Novel diagnosis of fungal endophthalmitis by broad-range real-time PCR detection of fungal 28S ribosomal DNA

Aim

To detect the fungal genome in the ocular fluids of patients with fungal endophthalmitis by using a novel broad-range polymerase chain reaction (PCR) system.

Methods

After informed consent was obtained, ocular fluid samples (aqueous humor or vitreous fluids) were collected from 497 patients (76 patients with infectious endophthalmitis including clinically suspected bacterial and fungal endophthalmitis and 421 patients with infectious or non-infectious uveitis). Forty ocular samples from non-infectious patients without ocular inflammation were collected as controls. Fungal ribosomal DNA (28?S rDNA) was measured by a quantitative real-time PCR assay.

Results

Fungal 28?S rDNA of the major fungal species, such as Candida, Aspergillus, and Cryptococcus, were detected by novel broad-range real-time PCR examination (>10¹ copies/ml). Fungal 28?S rDNA was detected in the ocular fluids of 11 patients with endophthalmitis or uveitis (11/497, 2.2%). All 11 positive samples were detected in the infectious endophthalmitis patients (11/76, 14.5%). These PCR-positive ocular fluids had high copy numbers of fungal 28?S rDNA (range, 1.7 × 10³ to 7.9 × 10⁶ copies/ml), which indicated the presence of fungal infection. Of the 11 patients who were PCR positive, further examinations led to a diagnosis of fungal endophthalmitis in ten patients. The fungal 28?S rDNA was detected in one non-infectious case (a false-positive case). In addition, there were two PCR false-negative cases that were clinically suspected of having fungal endophthalmitis.

Conclusions

This novel quantitative broad-range PCR of fungal 28?S rDNA is a useful tool for diagnosing endophthalmitis related to fungal infections.     

Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis

PurposeInterleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection.MethodsGene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans–infected corneas. B6 CD11c–DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively.ResultsIL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1β levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1β, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas.ConclusionsIL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection–induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.     

Periodic acid–Schiff staining demonstrates fungi in chronic anterior blepharitis

Purpose

To evaluate the presence of fungi in patients with chronic anterior blepharitis with periodic acid–Schiff (PAS) staining of the eyelashes in addition to the conventional methods of fungal cultures and direct microscopy.

Methods

Nineteen patients with chronic anterior blepharitis of seborrheic or mixed seborrheic/staphylococcal type and 11 healthy age- and sex-matched controls were included in this prospective, nonrandomized, cross-sectional study. Blepharitis was diagnosed based on clinical evidence of greasy scales between the cilia, lid margin erythema, conjunctival hyperemia, telangiectasia, thickening, or irregularity of the eyelid margins by slit-lamp biomicroscopy. Eyelash samples were obtained by epilation with a sterile forceps and evaluated with PAS staining, fungal cultures, and direct microscopy.

Results

We demonstrated fungal elements with PAS staining in 79% of the blepharitis group (hyphae and/or spores) and 18% of the control group. The difference was statistically significant (P=0.002). Four patients in the blepharitis group (21%) had positive cultures for fungi. The isolated fungi were Penicillium species (2 cases), Candida species (1 case), and Trichophyton verrucosum (1 case). Direct microscopic examination revealed Demodex mites in 42.1% of the blepharitis group. No culture growth or Demodex mites were observed in the control group.

Conclusions

We have shown fungi with PAS staining in the majority of patients with chronic anterior blepharitis. Further controlled studies are necessary to clarify the role of fungi in the etiopathogenesis of blepharitis.     

Epidemiological and microbiological profile of infective keratitis in Ahmedabad

Context:

Study of patients attending tertiary care ophthalmology institute at Ahmedabad.

Aims:

To study the microbiological etiology and epidemiological factors associated with suppurative keratitis.

Settings and Design:

A total of 150 corneal scrapings were evaluated from patients presenting with corneal ulcers at a tertiary ophthalmology center, Ahmedabad from July 2007 to June 2008.

Materials and Methods:

Scrapings were subjected to Gram stain, potassium hydroxide preparation and culture for bacterial and fungal pathogens. Socio-demographic data and risk factors were recorded.

