Elucidating the clinical, microbiological and molecular diagnostic aspects of Macrophomina phaseolina keratitis.

This study reports the clinico-microbiological features of Macrophomina phaseolina keratitis. Clinically diagnosed as microbial keratitis, six patients underwent microbiological evaluation. Fungal culture isolates from cornea were subjected to DNA sequencing of the ITS region, phylogenetic analysis and reconfirmation by polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) of six antifungal drugs were determined by microbroth dilution method against the six isolates. All patients were treated with antifungals. Failed medical therapy necessitated therapeutic penetrating keratoplasty (TPK). Corneal buttons were processed for histopathology. In all patients, the corneal scraping showed septate hyaline fungal filaments. The BLAST analysis for ITS sequences of all six fungal isolates suggested M. phaseolina, however, when limited to sequences from type material, they matched M. pseudophaseolina. Phylogenetic analysis could not differentiate between these two species and clustered in a single clade. PCR assay of specific gene sequence [MpCal (calmodulin)] reconfirmed all isolates as M. phaseolina. The MICs of voriconazole and posaconazole were lowest (0.03 to 2 and 0.1 to 2µg/mL respectively) and all isolates were susceptible to natamycin. Except for case 1, which healed with a scar on treatment, all other cases worsened, despite medical treatment, necessitating TPK. Histopathology of 3 out of 4 buttons showed the presence of fungal filaments. While direct microscopic examination of corneal scrapings is helpful in diagnosis, identification of M. phaseolina in culture is challenging. Although MICs of commonly used antifungals are low response to medical therapy is not encouraging; patients may require TPK for resolution of infection in M. phaseolina keratitis.

DNA sequencing, phylogenetic analysis and specific PCR confirmed Macrophomina phaseolina keratitis in six patients. Although antifungal susceptibility showed the organisms to be susceptible to natamycin five patients did not respond to treatment and needed keratoplasty.

Linezolid shows high safety and efficacy in the treatment of Pythium insidiosum keratitis in a rabbit model.

Efficacy and safety of three antibiotics (Linezolid-LZ, 0.2%; Azithromycin-AZ, 1%; Tigecycline-TG, 1%) were determined in the treatment of Pythium insidiosum keratitis in rabbits. Infection of right eye of 38 rabbits was induced by standard intracorneal injection of P. insidiosum zoospores (left eye, intracorneal saline). Corneal infection developed in all right eyes. One hourly eye drops of one of the three antibiotics was instilled in both eyes (3 groups of 12 rabbits each) except in controls. Half of the rabbits in each group received intracorneal injection of the respective antibiotic after 4 days of starting eye drops. Clinical scoring of eyes was done over next 3 weeks. The reduction in scores post-treatment was significant for each drug (LZ: p < 0.025, AZ: p < 0.025, TG: p < 0.01). Scores with LZ (median change of 3) was significantly (p = 0.013) higher than TG (median change of 2) and comparable (p = 0.06) to AZ (median change of 3). Reduction in clinical scores in eyes receiving intracorneal antibiotics was not significantly different from the eyes that did not receive intracorneal antibiotics (p = 0.73). While no adverse effect of LZ was seen in the control corneas, 66-100% of rabbits showed reaction to AZ and TG. Histopathology showed severe inflammation in all infected corneas and intraocular extension in some of the rabbits with poor response. The success rate was noted to be 16.7%, 25% and 50% in AZ, TG and LZ respectively (p = 0.45). LZ demonstrated superior efficacy and safety and can be considered for trial in human disease.

Randomized Double-Masked Placebo-Controlled Trial for the Management of Pythium Keratitis: Combination of Antibiotics Versus Monotherapy.

