Kredics Et Al 2015 Mycoses

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
Kredics,1 Venkatapathy Narendran,2 Coimbatore Subramanian Shobana,3 Csaba
La
gvo
lgyi,1,4 Palanisamy Manikandan2,5 and Indo-Hungarian Fungal Keratitis Working Groupa
Va
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 specieslevel 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.
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
2015 Blackwell Verlag GmbH
usually ulcerative corneal infection which may result

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 particularly from developing countries. Although yeast
infections occur more frequently in mild climates, filamentous 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 people involved in soil-related work such as agriculture
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 frequently 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 therapy.4 In this context, although most cases of fungal
keratitis exhibit uniform symptoms and clinical features, 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 therapyrefractive 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 overview 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 antifungal 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)2,3,5,7106 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 keratitis 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 largescale studies were reported from India,2,5,9,11,12,14,16,20
23,27,29,30,32,35,38,39,4548,50,54,5961,63,65,66,70,7680,83,
8894,9699,101,102,104,105
followed by the USA,7,8,17,24,

China
and Brazil,26,37,
whereas there is a lack of large
34,41,43,44,49,73,100
68,71,72,82
25,31,42,52,67,75
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 keratitis 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 cornerstone of the diagnosis of most cases of fungal keratitis,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 inappropriate culture methods.95 In many cases the fungal
nature of the infection was determined only in pathological sections.
Earlier, isolation of the causative fungus was the
only tool available for the confirmation and the findings were applied as a key for therapy8 without identifying 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 identified at the genus level only.2224 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.2,1921 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 keratomycosis (Table 1). This is distressing, as the antifungal
susceptibility patterns may vary between different species 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 predominant 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.
Country
Period of study
USA, Texas
USA, Florida
India, Jamnagar
Bangladesh, Chittagong
India, Madras (south)
India, Madras
Paraguay
India, Karnataka
1962
January 1968July 1970
1985
1987
19801982
NA
April 1988April 1989
October
1985September 1988
11 months
Bangladesh,
Chittagong
India, Chandigarh
(north)
USA, Miami
Ghana, Accra
Singapore
India, Madurai (south)
India, Madurai
India, Mumbai (west)
India, Hyderabad
India, New Delhi
USA, Pennsylvania,
China
Brazil
Ghana
Thailand (central)
India, Hyderabad
(south)
India, Tiruchirapalli
(south)
India, Tirunelveli
(south)
China, Hong Kong
India, Tirunelveli
Paraguay
USA, Florida
India, Madurai
Taiwan, Taipei
Brazil
India, New Delhi
India, West Bengal
Nepal, Dharan
USA, Kentucky
China
USA, Florida
USA, Florida
India, Coimbatore
(south, children)
India, New Delhi
India, Tirunelveli
India, Tiruchirappalli
USA, New York
6 years
January 1982January 1992
NA
January 1991December 1995
January 1994March 1994
NA
19881996
February 1991June 1995
NA
January 1996 and
December 1999
19831997
June 1999May 2001
Number
of fungal
isolates
10
33
37
7
68
322
26
67
Fusarium
(%)
3
15
12
1
8
44
11
(30.0)
(45.5)
(32.5)
(14.3)
(11.8)
(13.7)
(42.3)
Aspergillus
(%)
2
11
10
2
36
55
5
23
(20.0)
(33.3)
(27.0)
(28.6)
(52.9)
(17.1)
(19.2)
(34.3)
Curvularia
(%)
1
2
1
2
(3.0)
(5.4)
(5.9)
(3.9)
(3.0)
Other
fungi
(%)
5
6
13
4
20
223
9
42
Reference
Year
(50.0)
(18.2)
(35.1)
(57.1)
(29.4)
(69.2)
(34.6)
(62.7)
7
8
9
10
11
12
13
14
1962
1971
1985
1987
1989
1989
1991
1992
51
10 (19.6)
19 (37.3)
6 (11.8)
16 (31.3)
15
1994
61
10 (16.4)
25 (41.0)
5 (8.2)
21 (34.4)
16
1994
127
65
29
155
63
387
21
13
24
97
79
34
15
73
22
33
3
4
6
63
(62.2)
(52.3)
(51.7)
(47.1)
(34.9)
(8.5)
(14.3)
(30.8)
(25)
(65.0)
5
10
4
25
22
219
7
6
1
14
(4.0)
(15.4)
(13.8)
(16.1)
(34.9)
(56.6)
(33.3)
(46.1)
(4.2)
(14.4)
11
2
1
6
5
10
3
2
(8.7)
(3.1)
(3.5)
(3.9)
(8.0)
(2.6)
(14.3)
(15.4)
32
19
9
51
14
125
8
1
17
20
(25.1)
(29.2)
(31.0)
(32.9)
(22.2)
(32.3)
(38.1)
(7.7)
(70.8)
(20.6)
17
18
19
2
20
21
22
23
24
25
1994
1995
1997
1997
1998
1999
2000
2000
2000
2001
25
109
34
1360
8
46
12
506
(32.0)
(42.2)
(35.3)
(37.2)
4
38
7
417
(16.0)
(34.9)
(20.6)
(30.7)
1 (0.9)
7 (20.6)
54 (4.0)
13
24
8
383
(52.0)
(22.0)
(23.5)
(28.1)
26
27
28
29
2001
2002
2002
2002
June 1999May 2001
353
141 (40.0)
76 (21.5)
36 (10.2)
100 (28.3)
27
2002
September 1999March 2001
554
254 (45.8)
135 (24.4)
35 (6.3)
130 (23.5)
30
2002
April 1997August 1998

