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.

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

Ocular Chlamydia trachomatis infections in patients attending a tertiary eye care hospital in north India A twelve year study.

Background & objectives: Ocular infection with Chlamydia trachomatis is a major public health problem in densely populated countries like India. The true prevalence of such infections is uncertain due to insufficient data available from India. The aim of this study was to do a retrospective analysis of C. trachomatis eye infections in patients attending the outpatient department of Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, over a period of 12 years. Methods: From 1997 to 2008, the Chlamydia laboratory received conjunctival swabs from 1281 consecutive patients for C. trachomatis detection after thorough clinical examination. Specimens were subjected to direct fluorescent antigen detection assay using monoclonal antibody based commercial kit to detect the presence of C. trachomatis antigen. Results: Antigen positivity varied between 22-28 per cent. Children below 11 yr and people above the age of 60 yr showed comparatively higher antigen positivity (25.7 and 27.8%, respectively). As compared to males significantly (P<0.05) higher number of females in the age group of 31-60 yr were positive for C. trachomatis antigen. Patients with the clinical diagnosis of follicular/allergic conjunctivitis and trachoma showed higher rate of antigen positivity. Interpretation & conclusions: Northern India having dry and arid climatic conditions in most parts of the year was considered in the past as one of the trachoma hyper-endemic foci. The study indicated that laboratory proven C. trachomatis eye infection still persisted in this part of the country throughout the study period of 12 years.

A pilot study for diagnosis of genital Chlamydia trachomatis infections by polymerase chain reaction among symptomatic Indian women.

Background: Chlamydia trachomatis is the most common bacterial etiology of sexually transmitted infection. Aim : A pilot study was designed using PCR for amplification and detection of a specific 517 bp sequence of the common endogenous plasmid of C. trachomatis from clinical swab specimens obtained from symptomatic female patients attending STD clinics of AIIMS and Regional STD Teaching, Training & Research Center, Safdarjang Hospital, New Delhi. Methods: 97 patients were recruited in the study, and endocervical swabs were collected following standard procedures. The samples were analyzed by PCR and direct fluorescence antibody (DFA) for detection of C. trachomatis, and the sensitivity, specificity, PPV and NPV of PCR were calculated taking DFA as gold standard. Results: Out of 97 samples tested, 9 were positive for C. trachomatis by PCR. 1 PCR positive patient was negative by DFA although a total of 11 patients were positive by DFA. The sensitivity, specificity, PPV and NPV of PCR with reference to DFA was 72.73%, 98.84%, 88.89% and 96.59%, respectively. This PCR had high specificity and NPV for detection of C.trachomatis. Conclusions : In light of the introduction of enhanced syndromic approach, which involves the use of laboratory techniques (wherever possible) to confirm clinical diagnosis, a diagnostic PCR with high specificity and NPV is particularly valuable for determination of etiological diagnosis and hence contribute to judicious use of antimicrobials in the community.

Molecular characterization of drug-resistant and drug-sensitive Aspergillus isolates causing infectious keratitis.

Purpose: To study the susceptibilities of Aspergillus species against amphotericin B in infectious keratitis and to find out if drug resistance had any association with the molecular characteristics of the fungi. Materials and Methods: One hundred and sixty Aspergillus isolates from the corneal scrapings of patients with keratitis were tested for susceptibilities to amphotericin B by broth microdilution method. These included Aspergillus flavus (64 isolates), A. fumigatus (43) and A. niger (53). Fungal DNA was extracted by glass bead vertexing technique. Polymerase chain reaction (PCR) assay was standardized and used to amplify the 28S rRNA gene. Single-stranded conformational polymorphism (SSCP) of the PCR product was performed by the standard protocol. Results: Of the 160 isolates, 84 (52.5%) showed low minimum inhibitory concentration (MIC) values (≤ 1.56 μg/ml) and were designated as amphotercin B-sensitive. Similarly, 76 (47.5%) had high MICs (≥ 3.12 μg/ml) and were categorized as amphotericin B-resistant. MIC50 and MIC90 values ranged between 3.12-6.25 μg/ml and 3.12-12.5 μg/ml respectively. A. flavus and A. niger showed higher MIC50 and MIC90 values than A. fumigatus. The SSCP pattern exhibited three extra bands (150 bp, 200 bp and 250 bp each) in addition to the 260 bp amplicon. Strains (lanes 1 and 7) lacking the 150 bp band showed low MIC values (≤ 1.56 μg/ml). Conclusion: A. niger and A. flavus isolates had higher MICs compared to A. fumigatus, suggesting a high index of suspicion for amphotericin B resistance. PCR-SSCP was a good molecular tool to characterize Aspergillus phenotypes in fungal keratitis.

