Medical Mycology, 2015, 0, 1–8doi: 10.1093/mmy/myv105

Advance Access Publication Date: 0 2015Original Article

Original Article

Antifungal Susceptibility and Phylogeny of

Opportunistic Members of the Genus Fusarium

Causing Human Keratomycosis in South India

Anamangadan Shafeeq Hassan1,†, Abdullah M.S. Al-Hatmi2,3,4,†,Coimbatore Subramanian Shobana5, Anne D. van Diepeningen2,

Laszlo Kredics6, Csaba Vagvolgyi6,7, Monika Homa6, Jacques F. Meis8,9,

G. Sybren de Hoog2,3,10, Venkatapathy Narendran11,

Palanisamy Manikandan11,12,13,∗ and IHFK Working Group

1Department of Microbiology, Dr. G.R. Damodaran College of Science, Coimbatore, India, 2CBS-KNAWFungal Biodiversity Centre, Utrecht, the Netherlands, 3Institute of Biodiversity and Ecosystem Dynamics,University of Amsterdam, Amsterdam, the Netherlands, 4Directorate General of Health Services, Ministryof Health, Ibri Hospital, Ibri, Oman, 5Department of Microbiology, PSG College of Arts & Science, Coim-batore, India, 6Department of Microbiology, Faculty of Science and Informatics, University of Szeged,Szeged, Hungary, 7Botany and Microbiology Department, King Saud University, Riyadh, Kingdom of SaudiArabia, 8Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital,Nijmegen, the Netherlands, 9Department of Medical Microbiology, Radboud University Medical Center,Nijmegen, the Netherlands, 10Basic Pathology Department, Federal University of Parana State, Curitiba,Parana, Brazil, 11Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, India,12Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah Univer-sity, Kingdom of Saudi Arabia and 13Greenlink Analytical and Research Laboratory India Private Ltd.,Coimbatore, India.∗To whom correspondence should be addressed. P. Manikandan, Department of Microbiology, Aravind Eye Hospital andPostgraduate Institute of Ophthalmology, Coimbatore, Tamilnadu, India, Department of Medical Laboratory Sciences,College of Applied Medical Sciences, Majmaah University, Kingdom of Saudi Arabia and Greenlink Analytical andResearch Laboratory India Private Ltd., Coimbatore, India. Tel: +91 422 4360400; E-mail:manikandanpalanisamy@gmail.com†These authors contributed equally to this work.

Received 26 August 2015; Revised 11 November 2015; Accepted 15 November 2015

Abstract

Fusarium species are reported frequently as the most common causative agents of fungalkeratitis in tropical countries such as India. Sixty-five fusaria isolated from patients weresubjected to multilocus DNA sequencing to characterize the spectrum of the species asso-ciated with keratitis infections in India. Susceptibilities of these fusaria to ten antifungalswere determined in vitro by the broth microdilution method. An impressive phylogeneticdiversity of fusaria was reflected in susceptibilities differing at species level. Typing re-sults revealed that the isolates were distributed among species in the species complexes

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(SCs) of F. solani (FSSC; n = 54), F. oxysporum (FOSC; n = 1), F. fujikuroi (FFSC; n = 3),and F. dimerum (FDSC; n = 7). Amphotericin B, voriconazole, and clotrimazole proved tobe the most effective drugs, followed by econazole.

Key words: Keratitis, Bisifusarium, Neocosmospora, treatment options.

Introduction

Diseases affecting the cornea are a major cause of blindnessworldwide and comprise a large variety of infectious and in-flammatory eye diseases.1 In tropical southern India, whichlocally has wet or dry climatic conditions and is semi-aridin certain regions, the majority of the people are engaged inagriculture. This profession carries a risk for corneal fungalinfection from plant associated sources. Species of Fusar-ium are commonly occurring environmental fungi that po-tentially cause eye infections in humans, with keratitis beingone of the most frequent clinical manifestations.2,3,4,5

