Hindawi Publishing CorporationBioMed Research InternationalVolume 2013, Article ID 605308, 10 pageshttp://dx.doi.org/10.1155/2013/605308Clinical StudyMicroscopic Evaluation, Molecular Identification, AntifungalSusceptibility, and Clinical Outcomes in Fusarium, Aspergillusand, Dematiaceous KeratitisDevarshi U. Gajjar,1,2Anuradha K. Pal,2Bharat K. Ghodadra,2and Abhay R. Vasavada21Department of Microbiology and Biotechnology Centre, Faculty of Science, M. S. University of Baroda, Vadodara 390 002, India2Iladevi Cataract and IOL Research Centre, Ahmedabad, Gujarat 380052, IndiaCorrespondence should be addressed to Devarshi U. Gajjar; devarshimistry@yahoo.comReceived 30 April 2013; Revised 22 July 2013; Accepted 16 August 2013Academic Editor: Kelvin ToCopyright 2013 Devarshi U. Gajjar et al. Tis 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 countries.Hereinwereportaprospectivestudyonfungal keratitiscausedbythesethreefungal species. Methodology. Aprospectiveinvestigation was undertaken to evaluate eyes with presumed fungal keratitis. All the fungal isolates ( = 73) obtained fromkeratitisinfections were identifed using morphological and microscopic characters. Molecular identifcation using sequencing of the ITSregion and antifungal susceptibility tests using microdilution method were done. Te fnal clinical outcome was evaluated in termsof the time taken for resolution of keratitis and the fnal visual outcome. Te results were analyzed afer segregating the cases intothree groups, namely, Fusarium, Aspergillus, and Dematiaceous keratitis. Results. Diagnosis of fungal keratitis was established in 73(35.9%) cases out of 208 cases. Te spectra of fungi isolated were Fusariumspp. (26.6%), Aspergillus spp. (21.6%), and Dematiaceousfungi (11.6%). Te sequence of the ITS region could identify the Fusarium and Aspergillus species at the species complex level,and the Dematiaceous isolates were accurately identifed. Using antifungal agents such as fuconazole, natamycin, amphotericinB, and itraconazole, the minimum inhibitory concentrations (MICs) for Fusarium spp. were >32 g/mL, 48 g/mL, 0.51 g/mL,and >32 g/mL, 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 efective antifungal agents. Te comparative clinicaloutcomes in all cases showed that the healing response in terms of visual acuity of the Dematiaceous group was signifcantly goodwhen compared with the Fusarium and Aspergillus groups ( < 0.05). Te time required for healing in the Fusarium group wasstatistically signifcantly less when compared with the Aspergillus and Dematiaceous groups. Conclusion. Tis study demonstratesimportant diferences in microscopic features of scraping material and antifungal susceptibility between the three groups. Earlyand accurate identifcation coupled with the MIC data, and thereby appropriate treatment is crucial for complete recovery.1. IntroductionMycotic keratitis is an important ophthalmic problem caus-ing visual disability due to its protracted course and unfa-vorable responses. Te incidence of fungal keratitis has beenreported to range between 25.6% and 36.7% in various partsof India [14]. 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, 57]. Studies on their molecular identifcation, antifungalsusceptibility, and comparisons with the clinical outcomeswould be of great importance, as the pathogenic potentialmay vary between these genera. Te 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 identifcation[8].Molecular identifcation of keratitis causing FusariumandAspergillus isolates has beenreportedearlier [912].Fungal ulcers are commonly treated empirically; drugs aretypically selected without regard to susceptibility data. Tisis because the antifungal susceptibility testing takes time and2 BioMed Research Internationalneeds trained personnel to perform the testing. Tere arefour studies that report the in vitro antifungal susceptibilitypatterns among keratitis-causing fungal isolates from India[1316]. OntheotherhandnonophthalmicFusariahavebeenreportedtoexhibit greater resistancetoantifungalagents continuously over a period of time [17, 18]. Hence,periodicreportsfromdiferent geographical areaswouldhelp record the variations over a period of time and at thesame time help in modulating the current treatment options.We report a prospective study to compare diferent aspectsof fungal keratitis suchas its clinical features, microbialevaluation, molecular identifcation, antifungal