Filamentous Fungal Keratitis Research Articles

Association between in vitro susceptibility and clinical outcomes in fungal keratitis

PurposeThe purpose of this study was to assess the association between antifungal susceptibility as measured by minimum inhibitory concentration (MIC) and clinical outcomes in fungal keratitis.MethodsThis pre-specified secondary analysis of the Mycotic Ulcer Treatment Trial II (MUTT II) involved patients with filamentous fungal keratitis presenting to Aravind Eye Hospitals in South India. Antifungal susceptibility testing for natamycin and voriconazole was performed on all samples with positive fungal culture results according to Clinical and Laboratory Standards Institute Guidelines. The relationship between MIC and clinical outcomes of best-corrected visual acuity, infiltrate or scar size, corneal perforation, need for therapeutic penetrating keratoplasty, and time to re-epithelialization were assessed.ResultsWe obtained MIC values from 141 patients with fungal keratitis. The most commonly cultured organisms were Aspergillus (46.81%, n = 66) and Fusarium (44.68%, n = 63) species. Overall, there was no association between antifungal MICs and clinical outcomes. Subgroup analysis revealed that among Fusarium-positive cases, higher voriconazole MIC was correlated with worse three-month best-corrected visual acuity (p = 0.03), increased need for therapeutic penetrating keratoplasty (p = 0.04), and time to re-epithelialization (p = 0.03). No significant correlations were found among Aspergillus-positive cases. There were no significant correlations found between natamycin MIC and clinical outcomes among organism subgroups.ConclusionsDecreased susceptibility to voriconazole was associated with increased odds of requiring a therapeutic penetrating keratoplasty in Fusarium-positive cases. Susceptibility to natamycin was not associated with any of the measured outcomes.

Overview of Mycotic Keratitis.

Keratomycosis is a serious corneal infection associated with high ocular morbidity that can lead to severe vision loss. It is estimated to affect more than 1 million patients annually, most commonly occurring in tropical climates, and represents a growing threat to patients worldwide. Despite aggressive medical management, fungal infections have a higher rate of perforation requiring surgical intervention compared with other infectious etiologies. Early diagnosis and appropriate treatment are keys to preserving vision and saving patients' eyes.Timely diagnosis of fungal keratitis helps minimize corneal damage and scarring and increases the likelihood of a favorable outcome. Studies have shown that correct identification of fungal infections is often delayed up to 2 to 3 weeks after initial presentation. This leads to incorrect or ineffective treatment for many patients. Diagnostic techniques explored in this study include corneal scrapings with staining and culture, visualization with in vivo confocal microscopy, molecular diagnostic techniques including polymerase chain reaction, and recently developed omics-based technologies.Treatment of fungal keratitis begins with topical antifungals. Medical management has been proven to be effective, but with limitations including poor drug penetration and low bioavailability. Cases that do not respond to topical therapy require more invasive and novel treatments to control the infection. We review the clinical trials that have shaped current practice patterns, with focus on the efficacy of topical natamycin as the primary therapy for filamentous fungal keratitis. We explore additional management strategies such as localized intrastromal and intracameral injections of antifungal medications, photodynamic therapy, and surgical intervention.

Coniochaeta hoffmannii and mutabilis Invasive Fungal Keratitis: A Case Series and Literature Review

Purpose: To describe 2 Coniochaeta species (Coniochaeta hoffmannii and Coniochaeta mutabilis) as rare causes of invasive fungal keratitis resulting in enucleations and to provide a review of ocular infections caused by the genus Coniochaeta and underscore changes to its classification over the past 3 decades, which may clarify previous reports with outdated nomenclature. Methods: This is a small case series from a single academic institution (Casey Eye Institute) with a summary of the literature. Results: The first case describes a 53-year-old man who had an indolent but persistent anterior chamber reaction after repair of a penetrating nail injury. Cultures from a second penetrating keratoplasty identified C. hoffmannii, which was determined through phenotypic characterization and DNA sequencing. Despite surgical management, cultures were persistently positive for C. hoffmannii. Two years post-injury, he developed a funnel retinal detachment with proliferative vitreoretinopathy and elected for enucleation. The second case describes a 54-year-old woman with poor contact lens hygiene with a presumed filamentous fungal keratitis that remained refractory to topical natamycin. Broad-range polymerase chain reaction of an anterior chamber aspirate identified C. mutabilis. Therapeutic keratoplasty was performed for worsening infection; however, because of intractable pain, she elected for enucleation. Pathology from the enucleation specimen demonstrated persistent, but sparse, fungal elements. Conclusions: This is the first case series characterizing ocular infections caused by Coniochaeta, a rare cause of invasive fungal keratitis resulting in devastating outcomes for our patients. Identification of Coniochaeta was initially difficult by traditional culturing techniques, and subsequent molecular diagnostic testing proved useful in detection. Our review of ocular Coniochaeta clarifies previous reports with outdated nomenclature.