Results:

Ninety percent (135/150) people with corneal ulcers had trauma as predisposing factor for keratitis. Trauma due to wooden objects was the leading cause (46/135) followed by vegetable matter and stone injury (23/135). Microbial etiology was established in 59.3% (89/150) of scrapings. Out of 89 positive isolates, 65.1% (58/89) were bacterial while 34.9% (31/89) were fungal. Among the bacterial isolates, 60.3% (35/58) were Gram-positive cocci while 39.7% (23/58) were Gram-negative bacilli. The most common bacterial isolate was Staphylococus aureus (32.7%, 19/58) followed by coagulase-negative Staphylococci (25.8%, 15/58) and Pseudomonas (18.9%, 11/58). Among the 31 fungal pathogens, Aspergillus species was the most common (35.4%11/31), followed by Fusarium species (22.5%, 7/31).

Conclusion:

Trauma with wooden material is the most common predisposing factor for suppurative keratitis. Males were more affected than females. Bacterial ulcers were more common than fungal in areas in and around Ahmedabad. Staphylococcus aureus and Aspergillus were the commonest bacterial and fungal isolates respectively. Geographical variation persists in microbial etiology of suppurative keratitis.     

Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, south India

AIMS/BACKGROUND—To determine the epidemiological characteristics and risk factors predisposing to corneal ulceration in Madurai, south India, and to identify the specific pathogenic organisms responsible for infection.
METHODS—All patients with suspected infectious central corneal ulceration presenting to the ocular microbiology and cornea service at Aravind Eye Hospital, Madurai, from 1 January to 31 March 1994 were evaluated. Sociodemographic data and information pertaining to risk factors were recorded, all patients were examined, and corneal cultures and scrapings were performed.
RESULTS—In the 3 month period 434 patients with central corneal ulceration were evaluated. A history of previous corneal injury was present in 284 patients (65.4%). Cornea cultures were positive in 297 patients (68.4%). Of those individuals with positive cultures 140 (47.1%) had pure bacterial infections, 139 (46.8%) had pure fungal infections, 15 (5.1%) had mixed bacteria and fungi, and three (1.0%) grew pure cultures of Acanthamoeba. The most common bacterial pathogen isolated was Streptococcus pneumoniae, representing 44.3% of all positive bacterial cultures, followed by Pseudomonas spp (14.4%). The most common fungal pathogen isolated was Fusarium spp, representing 47.1% of all positive fungal cultures, followed by Aspergillus spp (16.1%).
CONCLUSIONS—Central corneal ulceration is a common problem in south India and most often occurs after a superficial corneal injury with organic material. Bacterial and fungal infections occur in equal numbers with Streptococcus pneumoniae accounting for the majority of bacterial ulcers and Fusarium spp responsible for most of the fungal infections. These findings have important public health implications for the treatment and prevention of corneal ulceration in the developing world.

     

Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: Experience of over a decade

Purpose:

To review the epidemiological characteristics, microbiological profile, and treatment outcome of patients with suspected microbial keratitis.

Materials and Methods:

Retrospective analysis of a non-comparative series from the database was done. All the patients presenting with corneal stromal infiltrate underwent standard microbiologic evaluation of their corneal scrapings, and smear and culture-guided antimicrobial therapy.

Results:

Out of 5897 suspected cases of microbial keratitis 3563 (60.4%) were culture-proven (bacterial – 1849, 51.9%; fungal – 1360, 38.2%; Acanthamoeba – 86, 2.4%; mixed – 268, 7.5%). Patients with agriculture-based activities were at 1.33 times (CI 1.16–1.51) greater risk of developing microbial keratitis and patients with ocular trauma were 5.33 times (CI 6.41–6.44) more likely to develop microbial keratitis. Potassium hydroxide with calcofluor white was most sensitive for detecting fungi (90.6%) and Acanthamoeba (84.0%) in corneal scrapings, however, Gram stain had a low sensitivity of 56.6% in detection of bacteria. Majority of the bacterial infections were caused by Staphylococcus epidermidis (42.3%) and Fusarium species (36.6%) was the leading cause of fungal infections. A significantly larger number of patients (691/1360, 50.8%) with fungal keratitis required surgical intervention compared to bacterial (799/1849, 43.2%) and Acanthamoeba (15/86, 17.4%) keratitis. Corneal healed scar was achieved in 75.5%, 64.8%, and 90.0% of patients with bacterial, fungal, and Acanthamoeba keratitis respectively.