Historically, the genus Fusarium has developed intoa large genus with multiple species complexes, some ofwhich can cause opportunistic infections in humans. Es-pecially members of the Fusarium solani, F. oxysporum,F. fujikuroi, F. dimerum, F. chlamydosporum, and F.incarnatum-equiseti species complexes have been involvedin human and animal infections.6,7 In 2013, Geiser8 andmany coauthors from clinical and phytopathological back-grounds have launched a plea for nomenclatural stabilityand the preservation of the name Fusarium for the clini-cally relevant species complexes. However, recently Lom-bard et al.9 moved F. solani to the genus Neocosmospora,while the F. dimerum species complex was transferred to thenew genus Bisifusarium. A counterproposal for Fusicolla asgenus name for the F. solani complex is in preparation. Topreserve the nomenclature stability, we adhere in this paperto Fusarium as best known descriptor for the etiologicalagents observed in the study.

Although culture techniques remain the cornerstone forthe diagnosis of most cases of fungal keratitis,2 misdiagnosisor lack of advanced investigative tools to accurately identifythe causal agents may potentially lead an ophthalmologistto initiate inappropriate antifungal therapy.10 In this con-text, it is becoming increasingly evident that efficient ther-apy of keratitis relies on precise identification of the fungalpathogens to the species level using advanced and reliablemolecular methods. Furthermore, treatment is limited bythe restricted number of prescribed antifungals for specificophthalmic use and especially local (un)availability of thecompounds themselves. Conventional treatment varies percountry and depends on obtainability and registration ofantifungal drugs. In India, Fusarium keratomycosis is oftentreated with topical natamycin, while for instance in theNetherlands natamycin is restricted for use as food preser-

vative and hence amphotericin B and voriconazole are thedrugs of choice there.

Antifungal susceptibilities of the different Fusarium SCsvary, and fusaria show remarkable resistance to most clini-cally applied antifungal drugs.11,12,13 Therefore, the presentstudy was undertaken with the following objectives: (i) todetermine the species complex level of Fusarium isolatesfrom human keratomycosis (South India) based on the par-tial sequences of the second-largest subunit of RNA poly-merase II (RPB2) and translation elongation factor 1 α

(TEF1); and (ii) to determine the in vitro susceptibilitiesof Fusarium isolates to ten commonly used antifungals.

Materials and methods

Isolates

A total of 1628 corneal scrapings of human keratomycosispatients attending Aravind Eye Hospital and Postgradu-ate Institute of Ophthalmology (Coimbatore, Tamilnadu,India) during the years 2012 and 2013 were processed forisolation of the causative agent as described before.14 Ini-tial identification of Fusarium isolates was based on colonymorphology on potato dextrose agar (PDA - 250 g of potatoslices, 15 g agar, 10 g dextrose, and 1000 ml distilled wa-ter) plates and microscopic feature in a lactophenol wetmount preparation.15 Isolates that grew after subculturingtwice on PDA were deposited in the reference collection ofthe CBS-KNAW Fungal Biodiversity Centre in Utrecht, TheNetherlands.

DNA extraction

DNA extraction was performed using glass beads (SigmaG9143, Sigma-Aldrich Co, Missouri, USA) according tothe protocol described by Al-Hatmi et al.16

DNA amplification and sequencing

Two gene regions were amplified directly from the ex-tracted genomic DNA for multilocus sequencing. Theprimer pairs for TEF1 and RNA polymerase II were EF1and EF217 and RPB2-7cR and RPB2 -5F, respectively.18

Polymerase chain reaction (PCR) products were visualizedand checked by electrophoresis on a 1% (w/v) agarose gel.

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Amplicons were purified using exoSAP. The ABI PrismHBig DyeTM Terminator v. 3.0 Ready Reaction Cycle Se-quencing Kit (Applied Biosystems, Foster City, USA) wasused for sequencing PCR. Sequencing reaction was per-formed as follows: 1 min at 95◦C, followed by 30 cyclesconsisting of 10 sec at 95◦C, 5 sec at 50◦C, and 2 min60◦C. Reactions were purified with Sephadex G-50 fine(GE Healthcare Bio-Sciences, Uppsala, Sweden), and se-quencing was done on an ABI 3730XL automatic sequencer(Applied Biosystems, Foster City, USA) with ABI PRISMBigDyeTM terminator cycle sequencing kit. A BLASTsearch of TEF1 and RPB2 sequences against the databaseFUSARIUM-ID (http://isolate.fusariumdb.org), the Fusar-ium MLST (http://www.cbs.knaw.nl/fusarium) and Gen-Bank databases (www.ncbi.nlm.nih.gov) were used as aninitial step to identify isolates to species and/or species com-plex. The TEF1 and RPB2 nucleotide sequences for all theisolates were deposited in GenBank.