Vision-Related Quality of Life Outcomes in Patients Treated for Filamentous Fungal Keratitis in the CLAIR Trial

Purpose To analyze vision-related quality of life in patients with fungal keratitis treated with topical antifungal medications and adjuvant crosslinking in the Cross-Linking Assisted Infection Reduction (CLAIR) trial Methods Study participants were randomized to one of four treatment combinations: (1) topical natamycin 5%, (2) topical natamycin 5% plus CXL, (3) topical amphotericin 0.15%, and (4) topical amphotericin 0.15% plus CXL. All participants responded to the Indian visual function questionnaire (IND-VFQ) at the baseline visit and 3 months after enrollment. Responses were averaged within four subscales (mobility, activity limitation, psychosocial impact, and visual function). Scores were compared between the acute phase of infection and after treatment. Linear regressions were then performed to compare IND-VFQ results between the four arms. Results IND-VFQ scores improved in patients three months after initiating treatment compared to baseline (P<0.01). Participants treated with amphotericin had a VFQ score 2.60 points (95% CI, -6.90-12.10) higher than those treated with natamycin (P=0.59). Participants treated with CXL had an average VFQ score 4.15 points (95% CI, -5.43-13.70) higher than those treated with medication only (P=0.29). Subscale analysis did not show significant differences between treatment groups. Conclusions All patients reported improved vision-related quality of life after treatment of fungal keratitis. Treatment with natamycin and amphotericin B produced similar results. Primary adjunctive CXL did not benefit patients with respect to vision-related quality of life in fungal keratitis patients. These results reflected primary results of CLAIR regarding microbiological cure, infiltrate and/or scar size, percent epithelialization, and frequency of adverse events.

FUNGAL KERATITIS - AN UPDATE ON THE GLOBAL THREAT

Fungal keratitis (FK) is a sight-threatening disease caused by infection of the cornea by filamentous fungi or yeasts. FK can be challenging to diagnose and treat. In tropical, low, and middle-income countries, it accounts for the majority of cases of microbial keratitis (MK). Filamentous fungi, in particular, Fusarium spp., the aspergilli, and dematiaceous fungi are responsible for the most significant burden of disease. The predominant risk factor for filamentous fungal keratitis is trauma, typically with organic, plant-based material. In 2020, the incidence of FK was estimated to be over 1 million cases per year, with a significant geographical variation. Diagnosing FK is challenging accurate diagnosis relies on reliable microscopy and culture. Current topical antifungals are not very effective infections can progress despite prompt treatment. Antifungal drops are often unavailable. When available, natamycin is usually the first-line treatment. However, infections may progress to perforation in ~25% of cases. Future work needs to be directed at addressing these challenges and unmet needs. This review discusses the epidemiology, clinical features, diagnosis, management, and etiology of FK.

An artificial intelligence approach to classify pathogenic fungal genera of fungal keratitis using corneal confocal microscopy images.

Fungal keratitis is a common cause of blindness worldwide. Timely identification of the causative fungal genera is essential for clinical management. In vivo confocal microscopy (IVCM) provides useful information on pathogenic genera. This study attempted to apply deep learning (DL) to establish an automated method to identify pathogenic fungal genera using IVCM images. Deep learning networks were trained, validated, and tested using a data set of 3364 IVCM images that collected from 100 eyes of 100 patients with culture-proven filamentous fungal keratitis. Two transfer learning approaches were investigated: one was a combined framework that extracted features by a DL network and adopted decision tree (DT) as a classifier; another was a complete supervised DL model which used DL-based fully connected layers to implement the classification. The DL classifier model revealed better performance compared with the DT classifier model in an independent testing set. The DL classifier model showed an area under the receiver operating characteristic curves (AUC) of 0.887 with an accuracy of 0.817, sensitivity of 0.791, specificity of 0.831, G-mean of 0.811, and F1 score of 0.749 in identifying Fusarium, and achieved an AUC of 0.827 with an accuracy of 0.757, sensitivity of 0.756, specificity of 0.759, G-mean of 0.757, and F1 score of 0.716 in identifying Aspergillus. The DL model can classify Fusarium and Aspergillus by learning effective features in IVCM images automatically. The automated IVCM image analysis suggests a noninvasive identification of Fusarium and Aspergillus with clear potential application in early diagnosis and management of fungal keratitis.