Post-operative endophthalmitis: Antibiogram & genetic relatedness between Pseudomonas aeruginosa isolates from patients & phacoemulsifiers.

Background & objectives: Though not frequently but there are reports showing phacoemulsifiers as a potent source of infection in post-operative cases of endophthalmitis. This study was carried out to find antibiogram and genetic relatedness between Pseudomonas aeruginosa isolates from a post-cataract surgery endophthalmitis outbreak (3 patients) and internal tubings of 5 phacoemulsifiers. Methods: In vitro antimicrobial sensitivity patterns of the 8 bacterial isolates were observed. Genetic analysis of the bacterial isolates was done using random amplification of polymorphic DNA (RAPD) assay and PCR ribotyping. The resulting DNA band patterns were examined visually and by computer assisted analysis using unweighted pair group method. Results: The three P. aeruginosa patient isolates were found to be different from the five phacoemulsifier isolates in sensitivity towards 3 antibiotics and by genetic analysis (33 and 44% homology by RAPD assay and PCR ribotyping). Two of the patient isolates shared 100 per cent genetic homology by RAPD assay and another pair shared 100 per cent homology by PCR ribotyping. The five isolates from phacoemulsifiers did not share significant genetic homology. There was significant genetic variation between bacterial isolates from patients and phaco emulsifiers. Interpretation & conclusion: Though the three P. aeruginosa isolates obtained from the patients were phenotypically similar and genetically close, they differed from the phaco-machine isolates both genetically, and in their antibiogram profile. However, the five phacoemulsifier isolates were genetically diverse though they shared the same antibiogram profile. Therefore the Ringer’s lactate from phacomachines could not be conclusively proven to be the source of infection.

Detection of Chlamydophila pneumoniae (Chlamydia pneumoniae) in endarteroctomy specimens of coronary heart diseases patients.

Background & objectives: Association of Chlamydia pneumoniae with atherosclerosis and coronary artery disease is debated. Increased antibody levels against C. pneumoniae in patients with coronary artery disease is widely reported. Direct evidence would be demonstration of C. pneumoniae, its antigen or genome in the diseased arterial tissue. This study was thus conducted to look for antigen or genome of C. pneumoniae in coronary artery specimens from patients with coronary artery disease along with serology. Methods: Sixty two end arteriotomy specimens of discarded coronary arteries from patients of coronary heart disease were tested for presence of C.pnuemoniae genome using 2 nested PCR assays and antigen detection by immuno-fluorescence assay. Presence of species specific antibodies were also tested in the patients. Results: C. pneumoniae could not be detected by PCR or immunofluorescence assay in any specimen. C. pnuemoniae Ig G antibody was detected in 42 of the 62 (67.7%) patients studied, compared to 10 of the 23 (43.47%) of controls. Moreover 18 of 62 (29%) patients compared to 4 of 23 (17.39%) controls possessed IgA antibodies. Interpretation & conclusions: Association of C.pneumoniae and coronary artery disease would not be established by genome or antigen detection. However, C. pneumoniae antibodies were detected in more number of patients than controls. More studies are required to reach to a conclusion.

Detection of Chlamydophila pneumoniae (Chlamydia pneumoniae) in endarteroctomy specimens of coronary heart diseases patients.

BACKGROUND & OBJECTIVE: Association of Chlamydia pneumoniae with atherosclerosis and coronary artery disease is debated. Increased antibody levels against C. pneumoniae in patients with coronary artery disease is widely reported. Direct evidence would be demonstration of C. pneumoniae, its antigen or genome in the diseased arterial tissue. This study was thus conducted to look for antigen or genome of C. pneumoniae in coronary artery specimens from patients with coronary artery disease along with serology. METHODS: Sixty two end arteriotomy specimens of discarded coronary arteries from patients of coronary heart disease were tested for presence of C.pnuemoniae genome using 2 nested PCR assays and antigen detection by immuno-fluorescence assay. Presence of species specific antibodies were also tested in the patients. RESULTS: C. pneumoniae could not be detected by PCR or immunofluorescence assay in any specimen. C. pnuemoniae Ig G antibody was detected in 42 of the 62 (67.7%) patients studied, compared to 10 of the 23 (43.47%) of controls. Moreover 18 of 62 (29%) patients compared to 4 of 23 (17.39%) controls possessed IgA antibodies. INTERPRETATION & CONCLUSION: Association of C.pneumoniae and coronary artery disease would not be established by genome or antigen detection. However, C. pneumoniae antibodies were detected in more number of patients than controls. More studies are required to reach to a conclusion.

