pathogens in a single-well
diagnostic assay using flow cytometry and fluorescent
microsphere technology.151
Antifungals for the therapy of mycotic
keratitis
Topical steroids along with antifungals (polyenes and
triazoles) are commonly applied for the treatment of
fungal keratitis. Polyenes include natamycin (NTM,
5% suspension) and amphotericin B (AMB), the former
being considered as the drug of choice against filamen-
tous fungi179 in many cases. Although NTM exhibits
relatively poor penetration through the corneal layers,
its bioavailability is stated to be sufficient for antifun-
gal activity and for effective treatment of Fusarium ker-
atitis in vivo.169 Topical NTM (5%) and/or topical
AMB (0.5%) were the first-line treatments of fungal
keratitis in Singapore, where Fusarium and Aspergillus
spp. were the most commonly isolated organisms.19
Iyer et al. [44] also reported treatment of fungal kera-
titis with NTM and AMB. A prospective study by Pra-
jna et al. [180] described that 2% econazole (ECZ) can
be a substitution for 5% NTM in certain cases. In the
study of Kalavathy et al. [181] topical NTM (5%) was
shown to be superior to topical itraconazole (ITZ, 1%)
in the management of fungal keratitis due to filamen-
tous fungal species including fusaria. The authors also
stated that although NTM and AMB exhibited rela-
tively poor penetration through the corneal layers, the
bioavailability of NTM was sufficient for antifungal
activity and it was effective for the treatment of Fusari-
um keratitis in vivo. ECZ and clotrimazole (CLZ) are
available as 1% oil, drops and ointment for the topical
treatment of fungal keratitis, however, CLZ is not rec-
ommended for monotherapy, its combination with a
polyene derivative should be considered.182 Other
azole compounds such as imidazole (IMZ), miconazole
(MCZ) and ketoconazole (KTZ) are inferior to AMB
despite having less systemic toxicity to the host.
9
L. Kredics et al.
Because of relatively reduced systemic toxicity these
compounds can be used systemically for keratomyco-
sis.183 Thomas [1] reported varying positive response
in fungal keratitis patients treated with KTZ (69%),
ITZ (66%), AMB (53%) and NTM (56%). Fluconazole
(FLZ), a fungistatic bis-triazole was also considered as
topical and systemic agent in the treatment of fungal
keratitis due to Candida spp. and Aspergillus spp.184
FLZ and corticosteroids were used for the treatment of
experimental C. albicans keratomycosis185; however, it
did not show encouraging results against Aspergillus
and Fusarium spp.186 Voriconazole (VRZ) – an azole
antifungal agent derived from FLZ with more effective
action than IMZ – showed a broad spectrum of activity
against Candida, Aspergillus and Fusarium spp.187 In
severe cases with impending or presenting corneal per-
foration or scleral extension, systemic antifungals such
as FLZ and ITZ are added. Second-line treatment with
topical and systemic VRZ for non-responsive cases was
also instituted, because this agent was reported to
have a broad-spectrum antifungal activity, greater bio-
availability, and higher aqueous and vitreous levels in
the eye.34,188
Another promising strategy for the improvement of
the therapy of fungal keratitis is the screening for new
antifungals. Rahman et al. [20] suggested based on
the results of a clinical trial that chlorhexidine has the
potential as an inexpensive topical agent for Aspergillus
and Fusarium keratitis. However, Johnson [189]
reported that treatment of patients with this com-
pound in two locations in Africa did not have encour-
aging results. In experimental Aspergillus keratitis,
polyhexamethylene biguanide (0.02%) was found to
be a moderately effective drug, whereas 1% povidone
iodine was not effective.190 Similarly, fluorinated pyr-
imidines may also not have significant roles in the
management of Aspergillus keratitis. In a recent study
the antifungal effects of essential oils derived from nine
herbal plants (Cinnamomum zeylanicum, Citrus limon,
Juniperus communis, Eucalyptus citriodora, Gaultheria
procumbens, Melaleuca alternifolia, Origanum majoranna,
Salvia sclarea and Thymus vulgaris) were examined
against Fusarium isolates from keratomycosis cases.191
The lowest MIC values were observed in the case of
Cinnamomum zeylanicum essential oil (CZEO), the main
component of which, trans-cinnamaldehyde (tCA)
showed the same activity. Microscopic observations
revealed that CZEO and tCA inhibit conidial germina-
tion and reduce the cellular metabolism. Based on
these results, CZEO and tCA provide a promising basis
for the development of a novel strategy for the treat-
ment of Fusarium keratitis.191
10
Significance of susceptibility testing in the
management of fungal keratitis
Although in vitro antifungal susceptibility testing may
not always accurately predict the clinical response of
individual fungal keratitis patients, literature data
about the minimum inhibitory concentrations (MICs)
of antifungals against various isolates of filamentous
fungi causing keratitis are highly valuable in guiding
the clinicians to select the appropriate therapeutic
agents.192 Due to the fact that the practice of subject-
ing fungal isolates to antifungal susceptibility tests
remains uncommon across the diagnostic microbiology
laboratories, and that the susceptibility pattern is
depending from the involved species of Aspergillus/
Fusarium as well as the nature and concentration of
the drug, it is further emphasised that the isolates
should compulsorily be examined for their susceptibil-
ity to increase the chances of an accurate therapy.
