Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 605308 10 pageshttpdxdoiorg1011552013605308

Clinical StudyMicroscopic Evaluation Molecular Identification AntifungalSusceptibility and Clinical Outcomes in Fusarium Aspergillusand Dematiaceous Keratitis

Devarshi U Gajjar12 Anuradha K Pal2 Bharat K Ghodadra2 and Abhay R Vasavada2

1 Department of Microbiology and Biotechnology Centre Faculty of Science M S University of Baroda Vadodara 390 002 India2 Iladevi Cataract and IOL Research Centre Ahmedabad Gujarat 380052 India

Correspondence should be addressed to Devarshi U Gajjar devarshimistryyahoocom

Received 30 April 2013 Revised 22 July 2013 Accepted 16 August 2013

Academic Editor Kelvin To

Copyright copy 2013 Devarshi U Gajjar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Purpose Fusarium Aspergillus and Dematiaceous are the most common fungal species causing keratitis in tropical countriesHerein we report a prospective study on fungal keratitis caused by these three fungal species Methodology A prospectiveinvestigationwas undertaken to evaluate eyes with presumed fungal keratitis All the fungal isolates (119899 = 73) obtained from keratitisinfections were identified using morphological and microscopic characters Molecular identification using sequencing of the ITSregion and antifungal susceptibility tests using microdilution method were doneThe final clinical outcome was evaluated in termsof the time taken for resolution of keratitis and the final visual outcome The results were analyzed after segregating the cases intothree groups namely Fusarium Aspergillus and Dematiaceous keratitis Results Diagnosis of fungal keratitis was established in 73(359) cases out of 208 casesThe spectra of fungi isolated were Fusarium spp (266)Aspergillus spp (216) andDematiaceousfungi (116) The sequence of the ITS region could identify the Fusarium and Aspergillus species at the species complex leveland the Dematiaceous isolates were accurately identified Using antifungal agents such as fluconazole natamycin amphotericinB and itraconazole the minimum inhibitory concentrations (MICs) for Fusarium spp were gt32 120583gmL 4ndash8 120583gmL 05ndash1 120583gmLand gt32 120583gmL respectively Antifungal susceptibility data showed that Curvularia spp was highly resistant to all the antifungalagents Overall natamycin and amphotericin B were found to be the most effective antifungal agents The comparative clinicaloutcomes in all cases showed that the healing response in terms of visual acuity of the Dematiaceous group was significantly goodwhen compared with the Fusarium and Aspergillus groups (119875 lt 005) The time required for healing in the Fusarium group wasstatistically significantly less when compared with the Aspergillus and Dematiaceous groups Conclusion This study demonstratesimportant differences in microscopic features of scraping material and antifungal susceptibility between the three groups Earlyand accurate identification coupled with the MIC data and thereby appropriate treatment is crucial for complete recovery

1 Introduction

Mycotic keratitis is an important ophthalmic problem caus-ing visual disability due to its protracted course and unfa-vorable responses The incidence of fungal keratitis has beenreported to range between 256 and 367 in various partsof India [1ndash4] It is evident that Aspergillus and Fusariumare the most common species causing keratitis in tropicalcountries including India whereas pigmented Dematiaceousfungi are the third most common cause of mycotic keratitis[1 5ndash7] Studies on their molecular identification antifungal

susceptibility and comparisons with the clinical outcomeswould be of great importance as the pathogenic potentialmay vary between these genera The most widely sequencedDNA region in fungi is the ITS region and the InternationalSub-Commission of fungal bar coding has proposed the ITSregion as the prime fungal bar code for species identification[8] Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]Fungal ulcers are commonly treated empirically drugs aretypically selected without regard to susceptibility data Thisis because the antifungal susceptibility testing takes time and

2 BioMed Research International

needs trained personnel to perform the testing There arefour studies that report the in vitro antifungal susceptibilitypatterns among keratitis-causing fungal isolates from India[13ndash16] On the other hand nonophthalmic Fusaria havebeen reported to exhibit greater resistance to antifungalagents continuously over a period of time [17 18] Henceperiodic reports from different geographical areas wouldhelp record the variations over a period of time and at thesame time help in modulating the current treatment optionsWe report a prospective study to compare different aspectsof fungal keratitis such as its clinical features microbialevaluationmolecular identification antifungal susceptibilityand clinical outcomes

2 Materials and Methods

Patients were recruited after an informed consent wasobtained from all subjectsThe study followed the declarationof tenets of Helsinki and was approved by the InstitutionalEthical Review Board

21 Clinical Examination A prospective study of patientswith keratitis was conducted during the period from June2009 toMay 2012 All the patients were examinedwith a stan-dard written protocol that included a detailed history withregard to the duration of symptoms predisposing factors theexact nature of trauma immediate treatment administeredcontact lens usage previous history of ocular surgerieshistory of diabetes and usage of topical or systemic steroidsThe same consultant doctor as per the standard protocolapproved by the institutional review board performed athorough examination of the involved and fellow eyes Thesame consultant ophthalmologist filled a form In this formthe following aspects were documented the presence orabsence and form of the following clinical features elevationof slough (raised or flat) texture of slough (wet or dry)ulcer margins (serrated or well defined) size of the abscesspigmentation Descemetrsquos folds satellite lesions dendriticlesions immune ring hypopyon fibrin flare or cells inthe anterior chamber deep lesions and endothelial plaqueClinical photographs were taken using the Haag Straight slitlamp microscope with a photo slit attachment

22 Clinical Specimens and Microbiological InvestigationsCorneal scrapings were taken from patients when at leastone of the following was present size of the infiltration wasgt2mm with an epithelial defect depth of the infiltrate wasgt20 of the corneal thickness the anterior chamber reactionwas gt grade 2 evidence of any organic trauma or failure toregress in 24 hours Local anesthetic eye drops (proparacaine05) were instilled to the affected eye to minimize oculardiscomfort and facilitate the corneal scraping procedureScrapings were obtained aseptically from the base and edgesof each ulcer using a disposable blade A part of the scrapingmaterial was examined for the presence of fungi bacteriaor acanthamoeba by using 10 KOHndash005 calcofluor white

stain wet mounts and Gram staining [19]The scraping mate-rial was also directly inoculated in blood agar Sabouraudrsquosdextrose agar (SDA) and chocolate agar media (HimediaHimedia Pvt Ltd Mumbai) which were incubated at 37∘Cand 28∘C and in 5 CO

2 respectively A diagnosis of fungal

keratitis was made when at least one of the following wasconfirmed a corneal scraping examination revealed fungalhyphae in wet mounts or smears the same fungus grew inthe two culture media used or the fungus grew confluentlyat all the inoculated sites on a single media Microscopicpictures of the KOH wet mounts for all samples were takenunder the lightmicroscope and fluorescencemicroscopeTheaverage width of 25 randomly selected hyphae the distancebetween the septa and the diameter of the chlamydospore-like structures whenever present were measured from digi-talized photographs at 400xmagnification using Biovis ImagePlus Software v411 (Expert Vision Mumbai India) Picturesof media plates showing fungal growth were taken at 24-hour growth 48-hour growth and 72-hour growth Colourdiameter and presence or absence of spores were observedfor all fungal colonies on SDA and blood agar plates

23 Morphological and Molecular Identification Pure cul-tures of all isolates were maintained on Potato Dextroseagar (PDA) Cultures were examined using the lactophenolblue mount for sporulation at the end of 10 20 and 30days The morphological and microscopic identification wasdone by growth characteristics and microscopic charac-teristics respectively All morphological and microscopiccharacteristics were confirmed by comparing them with thecharacters given in the ldquoAtlas of clinical fungirdquo [20] Culturesthat failed to sporulate on SDA and PDA were subculturedon oatmeal agar and carnation leaf agar For molecularidentification of the fungus sequencing of the ITS (internaltranscribed spacer) region was doneTheDNAwas extractedfrom the pure culture of the fungus grown on SDA usingZymo Research DNA isolation kit After extraction the DNAwas amplified using ITS 1 (F-51015840-TCCGTAGGTGAACC-31015840)and ITS 4 primers (R-51015840TCCTCCGCTTATTGATATGA-31015840)which amplify the following genes of the fungal genomepartial 18S rRNA gene complete ITS1 58S rRNA geneand ITS2 regions and partial 28S rRNA gene Annealingtemperature was 55∘C for 1 minute The size of ampliconproduced after PCR reaction was around 500ndash600 basepairs for all fungi used in the present study Sequencingof the ITS region was done at First Base Laboratories SdnBhd Malaysia using primers ITS1 and ITS4 Sequenceswere obtained using both forward and reverse primersChromatogram processing quality control and editing ofthe sequences were done using BioEdit Software Bothsequences were aligned and a final sequence was createdThis final sequence was used for the BLASTN similaritysearch (httpwwwncbinlmnihgovBLAST) and was alsosubmitted to NCBI For identification only complete ITS1-58S-ITS2 entries of reference isolates in the BLAST databasewere taken into consideration Complete identification wasconsidered when a percent sequence similarity of gt98 witha BLAST search expected value of zero was obtained

BioMed Research International 3

24 Antifungal Susceptibility Testing In vitro antifungal sus-ceptibility testing was done against natamycin (Natamet5 suspension Sun Pharmaceuticals Ind Ltd Halol India)itraconazole (Itral 1 suspension Jawa PharmaceuticalsGurgaon India) fluconazole (Nufl ucon 03 suspensionNuLife Pharmaceuticals Pune India) and amphotericin B(RM 462 Himedia Labs Ltd Mumbai India) using themicrodilutionmethod and following theClinical andLabora-tory Standards Institute (CLSI) guidelines [21] All antifungalagents were dissolved in DMSO and fluconazole was dis-solved inwaterThe inoculumswere prepared by covering the7-day-old culture plate with normal saline (085NaCl)Thiswas followed by gentle probing of the colonies with the help ofa pipette and adjusting the densities of the suspension (read at530 nm) to a final inoculum of 05 McFarland standard Thefinal drug concentration range prepared using serial dilutionwere 0008 to 132 120583gmL for all the four antifungal agents Allthe antifungal agents were tested in RPMI 1640 media with2 glucose and without sodium carbonate

25 Treatment Regime and Evaluation of Clinical OutcomesSubsequent to the microscopic examination if a positivereport of fungal filaments was received antifungal topicaltherapy with 5 natamycin was started for all cases immedi-ately One-hourly topical eye drops were applied around theclock for the first three days followed by two-hourly dropsduring waking hours until resolution of the ulcers Patientsalso received 1 atropine sulphate eye drops Systemic flu-conazole (150mg once a day) was prescribed for all patientswith corneal stromal infiltrate extending beyond one-thirdof the cornea After treatment an ulcer was considered tobe healed when the epithelial defect was lt1mm in diameterwith a visible scar under slit lamp biomicroscopy A healingtime of less than 3 weeks from presentation was considereda good result and healing time of more than three weeks wasconsidered a poor responseThe responses to treatment werecategorized into three groups as follows perception of light tothe Snellenrsquos chart (good response) no change in visual acuityafter treatment (poor response) and slight change in thevisual acuity (slight response) Initial and final visual acuitywas comparedfollowingtreatment using statistical analysisThe time for complete healing was also compared among theisolates The follow-up best-corrected visual acuity (BCVA)of patients treated with topical and oral antimicrobial agentswas the visual acuity measured when the patient was cured(inactive corneal scar with intact epithelium) Visual acuitiesobtained using Snellenrsquos chart were converted into logarithmsof the minimum angle of resolution (logMAR) for dataanalysis