Characterization of Streptococcus pneumoniae ophthalmic, systemic & commensal isolates by pulsed-field gel electrophoresis & ribotyping.

BACKGROUND & OBJECTIVE: Streptococcus pneumoniae is common in ocular and systemic infections and is a part of normal nasopharyngeal flora. Very few studies regarding genetic analysis of S. pneumoniae isolates causing eye infections are available. This study was undertaken to do pulse field gel electrophoresis (PFGE) analysis and ribotyping of S. pneumoniae isolates obtained from eye infections, systemic infections and nasopharyngeal flora. METHODS: Sixty one well characterized S. pneumoniae isolates (38 from ophthalmic infections, 9 from systemic infections and 14 commensals) were characterized using PFGE of the whole genome after SmaI, restriction enzyme digestion and conventional ribotyping using Escherichia coli rRNA operon as the probe. Phylogenetic tree was drawn using unweighted pair group method analysis (UPGMA). RESULTS: The 38 S. pneumoniae isolates from eye infections belonging to 15 serotypes were placed in to 11 PFGE types and 15 ribotypes. The 9 systemic isolates (7 seotypes) were distributed in 7 PFGE types and 6 ribotypes. The 14 commensal isolates were placed in 11 serotypes, 5 PFGE types and 6 ribotypes. Most of the PFGE types and ribotypes consisting of ocular isolates also contained systemic and commensal isolates. INTERPRETATION & CONCLUSION: Considerable genetic similarity was observed between the isolates from ocular and systemic infections and those colonized in nasopharynx. PFGE analysis could differentiate majority of the isolates according to site of infections. There was a considerable DNA polymorphism within the studied bacterial population.

Phenotypic and plasmid pattern analysis of Staphylococcus epidermidis in bacterial keratitis.

BACKGROUND: Staphylococcus epidermidis, a commensal of the conjunctival sac has been incriminated as the commonest etiological agent of bacterial keratitis. However, the pathogenic potential of this commensal organism is not clearly known. AIM: To determine any phenotypic, molecular markers of S. epidermidis pathogenicity in bacterial keratitis. MATERIALS AND METHODS: A total of 382 corneal ulcer isolates of S. epidermidis and 87 S. epidermidis isolates from healthy eyes (controls) were studied. Speciation, biotyping and antibiotic sensitivity testing were performed by conventional methods. Tube slime and adherence tests were carried out by recommended techniques. Plasmid analysis was conducted by a standard protocol. STATISTICAL ANALYSIS: Chi-square test was employed for calculations. RESULTS: Out of 382 corneal ulcer isolates (Pathogens) 284 (74.3%) belonged to biotypes I and II. Slime was detected in 164 (42.9%) of 382 pathogens vs. 21 (24.1%) of 87 controls (P<0.001). Sixty-five (39.6%) of 164 slime positive isolates were multidrug-resistant as compared to only 49 (22.4%) of 218 slime negative isolates (P<0.001). A significantly higher number i.e, 73.1% (120/164) of slime-producers possessed a 21 Kb plasmid in contrast to only 53.2% (116/218) of nonslime-producers (P<0.001). Presence of this plasmid had a statistical correlation of low significance with multidrug resistance (P=0.04). One hundred and seventy-two (45.0%) of 382 pathogens and 24 (27.6%) of the 87 controls were adherent to artificial surfaces (P=0.003) and the majority of the adherent organisms (99/172, 57.6%) were slime producers (P<0.001). CONCLUSIONS: Slime was associated with multidrug resistance in corneal ulcer isolates of S. epidermidis. The 21 Kb plasmid could determine virulence as it was responsible for slime production and adherence.