MIC data generated by facilities where the determina-
tion of fungal susceptibility is possible can be used as
a basis for the treatment of patients elsewhere. In this
line, the following overview of relevant literature sum-
marises the data already available at national and glo-
bal levels about the antifungal susceptibilities of fungal
keratitis isolates.
NTM, also known as pimaricin is reported to have a
broad spectrum of activity against various fungi,
including Fusarium and Aspergillus spp.193,194 Lalitha
et al.195 applied broth macrodilution and reported that
NTM had a good activity against species of Fusarium
with similar MIC90 values. They also stated that Asper-
gillus isolates had slightly higher MIC values. In a sub-
sequent study196 performed by the reference method
of the Clinical and Laboratory Standards Institute
(CLSI),197 NTM had good activities against both Fusa-
rium and Aspergillus spp. with slightly higher MICs
against Aspergillus spp. Nayak et al. [198] reported
that Asp. niger and Asp. flavus had higher MICs for
AMB compared to Asp. fumigatus suggesting a high
index of suspicion for AMB resistance. Alfonso [199]
reported MIC90 of Fusarium as 4.8 lg ml
1 by the
CLSI method. Conversely, Xuguang et al. [52] reported
from China that 93% of the Fusarium and 72.4% of
the Aspergillus isolates were sensitive to NTM by the
disc diffusion method. Rahman et al. [20] stated that
the majority of the Aspergillus spp. was susceptible to
NTM between 16 and 32 lg ml
1. Xie et al. [200]
reported that 88.9% of both Asp. flavus and Asp. fumig-
atus were susceptible to NTM, whereas 94.2%, 92%
and 91.3% of FSSC members, Fus. moniliforme and
FOSC members, respectively, were susceptible to NTM
© 2015 Blackwell Verlag GmbH

by the CLSI method. A study by Pradhan et al. [201]
performed by microbroth dilution concluded that NTM
showed poor outcome in patients with advanced fun-
gal keratitis.
In the case AMB, the other polyene compound fre-
quently used for the therapy of keratitis, Xie et al.
[200], Alastruey-Izquierdo et al. [202] and Lalitha
et al. [196] differentiated that fusaria were more sensi-
tive than Aspergillus spp. O’Day et al. [203] stated that
this drug was not effective against Fusarium spp. Simi-
larly, Pujol et al. [204] reported MIC50 as
2.31 lg ml
1 and MIC90 as 4.62 lg ml
1 for AMB.
Xie et al. [200] stated that the poor penetration into
the cornea and the obvious stimulative symptoms
make the topical preparation of AMB unsuitable to be
administered with a large dosage and for a long time.
Galarreta et al. [3] reported that 20.8% of the exam-
ined filamentous fungi were resistant to AMB. Therese
et al. [205] applied the agar dilution method and
reported Asp. fumigatus being more sensitive to AMB
than other Aspergillus species.
ITZ is a broad-spectrum triazole antifungal agent with
a high degree of efficacy against filamentous fungi. The
guidelines of CLSI197 classify isolates with ITZ MICs of
≥8 lg ml
1 as ‘resistant’. Galarreta et al. [3] found only
25% of filamentous fungi isolated from corneal ulcers as
ITZ resistant. Fusaria were reported totally resistant,195
whereas aspergilli 100% susceptible206 to the drug.