susceptibility,and clinical outcomes.2. Materials and MethodsPatients were recruited afer an informed consent wasobtained fromall subjects. Te study followed the declarationof tenets of Helsinki and was approved by the InstitutionalEthical Review Board.2.1. Clinical Examination. Aprospectivestudyofpatientswith keratitis was conducted during the period from June2009 to May 2012. All the patients were examined with 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 historyof ocular surgeries,history of diabetes, and usage of topical or systemic steroids.Te same consultant doctor,as per the standard protocolapprovedbytheinstitutional reviewboard, performedathorough examination of the involved and fellow eyes. Tesame consultant ophthalmologist flled a form. In this form,thefollowingaspects weredocumented: thepresenceorabsence and form of the following clinical features, elevationofslough(raisedorfat), textureofslough(wetordry),ulcer margins (serrated or well defned), size of the abscess,pigmentation, Descemetsfolds, satellitelesions, dendriticlesions, immunering, hypopyon, fbrin, fareor cells inthe anterior chamber, deep lesions, and endothelial plaque.Clinical photographs were taken using the Haag Straight slitlamp microscope with a photo slit attachment.2.2. Clinical Specimens andMicrobiological Investigations.Corneal scrapings were taken from patients when at leastone of the following was present: size of the infltration was>2 mm with an epithelial defect, depth of the infltrate was>20%of the corneal thickness, the anterior chamber reactionwas > grade 2, evidence of any organic trauma, or failure toregress in 24 hours. Local anesthetic eye drops (proparacaine0.5%) were instilled to the afected eye to minimize oculardiscomfort andfacilitatethecorneal scrapingprocedure.Scrapings 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, bacteria,or acanthamoeba by using 10% KOH0.05% calcofuor whitestain wet mounts and Gram staining [19]. Te scraping mate-rial was also directly inoculated in blood agar, Sabouraudsdextrose agar (SDA), and chocolate agar media (Himedia,Himedia Pvt Ltd, Mumbai) which were incubated at 37Cand 28C and in 5% CO2, respectively. A diagnosis of fungalkeratitis was made when at least one of the following wasconfrmed: a corneal scraping examination revealed fungalhyphae in wet mounts or smears; the same fungus grew inthe two culture media used; or the fungus grew confuentlyat allthe inoculated sites on a single media.Microscopicpictures of the KOH wet mounts for all samples were takenunder the light microscope and fuorescence microscope. Teaverage width of 25 randomly selected hyphae, the distancebetween the septa, and the diameter of the chlamydospore-like structures whenever present were measured from digi-talizedphotographs at 400x magnifcationusing Biovis ImagePlus Sofware v.4.11 (Expert Vision, Mumbai, India). Picturesof media plates showing fungal growth were taken at 24-hour growth, 48-hour growth, and 72-hour growth. Colour,diameter, and presence or absence of spores were observedfor all fungal colonies on SDA and blood agar plates.2.3. Morphological andMolecularIdentifcation. Purecul-turesof all isolatesweremaintainedonPotatoDextroseagar (PDA). Cultures were examined using the lactophenolbluemount forsporulationat theendof 10, 20, and30days. Te morphological and microscopic identifcation wasdonebygrowthcharacteristics, andmicroscopiccharac-teristics, respectively. All morphological andmicroscopiccharacteristics were confrmed by comparing them with thecharacters given in the Atlas of clinical fungi [20]. Culturesthat failed to sporulate on SDA and PDA were subculturedonoatmeal agar andcarnationleaf agar. For molecularidentifcation of the fungus, sequencing of the ITS (internaltranscribed spacer) region was done. Te DNAwas extractedfrom the pure culture of the fungus grown on SDA usingZymo Research DNAisolation kit. Afer extraction, the DNAwas amplifed using ITS 1 (F-5

-TCCGTAGGTGAACC-3

)and ITS 4 primers (R-5

TCCTCCGCTTATTGATATGA-3