Phylogenetic analyses

Sequences were edited using SeqMan in the Lasergenepackage (DNAstar, Madison, USA). A phylogenetic ap-proach was used to investigate relationship between 65strains of Fusarium species including type and referencestrains. The sequences were aligned using MAFFT v.7.127 (http://mafft.cbrc.jp/alignment/software/), followedby manual adjustments with MEGA v. 6.19 A combinedalignment was constructed for TEF1 and RPB2 for both thereference and test strains. The best-fit model of evolutionwas determined by jModelTest v. 0.1.1.20 A Maximum-Likelihood tree was constructed by using RAxML (7.6.6)21

with 1000 bootstrap replicates and edited with MEGA v. 6software 6.19

In vitro susceptibility testing

The following clinically used and commercially availableantifungals were employed in the antifungal susceptibilitytests: amphotericin B (AMB), nystatin (NYT), ketocona-zole (KTZ), fluconazole (FLZ), and miconazole (MCZ)[HiMedia, Mumbai, India]; voriconazole (VRZ), econazole(ECZ) and clotrimazole (CLZ) [Aurolab, Madurai, India];natamycin (NTM) and itraconazole (ITZ) [Sigma-Aldrich,St. Louis, MO, USA]. RPMI 1640 medium was used forpreparing the drug solutions and the final drug concentra-tions ranged from 64 to 0.125 μg ml−1. Stocks were storedat −20◦C until needed. Inoculum preparation and determi-nation of the minimum inhibitory concentrations (MICs)were done following the guidelines of the Clinical and Lab-oratory Standards Institute (CLSI) M38-A2 document.22

Experiments were performed in triplicate. Aspergillus flavusATCC 204304 was included in the tests as a quality controlstrain.

Results

Identification of Fusarium isolates and phylogeny

Of 1628 specimens, a total of 624 (38.3%) were culturepositive for different microbial pathogens and 417 (66.8%of 624) keratitis cases were found to be caused by fungalisolates, out of which 196 isolates (47% of 417) were con-firmed as Fusarium spp. A total of 65 Fusarium isolateswere chosen for further analyses. Upon molecular identifi-cation based on TEF1 and RPB2 partial genes analysis, themajority of Fusarium isolates were found to be membersof FSSC: F. falciforme (n = 45) followed by F. keratoplas-ticum (n = 8), and F. lichenicola (n = 1). We also identifiedmembers of other species complexes: F. oxysporum (n =1) in FOSC, F. nygamai (n = 1) and F. sacchari (n = 2) inFFSC, as well as F. delphinoides (n = 6) and F. dimerum(n = 1) in FDSC.

In order to obtain understanding of the phylogenetic po-sitions of species found in this study, they were compared toreference sequences from the GenBank and CBS databases(n = 9). A phylogenetic tree (Fig. 1) was constructed with atotal of 74 sequences for two genes, with F. dimerum (syn-onym Bisifusarium dimerum) as outgroup as other studiesconfirm this group as the most basal lineage.8 The combinedphylogenetic analysis of TEF1 and RPB2 (1215-bp align-ment) of 74 strains could be aligned confidently over theentire genus Fusarium. The tree topologies of these two lociappeared to be strictly concordant and effective for geno-typing all species concerned. The generated tree separatedinto four clades. Clade 1 included all members of FSSC,clade 2 represented FOSC, clade 3 the FFSC, while clade 4the FDSC. The final result of the identification process wasthat 54 isolates were confirmed as members of FSSC, oneisolate belonged to FOSC, three isolates to FFSC and sevento FDSC.

