The role of fungi in fungal keratitis.

Fungal keratitis (FK) accounts for approximately half of the microbial keratitis encountered in low middle income countries (LMICs) and predominantly affect the working rural-poor. FK causes significant morbidity with the majority of patients left with moderate or worse visual impairment and approximately 25% requiring expensive and often unsuccessful surgical interventions. The severity of FK and the resultant corneal damage or resolution can be attributed to i) the virulence and bioburden of the fungal pathogen, ii) the host defense mechanism and immune response and iii) sub-optimal diagnostics and anti-fungal treatment strategies. This review provides a comprehensive overview of the multifaceted components that drive FK progression and resolution, highlighting where knowledge gaps exist and areas that warrant further research.

Ten-year trends in the incidence, clinical profile and outcomes of acute-onset endophthalmitis following combined pars plana vitrectomy and sutureless, glueless and flapless scleral fixation of intraocular lenses.

PURPOSE: To evaluate the frequency and outcomes of acute-onset endophthalmitis following combined pars plana vitrectomy and scleral fixation of intraocular lens. METHODS: We evaluated patients undergoing a sutureless, glueless, flapless technique of scleral fixation of intraocular lenses (SFIOL) implantation for various causes of aphakia and documented the clinico-demographic data, microbiological profile and final outcome after acute endophthalmitis in this cohort of eyes. RESULTS: The frequency of suspected acute endophthalmitis diagnosed post-surgery was 0.112% (4/3541 eyes), with culture-positive endophthalmitis frequency being 0.028% (1 eye), showing growth of Pseudomonas aeruginosa. Mean age of patients with endophthalmitis was 51.75 ± 9.28 years, and mean interval between surgery and acute endophthalmitis presentation was 10.25 ± 9.6 days. Patients were managed with intravitreal antibiotics with or without core vitrectomy. Visual acuity of patients increased from baseline 1.43 ± 0.32 logMAR (Snellen equivalent = 6/150) to 0.79 ± 0.16 logMAR (Snellen equivalent = 6/36) after an average follow-up of 11 ± 2 weeks. CONCLUSION: Endophthalmitis is a rare complication following SFIOL surgery, and all ophthalmic surgeons must be aware of this inadvertent possibility, since SFIOLs are gaining wider acceptability recently. Moreover, these cases of endophthalmitis may show a different pattern of microorganisms than post-cataract surgery endophthalmitis; however, with prompt diagnosis and effective timely management, favorable outcomes can be achieved.

Development and Clinical Evaluation of a CRISPR/Cas12a-Based Nucleic Acid Detection Platform for the Diagnosis of Keratomycoses.

Objective: The objective of this study was to develop a rapid and accurate clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based molecular diagnostic assay (Rapid Identification of Mycoses using CRISPR, RID-MyC assay) to detect fungal nucleic acids and to compare it with existing conventional mycologic methods for the diagnosis of fungal keratitis (FK). Design: This study was structured as a development and validation study focusing on the creation and assessment of the RID-MyC assay as a novel diagnostic modality for FK. Subjects: Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India. Methods: The RID-MyC assay utilized recombinase polymerase amplification targeting the 18S ribosomal RNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction for the diagnosis of FK. Main Outcome Measures: The primary outcome measures focused on the analytical sensitivity and specificity of the RID-MyC assay in detecting fungal nucleic acids. Secondary outcomes measured the assay's diagnostic sensitivity and specificity for FK, including its concordance with conventional diagnostic methods. Results: The RID-MyC assay exhibited a detection limit ranging from 13.3 to 16.6 genomic copies across 4 common fungal species. In patients with microbial keratitis, the RID-MyC assay showed substantial agreement with microscopy (kappa = 0.714) and fair agreement with culture (kappa = 0.399). The assay demonstrated a sensitivity of 93.27% (95% confidence interval [CI], 86.62%-97.25%) and a specificity of 89.47% (95% CI, 66.86%-98.70%) for FK diagnosis, with a median diagnostic time of 50 minutes (range, 35-124 minutes). Conclusions: The RID-MyC assay, utilizing CRISPR-Cas12a technology, offers high diagnostic accuracy for FK. Its potential for point-of-care use could expedite and enhance the precision of fungal diagnostics, presenting a promising solution to current diagnostic challenges. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Microbiological Profile of Post-cataract Surgery Endophthalmitis with Usage of Prophylactic Intracameral Moxifloxacin.