),whichamplify the followinggenesofthe fungalgenome:partial 18SrRNAgene, complete ITS1, 5.8SrRNAgeneand ITS2 regions,and partial28S rRNA gene.Annealingtemperature was 55C for 1 minute.Te size of ampliconproducedafer PCRreactionwas around500600basepairsforall fungi usedinthepresent study. Sequencingof the ITS region was done at First Base Laboratories Sdn.Bhd, Malaysia, usingprimers ITS1 andITS4. Sequenceswere obtainedusingbothforwardandreverse primers.Chromatogram processing,quality control,and editing ofthe sequences were done using BioEdit Sofware. Bothsequences were aligned, and a fnal sequence was created.Tisfnal sequencewasusedfortheBLASTNsimilaritysearch (http://www.ncbi.nlm.nih.gov/BLAST) and was alsosubmitted to NCBI. For identifcation, only complete ITS1-5.8S-ITS2 entries of reference isolates in the BLAST databasewere taken into consideration. Complete identifcation wasconsidered when a percent sequence similarity of >98% witha BLAST search expected value of zero was obtained.BioMed Research International 32.4. Antifungal Susceptibility Testing. In vitro antifungal sus-ceptibilitytestingwas doneagainst natamycin(Natamet;5% suspension; Sun Pharmaceuticals Ind. Ltd, Halol, India),itraconazole(Itral; 1%suspension; JawaPharmaceuticals,Gurgaon, India), fuconazole (Nuf ucon; 0.3% suspension;NuLife Pharmaceuticals, Pune, India), and amphotericin B(RM462, HimediaLabs Ltd, Mumbai, India) usingthemicrodilutionmethodandfollowing the Clinical andLabora-tory Standards Institute (CLSI) guidelines [21]. All antifungalagents were dissolved in DMSO and fuconazole was dis-solved in water. Te inoculums were prepared by covering the7-day-old culture plate with normal saline (0.85%NaCl). Tiswas followed by gentle probing of the colonies with the help ofa pipette and adjusting the densities of the suspension (read at530 nm) to a fnal inoculum of 0.5 McFarland standard. Tefnal drug concentration range prepared using serial dilutionwere 0.008 to 132 g/mL for all the four antifungal agents. Allthe antifungal agents were tested in RPMI 1640 media with2% glucose and without sodium carbonate.2.5. Treatment Regime and Evaluation of Clinical Outcomes.Subsequent tothemicroscopicexamination, if apositivereport of fungal flaments 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 frst three days followed by two-hourly dropsduring waking hours until resolution of the ulcers. Patientsalso received 1% atropine sulphate eye drops. Systemic fu-conazole (150 mg once a day) was prescribed for all patientswith corneal stromal infltrate extending beyond one-thirdof the cornea. Afer treatment, an ulcer was considered tobe healed when the epithelial defect was 4 mm) in the central visual axis inall the three groups. Further, the presence of satellite lesions,ring infltrate, dry appearance, and stromal involvement wasevident in most of the cases. Te presence of hypopyon andpigmentation was mainly associated with the Dematiaceousgroup, and this attained statistical signifcance. Te presenceof endothelial plaque was mainly associated with FusariuminfectionswhereasdendriticlesionsandDescemetsfoldswere mainly observed in the Aspergillus group.3.2. Microscopic Evaluation and Growth Characteristics of theScraping Material. All the samples of scraping material takenfromthe three groups showed the presence of large quantitiesof fungal flaments when seen under light and fuorescencemicroscopes (Figure 1).Samples from Fusarium infections(Figures 1(a) and 1(b)) showed the presence of fungal hyphaewithanaveragethickness 3.87 0.6 m. Septawerenotvisibleunderthelight microscope(Figure 1(a))but wereclearly seen under the fuorescence microscope (Figure 1(b)).Tedistancebetweentheseptawas 21.65 4.2 m. Teaverage hyphal thickness in the Aspergillus group (Figures1(c) and 1(d)) was 4.13 0.65 m, and this was almost similarto the hyphalthicknessin the Fusariumgroup.However,thedistancebetweentheseptawas 12.84 1.9 m. Teaveragehyphal thicknessintheDematiaceousgroupwas8.79 0.9 m (Figures 1(e) and 1(f)). Terminal and internalchlamydospore-likestructureswithadiameterof 9.44 1.11 m were seen exclusively in all four samples from theFusariumdelphinoidesgroup(Figures2(a)and2(b)). Tescraping materialfrom Curvulariainfections also showedlargequantitiesof chlamydospore-likestructureswithanaverage diameter of 11.15 1.58 m (Figures 2(c) and 2(d)).Tese structures were absent in all the remaining samples ofthe Dematiaceous group. A huge variation in microscopicfeatureswasnoticedintheDematiaceousgroup(Figures3(a)3(d)). Scrapingmaterial, other thanthe Curvulariainfection, showed a typical arrangement of septa.4 BioMed Research InternationalTable 1: Epidemiologic characteristics, risk factors, and clinical features.Variable Fusarium spp. ( = 26) Aspergillus spp. ( = 15) Dematiaceous isolates ( = 12)Age (in years) 41.84 52.46 