Secondary bacterial corneal infection caused by Myroides species in primary fungal keratitis.

A middle-aged male patient presented with a central corneal perforation in a deep stromal infiltrate in his left eye. An emergency therapeutic penetrating keratoplasty was performed. Microbiological evaluation of the corneal scraping specimen revealed septate fungal filaments on stains. However, culture reports after 24 hours from the scraping sample and the excised half corneal button showed growth of gram-negative bacilli. This pathogen was identified as an aerobic, non-fermentative, gram-negative, bacillus by conventional microbiology and confirmed as Myroides species by the VITEK 2 Compact system (bioMérieux, Marcy l'Etoile, France). Susceptibility to chloramphenicol was noted based on which the patient was treated with topical chloramphenicol 0.5%. No recurrence of the infection was noted. This is the first reported case of corneal infection with the Myroides species of bacteria which, heretofore, have been known to cause endocarditis and urinary tract infections.

Green synthesis of copper nanoparticles by using pineapple peel waste: in vitro characterizations and antibacterial potential.

A considerable amount of fruit waste is being produced every day worldwide. The green synthesis of metal nanoparticles from fruit peel waste can be an innovative, cost-effective, and eco-friendly alternative to traditional methods. Copper nanoparticles (CuNPs) were synthesized by a green method using the pineapple peels extract (PLX) and copper sulfate pentahydrate. The formation of CuNPs was visually identified and detected by UV-Visible spectroscopy. The CuNPs were characterized by Fourier-transform infrared (FTIR) spectroscopy, particle size analyzer, scanning electron microscopy (SEM), and X-ray diffraction (XRD). The antioxidant and reducing power of CuNPs were conducted by %DPPH scavenging and electron transfer-based ferric reducing antioxidant power (FRAP) assay, respectively. The antibacterial properties of CuNPs were determined in gram-positive, and gram-negative bacteria. The results showed that the CuNPs were spherical in shape with mean particle size 290.5 nm. The zeta potential of the nanoparticles was found to be - 12.3 mV indicating the instability in the colloidal state. The FTIR study confirmed the peaks of phytochemicals present in the PLX and the nanoparticles supporting the use of pineapple peels as stabilizing, reducing and capping agents. Both the DPPH and reducing power assay depicted that the synthesized CuNPs had significant antioxidant activity. However, the synthesized CuNPs had strong inhibitory capacity against both gram-positive and gram-negative test organisms. Thus, the CuNPS could be used for its viable antibacterial potential to preserve fruits, flowers, and vegetables from bacterial contamination.

Clinico-Microbiological Correlation in Salmonella Endophthalmitis: Case Series and Review of Literature.

PURPOSE: To report two rare cases of Salmonella endogenous endophthalmitis in an immunocompromised premature baby and an immunocompetent adult and do a brief literature review of related cases. Diagnosis in both cases was confirmed only after the pathogen grew from ocular samples, in the absence of clear signs of enteric fever. METHODS: Retrospective analysis of medical and microbiology records. RESULTS: Both of our cases of Salmonella endophthalmitis had poor visual outcome, despite timely and aggressive management and irrespective of immune status of the patient. Salmonella infection being a rare cause of endophthalmitis was not initially suspected as the adult had minimal systemic symptoms 2 weeks before presentation, while the preterm baby was still on milk feeds. These were just two microbiologically confirmed cases of Salmonella endophthalmitis at our institute over the past 10 years, though enteric fever due to Salmonella species is endemic in Asian countries. CONCLUSIONS: Salmonella endophthalmitis, though rare, leads to poor visual outcomes despite early recognition and aggressive management and may be confused with other infections or non-infectious entities such as necrotizing retinoblastoma in babies, in the absence of clear systemic signs of the disease.

Pseudopithomyces Maydicus and Phialophora Chinensis Rare Fungal Keratitis: Case Series, DNA Sequencing, Antifungal Susceptibility Patterns.