PURPOSE: To study the effect of prophylactic intracameral moxifloxacin (ICM) on microbiological profile and antimicrobial sensitivity of culture-proven postoperative endophthalmitis (POE). METHODS: This study evaluated culture-proven POE diagnosed within 6 weeks of cataract surgery over two periods, period-1 (January 2010-March 2015) and period-2 (April 2015-December 2019), before and after introduction of prophylactic ICM, respectively. RESULTS: In period-1, 100 cases of culture-positive POE were reported (1 in 4879, 0.02%), while 20 cases (1 in 24635, 0.004%) were reported in period-2 (5-fold reduction, p < .001). The cumulative culture positivity rate decreased from 27.6% to 17.1% (1.6-fold) . Coagulase-negative Staphylococci (CoNS) were significantly reduced (p = .005). CONCLUSIONS: ICM reduced the incidence of culture-proven POE, CoNS in particular. In future, POE caused by rarer pathogens may become more prevalent due to reduction in the rate of commoner and more virulent pathogens with use of intracameral antibiotics. The moxifloxacin sensitivity of CONS did not show change with the use of ICM. Studies with larger population of POE will be more helpful to understand the trend better.

Incidence, Clinical Profile, and Management of Keratitis Caused by Uncommon Species of Pseudomonas at a Tertiary Eye Care Center.

PURPOSE: The purpose of this study was to study the incidence, demographic features, clinical course, profiling, and management of uncommon species of Pseudomonas keratitis (other than Pseudomonas aeruginosa ) at a tertiary eye care center. METHODS: Thirty cases of culture-proven uncommon species of Pseudomonas keratitis between January 2017 and December 2021 were retrospectively studied. The incidence, demographic and clinical profile, predisposing factors, microbial results, treatment, and visual outcomes were analyzed. We evaluated the risk factors for poor treatment outcomes. RESULTS: Among bacterial keratitis cases, uncommon species of Pseudomonas keratitis occurred at a rate of 2.2%. The mean age at presentation was 51.37 years, and the most common predisposing factor was corneal trauma (36.7%). The mean best corrected visual acuity (BCVA) [in log of minimum angle of resolution (logMAR)] at presentation was 1.99, and the mean ulcer size was 5.75 mm. On culture, 56.7% of the cases were identified as Pseudomonas putida , 26.7% as Pseudomonas stutzeri , 10% as Pseudomonas mendocina, and 3.3% each of Pseudomonas oryzihabitans and Pseudomonas alcaligenes . We recorded good treatment responses in 66.7% of cases with the medical therapy of a combination of broad-spectrum antibiotics, whereas 33.3% of cases required surgical intervention. The risk factors for poor clinical outcome were older age, ocular trauma, previous ocular surgeries, poor BCVA at presentation, large ulcer size, delayed treatment, hypopyon, and early complications such as perforation, limbal involvement, and total ulcer. CONCLUSIONS: Uncommon species of pseudomonas keratitis was more closely related to predisposing factors such as corneal trauma and other factors such as previous ocular surgeries, older age, large ulcers, longer duration of treatment, early surgical intervention in complicated cases, and poor visual outcome.

Challenges in Post-cataract Surgery Nocardia Endophthalmitis: Management Strategies and Clinical Outcomes.

PURPOSE: To describe the clinical and microbiological features, management strategies, outcomes, and challenges in post-cataract surgery nocardia endophthalmitis. METHODS: We evaluated 28 eyes with culture-confirmed Nocardia endophthalmitis following cataract surgery. Data were collected regarding age, gender, clinical presentation, management modalities used, and final outcomes. RESULTS: Mean age of the patients was 66.07 + -8.6 years. Four eyes (14.3%) underwent early pars plana core vitrectomy, while 18 eyes (64.3%) underwent tap/inject regimen followed by observation for 48 h, with or without a delayed vitrectomy. Thirteen eyes (46.4%) received amikacin treatment. Visual acuity (VA) improved from presentation in 9/28 (32.1%) eyes, worsened in 14/28 (50%) eyes, and showed no change in 5/28 eyes (17.9%). CONCLUSIONS: We did not find any significant association between functional outcome and time of surgery or presenting VA. Additional amikacin may be considered if there is a strong suspicion of nocardia endophthalmitis, as it may help in achieving better anatomical outcomes.

