agar and Keratitis

Pythium insidiosum is an oomycete and is also called "parafungus" as it closely mimics fungal keratitis. The last decade saw an unprecedented surge in Pythium keratitis cases, especially from Asia and India, probably due to growing research on the microorganism and improved diagnostic and treatment modalities. The clinical features such as subepithelial infiltrate, cotton wool-like fluffy stromal infiltrate, satellite lesions, corneal perforation, endoexudates, and anterior chamber hypopyon closely resemble fungus. The classical clinical features of Pythium that distinguish it from other microorganisms are reticular dots, tentacular projections, peripheral furrowing, and early limbal spread, which require a high index of clinical suspicion. Pythium also exhibits morphological and microbiological resemblance to fungus on routine smearing, revealing perpendicular or obtuse septate or aseptate branching hyphae. Culture on blood agar or any other nutritional agar is the gold standard for diagnosis. It grows as cream-colored white colonies with zoospores formation, further confirmed using the leaf incarnation method. Due to limited laboratory diagnostic modalities and delayed growth on culture, there was a recent shift toward various molecular diagnostic modalities such as polymerase chain reaction, confocal microscopy, ELISA, and immunodiffusion. As corneal scraping (10% KOH, Gram) reveals fungal hyphae, antifungals are started before the culture results are available. Recent in vitro molecular studies have suggested antibacterials as the first-line drugs in the form of 0.2% linezolid and 1% azithromycin. Early therapeutic keratoplasty is warranted in nonresolving cases. This review aims to describe the epidemiology, clinical features, laboratory and molecular diagnosis, and treatment of Pythium insidiosum keratitis.

Topics: Agar; Animals; Corneal Ulcer; Humans; Keratitis; Pythiosis; Pythium

The purpose of this study was to report the first case of keratitis caused by Cladorrhinum samala and review of the literature.. This was a case report and literature review.. A 35-year-old immunocompetent man presented with pain, redness, and watering in the right eye 7 days after trauma with some foreign body. He was diagnosed with infectious keratitis, and a thorough microbiological workup was performed. Corneal scrapings were subjected to a potassium hydroxide (KOH) examination, Gram staining, bacterial (blood agar and Robertson cooked meat broth), and fungal culture (Sabouraud dextrose agar and brain-heart infusion agar). The KOH mount revealed septate fungal hyphae with irregular margins. Yellow-white nonsporulating mycelial growth was noted on the Sabouraud dextrose agar, which was identified as C. samala by sequencing. The patient responded to 5% natamycin and 1% voriconazole eye drops, and there was a formation of a corneal opacity in a period of 3 weeks.. We report the first case of keratitis by C. samala, highlighting the emergence of a rare dematiaceous fungi causing keratitis and the role of molecular modalities in the diagnosis of nonsporulating fungi in suspected cases.

Topics: Adult; Agar; Corneal Ulcer; Culture Media; Eye Infections, Fungal; Glucose; Humans; Keratitis; Male; Natamycin; Ophthalmic Solutions; Sordariales; Voriconazole

Topics: Acrylamides; Agar; Chlorobutanol; Critical Care; Critical Illness; Drug Combinations; Humans; Keratitis; Lanolin; Mineral Oil; Petrolatum; Prospective Studies; Severity of Illness Index