Management of Filamentous Fungal Keratitis: A Pragmatic Approach.

Filamentous fungal infections of the cornea known as filamentous fungal keratitis (FK) are challenging to treat. Topical natamycin 5% is usually first-line treatment following the results of several landmark clinical trials. However, even when treated intensively, infections may progress to corneal perforation. Current topical antifungals are not always effective and are often unavailable. Alternatives topical therapies to natamycin include voriconazole, chlorhexidine, amphotericin B and econazole. Surgical therapy, typically in the form of therapeutic penetrating keratoplasty, may be required for severe cases or following corneal perforation. Alternative treatment strategies such as intrastromal or intracameral injections of antifungals may be used. However, there is often no clear treatment strategy and the evidence to guide therapy is often lacking. This review describes the different treatment options and their evidence and provides a pragmatic approach to the management of fungal keratitis, particularly for clinicians working in tropical, low-resource settings where fungal keratitis is most prevalent.

Clinical differentiation of Pythium keratitis from fungal keratitis and development of a scoring system.

To differentiate Pythium keratitis from fungal keratitis using clinical signs, to explore usefulness of various signs as diagnostic prognosticators, and develop a clinical scoring system. A retrospective review of medical records and archived clinical photographs of patients with culture-positive Pythium keratitis and hyaline filamentous fungal keratitis was conducted at a tertiary eye institute to explore characteristics of ulcers that may aid diagnosis. Full-thickness corneal stromal keratitis (P = 0.055), a dry ulcer surface (P = 0.010), tentacles (P < 0.0001), intrastromal dots (P < 0.0001), ring infiltrates (P = 0.024), reticular patterns (P < 0.0001), and peripheral furrows (P < 0.0001) were clinical signs associated with Pythium keratitis. Multiple regression analysis identified tentacles (odds ratio: 24.1, 95% confidence interval (CI): 3.8-158.1, P = 0.001) and peripheral furrows (odds ratio: 60.6, 95% CI: 5.1-712.3, P = 0.001) as independent diagnostic prognosticators for Pythium keratitis. The positive and negative likelihood ratios of a dry ulcer surface, tentacles, intrastromal dots, ring infiltrates, reticular patterns, and peripheral furrows predicting Pythium keratitis were 1.6, 13.6, 17.9, 4.3, 30.7, 15.3 and 0.4, 0.4, 0.7, 0.9, 0.6 and 0.8, respectively. The presence of two or more of these clinical signs (excluding a dry ulcer surface) had a sensitivity of 55.6% and a false positive rate of 1.4%. Tentacles, intrastromal dots, ring infiltrates, reticular patterns, and peripheral furrows are clinical signs to be considered for the diagnosis of Pythium keratitis and the presence of two or more signs has a very low false positive rate.

Diagnosis of Fungal Keratitis in Low-Income Countries: Evaluation of Smear Microscopy, Culture, and In Vivo Confocal Microscopy in Nepal.

Clinically diagnosing fungal keratitis (FK) is challenging; diagnosis can be assisted by investigations including in vivo confocal microscopy (IVCM), smear microscopy, and culture. The aim of this study was to estimate the sensitivity in detecting fungal keratitis (FK) using IVCM, smear microscopy, and culture in a setting with a high prevalence of FK. In this cross-sectional study nested within a prospective cohort study, consecutive microbial keratitis (MK) patients attending a tertiary-referral eye hospital in south-eastern Nepal between June 2019 and November 2020 were recruited. IVCM and corneal scrapes for smear microscopy and culture were performed using a standardised protocol. Smear microscopy was performed using potassium hydroxide (KOH), Gram stain, and calcofluor white. The primary outcomes were sensitivities with 95% confidence intervals [95% CI] for IVCM, smear microscopy and culture, and for each different microscopy stain independently, to detect FK compared to a composite referent. We enrolled 642 patients with MK; 468/642 (72.9%) were filamentous FK, 32/642 (5.0%) were bacterial keratitis and 64/642 (10.0%) were mixed bacterial-filamentous FK, with one yeast infection (0.16%). No organism was identified in 77/642 (12.0%). Smear microscopy had the highest sensitivity (90.7% [87.9–93.1%]), followed by IVCM (89.8% [86.9–92.3%]) and culture (75.7% [71.8–79.3%]). Of the three smear microscopy stains, KOH had the highest sensitivity (85.3% [81.9–88.4%]), followed by Gram stain (83.2% [79.7–86.4%]) and calcofluor white (79.1% [75.4–82.5%]). Smear microscopy and IVCM were the most sensitive tools for identifying FK in our cohort. In low-resource settings we recommend clinicians perform corneal scrapes for microscopy using KOH and Gram staining. Culture remains an important tool to diagnose bacterial infection, identify causative fungi and enable antimicrobial susceptibility testing.