Prevalence and Molecular Characterization of Metallo ß-Lactamase Producing Gram-Negative Pathogens Causing Eye Infections.

Purpose: Metallo ß-lactamases (MßL) production is a worldwide problem, particularly in gram-negative bacteria. As scanty data is available on the prevalence of MBL, the present study is being undertaken to determine the prevalence, antibacterial sensitivity patterns, and molecular characterization of MßL associated resistant genes in gram-negative bacteria isolated from ocular infections. Material and Methods: At a tertiary eye care center in south India, 359 gram-negative pathogens, 200 isolates from eye infections, and 159 isolates from normal flora of the eye were studied. A gold standard microbiology method was used to identify the isolates. An antibiotic double disc synergy test and a combination disc test were used to detect MßL production. Multiplex PCR was used to investigate the molecular characteristics of the MßL encoding genes blaVIM, blaIMP, and blaNDM. Results: Of the 359 gram-negative bacterial pathogens, Pseudomonas aeruginosa 108 (30.1%) and Enterobacter agglomerans 46 (12.8%) were commonly isolated. High prevalence of P. aeruginosa 81% (17 strains) was detected as an MßL producer and it shows 100% resistance to 2nd and 3rd generation cephalosporins and meropenem. Multiplex PCR detected only the blaVIM gene in 56 (28%) of various eye infections and 27 (17%) of normal flora of the gram-negative bacteria (GNB). The blaVIM gene is detected predominantly in 51.8% of keratitis and 21.4% of postoperative endophthalmitis. High prevalence of the gene was detected in P. aeruginosa 42.9% (24 of 56) and Alcaligens denitrificans 10.7% (6 of 56) from eye infections. Whereas, in the control group, P. aeruginosa and E. coli each had 14.8% (4 of 27) that were shown positive. Conclusion: The emerging MßLs mediated resistance among P. aeruginosa is a challenging task for ophthalmologists, especially in patients with endophthalmitis and bacterial keratitis. This local knowledge will aid in advising appropriate antibiotic use and avoiding unnecessary antibiotic prescriptions, which are highly warranted.

Incidence and outcomes of endophthalmitis with in-house compounded intravitreal bevacizumab injections: A multicentric study.

PURPOSE: To evaluate the incidence rate and outcomes of endophthalmitis after intravitreal injection of in-house compounded bevacizumab. METHODS: In this multicentric study performed from 2014 to 2018, patients who were injected with in-house compounded and sterilized bevacizumab were observed for endophthalmitis. In-house compounded syringes were prepared in compounding pharmacy using sterile standard operating guidelines. All cases of acute endophthalmitis following intravitreal injection were recorded and followed up for final anatomical and visual outcome after management. RESULTS: A total of 50,361 bevacizumab injections were administered. Incidence of post-injection endophthalmitis was 0.0377%. Incidence of culture-positive endophthalmitis was 0.0178%, with 55.5% culture positivity for Staphylococcus species. A significant improvement in the final visual outcome was noted (p<0.05), and overall five patients (26.3%) achieved visual acuity of 6/18 or more. CONCLUSIONS: In-house compounded injections of bevacizumab can reduce post-injection endophthalmitis to a minimum, with maintenance of proper asepsis and strict protocols by the compounding pharmacy.

Post-cataract Surgery Fungal Endophthalmitis: Management Outcomes and Prognostic Factors.