PURPOSE: The aim of this study was to compare the efficacy of monotherapy (topical linezolid 0.2%) versus a combination of antibiotics (topical linezolid 0.2% and topical azithromycin 1%) for the treatment of Pythium insidiosum keratitis. METHODS: Cases of P. insidiosum keratitis were prospectively randomized into group A on topical 0.2% linezolid along with topical placebo (sodium carboxymethyl cellulose [CMC] 0.5%) and group B on a combination of topical 0.2% linezolid and topical 1% azithromycin. Both groups were compared by proportion of both clinical resolution and worsening of keratitis along with the number of therapeutic penetrating keratoplasty (TPK) performed at 3 months. RESULTS: We initially planned N = 66 patients but later limited to 20 (N = 10 in each group) patients owing to one interim analysis. The average size of the infiltrate in group A and B was 5.6 ± 1.5 mm and 4.8 ± 2.0 mm, respectively, with a mean Logarithm of the Minimum Angle of Resolution (logMAR) visual acuity of 2.74 ± 0.55 and 1.79 ± 1.19. At 3 months, from group A, 7 (70%) patients needed TPK and 2 patients had signs of resolution, whereas from group B, 6 (60%) patients achieved complete resolution ( P = 0.0003) and 2 were improving while only 1 needed TPK ( P = 0.02). The median duration of treatment in group A and B, with the study drugs, was 31 days (17.8-47.8) and 101.5 days (80-123.3), P value = 0.003, respectively. Final visual acuity at 3 months was 2.50 ± 0.81 and 0.75 ± 0.87, P = 0.02, respectively. CONCLUSIONS: A combination of topical linezolid and topical azithromycin was found to have superior efficacy than the monotherapy with topical linezolid for the management of Pythium keratitis.

Elevated Levels of Interleukins, Leukocyte Protein and Cathelicidin Antimicrobial Peptide are Strongly Associated with Early to Mid-Stage of Pythium insidiosum Infection in Rabbit Corneas.

PURPOSE: Corneal infection in humans caused by Pythium insidiosum can lead to blindness and the host ocular immune response to it is less studied. Herein, we investigate the expression of mediators of innate and adaptive immune responses in a rabbit model. METHODS: P. insidiosum zoospores were injected intracorneally in right eye of the nine New Zealand White rabbits while left eye was injected with 1XPBS. RT-qPCR and multiplex ELISA (mELISA) were used to study the expression of antimicrobial peptides (AMPs) and immune mediators in infected cornea on 3rd, 7th and 9th day of post-infection(PI). STRING-11.0 analysis was used to predict the interactions of immune mediators. mRNA expressions of pathogen recognition receptors (PRRs) were determined in human corneal epithelial cells (HCECs) stimulated with P. insidiosum zoospores. Data was analyzed using one-way ANOVA with Post-Hoc Tukey HSD test and p-value <0.05 was considered significant. RESULTS: mRNA expression assay for IL-1ß, IL-6, IL-8 and Cathelicidin antimicrobial peptide (CAP)-18 showed significant upregulation (p-value < 0.05) on 7th day post-infection (PI) compared to 3rd and 9th day while Leukocyte protein (LeukoP) was elevated significantly on 3rd day followed by 7th and 9th day PI . Only IL-17A among other adaptive immune cytokines showed significant upregulation on 7th day compared to 9th day PI. Expressed in pg/mL, mELISA showed significant higher levels (p-value < 0.05) of IL-1ß, IL-8 in infected tissue in each of the time points compared to control. STRING analysis revealed co-expression of IL-1ß, IL-8 and IL-6. Among PRRs, Dectin 1 and TLR4 showed significant upregulation in HCECs at 12 hrs compared to 6 hrs. CONCLUSION: In the rabbit P. insidiosum keratitis model, innate immune mediators: IL-1ß, IL-8, IL-6, AMPs: LeukoP and CAP-18 are strongly associated in early to mid-stage of corneal infection. Dectin 1 and TLR4 were observed to be associated with recognition of P. insidiosum.

Clinical Outcomes of Rose Bengal Mediated Photodynamic Antimicrobial Therapy on Fungal Keratitis with Their Microbiological and Pathological Correlation.