26 Statistical Analysis The test of proportion was used toevaluate the epidemiological features and risk factors In vitrosusceptibility results obtained from the three groups werestatistically analyzed using the Kruskal-Wallis test A post hocpair wise comparison was also done The Wilcoxon SignedRanks Test was used to compare visual acuity before and aftertreatment in each group

3 Results

31 Epidemiological Characteristics and Clinical Features Atotal of 208 patients with keratitis were recruited during theperiod fromMay 2009 to June 2012 In all 73 patients who hadculture-proven mycotic keratitis the incidence of culture-proven mycotic keratitis was 350 Out of these 73 patients26 (356) were infected with Fusarium spp 15 (205)were infected with Aspergillus species and 11 (150) wereinfected with Dematiaceous fungi The average age of thepatients was 4184 years in the Fusarium group 5246 yearsin theAspergillus group and 4653 years in the Dematiaceousfungi group There were more males than females in all threegroupsThe seasonal distribution showed that infection of allthe three types of keratitis was highest in winter and attainedstatistical significance (Table 1) The risk factors such astrauma to the eyeocular surgery were predominantly seen inthe Aspergillus keratitis group whereas the incidence of entryof vegetative foreign bodies was mainly seen in the Fusariumgroup (Table 1) Table 1 shows the comparative evaluationof the clinical features in all the three groups The area ofinfiltration was large (gt4mm) in the central visual axis inall the three groups Further the presence of satellite lesionsring infiltrate dry appearance and stromal involvement wasevident in most of the cases The presence of hypopyon andpigmentation was mainly associated with the Dematiaceousgroup and this attained statistical significance The presenceof endothelial plaque was mainly associated with Fusariuminfections whereas dendritic lesions and Descemetrsquos foldswere mainly observed in the Aspergillus group

32 Microscopic Evaluation and Growth Characteristics of theScrapingMaterial All the samples of scrapingmaterial takenfrom the three groups showed the presence of large quantitiesof fungal filaments when seen under light and fluorescencemicroscopes (Figure 1) Samples from Fusarium infections(Figures 1(a) and 1(b)) showed the presence of fungal hyphaewith an average thickness 387 plusmn 06 120583m Septa were notvisible under the light microscope (Figure 1(a)) but wereclearly seen under the fluorescence microscope (Figure 1(b))The distance between the septa was 2165 plusmn 42 120583m Theaverage hyphal thickness in the Aspergillus group (Figures1(c) and 1(d)) was 413plusmn 065 120583m and this was almost similarto the hyphal thickness in the Fusarium group Howeverthe distance between the septa was 1284 plusmn 19 120583m Theaverage hyphal thickness in the Dematiaceous group was879 plusmn 09 120583m (Figures 1(e) and 1(f)) Terminal and internalchlamydospore-like structures with a diameter of 944 plusmn111 120583m were seen exclusively in all four samples from theFusarium delphinoides group (Figures 2(a) and 2(b)) Thescraping material from Curvularia infections also showedlarge quantities of chlamydospore-like structures with anaverage diameter of 1115 plusmn 158 120583m (Figures 2(c) and 2(d))These structures were absent in all the remaining samples ofthe Dematiaceous group A huge variation in microscopicfeatures was noticed in the Dematiaceous group (Figures3(a)ndash3(d)) Scraping material other than the Curvulariainfection showed a typical arrangement of septa

4 BioMed Research International

Table 1 Epidemiologic characteristics risk factors and clinical features

Variable Fusarium spp (119899 = 26) Aspergillus spp (119899 = 15) Dematiaceous isolates (119899 = 12)Age (in years) 4184 5246 465Gender ratio (male female) 271 2 14Risk factors

Trauma other than vegetative body 1 (38) 4 (266) 0Trauma with vegetative body 11 (423)lowast 0 4 (333)Prior ocular surgeryinfection 1 (38) 5 (333) 1 (83)

Seasonal distributionSummer 6 (23) 3 (20) 1 (83)Monson 4 (153) 7 (466) 3 (25)Winter 14 (538) 7 (466) 6 (50)

Clinical featuresCentral location 23 (884) 12 (80) 11 (916)Size (gt10mm) 16 (615) 9 (60) 11 (916)Hypopyon 10 (384) 5 (333) 9 (75)lowast

Elevated slough 19 (730) 6 (40) 10 (833)Dry texture 20 (769) 7 (466) 9 (75)Serrated ulcer margins 21 (807) 9 (60) 8 (666)Pigmentation 0 1 (66) 5 (416)Endothelial plaque 4 (153) 0 0Dendritic lesions 0 2 (133) 0Satellite lesions 15 (576) 8 (533) 3 (25)Mean number of days for complete healing 574lowast 1148 1256

lowastGroup significantly higher compared to other

A total of 47 (8867) out of 53 samples showed visiblegrowth on all the media inoculated at 24 hours (Figure 4(a))In the Fusarium group 23 (884) samples showed growthwithin 24 hours while 3 samples showed growth within 48hours In the Aspergillus group 13 (8666) samples showedgrowth within 24 hours and the remaining 2 samples showedgrowth within 48 and 72 hours respectively The growth ofboth Fusarium and Aspergillus samples on SDA at 48 hourswas similar with respect to the growth rate (Figures 4(b)and 4(c)) In the Dematiaceous group 1112 (916) samplesshowed growth on SDA within 24 hours All the samples ofthe Dematiaceous group showed the presence of a peculiarcolor for example pink or light brown in case of Curvulariaspp (Figure 4(d)) yellow for Papulaspora spp (Figure 4(e))and dark brown for Exserohilum spp (Figure 4(f)) A sampleof Lasiodiplodia theobromae obtained from the scrapingmaterial was grown for 48 hours and a gray fluffy growthwithabundant aerial mycelia was visible as seen in Figure 4(g) Itwas further observed that SDA did not support sporulationof Curvularia spp and Lasiodiplodia spp in all the samples

33 Microscopic and Molecular Identification of FusariumAspergillus and Dematiaceous Spp All isolates in the Fusar-ium and Aspergillus groups were identified to the genuslevel by means of their morphological characteristics Themorphological evaluation of Fusarium solani appeared to bestraight forward and this was further confirmed using theITS sequences However when the sequences were evaluated

using the Fusarium MLST website the match was to theFusarium solani species complex and not to Fusarium solaniper se Hence all Fusarium isolates were named as membersof the Fusarium solani species complex All other isolatesof Fusarium (119899 = 4) were identified as Fusarium dimerumusing their morphological features However identificationusing the ITS sequences at NCBI BLAST was F delphinoidesisolates (119899 = 4) Fusarium dimerum (119899 = 1) and Fusariumdelphinoides (119899 = 3) using the MLST database In theAspergillus group A niger A flavus A terreus and Afumigatus were identified using morphological features Atamarii A tubingensis A versicolor and A sydowii wereonly identified when the ITS sequences were available Inthe Dematiaceous group Curvularia lunata and Exserohilumrostratum were identified using their growth characteristicsand typical spores and this was confirmed using the ITSsequence Using ITS sequences all the other dematiaceousisolates were identified as Lasiodiplodia theobromae Clador-rhinum bulbilosum and Cladosporium cladosporioides TheITS sequence misidentified only one isolate as Chaetomiumspp It was later identified as Papulaspora spp on the basis ofits typical microscopic features

34 In Vitro Antifungal Susceptibility Table 2 shows the re-sults of antifungal susceptibility testing of all isolates Anti-fungal results showed that amphotericin B and natamycinare the most effective antifungal agents against Fusariumspp In the Aspergillus group amphotericin B and itracona-zole showed the lowest MIC against A flavus A terreus

BioMed Research International 5

(a) (b)

(c) (d)

(e) (f)

Figure 1 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium infections (c and d)Scrapping material from Aspergillus infections (e and f) Scrapping material from Exserohilum infections

A tamarii and A tubingensis whereas natamycin andamphotericin B showed good in vitro activity against Aniger and A sydowii In the Dematiaceous group exceptCurvularia all the other isolates were highly susceptible tothe antifungal agents tested Amphotericin B and natamycinshowed good in vitro activity against Curvularia lunata

35 Evaluation of Clinical Outcomes Table 3 shows the com-parative clinical outcomes in all cases In the Fusarium group20 cases healed and 3 worsened while 3 were lost to follow-up The minimum time required for healing was 14 dayswhereas the maximum time taken to heal in the case of

one patient was 300 days In the Aspergillus group 12 outof 15 cases healed with topical and oral antifungal medicaltreatment and two cases required therapeutic keratoplastywhereas one case caused by A tamarii worsened Theminimum time required for healing was 30 days and themaximum time required was 330 days In the Dematiaceousgroup 11 cases healed and one was lost to follow-up Theminimum time taken to heal was 7 days while the maximumtime was 300 days The healing response in terms of visualacuity of the Dematiaceous group was significantly goodwhen compared with the Fusarium and Aspergillus groups(119875 lt 005) The time required for healing in the Fusarium

6 BioMed Research International

(a) (b)

(c) (d)

Figure 2 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium delphinoides infections(c and d) Scrapping material from Curvularia infections

group was statistically significantly less when compared withthe Aspergillus and Dematiaceous groups

4 Discussion

In the present study of 208 keratitis patients fungal etiologywas confirmed in 35 of the cases where Fusarium sppwas the most common isolate followed by Aspergillus andDematiaceousThis is comparable to most studies from India[1ndash5] In India Aspergillus is mainly reported as the mostcommon isolate in the Northern region [1 2 22 23] whileFusarium is mainly reported in the Southern region [4 24]and Dematiaceous fungi are reported to be the third mostcommon fungi We found two reports from AhmedabadFusarium was reported in a study conducted in 2003ndash2005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26] We have obtained adefinite history of trauma with vegetativeagricultural bodieslargely in patients with Fusarium keratitis whereas traumadue to a factor other than vegetative material or any otherocular surgery was found to be largely associated with theAspergillus group Among the traumatic agents plants andagricultural material like hay have contributed to 76 785and 612 of cases of keratitis in studies from Assam [27]Gujarat [25] and Tamil Nadu [28] Fungal keratitis is more

frequently reported in winter with a humid climate favoringfungal growth [29 30]

