Exophiala dermatitidis Endophthalmitis: Case Report and Literature Review.

We report a case of a 59-year-old male patient with a postoperative fungal infection of the left eye. A dark-pigmented yeast, Exophiala dermatitidis (previously known as Wangiella dermatitidis), was identified from the culture of the biopsy taken from the posterior capsule. The infection was successfully eradicated by a combination of surgical and medical (i.e., voriconazole and fluconazole) treatment. This is the first report of successfully treated E. dermatitidis endophthalmitis, which demonstrates that a prompt and aggressive antifungal therapy combined with surgical intervention is necessary to prevent vision loss in cases of endophthalmitis due to Exophiala species. Beside the case description, we also aim to provide a literature review of previously reported eye infections caused by Exophiala species in order to help the future diagnosis and management of the disease.

Antifungal Susceptibility and Phylogeny of Opportunistic Members of the Genus Fusarium Causing Human Keratomycosis in South India.

Fusarium species are reported frequently as the most common causative agents of fungal keratitis in tropical countries such as India. Sixty-five fusaria isolated from patients were subjected to multilocus DNA sequencing to characterize the spectrum of the species associated with keratitis infections in India. Susceptibilities of these fusaria to ten antifungals were determined in vitro by the broth microdilution method. An impressive phylogenetic diversity of fusaria was reflected in susceptibilities differing at species level. Typing results revealed that the isolates were distributed among species in the species complexes (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 to be the most effective drugs, followed by econazole.

Antifungal Effect of Essential Oils against Fusarium Keratitis Isolates.

The present study was carried out to investigate the antifungal effects of Cinnamomum zeylanicum, Citrus limon, Juniperus communis, Eucalyptus citriodora, Gaultheria procumbens, Melaleuca alternifolia, Origanum majorana, Salvia sclarea, and Thymus vulgaris essential oils against Fusarium species, the most common etiologic agents of filamentous fungal keratitis in South India. C. zeylanicum essential oil showed strong anti-Fusarium activity, whereas all the other tested essential oils proved to be less effective. The main component of C. zeylanicum essential oil, trans-cinnamaldehyde, was also tested and showed a similar effect as the oil. The in vitro interaction between trans-cinnamaldehyde and natamycin, the first-line therapeutic agent of Fusarium keratitis, was also investigated; an enhanced fungal growth inhibition was observed when these agents were applied in combination. Light and fluorescent microscopic observations revealed that C. zeylanicum essential oil/trans-cinnamaldehyde reduces the cellular metabolism and inhibits the conidia germination. Furthermore, necrotic events were significantly more frequent in the presence of these two compounds. According to our results, C. zeylanicum essential oil/trans-cinnamaldehyde provides a promising basis to develop a novel strategy for the treatment of Fusarium keratitis.

Filamentous fungal infections of the cornea: a global overview of epidemiology and drug sensitivity.

Fungal keratitis is a serious suppurative, usually ulcerative corneal infection which may result in blindness or reduced vision. Epidemiological studies indicate that the occurrence of fungal keratitis is higher in warm, humid regions with agricultural economy. The most frequent filamentous fungal genera among the causal agents are Fusarium, Aspergillus and Curvularia. A more successful therapy of fungal keratitis relies on precise identification of the pathogen to the species level using molecular tools. As the sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster (rDNA) is not discriminative enough to reveal a species-level diagnosis for several filamentous fungal species highly relevant in keratitis infections, analysis of other loci is also required for an exact diagnosis. Molecular identifications may also reveal the involvement of fungal species which were not previously reported from corneal infections. The routinely applied chemotherapy of fungal keratitis is based on the topical and systemic administration of polyenes and azole compounds. Antifungal susceptibility testing of the causal agents is of special importance due to the emergence and spread of resistance. Testing the applicability of further available antifungals and screening for new, potential compounds for the therapy of fungal keratitis are of highlighted interest.

Epidemiology of Aspergillus keratitis at a tertiary care eye hospital in South India and antifungal susceptibilities of the causative agents.

