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 ~  Abstract
 ~ Introduction
 ~ Patients and Methods
 ~ Results
 ~ Discussion
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 564-568
 

Exserohilum keratitis: Clinical profile of nine patients and comparison of morphology versus ITS-Based DNA sequencing for species identification of the fungal isolates


1 Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, Hyderabad, Telangana, India
2 Tej Kohli Cornea Institute, Hyderabad, Telangana, India
3 Ophthalmic Pathology Laboratory, Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, Telangana, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Savitri Sharma
Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Hyderabad - 500 034, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_51

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 ~ Abstract 

Purpose: The objective of this study was to describe the microbiological and clinical features of nine cases of Exserohilum keratitis. Patients and Methods: Fungal isolates from corneal scrapings were identified based on macroscopic and microscopic characteristics of the colonies and DNA sequencing of ITS1-5.8S-ITS2 region in the rRNA gene. All patients were treated with topical and if required systemic antifungals. Therapeutic penetrating keratoplasty (TPK) was done in case of failed medical therapy. Results: Morphologically, all fungal isolates were Exserohilum rostratum except one Exserohilum mcginnisii. Based on the BLAST analysis, 6 isolates showed 100% similarity to Setosphaeria rostrata (CBS 112815) and E. mcginnisii (CBS 20308). The other three isolates were Setosphaeria holmii (CBS 128053), not reported earlier in fungal keratitis. Three patients were lost to follow-up and response to medical therapy was good (Healed scar – 4 patients). Two out of nine patients were advised TPK. Conclusions: Diagnosis and clinical features of Exserohilum keratitis are akin to other dematiaceous keratitis. The two morphological species of E. mcginnisii and E. rostratum are indistinguishable from Setosphaeria rostratum at DNA sequence level, which justifies the retention of the latter nomenclature.


Keywords: DNA sequencing, Exserohilum, keratitis, phylogenetic analysis, Setosphaeria holmii, Setosphaeria rostrata, treatment


How to cite this article:
Jayasudha R, Sharma S, Kalra P, Mishra DK. Exserohilum keratitis: Clinical profile of nine patients and comparison of morphology versus ITS-Based DNA sequencing for species identification of the fungal isolates. Indian J Med Microbiol 2018;36:564-8

How to cite this URL:
Jayasudha R, Sharma S, Kalra P, Mishra DK. Exserohilum keratitis: Clinical profile of nine patients and comparison of morphology versus ITS-Based DNA sequencing for species identification of the fungal isolates. Indian J Med Microbiol [serial online] 2018 [cited 2019 Jul 17];36:564-8. Available from: http://www.ijmm.org/text.asp?2018/36/4/564/254409



 ~ Introduction Top


Association of dematiaceous fungi with human keratitis (corneal ulcer) has been reported way back in 1975, wherein three cases seen before 1969 have been described.[1] The authors found dematiaceous fungi to be the second most common cause of fungal keratitis after Fusarium solani in South Florida, USA, and described clinical features and management of 16 patients. A number of species were identified using colony characteristics and microscopic appearance of the spores. The literature on fungal keratitis thereafter is replete with reports of keratitis caused by pigmented fungi.[2],[3],[4],[5],[6],[7] The most common pigmented fungal species reported has been Curvularia lunata followed by Alternaria species. Reports of Exserohilum species have been relatively rare. Difficulties with identification of this group of fungi were mitigated to some extent by the publication of morphological details of emerging agents of phaeohyphomycosis including species of Exserohilum.[8] The paper dealt with the morphology-based taxonomy and nomenclature of fungal genera such as Bipolaris, Exserohilum, Drechslera and Helminthosporium. Two earliest reports of Exserohilum rostratum keratitis were from India.[9],[10] In a retrospective analysis of 1360 fungi isolated from patients with fungal keratitis between 1991 and 2001, we have reported a prevalence of Exserohilum keratitis at 0.7%.[5]

With the advancement in molecular techniques, fungal identification is currently revolutionised. The non-sporulating fungi, especially the pigmented species, are now receiving new nomenclature to the delight of mycologists. Molecular targets such as internal transcribed spacer (ITS) region of ribosomal DNA and translation elongation factor-1α gene are being used for DNA sequence-based or multilocus sequence-based typing.[11],[12] Phylogenetic analysis adds strength to the species determination within a genus in relation to type strains, like never before. It also brings forth the limitations in the current database of fungal genome.