The generally accepted clinical features for the diagnosisof mycotic keratitis are the presence of a dry raised ulcer witha feathery or hyphate border satellite lesions and recurrenthypopyon [31] Our results are similar to reports from Delhi[2] and Madurai [30 32] The pigmented plaque like thepresentation seen in 42 of our cases in the Dematiaceousgroup is similar to the series reported by Garg and associatesin 2000 and 2004 respectively [33 34]

Directmicroscopy is an important diagnosticmodality ininvestigating microbial keratitis and a highest sensitivity at99 is reported [35]The addition of calcofluor white (CFW)stain to the diagnostic armamentarium has significantlyincreased the sensitivity of smear examination on directmicroscopy [19] However it is difficult to determine thegenus of fungi from KOH mounts [35] Preliminary identifi-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36]Identification of Fusarium from scraping material is reportedby the detection of adventitious sporulation [37] The pres-ence of a brown colored fungal hyphae in the scraping mate-rial raised the possibility of the presence of a Dematiaceousmold [33] Morphologically we did not find any remarkabledifferences between the Fusarium and Aspergillus groupsexcept that the distance between the septa was larger in

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 BioMed Research International

needs trained personnel to perform the testing There arefour studies that report the in vitro antifungal susceptibilitypatterns among keratitis-causing fungal isolates from India[13ndash16] On the other hand nonophthalmic Fusaria havebeen reported to exhibit greater resistance to antifungalagents continuously over a period of time [17 18] Henceperiodic reports from different geographical areas wouldhelp record the variations over a period of time and at thesame time help in modulating the current treatment optionsWe report a prospective study to compare different aspectsof fungal keratitis such as its clinical features microbialevaluationmolecular identification antifungal susceptibilityand clinical outcomes

2 Materials and Methods

Patients were recruited after an informed consent wasobtained from all subjectsThe study followed the declarationof tenets of Helsinki and was approved by the InstitutionalEthical Review Board

21 Clinical Examination A prospective study of patientswith keratitis was conducted during the period from June2009 toMay 2012 All the patients were examinedwith a stan-dard written protocol that included a detailed history withregard to the duration of symptoms predisposing factors theexact nature of trauma immediate treatment administeredcontact lens usage previous history of ocular surgerieshistory of diabetes and usage of topical or systemic steroidsThe same consultant doctor as per the standard protocolapproved by the institutional review board performed athorough examination of the involved and fellow eyes Thesame consultant ophthalmologist filled a form In this formthe following aspects were documented the presence orabsence and form of the following clinical features elevationof slough (raised or flat) texture of slough (wet or dry)ulcer margins (serrated or well defined) size of the abscesspigmentation Descemetrsquos folds satellite lesions dendriticlesions immune ring hypopyon fibrin flare or cells inthe anterior chamber deep lesions and endothelial plaqueClinical photographs were taken using the Haag Straight slitlamp microscope with a photo slit attachment

22 Clinical Specimens and Microbiological InvestigationsCorneal scrapings were taken from patients when at leastone of the following was present size of the infiltration wasgt2mm with an epithelial defect depth of the infiltrate wasgt20 of the corneal thickness the anterior chamber reactionwas gt grade 2 evidence of any organic trauma or failure toregress in 24 hours Local anesthetic eye drops (proparacaine05) were instilled to the affected eye to minimize oculardiscomfort and facilitate the corneal scraping procedureScrapings were obtained aseptically from the base and edgesof each ulcer using a disposable blade A part of the scrapingmaterial was examined for the presence of fungi bacteriaor acanthamoeba by using 10 KOHndash005 calcofluor white

stain wet mounts and Gram staining [19]The scraping mate-rial was also directly inoculated in blood agar Sabouraudrsquosdextrose agar (SDA) and chocolate agar media (HimediaHimedia Pvt Ltd Mumbai) which were incubated at 37∘Cand 28∘C and in 5 CO

2 respectively A diagnosis of fungal

keratitis was made when at least one of the following wasconfirmed a corneal scraping examination revealed fungalhyphae in wet mounts or smears the same fungus grew inthe two culture media used or the fungus grew confluentlyat all the inoculated sites on a single media Microscopicpictures of the KOH wet mounts for all samples were takenunder the lightmicroscope and fluorescencemicroscopeTheaverage width of 25 randomly selected hyphae the distancebetween the septa and the diameter of the chlamydospore-like structures whenever present were measured from digi-talized photographs at 400xmagnification using Biovis ImagePlus Software v411 (Expert Vision Mumbai India) Picturesof media plates showing fungal growth were taken at 24-hour growth 48-hour growth and 72-hour growth Colourdiameter and presence or absence of spores were observedfor all fungal colonies on SDA and blood agar plates

23 Morphological and Molecular Identification Pure cul-tures of all isolates were maintained on Potato Dextroseagar (PDA) Cultures were examined using the lactophenolblue mount for sporulation at the end of 10 20 and 30days The morphological and microscopic identification wasdone by growth characteristics and microscopic charac-teristics respectively All morphological and microscopiccharacteristics were confirmed by comparing them with thecharacters given in the ldquoAtlas of clinical fungirdquo [20] Culturesthat failed to sporulate on SDA and PDA were subculturedon oatmeal agar and carnation leaf agar For molecularidentification of the fungus sequencing of the ITS (internaltranscribed spacer) region was doneTheDNAwas extractedfrom the pure culture of the fungus grown on SDA usingZymo Research DNA isolation kit After extraction the DNAwas amplified using ITS 1 (F-51015840-TCCGTAGGTGAACC-31015840)and ITS 4 primers (R-51015840TCCTCCGCTTATTGATATGA-31015840)which amplify the following genes of the fungal genomepartial 18S rRNA gene complete ITS1 58S rRNA geneand ITS2 regions and partial 28S rRNA gene Annealingtemperature was 55∘C for 1 minute The size of ampliconproduced after PCR reaction was around 500ndash600 basepairs for all fungi used in the present study Sequencingof the ITS region was done at First Base Laboratories SdnBhd Malaysia using primers ITS1 and ITS4 Sequenceswere obtained using both forward and reverse primersChromatogram processing quality control and editing ofthe sequences were done using BioEdit Software Bothsequences were aligned and a final sequence was createdThis final sequence was used for the BLASTN similaritysearch (httpwwwncbinlmnihgovBLAST) and was alsosubmitted to NCBI For identification only complete ITS1-58S-ITS2 entries of reference isolates in the BLAST databasewere taken into consideration Complete identification wasconsidered when a percent sequence similarity of gt98 witha BLAST search expected value of zero was obtained

BioMed Research International 3

24 Antifungal Susceptibility Testing In vitro antifungal sus-ceptibility testing was done against natamycin (Natamet5 suspension Sun Pharmaceuticals Ind Ltd Halol India)itraconazole (Itral 1 suspension Jawa PharmaceuticalsGurgaon India) fluconazole (Nufl ucon 03 suspensionNuLife Pharmaceuticals Pune India) and amphotericin B(RM 462 Himedia Labs Ltd Mumbai India) using themicrodilutionmethod and following theClinical andLabora-tory Standards Institute (CLSI) guidelines [21] All antifungalagents were dissolved in DMSO and fluconazole was dis-solved inwaterThe inoculumswere prepared by covering the7-day-old culture plate with normal saline (085NaCl)Thiswas followed by gentle probing of the colonies with the help ofa pipette and adjusting the densities of the suspension (read at530 nm) to a final inoculum of 05 McFarland standard Thefinal drug concentration range prepared using serial dilutionwere 0008 to 132 120583gmL for all the four antifungal agents Allthe antifungal agents were tested in RPMI 1640 media with2 glucose and without sodium carbonate

25 Treatment Regime and Evaluation of Clinical OutcomesSubsequent to the microscopic examination if a positivereport of fungal filaments was received antifungal topicaltherapy with 5 natamycin was started for all cases immedi-ately One-hourly topical eye drops were applied around theclock for the first three days followed by two-hourly dropsduring waking hours until resolution of the ulcers Patientsalso received 1 atropine sulphate eye drops Systemic flu-conazole (150mg once a day) was prescribed for all patientswith corneal stromal infiltrate extending beyond one-thirdof the cornea After treatment an ulcer was considered tobe healed when the epithelial defect was lt1mm in diameterwith a visible scar under slit lamp biomicroscopy A healingtime of less than 3 weeks from presentation was considereda good result and healing time of more than three weeks wasconsidered a poor responseThe responses to treatment werecategorized into three groups as follows perception of light tothe Snellenrsquos chart (good response) no change in visual acuityafter treatment (poor response) and slight change in thevisual acuity (slight response) Initial and final visual acuitywas comparedfollowingtreatment using statistical analysisThe time for complete healing was also compared among theisolates The follow-up best-corrected visual acuity (BCVA)of patients treated with topical and oral antimicrobial agentswas the visual acuity measured when the patient was cured(inactive corneal scar with intact epithelium) Visual acuitiesobtained using Snellenrsquos chart were converted into logarithmsof the minimum angle of resolution (logMAR) for dataanalysis

26 Statistical Analysis The test of proportion was used toevaluate the epidemiological features and risk factors In vitrosusceptibility results obtained from the three groups werestatistically analyzed using the Kruskal-Wallis test A post hocpair wise comparison was also done The Wilcoxon SignedRanks Test was used to compare visual acuity before and aftertreatment in each group

3 Results

31 Epidemiological Characteristics and Clinical Features Atotal of 208 patients with keratitis were recruited during theperiod fromMay 2009 to June 2012 In all 73 patients who hadculture-proven mycotic keratitis the incidence of culture-proven mycotic keratitis was 350 Out of these 73 patients26 (356) were infected with Fusarium spp 15 (205)were infected with Aspergillus species and 11 (150) wereinfected with Dematiaceous fungi The average age of thepatients was 4184 years in the Fusarium group 5246 yearsin theAspergillus group and 4653 years in the Dematiaceousfungi group There were more males than females in all threegroupsThe seasonal distribution showed that infection of allthe three types of keratitis was highest in winter and attainedstatistical significance (Table 1) The risk factors such astrauma to the eyeocular surgery were predominantly seen inthe Aspergillus keratitis group whereas the incidence of entryof vegetative foreign bodies was mainly seen in the Fusariumgroup (Table 1) Table 1 shows the comparative evaluationof the clinical features in all the three groups The area ofinfiltration was large (gt4mm) in the central visual axis inall the three groups Further the presence of satellite lesionsring infiltrate dry appearance and stromal involvement wasevident in most of the cases The presence of hypopyon andpigmentation was mainly associated with the Dematiaceousgroup and this attained statistical significance The presenceof endothelial plaque was mainly associated with Fusariuminfections whereas dendritic lesions and Descemetrsquos foldswere mainly observed in the Aspergillus group