Purpose: To describe the clinical and microbiological features, management outcomes, and prognostic factors of visual improvement in post-cataract surgery fungal endophthalmitis.Methods: We evaluated 17 patients with culture-confirmed fungal endophthalmitis following cataract surgery. Data were collected regarding intravitreal antibiotics and antifungals, pars plana vitrectomy (PPV), intraocular lens (IOL) explantation, and scleral-fixated IOL (SFIOL) implantation. Clinical outcomes were recorded in terms of visual acuity and management strategies.Results: PPV was performed as a primary procedure at first presentation in three eyes. Fourteen eyes (82.3%) received intravitreal voriconazole. IOL explantation was performed in four eyes, followed by an SFIOL implantation in three eyes. Six eyes additionally presented with keratitis. At the final follow-up, cases having visual acuity ≥6/60 doubled from 17.6% to 35.3%. Eyes receiving antifungals had a trend toward worse visual outcome.Conclusions: Corneal involvement in addition to endophthalmitis and the presence of Aspergillus terreus were found to be poor prognostic markers.

Comparative Study of Microbiological Profile and Management Outcomes of Acute Endophthalmitis after Microincision Vitrectomy Surgery versus Intravitreal Injections.

Purpose: The aim of this study was to evaluate and compare incidence and outcomes of management of acute endophthalmitis after microincision vitrectomy surgery (MIVS) and intravitreal injections (IVIs).Methods: Medical records were retrospectively reviewed from January 2012 to December 2017, and the incidence, clinical and microbiological profiles of acute endophthalmitis were documented.Results: Of 26,332 MIVS and 24,143 IVI performed, incidence of acute endophthalmitis in MIVS group was 0.027% (1 in 3761 cases) against 0.054% (1 in 1857 cases) in IVI. Gram-positive organisms were causative in post IVI group as against gram-negative organisms in MIVS group.Conclusion: Incidence of endophthalmitis after IVI is almost twice that after MIVS. A trend toward poorer outcomes in MIVS eyes was observed. Both MIVS and IVI being pars plana procedures warrant similar kind of aseptic precautions.

Seroprevalence of SARS-CoV-2 specific IgG antibodies among eye care workers in South India.

PURPOSE: Health care workers are at higher risk of acquiring the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This study aims to understand the seroprevalence of anti-SARS-CoV-2 IgG antibody among the eye care workers in South India. METHODS: The participants included eye care workers from the nine eye care centres. All the participants were interviewed with a questionnaire to obtain essential information about socio-demographics, past contact with COVID-19 patients and additional information as recommended by Indian Council of Medical Research, India. Serum samples were tested for anti-SARS-CoV-2 IgG antibodies by ELISA. RESULTS: A total of 1313 workers were included and 207 (15.8%) were positive for the SARS-CoV-2 IgG antibody. The seropositivity was higher in the moderate risk group (19.5%) followed by low (18.6%) and high risk (13.7%) groups. The seropositivity was significantly higher among i) day scholars compared to hostellers (OR - 2.22, 1.56 to 3.15, P â€‹< â€‹0.0001), ii) individuals with history of flu-like illness (4.57, 3.08-6.78, P â€‹< â€‹0.001) or who were symptomatic or in contact with COVID 19 positive cases (2.2, 1.02-4.75, P - 0.043) and iii) individuals with history of systemic illness (2.11, 1.39-3.21, P â€‹< â€‹0.001). Individuals (11.97%) who had no history of contact or any illness were also seropositive. CONCLUSIONS: The effectiveness of the protective measures taken against COVID infection was evident from the lower percentage of seropositivity in the high risk group. The study highlighted the need to create awareness among individuals to follow strict safety measures even in non-work hours and also in social circles.

Identification of Bacterial and Fungal Pathogens by rDNA Gene Barcoding in Vitreous Fluids of Endophthalmitis Patients.