Topical Chlorhexidine 0.2% versus Topical Natamycin 5% for the Treatment of Fungal Keratitis in Nepal: A Randomized Controlled Noninferiority Trial

PurposeTo investigate if topical chlorhexidine 0.2%, which is low cost and easy to formulate, is noninferior to topical natamycin 5% for the treatment of filamentous fungal keratitis.DesignRandomized controlled, single-masked, noninferiority clinical trial.ParticipantsAdults attending a tertiary-level ophthalmic hospital in Nepal with filamentous fungal infection confirmed on smear or confocal microscopy.MethodsParticipants were randomly allocated to receive topical chlorhexidine 0.2% or topical natamycin 5%. Primary analysis (intention-to-treat) was by linear regression, using baseline logarithm of the minimum angle of resolution (logMAR) best spectacle-corrected visual acuity (BSCVA) and treatment arm as prespecified covariates. Mixed fungal-bacterial infections were excluded from the primary analysis but included in secondary analyses and secondary safety-related outcomes. The noninferiority margin was 0.15 logMAR. This trial was registered with ISRCTN, number ISRCTN14332621.Main Outcome MeasuresThe primary outcome measure was BSCVA at 3 months. Secondary outcome measures included perforation or therapeutic penetrating keratoplasty by 90 days.ResultsBetween June 3, 2019, and November 9, 2020, 354 eligible participants were enrolled and randomly assigned: 178 to chlorhexidine and 176 to natamycin. Primary outcome data were available for 153 and 151 of the chlorhexidine and natamycin groups, respectively. Of these, mixed bacterial-fungal infections were found in 20 cases (12/153 chlorhexidine, 8/151 natamycin) and excluded from the primary analysis. Therefore, 284 patients were assessed for the primary outcome (141 chlorhexidine, 143 natamycin). We did not find evidence to suggest chlorhexidine was noninferior to natamycin and in fact found strong evidence to suggest that natamycin-treated participants had significantly better 3-month BSCVA than chlorhexidine-treated participants, after adjusting for baseline BSCVA (regression coefficient, −0.30; 95% confidence interval [CI], −0.42 to −0.18; P < 0.001). There were more perforations and emergency corneal grafts in the chlorhexidine arm (24/175, 13.7%) than in the natamycin arm (10/173, 5.8%; P = 0.018, mixed infections included), whereas natamycin-treated cases were less likely to perforate or require an emergency corneal graft, after adjusting for baseline ulcer depth (odds ratio, 0.34; 95% CI, 0.15–0.79; P = 0.013).ConclusionsTreatment with natamycin is associated with significantly better visual acuity, with fewer adverse events, compared with treatment with chlorhexidine. Natamycin remains the preferred first-line monotherapy treatment for filamentous fungal keratitis.

Mycotic Keratitis-A Global Threat from the Filamentous Fungi.