32 Microscopic Evaluation and Growth Characteristics of theScrapingMaterial All the samples of scrapingmaterial takenfrom the three groups showed the presence of large quantitiesof fungal filaments when seen under light and fluorescencemicroscopes (Figure 1) Samples from Fusarium infections(Figures 1(a) and 1(b)) showed the presence of fungal hyphaewith an average thickness 387 plusmn 06 120583m Septa were notvisible under the light microscope (Figure 1(a)) but wereclearly seen under the fluorescence microscope (Figure 1(b))The distance between the septa was 2165 plusmn 42 120583m Theaverage hyphal thickness in the Aspergillus group (Figures1(c) and 1(d)) was 413plusmn 065 120583m and this was almost similarto the hyphal thickness in the Fusarium group Howeverthe distance between the septa was 1284 plusmn 19 120583m Theaverage hyphal thickness in the Dematiaceous group was879 plusmn 09 120583m (Figures 1(e) and 1(f)) Terminal and internalchlamydospore-like structures with a diameter of 944 plusmn111 120583m were seen exclusively in all four samples from theFusarium delphinoides group (Figures 2(a) and 2(b)) Thescraping material from Curvularia infections also showedlarge quantities of chlamydospore-like structures with anaverage diameter of 1115 plusmn 158 120583m (Figures 2(c) and 2(d))These structures were absent in all the remaining samples ofthe Dematiaceous group A huge variation in microscopicfeatures was noticed in the Dematiaceous group (Figures3(a)ndash3(d)) Scraping material other than the Curvulariainfection showed a typical arrangement of septa

4 BioMed Research International

Table 1 Epidemiologic characteristics risk factors and clinical features

Variable Fusarium spp (119899 = 26) Aspergillus spp (119899 = 15) Dematiaceous isolates (119899 = 12)Age (in years) 4184 5246 465Gender ratio (male female) 271 2 14Risk factors

Trauma other than vegetative body 1 (38) 4 (266) 0Trauma with vegetative body 11 (423)lowast 0 4 (333)Prior ocular surgeryinfection 1 (38) 5 (333) 1 (83)

Seasonal distributionSummer 6 (23) 3 (20) 1 (83)Monson 4 (153) 7 (466) 3 (25)Winter 14 (538) 7 (466) 6 (50)

Clinical featuresCentral location 23 (884) 12 (80) 11 (916)Size (gt10mm) 16 (615) 9 (60) 11 (916)Hypopyon 10 (384) 5 (333) 9 (75)lowast

Elevated slough 19 (730) 6 (40) 10 (833)Dry texture 20 (769) 7 (466) 9 (75)Serrated ulcer margins 21 (807) 9 (60) 8 (666)Pigmentation 0 1 (66) 5 (416)Endothelial plaque 4 (153) 0 0Dendritic lesions 0 2 (133) 0Satellite lesions 15 (576) 8 (533) 3 (25)Mean number of days for complete healing 574lowast 1148 1256

lowastGroup significantly higher compared to other

A total of 47 (8867) out of 53 samples showed visiblegrowth on all the media inoculated at 24 hours (Figure 4(a))In the Fusarium group 23 (884) samples showed growthwithin 24 hours while 3 samples showed growth within 48hours In the Aspergillus group 13 (8666) samples showedgrowth within 24 hours and the remaining 2 samples showedgrowth within 48 and 72 hours respectively The growth ofboth Fusarium and Aspergillus samples on SDA at 48 hourswas similar with respect to the growth rate (Figures 4(b)and 4(c)) In the Dematiaceous group 1112 (916) samplesshowed growth on SDA within 24 hours All the samples ofthe Dematiaceous group showed the presence of a peculiarcolor for example pink or light brown in case of Curvulariaspp (Figure 4(d)) yellow for Papulaspora spp (Figure 4(e))and dark brown for Exserohilum spp (Figure 4(f)) A sampleof Lasiodiplodia theobromae obtained from the scrapingmaterial was grown for 48 hours and a gray fluffy growthwithabundant aerial mycelia was visible as seen in Figure 4(g) Itwas further observed that SDA did not support sporulationof Curvularia spp and Lasiodiplodia spp in all the samples

33 Microscopic and Molecular Identification of FusariumAspergillus and Dematiaceous Spp All isolates in the Fusar-ium and Aspergillus groups were identified to the genuslevel by means of their morphological characteristics Themorphological evaluation of Fusarium solani appeared to bestraight forward and this was further confirmed using theITS sequences However when the sequences were evaluated

using the Fusarium MLST website the match was to theFusarium solani species complex and not to Fusarium solaniper se Hence all Fusarium isolates were named as membersof the Fusarium solani species complex All other isolatesof Fusarium (119899 = 4) were identified as Fusarium dimerumusing their morphological features However identificationusing the ITS sequences at NCBI BLAST was F delphinoidesisolates (119899 = 4) Fusarium dimerum (119899 = 1) and Fusariumdelphinoides (119899 = 3) using the MLST database In theAspergillus group A niger A flavus A terreus and Afumigatus were identified using morphological features Atamarii A tubingensis A versicolor and A sydowii wereonly identified when the ITS sequences were available Inthe Dematiaceous group Curvularia lunata and Exserohilumrostratum were identified using their growth characteristicsand typical spores and this was confirmed using the ITSsequence Using ITS sequences all the other dematiaceousisolates were identified as Lasiodiplodia theobromae Clador-rhinum bulbilosum and Cladosporium cladosporioides TheITS sequence misidentified only one isolate as Chaetomiumspp It was later identified as Papulaspora spp on the basis ofits typical microscopic features

34 In Vitro Antifungal Susceptibility Table 2 shows the re-sults of antifungal susceptibility testing of all isolates Anti-fungal results showed that amphotericin B and natamycinare the most effective antifungal agents against Fusariumspp In the Aspergillus group amphotericin B and itracona-zole showed the lowest MIC against A flavus A terreus

BioMed Research International 5

(a) (b)

(c) (d)

(e) (f)

Figure 1 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium infections (c and d)Scrapping material from Aspergillus infections (e and f) Scrapping material from Exserohilum infections

A tamarii and A tubingensis whereas natamycin andamphotericin B showed good in vitro activity against Aniger and A sydowii In the Dematiaceous group exceptCurvularia all the other isolates were highly susceptible tothe antifungal agents tested Amphotericin B and natamycinshowed good in vitro activity against Curvularia lunata

35 Evaluation of Clinical Outcomes Table 3 shows the com-parative clinical outcomes in all cases In the Fusarium group20 cases healed and 3 worsened while 3 were lost to follow-up The minimum time required for healing was 14 dayswhereas the maximum time taken to heal in the case of

one patient was 300 days In the Aspergillus group 12 outof 15 cases healed with topical and oral antifungal medicaltreatment and two cases required therapeutic keratoplastywhereas one case caused by A tamarii worsened Theminimum time required for healing was 30 days and themaximum time required was 330 days In the Dematiaceousgroup 11 cases healed and one was lost to follow-up Theminimum time taken to heal was 7 days while the maximumtime was 300 days The healing response in terms of visualacuity of the Dematiaceous group was significantly goodwhen compared with the Fusarium and Aspergillus groups(119875 lt 005) The time required for healing in the Fusarium

6 BioMed Research International

(a) (b)

(c) (d)

Figure 2 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium delphinoides infections(c and d) Scrapping material from Curvularia infections

group was statistically significantly less when compared withthe Aspergillus and Dematiaceous groups

4 Discussion

In the present study of 208 keratitis patients fungal etiologywas confirmed in 35 of the cases where Fusarium sppwas the most common isolate followed by Aspergillus andDematiaceousThis is comparable to most studies from India[1ndash5] In India Aspergillus is mainly reported as the mostcommon isolate in the Northern region [1 2 22 23] whileFusarium is mainly reported in the Southern region [4 24]and Dematiaceous fungi are reported to be the third mostcommon fungi We found two reports from AhmedabadFusarium was reported in a study conducted in 2003ndash2005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26] We have obtained adefinite history of trauma with vegetativeagricultural bodieslargely in patients with Fusarium keratitis whereas traumadue to a factor other than vegetative material or any otherocular surgery was found to be largely associated with theAspergillus group Among the traumatic agents plants andagricultural material like hay have contributed to 76 785and 612 of cases of keratitis in studies from Assam [27]Gujarat [25] and Tamil Nadu [28] Fungal keratitis is more

frequently reported in winter with a humid climate favoringfungal growth [29 30]

The generally accepted clinical features for the diagnosisof mycotic keratitis are the presence of a dry raised ulcer witha feathery or hyphate border satellite lesions and recurrenthypopyon [31] Our results are similar to reports from Delhi[2] and Madurai [30 32] The pigmented plaque like thepresentation seen in 42 of our cases in the Dematiaceousgroup is similar to the series reported by Garg and associatesin 2000 and 2004 respectively [33 34]

Directmicroscopy is an important diagnosticmodality ininvestigating microbial keratitis and a highest sensitivity at99 is reported [35]The addition of calcofluor white (CFW)stain to the diagnostic armamentarium has significantlyincreased the sensitivity of smear examination on directmicroscopy [19] However it is difficult to determine thegenus of fungi from KOH mounts [35] Preliminary identifi-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36]Identification of Fusarium from scraping material is reportedby the detection of adventitious sporulation [37] The pres-ence of a brown colored fungal hyphae in the scraping mate-rial raised the possibility of the presence of a Dematiaceousmold [33] Morphologically we did not find any remarkabledifferences between the Fusarium and Aspergillus groupsexcept that the distance between the septa was larger in

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 3

24 Antifungal Susceptibility Testing In vitro antifungal sus-ceptibility testing was done against natamycin (Natamet5 suspension Sun Pharmaceuticals Ind Ltd Halol India)itraconazole (Itral 1 suspension Jawa PharmaceuticalsGurgaon India) fluconazole (Nufl ucon 03 suspensionNuLife Pharmaceuticals Pune India) and amphotericin B(RM 462 Himedia Labs Ltd Mumbai India) using themicrodilutionmethod and following theClinical andLabora-tory Standards Institute (CLSI) guidelines [21] All antifungalagents were dissolved in DMSO and fluconazole was dis-solved inwaterThe inoculumswere prepared by covering the7-day-old culture plate with normal saline (085NaCl)Thiswas followed by gentle probing of the colonies with the help ofa pipette and adjusting the densities of the suspension (read at530 nm) to a final inoculum of 05 McFarland standard Thefinal drug concentration range prepared using serial dilutionwere 0008 to 132 120583gmL for all the four antifungal agents Allthe antifungal agents were tested in RPMI 1640 media with2 glucose and without sodium carbonate

