Clinical Profile, Antibiotic Resistance and Outcomes in Bacterial Endophthalmitis: Coagulase-Negative Staphylococcus Endophthalmitis as Compared to Other Organisms.

BACKGROUND: To evaluate the clinical profile, outcomes and antibiotic resistance in bacterial endophthalmitis. METHODS: This was a post-hoc analysis of a study conducted at a tertiary centre, where 60 consecutive cases of culture-proven bacterial endophthalmitis were included prospectively. Group 1 included coagulase-negative Staphylococcus endophthalmitis (CNSE), while group 2 included the remaining cases. Clinical features, antibiotic resistance and visual outcomes were compared. Visual acuity >3/60 at six months of follow-up was defined as a good visual outcome. RESULTS: Group 1 had 31 cases, while group 2 had 29. Group 2 included 12 gram-positive and 17 gram-negative isolates. Among the groups, group 2 had more patients with presenting visual acuity below hand motions close to the face (25 vs. 12, p<0.001), poor visual outcomes (26 vs. 3, p<0.001) and retinal detachment (RD) (10 vs. 2, p=0.007). Pseudomonas was most commonly resistant to antibiotics, and ceftazidime (p=0.005) and cefazolin (p=0.009) resistance were higher in group 2 isolates. In group 1, five isolates were resistant to any one of the antibiotics, whereas in group 2, 13 isolates were resistant to any one of the antibiotics (p=0.024). CONCLUSIONS: In the current study, eyes in the group of endophthalmitis caused by CNSE achieved better visual acuities at the last follow-up compared to eyes with endophthalmitis caused by other bacteria. Antibiotic resistance in isolates other than CNSE is a cause of concern.

Central Line-Associated Bloodstream Infections: Effect of Patient and Pathogen Factors on Outcome.

Introduction: Patients on central lines are often having multiple morbidities, and invasive devices provide a niche for biofilm formation, which makes central line-associated bloodstream infections (CLABSIs), a serious concern in health-care settings, as the infections difficult to treat. In this study, we evaluated the common bacteria causing CLABSI, and various patient and pathogen factors affecting the clinical outcome. Methods: In the prospective observational study, patients diagnosed with CLABSI were recruited. Extensive clinical, microbiological, and other laboratory workup was done, and observations were recorded. Congo red agar method, tube test, and microtiter plate assay were used for eliciting the biofilm-forming attributes of the bacterial pathogens. Results: Klebsiella pneumoniae was responsible for 48% of CLABSI, followed by Coagulase-negative Staphylococci (16%) and Staphylococcus aureus and Acinetobacter baumannii (12% each). Fifty-six percent of the isolates produced biofilms. The median (interquartile range) duration of hospital stay till death or discharge was 30 (20, 43) days. The all-cause mortality was 44%. Patients having a deranged liver function on the day of diagnosis (P value for total bilirubin 0.001 and for aspartate transaminase 0.02), and those infected with multidrug-resistant organisms (P value = 0.04) had significantly poor prognosis. The difference in the demographic, clinical, laboratory profile, and outcome of patients infected with biofilm producers and nonproducers was not found to be statistically significant. Conclusion: The study throws light on various host and pathogen factors determining the cause and outcome of CLABSI patients. To the best of our knowledge, this is the first study trying to decipher the role of biofilm formation in the virulence of pathogens and the prognosis of CLABSI.

Evaluation of liposomal amphotericin B for the treatment of fungal keratitis in a tertiary eye care hospital.