Hahn et al. [207] also suggested that the use of ITZ
should be a primary consideration in the treatment of
Aspergillus keratitis, as the growth of Asp. fumigatus,
Asp. niger, Asp. terreus and Asp. tamarii were completely
restricted at an ITZ concentration of ≤0.5 lg ml
1.
Based on the E-test commercial antifungal susceptibility
testing system (AB Biodisk, Solna, Sweden), Qiu et al.
[208] determined 100% (n=9) of the examined asper-
gilli as susceptible to ITZ. Using the Sensititre Yeast One
Method, Marangon et al. [34] reported the MIC values
of four Aspergillus isolates between 0.256 and
1 lg ml
1. However, Xie et al. [200] stated that 77.8%
and 80% of Asp. flavus and Asp. fumigatus, respectively,
were resistant to ITZ. Heidemann et al. [209], Thomas
et al. [210] and Kaushik et al. [211] had reasoned that
the unresponsiveness of ITZ against certain strains of
Aspergillus could be due to drug resistance developed
among the strains. Xie et al. [200] stated that more
number of the tested fusaria – FSSC members (85.3%),
Fus. moniliforme (64%) and FOSC members (87%) were
resistant to ITZ.
Marangon et al. [34] and Johnson et al. [189]
reported that VRZ was more effective against Aspergil-
lus spp. Lalitha et al. [195] reported MIC90 against
© 2015 Blackwell Verlag GmbH
Epidemiology of filamentous fungal keratitis
Aspergillus and Fusarium as 1 and 8 lg ml
1 respec-
tively. Similarly, Pfaller et al. [212] and Lass-Fl€ orl
et al. [213] determined comparatively lower VRZ con-
centrations required to inhibit the growth of aspergilli.
Conversely, Alfonso [199] stated 16 lg ml
1 as the
MIC90 against Fus. solani.
A study reported that more than half (58%) of the
filamentous fungi isolated from fungal keratitis
patients were sensitive to ECZ.3 Guinet and Mazoyer
[214] stated that ECZ exhibited the best in vitro activ-
ity against 96% of the Aspergillus strains with MICs of
≤3.12 lg ml
1. However, Gonawardena et al. [215]
reported decreased susceptibility of keratitis fungi to
this drug. Although Bernauer et al. [216] insisted on
using ECZ with other antifungals, Prajna et al. [35]
reported that concurrent use of 5% NTM and 2% ECZ
was not an additional benefit for the management of
fungal keratitis.
A very early report described that in vitro suscepti-
bility tests indicated good antifungal activity of CLZ
and ECZ against Asp. fumigatus.22 In the study by
Hahn et al. [207], it was reported that the Aspergillus
isolates from mycotic keratitis showed a higher suscep-
tibility to CLZ than AMB. However, more recently, the
utility of CLZ was reported to be limited, since only
40% of filamentous fungi were sensitive to this anti-
fungal agent according to Galarreta et al. [3].
Although the interpretive MIC breakpoints of KTZ
against fungal isolates were to be defined by the CLSI,
Therese et al. [205] reported filamentous fungi with
the KTZ MIC value of ≤0.8 lg ml
1 as ‘susceptible’.
Pujol et al. [204] reported KTZ MIC90 ≥
51.20 lg ml
1 for fusaria. Xie et al. [200] reported
the MIC90 of Fus. solani as 16 lg ml
1 and that of
Asp. fumigatus as 2 lg ml
1, and that KTZ is more
effective against Asp. flavus than against Asp.
fumigatus.
Studies in the literature comparing the susceptibili-
ties of large sets of fungal corneal isolates to a series of
different antifungal agents are especially informative.
Pujol et al. [204] applied a broth microdilution method
and reported that most of the examined Fusarium
isolates were resistant in vitro to all tested antifungals,
AMB, KTZ, MCZ, ITZ, FLZ and flucytosine (5FC). Qiu
et al. [208] applied the E-test method to fungi isolated
from keratitis to determine the MIC values of ITZ, FLZ
and AMB. The results showed that 60.6% of the
Fusarium isolates were susceptible to AMB and all of
them were resistant to ITZ and FLZ. On the other
hand, all the Aspergillus isolates were susceptible to
ITZ, 44.4% of them to AMB and 22.2% of them to
FLZ.