25 Treatment Regime and Evaluation of Clinical OutcomesSubsequent to the microscopic examination if a positivereport of fungal filaments was received antifungal topicaltherapy with 5 natamycin was started for all cases immedi-ately One-hourly topical eye drops were applied around theclock for the first three days followed by two-hourly dropsduring waking hours until resolution of the ulcers Patientsalso received 1 atropine sulphate eye drops Systemic flu-conazole (150mg once a day) was prescribed for all patientswith corneal stromal infiltrate extending beyond one-thirdof the cornea After treatment an ulcer was considered tobe healed when the epithelial defect was lt1mm in diameterwith a visible scar under slit lamp biomicroscopy A healingtime of less than 3 weeks from presentation was considereda good result and healing time of more than three weeks wasconsidered a poor responseThe responses to treatment werecategorized into three groups as follows perception of light tothe Snellenrsquos chart (good response) no change in visual acuityafter treatment (poor response) and slight change in thevisual acuity (slight response) Initial and final visual acuitywas comparedfollowingtreatment using statistical analysisThe time for complete healing was also compared among theisolates The follow-up best-corrected visual acuity (BCVA)of patients treated with topical and oral antimicrobial agentswas the visual acuity measured when the patient was cured(inactive corneal scar with intact epithelium) Visual acuitiesobtained using Snellenrsquos chart were converted into logarithmsof the minimum angle of resolution (logMAR) for dataanalysis

26 Statistical Analysis The test of proportion was used toevaluate the epidemiological features and risk factors In vitrosusceptibility results obtained from the three groups werestatistically analyzed using the Kruskal-Wallis test A post hocpair wise comparison was also done The Wilcoxon SignedRanks Test was used to compare visual acuity before and aftertreatment in each group

3 Results

31 Epidemiological Characteristics and Clinical Features Atotal of 208 patients with keratitis were recruited during theperiod fromMay 2009 to June 2012 In all 73 patients who hadculture-proven mycotic keratitis the incidence of culture-proven mycotic keratitis was 350 Out of these 73 patients26 (356) were infected with Fusarium spp 15 (205)were infected with Aspergillus species and 11 (150) wereinfected with Dematiaceous fungi The average age of thepatients was 4184 years in the Fusarium group 5246 yearsin theAspergillus group and 4653 years in the Dematiaceousfungi group There were more males than females in all threegroupsThe seasonal distribution showed that infection of allthe three types of keratitis was highest in winter and attainedstatistical significance (Table 1) The risk factors such astrauma to the eyeocular surgery were predominantly seen inthe Aspergillus keratitis group whereas the incidence of entryof vegetative foreign bodies was mainly seen in the Fusariumgroup (Table 1) Table 1 shows the comparative evaluationof the clinical features in all the three groups The area ofinfiltration was large (gt4mm) in the central visual axis inall the three groups Further the presence of satellite lesionsring infiltrate dry appearance and stromal involvement wasevident in most of the cases The presence of hypopyon andpigmentation was mainly associated with the Dematiaceousgroup and this attained statistical significance The presenceof endothelial plaque was mainly associated with Fusariuminfections whereas dendritic lesions and Descemetrsquos foldswere mainly observed in the Aspergillus group

32 Microscopic Evaluation and Growth Characteristics of theScrapingMaterial All the samples of scrapingmaterial takenfrom the three groups showed the presence of large quantitiesof fungal filaments when seen under light and fluorescencemicroscopes (Figure 1) Samples from Fusarium infections(Figures 1(a) and 1(b)) showed the presence of fungal hyphaewith an average thickness 387 plusmn 06 120583m Septa were notvisible under the light microscope (Figure 1(a)) but wereclearly seen under the fluorescence microscope (Figure 1(b))The distance between the septa was 2165 plusmn 42 120583m Theaverage hyphal thickness in the Aspergillus group (Figures1(c) and 1(d)) was 413plusmn 065 120583m and this was almost similarto the hyphal thickness in the Fusarium group Howeverthe distance between the septa was 1284 plusmn 19 120583m Theaverage hyphal thickness in the Dematiaceous group was879 plusmn 09 120583m (Figures 1(e) and 1(f)) Terminal and internalchlamydospore-like structures with a diameter of 944 plusmn111 120583m were seen exclusively in all four samples from theFusarium delphinoides group (Figures 2(a) and 2(b)) Thescraping material from Curvularia infections also showedlarge quantities of chlamydospore-like structures with anaverage diameter of 1115 plusmn 158 120583m (Figures 2(c) and 2(d))These structures were absent in all the remaining samples ofthe Dematiaceous group A huge variation in microscopicfeatures was noticed in the Dematiaceous group (Figures3(a)ndash3(d)) Scraping material other than the Curvulariainfection showed a typical arrangement of septa

4 BioMed Research International

Table 1 Epidemiologic characteristics risk factors and clinical features

Variable Fusarium spp (119899 = 26) Aspergillus spp (119899 = 15) Dematiaceous isolates (119899 = 12)Age (in years) 4184 5246 465Gender ratio (male female) 271 2 14Risk factors

Trauma other than vegetative body 1 (38) 4 (266) 0Trauma with vegetative body 11 (423)lowast 0 4 (333)Prior ocular surgeryinfection 1 (38) 5 (333) 1 (83)

Seasonal distributionSummer 6 (23) 3 (20) 1 (83)Monson 4 (153) 7 (466) 3 (25)Winter 14 (538) 7 (466) 6 (50)

Clinical featuresCentral location 23 (884) 12 (80) 11 (916)Size (gt10mm) 16 (615) 9 (60) 11 (916)Hypopyon 10 (384) 5 (333) 9 (75)lowast

Elevated slough 19 (730) 6 (40) 10 (833)Dry texture 20 (769) 7 (466) 9 (75)Serrated ulcer margins 21 (807) 9 (60) 8 (666)Pigmentation 0 1 (66) 5 (416)Endothelial plaque 4 (153) 0 0Dendritic lesions 0 2 (133) 0Satellite lesions 15 (576) 8 (533) 3 (25)Mean number of days for complete healing 574lowast 1148 1256

lowastGroup significantly higher compared to other

A total of 47 (8867) out of 53 samples showed visiblegrowth on all the media inoculated at 24 hours (Figure 4(a))In the Fusarium group 23 (884) samples showed growthwithin 24 hours while 3 samples showed growth within 48hours In the Aspergillus group 13 (8666) samples showedgrowth within 24 hours and the remaining 2 samples showedgrowth within 48 and 72 hours respectively The growth ofboth Fusarium and Aspergillus samples on SDA at 48 hourswas similar with respect to the growth rate (Figures 4(b)and 4(c)) In the Dematiaceous group 1112 (916) samplesshowed growth on SDA within 24 hours All the samples ofthe Dematiaceous group showed the presence of a peculiarcolor for example pink or light brown in case of Curvulariaspp (Figure 4(d)) yellow for Papulaspora spp (Figure 4(e))and dark brown for Exserohilum spp (Figure 4(f)) A sampleof Lasiodiplodia theobromae obtained from the scrapingmaterial was grown for 48 hours and a gray fluffy growthwithabundant aerial mycelia was visible as seen in Figure 4(g) Itwas further observed that SDA did not support sporulationof Curvularia spp and Lasiodiplodia spp in all the samples

33 Microscopic and Molecular Identification of FusariumAspergillus and Dematiaceous Spp All isolates in the Fusar-ium and Aspergillus groups were identified to the genuslevel by means of their morphological characteristics Themorphological evaluation of Fusarium solani appeared to bestraight forward and this was further confirmed using theITS sequences However when the sequences were evaluated

using the Fusarium MLST website the match was to theFusarium solani species complex and not to Fusarium solaniper se Hence all Fusarium isolates were named as membersof the Fusarium solani species complex All other isolatesof Fusarium (119899 = 4) were identified as Fusarium dimerumusing their morphological features However identificationusing the ITS sequences at NCBI BLAST was F delphinoidesisolates (119899 = 4) Fusarium dimerum (119899 = 1) and Fusariumdelphinoides (119899 = 3) using the MLST database In theAspergillus group A niger A flavus A terreus and Afumigatus were identified using morphological features Atamarii A tubingensis A versicolor and A sydowii wereonly identified when the ITS sequences were available Inthe Dematiaceous group Curvularia lunata and Exserohilumrostratum were identified using their growth characteristicsand typical spores and this was confirmed using the ITSsequence Using ITS sequences all the other dematiaceousisolates were identified as Lasiodiplodia theobromae Clador-rhinum bulbilosum and Cladosporium cladosporioides TheITS sequence misidentified only one isolate as Chaetomiumspp It was later identified as Papulaspora spp on the basis ofits typical microscopic features

34 In Vitro Antifungal Susceptibility Table 2 shows the re-sults of antifungal susceptibility testing of all isolates Anti-fungal results showed that amphotericin B and natamycinare the most effective antifungal agents against Fusariumspp In the Aspergillus group amphotericin B and itracona-zole showed the lowest MIC against A flavus A terreus

BioMed Research International 5

(a) (b)

(c) (d)

(e) (f)

Figure 1 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium infections (c and d)Scrapping material from Aspergillus infections (e and f) Scrapping material from Exserohilum infections

A tamarii and A tubingensis whereas natamycin andamphotericin B showed good in vitro activity against Aniger and A sydowii In the Dematiaceous group exceptCurvularia all the other isolates were highly susceptible tothe antifungal agents tested Amphotericin B and natamycinshowed good in vitro activity against Curvularia lunata

35 Evaluation of Clinical Outcomes Table 3 shows the com-parative clinical outcomes in all cases In the Fusarium group20 cases healed and 3 worsened while 3 were lost to follow-up The minimum time required for healing was 14 dayswhereas the maximum time taken to heal in the case of

one patient was 300 days In the Aspergillus group 12 outof 15 cases healed with topical and oral antifungal medicaltreatment and two cases required therapeutic keratoplastywhereas one case caused by A tamarii worsened Theminimum time required for healing was 30 days and themaximum time required was 330 days In the Dematiaceousgroup 11 cases healed and one was lost to follow-up Theminimum time taken to heal was 7 days while the maximumtime was 300 days The healing response in terms of visualacuity of the Dematiaceous group was significantly goodwhen compared with the Fusarium and Aspergillus groups(119875 lt 005) The time required for healing in the Fusarium

6 BioMed Research International

(a) (b)

(c) (d)

Figure 2 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium delphinoides infections(c and d) Scrapping material from Curvularia infections

group was statistically significantly less when compared withthe Aspergillus and Dematiaceous groups

4 Discussion

In the present study of 208 keratitis patients fungal etiologywas confirmed in 35 of the cases where Fusarium sppwas the most common isolate followed by Aspergillus andDematiaceousThis is comparable to most studies from India[1ndash5] In India Aspergillus is mainly reported as the mostcommon isolate in the Northern region [1 2 22 23] whileFusarium is mainly reported in the Southern region [4 24]and Dematiaceous fungi are reported to be the third mostcommon fungi We found two reports from AhmedabadFusarium was reported in a study conducted in 2003ndash2005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26] We have obtained adefinite history of trauma with vegetativeagricultural bodieslargely in patients with Fusarium keratitis whereas traumadue to a factor other than vegetative material or any otherocular surgery was found to be largely associated with theAspergillus group Among the traumatic agents plants andagricultural material like hay have contributed to 76 785and 612 of cases of keratitis in studies from Assam [27]Gujarat [25] and Tamil Nadu [28] Fungal keratitis is more

frequently reported in winter with a humid climate favoringfungal growth [29 30]