Antifungal susceptibility

Ten polyene and azole antifungal compounds used beforeto treat keratomycoses were tested in our study: PolyenesAMB and NTM are both commonly used to treat fungalkeratitis, while NYT is somewhat less potent and hence lesscommonly used clinically. AMB is used both for treatmentof yeasts and filamentous fungi, while NTM proves to havea broader spectrum against filamentous fungi and has a bet-ter penetration of the intact corneal epithelium. The otherstudied antifungal drugs are all azoles.

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Figure 1. Phylogenetic tree resulting from RAxML analysis for the TEF1 and RPB2 genes (values over 70% for maximum likelihood are shown).List of the 65 isolates examined in this study including their Genbank accession numbers, CBS number and others. FDSC or Fusarium dimerumspecies complex corresponds to the genus Bisifusarium, while FSSC or Fusarium solani species complex corresponds to Neocosmospora accordingto Lombard et al.9 As outgroup the FDSC group has been used including the epitype (ET) strain of F. dimerum. CBS 108944.

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Table 1. Distribution of the tested Fusarium species complexes according to their minimum inhibitory concentrations (MICs).

MIC (μg ml−1)

Species complex1 Antifungal agent 0.125 0.25 0.5 1 2 4 8 16 32 64

FSSC (n = 54) Amphotericin B 4 8 12 20 10Natamycin 1 12 38 3Nystatin 37 17Voriconazole 1 1 11 28 13Econazole 7 20 27Clotrimazole 1 10 31 11 1Ketoconazole 1 2 11 14 26Itraconazole 1 6 6 17 24Miconazole 1 3 19 31Fluconazole 16 38

FOSC (n = 1) Amphotericin B 1Natamycin 1Nystatin 1Voriconazole 1Econazole 1Clotrimazole 1Ketoconazole 1Itraconazole 1Miconazole 1Fluconazole 1

FFSC (n = 3) Amphotericin B 1 2Natamycin 1 2Nystatin 2 1Voriconazole 1 2Econazole 1 2Clotrimazole 3Ketoconazole 1 1 1Itraconazole 2 1Miconazole 3Fluconazole 1 2

FDSC (n = 7) Amphotericin B 1 6Natamycin 1 6Nystatin 6 1Voriconazole 2 3 2Econazole 1 3 1 1 1Clotrimazole 1 5 1Ketoconazole 1 1 1 1 2 1Itraconazole 1 1 2 2 1Miconazole 1 3 1 2Fluconazole 3 4

1F. solani species complex (FSSC); F. oxysporum species complex (FOSC); F. fujikuroi species complex (FFSC); F. dimerum species complex (FDSC) isolates.

The lowest MIC of 0.5 μg ml−1 AMB and VRZ wasrecorded for four (F. falciforme strains ASH 134, ASH 139,ASH 144) and one (F. falciforme strain ASH 142) FSSCisolates, respectively. NTM and FLZ inhibited most of theFSSC isolates (38, 70%) at a concentration of 16 μg ml−1.The FOSC member (n = 1) was inhibited at 1 μg ml−1

of AMB. For FFSC isolates, 4 μg ml−1 of CLZ followed byAMB and VRZ was found to be effective. Most of the FDSCmembers were inhibited at 4 μg ml−1 of CLZ (n = 5) and8 μg ml−1 of NYT (n = 6) (Table 1). Approximately 80%of the Fusarium isolates were susceptible to concentrationsof ≤ 4 μg ml−1 of AMB (84%), VRZ (80%), CLZ (78%).

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Nearly half of the isolates were susceptible to concentra-tions ≤ 4 μg ml−1 of ECZ (53%), and MCZ (43%), whilelesser number of isolates showed susceptibility to similarlevels of KTZ (29%), ITZ (13%) and NTM (1.5%).

Discussion

Fungal keratitis occurs more frequently in dry and warmenvironments, especially people with outdoor professions,such as agricultural workers are prone to this infection.23

In India, Fusarium24 and Aspergillus25 species are reportedto be the most common causative agents of fungal kerati-tis. In this study, Fusarium species obtained after processingcorneal scrapings from human keratomycosis cases were in-vestigated. Molecular identification was performed for 65clinical Fusarium isolates using TEF1 and RPB2. Both genespossessed enough polymorphism to serve as excellent mark-ers with 99–100% accuracy for identification of Fusariumat the species level with sequences deposited in GenBank,the Fusarium-ID and the Fusarium MLST databases.