11
L. Kredics et al.
A study from China200 applying the CLSI method
reported AMB and terbinafine (TRB) with lowest MICs
against Fusarium and Aspergillus spp., respectively.
Common species of Fusarium and Aspergillus were the
most sensitive to NTM (92%), followed by AMB (74%)
and TRB (74%), whereas the sensitivities to KTZ, MCZ,
ITZ, FLZ and 5FC were rather low. Alastruey-Izquierdo
et al. [202] applied the CLSI method and reported that
AMB was the most active agent against Fusarium spp.
with its geometric mean MIC being 1.15 mg l
1. The
numbers of isolates for which MICs of AMB were
≥2 mg l
1 differed depending on the taxa: 12 out of
22 (54.6%) FSSC members, 9 out of 14 (64.3%) Fus.
proliferatum, four out of 13 (30.8%) Fus. verticilloides
and 1 out of 14 (7.1%) FOSC members had MICs
≥2 mg l
1. According to Lalitha et al. [196], the CLSI
method revealed that triazoles and caspofungin (CSF)
had the lowest MICs against Aspergillus species, VRZ,
AMB and posaconazole (PSZ) had the lowest MICs
against Fusarium species, whereas none of the Fusari-
um species were inhibited by ITZ or CSF. Alfonso
[199] stated based on results achieved by the CLSI
method that members of the FSSC tend to be more
resistant to azoles.
Homa et al. [154] studied the MIC values of AMB,
CLZ, ECZ, ITZ, NTM, TRB and VRZ towards the Fusari-
um keratitis isolates by the CLSI method. AMB, NTM
and TRB proved to be the most effective drugs. The com-
bination of the latter two showed similar – or even bet-
ter – synergistic activity against fusaria when tested by
the checkerboard microdilution method. TRB is known
to efficiently penetrate through the corneal epithelium
and accumulate in the eye tissues,217 whereas NTM
shows poor penetration, therefore it is more effective in
the therapy of superficial keratitis.201,218 Based on the
detected synergism between these two antifungals, their
combination can be suggested as a possible strategy for
the efficient treatment of Fusarium keratitis.
As previous studies suggested that the susceptibility
levels of Aspergillus strains to various antifungal com-
pounds may be variable among different isolates,198,201
the MIC values of 8 antifungal agents (AMB, CLZ, ECZ,
FLZ, ITZ, KTZ, NTM and VRZ) were determined towards
Aspergillus isolates from keratitis by the CLSI method.5
CLZ, ECZ and KTZ proved to be active against Asp. fla-
vus, the most frequently occurring keratitis pathogen of
the genus, whereas Asp. fumigatus exhibited higher MIC
values against azoles indicating an emergence in azole
resistance. In contrast, AMB was more effective against
Asp. fumigatus than against Asp. flavus. Importantly, all
the examined Aspergillus isolates proved to be com-
pletely resistant to FLZ.5
12
As it is frequently reported that the resistance to
conventional antifungal agents is rapidly spreading
among human pathogenic Fusarium species, the anti-
fungal susceptibilities of Fusarium strains isolated at
the Aravind Eye Hospital, Coimbatore to conventional
antifungal drugs were compared between two different
periods: 2004–2005 and 2010–2011.155 Based on the
observations the in vitro susceptibilities to azoles, such
as CLZ, ECZ and ITZ have unambiguously decreased
from 2004–2005 to 2010–2011: most of the isolates
from the years 2010–2011 showed high MIC values
(≥64 lg ml
1) to these drugs. The detected shift of the
obtained MIC value ranges towards higher values sug-
gests the spreading of azole resistance. On the other
hand, no changes could be observed in the case of the
MICs of NTM and TRB.155
Besides Aspergillus and Fusarium strains, fungal ker-
atitis isolates belonging to other genera (Exserohilum,
Alternaria, Exophiala) were also involved in a recent
study aimed at the evaluation of the efficacy of azole
drugs (CLZ, ECZ, ITZ and VRZ) for the therapy of fila-
mentous fungal keratitis by the CLSI method.219 A
variation in the overall activities of the azole drugs
was observed depending on the type of the fungal
species and the drug concentration. Twenty-two of
30 Fusarium isolates were inhibited at a CLZ concen-