Mycotic or fungal keratitis (FK) is a sight-threatening disease, caused by infection of the cornea by filamentous fungi or yeasts. In tropical, low and middle-income countries, it accounts for the majority of cases of microbial keratitis (MK). Filamentous fungi, in particular Fusarium spp., the aspergilli and dematiaceous fungi, are responsible for the greatest burden of disease. The predominant risk factor for filamentous fungal keratitis is trauma, typically with organic, plant-based material. In developed countries, contact lens wear and related products are frequently implicated as risk factors, and have been linked to global outbreaks of Fusarium keratitis in the recent past. In 2020, the incidence of FK was estimated to be over 1 million cases per year, and there is significant geographical variation; accounting for less than 1% of cases of MK in some European countries to over 80% in parts of south and south-east Asia. The proportion of MK cases is inversely correlated to distance from the equator and there is emerging evidence that the incidence of FK may be increasing. Diagnosing FK is challenging; accurate diagnosis relies on reliable microscopy and culture, aided by adjunctive tools such as in vivo confocal microscopy or PCR. Unfortunately, these facilities are infrequently available in areas most in need. Current topical antifungals are not very effective; infections can progress despite prompt treatment. Antifungal drops are often unavailable. When available, natamycin is usually first-line treatment. However, infections may progress to perforation in ~25% of cases. Future work needs to be directed at addressing these challenges and unmet needs. This review discusses the epidemiology, clinical features, diagnosis, management and aetiology of FK.

Identification and molecular characterization of Subramaniula asteroides causing human fungal keratitis: a case report

BackgroundKeratitis due to by filamentous fungi are not easy to diagnose thus causing a delay in correct therapy. There are many descriptions of keratitis due to Candida, Fusarium and Aspergillus genera. Subramaniula genus has only recently been reported to cause human infections and there are few descriptions of eye infections due to this filamentous fungus. Diagnosis of fungal keratitis is usually based on microscopic and cultural techniques of samples obtained by corneal swabbing or scraping. Considering the amount of time required to obtain culture results it is wise to use other diagnostic methods, such as molecular analyses. Therapeutic options against these fungi are limited by low tissue penetration in the eye due to ocular barriers. We describe the first case of S. asteroides human keratitis treated with isavuconazole.Case presentationWe describe a rare case of fungal keratitis unresponsive to antimicrobial treatment in a 65-year-old male patient without a history of diabetes or immunological diseases. He reported that the onset of symptoms occurred during a long holiday in Cape Verde Island. Initial treatment with topical antibiotics associated to steroids were ineffective, allowing a slow clinical progression of disease to corneal perforation. On admission in our Hospital, slit-lamp examination of the left eye showed conjunctival congestion and hyperemia, a large inferior corneal ulceration with brown pigment, corneal edema, about 3 mm of hypopyon and irido-lenticular synechiae. The slow clinical progression of the disease to corneal perforation and the aspect of the ulcer were consistent with a mycotic etiology. Molecular methods used on fungal colonies isolated by Sabouraud’s dextrose agar cultures allowed the identification of Subramaniula asteroids from corneal scraping. Antimicrobial test showed a good susceptibility of this filamentous fungus to voriconazole and isavuconazole. Moreover, this fungal keratitis was successfully treated with isavuconazole, without side effects, observing a progressive clinical improvement.ConclusionsMolecular methods may be useful for the identification of filamentous fungal keratitis on scraping samples thus shortening the time of diagnosis. Systemic therapy by isavuconazole could be useful to treat the filamentous fungal keratitis, reducing the possible adverse effects due to the use of voriconazole by systemic administration.

Determination of surgical outcomes with a novel formulation of intrastromal natamycin in recalcitrant fungal keratitis: A pilot study.

Purpose:To evaluate the outcomes of water-soluble intrastromal natamycin (IS-NTM) as an adjunct therapy for recalcitrant fungal keratitis.Methods:This was a prospective interventional pilot study in the setting of a tertiary eye-care center. Twenty eyes of 20 consecutive patients with microbiologically proven recalcitrant fungal keratitis (ulcer size >2 mm, depth >50%, and not responding to topical NTM for 2 weeks) were recruited. The selected patients were injected with a novel composition of IS-NTM (10 ug/0.1 mL, soluble natamycin) prepared aseptically in the ocular pharmacology department. All the patients continued using topical NTM suspension 5% 4-hourly until the ulcer healed. Repeat injections were undertaken after 72 h depending on the clinical response and all the patients were followed till 6 months.Results:The mean age of the patients was 40.42 ± 10.09 years. The mean duration of the presentation was 20.8 ± 5.1 days. The most commonly isolated organisms were Aspergillus sp. (12/20, 60%) and Fusarium sp. (8/20, 40%). No patient had iatrogenic perforation or precipitate formation after IS-NTM injection. The overall cure rate with IS-NTM was 95% (19/20 patients). The number of patients who healed with the 1st, 2nd, and 3rd injection was 13, 5, and 1, respectively. One (5%) had no response to treatment and was subjected to penetrating keratoplasty. The average time taken for the resolution of the epithelial defect, stromal infiltrates, and hypopyon was 34 ± 5.2 days, 35.3 ± 6.4 days, and 15 ± 2.5 days. Healing with deep vascularization and cataract was noted in 6/19 eyes (31%) and 13/19 eyes (68.42%), respectively.Conclusion:Intrastromal injection of a novel formulation of NTM holds a promising role as adjunctive therapy to topical NTM in the management of recalcitrant filamentous fungal keratitis. The preliminary results are encouraging and further studies are required to validate the results.