46.5Gender ratio (male : female) 2.71 2 1.4Risk factorsTrauma other than vegetative body 1 (3.8%) 4 (26.6%) 0Trauma with vegetative body 11 (42.3%)0 4 (33.3%)Prior ocular surgery/infection 1 (3.8%) 5 (33.3%) 1 (8.3%)Seasonal distributionSummer 6 (23%) 3 (20%) 1 (8.3%)Monson 4 (15.3%) 7 (46.6%) 3 (25%)Winter 14 (53.8%) 7 (46.6%) 6 (50%)Clinical featuresCentral location 23 (88.4%) 12 (80%) 11 (91.6%)Size (>10 mm) 16 (61.5%) 9 (60%) 11 (91.6%)Hypopyon 10 (38.4%) 5 (33.3%) 9 (75%)Elevated slough 19 (73.0%) 6 (40%) 10 (83.3%)Dry texture 20 (76.9%) 7 (46.6%) 9 (75%)Serrated ulcer margins 21 (80.7%) 9 (60%) 8 (66.6%)Pigmentation 0 1 (6.6%) 5 (41.6%)Endothelial plaque 4 (15.3%) 0 0Dendritic lesions 0 2 (13.3%) 0Satellite lesions 15 (57.6%) 8 (53.3%) 3 (25%)Mean number of days for complete healing 57.4114.8 125.6Group signifcantly higher compared to other.A total of 47 (88.67%) out of 53 samples showed visiblegrowth on all the media inoculated at 24 hours (Figure 4(a)).In the Fusarium group, 23 (88.4%) samples showed growthwithin 24 hours, while 3 samples showed growth within 48hours. In the Aspergillus group, 13 (86.66%) samples showedgrowth within 24 hours and the remaining 2 samples showedgrowth within 48 and 72 hours, respectively. Te 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, 11/12 (91.6%) 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 Lasiodiplodiatheobromae obtainedfromthe scrapingmaterial was grown for 48 hours and a gray fufy growth withabundant 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.3.3. MicroscopicandMolecularIdentifcationof Fusarium,Aspergillus and Dematiaceous Spp. All isolates in the Fusar-iumandAspergillus groups wereidentifedtothegenuslevel by means of their morphological characteristics. Temorphological evaluation of Fusarium solani appeared to bestraight forward and this was further confrmed using theITS sequences. However, when the sequences were evaluatedusingtheFusariumMLSTwebsite, thematchwastotheFusarium 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 ( = 4) were identifed as Fusarium dimerumusing their morphological features. However, identifcationusing the ITS sequences at NCBI BLAST was F. delphinoidesisolates ( = 4), Fusarium dimerum ( = 1), and Fusariumdelphinoides ( = 3) usingtheMLSTdatabase. IntheAspergillus group, A. niger, A. favus, A. terreus, andA.fumigatus were identifed using morphological features. A.tamarii,A.tubingensis,A.versicolor,and A.sydowii wereonly identifed when the ITS sequences were available. Inthe Dematiaceous group, Curvularia lunata and Exserohilumrostratum were identifed using their growth characteristicsand typical spores,and this was confrmed using the ITSsequence. Using ITS sequences, all the other dematiaceousisolates were identifed as Lasiodiplodia theobromae, Clador-rhinum bulbilosum, and Cladosporium cladosporioides. TeITS sequence misidentifed only one isolate as Chaetomiumspp. It was later identifed as Papulaspora spp. on the basis ofits typical microscopic features.3.4. 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 natamycinarethemost efectiveantifungal agentsagainstFusariumspp. In the Aspergillus group, amphotericin B and itracona-zole showed the lowest MIC against A.favus,A.terreus,BioMed Research International 5(a) (b)(c) (d)(e) (f)Figure 1: 10% KOH mount of the scrapping material showing fungal hyphae, magnifcation 400. Lef panel: light microscopic picture, rightpanel: fuorescent microscopic picture taken afer calcofuor 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 andamphotericinBshowedgoodinvitroactivityagainst A.nigerandA. sydowii. IntheDematiaceousgroup, exceptCurvularia, all the other isolates were highly susceptible tothe antifungal agents tested. Amphotericin B and natamycinshowed good in vitro activity against Curvularia lunata.3.5. Evaluation of Clinical Outcomes. Table 3 shows the com-parative clinical outcomes in all cases. In the Fusariumgroup,20 cases healed and 3 worsened, while 3 were lost to follow-up.Te minimum time required for healing was 14 days,whereasthemaximumtimetakentoheal inthecaseofone patient was 300 days. In the Aspergillus group, 12 outof 15 cases healed with topical and oral antifungal medicaltreatment and two cases required therapeutic keratoplasty,whereas one case caused by A. tamarii worsened. Teminimum 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. Teminimum time taken to heal was 7 days while the maximumtime was 300 days. Te healing response in terms of visualacuityof theDematiaceousgroupwassignifcantlygoodwhen compared with the Fusarium and Aspergillus groups( < 0.05). Te time required for healing in the Fusarium6 BioMed Research International(a) (b)(c) (d)Figure 2: 10%KOHmount of the scrapping material showing fungal hyphae, magnifcation 400. Lef panel: light microscopic picture, rightpanel: fuorescent microscopic picture taken afer calcofuor white stain. (a and b) Scrapping material from Fusarium delphinoides infections.