The largest part of the identified strains (n = 54) provedto belong to FFSC. Of these, 45 were identified as F. fal-ciforme and 8 as F. keratoplasticum, suggesting a signif-icant pathogenic potential of these species and/or a com-mon prevalence in the environment.26,27 As shown by themolecular analysis of combined sequence data of TEF1 re-gions and RPB2 (Fig. 1), F. lichenicola formed a distinctgroup. This species was first described as Cylindrocarponbut using phylogenetic data, Summerbell and Schroers28

showed a relationship with F. keratoplasticum and F. falci-forme (separated at 94% bootstrap support). Support val-ues above 75% were found between other species in Fusar-ium. The present study confirms the genetic diversity of clin-ical strains in the FSSC, as reported in earlier studies.29,30

In addition to the FSSC in our phylogenetic analysis, somestrains were nested within known phylogenetic species ofFOSC, FFSC, and FDSC. Homa et al.24 reported similar dis-tributions; most Fusarium isolates from human keratomy-cosis are proven to be members of FSSC (n = 53), while oc-casional isolates belonging to FDSC (n = 6), FFSC (n = 6),FOSC (n = 3), and F. incarnatum- equiseti SC (n = 2) werealso encountered.

Species delimitation within FOSC has been studied in-tensively by O’Donnell et al.31 We observed only a singleFOSC infection in our study, suggesting that these speciesoccur only at low frequency in India. Seventy-three Fusar-ium species have been reported to cause human infection,15 of which belonged to FFSC including F. temperatum andF. musae.32 In this study we report two species belonging tothe FFSC complex, F. nygamai and F. sacchari. Accordingto Guarro et al.6 three recognized members of FDSC viz.,

F. dimerum, F. penzigii, and F. delphinoides and two un-named lineages are also involved in human infections: thecurrent study yielded one isolate of F. dimerum and 6 of F.delphinoides belonging to FDSC.

Antifungal susceptibilities of clinical isolates were ana-lyzed and the results were comparable to those reported inthe current literature. In the present study 82% of FSSCisolates were inhibited by ≤ 4 μg ml−1 of AMB followedby CLZ (78%) and VRZ (76%). ITZ was found to be themost ineffective antifungal agent, which corresponds withthe findings of Homa et al.24, Xie et al.33, and Al-Hatmiet al.12. In contrast to our findings, O’Day et al.34 statedthat AMB was ineffective against Fusarium. Among theFSSC members, F. falciforme (n = 3) and F. keratoplasticum(n = 1) were inhibited at the lowest concentration of 0.5 μgml−1 of AMB and one isolate of F. falciforme was inhibitedat 0.5 μg ml−1 of VRZ. Of FOSC and FFSC, only one andthree clinical isolates, respectively, were available in thisstudy. Due to the small sample size and high variability ofMIC data it was not possible to make an overall statementabout the susceptibility profile of these three species. How-ever, one isolate of F. sacchari had a MIC of 0.5 μg ml−1

of VRZ and 1 μg ml−1 of AMB was effective against F.oxysporum. Only 1.5% of all isolates tested in the currentstudy were susceptible (MIC ≤ 4 μg ml−1) to NTM. Con-trary to this, Xie et al.33 stated that 94.2% and 91.3% ofFSSC and FOSC members, respectively, were susceptible toNTM. Lalitha et al.35 and Xuguang et al.36 also reportedgood activity of NTM against Fusarium. However, in thepresent study, 95.38% of all the isolates tested were sus-ceptible to the NTM concentration ≤ 16 μg ml−1. Topicaluse of NTM is common practice in India but also approvedfor ophthalmic use in the United States. The discrepancybetween the high in vitro MICs and clinical success maybe explained by the effective corneal penetration of the an-tifungal and the resulting higher local concentration com-pared with many other drugs.