PURPOSE: We report two cases of fungal keratitis due to rare melanized fungal pathogens in ocular infection, one is the first case report of keratitis due to Pseudopithomyces maydicus and the second is keratitis due to rare fungal pathogen in ocular infections Phialophora chinensis. METHOD: Conventional mycology during routine diagnostics helped in identifying these rare fungal isolates, following which we proceeded for the confirmation of identification by DNA sequencing and did in-vitro antifungal susceptibility test to understand their susceptibility pattern. The clinical information for these two patients were collected from hospital electronic medical records. RESULTS: We discuss the clinical presentation, treatment given, and clinical outcome in these patients and correlate these with the conventional microbiology and sequencing techniques, which helped in identifying the pathogen and the in-vitro antifungal susceptibility of these rare isolates. We also do a brief literature review for these two rare fungal pathogens. CONCLUSIONS: Pseudopithomyces maydicus and Phialophora chinensis are rare causes of fungal keratitis due to melanized fungi. Both of this fungal keratitis respond well to medical therapy alone if diagnosed and treated early.

Practical tips and common mistakes in ocular microbiology sampling and processing.

Ocular microbiology deals with miniscule samples from ocular infections, which are difficult to collect, process, and analyze, necessitating special skills, and the knowledge of troubleshooting errors to reach a specific diagnosis. In this article, we highlight several practical points in ocular microbiology, common mistakes, and various approaches to resolve them. We have covered sample collection from different ocular compartments, processing for smear preparation and culture, transport of samples, staining and reagents issues, artifacts and contaminants, and interpretation of in-vitro antimicrobial susceptibility testing reports. This review aims to help both ophthalmologists and microbiologists in making the practice of ocular microbiology and the interpretation of reports more reliable, hassle-free, and precise.

Case Report: Ocular Tissue Diagnosis of Previously Undiagnosed, Extensively Drug-Resistant Pulmonary Tuberculosis.

We describe a patient with concurrent ocular and pulmonary tuberculosis (TB) in whom the diagnosis of extensively drug-resistant TB was made through phenotypic drug-sensitivity testing of an ocular fluid sample after sputum testing yielded incomplete results. Our results are remarkable, because culture-based diagnosis of TB in ocular fluid is unusual. We not only overcame this limitation, but also were able to create a complete drug-sensitivity testing profile from ocular samples, which led to effecting appropriate therapy for the patient.

Lacrimal Drainage Infections with Sphingomonas paucimobilis: Clinical Presentations, Complications and Outcomes.

PURPOSE: To report the clinical presentations, complications, and outcomes of Sphingomonas paucimobilis-specific lacrimal drainage infections. METHODS: A retrospective chart review of all patients diagnosed with Sphingomonas paucimobilis lacrimal infections and managed at a tertiary care Dacryology Service from November 2015 to May 2022 over a 6.5-year period were recruited and analyzed. Data collected include demographic details, clinical presentation, microbiological diagnosis, antibiotic susceptibility profiles, management, complications, and outcomes. The microbiological techniques employed were aerobic and anaerobic cultures, phenotypic identification with VITEK 2R system, polymerase chain reaction, antibiotic sensitivity profile and minimal inhibitory concentration. RESULTS: Twelve Sphingomonas-specific lacrimal drainage infections of 11 patients were identified. Of these five were canaliculitis and seven were acute dacryocystitis. All the seven acute dacryocystitis presented in an advanced stage; five were with lacrimal abscess and two with orbital cellulitis. The antibiotic susceptibility profile of canaliculitis and acute dacryocystitis were comparable and the organism was sensitive to several classes of antibiotics. The outcomes of punctal dilatation and nonincisional curettage were effective for canaliculitis. Patients with acute dacryocystitis had advanced clinical stage at presentation but responded well to intensive systemic management and had excellent anatomical and functional outcomes with dacryocystorhinostomy. CONCLUSION: Sphingomonas-specific lacrimal sac infections can have aggressive clinical presentations and need early and intensive therapy. The outcomes are excellent with multimodal management.

Multidrug-resistant keratitis: challenging yet manageable.