The generally accepted clinical features for the diagnosisof mycotic keratitis are the presence of a dry raised ulcer witha feathery or hyphate border satellite lesions and recurrenthypopyon [31] Our results are similar to reports from Delhi[2] and Madurai [30 32] The pigmented plaque like thepresentation seen in 42 of our cases in the Dematiaceousgroup is similar to the series reported by Garg and associatesin 2000 and 2004 respectively [33 34]

Directmicroscopy is an important diagnosticmodality ininvestigating microbial keratitis and a highest sensitivity at99 is reported [35]The addition of calcofluor white (CFW)stain to the diagnostic armamentarium has significantlyincreased the sensitivity of smear examination on directmicroscopy [19] However it is difficult to determine thegenus of fungi from KOH mounts [35] Preliminary identifi-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36]Identification of Fusarium from scraping material is reportedby the detection of adventitious sporulation [37] The pres-ence of a brown colored fungal hyphae in the scraping mate-rial raised the possibility of the presence of a Dematiaceousmold [33] Morphologically we did not find any remarkabledifferences between the Fusarium and Aspergillus groupsexcept that the distance between the septa was larger in

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

Table 1 Epidemiologic characteristics risk factors and clinical features

Variable Fusarium spp (119899 = 26) Aspergillus spp (119899 = 15) Dematiaceous isolates (119899 = 12)Age (in years) 4184 5246 465Gender ratio (male female) 271 2 14Risk factors

Trauma other than vegetative body 1 (38) 4 (266) 0Trauma with vegetative body 11 (423)lowast 0 4 (333)Prior ocular surgeryinfection 1 (38) 5 (333) 1 (83)

Seasonal distributionSummer 6 (23) 3 (20) 1 (83)Monson 4 (153) 7 (466) 3 (25)Winter 14 (538) 7 (466) 6 (50)

Clinical featuresCentral location 23 (884) 12 (80) 11 (916)Size (gt10mm) 16 (615) 9 (60) 11 (916)Hypopyon 10 (384) 5 (333) 9 (75)lowast

Elevated slough 19 (730) 6 (40) 10 (833)Dry texture 20 (769) 7 (466) 9 (75)Serrated ulcer margins 21 (807) 9 (60) 8 (666)Pigmentation 0 1 (66) 5 (416)Endothelial plaque 4 (153) 0 0Dendritic lesions 0 2 (133) 0Satellite lesions 15 (576) 8 (533) 3 (25)Mean number of days for complete healing 574lowast 1148 1256

lowastGroup significantly higher compared to other

A total of 47 (8867) out of 53 samples showed visiblegrowth on all the media inoculated at 24 hours (Figure 4(a))In the Fusarium group 23 (884) samples showed growthwithin 24 hours while 3 samples showed growth within 48hours In the Aspergillus group 13 (8666) samples showedgrowth within 24 hours and the remaining 2 samples showedgrowth within 48 and 72 hours respectively The growth ofboth Fusarium and Aspergillus samples on SDA at 48 hourswas similar with respect to the growth rate (Figures 4(b)and 4(c)) In the Dematiaceous group 1112 (916) samplesshowed growth on SDA within 24 hours All the samples ofthe Dematiaceous group showed the presence of a peculiarcolor for example pink or light brown in case of Curvulariaspp (Figure 4(d)) yellow for Papulaspora spp (Figure 4(e))and dark brown for Exserohilum spp (Figure 4(f)) A sampleof Lasiodiplodia theobromae obtained from the scrapingmaterial was grown for 48 hours and a gray fluffy growthwithabundant aerial mycelia was visible as seen in Figure 4(g) Itwas further observed that SDA did not support sporulationof Curvularia spp and Lasiodiplodia spp in all the samples

33 Microscopic and Molecular Identification of FusariumAspergillus and Dematiaceous Spp All isolates in the Fusar-ium and Aspergillus groups were identified to the genuslevel by means of their morphological characteristics Themorphological evaluation of Fusarium solani appeared to bestraight forward and this was further confirmed using theITS sequences However when the sequences were evaluated

using the Fusarium MLST website the match was to theFusarium solani species complex and not to Fusarium solaniper se Hence all Fusarium isolates were named as membersof the Fusarium solani species complex All other isolatesof Fusarium (119899 = 4) were identified as Fusarium dimerumusing their morphological features However identificationusing the ITS sequences at NCBI BLAST was F delphinoidesisolates (119899 = 4) Fusarium dimerum (119899 = 1) and Fusariumdelphinoides (119899 = 3) using the MLST database In theAspergillus group A niger A flavus A terreus and Afumigatus were identified using morphological features Atamarii A tubingensis A versicolor and A sydowii wereonly identified when the ITS sequences were available Inthe Dematiaceous group Curvularia lunata and Exserohilumrostratum were identified using their growth characteristicsand typical spores and this was confirmed using the ITSsequence Using ITS sequences all the other dematiaceousisolates were identified as Lasiodiplodia theobromae Clador-rhinum bulbilosum and Cladosporium cladosporioides TheITS sequence misidentified only one isolate as Chaetomiumspp It was later identified as Papulaspora spp on the basis ofits typical microscopic features

34 In Vitro Antifungal Susceptibility Table 2 shows the re-sults of antifungal susceptibility testing of all isolates Anti-fungal results showed that amphotericin B and natamycinare the most effective antifungal agents against Fusariumspp In the Aspergillus group amphotericin B and itracona-zole showed the lowest MIC against A flavus A terreus

BioMed Research International 5

(a) (b)

(c) (d)

(e) (f)

Figure 1 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium infections (c and d)Scrapping material from Aspergillus infections (e and f) Scrapping material from Exserohilum infections

A tamarii and A tubingensis whereas natamycin andamphotericin B showed good in vitro activity against Aniger and A sydowii In the Dematiaceous group exceptCurvularia all the other isolates were highly susceptible tothe antifungal agents tested Amphotericin B and natamycinshowed good in vitro activity against Curvularia lunata

35 Evaluation of Clinical Outcomes Table 3 shows the com-parative clinical outcomes in all cases In the Fusarium group20 cases healed and 3 worsened while 3 were lost to follow-up The minimum time required for healing was 14 dayswhereas the maximum time taken to heal in the case of

one patient was 300 days In the Aspergillus group 12 outof 15 cases healed with topical and oral antifungal medicaltreatment and two cases required therapeutic keratoplastywhereas one case caused by A tamarii worsened Theminimum time required for healing was 30 days and themaximum time required was 330 days In the Dematiaceousgroup 11 cases healed and one was lost to follow-up Theminimum time taken to heal was 7 days while the maximumtime was 300 days The healing response in terms of visualacuity of the Dematiaceous group was significantly goodwhen compared with the Fusarium and Aspergillus groups(119875 lt 005) The time required for healing in the Fusarium

6 BioMed Research International

(a) (b)

(c) (d)

Figure 2 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium delphinoides infections(c and d) Scrapping material from Curvularia infections

group was statistically significantly less when compared withthe Aspergillus and Dematiaceous groups

4 Discussion

In the present study of 208 keratitis patients fungal etiologywas confirmed in 35 of the cases where Fusarium sppwas the most common isolate followed by Aspergillus andDematiaceousThis is comparable to most studies from India[1ndash5] In India Aspergillus is mainly reported as the mostcommon isolate in the Northern region [1 2 22 23] whileFusarium is mainly reported in the Southern region [4 24]and Dematiaceous fungi are reported to be the third mostcommon fungi We found two reports from AhmedabadFusarium was reported in a study conducted in 2003ndash2005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26] We have obtained adefinite history of trauma with vegetativeagricultural bodieslargely in patients with Fusarium keratitis whereas traumadue to a factor other than vegetative material or any otherocular surgery was found to be largely associated with theAspergillus group Among the traumatic agents plants andagricultural material like hay have contributed to 76 785and 612 of cases of keratitis in studies from Assam [27]Gujarat [25] and Tamil Nadu [28] Fungal keratitis is more

frequently reported in winter with a humid climate favoringfungal growth [29 30]

The generally accepted clinical features for the diagnosisof mycotic keratitis are the presence of a dry raised ulcer witha feathery or hyphate border satellite lesions and recurrenthypopyon [31] Our results are similar to reports from Delhi[2] and Madurai [30 32] The pigmented plaque like thepresentation seen in 42 of our cases in the Dematiaceousgroup is similar to the series reported by Garg and associatesin 2000 and 2004 respectively [33 34]

Directmicroscopy is an important diagnosticmodality ininvestigating microbial keratitis and a highest sensitivity at99 is reported [35]The addition of calcofluor white (CFW)stain to the diagnostic armamentarium has significantlyincreased the sensitivity of smear examination on directmicroscopy [19] However it is difficult to determine thegenus of fungi from KOH mounts [35] Preliminary identifi-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36]Identification of Fusarium from scraping material is reportedby the detection of adventitious sporulation [37] The pres-ence of a brown colored fungal hyphae in the scraping mate-rial raised the possibility of the presence of a Dematiaceousmold [33] Morphologically we did not find any remarkabledifferences between the Fusarium and Aspergillus groupsexcept that the distance between the septa was larger in

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

(a) (b)

(c) (d)

(e) (f)

Figure 1 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium infections (c and d)Scrapping material from Aspergillus infections (e and f) Scrapping material from Exserohilum infections

A tamarii and A tubingensis whereas natamycin andamphotericin B showed good in vitro activity against Aniger and A sydowii In the Dematiaceous group exceptCurvularia all the other isolates were highly susceptible tothe antifungal agents tested Amphotericin B and natamycinshowed good in vitro activity against Curvularia lunata

35 Evaluation of Clinical Outcomes Table 3 shows the com-parative clinical outcomes in all cases In the Fusarium group20 cases healed and 3 worsened while 3 were lost to follow-up The minimum time required for healing was 14 dayswhereas the maximum time taken to heal in the case of

one patient was 300 days In the Aspergillus group 12 outof 15 cases healed with topical and oral antifungal medicaltreatment and two cases required therapeutic keratoplastywhereas one case caused by A tamarii worsened Theminimum time required for healing was 30 days and themaximum time required was 330 days In the Dematiaceousgroup 11 cases healed and one was lost to follow-up Theminimum time taken to heal was 7 days while the maximumtime was 300 days The healing response in terms of visualacuity of the Dematiaceous group was significantly goodwhen compared with the Fusarium and Aspergillus groups(119875 lt 005) The time required for healing in the Fusarium

6 BioMed Research International

(a) (b)

(c) (d)

Figure 2 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium delphinoides infections(c and d) Scrapping material from Curvularia infections

group was statistically significantly less when compared withthe Aspergillus and Dematiaceous groups