Cross-Linking Assisted Infection Reduction: One-year Follow-up of a Randomized Clinical Trial Evaluating Cross-Linking for Fungal Keratitis

Although the evidence for corneal cross-linking (CXL) as a treatment for fungal keratitis is not robust, clinicians have used adjunctive CXL in hopes that there will be some benefit for this challenging disease. Cross-Linking Assisted Infection Reduction was a randomized, outcome-masked clinical trial that found no benefit to CXL in the primary treatment of filamentous fungal keratitis at 3 months.1 We report on the 12-month outcomes of the Cross-Linking Assisted Infection Reduction trial.

Filamentous Fungal Keratitis in Taiwan: Based on Molecular Diagnosis.

PurposeTo analyze the epidemiological pattern, demographics, risk factors, and treatment outcomes of filamentous fungal keratitis at a tertiary hospital in Taiwan.MethodsWe recruited 65 patients (65 eyes) with culture-proven filamentous fungal keratitis who received diagnosis and treatment at Chang Gung Memorial Hospital between 2015 and 2018. All isolates were examined through conventional morphological identification and subjected to molecular identification with internal transcribed spacer sequencing. Data on patient demographics, predisposing factors, and treatment outcomes were collected.ResultsIn total, filamentous fungi belonged to 16 genera were identified. Fusarium spp. (29 cases [44.6%]) was the most commonly isolated organism overall, followed by Colletotrichum spp. and Purpureocillium linacinum (seven cases [10.8% for each]), and Aspergillus spp. (six cases [9.2%]). Some fungi that have not been regarded as human pathogens were also identified, such as Paracremonium and Phellinum. Among 52 (80%) patients with predisposing factors, 30 (46.2%) had trauma. The ulcers of 33 (50.8%) patients resolved with medical treatment only. Additionally, six patients (9.2%) had corneal perforation, and nine patients (13.9%) required therapeutic/destructive surgical interventions including therapeutic penetrating keratoplasty (seven patients) or evisceration (two patients). Only 16 patients (24.6%) had final visual acuity of 20/40 or better.ConclusionsThrough molecular diagnosis, a high diversity of fungal pathogens was revealed along with an increasing incidence of Colletotrichum spp. and Purpureocillium spp. in Taiwan. The most common risk factor for filamentous fungal keratitis was trauma. The visual outcomes were guarded.Translational RelevanceThe molecular diagnosis provides insight into accurate identification, which affects the epidemiology and diversity of pathogens of filamentous fungal keratitis.

The Prognostic Value of Persistent Culture Positivity in Fungal Keratitis in the Mycotic Antimicrobial Localized Injection Trial

To evaluate the utility of repeat cultures at days 3 and 7 after starting antifungal medications for predicting outcomes in fungal keratitis. Prespecified secondary analysis of the randomized clinical Mycotic Antimicrobial Localized Injection trial. Patients presenting to Aravind Eye Hospital, Pondicherry, India, with fungal keratitis and visual acuity worse than 20/70 received topical natamycin and were randomized to either receive intrastromal injection of voriconazole or topical therapy alone. All subjects received corneal cultures at date of presentation, day 3, and day 7. Outcome measures included 3-week and 3-month visual acuity and scar size, corneal perforation, and/or the need for therapeutic penetrating keratoplasty (TPK). Visual acuity and scar size were analyzed with multiple linear regression controlling for baseline measures. Survival analysis was used to analyze the risk of corneal perforation and/or need for TPK. Of the 70 study subjects with fungal keratitis, 25 of 69 (36%) remained culture positive at day 3, and 20 of 62 (32%) were culture positive at day 7. Culture positivity at day 3 conferred a hazard ratio of 2.8 for requiring TPK (P= .03) but was not a statistically significant predictor of perforation, scar size, or final visual acuity. Culture positivity at day 7 had a hazard ratio of 3.5 for requiring TPK (P= .003). Those with positive cultures at day 7 had on average 3 logMAR lines worse visual acuity at 3months (95% confidence interval 0.9 to 5.2 logMAR lines, P= .006) and 1.1mm larger scar size at 3months after controlling for baseline measures (95% confidence interval 0.1 to 2.2mm; P= .03). While not as predictive as day 7 cultures, culture positivity at day 3 after starting treatment is a significant predictor of the need for TPK in patients with moderate-to-severe filamentous fungal keratitis. This has applications for risk stratification, and may facilitate earlier consideration of TPK in high-risk patients.