PURPOSE: To study the incidence, clinical features and outcomes of multidrug-resistant (MDR) bacterial keratitis. METHODS: All cases of MDR-bacterial keratitis presenting to our institute over a period of 2 years were retrospectively analysed. Details of risk factors, size and depth of infiltrate, treatment, and outcome were noted. Antibiotic susceptibility tests were done on the ocular isolates from the culture of samples obtained from ocular infections, and resistance or sensitivity of the organisms to the commonly used antibiotics was studied. RESULTS: Forty patients were diagnosed with MDR-bacterial keratitis in the study period. The mean age of patients was 50.9±25.4 years. Most common risk factors were vegetative trauma (n=12, 30.0%), followed by corneal transplantation (n=7, 17.5%) and systemic comorbidities (n=7, 17.5%). Infiltrate was small (<6 mm) in 22 (55%) and large (>6 mm) in 18 (45%) patients. It involved the superficial, mid and deep stroma in 11 (27.5%), 9 (22.5%) and 15 (37.5%) cases, respectively. Gram-negative bacilli (n=18, 45%) were the maximum, among which Pseudomonas aeruginosa (15%) was the most common. Resistance to 3 (n=17, 42.5%) and 4 (n=17, 42.5%) classes of antibiotics was the most commonly observed. One (2.5%) patient showed resistance to all seven classes of drugs tested. Complete resolution of infection was seen in 15 (37.5%) MDR patients on medical management alone. Five (12.5%) patients underwent therapeutic penetrating keratoplasty. Size of the infiltrate was found to have a significant correlation with the outcome (p=0.002). CONCLUSION: MDR keratitis, despite being a challenge to treat, can be successfully managed by medical therapy alone, if appropriate therapy is started early in the clinical course.

Case Report: Poor Outcome Despite Aggressive Management in Pythium insidiosum Endophthalmitis.

Pythium insidiosum is a parasitic oomycete that can cause keratitis and closely resembles fungus, both clinically and morphologically. It requires a trained microbiologist for its differentiation from fungal filaments and has poor response to antifungal therapy. We present a case of primary isolation of the organism from the vitreous cavity in a case of endophthalmitis. The infection spread quickly and involved all the ocular tissues. The eye had poor visual outcome despite a sequence of rapidly completed retinal and corneal surgeries along with initiation of anti-Pythium therapy.

Acremonium keratitis: Risk factors, clinical characteristics, management, and outcome in 65 cases.

Purpose: To study the risk factors, clinical presentation, management options, and outcomes in cases of culture-proven Acremonium keratitis. Methods: Medical and microbiology records of culture-proven Acremonium keratitis from Jan 2007 to Dec 2019 at a tertiary eye care center were reviewed. Details of clinical findings on each visit and operating notes were reviewed from the medical records. All cases were subjected to corneal scraping at the first visit for microbiological investigation consisting of direct smear examination and culture. Topical natamycin 5% was the mainstay of medical treatment. Surgical treatment was considered for nonresponding patients. Results: During the 13-year study period, 65 cases of culture-proven Acremonium keratitis were identified out of 1605 cases of fungal keratitis. Trauma was the most common predisposing factor in 32 cases (49.2%). The average area of the corneal stromal infiltrate was 24.8 mm2 at the initial presentation. Hypopyon at the time of presentation was evident in 28 (43.1%) cases. Staphylococcus spp. was the most common (n = 22, 33.8%) organism coexistent with Acremonium. Direct microscopy of corneal scraping was positive for fungal filaments in 57/65 (87.6%) cases. Medical management alone was given in 44 patients (67.6%). Age (>50 years) and treatment delay (>15 days) were found to be independent risk factors for the poor final visual outcome (VA <20/60). Conclusion: When treated early, Acremonium keratitis responds well to medical therapy with currently available topical antifungals. However, advanced and nonresponding cases require surgical intervention for resolution of the infection.

Use of different stains for microscopic evaluation for the diagnosis of Pythium keratitis.

PURPOSE: To evaluate the efficacy of various staining techniques for detection of Pythium in keratitis cases. METHODS: Data of nineteen consecutive culture-positive cases of Pythium keratitis were retrospectively analysed. Corneal scrapings and corneal buttons (in the cases which underwent therapeutic penetrating keratoplasty [TPK]) were sent for microbiological and histopathological examination. The direct smears were stained with Potassium hydroxide and calcofluor white (KOH â€‹+ â€‹CFW), Gram and Iodine-Potassium Iodide-Sulphuric Acid (IKI-H2SO4) stains. The corneal buttons were stained with Gomori's Methanamine Silver (GMS), Periodic Acid-Schiff (PAS) and Iodine-Potassium Iodide-Sulphuric Acid (IKI-H2SO4) stains. The positivity of various stains in detecting Pythium was studied. RESULTS: Gram and KOH â€‹+ â€‹CFW staining from smear was done in 16 out of 19 (84.2%) cases. KOH â€‹+ â€‹CFW and Gram stains were suggestive of Pythium in 10 (62.5%) and 7 (43.8%) cases, respectively. IKI-H2SO4 staining in scraping samples was positive for Pythium in all the 4 (100%) cases in which it was performed. Half corneal buttons were positive for Pythium with IKI-H2SO4 stain as well as GMS stain in all the 18 cases that underwent TPK (100%). PAS stain showed weak to faint pink staining of Pythium filaments in 7 out of 18 cases (38.9%). CONCLUSION: IKI-H2SO4 stain followed by KOH â€‹+ â€‹CFW stain detects Pythium filaments most accurately in corneal scraping samples from keratitis patients, although the differences were not statistically significant. The positivity of the stains depends on astute observation by an experienced ocular microbiologist and pathologist.