(c and d) Scrapping material from Curvularia infections.group was statistically signifcantly less when compared withthe Aspergillus and Dematiaceous groups.4. DiscussionIn the present study of 208 keratitis patients, fungal etiologywasconfrmedin35%ofthecases, whereFusariumspp.was the most common isolate followed by Aspergillus andDematiaceous. Tis is comparable to most studies fromIndia[15]. 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 mostcommonfungi. WefoundtworeportsfromAhmedabad,Fusarium was reported in a study conducted in 20032005[25] while Aspergillus was the most common isolate reportedin a study conducted in 2007-2008 [26]. We have obtained adefnite history of trauma with vegetative/agricultural 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%, 78.5%,and 61.2% of cases of keratitis in studies from Assam [27],Gujarat [25], and Tamil Nadu [28]. Fungal keratitis is morefrequently reported in winter with a humid climate favoringfungal growth [29, 30].Te 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]andMadurai [30, 32]. Tepigmentedplaquelikethepresentation 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].Direct microscopy is an important diagnostic modality ininvestigating microbial keratitis, and a highest sensitivity at99%is reported [35]. Te addition of calcofuor white (CFW)staintothe diagnostic armamentariumhas signifcantlyincreasedthesensitivityof smear examinationondirectmicroscopy[19]. However, itisdifculttodeterminethegenus of fungi from KOH mounts [35]. Preliminary identif-cation of Fusarium and Aspergillus species using microscopicfeatures in histological specimens has been reported [36].Identifcation of Fusarium fromscraping material is reportedby the detection of adventitious sporulation [37]. Te 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 fnd any remarkablediferencesbetweentheFusariumandAspergillus groupsexcept that thedistancebetweentheseptawaslargerinBioMed Research International 7(a) (b)(c) (d)Figure 3: Light microscopic pictures of 10% KOH mount of the scrapping material showing fungal hyphae, magnifcation 400. (ad)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-MIC (range)Natamycin Amphotericin B Fluconazole ItraconazoleFusarium solani ( = 22) 29.3 (8128) 6.3 (132) 128 (128) 113.7 (64128)Fusarium delphinoides ( = 4) 12 (816) 12 (816) 128 (128) 128 (128)Aspergillus favus ( = 6) 32 (32) 0.35 (0.20.5) 128 (128) 1.5 (12)Aspergillus terreus ( = 3) 13.3 (816) 2.5 (13) 26.6 (1632) 0.25 (0.25)Aspergillus niger ( = 1) 8 0.06 128 64Aspergillus tamarii ( = 1) 64 0.25 128 0.25Aspergillus tubingensis ( = 1) 8 2 32 0.25Aspergillus sydowii ( = 1) 4 4 128 128Aspergillus versicolor ( = 1) 4 2 32 0.25Curvularia lunata ( = 4) 12.5 (232) 16 (0.2532) 128 (128) 128 (128)Exherohelum rostratum ( = 2) 2 (24) 1 (0.52) 32 (32) 0.25 (0.250.5)Cladorrhinum bulbilossum ( = 1) 0.064 0.064 0.32 0.016Lasiodiplodia theobromae ( = 1) 0.064 0.064 0.32 0.016Papulaspora spp. ( = 1) 0.064 0.064 0.32 0.016Geometric 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.Molecularidentifcationof keratitiscausingFusariumandAspergillus isolates has beenreportedearlier [912].8 BioMed Research International(a) (b)(c) (d)(e) (f)(g)Figure 4: Growth of fungi from scrapping material afer (a) 24 hrs, (b and c) Fusarium and Aspergillus samples afer 48 hrs, (d and f)Dematiaceous samples, and (g) Lasiodiplodia sample.Table 3: Clinical outcomes of Fusarium, Aspergillus, and Dematiaceous keratitis.GroupsMean visual acuitybefore treatment(LOGMAR)Mean visual acuity afertreatment (LOGMAR)Change in visual acuityWorsenedPoor response(No PL to PL/PL to PR)Slight response(improved Snelens/CF to Snelens)Good response(PL to CF/Snelens)Fusarium 1.82 (0.83) 1.17 (03) 3 (20%) 8 (53.3%) 3 (20%) 1 (6.7%)Aspergillus 2.08 (0.483) 1.68 (03) 4 (16.7%) 12 (49.9%) 5 (20.9%) 3 (12.5%)Dematiaceous 2.45 (0.483) 0.91 (02) 0 3 (30%) 7 (70%)0P value