Serotype distribution of Streptococcus pneumoniae isolates from ophthalmic and systemic infections and of commensal origin.

BACKGROUND AND OBJECTIVES: Information regarding serotype distributions of Streptococcus pneumoniae causing ophthalmic infections is scanty. This study was therefore undertaken to determine the antimicrobial susceptibility status and serotypes of S. pneumoniae isolated from various ophthalmic infections and to compare with those isolated from systemic infections and commensal nasopharyngeal flora. METHODS: Thirty eight of S. pneumoniae isolates from ophthalmic infections, 9 from systemic infections and 14 from the nasopharynx of apparently healthy school children were biochemically characterized and tested for in vitro antimicrobial susceptibility to various antibiotics. Serotyping of these 61 isolates was done by a rapid co-agglutination method. RESULTS: All the 61 isolates were sensitive to oxacillin (penicillin) and susceptibility against other antimicrobials was variable. No multidrug resistance was observed. The 38 ophthalmic isolates were distributed in 15 different serotypes. Most prevalent serotypes were 14, followed by 8 and 19F. The 9 systemic and 14 commensal. isolates of S. pneumoniae were distributed in 7 and 11 serotypes respectively. Three of the systemic and six of the commensal serotypes were observed in ophthalmic infections whereas four of the commensal serotypes were observed in systemic infections. INTERPRETATION AND CONCLUSION: Resistance to penicillin was not observed. In ophthalmic infections, a wide range of serotypes of S. pneumoniae were observed. More than half of the commensal serotypes obtained in the study as well as majority of the systemic serotypes were observed in ophthalmic infections.

Immunocomb Chlamydia bivalent assay to study Chlamydia species-specific antibodies in patients with coronary heart disease.

BACKGROUND & OBJECTIVE: Serological evidences suggested an association between Chlamydia pneumoniae infection and coronary heart disease (CHD). Efficacy of available serological tests for detection of C. pneumoniae antibody has been debated. The present study was carried-out to assess the efficacy of Immunocomb Chlamydia bivalent IgG assay vis-à-vis micro immunofluorescence (MIF) test in detecting C. pneumoniae and C. trachomatis--specific antibodies in patients with CHD. METHODS: Serum samples collected from clinically confirmed cases of CHD (n=114) were subjected to Immunocomb Chlamydia bivalent assay and the standard MIF test. Antibodies specific to C. pneumoniae and C. trachomatis were detected quantitatively. RESULTS: Though Immunocomb Chlamydia bivalent test yielded 73.7 per cent positivity for C. pneumoniae- specific IgG antibody (compared to 50.8% by MIF), the specificity of Immunocomb was found only 32.14 per cent. Positive and negative predictive values of Immunocomb assay were 54.8 and 60.0 per cent respectively. INTERPRETATION & CONCLUSION: The findings of the present study indicated that though Immunocomb assay was inferior to MIF, it can be used as a method for presumptive serology due to its rapidity and ease of performance. Wherever possible, one or more additional tests should also be performed to increase the specificity of such studies.

Microbiological study of neonatal conjunctivitis with special reference to Chlamydia trachomatis.

PURPOSE: To study the microbial agents, chiefly Chlamydia trachomatis and other bacteria, in neonatal conjunctivitis. METHODS: Conjunctival specimens from 70 newborns with conjunctivitis were subjected to bacterial culture and sensitivity testing, monoclonal antibody based C. trachomatis antigen detection test and species-specific Chlamydia antibody detection in the sera of babies and their mothers, by micro-immunofluorescence assay. RESULTS: Bacteria were isolated from 35 (50%) babies; the majority (20, 57.14%) were Staphylococcus epidermidis. C. trachomatis antigen was detected in conjunctival smears of 17 (24%) babies, and 6 (35.29%) of them were positive for other bacteria. Six babies and their mothers tested positive for C. trachomatis Ig G antibodies. At follow-up after 14 weeks, 6 (35.29%) of the Chlamydia antigen-positive babies were found to have developed recurrent conjunctivitis. CONCLUSION: C. trachomatis is responsible for almost a quarter of all cases of neonatal conjunctivitis, with recurrences in 35% of cases. Bacteria could be isolated from 50% of the patients though the exact role of Staphylococcus epidermidis, isolated from 28.65% of the neonatal conjunctivitis cases, remains unclear.