September 1999August 2002
19882001
December 2002June 2003
19752003
January 1999June 2001
August 1998July 2001
January 1981December 2004.
January 1999June 2006.
February 1997January 2004
5
1100
209
421
100
34
233
191
710
200
89
596
122
59
37
3
471
41
208
63
10
137
24
132
45
18
437
66
24
17
286
37
30
26
5
28
78
373
75
9
72
19
7
11
55
15
34
3
1
63
27
9
29
9
2
2
288
116
149
8
19
67
26
205
53
53
87
8
19
7
(40.0)
(26.2)
(55.5)
(35.4)
(8.0)
(55.9)
(28.8)
(13.6)
(28.9)
(26.5)
(59.6)
(14.6)
(6.5)
(32.2)
(18.9)
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
2002
2003
2004
2004
2004
2004
2005
2005
2005
2005
2006
2006
2006
2006
2006

September
1999September 2002
January 1December 31, 2003
January 1987June 2003
77
1226
6 (7.8)
511 (41.7)
43 (55.8)
305 (24.9)
2 (2.6)
81 (6.6)
26 (33.8)
329 (26.8)
46
47
2006
2006
93
61
32 (34.4)
6 (9.8)
29 (31.2)
7 (11.5)
9 (9.7)
23 (24.7)
48 (78.7)
48
49
2006
2006
(60.0)
(42.8)
(19.6)
(49.4)
(63.0)
(29.4)
(58.8)
(12.6)
(18.6)
(22.5)
(20.2)
(73.3)
(54.1)
(40.7)
(46.0)
(26.0)
(17.7)
(7.1)
(26.0)
(14.7)
(12.0)
(40.8)
(52.5)
(37.5)
(10.1)
(12.1)
(15.6)
(11.9)
(29.7)
(5.0)
(7.2)
(8.1)
(3.0)
(0.4)
(33.0)
(13.5)
(10.1)
(23.8)
(15.2)
(5.4)
(continued)
L. Kredics et al.
Table 1 (continued)
Country
Period of study
India, New Delhi