Colletotrichum keratitis: A rare yet important fungal infection of human eyes.

Colletotrichum is a rare cause of human infection. Previous reports about Colletotrichum keratitis were limited, and most diagnoses from past reports were based on morphological distinction, which could have led to underestimation of the prevalence of Colletotrichum species. We reported phylogenetic analysis, clinical feature and treatment outcome of molecularly diagnosed Colletotrichum keratitis in our hospital. We recruited 65 patients with culture-proven filamentous fungal keratitis between January 1, 2015 and December 30, 2018. Through molecular sequencing including internal transcribed spacer (ITS) and multi-locus phylogenetic analysis of fungal DNA, seven patients were verified as infected with Colletotrichum species, and their medical records were reviewed to determine the clinical characteristics. Six of seven patients had predisposing factors including trauma (5) and immunosuppressive status (1). Six isolates were initially misidentified as other fungi through morphological identification. ITS sequencing identified the isolates belonged to two species complex (SC): C.truncatum and C.gloeosporioides; multi-locus phylogenetic analysis enabled species identification including C.tropicale (3), C.fructicola (2), C.truncatum (1) and C.fusiforme (1). Five patients with C.gloeosporioides SC responded well to medical treatment and two patients with Ctruncatum SC underwent evisceration because of either no visual potential or intractable pain. The molecular approach provides accurate diagnosis and raises epidemiological awareness of Colletotrichum keratitis. Through multi-locus phylogenetic analysis, we report the human infections caused by C.tropicale, C.fructicola and C.fusiforme. We also highlight the different clinical outcomes between C.gloeosporioides SC and C.truncatum SC.

Cross-Linking–Assisted Infection Reduction: A Randomized Clinical Trial Evaluating the Effect of Adjuvant Cross-Linking on Outcomes in Fungal Keratitis

To determine if there is a benefit to adjuvant corneal crosslinking (CXL) and to compare natamycin versus amphotericin B for filamentous fungal keratitis. Outcome-masked, 2×2 factorial design, randomized controlled clinical trial. Consecutive patients presenting with moderate vision loss from a smear-positive fungal ulcer at Aravind Eye Hospital, Madurai, India. Study eyes were randomized to 1 of 4 treatment combinations using an adaptive randomization protocol. The treatment arms included (1) topical natamycin 5% alone, (2) topical natamycin 5% plus CXL, (3) topical amphotericin B 0.15% alone, and (4) topical amphotericin 0.15% plus CXL. The primary outcome of the trial was microbiological cure at 24 hours on repeat culture. Secondary outcomes included best spectacle-corrected visual acuity (BSCVA) at 3 weeks and 3 months, percentage of study participants with epithelial healing at 3 days, 3 weeks, and 3 months, infiltrate or scar size at 3 weeks and 3 months, 3-day smear and culture, and adverse events. Those randomized to CXL regardless of medication (topical natamycin or amphotericin) had 1.32-fold increased odds of 24-hour culture positivity, although this was not statistically significant (95% confidence interval [CI], 0.57-3.06; P= 0.51). We were also unable to find a difference in 24-hour culture positivity between those randomized to amphotericin and those randomized to natamycin when evaluating as a group regardless of whether or not they received CXL (coefficient 1.10; 95% CI, 0.47-2.54; P= 0.84). The BSCVA was approximately 0.22 logarithm of the minimum angle of resolution (logMAR) (2.2 Snellen lines) worse on average at 3 weeks among those receiving CXL regardless of medication (95% CI,-0.04 to 0.40; P= 0.04) and 0.32 logMAR (3.2 Snellen lines) worse visual acuity at 3 months after controlling for baseline visual acuity (95% CI, 0.03-0.54; P= 0.02). There was no difference in infiltrate or scar size, percentage of epithelialized or adverse events when comparing CXL with no CXL or the 2 topical medications. There appears to be no benefit of adjuvant CXL in the primary treatment of moderate filamentous fungal ulcers, and it may result in decreased visual acuity.