AMB was found to be the most effective drug againstFDSC isolates. Among six isolates of F. delphinoides, onehad low MIC (0.5 μg ml−1) values for ECZ, KTZ, ITZ,and MCZ. In the present study, most of the FSSC members(n = 24) were inhibited at 32 μg ml−1 of ITZ. Lalithaet al.35 and Alfonso et al.37 reported that the MIC90 ofITZ for Fusarium sp. was > 8 μg ml−1 and 16 μg ml−1,respectively. A total of 53% and 78% of Fusarium strainstested had low MICs of ≤ 4 μg ml−1 of ECZ and CLZ,respectively. The results correlated with those of Shobanaet al.38 Overall, 84%, 80% and 78% of the tested fusariawere inhibited at ≤ 4 μg ml−1 of AMB, VRZ, and CLZ,respectively. Twenty-six isolates of FSSC had MICs of 16 μgml−1 of KTZ. Pujol et al.39 and Xie et al.33 reported higher

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MICs of KTZ in Fusarium. In our study, 69% of the isolateswere inhibited at 8 μg ml−1 and 16 μg ml−1 of NYT andFLZ, respectively. A total of 37 isolates had 8 μg ml−1 MICfor MCZ.

It is concluded that the majority of Fusarium isolatestested belong to FSSC. Based on the observed MIC data inthe present study, we report that AMB, VRZ, and CLZ at≤ 4 μg ml−1 and FLZ and NTM ≤ 16 μg ml−1 are potentialantifungal agents for the treatment of human keratomycosiscaused by Fusarium. However, application of the drugs andtheir penetration into the affected area should be taken intoaccount as well.

Acknowledgements

This study was partially funded by the Ministry of Health, Oman(Formal Agreement no. 28/2014). Abdullah M. S. AL-Hatmi receiveda PhD scholarship from the Ministry of Health, Oman. The studywas also supported by the Indian National Science Academy andthe Hungarian Academy of Sciences within the frames of the Indo–Hungarian bilateral exchange program (INSA-HAS Project 2013–2015/189). Csaba Vagvolgyi thanks the visiting professor program,Deanship of Scientific Research at King Saud University, Riyadh.

The Indian research group was supported by the UniversityGrants Commission (UGC), Bahadur Shah Zafar Marg, New Delhi-110 002, India [F. No. 42–469/2013 (SR)].

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and the writing of the paper.

References

1. Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness:a global perspective. Bull World Health Organ 2001; 79: 214–221.

2. Thomas PA. Fungal infections of the cornea. Eye 2003; 17: 852–862.

3. Gopinathan U, Sharma S, Garg P et al. Review of epidemiolog-ical features, microbiological diagnosis and treatment outcomeof microbial keratitis: Experience of over a decade. Indian JOphthalmol 2009; 57: 273–279.

4. Nath R, Baruah S, Saikia L et al. Mycotic corneal ulcers in upperAssam. Indian J Ophthalmol 2011; 59: 367–371.

5. Rautaraya B, Sharma S, Kar S et al. Diagnosis and treatmentoutcome of mycotic keratitis at a tertiary eye care center in east-ern India. BMC Ophthalmol 2011; 11: 39. doi: 10.1186/1471-2415-11-39.

6. Guarro J. Fusariosis, a complex infection caused by a high di-versity of fungal species refractory to treatment. Eur J Clin Mi-crobiol Infect Dis 2013; 32: 1491–1500.

7. van Diepeningen AD, Al-Hatmi AMS, Brankovics B et al. Tax-onomy and clinical spectra of Fusarium species: Where do westand in 2014? Clin Microbiol Rep 2014; 1: 10–18.

8. Geiser DM, Aoki T, Bacon CW et al. One fungus, one name:defining the genus Fusarium in a scientifically robust way thatpreserves longstanding use. Phytopathology 2013; 103: 400–408.

9. Lombard L, van der Merwe NA, Groenewald JZ et al. Genericconcepts in Nectriaceae. Stud Mycol 2015; 80: 189–245.

10. Manikandan P, Doczi I, Kocsube S et al. Aspergillus species inhuman keratomycosis. In: Varga J, Samson RA (eds), Aspergillusin the genomic era. Wageningen: Wageningen Academic Publish-ers, 2008: 293–328.