Keratitis following descemet's stripping endothelial keratoplasty (DSEK): Clinical features and outcome in early vs late presentation.

OBJECTIVE: To compare the clinical presentation, risk factors, causative organisms, and final outcome of early- and late- onset microbial keratitis following Descemet's Stripping Endothelial Keratoplasty (DSEK). MATERIALS AND METHODS: In a retrospective, comparative, case series, twenty-three (10 early-onset keratitis, 13 late-onset keratitis) cases were included for analysis from a single centre tertiary eye care setup. Data about demography, indication, clinical features, and outcome were collected for both the groups. Comparison of clinical presentation, risk factors, microbiology and final outcome were carried out. RESULTS: The most common indication was failed graft. While all patients with late-onset keratitis presented with ulcerative keratitis, 60% of early-onset keratitis presented with ulcerative keratitis. Four patients in the early-onset group had interface keratitis. Associated endophthalmitis was more in the early-onset compared to the late-onset group. Early-onset interface keratitis cases are mostly associated with donor-related infection. Gram-positive bacteria were the most common organisms isolated from both early- and late- onset infection. Major surgical procedures in both early- and late- onset keratitis included lenticule removal (n = 4) and therapeutic penetrating keratoplasty (n = 5). CONCLUSIONS: Failed graft is a major risk factor in post-DSEK keratitis. Interface keratitis and endophthalmitis are commonly observed in early-onset keratitis. A majority of the patients need surgical intervention.

Effect of polymicrobial interactions on antimicrobial resistance: an in vitro analysis in human ocular infections.

Purpose: Investigate the effect of polymicrobial interactions on antimicrobial resistance (AMR) of ocular pathogens in polymicrobial settings, compared with monomicrobial infections. Methods: Polymicrobial interactions were labeled as antagonistic, synergistic or indifferent based on a reduction, an increase or no change, respectively, in antibiotics' MIC by the Vitek 2 compact system, compared with monomicrobial pathogens. Results:Staphylococcus epidermidis showed antagonistic polymicrobial interactions (22.6%); Pseudomonas aeruginosa showed synergistic interactions (62.5%); multidrug-resistant Acinetobacter baumannii showed increased susceptibility to select antibiotics; Serratia ficaria (inherently colistin resistant) became colistin-susceptible in polymicrobial combinations. Conclusion: Both antagonistic and synergistic interactions exist among human pathogens in polymicrobial settings. Gram-positive pathogens had significantly higher antagonistic polymicrobial interactions (increased MICs: 20.4%) compared with Gram-negative ones (synergistic: 59.4%).

Sensitivity and specificity of potassium hydroxide and calcofluor white stain to differentiate between fungal and Pythium filaments in corneal scrapings from patients of Pythium keratitis.

PURPOSE: To assess the sensitivity of potassium hydroxide and calcofluor white (KOH+CFW) mount in the diagnosis of Pythium keratitis and concordance among microbiologists. METHODS: Three microbiologists evaluated the microscopic images of KOH + CFW mounts of confirmed cases of Pythium and fungal keratitis seen between January 2019 and February 2021. The filaments were compared using specific differentiating features. The sensitivity and specificity of KOH + CFW in diagnosing Pythium infection were evaluated along with concordance among the microbiologists. RESULTS: Sixty consecutive cases with confirmed growth of fungus or Pythium insidiosum (n = 29) were evaluated. The sensitivity of KOH + CFW in the correct identification of Pythium filaments ranged from 79.3% to 96.5% among three microbiologists. There was good interobserver (k = 0.76-0.90) and intraobserver (k = 0.70-0.97) agreements among three microbiologists. The differentiating findings (P < 0.0001) suggestive of Pythium filaments were the absence of septae in 23 (79.3%) and collapsed walls in 22 (75.9%) cases. CONCLUSION: KOH + CFW has good sensitivity and specificity in the diagnosis of Pythium keratitis with good interobserver and intraobserver concordance.