Paraguay
China
Australia, Melbourne
India, New Delhi
Turkey, West Anatolia
Japan, Tokyo
UK, London
Malaysia
Thailand
India, Chandigarh
(north)
India, New Delhi
India, Ujjain
Australia,
Queensland
India, Chennai
(south)
Nepal, Dharan
India, Hyderabad
(south)
India, Mumbai
China, Zhengzhou
Brazil, Southeastern
UK, London
NA
20012004
July 1996May 2004
December 2005
December 1993January 2007
January 2004April 2005
India, Varanasi
Brazil, S~
ao Paulo
Brazil, Uberlandia
USA, Philadelphia
Sierra Leone
China
India, Amristar
India, Assam
India, Bhubaneswar
India, Gujarat
India, Pondychery
South Korea,
Jeonbuk
Brazil, S~
ao Paulo
India, Maharashtra
Saudi Arabia, Riyadh
Nepal
Saudi Arabia
Vietnam
India, Ahmedabad
India, Maharashtra
India,
India,
India,
India,
West Bengal
Karnataka
Delhi
West Bengal
Number
of fungal
isolates
9
25
681
49
486
50
6
66
4
49
34
Fusarium
(%)
2
6
395
6
71
25
12
2
13
8
(22.2)
(24.0)
(58.0)
(12.2)
(14.6)
(50.0)
(18.2)
(50.0)
(26.5)
(23.5)
Aspergillus
(%)
5
2
116
7
268
10
7
1
9
14
(55.6)
(8.0)
(17.0)
(14.3)
(55.1)
(20.0)
(10.6)
(25.0)
(18.4)
(41.2)
Curvularia
(%)
1
4
15
2
4
(11.1)
(16.0)
(3.1)
(4.1)
(11.8)
Other
fungi
(%)
1
13
170
36
132
15
6
47
1
25
8
(11.1)
(52.0)
(25.0)
(73.5)
(27.2)
(30.0)
(100.0)
(71.2)
(25.0)
(51.0)
(23.5)
Reference
Year
50
51
52
53
54
55
56
3
57
58
59
2006
2006
2007
2007
2007
2007
2007
2007
2008
2008
2008
NA
April 2006November 2007
19982008
39
37
16
2 (5.1)
2 (5.4)
8 (50.0)
22 (56.4)
20 (54.1)
2 (12.5)
7 (18.0)
6 (16.2)
2 (12.5)
8 (20.5)
9 (24.3)
4 (25.0)
60
61
62
2008
2008
2008
July 2006May 2008
20
7 (35.0)
8 (40.0)
2 (10.0)
3 (15.0)
63
2009
January 2007July 2008

February 1991June 2001
12
1648
8 (66.6)
588 (35.7)
2 (16.7)
478 (29.0)
2 (16.7)
106 (6.4)
0
476 (28.9)
64
65
2009
2009
NA
20002004
December
2003November 2005
19752007
July 2001August 2004
April 1999December 2008
October
2004September 2009
NA
April 2007March 2009
July 2006December 2009
September 2003June 2005
January 2009August 2008
January 2000 and
December 2007
July 1975September 2007
January 2006 and
December 2009
2008
July 2007June 2008
December
2004December 2009
February 2007January 2011
September 2006August 2007
April 2009April 2010
NA
40
1458
66
39
6
1076
44
2
(15.0)
(73.8)
(66.7)
(5.1)
20
195
7
6
(50.0)
(13.4)
(10.6)
(15.4)
4 (10.0)
10
187
15
31
(25.0)
(12.8)
(22.7)
(79.5)
66
67
68
69
2009
2009
2009
2009
36
94
18
66
26
139
7
28
11
29
1
67
(19.5)
(29.8)
(61.1)
(44.0)
(3.8)
(48.2)
17
9
3
2
4
26
(47.2)
(9.6)
(16.7)
(3.0)
(15.4)
(18.7)
1
2
9
57
3
33
21
45
(25.0)
(60.6)
16.6)
(50.0)
(80.8)
(32.4)
70
71
72
73
74
75
2010
2010
2010
2010
2010
2011
86
184
215
57
373
34
11
46
50
17
134
16
(12.8)
(25.0)
(23.3)
(29.8)
(35.9)
(47.0)
36
27
60
12
97
2
(41.9)
(14.7)
(27.9)
(21.1)
(26.0)
(6.0)
7
13
10
6
84
(8.1)
(7.1)
(4.7)
(10.5)
(22.5)
32
98
95
22
58
16
(37.2)
(53.2)
(44.1)
(38.6)
(15.6)
(47.0)
76
77
78
79
80
81
2011
2011
2011
2011
2011
2011
364
25
106
150
87
189
4
12
19
24
(51.9)
(16.0)
(11.3)
(12.7)
(27.6)
33
14
28
50
15
(9.1)
(56.0)
(26.4)
(33.3)
(17.2)
2 (8.0)
15 (10.0)
1 (1.2)
142
5
66
66
47
(39.0)
(20.0)
(62.3)
(44.0)
(54.0)
82
83
84
85
86
2011
2011
2011
2012
2012
351
31
311
143 (40.7)
7 (22.6)
109 (35.0)
91 (25.9)
11 (35.5)
56 (18.0)
9 (2.6)
5 (16.1)
10 (3.2)
108 (30.8)
8 (25.8)
136 (43.8)
87
88
89
2012
2012
2012
399
28
22
15
81 (20.3)
16 (57.2)
4 (26.7)
41 (10.3)
1 (3.6)
8 (36.4)
126
2
8
7
90
91
92
93
2012
2012
2012
2012
151
9
6
4
(37.8)
(32.1)
(27.2)
(26.7)
(8.3)
(5.6)
(3.0)
(0.7)
(31.6)
(7.1)
(36.4)
(46.6)
(continued)
Table 1 (continued)
Country
Period of study
India, Pondicherry
India, Coimbatore
Thailand
India, Baroda
India, Gujarat
(west)
India, Karnataka
India, Karnataka
20032009
20052008
July 2007May 2009
June 2009May 2012
September
2006February 2008
JanuaryJune 2012
December
2009February 2011
July 2001June 30, 2011
JanuaryJune, 2011
USA, Kansas
India,
Bhubaneswar
India, Karnataka
Tunisia, Sfax
India, Madurai
India, New Delhi
Egypt, Assiut
July 2009June 2010