Conclusions:

While diagnostic and treatment modalities are well in place the final outcome is suboptimal in fungal keratitis. With more effective treatment available for bacterial and Acanthamoeba keratitis, the treatment of fungal keratitis is truly a challenge.     

Ocular mycosis at a referral center in Saudi Arabia: A 20-year study

Purpose

To review the clinical experience of fungal keratitis cases at King Khaled Eye Specialist Hospital (KKESH) in Riyadh, Saudi Arabia.

Methods

Retrospective observational review and analysis of 124 patient charts with confirmed diagnosis of fungal keratitis between 1984 and 2004.

Results

One hundred and twenty four eyes of 124 patients had proven fungal infection; 101 eyes had fungal keratitis and 23 eyes had fungal endophthalmitis complicating keratitis. Estimated proportion of fungal keratitis and endophthalmitis was 10.3%. Mean age was 55 years with male predominance (79.0%). Commonly associated factors were previous intraocular surgery (38.7%) and trauma (20.9%). Major risk factor for progressing to endophthalmitis was previous intraocular surgery (65.2%), p < 0.001. Initial laboratory results were fungal positive only in 30.6% (p < 0.001). Commonest organisms isolated were Aspergillus spp. (29.8%) followed by Trichophyton sp. (16.1%), then Candida and Fusarium sp. Comparison of both phases of the study showed improvement in the rate of successfully treated cases from 34.6% to 58.3%, and a decline in cases progressing to endophthalmitis from 25.0% to 13.9%. Therapeutic penetrating keratoplasty increased from 26.9% to 73.6% (p < 0.001). Thirteen eyes required enucleation or evisceration.

Conclusions

In contrast to other studies on fungal keratitis, Aspergillusspp. and Trichophyton sp. were the most commonly isolated fungal pathogens; the former carries the worst prognosis. Risk factors included previous intraocular surgery and trauma. Poor outcome was associated with Aspergillus spp., delayed presentation, previous intraocular surgery and late surgical intervention. This study recommends early surgical intervention to improve the outcome.     

Corneal Phaeohyphomycosis Caused by Bipolaris hawaiiensis

PurposeTo report a rare case of keratitis infected by Bipolaris hawaiiensis.MethodsA patient who was diagnosed as fungal keratitis caused by B. hawaiiensis was retrospectively reviewed for history, clinical characteristics, risk factors, laboratory findings, treatments, and outcomes.ResultsA 63-year-old man with a history of trauma and saw dust in the left eye presented with a corneal ulcer. Eye examination revealed whitish infiltration with a feathery edge and small brownish deposits in the anterior stroma of the left cornea. Numerous septate hyphal fragments were detected in a corneal specimen, and nucleotide sequence analysis identified B. hawaiiensis. Treatment was started with 5% natamycin eyedrops and oral itraconazole. Subsequently, a corneal plaque developed which did not respond to medication and debridement. The patient underwent therapeutic penetrating keratoplasty.ConclusionsB. hawaiiensis is a rare cause of corneal phaeohyphomycosis. A brownish pigmented infiltration is an important diagnostic clue, however microbiologic studies are required to obtain a definite diagnosis. Although antifungal medication and debridement are the mainstay of most corneal fungal infection, therapeutic penetrating keratoplasty can prevent morbidity related to this fungal infection.Key Words: Bipolaris hawaiiensis, Phaeohyphomycosis, Keratitis, Dematiaceous fungus     