In this paper, we describe nine cases of culture-proven Exserohilum keratitis that were diagnosed based on morphological descriptions of the spores. DNA sequencing and phylogenetic analysis further characterised the isolates. Clinical features and treatment outcome of the patients were analysed.


 ~ Patients and Methods Top


The study was done at L. V. Prasad Eye Institute, Hyderabad, India. As per the institutional protocol, complete clinical examination including slit-lamp biomicroscopy and microbiological investigation of the corneal scrapings was done for all patients presenting to the cornea clinic with suspected microbial keratitis. We have described our institutional microbiological procedures for processing of corneal scrapings earlier.[5] Nine fungal isolates that were identified based on the colony characteristics and microscopic features of the spores [8] to be Exserohilum species were stocked in potato dextrose agar at room temperature and analysed later. DNA was isolated using QIAamp DNA kit (Qiagen, Germany) and ITS1-5.8S-ITS2 regions of the rRNA gene were amplified by polymerase chain reaction (PCR) as described earlier,[13] and the purified PCR products were sequenced using the same primers. The resultant forward and reverse sequences were assembled with Lasergene Seqman Pro software (DNASTAR Inc., Madison, Wisconsin, USA) and verified manually. Sequences were compared to identify closest neighbours in NCBI nucleotide sequence database using BLASTn algorithm. Subsequently, reference strains of closely related species were retrieved from the database and aligned using BioEdit software through ClustalW multiple alignment. The obtained alignments were used to determine the phylogenetic relationship using neighbor-joining algorithm of MEGA software version 6.06[14] with 1000 bootstrap replications. The sequences were deposited in the GenBank database under accession numbers MH319023 to MH319031.

All patients were treated with topical 5% natamycin eye drop with or without topical 1% voriconazole and systemic antifungals except Patient No. 2 who was treated for chemical injury. Based on response to medical therapy, for non-responding cases, surgical treatment included therapeutic penetrating keratoplasty (TPK). Corneal tissue from patient undergoing TPK was submitted for histopathology evaluation.


 ~ Results Top


[Table 1] shows the demographic, clinical and microbiological findings of all patients included in the study. There were six male patients and three females and the right eye was involved in all except two patients. The predisposing factor was not known in two patients, while in the remaining patients, it was fall of known or unknown foreign body in the eye with one patient (no. 2) sustaining chemical injury. This patient was treated for the surface pathology mainly and did not receive antifungal therapy. The mean follow-up period was 63.3 days (excluding Patient No. 2) and three patients were lost to follow-up (LTFU). Two patients had shown resolving corneal ulcer at the last visit before they were LTFU and one patient declined TPK. Only one patient (No. 1) had undergone TPK in this series. The infection resolved in 4 remaining patients on medical therapy. Slit-lamp photograph of Patient No. 6 at presentation and 17 days into treatment is shown in [Figure 1].
Table 1: Clinical and laboratory data of nine patients of Exserohilum keratitis

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Figure 1: Slit-lamp photographs of Patient No. 6: at presentation (a and b) showing anterior to mid-stromal greyish white patchy infiltrates (5.2 mm × 5.0 mm) with active margins in ring-like pattern and feathery extensions along with surrounding oedema. After 17 days of treatment (c and d) showing reduced infiltrates, reduced oedema and blunting of feathery extensions