January
1995December 2012
20062011
January
2004January 2012
NA
Number
of fungal
isolates
1224
1087
12
73
26
431
554
5
26
3
(35.2)
(50.9)
(41.7)
(35.6)
(11.5)
Aspergillus
(%)
Curvularia
(%)
Other
fungi
(%)
353
200
1
14
20
123
79
1
4
1
317
254
5
29
2
(28.9)
(18.3)
(8.3)
(19.2)
(76.9)
(10.0)
(7.2)
(8.3)
(5.5)
(3.9)
Reference
Year
(25.9)
(23.3)
(41.7)
(39.7)
(7.7)
94
5
95
96
97
2012
2012
2012
2013
2013
36
38
7 (19.4)
16 (42.1)
16 (44.5)
11 (28.9)
4 (11.1)
2 (5.3)
9 (25.0)
9 (23.7)
98
99
2013
2013
50
62
19 (38.0)
15 (24.2)
10 (20.0)
18 (29.0)
4 (6.5)
21 (42.0)
25 (40.3)
100
101
2013
2013
23
60
4 (17.5)
29 (48.3)
17 (73.9)
13 (21.7)
1 (4.3)
1 (4.3)
18 (30.0)
102
103
2013
2014
103
63
32 (31.1)
10 (15.9)
26 (25.2)
32 (50.8)
11 (10.7)
1 (1.6)
34 (33.0)
20 (31.7)
104
105
2014
2014
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 informations about the causal agents of filamentous fungal
keratitis. This, however, is not possible from epidemiological and antifungal susceptibility studies where the
causal agents were identified at the genus level only,
or the identifications were carried out based on morphological 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 nucleotidenucleotide BLAST (Basic
Local Alignment Search Tool)108 search in the nucleotide sequence collection of the National Center for Biotechnology 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).109146 These included Lagenidium
and Pythium species from the Oomycota division, a series of species from the Botryosphaeriales, Capnodiales,
Diaporthales, Eurotiales, Glomerellales, Hypocreales,
Fusarium
(%)
Microascales, Pleosporales, Sordariales and Xilariales

orders of the Ascomycota division, as well as Schizophyllum commune from Basidiomycota. The initial reports
applying sequence-based approaches for the specieslevel diagnosis of filamentous fungal keratitis cases
were published during the first years of the 21st century and reported the occurrence of Pythium insidiosum,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 identification 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 potential DNA barcodes for the kingdom Fungi revealed that
the ITS region has the highest probability of successful
identification for the broadest range of fungi147 including non-sporulating molds.113 However, there is a
problem with ITS-based strategies in relation with filamentous 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 keratitis 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
L. Kredics et al.
Table 2 Causal agents of filamentous fungal keratitis recognised in case reports by sequence-based identification.
Species
Lagenidium sp.
Pythium insidiosum
Pythium insidiosum
Auerswaldia lignicola
Taxonomic status
(division, order, family)
Cladosporium cladosporioides
Phomopsis phoenicicola
Aspergillus viridinutans
Aspergillus tamarii
Oomycota, Lagenidiales, Lagenidiaceae