Purpose: To identify the bacterial and fungal pathogens in ocular samples of clinically suspected endophthalmitis patients by conventional culture methods and 16S and 28S rDNA gene sequencing respectively. Methods: A total of 88 patients with clinically suspected endophthalmitis were included in this study. Under sterile operating conditions, a vitreous fluid (0.1-0.2 ml) was obtained by pars plana vitrectomy procedure. The samples were processed for conventional microbiology methods and PCR. PCR targeting 16S rDNA gene for bacteria and 28S rDNA gene for fungus were performed individually using the MightyAmp DNA Polymerase Ver. 2 (TaKaRa China) kit. The PCR amplified samples were sequenced and aligned using CLUSTAL-W tool. The phylogenetic tree was constructed by Neighborhood joining along with the reference sequences downloaded from NCBI database using MEGA X software. Results: 67 Post-operative, 12 Endogenous and 9 traumatic endophthalmitis patients were included as study subjects. By the direct culturing bacterial growth was observed in 17 samples and fungal growth in three samples. PCR was positive for all the culture positive samples, in addition 14 were positive in culture negative samples. The predominant species identified in gram-positive bacteria were Staphylococcus spp., and Pseudomonas spp. in the gram-negative group. Both PCR and culture identified only three samples positive for fungal pathogens which were identified as Aspergillus fumigatus, Candida albicans, and Exerohilum rostratum. Conclusions: PCR based molecular diagnosis is more sensitive than the conventional gold standard culture methods in endophthalmitis. Bacterial pathogens were found to be the predominant in causing endophthalmitis than fungal pathogens.

A rare case of Myrothecium species causing mycotic keratitis: Diagnosis and management.

We report two cases of keratitis caused by a rare fungus Myrothecium species. Clinical presentation, identification, and management were studied. Both fungi were grown from corneal scraping and identified as M. verrucaria and M. gramineum based on the microbiological techniques and DNA sequencing analysis. Both patients were treated with topical natamycin and or voriconazole/econazole. In the first patient, there was total healing of the with scar formation, however, the prognosis was poor in the second patient.

Pythium keratitis in South India: Incidence, clinical profile, management, and treatment recommendation.

PURPOSE: To study the demographic profile, clinical features, treatment outcome, and ocular morbidity of microbiologically proven Pythium keratitis in South India. METHODS: A retrospective analysis of clinical records of microbiologically proven Pythium keratitis at a tertiary eye care referral center in South India from January 2016 to November 2017 was performed. Demographic details, predisposing risk factors, microbiological investigations, clinical course, and visual outcome were analyzed. RESULTS: Seventy-one patients with microbiologically proven Pythium keratitis were identified. The mean age was 44(±18.2) years with an increase in male preponderance and 50% were farmers. Duration of delay at time of presentation to the hospital was a mean of 14(±7.2) days. The visual acuity at baseline ranged from 6/6 to no light perception (median 2.1 logMAR). A combination of 5% natamycin and 1% voriconazole was given to 42% patients, and natamycin alone was given to 39.4% patients. 1% itraconazole eye drops alone was initiated in 7 (10%) patients and 3 among this group responded. Therapeutic keratoplasty (TPK) was performed in 48 (67.6%) patients. None of the primary grafts remained clear after a period of 1 month. Twenty-six eyes (54.2%) had graft reinfection and all these eyes either developed anterior staphyloma (4) or were eviscerated (3) and 13 eyes became phthisical. The remaining 22 patients who had TPK resulted in failed graft. Among these, re-grafts were performed in 6 patients, of which 5 were doing well at the last follow-up. CONCLUSION: We report a large series of patients with Pythium keratitis. Promoting early and differential diagnosis, awareness of clinicians and specific treatment options are needed for this devastating corneal disease.

Fungal keratitis caused by Podospora austroamericana: A first case report.

We report a case of keratitis caused by a rare fungus Podospora austroamericana. Clinical and microbiological evaluation of the corneal ulcer was done and the treatment outcome was studied. The fungus was grown from the corneal scraping, and it was identified as P. austroamericana based on DNA sequence and analysis of the internal transcribed spacer region. The patient was treated with topical azithromycin, natamycin and voriconazole. Despite maximum medical therapy, the ulcer progressed very rapidly and the patient developed panophthalmitis and evisceration of the eye had to be done. This is the first reported case of keratitis caused by P. austroamericana.

Incidence of Endophthalmitis after Intravitreal Injections: Risk Factors, Microbiology Profile, and Clinical Outcomes.