In recent years, Aspergillus species are reported frequently as aetiological agents of fungal keratitis in tropical countries such as India. Our aim was to evaluate the epidemiological features of Aspergillus keratitis cases over a 3-year period in a tertiary eye care hospital and to determine the antifungal susceptibilities of the causative agents. This study included culture proven Aspergillus keratitis cases diagnosed between September 2005 and August 2008. Data including prevalence, predisposing factors and demography were recorded, the isolates were identified by morphological and molecular methods and the minimum inhibitory concentration values of antifungal agents towards the isolates were determined by the microdilution method. Two hundred Aspergillus isolates were identified among 1737 culture proven cases. Most of the aspergilli (75%) proved to be A. flavus, followed by A. fumigatus (11.5%). Sixteen (8%) isolates belonged to species that are recently identified causative agents of mycotic keratitis. Most of the infected patients (88%) were adults ranging from 21 to 70 years of age. Co-existing ocular disease was confirmed in 16.5% of the patients. Econazole, clotrimazole and ketoconazole were notably active against A. flavus. Aspergillus keratitis is a significant problem in patients with ocular lesions in South-Indian States, warranting early diagnosis and initiation of specific antifungal therapy to improve outcome.

Molecular identification, phylogeny and antifungal susceptibilities of dematiaceous fungi isolated from human keratomycosis.

PURPOSE: To investigate the dematiaceous fungal profile of patients with ocular mycoses attending a tertiary eye care hospital in Coimbatore, India METHODS: The identification of dematiaceous fungus based on their morphology, their genotypes, and the measurement of the minimum inhibitory concentrations (MICs) using microdilution method of routinely used antifungal drugs were all compared. RESULTS: A total of 148 dematiaceous fungi were isolated during a study period of 27 months. Isolates were confirmed as Curvularia spp. (n = 98), Exserohilum spp. (n = 32), Alternaria spp. (n = 14), Exophiala spp. (n = 2), Cladosporium sp. (n = 1) and Aureobasidium sp. (n = 1). Out of 50 well grown isolates characterized genotypically based on the amplification and sequencing of the ITS region of the ribosomal RNA gene cluster and subsequent BLAST analysis, Curvularia lunata (n = 24), C. aeria (n = 1), C. spicifera (n = 8), C. hawaiiensis (n = 1), C. maydis (n = 2), C. papendorfii (n = 2), C. geniculata (n = 3), C. tetramera (n = 2) and Exs. rostratum (n = 7) were identified. In vitro antifungal susceptibilities of the most tested dematiaceous isolates showed that voriconazole had a MIC50 of 0.25 µg ml-1, while amphotericin B had a MIC50 of 0.25 µg ml-1 for Curvularia spp. and Alternaria spp. CONCLUSION: Voriconazole proved to be the most effective drug against the pigmented filamentous fungi, followed by amphotericin B, itraconazole and econazole.

In-vitro assessment of first-line antifungal drugs against Aspergillus spp. caused human keratomycoses.

BACKGROUND AND OBJECTIVES: Aspergillus keratitis are in the increasing trend and reported as the second most common cause of mycotic keratitis in developing countries. The present study was designed to isolate, identify Aspergillus spp. from the keratits/corneal ulcer patients attending a tertiary care eye hospital, Coimbatore, South India and to assess the minimum inhibitory concentrations (MICs) against ten clinically used first-line antifungal drugs. METHODS: A total of seventy-three Aspergillus strains isolated from corneal scrapings were included and assessed for a period of one year. All isolates were identified up to the species level by morphological observations. Antifungal drug susceptibilities were determined against a standard panel of antifungal agents. CONCLUSIONS: Five different species of aspergilli, A. flavus (n=53), A. fumigatus (n=14), A. terreus (n=9), A. tamarii (n=6) and A. niger (n=3) were identified based on morphological features. Minimum inhibitory concentration analyses indicated that, voriconazole, natamycin, itraconazole, clotrimazole, econazole followed by ketoconazole shall be the order of choices for the effective treatment for Aspergillus keratitis.

Evaluation of in vitro activities of extracellular enzymes from Aspergillus species isolated from corneal ulcer/keratitis.