Voriconazole-refractory fungal infection of phacoemulsification tunnel

A 44-year-old man presented 28 days after cataract surgery (phacoemulsification) in right eye with multiple pinpoint infiltrates in posterior stroma at cataract surgery wound site. Visual acuity was 20/60. Corneal scraping from the floor of the corneal tunnel revealed fungus which was later identified to be Aspergillus flavus. The patient was started on oral voriconazole 200 mg twice daily and topical voriconazole 1% every hour. Two intracameral injections of voriconazole (50 micrograms/ 0.1 ml) were given 72 h apart, five days after starting initial therapy. Infiltrates increased in size and density in spite of 20 days of voriconazole therapy. Full-thickness patch graft was done to arrest progressive necrosis. Four months after surgery, patient had 20/60 best-corrected visual acuity. There was no recurrence in one-year follow-up. Present case illustrates the therapeutic challenge in fungal tunnel infections and possibility of voriconazole-resistant Aspergillus species.     

Randomised trial of 0.2% chlorhexidine gluconate and 2.5% natamycin for fungal keratitis in Bangladesh

AIM—The management of suppurative keratitis due to filamentous fungi presents severe problems in tropical countries. The aim was to demonstrate the efficacy of chlorhexidine 0.2% drops as an inexpensive antimicrobial agent, which could be widely distributed for fungal keratitis.
METHODS—Successive patients presenting to the Chittagong Eye Institute and Training Complex with corneal ulcers were admitted to the trial when fungal hyphae had been seen on microscopy. They were randomised to drop treatment with chlorhexidine gluconate 0.2% or the standard local treatment natamycin 2.5%. The diameters, depths, and other features of the ulcers were measured and photographed at regular intervals. The outcome measures were healing at 21 days and presence or absence of toxicity. If there was not a favourable response at 5 days, "treatment failure" was recorded and the treatment was changed to one or more of three options, which included econazole 1% in the latter part of the trial.
RESULTS—71 patients were recruited to the trial, of which 35 were randomised to chlorhexidine and 36 to natamycin. One allocated to natamycin grew bacteria and therefore was excluded from the analysis. None of the severe ulcers was fully healed at 21 days of treatment, but three of those allocated to chlorhexidine eventually healed in times up to 60 days. Of the non-severe ulcers, 66.7% were healed at 21 days with chlorhexidine and 36.0% with natamycin, a relative efficacy (RE) of 1.85 (CL 1.01-3.39, p = 0.04). If those ulcers were excluded where fungi were seen in the scraping but did not grow on culture, the estimated efficacy ratio does not change but becomes less precise because of smaller numbers. Equal numbers of Aspergillus (22) and Fusarium (22) were grown. The Aspergillus were the most resistant to either primary treatment.
CONCLUSIONS—Chlorhexidine may have potential as an inexpensive topical agent for fungal keratitis and warrants further assessment as a first line treatment in situations where microbiological facilities and a range of antifungal agents are not available.

Keywords: fungal keratitis; corneal ulcers; chlorhexidine; Bangladesh     

Spectral domain anterior segment optical coherence tomography in microbial keratitis

Purpose

To investigate the spectral domain anterior segment optical coherence tomography (SDAS-OCT) patterns in microbial keratitis (fungal and bacterial keratitis).

Design

Prospective, cross-sectional, observational study.

Methods

Twenty eyes of 20 patients with proven fungal and bacterial microbial keratitis, at different stages of the disease, underwent SDAS-OCT imaging.

Results

Eight eyes presented with proven bacterial keratitis (3 Staphylococcus Aureus, 2 Pseudomonas Aeruginosa and 3 Staphylococcus Epidermidis). Twelve eyes presented with proven fungal keratitis of Aspergillus species. Twelve different SDAS-OCT presentations of fungal and bacterial keratitis were found in this study. Our findings in fungal keratitis grasped two unique patterns of early localized and diffuse necrotic stromal cystic spaces.

Conclusion

SDAS-OCT imaging provided a range of characteristic patterns that could be used as an additional tool in diagnosis and management of bacterial and fungal microbial keratitis.     

CLEC-1 Acts as a Negative Regulator of Dectin-1 Induced Host Inflammatory Response Signature in Aspergillus fumigatus Keratitis

PurposeC-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro.MethodsQuantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages.ResultsThe expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1β production.ConclusionsThese findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1β production in response to A. fumigatus keratitis.     