Oomycota, Pythiales, Pythiaceae
Oomycota, Pythiales, Pythiaceae
Ascomycota, Botryosphaeriales,
Botryosphaeriaceae
Ascomycota, Botryosphaeriales,
Botryosphaeriaceae
Ascomycota, Capnodiales, Cladosporiaceae
Ascomycota, Diaporthales, Diaporthaceae
Ascomycota, Eurotiales, Aspergillaceae
Aspergillus pseudotamarii
Aspergillus nomius

Aspergillus tubingensis
Aspergillus brasiliensis
Neosartorya udagawae
Colletotrichum gloeosporioides
Colletotrichum truncatum
Plectosporium tabacinum

Ascomycota, Glomerellales, Glomerellaceae
Ascomycota, Glomerellales, Glomerellaceae
Ascomycota, Glomerellales,
Plectosphaerellaceae
Ascomycota, Hypocreales, Cordycipitaceae
Ascomycota, Hypocreales, Nectriaceae
Ascomycota, Hypocreales,
Ophiocordycipitaceae
Ascomycota, Microascales, Microascaceae
Ascomycota, Ophiostomatales,
Ophiostomataceae
Botryosphaeria rhodina
Beauveria bassiana
Fusarium equiseti
Fusarium polyphialidicum
Fusarium proliferatum
Fusarium verticillioides
Neocosmospora vasinfecta
Fusarium temperatum
Purpureocillium lilacinum
Scedosporium apiospermum
Sporothrix pallida
Corynespora cassiicola
Alternaria alternata
Alternaria infectoria
Edenia gomezpompae
Pyrenochaeta keratinophila1
Ulocladium sp.2
Chaetomium atrobrunneum
Chaetomium sp.
Thielavia subthermophila
Cladorrhinum bulbillosum
Pestalotiopsis clavispora
Phaeoisaria sp.3
Ascomycota, Pleosporales,
Corynesporascaceae
Ascomycota, Pleosporales, Pleosporaceae
Ascomycota, Sordariales, Chaetomiaceae
Ascomycota, Sordariales, Lasiosphaeriaceae
Ascomycota, Xilariales, Amphisphaeriaceae
Ascomycota, Xilariales, Diatrypaceae
Schizophyllum commune
Basidiomycota, Agaricales, Schizophyllaceae
Sequenced locus (GenBank ID)
Reference
Year
ITS (JX646749)
ITS (NA)
ITS (GU584093)
18S rDNA gene (KC866317.1)
109
110
111
112
2013
2001
2011
2013
ITS (EF446281)
113
2008
ITS (NA)
ITS (100% identity with FJ 889452)
b-tub (NA)
ITS (EF525554) b-tubulin (EF525555)
calmodulin (EF525556)
Calmodulin (KC202290)
ITS (GQ221261) b-tubulin (GQ221262)
calmodulin (GQ221263)
b-tubulin (EU600389 and EU600388)
b-tubulin (EU600386 and EU600387)
ITS (100% identity with AB250781)
ITS1 (NA)
ITS (100% identity with GU227878)
ITS (100% identity with AM408781)
114
115
116
117
2009
2011
2012
2007
118
119
2013
2009
120
121
122
123
124
125
2009
2010
2011
2009
2011
2012
ITS (HF675188), 28S rRNA (HF675189)

ITS (100% identity with GQ505694)
ITS (99% identity with X94172)
ITS (EF446290)
ITS (100% identity with AY533376)
ITS (EF373539)
tef1 (KF956084) b-tub (KF956080)
ITS (AB808481 and AB909369)
126
127
128
113
127
129
130
131
2014
2012
2003
2008
2012
2008
2014
2014