11. Azor M, J Gene, Cano J et al. Less-frequent Fusarium species ofclinical interest: correlation between morphological and molec-ular identification and antifungal susceptibility. J Clin Microbiol2009; 47: 1463–1468.

12. Al-Hatmi AM, van Diepeningen AD, Curfs-Breuker I et al.Specific antifungal susceptibility profiles of opportunists in theFusarium fujikuroi complex. J Antimicrob Chemother 2015; 70:1068–1071.

13. Wang H, Xiao M, Kong F et al. Accurate and practicalidentification of 20 Fusarium species by seven-locus sequenceanalysis and reverse line blot hybridization, and an in vitroantifungal susceptibility study. J Clin Microbiol 2011; 49:1890–1898.

14. Manikandan P, J Varga, Kocsube S et al. Epidemiology of As-pergillus keratitis at a tertiary care eye hospital in South Indiaand antifungal susceptibilities of the causative agents. Mycoses2013; 56: 26–33.

15. Harris JL. Safe, low-distortion tape touch method for fungalslide mounts. J Clin Microbiol 2000; 38: 4683–4684.

16. Al-Hatmi AM, Bonifaz A, de Hoog GS et al. Keratitis by Fusar-ium temperatum, a novel opportunist. BMC Infect Dis 2014Nov 12; 14: 588. doi: 10.1186/s12879-014-0588-y.

17. O’Donnell K, Kistler HC, Cigelnik E et al. Multiple evolutionaryorigins of the fungus causing Panama disease of banana: concor-dant evidence from nuclear and mitochondrial gene genealogies.Proc Natl Acad Sci USA 1998; 95: 2044–2049.

18. Reeb V, Lutzoni F, Roux C. Contribution of RPB2 to multilo-cus phylogenetic studies of the euascomycetes (Pezizomycotina,Fungi) with special emphasis on the lichen-forming Acarospo-raceae and evolution of polyspory. Mol Phylogenet Evol 2004;32: 1036–1060.

19. Tamura K, Stecher G, Peterson D et al. MEGA6: MolecularEvolutionary Genetics Analysis Version 6.0. Mol Biol Evol 2013;30: 2725–2729.

20. Posada D. JModelTest Phylogenetic Model Averaging. Mol BiolEvol 2008: 25: 1253–1256.

21. Stamatakis A. RAxML-VI-HPC: maximum likelihood-basedphylogenetic analyses with thousands of taxa and mixed models.Bioinformatics 2006; 22: 2688–2690.

22. Clinical and Laboratory Standards Institute. Reference Methodfor Broth Dilution Antifungal Testing of Filamentous Fungi;Approved Standard-Second Edition. CLSI document M38–A2.Wayne, PA: 2008.

23. Srinivasan M, Gonzales CA, George C et al. Epidemiology andaetiological diagnosis of corneal ulceration in Madurai, SouthIndia. Br J Ophthalmol 1997; 81: 965–971.

24. Homa M, Shobana CS, Singh YR et al. Fusarium keratitis inSouth India: causative agents, their antifungal susceptibilities

by guest on Decem

ber 24, 2015http://m

my.oxfordjournals.org/

Dow

nloaded from

8 Medical Mycology, 2015, Vol. 00, No. 00

and a rapid identification method for the Fusarium solani speciescomplex. Mycoses 2013; 56: 501–511.

25. Saha R, Das S. Mycological profile of infectious keratitis fromDelhi. Indian J Med Res 2006; 123: 159–164.

26. Zhang N, O’Donnell K, Sutton DA et al. Members of the Fusar-ium solani species complex that cause infections in both humansand plants are common in the environment. J Clin Microbiol2006; 44: 2186–2190.

27. O’Donnell K. Molecular phylogeny of the Nectriahaematococca-Fusarium solani species complex. Mycolo-gia 2000; 92: 919–938.

28. Summerbell RC, Schroers HJ. Analysis of phylogenetic relation-ship of Cylindrocarpon lichenicola and Acremonium falciformeto the Fusarium solani species complex and a review of similar-ities in the spectrum of opportunistic infections caused by thesefungi. J Clin Microbiol 2002; 40: 2866–2875.