Purpose: To evaluate the efficacy of liposomal amphotericin B (L-AMB) for the treatment of fungal keratitis. Methods: Patients with fungal keratitis confirmed by potassium hydroxide (KOH) smear and/or confocal microscopy were administered topical L-AMB and randomized into three groups treated with three different formulations. The medication was administered two hourly till clinical improvement was achieved, followed by six hourly till complete resolution. The outcome measures were time to clinical improvement, resolution of epithelial defect, stromal infiltrate, hypopyon, extent and density of corneal opacity, neovascularization, and best corrected visual acuity (BCVA) at 3 months. Results: Mean age of the patients was 46.6 ± 14.8 years, and trauma with vegetative matter was the most common predisposing factor. Aspergillus flavus (36%) was the most common fungus cultured, followed by Fusarium (23%). Mean time to clinical improvement, time to resolution of epithelial defect, mean time to resolution of infiltrate, and time to resolution of hypopyon were 3.45 ± 1.38, 25.35 ± 8.46, 37.97 ± 9.94, and 13.33 ± 4.90 days, respectively, and they were comparable among the three groups. There was a significant difference between treatment failure and success cases in terms of days of presentation (P < 0.01), size of the epithelial defect (P-value 0.04), and infiltrate size at presentation (P-value 0.04). At 3 months follow-up, no statistically significant difference was noted in BCVA and mean scar size among groups. Conclusion: L-AMB in a gel form is an effective antifungal agent that promotes the healing of fungal ulcers with notably least vascularization and better tolerance. Trial registration number: CTRI/2020/04/024550.

Spectrum of bacterial keratitis in North India: A retrospective analysis of six years at a tertiary care eye center.

Purpose: To analyze the pattern of bacterial pathogens causing infective keratitis and their resistance to the recommended antibiotics over six years. Methods: It was a retrospective study of 9,357 cases of bacterial keratitis from January 2015 to December 2020, at a tertiary care ophthalmic center. A total of 9,547 corneal specimens were obtained from the study subjects. Demographic details of the patients, pathogenic bacteria isolated, and their antimicrobial susceptibility were noted and analyzed. Results: Bacterial pathogens were identified in 23.52% of the specimens. The most common isolates were coagulase-negative Staphylococci (60.75%), followed by Pseudomonas aeruginosa (14.23%), Staphylococcus aureus (13.92%), gram negative bacilli of the family Enterobacterales (8.64%), Streptococcus spp. (1.72%), Acinetobacter spp. (0.13%), and other non-fermenting gram-negative bacilli (0.57%). In Staphylococci, 55-80% of isolates were resistant to erythromycin, and 40-70% to fluoroquinolones, while no resistance was observed against vancomycin. 40-60% of isolates of P. aeruginosa were resistant to cephalosporins, 40-55% to fluoroquinolones, and 30-60% to aminoglycosides. Also, 40-80% of isolates of Enterobacterales were resistant to cephalosporins, and 50-60% to fluoroquinolones. Most gram-negative isolates were susceptible to carbapenems and polymyxin B. Conclusion: To the best of our knowledge, our study is the largest compilation of microbiological profile of bacterial keratitis from North India. It highlights the current trend of the bacterial pathogens that cause infectious keratitis. Staphylococci and Pseudomonas were found to be the most common pathogens. Increased resistance was seen against some of the commonly prescribed empirical antibiotics. Such evidence is useful for restructuring the empirical prescription practices from time to time.

Real-life comparison of three intravitreal antibiotic drug regimens in endophthalmitis.

Purpose: Real-life comparison of three intravitreal drug regimens used in cases of endophthalmitis at a tertiary care center in India. Methods: In this prospective, comparative study, patients of bacterial endophthalmitis were grouped according to intravitreal antibiotic drug regimens into Group 1 (ceftazidime and vancomycin), Group 2 (piperacillin + tazobactam and vancomycin), and Group 3 (imipenem and vancomycin). Forty-eight hours after injection nonresponding/worsening patients underwent vitrectomy. Vitreous samples were subjected to microbiological and pharmacokinetic tests. Results: A total of 64 patients were included and divided into Group 1: 29, Group 2: 20, and Group 3: 15 cases. Also, 75% of patients were post-surgical endophthalmitis, whereas 25% were post-traumatic. Improvement in vision (V90-0) and vision at 3 months (V90) were comparable between the three groups. Visual recovery was poorer in post-traumatic cases. In post-surgical cases, visual recovery was poorer in those presenting beyond 72 h of onset of symptoms (P = 0.0002). Polymerase chain reaction (PCR) positivity (66%) was higher than BACTECTM (33%) and culture (14%). Antibiotic resistance was comparable amongst the three groups. Most patients (62/64) further underwent vitrectomy. Ceftazidime and vancomycin achieved vitreous concentrations more than the minimum inhibitory concentration (MIC) at 48 h after the first injection. Conclusion: The choice of antibiotics did not affect the rate of vitrectomy and final vision in a real-life scenario. Ceftazidime and vancomycin can still be used as first-line intravitreal antibiotics owing to their comparable microbial sensitivity profile and adequate ocular bioavailability.

Acanthamoeba keratitis: Experience from a tertiary eye care center in North India.

The free-living amebae of genus Acanthamoeba are an important cause of microbial keratitis. The clinical appearance of Acanthamoeba keratitis (AK) usually mimics viral or fungal keratitis. Thus, microbiological workup plays a significant role in the diagnosis and timely treatment of such cases. We report a retrospective case series of seven culture-confirmed AK cases from a tertiary eye care center in North India. Various risk factors and triggers of infection, clinical presentations, microbiological findings, and management of AK are elucidated.

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.

Evaluation of various preservation media for storage of donor corneas.

PURPOSE: To compare the physical and microbiological characteristics of McCarey-Kaufman (MK), Cornisol, and Optisol-GS media and evaluate the outcomes of keratoplasty performed using corneas stored in these three media. METHODS: The study involved 60 donor corneas which were distributed in 3 groups: MK, Cornisol, and Optisol-GS. Corneas in these groups were further analyzed based on the type of keratoplasty performed (full thickness versus endothelial keratoplasty). At baseline, the endothelial cell density and death to preservation time of donor corneas were recorded. Following keratoplasty, patients were evaluated on day 1, at 1 month, 3 months, and 6 months follow-up. Outcomes were assessed in terms of corrected distance visual acuity (CDVA), endothelial cell density, percentage endothelial cell loss, and corneal thickness. The storage media were also assessed for their physical quality and their microbiological characteristics. RESULTS: Physical characteristics of all three media were found to be within normal limits. Mean CDVA was comparable among the 3 groups at 6-month follow-up. The absolute endothelial cell count values were significantly lower for corneas stored in MK medium (1873.7 ± 261.1 cells/mm2) compared to the Cornisol (2085.0 ± 230.3 cells/mm2) and Optisol-GS media [(2180.3 ± 217.2 cells/mm2) (P = <0.001)]. Corneas stored in Optisol-GS medium were significantly thinner at 1-month follow-up with no significant difference at 6 months (P = 0.66). CONCLUSION: Optisol-GS and Cornisol media were found to preserve endothelial cell density better and stabilize corneal thickness earlier as compared to the MK medium. However, the functional outcomes were comparable among the three groups.

Species identification, antifungal susceptibility profiles and biofilm formation attributes of Rhodotorula isolates from ocular infections.

BACKGROUND: Members of genus Rhodotorula are widely distributed in nature and have been traditionally considered non-pathogenic. Last few decades have seen the yeast as an emerging pathogen. We observed increase in numbers of Rhodotorula isolates from ocular infections in last few years, thus this prospective study was planned. OBJECTIVES: To identify the species of Rhodotorula isolates from ocular infections. To know the antifungal susceptibilities and study the biofilm formation attributes of the isolates. MATERIALS AND METHODS: Rhodotorula isolates were speciated using conventional methods, Matrix Assisted Laser Desorption and Ionisation - Time of Flight (MALDI- TOF) and sequencing of ITS region of ribosomal DNA. Antifungal susceptibility testing (AFST) was done using disc diffusion and E-test. Biofilm formation was studied using XTT [2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] assay. RESULTS: Twenty four isolates (92.3%) were identified as R. mucilaginosa and two as R. Minuta. AFST showed high MICs against Fluconazole, Amphotericin-B, Caspofungin, Micafungin and Flucytosine; MIC distribution from low to very high against Voriconazole, Itraconazole and Natamycin; and very low MICs against Posaconazole 57.7% of isolates were strong biofilm producers, 23.1% were moderate, and 19.2% were non producers. CONCLUSIONS: This is the first prospective study on species distribution, antifungal susceptibility and biofilm production attributes of Rhodotorula isolates from ocular infections; also first time demonstrating the utility of proteomics based MALDI-TOF in diagnosing Rhodotorula up to species level. The study has shown high MICs against the conventional azoles, Amphotericin-B and Flucytosine. However, low MICs against Posaconazole and Natamycin give a hope for their possible therapeutic use.

Comparison of Safety and Efficacy of Intrastromal Injections of Voriconazole, Amphotericin B and Natamycin in Cases of Recalcitrant Fungal Keratitis: A Randomized Controlled Trial.

OBJECTIVE: To compare the safety and efficacy of intrastromal voriconazole (IS-VCZ), amphotericin B (IS-AMB) and natamycin (IS-NTM) as an adjunct to topical natamycin (NTM) in cases of recalcitrant fungal keratitis. DESIGN: Prospective randomized trial. SETTING: Tertiary eye centre. PARTICIPANTS: Sixty eyes of 60 patients with microbiologically proven recalcitrant fungal keratitis (ulcer size >2 mm, depth >50% of stroma, and not responding to topical NTM therapy for two weeks) were recruited. METHODS: patients were randomized into three groups of 20 eyes, each receiving ISVCZ 50ug/0.1 mL, ISAMB, 5ug/0.1 mL and ISNTM 10ug/0.1 mL (prepared aseptically in ocular pharmacology). The patients in all three groups continued topical NTM 5% every four hours until the ulcer healed. Primary outcome measure was time taken till complete clinical resolution of infection, and secondary outcome measure was best corrected visual acuity (BCVA) at six months. RESULTS: All three groups had comparable baseline parameters. The mean duration of healing was significantly better (p=0.02) in the ISNTM group (34±5.2 days) as compared to the ISVCZ group (36.1±4.8 days) and the ISAMB group (39.2±7.2 days). About 95%, 90% and 95% patients healed successfully in the ISVCZ, ISAMB and ISNTM groups, respectively. In terms of healing, deep vascularization was significantly greater in the ISAMB group (55%, p=0.02) when compared to the ISVCZ and ISNTM groups (31% and 26%, respectively). There were fewer repeat injections in the ISNTM group (7/20 vs 8/20 and 9/20 in the ISVCZ and ISNTM groups, respectively). CONCLUSION: Intrastromal injections are a safe and effective adjunct to conventional therapy in the management of recalcitrant fungal keratitis. ISNTM had a similar visual outcome with faster healing while ISAMB had a higher rate of deep vascularization after healing.

Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis.

To study the feasibility of 16S rRNA metagenomics using next generation sequencing (NGS) along with broad range PCR assay for 762 bp region of 16S rRNA gene with Sanger's sequencing, in microbial diagnosis of culture negative endophthalmitis. Vitreous fluid from 16 culture negative and one culture positive endophthalmitis patients, admitted to a tertiary care hospital were processed for targeted metagenomics. NGS of 7 variable regions of 16S rRNA gene was done using Ion Torrent Personal Genome Machine (PGM). Sequence data were analyzed using Ion Reporter software using QIIME and BLSATN tools and Greengenes and NCBI-Genbank databases. Bacterial genome sequences were detected in 15 culture negative and culture positive vitreous specimens. The sequence reads varied between 25,245-540,916 with read length between 142bp-228bp and coverage depth was 41.0X and 81.2X. Operational taxonomic unit (OTUs) of multiple bacterial genera and species were detected in 13 culture negative vitreous specimens and OTUs of a single bacterial species were detected in 2 culture negative and 1 culture positive specimens; one negative specimen had no bacterial DNA. Maximum numbers of OTUs detected by NGS for a bacterial species from any vitreous specimen was the one which was detected and identified by Sanger's sequencing in broad range PCR. All the bacteria were belonging to clinically relevant species. Broad range PCR with sequencing failed to identify bacteria from 5 of the 16 (31.25%) culture negative vitreous specimens. Metagenomics could detect and identify bacterial pathogens in 15 of the 16 culture negative vitreous specimen's up to species level. With rapidly decreasing cost, metagenomics has a potential to be used widely in endophthalmitis diagnosis, in which culture negativity is usually high.

Role of therapeutic contact lens following Descemet's stripping automated endothelial keratoplasty: A randomized control trial.

Purpose: Therapeutic contact lenses (TCL) are known to help in epithelial healing and decreasing pain after various corneal surgeries. However, literature lacks any data describing their use following Descemet's stripping automated endothelial keratoplasty (DSAEK) where intraoperative epithelial debridement is commonly performed. Here we study the efficacy and safety of TCL in patients undergoing DSAEK. Methods: In this prospective, randomized, controlled clinical trial. 40 eyes of 40 patients of pseudophakic bullous keratopathy undergoing DSAEK were enrolled and randomized into two groups, control (no TCL) and test (TCL). Primary outcome was time taken for epithelial healing and secondary outcomes were postoperative pain score, graft attachment, best spectacle-corrected visual acuity, and endothelial cell loss at 3 months. Results: Average time taken for epithelial healing was 3.35 ± 0.49 days in the test group and 4.95 ± 1.05 days in the control group (P < 0.001). Average pain scores in first operative week were significantly lower in the test group as compared to control (P < 0.001). Graft detachment occurred in eight patients in control group and two in test group (P = 0.03). Both rebubbling rates and average endothelial cell loss at 3 months were higher in the control group with P = 0.07 and 0.06 respectively. No contact lens-related adverse effects were noted during the study period. Conclusion: Use of TCL in DSAEK leads to faster epithelial healing and lesser postoperative pain. In addition, it may also contribute to lower rebubbling rates and endothelial cell loss.

Evaluation of 16S rRNA broad range PCR assay for microbial detection in serum specimens in sepsis patients.

BACKGROUND: Early and accurate laboratory diagnosis and appropriate management of infection improves the survival rate in sepsis. In this study we evaluated broad range 16S rRNA and 16 S-23 S intergenic spacer region (ISR) PCR assays followed by nucleotide sequencing directly from patients' serum and automated blood culture for laboratory diagnosis in admitted sepsis patients. METHODS: A broad range 16S rRNA PCR and 16 S-23 S ISR PCR assay followed by nucleotide sequencing was used directly from patients' serum in hospital admitted patients in 62 sepsis and 16 suspected blood stream infection (sBSI) patients. Automated blood culture was also used in the same patients. Nucleotide sequences were analyzed against NCBI Genbank database and organisms were identified using CLSI MM18A guidelines. RESULTS: Bacterial culture were positive in 10/62 (16.12%) sepsis and 3/16 (18.75%) suspected BSI patients along with 3 detected fungi (2 in sepsis and 1 in suspected BSI group). PCR assay was positive in 36/62 (58.06%) sepsis and 6/16 (37.5%) suspected BSI patients respectively. All but 2 bacteria (both from culture negative patients) detected by PCR assay could be identified from nucleotide sequencing. Survival in sepsis patients was 77%. PCR assay could detect bacteria in 9/14 (64.28%) of sepsis patients with death. CONCLUSION: Broad range PCR assay was far superior for early diagnosis of infection. The bacteria which could not be detected by culture and were not commonly reported from this centre, were detected by the broad range PCR assays. Detection of these rare bacteria/fungi had significant clinical correlation with patient's underlying clinical conditions, immune status and prognosis. The tests could provide definitive diagnosis of infection in >58% of sepsis patients, which helped in patient management and better survival.

Ocular Surface Microbial Flora in Patients with Chronic Limbal Stem Cell Deficiency Undergoing Cultivated Oral Mucosal Epithelial Transplantation.

PURPOSE: The purpose of this study is to analyze the ocular surface microbial flora in patients with chronic limbal stem cell deficiency (LSCD) due to Stevens-Johnson Syndrome (SJS) and ocular chemical injury undergoing cultivated oral mucosal epithelial transplantation (COMET). METHODS: Patients of SJS and chemical injury who had bilateral total LSCD planned for COMET were studied. Conjunctival swab was taken before surgery. Parameters evaluated were organism cultured, sensitivity pattern, frequency of positive culture, and clinical impact on management strategy. RESULTS: Thirteen patients were included in which nine were males and four females. All patients had positive conjunctival swab culture. Most common organism isolated was Staphylococcus epidermidis, followed by Staphylococcus aureus and Pseudomonas aeruginosa. The staphylococcal species isolated were sensitive to all the conventional antibiotics while Pseudomonas cultured showed resistance to cefuroxime, ceftriaxone, and ceftazidime. Repeat conjunctival swab sent after a week of topical antibiotic therapy yielded positive culture of the same organism twice in 25% (3/12), thrice in 58.3% (7/12), and four times in 16.6% (2/12) of the patients. One patient had a polymicrobial flora with positive yield of S. aureus (thrice), S. epidermidis (twice), and P. aeruginosa (twice) in consecutive conjunctival swab culture in the absence of clinical infection. Two patients with persistent positive cultures had to undergo repeat oral mucosal harvesting as the transplantation of the cultivated explants had to be deferred. CONCLUSION: Ocular surface in LSCD patients yielded pathogenic organisms on culture. Poor ocular surface with absent tear film could be the contributing factors. It is important to perform the conjunctival swab culture before COMET surgery.

Microbial Keratitis in Stevens-Johnson Syndrome: A Prospective Study.

PURPOSE: To evaluate the microbiological profile and outcome in cases with infective keratitis in Stevens-Johnson syndrome (SJS). METHODS: Eighty-three eyes of 68 patients with SJS presenting with microbial keratitis were recruited and managed with standard antimicrobial therapy. RESULTS: Microbial keratitis developed in 34% of patients with SJS (83 eyes, 68 patients) over a period of 5 years. Four eyes (4.8%) had a history of concurrent topical steroid use at the onset of keratitis. Mean baseline best-corrected visual acuity was 1.8 ± 0.9 logMAR units. The site of corneal ulceration was central in 52 eyes (62.6%), paracentral in 17 eyes (20.5%), and peripheral in 14 eyes (16.8%). The mean ulcer area was 3.9 ± 2.7 mm. Approximately 15 of 24 (62.5%) culture-positive eyes had bacterial infection, most of which (80%) were caused by Gram-positive bacteria. Polymicrobial infection was noted in 7 of 24 eyes (29.1%). Although 57 of 83 (68.6%) eyes healed with medical therapy, 26 of 83 (31.3%) eyes had corneal perforation and were managed with cyanoacrylate glue application (30.7%) or therapeutic keratoplasty (69.3%). Systemic infection as an inciting factor of SJS and an early presentation for keratitis were the major risk factors associated with corneal perforation. Large mean ulcer size, paracentral ulcers, and punctal involvement were associated with a good visual outcome. CONCLUSIONS: Infective keratitis in SJS is common, and unlike routine cases, surgical intervention is often required. However, the antibiotic sensitivity pattern suggests that resistance is not that high.

Spectrum of mycotic keratitis in north India: Sixteen years study from a tertiary care ophthalmic centre.

INTRODUCTION: To analyse the fungal culture results of patients with fungal keratitis over sixteen years and look for variations in the trends over years and distribution across ages, gender and seasons. MATERIALS AND METHODS: Clinical and demographic records and microbiology reports of 18,898 patients of fungal keratitis from 2001 to 2016 were analysed. RESULTS: Overall fungal culture positivity was 21.5%. 67.3% were males and 32.7% were females. Maximum numbers of samples (17.9%) were received from age group 41-50 years, and maximum fungal culture positivity was seen in age group 31-40 years (30.8%). Most common fungus was Aspergillus species (31.1%), followed by Fusarium species (24.5%), Alternaria (10.5%), Curvularia (10.2%), Helminthosporium (5.7%), Bipolaris (5.4%), Penicillium (4.5%), Candida (4.4%), Acremonium (1.2%), Rhizopus (1.0%), Paecilomyces (0.8%), Rhodotorula (0.5%) and Mucor (0.2%). Fungal culture positivity and relative frequency of fungi remained almost stable over the study duration, except Rhodotorula spp, which showed a rise 2014 onwards. Highest numbers of culture proven fungal keratitis cases were seen in monsoon season. CONCLUSIONS: To the best of our knowledge, our study is the largest compilation of epidemiological and microbiological features of fungal keratitis, throwing light on important attributes relevant to management of mycotic keratitis patients.

Management Algorithm for Fungal Keratitis: The TST (Topical, Systemic, and Targeted Therapy) Protocol.

PURPOSE: To evaluate the efficacy of the topical, systemic and targeted therapy (TST) protocol in management of fungal keratitis. METHOD: All cases of treatment-naive smear- or culture-proven fungal keratitis presenting between June 2013 and May 2017 were recruited. The TST protocol included initial treatment with topical natamycin 5% with addition of oral ketoconazole or voriconazole in ulcers with size >5 mm, depth >50%, or impending perforation. Topical voriconazole 1% was included in case of poor response at 7 to 10 days. Intrastromal or intracameral antifungal injections were administered in case of poor response to combination therapy. Penetrating keratoplasty was performed in case of poor response to any of the regimen. RESULTS: The study included 223 cases of fungal keratitis with a mean age of 43.6 ± 15.3 years and a male-to-female ratio of 1.8:1. The mean area of the ulcer and infiltrate at presentation was 25.52 ± 19 and 25.7 ± 14.4 mm, respectively. Corrected distance visual acuity at presentation was 2.05 ± 0.43 logMAR that improved to 1.6 ± 0.4 logMAR at 3 months. Fusarium (42.2%) was the most common microorganism isolated, followed by Aspergillus (32.8%). The mean healing time was 41.5 ± 22.2 days, with a final scar size of 14.6 ± 8.2 mm. The treatment success rate with the TST protocol was 79.8%. Corneal perforation developed in 7% of cases (n = 15), and keratoplasty was performed for 20.2% of cases (n = 45). CONCLUSIONS: The TST protocol provides a stepwise treatment algorithm for management of cases of fungal keratitis with varying severity.

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital.

BACKGROUND: Endophthalmitis, a sight-threatening intraocular infection, can be of postsurgical, post-traumatic or endogenous origin. Laboratory diagnosis-based appropriate therapy can be vision-saving. Conventional culture-based laboratory diagnosis takes time and lacks sensitivity. In this study a broad-range PCR assay was assessed against conventional and automated culture methods in vitreous specimens for accurate microbiological diagnosis. AIMS: To use broad-range PCR assay targeting 16S ribosomal RNA (rRNA) region of bacteria and to assess its performance vis-à-vis conventional and automated culture methods in the laboratory diagnosis of endophthalmitis. METHODS: Vitreous specimens from 195 patients with clinically diagnosed endophthalmitis were processed for classical and automated culture methods, antimicrobial sensitivity and broad-range PCR assay targeting 762 bp region of 16S rRNA followed by nucleotide sequencing by Sanger's method. Causative agents were identified from the nucleotide sequences analysed against the GenBank database, and organisms were identified using the Clinical and Laboratory Standards Institute (CLSI) MM18A guidelines. RESULTS: Bacteria could be detected from 127 (65.13%) of the 195 vitreous specimens by broad-range PCR assay; bacterial isolation was possible from 17 (8.7%) and 60 (30.76%) of these specimens by conventional and automated culture methods, respectively (p<0.0001). PCR assay could detect two uncultured bacterium, and in five cases the bacterial identity could not be determined from NCBI database matching. CONCLUSION: Broad-range PCR assay could provide definitive microbial diagnosis within 24 hours in significantly more patients (p<0.0001). Some rare organisms could be detected, useful in treatment modalities. Automated culture was significantly more sensitive than conventional culture.

Spectral Domain Anterior Segment Optical Coherence Tomography in Fungal Keratitis.

PURPOSE: To evaluate the use of spectral domain anterior segment optical coherence tomography (SD-ASOCT) in fungal keratitis. METHODS: Fifty eyes of 50 patients with fungal keratitis were recruited. Serial ASOCT was performed on days 0, 7, 14, 21, 28, 42, and 56. Corneal thickness (CT) in the infiltrate area, infiltrate thickness (IT), and infiltrate width were measured at each follow-up. The presence of any specific feature on ASOCT was evaluated. RESULTS: Mean CT and IT at presentation were 650.5 ± 108 µm and 401.1 ± 91 µm, which reduced significantly at each follow-up [on days 7, 14, 28, and 42; 626.8 ± 113 µm (P < 0.001) and 367.3 ± 94 µm (P = 0.002), 601.4 ± 109 µm and 344.7 ± 94 µm (P < 0.001), 544.8 ± 103 µm and 305.1 ± 80 µm (P < 0.001), and 522.8 ± 97 µm and 291.4 ± 79 µm (P < 0.001), respectively]. The mean CT and scar depth at complete healing were 496.3 ± 101 µm and 283.2 ± 77 µm, respectively. In 10/50 (20%) eyes, the posterior border of the cornea was not clearly visible because of posterior shadowing; therefore, IT was measured along the maximum visible area of hyperreflectivity, whereas CT was measured just adjacent to the area of shadowing. The infiltrate width was measured in 35 eyes, and the mean values at days 0, 7, 14, 28, 42, and 56 were 5.5 ± 0.8 mm, 4.6 ± 0.7 mm, 4.4 ± 0.6 mm, 4.2 ± 0.6 mm, 4.1 ± 0.6 mm, and 4.1 ± 0.6 mm, respectively. A satellite lesion and endothelial plaque were seen in 30% (15/50) and 44% (22/50) eyes, respectively. CONCLUSIONS: ASOCT is a useful adjunct in monitoring fungal keratitis especially in cases with deep stromal involvement and endothelial plaques. In addition, it also provides insight into the activity of keratitis.

A 20-year experience of ocular herpes virus detection using immunofluorescence and polymerase chain reaction.

BACKGROUND: To detect the presence of herpes virus in corneal scrapings/corneal grafts of suspected herpetic keratitis patients attending the outpatient department/casualty of the Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi for the past 20 years with immunofluorescence assay and to analyse the efficacy of polymerase chain reaction over immunofluorescence for routine laboratory diagnosis in some of the specimens. METHODS: Corneal scrapings and corneal grafts were collected by the ophthalmologists from 1,926 suspected herpetic keratitis patients between 1996 and 2015, among whom 1,863 patients were processed with immunofluorescence assay and 302 patients were processed with polymerase chain reaction assay for the detection of herpes virus. Of the 302 patients, clinical specimens from 239 patients were analysed by both polymerase chain reaction and immunofluorescence assay. RESULTS: Of the 1,863 suspected herpetic keratitis patients diagnosed with immunofluorescence assay, 277 (14.9 per cent) were found positive for herpes simplex virus 1 antigen. Similarly, of the 302 suspected herpetic keratitis patients diagnosed by polymerase chain reaction, 70 (23.2 per cent) were found positive for herpes simplex virus DNA. Of the 239 patients diagnosed by both polymerase chain reaction and immunofluorescence assay, 35 (14.6 per cent) were found positive with immunofluorescence assay, 59 (24.7 per cent) were found positive with polymerase chain reaction, 30 (12.5 per cent) were positive with both immunofluorescence and polymerase chain reaction assay. CONCLUSION: Efficacy and accuracy of the polymerase chain reaction assay was greater compared to the immunofluorescence assay for detection of herpes virus in corneal scrapings/corneal grafts of suspected herpetic keratitis patients. Although the immunofluorescence assay is a rapid test for the detection of herpes virus in suspected herpetic keratitis patients, a combination of polymerase chain reaction with immunofluorescence assay will provide higher reliable and accurate results.