Fungal Endophthalmitis: Analysis of 730 Consecutive Eyes from 7 Tertiary Eye Care Centers in India.

PURPOSE: To evaluate the clinical and microbiological features of a large cohort with culture-confirmed fungal endophthalmitis across India. DESIGN: Cross-sectional, hospital-based, retrospective medical record review. PARTICIPANTS: Seven large tertiary eye care centers from different regions of India. METHODS: Patient data were pooled from electronic or physical medical records of each participating center. Fellowship-trained vitreoretinal specialists clinically managed all patients, and in-house microbiology laboratories performed all microbiological workups. The clinical and microbiological procedures were broadly uniform across all participating centers. The essential treatment consisted of vitreous surgery as well as intravitreal and systemic therapies with antifungal agents. MAIN OUTCOME MEASURES: Clinical outcome of the causative event and causative fungus. RESULTS: In the period from 2005 to 2020, 7 centers treated 3830 cases of culture-proven endophthalmitis, and of these, 19.1% (n = 730) were cases of culture-confirmed fungal endophthalmitis. It included 46.9% cases of postoperative (87.4% postcataract surgery), 35.6% of traumatic, and 17.5% of endogenous endophthalmitis. The fungi included 39.0% of Aspergillus (high prevalence in central, east, and south zones), 15.1% of Candida (high prevalence in west zone), and 15.9% of Fusarium (high prevalence in north and west zones). The time to symptom development was between 1 week and 4 weeks in more than one third of the patients, except in patients with traumatic endophthalmitis. Less than half of the patients had hypopyon on presentation. The presenting visual acuity (PVA) in most patients was <20/400. Nearly all patients needed vitrectomy and an average of 2 intravitreal injections of antifungal agents. At least 10% of eyes needed therapeutic keratoplasty, and up to 7% of eyes were eviscerated. After treatment, the final (best corrected) visual acuity (FVA) was >20/400 in 30.5% (n = 222) of eyes and >20/40 in 7.9% (n = 58) of eyes, and 12% (n = 88) of eyes lost light perception. A post hoc analysis showed the male sex to be significantly more associated with traumatic endophthalmitis than with postoperative (P < 0.0001) and endogenous (P = 0.001) endophthalmitis, more isolation of Candida species in patients with endogenous endophthalmitis than in those with postoperative (P = 0.004) and traumatic (P < 0.0001) endophthalmitis, better PVA in eyes with Candida species infection (P < 0.0001), and poorer FVA in eyes with Aspergillus species infection. CONCLUSIONS: Fungal endophthalmitis is not uncommon in India. The inclusion of antifungal agents with antibiotics as the first empirical intravitreal therapy before microbiological confirmation should be considered when a fungal infection is suspected.

Corynebacterium Keratitis: Pure Versus Mixed Infection and Antibiotic Susceptibility Patterns From Different Tertiary Eye Care Centers.

PURPOSE: The objective of this study was to compare the clinical and microbiological profiles of culture-proven pure Corynebacterium keratitis with mixed infection and their antibiotic susceptibility patterns over a 2-year period. METHODS: A retrospective analysis of culture-proven cases of Corynebacterium keratitis over a 2-year period was performed in 3 different tertiary eye care centers. All isolates were tested for antibiotic susceptibility in vitro using the disc-diffusion method for 7 antibiotics. RESULTS: Altogether 108 cases were identified as culture-positive Corynebacterium keratitis in 3 tertiary eye care centers. Of these, 60.2% (n = 65) and 39.8% (n = 43) of cases were due to pure Corynebacterium and mixed infection, respectively. The mean duration of symptoms was 23.2 ± 29.6 days. In the mixed-infection group, fungus was identified as the coexistent pathogen in 22 cases (51.1%). Ocular surface disorder was the most common risk factor (33.9%) in Corynebacterium keratitis. The most frequently isolated species was Corynebacterium amycolatum (22.2%) in both groups. Therapeutic keratoplasty was performed in 8.3% of cases. There was no significant difference in the outcome between the 2 groups. Cefazolin resistance was seen in 13.9% of patients, and all isolates were susceptible to vancomycin. The resistance pattern showed emerging resistance toward fluoroquinolone because the isolates were resistant to gatifloxacin (58.3%), moxifloxacin (47.2%), ciprofloxacin (54.6%), and ofloxacin (45.4%). CONCLUSIONS: Ocular surface disorder is the most common risk factor in Corynebacterium keratitis. Although fluoroquinolones are commonly used as first-line therapy in microbial keratitis, the in vitro resistance pattern indicates that these are less likely to be effective in infection with Corynebacterium species.