Tolerance to antibiotics may occur due to changes in bacterial growth patterns and can be a precursor to development of resistance. However, there is a lack of information on the ability of ocular bacteria isolates to develop tolerance. This paper explores the tolerance to 8 different antibiotics of 61 microbial keratitis isolates of Pseudomonas aeruginosa from Australia and India using the MBC/MIC ratio, with tolerance defined by a ratio ≥ 32, and tolerance to ciprofloxacin by an agar diffusion assay.. Antibiotics used were ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. Isolates were sourced from microbial keratitis infections in Australia and India. Minimum bactericidal and minimum inhibitory concentration (MBC and MIC) were obtained using broth microdilution and compared to breakpoints from the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) to determine bacterial susceptibility. Tolerance was assessed as MBC/MIC ≥ 32. An alternative method for tolerance detection (TD) was assessed with 13P. aeruginosa sensitive isolates by agar disk diffusion assay of ciprofloxacin followed by application of glucose to the agar and observation of re-growth of colonies.. Thirty-three isolates were resistant to imipenem, 20 to ciprofloxacin, 14 to tobramycin and piperacillin, 12 to levofloxacin and ceftazidime, 8 to gentamicin, and 5 to polymyxin B. The percentage of strains resistant to levofloxacin (7 vs 30 %; p = 0.023), gentamicin (0 vs 24 %; p = 0.005) and tobramycin (4 vs 33 %; p = 0.004) was significantly greater in isolates from India.On average, strains from India exhibited notably greater MIC and MBC values compared to strains obtained from Australia. Out of 61 isolates, none displayed an MBC/MIC ratio ≥ 32. However, three sensitive isolates had low tolerance, nine had medium tolerance and one had high tolerance to ciprofloxacin with the TDtest.. This study used two methods to determine whether P. aeruginosa strains could show tolerance to antibiotics. Using the MBC/MIC criteria no strain was considered tolerant to any of the eight antibiotics used. When 13 strains were tested for tolerance against ciprofloxacin, the most commonly used monotherapy for keratitis, one had high tolerance and nine had medium tolerance. This demonstrates the capacity of P. aeruginosa to develop tolerance which may result in therapeutic failures if inappropriate dosing regimens are used to treat keratitis.

Topics: Agar; Anti-Bacterial Agents; Ceftazidime; Ciprofloxacin; Gentamicins; Humans; Imipenem; Keratitis; Levofloxacin; Microbial Sensitivity Tests; Piperacillin; Polymyxin B; Pseudomonas aeruginosa; Pseudomonas Infections; Tobramycin

Fungal keratitis is a medical emergency that is among the most common causes of blindness in developing countries. The type of the agent may vary depending on the geographical conditions under which the patient lives, trauma exposure, the use of contact lenses and profession. Curvularia spp. is a saprophytic genus that rarely causes systemic disease in humans and has 250 species identified to date. They proliferate in soil and plants and spread to the environment with their spores and the formation of blackish and fluffy colonies is its most well-known morphological feature. There may be difficulties in cultivating brown (dematiaceous) fungi. Due to the similarity between the genera, conventional methods remain inadequate for diagnosis. In this report, a case of fungal keratitis associated with C.lunata was presented. Seventy-five years-old female patient admitted to the hospital with the symptoms of stinging pain, blurred vision, and swelling in the right eye. Her symptoms had begun four days ago after her eye was hit by a plant. The patient who had a history of peripheral neuropathy due to diabetes mellitus (DM) was hospitalized with a preliminary diagnosis of keratitis, and in the cultures of the patient's corneal scraping samples, the filamentous, black pigment-forming colonies of the pathogen growing on 5% sheep blood agar and potato dextrose agar showing an aerial hyphal structure, were stained with lactophenol cotton blue and examined under the microscope. The microscopic examination revealed geniculate conidiophores with brown pigmentation. On top of these structures were tetralocular macroconidia, one of which appeared to be larger than the main axis. The fungus was subjected to molecular identification with the prediagnosis of Curvularia/Bipolaris. DNA extraction of the ITS region polymerase chain reaction amplification and Sanger sequencing were performed for molecular identification. Sanger sequencing identified the agent to be Curvularia lunata with a similarity rate of 99.79% (NCBI-GenBank Nucleotide ID: OR365075). In vitro antifungal susceptibility of C.lunata was evaluated by microdilution method. Itraconazole and amphotericin B showed higher activity against C.lunata compared to other antifungals while fluconazole was the least active antifungal. Intrastromal and subconjunctival voriconazole injection was applied to the patient who was unresponsive to empirically initiated oral moxifloxacin and different topical treatments (vancomycin, ce

Topics: Agar; Aged; Antifungal Agents; Curvularia; Eye Infections, Fungal; Female; Humans; Keratitis

To report a new entity called "toxic non-inflammatory fungal keratitis.". Eyes manifesting infective keratitis with a history of prior administration of topical steroids were included in the study. The details pertaining to the type of injury, duration of injury, and primary treatment for corneal trauma were meticulously documented. The corneal tissues were scraped from the patients and were analyzed for fungal filaments by using a 10% KOH mount under a compound microscope. Moreover, these scraped materials were plated on blood agar and Sabouraud dextrose agar plates.. The corneal ulcers displayed a disproportionately reduced intensity of pain and improved visual acuity. Further, 10% KOH revealed profuse fungal filaments with few inflammatory cells in all the patients. The anterior chamber cells and flare were either reduced or entirely absent. There was no evidence of lid edema and surrounding corneal edema in any of the patients. The mean healing period was 28.8 days (standard deviation (SD): 10.05). The KOH mount revealed the presence of confluent fungal hyphae with a few inflammatory cell infiltrates. The Aspergillus species and Fusarium species were found in 47% and 40% of the cases, respectively.. Toxic non-inflammatory fungal keratitis following steroid therapy needs to be considered in fungal ulcers with disproportionately less pain and good visual acuity. The fungal ulcers with altered clinical signs of classical inflammation need to be assessed for topical steroid misuse.

Topics: Agar; Cornea; Corneal Ulcer; Eye Infections, Fungal; Fungi; Humans; Keratitis; Pain; Steroids; Ulcer

To evaluate the effects of two commercial silicone hydrogel contact lenses (CLs) soaked with natamycin (NA) or fluconazole (FL) on the growth of Candida albicans in an in vitro eye model.. Three-D printed molds were used as a cast for making eye-shaped models comprising potato dextrose agar. Senofilcon A (SA) and lotrafilcon B (LB) CLs were incubated with either 2 mL of NA or FL at a concentration of 1 mg/mL for 24 hr. To simulate a fungal infection, the eye models were coated with C. albicans. The drug-soaked lenses were placed on top of the eye models. Seven experimental conditions were examined: (1) NA-SA, (2) NA-LB, (3) FL-SA, (4) FL-LB, (5) SA, (6) LB, and (7) control-no lens. At specified time points (t=1, 8, 16, 24, 48 hr), the agar eyes from each experimental condition were removed from the incubator and photographed. The yeast cells from the 24 and 48 hr time point were also analyzed using light microscopy.. At 24 and 48 hr, there was considerable growth observed for all conditions except for the NA-SA and NA-LB conditions. When observed under the microscope at 24 and 48 hr, the morphology of the yeast cells in the FL-SA and SA condition were similar to that of the control (oval shaped). There was limited hyphae growth observed for LB and significant visible hyphae growth for the NA-LB group. For NA-SA, NA-LB, and FL-LB groups, the cells were significantly smaller compared with the control.. For NA-SA and NA-LB, there was limited growth of C. albicans observed on the eye models even after 48 hr. Under the microscope, the cell morphology differ noticeably between each testing condition, and is dependent on drug-lens combinations.

Topics: Agar; Antifungal Agents; Candida albicans; Candidiasis; Contact Lenses, Hydrophilic; Drug Delivery Systems; Eye Infections, Fungal; Fluconazole; Humans; Hydrogels; Keratitis; Models, Biological; Natamycin; Silicone Elastomers

Topics: Agar; Bacteriological Techniques; Colony Count, Microbial; Corynebacterium; Culture Media; Eye Infections, Bacterial; False Positive Reactions; Humans; Keratitis; Predictive Value of Tests; Prospective Studies; Reproducibility of Results; Sensitivity and Specificity; Staphylococcus; Streptococcus

To compare the blood agar (BA), sabouraud dextrose agar (SDA) and chocolate agar (CA) for the isolation of fungi in patients with mycotic keratitis. Corneal Scrapings of 229 patients with clinically diagnosed microbial keratitis were inoculated on BA, SDA, CA. The culture media were evaluated for the rate and time taken for the fungal growth. Seventy six of 229 patients had fungal keratitis. Fungus grew on BA in 60/76(78.9 %), on SDA in 76/76 (100 %), on CA in 40/76(52.6 %) patients. The fungi which grew on BA (60/76) also grown on SDA at the same time. The colony morphologies of different fungi were better on SDA than BA/CA. Among the different culture media, SDA is essential for the isolation fungi in patients with mycotic keratitis.