4 Discussion

In the present study of 208 keratitis patients fungal etiologywas confirmed in 35 of the cases where Fusarium sppwas the most common isolate followed by Aspergillus andDematiaceousThis is comparable to most studies from India[1ndash5] In India Aspergillus is mainly reported as the mostcommon isolate in the Northern region [1 2 22 23] whileFusarium is mainly reported in the Southern region [4 24]and Dematiaceous fungi are reported to be the third mostcommon fungi We found two reports from AhmedabadFusarium was reported in a study conducted in 2003ndash2005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26] We have obtained adefinite history of trauma with vegetativeagricultural bodieslargely in patients with Fusarium keratitis whereas traumadue to a factor other than vegetative material or any otherocular surgery was found to be largely associated with theAspergillus group Among the traumatic agents plants andagricultural material like hay have contributed to 76 785and 612 of cases of keratitis in studies from Assam [27]Gujarat [25] and Tamil Nadu [28] Fungal keratitis is more

frequently reported in winter with a humid climate favoringfungal growth [29 30]

The generally accepted clinical features for the diagnosisof mycotic keratitis are the presence of a dry raised ulcer witha feathery or hyphate border satellite lesions and recurrenthypopyon [31] Our results are similar to reports from Delhi[2] and Madurai [30 32] The pigmented plaque like thepresentation seen in 42 of our cases in the Dematiaceousgroup is similar to the series reported by Garg and associatesin 2000 and 2004 respectively [33 34]

Directmicroscopy is an important diagnosticmodality ininvestigating microbial keratitis and a highest sensitivity at99 is reported [35]The addition of calcofluor white (CFW)stain to the diagnostic armamentarium has significantlyincreased the sensitivity of smear examination on directmicroscopy [19] However it is difficult to determine thegenus of fungi from KOH mounts [35] Preliminary identifi-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36]Identification of Fusarium from scraping material is reportedby the detection of adventitious sporulation [37] The pres-ence of a brown colored fungal hyphae in the scraping mate-rial raised the possibility of the presence of a Dematiaceousmold [33] Morphologically we did not find any remarkabledifferences between the Fusarium and Aspergillus groupsexcept that the distance between the septa was larger in

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 BioMed Research International

(a) (b)

(c) (d)

Figure 2 10 KOHmount of the scrapping material showing fungal hyphae magnification times400 Left panel light microscopic picture rightpanel fluorescent microscopic picture taken after calcofluor white stain (a and b) Scrapping material from Fusarium delphinoides infections(c and d) Scrapping material from Curvularia infections

group was statistically significantly less when compared withthe Aspergillus and Dematiaceous groups

4 Discussion

In the present study of 208 keratitis patients fungal etiologywas confirmed in 35 of the cases where Fusarium sppwas the most common isolate followed by Aspergillus andDematiaceousThis is comparable to most studies from India[1ndash5] In India Aspergillus is mainly reported as the mostcommon isolate in the Northern region [1 2 22 23] whileFusarium is mainly reported in the Southern region [4 24]and Dematiaceous fungi are reported to be the third mostcommon fungi We found two reports from AhmedabadFusarium was reported in a study conducted in 2003ndash2005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26] We have obtained adefinite history of trauma with vegetativeagricultural bodieslargely in patients with Fusarium keratitis whereas traumadue to a factor other than vegetative material or any otherocular surgery was found to be largely associated with theAspergillus group Among the traumatic agents plants andagricultural material like hay have contributed to 76 785and 612 of cases of keratitis in studies from Assam [27]Gujarat [25] and Tamil Nadu [28] Fungal keratitis is more

frequently reported in winter with a humid climate favoringfungal growth [29 30]

The generally accepted clinical features for the diagnosisof mycotic keratitis are the presence of a dry raised ulcer witha feathery or hyphate border satellite lesions and recurrenthypopyon [31] Our results are similar to reports from Delhi[2] and Madurai [30 32] The pigmented plaque like thepresentation seen in 42 of our cases in the Dematiaceousgroup is similar to the series reported by Garg and associatesin 2000 and 2004 respectively [33 34]

Directmicroscopy is an important diagnosticmodality ininvestigating microbial keratitis and a highest sensitivity at99 is reported [35]The addition of calcofluor white (CFW)stain to the diagnostic armamentarium has significantlyincreased the sensitivity of smear examination on directmicroscopy [19] However it is difficult to determine thegenus of fungi from KOH mounts [35] Preliminary identifi-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36]Identification of Fusarium from scraping material is reportedby the detection of adventitious sporulation [37] The pres-ence of a brown colored fungal hyphae in the scraping mate-rial raised the possibility of the presence of a Dematiaceousmold [33] Morphologically we did not find any remarkabledifferences between the Fusarium and Aspergillus groupsexcept that the distance between the septa was larger in

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 7

(a) (b)

(c) (d)

Figure 3 Light microscopic pictures of 10 KOH mount of the scrapping material showing fungal hyphae magnification times400 (andashd)Scrapping material from Cladorrhinum Curvularia Papulaspora and Cladosporium infections respectively

Table 2 In vitro susceptibility of Fusarium Aspergillus and Dematiaceous isolates to antifungal agents

Isolate (number) Agent G-MIClowast (range)Natamycin Amphotericin B Fluconazole Itraconazole

Fusarium solani (119899 = 22) 293 (8ndash128) 63 (1ndash32) 128 (ge128) 1137 (64ndash128)Fusarium delphinoides (119899 = 4) 12 (8ndash16) 12 (8ndash16) 128 (ge128) 128 (ge128)Aspergillus flavus (119899 = 6) 32 (32) 035 (02ndash05) 128 (ge128) 15 (1ndash2)Aspergillus terreus (119899 = 3) 133 (8ndash16) 25 (1ndash3) 266 (16ndash32) 025 (025)Aspergillus niger (119899 = 1) 8 006 128 64Aspergillus tamarii (119899 = 1) 64 025 ge128 025Aspergillus tubingensis (119899 = 1) 8 2 32 025Aspergillus sydowii (119899 = 1) 4 4 ge128 ge128Aspergillus versicolor (119899 = 1) 4 2 32 025Curvularia lunata (119899 = 4) 125 (2ndash32) 16 (025ndash32) 128 (ge128) 128 (ge128)Exherohelum rostratum (119899 = 2) 2 (2ndash4) 1 (05ndash2) 32 (32) 025 (025ndash05)Cladorrhinum bulbilossum (119899 = 1) 0064 0064 032 0016Lasiodiplodia theobromae (119899 = 1) 0064 0064 032 0016Papulaspora spp (119899 = 1) 0064 0064 032 0016lowastGeometric mean of MIC

Fusarium specimens We established that hyphal thicknesswas greater in the Dematiaceous samples as compared to thespecimens from other groups Since brown coloration of thefungal cell wall may not be seen in all Dematiaceous cases

[33 38 39] we believe that hyphal thickness can be used asan indication of Dematiaceous fungi

Molecular identification of keratitis causing Fusariumand Aspergillus isolates has been reported earlier [9ndash12]

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 4 Growth of fungi from scrapping material after (a) 24 hrs (b and c) Fusarium and Aspergillus samples after 48 hrs (d and f)Dematiaceous samples and (g) Lasiodiplodia sample

Table 3 Clinical outcomes of Fusarium Aspergillus and Dematiaceous keratitis

Groups Mean visual acuitybefore treatment(LOGMAR)

Mean visual acuity aftertreatment (LOGMAR)

Change in visual acuityWorsened

Poor response(No PL to PLPL to PR)

Slight response(improved SnelensCF to Snelens)

Good response(PL to CFSnelens)

Fusarium 182 (08ndash3) 117 (0ndash3) 3 (20) 8 (533) 3 (20) 1 (67)Aspergillus 208 (048ndash3) 168 (0ndash3) 4 (167) 12 (499) 5 (209) 3 (125)Dematiaceous 245 (048ndash3) 091 (0ndash2) 0 3 (30) 7 (70)lowast 0lowastP value lt005 when compared to the other two groups

However in cases of Fusarium and Aspergillus it was estab-lished that the ITS region alone cannot discriminate betweenclose species [40] A comparative sequence analysis of othergenes such as EF-1 and RPB2 for Fusarium and 120573-tubulinfor Aspergillus is necessary for species identification withinthe complex There is only one such study from India wherecomplete identification of Aspergillus species was done [10]In a recent study on Fusarium keratitis where the ITS regionwas used most isolates belonged to the Fusarium solanispecies complex [12] In case of Fusarium the MLST web sitewas found to be more convenient than NCBI We found thata sequence comparison of the sole ITS region at the NCBIdatabase accurately identified the Dematiaceous isolates

Till date four studies have explored the in vitro antifungalsusceptibility patterns among keratitis-causing fungal isolatesfrom India [13ndash16] Our results are similar to those of Lalitha

et al [14] and show that amphotericin B and natamycin havethe lowest MICs for Fusarium and Aspergillus species Astudy on Fusarium keratitis isolates demonstrated high levelsof in vitro resistance to voriconazole amphotericin B andnatamycin [12] Our results corroborate with their findingsthat across the Fusarium solani species complex (FSSC)amphotericin B had the lowest MIC values Antifungalsusceptibility of Aspergillus species was recently reported ina study from India and our MIC data is similar in thecase of Fluconazole but slightly lower for Natamycin andAmphotericin B [10] Studies on Aspergillus isolates fromother infections also report low MIC ranges similar to oursforA fumigatusA flavus andA niger [41] In our study onlyone isolateA versicolor showed a highMIC value (16120583gmL)and these results are contradictory to the MIC values (1-2 120583gmL) reported previously [42] Antifungal susceptibility

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 9

toA terreus has been shown inmany studies with a very highvariability inMIC values [20] In a report on keratitis causingA terreus isolates ketoconazole was shown to be the mosteffective agent [43] Our results also show ketoconazole to bethe most effective agent against A terreus

We found a good response in the pigmented keratitisgroup and this was correlated with the lower MIC valuesHowever due to the small sample size the results werenot statistically significant A similar observation was madeearlier where in vitro susceptibility to natamycin correlatedwith a favourable clinical response [16] They attributed thegood treatment outcome in pigmented keratitis to the low vir-ulence of Dematiaceous fungi and their tendency to remainin the superficial tissues of the cornea In another studyeyes with pigmented keratitis and nonpigmented keratitisshowed hardly any difference in the medical response andvisual outcomes [44] Recently in a study from south India asignificant associationwasmade between corneal perforationand higher MIC values [13]

Despite the fact that we used a small sample size ineach group we noted pivotal dissimilarities between thegroups This study demonstrates important differences inmicroscopic features between Fusarium Aspergillus andDematiaceous molds from clinical samples The antifun-gal susceptibility results suggest that accurate identificationwould aid in specific treatment strategies