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In direct microscopy, the corneal scraping from all patients showed septate fungal filaments [Figure 2]a, [Figure 2]b, [Figure 2]c that were brown in 4 out of 9 cases. Morphological identification of the 9 isolates in culture is given in [Table 1]. Mixed infection of E. rostratum with Staphylococcus aureus was seen in one (No. 3) patient. The corneal tissue received post-TPK from case 1 showed intense inflammation throughout the cornea with broad septate fungal filaments [Figure 2]d, [Figure 2]e, [Figure 2]f.
Figure 2: Upper panel (Patient No. 3): microscopy of the corneal scrapings (a) fungal filaments with chlamydospores (KOH + CFW, ×400), (b) septate brown (arrow) fungal filaments (KOH, ×400), (c) brown, septate, broad (arrow) fungal filaments (Gram stain, ×1000). Lower panel (Patient No. 1): histopathology of the corneal button (d) perforated cornea with detached folded Descemet's membrane (arrow) (H and E, ×100), (e) broad, septate fungal filaments (arrow) in stroma (GMS, ×400), (f) perforated cornea with detached folded Descemet's membrane (arrow) and fungal filaments (PAS, ×100)

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[Table 2] shows the identification of the nine isolates through BLAST search and [Figure 3] is the phylogenetic analysis of the isolates. Amplification with ITS primers generated approximately 650-bp amplicon and Sanger sequencing of ITS region yielded an average of 589 ± 13 bp sequences per isolate. Based on BLAST analysis, the sequence similarity ranged from 99% to 100%. ITS sequences of L-1794/16 (No. 1), L-2546/16 (No. 3), L-2689/16 (No. 4), L-44/17 (No. 7), L-137/17 (No. 8) and L-319/17 (No. 9) isolates showed 100% similarity to both Setosphaeria rostrata and Exserohilum mcginnisii. All the six isolates formed a clade with S. rostrata (CBS 112815) and E. mcginnisii (CBS 20308) in phylogenetic tree [Figure 3]. The closest hit for the ITS sequences of L-2204/16 (No. 2), L-2718/16 (No. 5) and L-2963/16 (No. 6) isolates was Setosphaeria holmii (CBS 128053).
Table 2: Morphology and internal transcribed space sequence-based analysis of the nine isolates of corneal scrapings from Exserohilum keratitis

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Figure 3: Phylogenetic tree using neighbor-joining method generated on ITS sequences showing the clustering of 9 clinical isolates with their closest relatives (shown in bold). Bootstrap values (1000 replicates) of 50% are given at the nodes. Fusarium solani was used as an out-group. The ITS sequences of all the type strains were retrieved from NCBI database. Scale bar represents the number of substitutions per nucleotide position

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 ~ Discussion Top


The clinical features described in this case series are commensurate with the descriptions given earlier on keratitis due to dematiaceous fungi in a number of publications.[9],[10],[15] In one of the largest series, Garg et al. concluded that the corneal stromal characteristics of dry raised infiltrate with feathery margin may not be present in all cases.[15] Many of these reports have also mentioned about brown discoloration of the infiltrate in some cases. We found that the giveaway feature of brown infiltrate is not seen consistently in all cases and similarly brown discoloration of the fungal filaments in the corneal scrapings under the microscope may or may not be present. We could see brown corneal infiltrate in three out of nine cases [Table 1] and two of these showed brown filaments in the corneal scrapings [Figure 2].

Although a variety of staining techniques for the examination of corneal scrapings have been described, a rapid diagnosis of fungal keratitis is relatively simple with demonstration of the fungal elements in unstained wet mount of 10% potassium hydroxide.[9],[10] Direct microscopy of corneal scraping was positive in all our cases. All cases (except No. 2 with chemical injury) were treated with the standard hourly topical administration of 5% natamycin. Two patients (No. 1 and 7) received topical 1% voriconazole in addition to natamycin. Topical fortified cefazolin (5%) was given to three patients (No. 3, 6 and 8) for suspected concomitant bacterial infection. Although the value of systemic antifungal drugs in the treatment of fungal keratitis has been recently found to be unhelpful,[16] five of our patients received oral ketoconazole (200 mg, twice daily) owing to deep infiltrates. Response to medical therapy in dematiaceous fungal keratitis is reported to be good,[15] and the outcome in this series was similar with 4 patients showing complete resolution of infection. Two patients (No. 4 and 5) LTFU showed response to treatment at their last visit. Rate of TPK in fungal keratitis is variably reported from 15% to 38% in different studies. Patients reporting late and with deep stromal involvement are more likely to require TPK. Two out of nine patients were advised TPK, while it was done in one patient the other was LTFU. The outcome of TPK is often unsatisfactory [15] and the same was seen in Patient No. 1 who ended up with failed graft and required a second graft.