29. Short DP, O’Donnell K, Geiser DM. Clonality, recombination,and hybridization in the plumbing-inhabiting human pathogenFusarium keratoplasticum inferred from multilocus sequencetyping. BMC Evol Biol 2014 Apr 26; 14: 91. doi: 10.1186/1471-2148-14-91.

30. Kasson MT, O’Donnell K, Rooney AP et al. An inordinatefondness for Fusarium: phylogenetic diversity of fusaria cul-tivated by ambrosia beetles in the genus Euwallacea on av-ocado and other plant hosts. Fungal Genet Biol 2013; 56:147–157.

31. O’Donnell K, Gueidan C, Sink S et al. A two-locus DNA se-quence database for typing plant and human pathogens withinthe Fusarium oxysporum species complex. Fungal Genet Biol2009; 46: 936–948.

32. Al-Hatmi AM, Seifert KA, Gerrits van den Ende AHG et al.Two novel DNA barcodes for species recognition of op-portunistic pathogens in Fusarium. Fungal Biol 2015; doi:10.1016/j.funbio.2015.08.006.

33. Xie L, Zhai H, Zhao J et al. Antifungal susceptibility for commonpathogens of fungal keratitis in Shandong Province, China. AmJ Ophthalmol 2008; 146: 260–265.

34. O’Day DM, Head WS, Robinson RD et al. Corneal penetrationof topical amphotericin B and natamycin. Curr Eye Res 1986;5: 877–882.

35. Lalitha P, Shapiro BL, Srinivasan M et al. Antimicrobial sus-ceptibility of Fusarium, Aspergillus, and other filamentousfungi isolated from keratitis. Arch Ophthalmol 2007; 125:789–793.

36. Xuguang S, Zhixin W, Zhiqun W et al. Ocular fungal isolatesand antifungal susceptibility in northern China. Am J Ophthal-mol 2007; 143: 131–133.

37. Alfonso EC. Genotypic identification of Fusarium spp. from ocu-lar source: comparison to morphological classification and anti-fungal sensitivity testing. Trans Am Ophthalmol Soc 2008; 106:227–239.

38. Shobana CS, Mythili A, Homa M et al. In vitro susceptibility offilamentous fungi from mycotic keratitis to azole drugs. J MycolMed 2015; 25: 44–49.

39. Pujol I, Guarro J, Gene J et al. In vitro antifungal susceptibility ofclinical and environmental Fusarium spp. strains. J AntimicrobChemother 1997; 39: 163–167.

Appendix Indo-Hungarian Fungal Keratitis (IHFK)

Working Group

Janos Varga, Laszlo Galgoczy, Sandor Kocsube, Peter Kormoczi,Tamas Papp, Nikolett Baranyi, Andras Szekeres: Department of Mi-crobiology, Faculty of Science and Informatics, University of Szeged,Szeged, Hungary; Rajaraman Revathi, Raghavan Anita, Perumal Go-mathi: Aravind Eye Hospital and Postgraduate Institute of Ophthal-mology, Coimbatore, Tamilnadu, India; Arumugam Mythili: De-partment of Microbiology, Dr. G.R. Damodaran College of Science,Coimbatore, India; Thambidurai Punitha, Department of Microbi-ology, Vivekanandha College of Arts and Sciences for Women (A),Tiruchengode, Tamilnadu, India; Ramaswamy Rajendran: Depart-ment of Microbiology, PSG College of Arts & Science, Coimbatore,India; Kanesan Panneer Selvam: Department of Microbiology, MRGovernment Arts College, Mannargudi, India; Muthusamy Chan-drasekaran: Botany and Microbiology Department, King Saud Uni-versity, Riyadh, Kingdom of Saudi Arabia; Ilona Doczi: Departmentof Clinical Microbiology, Faculty of Medicine, University of Szeged,Szeged, Hungary; Balazs Leitgeb: Institute of Biophysics, Biologi-cal Research Centre of the Hungarian Academy of Sciences, Szeged,Hungary.

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