Experimental model of Fusarium solani keratitis in rats

AIM: To establish a repeatable rat model of Fusarium solani keratitis (F. solani keratitis) that mimicked fungal keratitis in humans. METHODS: Wistar rats’ corneas were scratched on the superficial stroma after scraping the unilateral corneal epithelia. Then, the corneal surface was inoculated with different inoculum dose of F. solani spore suspension. Doses ranged from 106 to 109 colony-forming unit per milliliter (CFU/mL). The treated corneas were covered by contact lenses that were made of Parafilm M membrane. Negative controls were inoculated with sterile phosphate-buffered saline (PBS). For statistical analysis, corneas were evaluated daily on a 12-point scale to check the state of corneal inflammation. Furthermore, the pathological characteristics of this model were investigated. RESULTS: The rat model of F. solani keratitis was established by the combination methods of corneal trauma and parafilm-made contact lens and inoculation of fungus spore suspension. 106 and 107CFU/mL of F. solani induced mild corneal infection, while 108CFU/mL of F. solani was sufficient to induce moderate infection that was consistent with human keratomycosis. Dose of 109CFU/mL of F. solani was excessive and led to perforated corneas. CONCLUSION: The rat model of F. solani keratitis, established by the combinational methods of corneal trauma, parafilm-made contact lens and the appropriate dose of inoculum, that imitates the developing processes of F. solani keratitis in human beings and provides a repeatable method of creating a rat model.     

Incidence and microbiological profile of mycotic keratitis in a tertiary care eye hospital: A retrospective analysis

Purpose

To determine the incidence and microbiological profile of mycotic keratitis seen at a tertiary care eye hospital.

Materials and methods

A retrospective review of microbiology records of patients presenting with suspected microbial keratitis seen between January 2006 and December 2009 was performed. Patients with positive fungal cultures were further analyzed for the type of fungus isolated and associated bacterial pathogens.

Results

Microbiology records of 2300 patients with suspected microbial keratitis were reviewed. A microbiological diagnosis of mycotic keratitis was established in 87 (3.8%) patients over a four year period based on positive fungal cultures. The yearly incidence of mycotic keratitis was 3.2% (2006), 4.9% (2007), 3.3% (2008) and 3.6% (2009). Filamentous fungi were isolated more often than yeasts. Aspergillus species followed by Fusarium species and Trichophyton species were the commonest filamentous fungi isolated while Candida albicans was the most frequently encountered yeast. Mixed infections due to fungal and bacterial pathogens were seen in 25/87 (28.7%) patients.

Conclusion

Cumulative incidence of mycotic keratitis was 3.8% over a four year period. Aspergillus species and Candida albicans were the most frequent pathogenic organisms causing mycotic keratitis in this part of the world. Mixed infections were seen in 28.7% of the patients. Knowledge of the “local” etiology within a region may be valuable in the management of mycotic keratitis in instituting an empirical therapy, especially when facilities for microscopy, cultures and antifungal susceptibility are not readily available. The baseline information presented will also be helpful in the planning of a corneal ulcer management strategy and for future studies on mycotic keratitis.     

Diagnosis of Fusarium keratitis in an animal model using the polymerase chain reaction

AIMS/BACKGROUND—The purpose of this study was apply the polymerase chain reaction (PCR) to develop a sensitive, specific, and rapid test to diagnose Fusarium keratitis. Fusarium is the most common cause of fungal corneal infection in some parts of the world. It is often difficult to establish that a keratitis is due to fungal infection.
METHODS—Fusarium solani keratitis was induced in three eyes of three rabbits by injection of a suspension of the fungus into the anterior corneal stroma. In one rabbit the contralateral eye served as a control. From four to 28 days after inoculation, the corneas were scraped for culture, then scraped and swabbed for PCR analysis. The PCR was performed with primers directed against a portion of the Fusarium cutinase gene, and the presence or absence of this amplified target sequence was determined by agarose gel.
RESULTS—The amplified DNA sequence was detected in 25 of 28 samples from the corneas infected with Fusarium, for a sensitivity of 89%. Only three of the 14 samples from these eyes with Fusarium keratitis were positive by culture, for a sensitivity of 21%. Seven of eight control samples were negative by the PCR based test, for a specificity of 88%.
CONCLUSION—This PCR based test holds promise of being an effective method of diagnosing Fusarium keratitis as well as Fusarium infections at other sites.