In an effort to further study the fungal isolates, we applied PCR-based DNA sequencing of the ITS region of all isolates that were morphologically E. rostratum or E. mcginnisii and found interesting results. Morphological differentiation of various species of Exserohilum has been tabulated in an earlier publication on the basis of the shape, number of septations and size of the macroconidia,[8] which formed the basis of our initial identification. The sequence analysis showed no difference in E. rostratum and E. mcginnisii. Six of the isolates showed 100% similarity to both S. rostrata and E. mcginnisii. All the six isolates formed a clade with S. rostrata (CBS 112815) and E. mcginnisii (CBS 20308) in phylogenetic tree [Figure 3]. Morphologically one of these isolates was E. mcginnisii and the rest were E. rostratum. It would thus appear that the morphological difference is arbitrary and not tenable at the genetic level. This result is in concordance with previous report of E. mcginnisii as a synonym of E. rostratum based on the comparison of sequences from ITS region, 28S ribosomal DNA (D1/D2), actin and elongation factor 1-alpha genes.[17] The use of intergenic spacer (IGS) region as molecular target might help in differentiating E. rostratum and E. mcginnisii as demonstrated in another study,[18] where IGS sequence analysis could clearly differentiate three closely related species of Curvularia (C. australiensis, C. hawaiiensis and C. spicifera), whereas ITS region was insufficient to discriminate them. Setosphaeria genus has been assigned to the teleomorph stage of Exserohilum; however, we believe that the nomenclature would be superior if it retained one name for one fungus, as has been suggested earlier.[19] The sequence similarity between two morphological species of Exserohilum and genotype S. rostrata would justify the retention of the latter name.

Three isolates in this study, morphologically considered E. rostratum, showed 99%–100% similarity and formed a distinct clade with S. holmii (CBS 128053) in the phylogenetic tree analysis [Figure 3]. Pairwise alignment in NCBI BLASTn with ITS sequence of S. holmii and E. rostratum showed 4% variation, which discloses that both species are not closely related, it was further evident by the formation of separate clades in phylogenetic analysis. This finding further points at the relative inadequacy of morphological criteria for species differentiation. Recent study by Hernández-Restrepo et al.[20] on multi-locus phylogeny of Exserohilum species also revealed that S. holmii and E. rostratum are not closely related. To the best of our knowledge, this is the first report of fungal keratitis caused by S. holmii. However, by the foregoing clinical descriptions, we realise that there is no clinical difference in terms of presentation and treatment of keratitis caused by different species of dematiaceous fungi.

In conclusion, the diagnosis and clinical features of Exserohilum keratitis are not very different from those caused by other dematiaceous fungi. Response to treatment is also similar. We found that the two morphological species of E. mcginnisii and E. rostratum are indistinguishable from Setosphaeria rostratum at DNA sequence level. Therefore, we recommend the name Setosphaeria rostratum for this group of fungi.

Financial support and sponsorship

The study was financially supported by the Hyderabad Eye Research Foundation, Hyderabad.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Tupaki-Sreepurna A, Thanneru V, Natarajan S, Sharma S, Gopi A, Sundaram M, et al. Phylogenetic diversity and in vitro susceptibility profiles of human pathogenic members of the Fusarium fujikuroi species complex isolated from South India. Mycopathologia 2018;183:529-40.  Back to cited text no. 12
    
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Prajna NV, Krishnan T, Rajaraman R, Patel S, Srinivasan M, Das M, et al. Effect of oral voriconazole on fungal keratitis in the mycotic ulcer treatment trial II (MUTT II): A randomized clinical trial. JAMA Ophthalmol 2016;134:1365-72.  Back to cited text no. 16
    
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Krizsán K, Tóth E, Nagy LG, Galgóczy L, Manikandan P, Chandrasekaran M, et al. Molecular identification and antifungal susceptibility of Curvularia australiensis, C. hawaiiensis and C. spicifera isolated from human eye infections. Mycoses 2015;58:603-9.  Back to cited text no. 18
    
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20.
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