Mycotic Antimicrobial Localized Injection: A Randomized Clinical Trial Evaluating Intrastromal Injection of Voriconazole

To determine if there is a benefit to adjuvant intrastromal voriconazole (ISV) injections for primary treatment of filamentous fungal keratitis. Outcome-masked, randomized controlled clinical trial. Patients with moderate vision loss resulting from a smear-positive fungal ulcer. Study eyes were randomized to topical natamycin plus ISV injection versus topical natamycin alone. The primary outcome of the trial was microbiological cure on 3-day repeat culture analysis. Secondary outcomes included microbiological cure on 7-day repeat culture analysis; 3-week and 3-month best spectacle-corrected visual acuity; infiltrate or scar size or both; rate of perforation; therapeutic penetrating keratoplasty (TPK); and other adverse events. A total of 151 patients with smear-positive ulcers were screened and 70 were enrolled at Aravind Eye Hospital, Pondicherry, India. Baseline cultures grew Fusarium in 19 samples (27%), Aspergillus in 17 samples (24%), and other filamentous fungi in 19 samples (27%) and showed negative results in 13 samples (19%). Those randomized to ISV injection had 1.82 times the odds of 3-day culture positivity after controlling for baseline culture status (95% confidence interval [CI], 0.65-5.23; P= 0.26, bias-corrected logistic regression) and 1.98 times the odds of positive 7-day culture results, after controlling for baseline culture status (95% CI, 0.69-5.91; P= 0.20, bias-corrected logistic regression). Those randomized to ISV injection showed 0.5 logMAR lines (approximately 0.5 Snellen lines) of decreased visual acuity (95% CI, -2.6 to 3.6 lines; P= 0.75) and 0.55 mm worse infiltrate or scar size or both at 3 months after controlling for baseline values (95% CI, -0.13 to 1.25; P= 0.11). Intrastromal voriconazole injections showed a 2.85-fold increased hazard of perforation after controlling for baseline infiltrate depth (95% CI, 0.76-10.75; P= 0.12) but no difference in the rate of TPK (hazard ratio, 0.95; 95% CI, 0.44-2.04; P= 0.90). There seems to be no benefit to adding ISV injections to topical natamycin in the primary treatment of moderate to severe filamentous fungal ulcers. Studies consistently suggest that voriconazole has a limited role in the treatment of filamentous fungal ulcers.

Therapeutic Penetrating Keratoplasty Button Cultures in The Mycotic Ulcer Treatment Trial II: A Randomized Trial Comparing Oral Voriconazole Versus Placebo

To compare oral voriconazole vs placebo in addition to topical antifungals in the treatment of filamentous fungal keratitis. Non-prespecified, secondary case-control analysis from a multicenter, double-masked, randomized placebo-controlled clinical trial. Study Participants: Patients with smear-positive filamentous fungal ulcers and visual acuity of 20/400 or worse who eventuated to therapeutic penetrating keratoplasty (TPK). Study participants were randomized to oral voriconazole vs oral placebo; all received topical antifungal drops. TPK button culture positivity. A total of 95 of 194 (49.5%) study participants enrolled at Madurai, Coimbatore, or Pondicherry, India eventuated to TPK in an average of 20.9days (standard deviation 15.2days, range 2-71days). TPK button cultures were available for 67 of 95 (71%) of the TPKs performed and were positive for filamentous fungus in 45 of 67 (67%) cases. For each 1-day increase in the time to TPK there was 0.94-fold decreased odds of fungalculture positivity (95% confidence interval [CI] 0.90-0.98, P= .005). Those randomized to oral voriconazole had 1.26-fold increased odds of TPK button culture positivity after controlling for time to TPK and baseline organism, but this was not statistically significant (95% CI 0.32-4.87; P= .74). Those who underwent TPK for lack of response to medical therapy were 10.64-fold more likely to be culture positive than if the indication for surgery was perforation and this was statistically significant (95% CI 2.16-51.70; P= .003). There appears to be no benefit to adding oral voriconazole to topical antifungal agents in the treatment of severe filamentous fungal ulcers. Infection rather than inflammation appears to be the reason for the worsening clinical picture in many of these patients.