ITS (100% identity with EF127880)
LSU (100% identity with EF139121)
b-tub (100% identity with EF139110)
132
133
2005
2013
134
2013
ITS (NA)
ITS (AY168773)
ITS (KC193601)
ITS (EU885415)
ITS (AY943384)
ITS (HQ222986)
ITS (HQ906667)
ITS (99% identity with AJ271575)
ITS (98% identity with FM955448)
ITS (100% identity with EF119336)
28S rDNA (97% identity
with JQ429231)
18S rDNA (JQ695912)
135
136
137
138
139
140
141
142
143
144
145
2002
2003
2013
2010
2006
2012
2012
2009
2011
2013
2010
146
2013
NA, not available.

1
Described as new species.
Reported as Ulocladium atrum.
Reported as Carpoligna sp.
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,116121
whereas b-tub and tef1 were applied for the identification of the novel opportunist Fus. temperatum as keratitis 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, vitreous 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. incarnatum-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 outbreaks of 2005 and 2006.149152 The method identified 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 identification 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
20102011. 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. Representatives 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%). Species-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
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.117121 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 Fumigati,157 Asp. sydowii and Asp. variecolor from section
Nidulantes, as well as Asp. neoniger 157 and Asp. welwitschiae (syn. Asp. awamori) from section Nigri5,157
were also detected as rarely occurring keratitis pathogens. Among the newly identified agents of keratitis,
Asp. tamarii proved later to be more frequent than initially 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 keratitis,158,159 sequences of the ITS and intergenic spacer
(IGS) regions of the nuclear ribosomal RNA gene cluster, 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 isolates.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 performing 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 successfully detect various fungal pathogens (Can. parapsilosis,
Alt. alternata, Scedosporium apiospermum, Asp. niger and
L. Kredics et al.
Asp. fumigatus) in ocular samples. In a study performed 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. (including Cur. lunata), Cladosporium spp. (including Cla. oxysporum), Penicillium brocae and Cochliobolus sp. Embong
et al. [163] also applied the same method for the diagnosis 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 during screening diagnosis tests when an early mycotic
keratitis is suspected. In a study from 2012, Tananuvat 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. (including 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 basidiomycetes Cryptococcus pseudolongus, Exidiopsis calcea,
Phanerochaete sordida and Hyphodontia sp., however,
the respective sequences were not submitted to the
GenBank database. Can. albicans, Alternaria sp., Curvularia 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
Corynespora spp. including Cor. cassiicola (the latter

three for the first time) as keratitis pathogens after
sequencing of cloned ITS fragments that were amplified from corneal scrapings.
During the BLAST searches performed with
sequences of the ITS region, fungi with sequences
showing 99100% 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 deposited under an incorrect species name, therefore, the
sequence records, and if available, the related publications should be thoroughly reviewed before the acceptance of a diagnosis. Possible pitfalls of BLAST-based
identifications were demonstrated by Badenoch et al.
[166] in a letter reflecting to the article of Bagyalakshmi et al. [113]: the identifications reported for
eight fungal keratitis cases (Thielavia tortuosa, Botryosphaeria dothidea, Glo. cingulata, Asp. terreus, Bipolaris sp.,
Rhizoctonia bataticola, Macrophomina phaseolina, Pythium insidiosum) were found to be uncertain. It was emphasised 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 haematococca, 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 identification 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 Phaeoisaria sp. (Xilariales) with 97% sequence identity. In
another case, the unique ITS sequence of a keratitis
isolate supported the proposal of a new species: Pyrenochaeta 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 services, which is substantially increasing the time and
costs of routine diagnosis. An alternate solution is the
application of diagnostic PCR methods based on specific primers, which require less time and costs, just
the availability of PCR and gel electrophoresis equipments. 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 keratitis 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 diagnostic 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 amplicons deriving from different rDNA regions was also
applied for the diagnosis of fungal keratitis on the
basis of size and primary structural differences,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.
Regarding further gemomic regions applied for PCRbased 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 suggested 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 medically 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 filamentous fungi179 in many cases. Although NTM exhibits
relatively poor penetration through the corneal layers,
its bioavailability is stated to be sufficient for antifungal activity and for effective treatment of Fusarium keratitis 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 keratitis with NTM and AMB. A prospective study by Prajna 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 filamentous fungal species including fusaria. The authors also
stated that although NTM and AMB exhibited relatively poor penetration through the corneal layers, the
bioavailability of NTM was sufficient for antifungal
activity and it was effective for the treatment of Fusarium 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 recommended 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.
L. Kredics et al.
Because of relatively reduced systemic toxicity these