Acknowledgments

This study was supported by a research Grant under theWomenrsquos Scientists Scheme (WOS-A) DST Government ofIndia (no SRWOS-ALS-1202008) and partly by ICMRGrant (53332010-ECD-I)

References

[1] J Chander N Singla N Agnihotri S K Arya and A DeepldquoKeratomycosis in and around Chandigarh a five-year studyfrom a north Indian tertiary care hospitalrdquo Indian Journal ofPathology and Microbiology vol 51 no 2 pp 304ndash306 2008

[2] A Chowdhary and K Singh ldquoSpectrum of fungal keratitis inNorth Indiardquo Cornea vol 24 no 1 pp 8ndash15 2005

[3] S D Deshpande and G V Koppikar ldquoA study of mycotic ker-atitis inMumbairdquo Indian Journal of Pathology andMicrobiologyvol 42 no 1 pp 81ndash87 1999

[4] U Gopinathan S Sharma P Garg and G N Rao ldquoReview ofepidemiological features microbiological diagnosis and treat-ment outcome of microbial keratitis experience of over adecaderdquo Indian Journal of Ophthalmology vol 57 no 4 pp 273ndash279 2009

[5] M J Bharathi R Ramakrishnan RMeenakshi S PadmavathyC Shivakumar and M Srinivasan ldquoMicrobial keratitis inSouth India Influence of risk factors climate and geographicalvariationrdquo Ophthalmic Epidemiology vol 14 no 2 pp 61ndash692007

[6] M Srinivasan M P Upadhyay B Priyadarsini R Mahalak-shmi and J P Whitcher ldquoCorneal ulceration in south-eastAsia III prevention of fungal keratitis at the village levelin south India using topical antibioticsrdquo British Journal ofOphthalmology vol 90 no 12 pp 1472ndash1475 2006

[7] M A G Tanure E J Cohen S Sudesh C J Rapuano and P RLaibson ldquoSpectrum of fungal keratitis at Wills eye hospitalPhiladelphia Pennsylvaniardquo Cornea vol 19 no 3 pp 307ndash3122000

[8] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcodemarker for Fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[9] M Azor J Gene J Cano and J Guarro ldquoUniversal in vitro anti-fungal resistance of genetic clades of the Fusarium solani speciescomplexrdquo Antimicrobial Agents and Chemotherapy vol 51 no4 pp 1500ndash1503 2007

[10] P Manikandan J Varga S Kocsube et al ldquoEpidemiology ofAspergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agentsrdquoMycosesvol 56 no 1 pp 26ndash33 2012

[11] K OrsquoDonnell D A Sutton A Fothergill et al ldquoMolecular phy-logenetic diversity multilocus haplotype nomenclature and invitro antifungal resistance within the Fusarium solani speciescomplexrdquo Journal of Clinical Microbiology vol 46 no 8 pp2477ndash2490 2008

[12] R A Oechsler M R Feilmeier D Miller et al ldquoFusarium ker-atitis genotyping in vitro susceptibility and clinical outcomesrdquoCornea vol 32 no 5 pp 667ndash673 2013

[13] P Lalitha N V Prajna C E Oldenburg et al ldquoOrganismminimum inhibitory concentration and outcome in a fungalcorneal ulcer clinical trialrdquo Cornea vol 31 no 6 pp 662ndash6672012

[14] P Lalitha B L Shapiro M Srinivasan et al ldquoAntimicrobial sus-ceptibility of Fusarium Aspergillus and other filamentous fungiisolated from keratitisrdquo Archives of Ophthalmology vol 125 no6 pp 789ndash793 2007

[15] P Lalitha R Vijaykumar N V Prajna and AW Fothergill ldquoInvitro natamycin susceptibility of ocular isolates of Fusarium andAspergillus species comparison of commercially formulatednatamycin eye drops to pharmaceutical-grade powderrdquo Journalof Clinical Microbiology vol 46 no 10 pp 3477ndash3478 2008

[16] L Pradhan S Sharma S Nalamada S K Sahu S Das andP Garg ldquoNatamycin in the treatment of keratomycosis corre-lation of treatment outcome and in vitro susceptibility of fungalisolatesrdquo Indian Journal of Ophthalmology vol 59 no 6 pp 512ndash514 2011

[17] A Alastruey-Izquierdo M Cuenca-Estrella A Monzon EMellado and J L Rodrıguez-Tudela ldquoAntifungal susceptibilityprofile of clinical Fusarium spp isolates identified by molecularmethodsrdquo Journal of Antimicrobial Chemotherapy vol 61 no 4pp 805ndash809 2008

[18] N J Iqbal A Boey B J Park andM E Brandt ldquoDeterminationof in vitro susceptibility of ocular Fusarium spp isolatesfrom keratitis cases and comparison of clinical and laboratorystandards institute M38-A2 and E test methodsrdquo DiagnosticMicrobiology and Infectious Disease vol 62 no 3 pp 348ndash3502008

[19] J Chander A Chakrabarti A Sharma J S Saini and DPanigarhi ldquoEvaluation of calcofluor staining in the diagnosis offungal corneal ulcerrdquoMycoses vol 36 no 7-8 pp 243ndash245 1993

[20] G S de Hoog J Guarro J Gene and M J Figueras Atlas ofClinical Fungi 3rd edition 2009

[21] Institute CLS Reference Method For Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi Clinical LaboratoryStandards Institute Wayne Pa USA 2nd edition 2008

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 BioMed Research International

[22] S L Sharma ldquoKeratomycosis in corneal sepsisrdquo Indian Journalof Ophthalmology vol 29 no 4 pp 443ndash445 1981

[23] O P Kulshrestha S Bhargava andMKDube ldquoKeratomycosisa report of 23 casesrdquo Indian Journal of Ophthalmology vol 21no 2 pp 51ndash55 1973

[24] M J Bharathi R Ramakrishnan S Vasu R Meenakshi and RPalaniappan ldquoEpidemiological characteristics and laboratorydiagnosis of fungal keratitis A three-year studyrdquo Indian Journalof Ophthalmology vol 51 no 4 pp 315ndash321 2003

[25] A Kumar S Pandya G Kavathia et al ldquoMicrobial keratitis inGujarat Western India findings from 200 casesrdquo The PanAfrican Medical Journal vol 10 no 48 2011

[26] A Tewari N Sood M M Vegad and D C Mehta ldquoEpidemi-ological and microbiological profile of infective keratitis inAhmedabadrdquo Indian Journal of Ophthalmology vol 60 no 4pp 267ndash272 2012

[27] R Nath S Baruah L Saikia B Devi A Borthakur and J Ma-hanta ldquoMycotic corneal ulcers in upper Assamrdquo Indian Journalof Ophthalmology vol 59 no 5 pp 367ndash371 2011

[28] M J Bharathi R Ramakrishnan RMeenakshi C Shivakumarand L L Raj ldquoAnalysis of the risk factors predisposing to fungalbacterial amp Acanthamoeba keratitis in south Indiardquo IndianJournal of Medical Research vol 130 no 6 pp 749ndash757 2009

[29] F M Polack H E Kaufman and E Newmark ldquoKeratomycosisMedical and surgical treatmentrdquoArchives ofOphthalmology vol85 no 4 pp 410ndash416 1971

[30] M Srinivasan C A Gonzales C George et al ldquoEpidemiologyand aetiological diagnosis of corneal ulceration in Maduraisouth Indiardquo British Journal of Ophthalmology vol 81 no 11pp 965ndash971 1997

[31] P AThomas A K Leck andM Myatt ldquoCharacteristic clinicalfeatures as an aid to the diagnosis of suppurative keratitis causedby filamentous fungirdquo British Journal of Ophthalmology vol 89no 12 pp 1554ndash1558 2005

[32] R Srinivasan R Kanungo and J L Goyal ldquoSpectrum of ocu-lomycosis in South Indiardquo Acta Ophthalmologica vol 69 no 6pp 744ndash749 1991

[33] P Garg U Gopinathan K Choudhary and G N Rao ldquoKer-atomycosis clinical and microbiologic experience with demati-aceous fungirdquoOphthalmology vol 107 no 3 pp 574ndash580 2000

[34] P Garg G K Vemuganti S Chatarjee U Gopinathan and GN Rao ldquoPigmented plaque presentation of dematiaceous fungalkeratitis a clinicopathologic correlationrdquo Cornea vol 23 no 6pp 571ndash576 2004

[35] M J Bharathi R Ramakrishnan R Meenakshi S Mittal CShivakumar and M Srinivasan ldquoMicrobiological diagnosis ofinfective keratitis comparative evaluation of direct microscopyand culture resultsrdquo British Journal of Ophthalmology vol 90no 10 pp 1271ndash1276 2006

[36] K Liu D N Howell J R Perfect and W A Schell ldquoMorpho-logic criteria for the preliminary identification of FusariumPaecilomyces and Acremonium species by histopathologyrdquoAmerican Journal of Clinical Pathology vol 109 no 1 pp 45ndash54 1998

[37] P A Thomas C A N Jesudasan P Geraldine and J Kaliam-urthy ldquoAdventitious sporulation in Fusarium keratitisrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 249no 9 pp 1429ndash1431 2011

[38] R K Forster G Rebell and L AWilson ldquoDematiaceous fungalkeratitis Clinical isolates and managementrdquo British Journal ofOphthalmology vol 59 no 7 pp 372ndash376 1975

[39] K R Wilhelmus and D B Jones ldquoCurvularia keratitisrdquo Trans-actions of the American Ophthalmological Society vol 99 pp111ndash130 2001

[40] S A Balajee A M Borman M E Brandt et al ldquoSequence-based identification of Aspergillus Fusarium and mucoralesspecies in the clinical mycology laboratory where are we andwhere should we go from hererdquo Journal of Clinical Microbiol-ogy vol 47 no 4 pp 877ndash884 2009

[41] M Murphy E M Bernard T Ishimaru and D ArmstrongldquoActivity of voriconazole (UK-109496) against clinical isolatesof Aspergillus species and its effectiveness in an experimen-tal model of invasive pulmonary aspergillosisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 696ndash698 1997

[42] D J Diekema S A Messer R J Hollis R N Jones and M APfaller ldquoActivities of caspofungin itraconazole posaconazoleravuconazole voriconazole and amphotericin B against 448recent clinical isolates of filamentous fungirdquo Journal of ClinicalMicrobiology vol 41 no 8 pp 3623ndash3626 2003

[43] SM Singh S Sharma andPKChatterjee ldquoClinical and exper-imental mycotic keratitis caused by Aspergillus terreus and theeffect of subconjunctival oxiconazole treatment in the animalmodelrdquoMycopathologia vol 112 no 3 pp 127ndash137 1990

[44] S Sengupta S Rajan P R Reddy et al ldquoComparative study onthe incidence and outcomes of pigmented versus non pig-mented keratomycosisrdquo Indian Journal of Ophthalmology vol59 no 4 pp 291ndash296 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom