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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.

Gunjan Saluja,1 Namrata Sharma,1 Rinky Agarwal,1 H P Sharma,2 Deepali Singhal,1 Prafulla Kumar Maharana,1 Rajesh Sinha,1 Tushar Agarwal,1 T Velpandian,2 Jeewan S Titiyal,1 Gita Satpathy3 1Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India; 2Ocular Pharmacology and Pharmacy Division, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India; 3Department of Ocular Microbiology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, IndiaCorrespondence: Namrata SharmaDepartment of Ophthalmology, Cornea, Cataract and Refractive Surgery Services, 4th Floor Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar East, New Delhi, Tel +91-11-26593144Fax +91-11-26588919Email namrata.sharma@gmail.comObjective: 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.Keywords: intrastromal injections, recalcitrant fungal keratitis

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.

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 =

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. Keywords: Keratomycosis, Fungal culture, Corneal scraping, Pathogenic fungi

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 1. To identify the species of Rhodotorula isolates from ocular infections. 2. 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-4nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] assay. Results 24 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.

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.

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.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.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.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.

Purpose: To determine the clinicomicrobiological profile of infectious agents and their antibiotic susceptibility in different type of endophthalmitis. Methods: A retrospective review of clinical and microbiological records from January 2001 to December 2010, was performed in 1110 patients diagnosed with different type of endophthalmitis (postoperative, posttraumatic, endogenous and post keratitis) to record the demographic details, clinical presentations; microbiological agents isolated with their antimicrobial sensitivity pattern. Antimicrobial susceptibility testing for various culture positive isolates (bacterial/fungal) was performed by the disc diffusion technique. Results: Out of the 1110 intra-ocular specimens processed, 384 (34.6%) were positive for bacteria. S epidermidis was the most predominant isolate accounting for 42.7% of all bacteria obtained, followed by Pseudomonas aeruginosa (24.5%). Besides Pseudomonas, Acinetobacter spp. were the next common gram negative bacilli detected (8.3%) followed by Klebsiella, E. coli, Enterobacter and Alkaligenes in 2.6%, 0.8%, 0.8% and 0.5% cases respectively. The predominant fungal species were Aspergillus spp., in 36.1%, followed by Fusarium spp. in 26.4% cases. Overall susceptibility pattern in our study showed that gram positive bacteria were most susceptible to glycopeptides like vancomycin (80-100%) and fluoroquinolones (87-91%). The sensitivity pattern of gram negative organisms like Pseudomonas and Klebsiella towards fluoroquinolones ranged between 61% - 82%. Conclusion: S epidermidis was the most common bacteria isolated in postoperative and posttraumatic endophthalmitis, Pseudomonas aeruginosa was the most common bacterial isolated in posttraumatic endophthalmitisAmongst fungi Aspergillus was the most common organism.

To evaluate the microbiological profile and outcome in cases with infective keratitis in Stevens-Johnson syndrome (SJS).Eighty-three eyes of 68 patients with SJS presenting with microbial keratitis were recruited and managed with standard antimicrobial therapy.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.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.

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.

Purpose To evaluate the conjunctival microbial flora in cases of ocular Stevens-Johnson Syndrome (SJS) in a tertiary eye care center. Methods This prospective study comprised 176 eyes of 88 patients with ocular SJS compared with 124 eyes of normal subjects. The conjunctival swabs were collected and sent for microbiological analysis for bacterial isolation and antibiotic sensitivity examination. The type of bacteria isolated and its antibiotic sensitivity pattern were studied. Results Of 176 eyes, 104 (59%) had positive cultures for bacteria in cases of SJS and 16 (12.9%) had positive culture in the control group, the difference being statistically significant (P = 0.001). In the SJS group, 14 different types of bacterial isolates were identified. The most common isolate was coagulase-negative staphylococci (CNS) (30/104, 28.8%) followed by Corynebacteria species (35/104, 33.6%) and Staphylococcus aureus (19/104, 18.2%). More than 1 bacteria were isolated in 7 eyes (6.7%). Most of the isolates showed resistance to ciprofloxacin with no resistance to gatifloxacin and moxifloxacin. In the control group, only 2 bacteria were isolated, which included CNS (14/16, 87.5%) and Streptococcus pneumoniae (2/16, 12.5%). CNS showed resistance to ciprofloxacin, and S. pneumoniae was resistant to tobramycin and gentamycin. Conclusions Ocular SJS is associated with alteration of the normal microbial flora residing in the conjunctival sac. The study of which is vital in cases of infection in these eyes with compromised ocular surface. Mixed flora are seen more often in cases of ocular SJS as compared with controls.

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.

BackgroundEndophthalmitis, 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.AimsTo 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.MethodsVitreous 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.ResultsBacteria 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 (pConclusionBroad-range PCR assay could provide definitive microbial diagnosis within 24 hours in significantly more patients (p

Introduction: Fungi can cause systemic or localised infections in human beings, among which mycotic keratitis and Invasive Fungal Infections (IFIs) are very important, which can lead to severe complications and fatal consequences. In some instances, fungal culture isolates may not be identified accurately on the basis of morphology and microscopy. In those cases, molecular characterisation of isolates becomes more helpful in identification, which is essential for providing appropriate antifungal therapy and avoiding bad prognosis. Aim: To identify the fungi collected from patients of mycotic keratitis and IFIs (diagnosed by conventional methods) using molecular methods and to compare the results with the results of conventional methods of identification. Materials and Methods: In the present prospective study, fungal culture isolates were taken from 24 keratomycosis and seven IFIs patients (diagnosed by conventional methods) reporting to the OPD and wards of AIIMS Hospital, Delhi, India (March 2016 to March 2017). Isolates were further subcultured for DNA isolation and amplification for the variable ITS1 and ITS2 regions, and processed for nucleotide sequencing. The obtained sequences for ITS1 and ITS2 regions were searched for homology with NCBI-BLAST program. Results of molecular diagnosis at species level were compared with the conventional methods. Results: Conventional methods could identify Fusarium spp., Alternaria spp., Bipolaris spp., Cladosporium spp., Penicillium spp., Rhizopus spp., up to genus level only, which could be further identified up to species level by molecular methods. Phenotypically identified isolates of Acremonium spp. was identified as Simplicillium spp., Alternaria spp. as Chaetomium globosum, Mucor spp. as Rhizopus oryzae by molecular methods. Conclusion: As prognosis and therapy varies among different genera of fungi and in some cases from species to species, correct species level identification is important; hence, molecular methods may be complemented to traditional methods. Identification of rare fungal isolates is also important in clinical mycology, so that they will not be discarded as contaminants, which is possible using molecular techniques.

Purpose To report a case of Pythium insidiosum keratitis leading to fatal cavernous sinus thrombophlebitis. Methods Case report. Results A 70-year-old man presented with excruciating pain, redness, and diminution of vision in his left eye for 2 weeks after washing his hair with tap water. A total corneal ulcer with surrounding infiltrates and associated corneal thinning was present. Corneal scraping revealed the presence of Gram-positive cocci. KOH wet mount and in vivo confocal microscopy revealed branching hyphae. Combined antibacterial and antifungal treatment was started, but 4 days later, the ulcer showed signs of worsening with perforation for which a large therapeutic penetrating keratoplasty was done. The host cornea showed branching septate hyphae on Sabarouds Dextrose Agar. Two weeks later, the patient developed left eye proptosis with associated extraocular movement restriction. Magnetic resonance imaging of the head and orbit revealed cavernous sinus thrombophlebitis. Lid sparing partial exenteration was performed. Polymerase chain reaction revealed P. insidiosum. The patient subsequently developed a cerebrovascular attack and died of its complications. Conclusions Ocular pythiosis may lead to cavernous sinus thrombophlebitis and can even be life threatening. Timely diagnosis and early radical surgery are of value. A high index of suspicion must be kept for P. insidiosum in cases with suspected fungal etiology not responding to conventional treatment.

The aim was to study the outcomes and results of therapeutic penetrating keratoplasty (Th PK) at a tertiary eye care hospital in northern India.In this retrospective interventional study, a cohort of 506 eyes that underwent a Th PK for microbial keratitis was evaluated. Th PK was performed in cases of recalcitrant microbial keratitis with impending perforation (descemetocele formation) or perforation (3 mm). Medical records were reviewed for demographic details, risk factors, ulcer and perforation size, microbiological investigations, size of donor and recipient beds, postoperative complications, and anatomical and visual outcomes.Anatomical success was seen in 454 eyes (89.7%). Preoperatively, the corrected distance visual acuity was3/60 in 495 eyes (97.8%); after performing the Th PK, the corrected distance visual acuity was3/60 in 249 eyes (49.2%), 3/60 to 6/60 in 182 eyes (35.9%), and6/60 in 75 eyes (14.8%). Eyes with smaller grafts (9 mm) had better anatomical and visual outcomes compared with eyes with larger grafts (9-11 mm; P = 0.03 and11 mm; P = 0.0). A higher success rate was achieved with pure bacterial or fungal organisms rather than with mixed infections. A higher incidence of secondary glaucoma was seen in eyes with perforated ulcers (29.36%; 111/378) than in eyes without perforation (11.71%; 15/128) (P.01) and in eyes with larger graft sizes (11 mm and 9-11 mm) than in eyes with smaller graft sizes (9 mm) (P0.01).Th PK has a definitive role in the management of severe and refractory keratitis with a high success in restoring anatomical integrity and providing useful vision. Better outcomes may be achieved with early intervention before perforation or limbal/scleral extension.

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.

PURPOSE The aim of this study was to detect fungal hyphae in a corneal scraping sample using a cost-effective assembly of smartphone and pocket magnifier. METHODS In this case report, a tissue sample was obtained by conventional corneal scraping from a clinically suspicious case of mycotic keratitis. The smear was stained with Gram stain, and a 10% potassium hydroxide mount was prepared. It was imaged using a smartphone coupled with a compact pocket magnifier and integrated light-emitting diode assembly at point-of-care. Photographs of multiple sections of slides were viewed using smartphone screen and pinch-to-zoom function. The same slides were subsequently screened under a light microscope by an experienced microbiologist. The scraping from the ulcer was also inoculated on blood agar and Sabouraud dextrose agar. RESULTS Smartphone-based digital imaging revealed the presence of gram-positive organism with hyphae. Examination under a light microscope also yielded similar findings. Fusarium was cultured from the corneal scraping, confirming the diagnosis of mycotic keratitis. The patient responded to topical 5% natamycin therapy, with resolution of the ulcer after 4 weeks. CONCLUSIONS Smartphones can be successfully used as novel point-of-care, cost-effective, reliable microscopic screening tools.

Objective To compare the efficacy of topical voriconazole and topical natamycin with that of intrastromal voriconazole and topical natamycin in patients with recalcitrant fungal keratitis. Design Randomized clinical trial. Participants Forty eyes of 40 patients with fungal keratitis (positive smear or culture results or both) larger than 2 mm, involving up to two thirds of the stromal depth, and not responding to topical natamycin therapy for 2 weeks were recruited. Intervention The patients were randomized to receive either topical 1% voriconazole therapy (n = 20) or intrastromal injections of voriconazole 50 μg/0.1 ml (n = 20). The patients in both groups continued topical natamycin 5% every 4 hours until the ulcer healed. Main Outcome Measures Primary outcome measure was best spectacle-corrected visual acuity (BSCVA) 3 months after intervention, and secondary outcome measures were time to healing and the size of the scar. Results The patients in both groups had comparable baseline parameters. The mean BSCVA after treatment was 1.295±0.5 logarithm of the minimum angle of resolution (logMAR) units in the topical group and 1.692±0.29 logMAR units in the intrastromal group. The visual acuity after treatment was significantly better in the topical voriconazole group ( P = 0.008). Nineteen patients receiving topical voriconazole and 16 patients who were given intrastromal voriconazole healed with therapy. Conclusions Topical voriconazole seems to be a useful adjunct to natamycin in fungal keratitis not responding to topical natamycin. Intrastromal injections did not offer any beneficial effect over topical therapy. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Objectives: To study the susceptibility patterns of clinical isolates of Aspergillus species to amphotericin B and voriconazole. Materials and Methods: Fifty isolates of Aspergillus species (21 A. flavus , 14 A. fumigatus and 15 A. niger ) from keratitis cases were tested for their susceptibilities to amphotericin B and voriconazole by a broth microdilution method, standardized in our laboratory. The relative MIC 50 units were calculated from the absolute MIC 50 (μg/ml) upon topical prescription dose in gm/100 ml. Results: The MIC values of amphotericin B for A. fumigatus and A. niger ranged between 0.2 mg/L to 6.25 mg/L (MIC 50; 1.56 mg/L and MIC 90; 6.25 mg/L) and for A. flavus between 0.78-6.25 mg/L (MIC 50 and MIC 90; 6.25 mg/L). MICs of voriconazole for A. fumigatus were in the ranges of 0.1 to 0.2 mg/L, for A. niger 0.05 to 0.2 mg/L (MIC 50 and MIC 90; 0.2 mg/L for both fungi) and against A. flavus 0.05 to 0.39 mg/L (MIC 50 ; 0.2 mg/L and MIC 90 ; 0.39 mg/L). The relative MIC 50 of voriconazole against all the three Aspergillus species was 0.002, compared to those of amphotericin B which were 0.104 against A. fumigatus and A. niger and 0.416 against A. flavus . Conclusion: Voriconazole had lower MIC values and relative MICs than amphotericin B against Aspergillus species, the commonest fungi causing keratitis in this part of the country. Combined with its reported higher bio-availability, it appears far superior to amphotericin B for the treatment of fungal keratitis.

Background Discovery of sessile mode of microbial existence (Biofilm state) focussed much interest, during the recent years, on the study of biofilms in many recurring and chronic infections. However, the exact role of microbial biofilms in chronic rhinosinusitis and orbital cellulitis were not elucidated earlier. The purpose of the present study was to look for the adherent property and biofilm producing ability of the clinical isolates in chronic rhinosinusitis and orbital cellulitis, and to look for the effects of antimicrobial agents on these biofilms by colorimetric assay and ultrastructural analysis. Methods Organisms were isolated and identified from various clinical samples in patients with chronic sinusitis and orbital cellulitis. Antimicrobial sensitivity testing was carried out by the standard protocol. Biofilms were developed; quantified and antimicrobial drug perfusion through the biofilm model was evaluated by the earlier devised procedure. Electronmicroscopic study of the biofilm was performed by the recommended technique. Results Of the total of 70 clinical samples processed, 48 i.e. 68.5 % grew bacteria and 13 i.e.(18.6 %) fungi. Staphylococcus aureus (20), S epidermidis (16) and Pseudomonas aeruginosa (6) accounted for the majority of the bacterial isolates. Aspergillus flavus (8), however was the commonest amongst the fungi. A total of 40 bacteria and 8 fungi could be tested for biofilm production. Eighteen (45 %) of the 40 bacterial isolates and 4(50 %) out of the 8 A flavus isolates were found to be biofilm producers. In vitro adherence testing revealed that majority i.e. 16 (88.8 %) of the 18 biofilm positive bacteria were adherent to artificial surfaces. Antimicrobial drug perfusion through the biofilm model was poor. Antimicrobial treatment was totally ineffective against strong biofilm producers, whose electron microscopic picture was quite similar to that observed for biofilm producers without any antimicrobial pre-treatment. Conclusions Filamentous fungi, like bacteria were capable of forming biofilms, which could be one of the important virulence factors in determining the pathogenic potential of these organisms in causing chronic rhinosinusitis and orbital cellulitis.

Background Acanthamoeba spp. are free-living ubiquitous protozoans capable of causing Acanthamoeba meningitis/meningoencephalitis (AME) of the central nervous system in humans. Acanthamoeba spp. are divided into 20 different genotypes (T1–T20) on the basis of variation in nucleotide sequences of the 18S rRNA gene. The objective of this study was to identify the genotypes of Acanthamoeba spp. in patients of Acanthamoeba meningitis/meningoencephalitis (AME) using 18S rRNA gene-based PCR assay. The present study provides information regarding the involvement of the most prevalent and predominant genotype of Acanthamoeba spp. in Acanthamoeba meningitis/meningoencephalitis infections in India. Methods Cerebrospinal fluid (CSF) was collected from 149 clinically suspected Acanthamoeba meningitis/meningoencephalitis (AME) patients reporting to the outpatient department/causality services of the Neurosciences Centre, AIIMS, New Delhi, India during the past five years. Samples were inoculated onto 2 % non-nutrient agar plates overlaid with E. coli and incubated at 30 °C for 14 days. Among 149 suspected patients, ten were found culture-positive for Acanthamoeba spp. out of which six isolates were established in axenic culture for molecular analysis. DNA was isolated and a PCR assay was performed for amplification of the Diagnostic fragment 3 (DF3) (~280 bp) region of the 18S rRNA gene from axenic culture of six Acanthamoeba spp. isolates. Rns genotyping was performed on the basis of the variation in nucleotide sequences of DF3 region of the 18S rRNA gene. Results In the phylogenetic analysis, all of the six Acanthamoeba spp. isolates were found to belong to genotype T4. The sequence homology search for these six isolates in the NCBI databank showed homology with the available strains of Acanthamoeba spp. The newly generated sequences are available in the GenBank database under accession numbers KT004416–KT004421. Conclusions In the present study, genotype T4 was found as the most prevalent and predominant genotype in Acanthamoeba meningitis/ meningoencephalitis infections. Hence further studies are needed to develop optimal therapeutic strategy against Acanthamoeba spp. of genotype T4 to combat against the infections.

Free-living amoebae of the genus Acanthamoeba are the causative agents of severe sight-threatening infection of the cornea. This study was designed to characterize the genotype of 20 Acanthamoeba spp. isolates obtained from corneal scrapings of 183 suspected Acanthamoeba keratitis patients reporting to the Outpatient Department/Casualty Services of Dr R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India during the period 2011–2015. Corneal scrapings were inoculated onto 2 % non-nutrient agar plates overlaid with Escherichia coli and incubated at 30 °C for 15 days. Amongst 183 suspected Acanthamoeba keratitis patients, 29 were found culture-positive for Acanthamoeba spp. out of which 20 samples were established in axenic culture for molecular analysis. DNA was isolated and PCR assay was performed for the amplification of the diagnostic fragment 3 (DF3) (∼280 bp) region of the 18S rRNA gene from axenic culture of 20 Acanthamoeba spp. isolates. Rns genotyping was performed on the basis of variation in nucleotide sequences of the DF3 region of the 18S rRNA gene. In the phylogenetic analysis, 16 of the 20 isolates were found to be of prevalent genotype T4, two were of genotype T10 and the remaining two isolates were of unassigned genotypes. Hence, it was concluded that genotype T4 was found as the most predominant genotype involved in Acanthamoeba keratitis infections. Genotype T10, which had not been reported from India, was detected for the first time in two patients. Two isolates were found to be unique, which shared

Purpose To evaluate and compare the pattern of resistance to topical moxifloxacin of ocular isolates recovered during comparable periods within 2 years. Methods Records of 611 consecutive cases of bacterial corneal ulcers that presented to our center between April and August 2007 were compared with records of 417 cases that presented in April and August 2006. Records of culture and sensitivity to cefazolin, tobramycin, gatifloxacin, and moxifloxacin at the time of presentation were analyzed. Parameters recorded included total number of cases in each period, culture-positive cases, organisms isolated, and sensitivity by Kirby-Bauer disk-diffusion test. The data from 2007 was compared with those from 2006 using χ test. Results Culture-positive rate was 70.2% in 2007 compared with 65.0% in 2006 (p = 0.143). The percentage of culture-positive cases resistant to moxifloxacin was 5.46% in 2007 compared with 5.42% in 2006 (p = 0.977). There was no significant difference between resistance against cefazolin during the two periods (p = 0.895). Resistance to gatifloxacin and tobramycin was significantly higher in samples tested in 2007 as compared with those tested in 2006 (gatifloxacin, p = 0.037; tobramycin, p = 0.031). Conclusions Resistance to moxifloxacin did not change significantly during a period of 2 years in our study. The static resistance pattern to moxifloxacin may indicate that such resistance to moxifloxacin is intrinsic in the antibiotic rather than new resistance stemming from mutations.