compounds can be used systemically for keratomycosis.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 perforation 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 bioavailability, 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 compound in two locations in Africa did not have encouraging 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 pyrimidines 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 germination 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 treatment 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 subjecting 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 susceptibility to increase the chances of an accurate therapy.
MIC data generated by facilities where the determination 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 summarises the data already available at national and global 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 Aspergillus isolates had slightly higher MIC values. In a subsequent study196 performed by the reference method
of the Clinical and Laboratory Standards Institute
(CLSI),197 NTM had good activities against both Fusarium 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 ml1 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 ml1. Xie et al. [200]
reported that 88.9% of both Asp. flavus and Asp. fumigatus were susceptible to NTM, whereas 94.2%, 92%
and 91.3% of FSSC members, Fus. moniliforme and
FOSC members, respectively, were susceptible to NTM
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 fungal keratitis.
In the case AMB, the other polyene compound frequently 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 sensitive than Aspergillus spp. ODay et al. [203] stated that
this drug was not effective against Fusarium spp. Similarly, Pujol et al. [204] reported MIC50 as
2.31 lg ml1 and MIC90 as 4.62 lg ml1 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 examined 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 ml1 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 ml1.
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 aspergilli 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 ml1. 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 Aspergillus spp. Lalitha et al. [195] reported MIC90 against
Aspergillus and Fusarium as 1 and 8 lg ml1 respectively. Similarly, Pfaller et al. [212] and Lass-Fl
orl
et al. [213] determined comparatively lower VRZ concentrations required to inhibit the growth of aspergilli.
Conversely, Alfonso [199] stated 16 lg ml1 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 activity against 96% of the Aspergillus strains with MICs of
3.12 lg ml1. 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 susceptibility 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 susceptibility 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 antifungal 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 ml1 as susceptible.
Pujol et al. [204] reported KTZ MIC90
51.20 lg ml1 for fusaria. Xie et al. [200] reported
the MIC90 of Fus. solani as 16 lg ml1 and that of
Asp. fumigatus as 2 lg ml1, and that KTZ is more
effective against Asp. flavus than against Asp.
fumigatus.
Studies in the literature comparing the susceptibilities 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 l1. The
numbers of isolates for which MICs of AMB were
2 mg l1 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 l1. 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 Fusarium 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 Fusarium keratitis isolates by the CLSI method. AMB, NTM
and TRB proved to be the most effective drugs. The combination of the latter two showed similar or even better 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 compounds 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. flavus, 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 completely 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 antifungal susceptibilities of Fusarium strains isolated at
the Aravind Eye Hospital, Coimbatore to conventional
antifungal drugs were compared between two different
periods: 20042005 and 20102011.155 Based on the
observations the in vitro susceptibilities to azoles, such
as CLZ, ECZ and ITZ have unambiguously decreased
from 20042005 to 20102011: most of the isolates
from the years 20102011 showed high MIC values
(64 lg ml1) to these drugs. The detected shift of the
obtained MIC value ranges towards higher values suggests 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 keratitis 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 filamentous 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 concentration of 4 lg ml1, whereas all other examined
fungi were inhibited at 2 lg ml1. 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 ml1, respectively,
with all Asp. flavus isolates being susceptible to
2 lg ml1 KTZ. Curvularia, Exserohilum and Alternaria
isolates could be characterised with KTZ MIC values
<8 lg ml1, whereas the sensitivity of the examined
Exophiala strain to this drug proved to be similar to
those recorded for fusaria (MIC = 16 lg ml1). For
ECZ and VRZ, 45% and 55% of the tested isolates
were inhibited by 1 lg ml1 and <2 lg ml1 concentrations of the drugs respectively. In the case of the
four examined Exserohilum isolates, the lowest MIC50
values were recorded for CLZ (0.12 lg ml1), ITZ,
ECZ and VRZ (each 0.06 lg ml1). CLZ proved especially efficient against Curvularia strains (MIC90
between 0.25 and 0.5 lg ml1). 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 filamentous 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
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 transfer by the initiation of collaborative projects between
eye clinics and research laboratories. Besides the data
available for polyenes and azoles, testing the applicability of additional antifungals (e.g. terbinafine and caspofungin), 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.
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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 Project/2007 & 2010). Csaba V
agv
olgyi thanks the visiting professor program, Deanship of Scientific Research
at King Saud University, Riyadh.
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Conflict of Interests
23
The authors declare that they have no conflict of