Mycotic/fungal keratitis is a suppurative, generally ulcerative infection of the cornea. The filamentous fungi, Aspergillus spp. are the second leading cause of mycotic keratitis, particularly in India. Aspergillus spp. produce a range of extracellular enzymes that are used to break down complex molecules and used for growth and reproduction, also for survival on/in host organism. The current study was designed with an objective to screen in vitro extracellular enzyme activity of Fusarium and Aspergillus isolates from mycotic keratitis patients and to correlate the same as a putative virulence factor. Extracellular enzymes viz., deoxyribonuclease (DNase), protease, lipase, elastase, keratinase, etc., produced by Aspergillus have key role in keratomycosis and hence their (n = 85) in vitro activities were investigated. It was found that, the majority of the Aspergillus isolates produced protease (n = 75; 88% of 85) followed by lipase (n = 59; 69% of 85), DNase (n = 35; 41% of 85), elastase (n = 26; 31% of 85) and keratinase (n = 13; 15% of 85). The enzyme activity indices (EAI) for DNase, elastase, protease and lipase ranged between 1.01 and 1.98, whereas elastase EAI varied between 1.26 and 1.92. DNase, protease and lipase showed a maximum EAI of 1.98 and lowest EAI value of 1.01, respectively. Extracellular enzymes of Aspergillus spp. may have potential role in the onset and progression of keratitis.

Characterization of Aspergillus tamarii Strains From Human Keratomycoses: Molecular Identification, Antifungal Susceptibility Patterns and Cyclopiazonic Acid Producing Abilities.

Aspergillus tamarii appears to be an emerging aetiological agent of human keratomycoses in South India. The investigated strains were isolated from six suspected fungal keratitis patients attending a tertiary care eye hospital in Coimbatore (Tamil Nadu, India), and were initially identified by the microscopic examinations of the scrapings and the cultures. Our data suggest that A. tamarii could be easily overlooked when identification is carried out based on morphological characteristics alone, while the sequence analysis of the calmodulin gene can be used successfully to recognize this species accurately. According to the collected clinical data, ocular trauma is a common risk factor for the infection that gradually developed from mild to severe ulcers and could be healed with an appropriate combined antifungal therapy. Antifungal susceptibility testing revealed that A. tamarii strains are susceptible to the most commonly used topical or systemic antifungal agents (i.e., econazole, itraconazole and ketoconazole) except for natamycin. Moreover, natamycin proved to be similarly less effective than the azoles against A. tamarii in our drug interaction tests, as the predominance of indifferent interactions was revealed between natamycin and econazole and between natamycin and itraconazole as well. Four and five isolates of A. tamarii were confirmed to produce cyclopiazonic acid (CPA) in RPMI-1640 - which is designed to mimic the composition of human extracellular fluids - and in yeast extract sucrose (YES) medium, respectively, which is a widely used culture medium for testing mycotoxin production. Although a ten times lower mycelial biomass was recorded in RPMI-1640 than in YES medium, the toxin contents of the samples were of the same order of magnitude in both types of media. There might be a relationship between the outcome of infections and the toxigenic properties of the infecting fungal strains. However, this remains to be investigated in the future.

Fungal Keratitis: Epidemiology, Rapid Detection, and Antifungal Susceptibilities of Fusarium and Aspergillus Isolates from Corneal Scrapings.

Fungal aetiology of keratitis/corneal ulcer is considered to be one of the leading causes of ocular morbidity, particularly in developing countries including India. More importantly, Fusarium and Aspergillus are reported commonly implicating corneal ulcer and against this background the present work was undertaken so as to understand the current epidemiological trend of the two fungal keratitis. During the project period, a total of 500 corneal scrapings were collected from suspected mycotic keratitis patients, of which 411 (82.2%) were culture positive for bacteria, fungi, and parasites. Among fungal aetiologies, Fusarium (216, 52.5% of 411) and Aspergillus (68, 16.5% of 411) were predominantly determined. While the study revealed a male preponderance with both the fungal keratitis , it further brought out that polyene compounds (natamycin and amphotericin B) and azoles were active, respectively, against Fusarium spp. and Aspergillus spp. Additionally, 94.1% of culture proven Fusarium keratitis and, respectively, 100% and 63.6% of A. flavus and A. fumigatus were confirmed by multiplex PCR. The sensitivity of the PCR employed in the present study was noted to be 10 fg/µl, 1 pg/µl, and 300 pg/µl of DNA, respectively, for Fusarium, A. flavus, and A. fumigatus. Alarming fact was that Fusarium and Aspergillus regionally remained to be the common cause of mycotic keratitis and the Fusarium isolates had a higher antifungal resistance than Aspergillus strains against most of the test drugs.

Keratomycosis Caused By Graphium eumorphum (Graphium State of Scedosporium apiospermum).