Efficacy of voriconazole and amphotericin B in corneal preservative media.

PURPOSE: To evaluate the efficacy of voriconazole and amphotericin B in McCarey-Kaufman (MK) media. METHODS: MK media vials were supplemented with either voriconazole at 1, 2, 20, 50, 100 µg/mL or amphotericin B at 0.5, 1, 2, 10, 20 µg/mL. The standard inoculum of the American Type Culture Collection (ATCC) strain of Candida albicans, Aspergillus flavus, and Fusarium keratinoplasticum was added to the set of vials. The efficacy outcomes were calculated as 'viable fungal colony counts' determined from the samples taken on Days 0 and 4. MK media containing fungal inoculum but without antifungal supplements were used as control. RESULTS: In the voriconazole arm, on Day 4, a reduction in the colony count was observed for Candida albicans (1 µg/mL, 36%; 100 µg/mL, 100%), Aspergillus flavus (1 µg/mL, 53.8%; 100 µg/mL, 80.4%), and Fusarium keratinoplasticum (1 µg/mL, 39.0%; 100 µg/mL, 72.2%). Similarly, in the amphotericin B arm, on Day 4, a reduction in the colony count was observed for Candida albicans (0.5 µg/mL; 99.9%; 20 µg/mL, 100%), Aspergillus flavus (0.5 µg/mL, 65.2%; 20 µg/mL, 84.8%), and Fusarium keratinoplasticum (0.5 µg/mL, 90.1%; 20 µg/mL, 100%). CONCLUSION: Compared to voriconazole, the addition of amphotericin B significantly reduces fungal contamination in MK media.

Hybridoma technology; advancements, clinical significance, and future aspects.

BACKGROUND: Hybridoma technology is one of the most common methods used to produce monoclonal antibodies. In this process, antibody-producing B lymphocytes are isolated from mice after immunizing the mice with specific antigen and are fused with immortal myeloma cell lines to form hybrid cells, called hybridoma cell lines. These hybridoma cells are cultured in a lab to produce monoclonal antibodies, against a specific antigen. This can be achieved by an in vivo or an in vitro method. It is preferred above all the available methods to produce monoclonal antibodies because antibodies thus produced are of high purity and are highly sensitive and specific. Monoclonal antibodies are useful in diagnostic, imaging, and therapeutic purposes and have a very high clinical significance. Once hybridoma cells become stable, these cell lines offer limitless production of homogenized antibodies. This method is also cost-effective. The antibodies produced by this method are highly sensitive and specific to the targeted antigen. It is an important tool used in various fields of research such as in toxicology, animal biotechnology, medicine, pharmacology, cell, and molecular biology. Monoclonal antibodies are used extensively in the diagnosis and therapeutic applications. Radiolabeled monoclonal antibodies are used as probes to detect tumor antigens in the living system; also radioisotope coupled antibodies are used for therapeutic target specific action on oncogenic cells. SHORT CONCLUSION: Presently, the monoclonal antibodies used are either raised in mice or rats; this poses a risk of disease transfer from mice to humans. There is no guarantee that antibodies thus created are entirely virus-free, despite the purification process. Also, there are some immunogenic responses observed against the antibodies of mice origin. Technologically advanced techniques such as genetic engineering helped in reducing some of these limitations. Advanced methods are under development to make lab-produced monoclonal antibodies as human as possible. This review discusses the advantages and challenges associated with monoclonal antibody production, also enlightens the advancement, clinical significance, and future aspects of this technique.