interest.
24
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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 Microbiology, Faculty of Science and Informatics, University
of Szeged, Szeged, Hungary; Raghavan Anita, Rajaraman Revathi, Perumal Gomathi: Aravind Eye
Hospital and Postgraduate Institute of Ophthalmology,
Coimbatore, Tamilnadu, India; Anamangadan Shafeeq Hassan, Yendrembam Randhir Babu Singh,
Arumugam Mythili: Department of Microbiology, Dr.
G.R. Damodaran College of Science, Coimbatore, India;
Kanesan Panneer Selvam: Department of Microbiology, MR Government Arts College, Mannargudi, India;
Ilona D
oczi: Department of Clinical Microbiology,
Faculty of Medicine, University of Szeged, Szeged, Hungary; Bal
azs Leitgeb: Institute of Biophysics, Biological Research Centre of the Hungarian Academy of
Sciences, Szeged, Hungary.

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mycoses

Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Filamentous fungal infections of the cornea: a global overview of

Fungal keratitis is a serious suppurative, usually ulcerative corneal infection which

Indo-Hungarian Fungal Keratitis (IHFK) Working Group authors are listed

2015 Blackwell Verlag GmbH

usually ulcerative corneal infection which may result

and construction have been frequently reported to be

Studies on the epidemiology of filamentous

followed by the USA,7,8,17,24,

epidemiological studies from European countries, which

2015 Blackwell Verlag GmbH

Epidemiology of filamentous fungal keratitis

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

June 1999May 2001

September 1999March 2001

April 1997August 1998

January 2000December 2004

2015 Blackwell Verlag GmbH

India, New Delhi

July 2006May 2008

January 2007July 2008

2015 Blackwell Verlag GmbH

Epidemiology of filamentous fungal keratitis

July 2009June 2010

number of fungal isolates have not missed these two

Importance of molecular identification

2015 Blackwell Verlag GmbH

Microascales, Pleosporales, Sordariales and Xilariales

Oomycota, Lagenidiales, Lagenidiaceae

Ascomycota, Eurotiales, Aspergillaceae

Ascomycota, Eurotiales, Aspergillaceae

Basidiomycota, Agaricales, Schizophyllaceae

Sequenced locus (GenBank ID)

ITS (HF675188), 28S rRNA (HF675189)

ITS (99% identity with AY213680)

NA, not available.

Described as new species.

Reported as Ulocladium atrum.

Reported as Carpoligna sp.

2015 Blackwell Verlag GmbH

Epidemiology of filamentous fungal keratitis

largest subunit (rpb2) or translation elongation factor

2015 Blackwell Verlag GmbH

keratitis.156 In another study from the same hospital

Corynespora spp. including Cor. cassiicola (the latter

2015 Blackwell Verlag GmbH

Epidemiology of filamentous fungal keratitis

corresponding sequences of Carpoligna pleurothecii

2015 Blackwell Verlag GmbH

Antifungals for the therapy of mycotic

Because of relatively reduced systemic toxicity these

Significance of susceptibility testing in the

2015 Blackwell Verlag GmbH

Epidemiology of filamentous fungal keratitis

by the CLSI method. A study by Pradhan et al. [201]

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A study from China200 applying the CLSI method

As it is frequently reported that the resistance to

2015 Blackwell Verlag GmbH

Epidemiology of filamentous fungal keratitis

The authors declare that they have no conflict of

Thomas PA. Current perspectives on ophthalmic mycoses. Clin

2015 Blackwell Verlag GmbH