PURPOSE: To report the incidence and characteristics of endophthalmitis after intravitreal injections (IVI) of antivascular endothelial growth factor agents and triamcinolone acetonide. METHODS: Patients' medical records were retrospectively reviewed from January 2009 to June 2016, and the incidence, risk factors, clinical and microbiological characteristics of post-IVI endophthalmitis were evaluated. RESULTS: The total number of intravitreal injections given, which included ranibizumab, bevacizumab, and triamcinolone acetonide, was 20,566, of which 27 cases developed endophthalmitis, giving an overall incidence of 0.131%. Significant reduction (p < 0.003) in incidence of endophthalmitis was observed in patients who received prefilled compounded bevacizumab injections (0.050%) compared to multiple bevacizumab injections from a single vial (0.235%). In the triamcinolone acetonide group, the incidence was 0.26%. Staphylococcus species were isolated from 18 cases (67%), and all strains were sensitive to vancomycin. CONCLUSIONS: Adherence to strict aseptic protocols and use of prefilled compounded bevacizumab injections reduces the rate of post-IVI endophthalmitis.

Fungal Keratitis Caused by Exserohilum, An Emerging Pathogen.

PURPOSE: To determine the epidemiology, risk factors, clinical features, and prognosis of fungal keratitis caused by Exserohilum. METHODS: All culture-proven cases of keratomycosis caused by Exserohilum presented from January 2012 to December 2014 were retrospectively analyzed using a computerized database. RESULTS: Among 1542 cases of keratomycosis, 47 (3%) were due to Exserohilum. The mean age of presentation was 50.4 years. Males (n = 32; 68%) were more commonly affected than females (n = 15; 32%). A history of trauma was present in most of the cases (n = 38; 80.9%). The mean time of the first visit to the hospital was 6.2 days after onset of symptoms. Most of the patients (n = 24; 72.3%) had central or paracentral ulcers. The infiltrate was confined to the superficial one-third of the stroma in 39 patients (83%). Most of the patients (89.4%) responded well to topical treatment (natamycin 5% for ulcers <5 mm; voriconazole 1% was added for ulcers >5 mm or with hypopyon); 4 patients (8.5%) required therapeutic penetrating keratoplasty and 1 patient (2.1%) progressed to endophthalmitis, requiring evisceration. The mean best-corrected visual acuity in the medically treated patients improved from 0.89 (logMAR) at presentation to 0.77 at the completion of treatment (P = 0.015). CONCLUSIONS: This study signifies the importance of this new emerging pathogen that was generally regarded as rare in the past. The infection seems to be more prevalent than previously thought, especially in hot and humid areas. Vision is likely to improve if the infection is diagnosed and appropriate topical treatment is started early.

Comparative evaluation of uniplex, nested, semi-nested, multiplex and nested multiplex PCR methods in the identification of microbial etiology of clinically suspected infectious endophthalmitis.

PURPOSE: This study is aimed to determine the utility of various polymerase chain reaction (PCR) methods in vitreous fluids (VFs) for detecting the infectious genomes in the diagnosis of infectious endophthalmitis in terms of sensitivity and specificity. METHODS: This prospective and consecutive analysis included a total of 66 VFs that were submitted for the microbiological evaluation, which were obtained from 66 clinically diagnosed endophthalmitis patients presented between November 2010 and October 2011 at the tertiary eye care referral centre in South India. Part of the collected VFs were subjected to cultures and smears, and the remaining parts were utilized for five PCR methods: uniplex, nested, semi-nested, multiplex and nested multiplex after extracting DNA, using universal eubacterial and Propionibacterium acnes species-specific primer sets targeting 16S rRNA gene in all bacteria and P. acnes, and panfungal primers, targeting 28S rRNA gene in all fungi. RESULTS: Of the 66 VFs, five (7.5%) showed positive results in smears, 16 (24%) in cultures and 43 (65%) showed positive results in PCRs. Among the 43 positively amplified VFs, 10 (15%) were positive for P. acnes genome, one for panfungal genome and 42 (62%) for eubacterial genome (including 10 P. acnes positives). Among 42 eubacterial-positive VFs, 36 were positive by both uniplex (first round) and multiplex (first round) PCRs, while nested (second round) and nested multiplex (second round) PCRs produced positive results in 42 and 41 VFs, respectively. Of the 43 PCR-positive specimens, 16 (37%) had positive growth (15 bacterial and one fungal) in culture. Of 50 culture-negative specimens, 27 (54%) were showed positive amplification, of which 10 were amplified for both P. acnes and eubacterial genomes and the remaining 17 were for eubacterial genome alone. CONCLUSIONS: Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.