tration of 4 lg ml
1, whereas all other examined
fungi were inhibited at 2 lg ml
1. The MICs of ECZ,
ITZ, KTZ and VRZ were in the ranges of 8-0.015,
32-0.06, 16-0.03 and 8-0.015 lg ml
1, respectively,
with all Asp. flavus isolates being susceptible to
2 lg ml
1 KTZ. Curvularia, Exserohilum and Alternaria
isolates could be characterised with KTZ MIC values
<8 lg ml
1, whereas the sensitivity of the examined
Exophiala strain to this drug proved to be similar to
those recorded for fusaria (MIC = 16 lg ml
1). For
ECZ and VRZ, 45% and 55% of the tested isolates
were inhibited by 1 lg ml
1 and <2 lg ml
1 concen-
trations of the drugs respectively. In the case of the
four examined Exserohilum isolates, the lowest MIC50
values were recorded for CLZ (0.12 lg ml
1), ITZ,
ECZ and VRZ (each 0.06 lg ml
1). CLZ proved espe-
cially efficient against Curvularia strains (MIC90
between 0.25 and 0.5 lg ml
1). As generally higher
concentrations of ITZ and KTZ were required for the
inhibition of the tested filamentous fungal isolates,
CLZ followed by VRZ and ECZ were suggested as
appropriate antifungal agents for the therapy of fila-
mentous fungal keratitis. The susceptibility patterns
were found to depend both from the fungal species
and from the nature and concentration of the applied
antifungal drug.219
© 2015 Blackwell Verlag GmbH

Conclusions
Misidentification of the causative agents of filametous
fungal keratitis cases and the subsequent application of
an inadequate antifungal therapy may result in the loss
of vision. The rapid, exact, species-level identification of
the pathogen is of highlighted importance; furthermore,
the antifungal susceptibilities of keratitis isolates should
also be determined in all cases to ensure the accurate
choice of drug enabling the successful therapy of the
patient. The recently available methodologies make
these possible, however, the necessary equipments are
not commonly available in eye clinics and the financial
resources are also lacking in many places, especially in
developing countries. A possible solution to this problem
is to establish the opportunity of direct knowledge trans-
fer by the initiation of collaborative projects between
eye clinics and research laboratories. Besides the data
available for polyenes and azoles, testing the applicabil-
ity of additional antifungals (e.g. terbinafine and caspo-
fungin), as well as screening for drug combinations and
further promising compounds for the therapy of fungal
keratitis could reveal new strategies for the treatment of
this vision-threatening disease.
Acknowledgements
This work was supported by the Indian National
Science Academy and the Hungarian Academy of
Sciences within the frames of the Indo-Hungarian
bilateral exchange program (Ref. IA/INSA-HAS Pro-
ject/2007 & 2010). Csaba V
agv€
olgyi thanks the visit-
ing professor program, Deanship of Scientific Research
at King Saud University, Riyadh.
Conflict of Interests
The authors declare that they have no conflict of
interest.
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Appendix
Indo-Hungarian Fungal Keratitis (IHFK) Working Group
J

anos Varga, L
aszl

o Galg
oczy, S
andor Kocsub
e,
Tibor Mih
aly N
emeth, Tam
as Papp, M
onika
Homa, Nikolett Baranyi, Andr
as Szekeres, P
eter
K€orm€oczi, Krisztina Krizs
an: Department of Micro-
biology, Faculty of Science and Informatics, University
of Szeged, Szeged, Hungary; Raghavan Anita, Rajar-
aman Revathi, Perumal Gomathi: Aravind Eye
Hospital and Postgraduate Institute of Ophthalmology,
Coimbatore, Tamilnadu, India; Anamangadan Sha-
feeq Hassan, Yendrembam Randhir Babu Singh,
Arumugam Mythili: Department of Microbiology, Dr.
G.R. Damodaran College of Science, Coimbatore, India;
Kanesan Panneer Selvam: Department of Microbiol-
ogy, MR Government Arts College, Mannargudi, India;
Ilona D
oczi: Department of Clinical Microbiology,
Faculty of Medicine, University of Szeged, Szeged, Hun-
gary; Bal
azs Leitgeb: Institute of Biophysics, Biologi-
cal Research Centre of the Hungarian Academy of
Sciences, Szeged, Hungary.
© 2015 Blackwell Verlag GmbH