PURPOSE: To report the clinical outcome of Photodynamic Antimicrobial Therapy (PDAT) with Rose Bengal (RB) used as an early adjuvant therapy in patients with fungal keratitis and their microbiological and pathological correlation. METHODS: Patients with microbiologically confirmed fungal keratitis underwent PDAT-RB along with topical natamycin 5% drops hourly and oral ketoconazole 200 mg twice a day. This was performed by applying rose bengal (0.1%) to the de-epithelialized cornea for 30 minutes, followed by irradiation with a 6 mW/cm2 custom-made green LED source for 15 minutes (5.4 J/cm2). The corresponding fungal isolates were tested in vitro using PDAT-RB and corneal buttons were evaluated for correlation. RESULTS: Following informed consent, seven patients (male-5, female-2, mean age 47.7 years) with fungal keratitis were recruited. There were 3 cases each of Fusarium and Aspergillus flavus and 1 case of Acremonium sp. The average vertical and horizontal diameters of the corneal infiltrate were 4.12 ± 0.55 and 3.99 ± 1.19 mm, respectively. The average depth of corneal involvement was 283 ± 75.27µ as measured by anterior segment OCT. The clinical resolution was achieved in the cases with Fusarium keratitis with an average time of 39 days. Three cases of A. flavus and a single patient with Acremonium keratitis worsened and needed therapeutic keratoplasty (TPK) for resolution. Post-TPK, the corneal tissues grew A. flavus in one out of three cases and Acremonium sp. in one case. In vitro PDAT-RB experiment was performed on the corresponding fungal isolates grown from the corneal scraping. PDAT-RB produced clear inhibition of Fusarium and Acremonium sp. with no effect on the growth of A. flavus. Histopathologically, 2 out of 4 (50%) corneal buttons showed fungal filaments. CONCLUSIONS: While the in vitro and in vivo results of PDAT-RB matched for Fusarium sp. and Aspergillus flavus keratitis being favourable in the former and non-favourable in the latter, these results were discrepant in Acremonium sp.

Clinical and Histopathological Evaluation of a Rabbit Model for Pythium insidiosum Keratitis.

Purpose: To describe the clinical characteristics and histopathological features in a rabbit model of Pythium insidiosum keratitis.Methods: Zoospores of P. insidiosum isolated from a patient with microbial keratitis were used for inoculation of the right eye of 48 New Zealand White rabbits in either low (LD) or high dose (HD). Apart from variable dosage the rabbits were grouped (6 rabbits per group) based on route of inoculation (topical on abraded cornea or intracorneal) and immunosuppression (subconjunctival steroid or no steroid). Left eye received phosphate buffered saline via route similar to the right eye. Daily clinical examination of the eye was done, the corneas were harvested on day 3, 7 and 9 and part of the cornea was preserved in 10% neutral buffered formalin for histopathological examination.Results: Left eye of all rabbits were clinically normal. Eyes with intracorneal injection of zoospores developed infection irrespective of dose of inoculation and administration of steroids. One of the consistent early signs of infection was ring like infiltrate in the peripheral cornea. On day 2, rabbits receiving HD developed significantly greater inflammation compared to LD [median clinical score in HD- 11 (IQR = 10-12), versus 9 (IQR = 8-9) in LD (p = 0.004)]. The density of inflammation showed temporal correlation (increase with time) when the inoculum was low. Of the rabbits that received topical inoculation one rabbit cornea showed mild infiltrate in steroid group while no eye was infected in the group without steroid. Sparsely septate to aseptate branching filaments were noted in the stroma of all infected corneas.Conclusions: We describe the first animal model of Pythium keratitis that holds promise for future studies. While topical inoculation of zoospores was unsuccessful in causing infection intracorneal inoculation without immunosuppression was sufficient to develop clinically severe keratitis in rabbits.