Graphium eumorphum is rarely associated with mycotic keratitis. We report the case of a 30-year-old female presented with complaints of redness and defective vision in the left eye for one month. Gram staining and 10% KOH wet mount of corneal smears revealed fungal filaments. On potato dextrose agar, fast growing greyish white colonies turning grayish black on maturity was obtained. Lactophenol cotton blue (LPCB) staining confirmed the isolate as Graphium eumorphum. The infection was resolved with the combination of natamycin, econazole and itraconazole.

In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil.

AIM: To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates. METHODS: The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation. Extracellular enzymes such as lipase, deoxyribonuclease (DNase), α-amylase, protease, cellulase and pectinase produced by the fungal isolates were screened on solid media supplemented with the corresponding substrates. Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungal isolates. RESULTS: A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these, Fusarium spp. (n=30), non sporulating molds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1) and Exophiala spp. (n=1) were identified and designated as FS1-30, NSM1-9, AF1-6, BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp., Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2 (EAI= 2.14). The DNase activity was higher in NSM9 (EAI=1.88). In case of protease, Fusarium spp. (FS9) had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i.e. NSM7 with EAI of 1.98 when compared to other corneal and soil isolates. The pectinase activity index was also prominent for corneal isolate NSM5 versus the soil isolates, SAF1, SFS1, SES1, SBS1 and SCS 1 as 1.76 versus 1.47, 1.38, 1.16, 1.11 and 1.14, respectively. CONCLUSION: The most common isolate was Fusarium spp. followed by Aspergillus, Curvularia, Exserohilum, Bipolaris, Exophiala and Alternaria species. Enzyme activity indices (EAI) of the enzymes analysed varied with the clinical and soil isolates with respect to protease and cellulase (P=0.01). Of all the strains compared it was noted that mean EAI was greater in many clinical fusarial isolates followed by non sporulating molds.

Virulence, Speciation and Antibiotic Susceptibility of Ocular Coagualase Negative Staphylococci (CoNS).

BACKGROUND: Coagulase negative Staphylococci (CoNS) are common inhabitants of human skin and mucous membranes. With the emergence of these organisms as prominent pathogens in patients with ocular infections, investigation has intensified in an effort to identify important virulence factors and to inform new approaches to treatment and prevention. AIM: To isolate CoNS from ocular specimens; to study the possible virulence factors; speciation of coagulase negative staphylococci (CoNS) which were isolated from ocular complications; antibiotic susceptibility testing of ocular CoNS. MATERIALS AND METHODS: The specimens were collected from the target patients who attended the Microbiology Laboratory of a tertiary care eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected to tube and slide coagulase tests for the identification of CoNS. All the isolates were subjected to screening for lipase and protease activities. Screening for other virulence factors viz., slime production on Congo red agar medium and haemagglutination assay with use of 96-well microtitre plates. These isolates were identified upto species level by performing biochemical tests such as phosphatase test, arginine test, maltose and trehalose fermentation tests and novobiocin sensitivity test. The isolates were subjected to antibiotic susceptibility studies, based on the revised standards of Clinical and Laboratory Standards Institutes (CLSI). RESULTS: During the one year of study, among the total 260 individuals who were screened, 100 isolates of CoNS were obtained. Lipolytic activity was seen in all the isolates, whereas 38 isolates showed a positive result for protease. A total of 63 isolates showed slime production. Of 100 isolates, 30 isolates were analyzed for haemagglutination, where 4 isolates showed the capacity to agglutinate the erythrocytes. The results of the biochemical analysis revealed that of the 100 isolates of CoNS, 43% were Staphylococcus epidermidis. The other isolates were identified as S. xylosus (n=8), S. captis (n=16), S. haemolyticus (n=10), S. saccharolyticus (n=2), S. hominis (n=5), S. saprophyticus (n=6) and S. intermedius (n=1). On the other hand, 9 isolates were not identified. In the antibiotic susceptibility analysis, it was found that most of the isolates were sensitive to vancomycin, amikacin and linczolid and resistant to cefatoxime, oxacillin, bacitracin and nalidixic acid. CONCLUSION: S. epidermidis was found to be predominant in causing the ocular complications. Slime production, heamagglutination, protease and lipase activities could be the putative virulence factors of CoNS. Antibiotic susceptibility patterns of CoNS against antibacterial agents revealed maximum resistance to beta lactam groups, and the resistance was found to be higher to oxacillin, and lowest to vancomycin.