Keywords: keratitis; Fusarium; ulcer; cornea; polymerase chain reaction     

Visual function assessment in simulated real-life situations in patients with age-related macular degeneration compared to normal subjects

Purpose

To evaluate visual function variations in eyes with age-related macular degeneration (AMD) compared to normal eyes under different light/contrast conditions using a time-dependent visual acuity testing instrument, the Central Vision Analyzer (CVA).

Methods

Overall, 37 AMD eyes and 35 normal eyes were consecutively tested with the CVA after assessing best-corrected visual acuity (BCVA) using ETDRS charts. The CVA established visual thresholds for three mesopic environments (M1 (high contrast), M2 (medium contrast), and M3 (low contrast)) and three backlight-glare environments (G1 (high contrast, equivalent to ETDRS), G2 (medium contrast), and G3 (low contrast)) under timed conditions. Vision drop across environments was calculated, and repeatability of visual scores was determined.

Results

BCVA significantly reduced with decreasing contrast in all eyes. M1 scores for BCVA were greater than M2 and M3 (P<0.001); G1 scores were greater than G2 and G3 (P<0.01). BCVA dropped more in AMD eyes than in normal eyes between M1 and M2 (P=0.002) and between M1 and M3 (P=0.003). In AMD eyes, BCVA was better using ETDRS charts compared to G1 (P<0.001). The drop in visual function between ETDRS and G1 was greater in AMD eyes compared to normal eyes (P=0.004). Standard deviations of test–retest ranged from 0.100 to 0.139 logMAR.

Conclusion

The CVA allowed analysis of the visual complaints that AMD patients experience with different lighting/contrast time-dependent conditions. BCVA changed significantly under different lighting/contrast conditions in all eyes, however, AMD eyes were more affected by contrast reduction than normal eyes. In AMD eyes, timed conditions using the CVA led to worse BCVA compared to non-timed ETDRS charts.     

Baicalein Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting TSLP-Induced Inflammatory Response

PurposeTo investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms.MethodsThe noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro.ResultsBaicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1β, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1β, IL-6, and TNF-α were significantly lower in the TSLP siRNA–treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1β, IL-6, and TNF-α.ConclusionsBaicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.     

A Case of Candida albicans Endophthalmitis with No Predisposing Risk Factors and a Distant Source of Infection

Purpose

To report a case of Candida albicans endophthalmitis with no identifiable predisposing risk factors.

Case Report

A 57-year-old male presented with a 3-day history of worsening floaters and reduced visual acuity. Fundoscopy and optical coherence tomography showed presence of fluffy white preretinal and intraretinal infiltrates. With no past medical history or evidence of immunosuppression but having travelled abroad and suffered from diarrhoea, fungal aetiology was thought to be unlikely and as a result, treatment was commenced for toxoplasma. Despite treatment, his vision did not improve. Initial investigations including inflammatory markers, serology for toxoplasmosis, blood culture, chest radiograph and aqueous sampling could not identify a source of infection. However, polymerase chain reaction results from vitreous sampling revealed C. albicans. As a result, the patient was treated with intravenous voriconazole and intravitreal amphotericin B. As initial clinical improvement was limited, a vitrectomy was performed with further intravitreal amphotericin B. Clinical improvement was rapid following vitrectomy. After repeated Gram staining and culture of infected toenails, Gram-positive yeast cells were isolated.

Conclusion

Although C. albicans is a frequent cause of endogenous endophthalmitis, patients often have one or more predisposing systemic condition assisting the diagnosis. The present case illustrates that (1) even in the absence of any predisposing risk factors, C. albicans should be considered as a possible differential diagnosis in recalcitrant uveitis, and (2) endogenous candida endophthalmitis can be a result of fungal infections from distant sites such as the toenails.Key Words: Candida albicans, Endophthalmitis, Amphotericin B, Voriconazole