Topics: Agar; Blood; Cacao; Culture Media; Eye Infections, Fungal; Fungi; Glucose; Humans; Keratitis

Slime is a major determinant of Staphylococcus epidermidis adherence. The established methods of laboratory detection of slime production by this organism i.e., Christensen's tube method and congo red agar plate method, can both yield inconclusive and/or intermediate results. We, therefore tried to find out electronmicroscopically the localization of slime in relation to the bacterial cell wall and look for the effect, if any of the slime location on the staphylococcal adherence as well as on the quantum of slime production.. A total of 132 coagulase negative staphylococci from cases of infectious keratitis were identified as S. epidermidis following the recommended protocol. Slime was detected both by Christensen's tube method and congo red agar plate method. Antibiotic sensitivity testing was performed by standardized disc diffusion method. Adherence of the organisms to artificial surfaces was determined by a quantitative method and transmission electron microscopy was carried out by the conventional techniques.. Of the total 132 isolates, 57 (43.2%) were slime positive and 75 (56.8%) were slime negative. Twenty seven (47.4%) of the 57 slime producing organisms were multi drug resistant as compared to only 12 (16%) of 75 nonslime-producing organisms (P<0.001). A majority i.e., 45 (78.9%) of 57 adherent organisms were slime producers as against 12 (16%) of 75 nonadherent organisms. Electron microscopic study revealed a thick viscid layer of slime anchoring to the bacterial cell wall, especially in adherent organisms and those yielding positive slime test. Some of the organisms showed loose nonadherent slime and those were mostly nonadherent to artificial surfaces.. Slime and multi drug resistance were the important virulence factors of S. epidermidis in bacterial keratitis. It was the adherent slime (i.e., slime in intimate association with the bacterial cell wall as shown by electron microscopy) only, which was responsible for resistance to multiple antibiotics and for the adhesion phenomenon observed in the quantitative slime test.

Topics: Agar; Animals; Anti-Bacterial Agents; Bacterial Adhesion; Cell Wall; Congo Red; Humans; Keratitis; Microbial Sensitivity Tests; Microscopy, Electron, Transmission; Staphylococcus epidermidis; Virulence Factors

To elucidate whether BACTEC Peds Plus F broth, usually used for culturing body fluids in paediatric departments, can be used for corneal cultures from cases with clinically suspected infectious keratitis, and to compare yields between this method and traditional methods (blood agar, chocolate agar, a fungal media, and swab transport media).. All cases with newly diagnosed, nonviral, clinically suspected infectious keratitis with no prior antibiotic therapy, were cultured both in the BACTEC Peds Plus F broth and the traditional method. McNemar's test was used for pairwise comparisons of the rates of positive growth between the two groups.. In total, 30 eyes were included in this study. The growth rates for the traditional method and the BACTEC broth were similar (50.0 and 53.33%, respectively, P=1.0). The overall growth rate for the two methods combined was 73.33%, which is 45.29% higher than the reported yield in the literature (average of 50.47%).. Our results show that BACTEC Peds Plus F broth can be used successfully in the work-up of clinically suspected infectious keratitis. The method has, apparently, several advantages over the 'Traditional method:' time-savings, as only one medium needs to be inoculated, transportation to the laboratory is simpler as there is no need for immediate incubation, and there is no need to keep and maintain a supply of fresh agar media. This method is especially suitable for office settings and remote clinics, but also can be used in hospital setting, as an adjunct, when available, to increase the growth yield.

Topics: Agar; Bacteria; Bacteriological Techniques; Culture Media; Eye Infections, Bacterial; Eye Infections, Fungal; Female; Humans; Keratitis; Male; Mycology

To compare the microbiologic yield of cultures obtained by direct inoculation of blood agar plates (BAP) from corneal ulcer swabbings versus indirect inoculation via transport media in a rabbit model of Streptococcus pneumoniae bacterial keratitis.. The corneas of 12 rabbits were inoculated with S. pneumoniae. Keratitis was confirmed 18 hours later. Sampling was performed at four 2.5-hour intervals. At each interval, corneal swabs were directly applied to BAP and placed into transport medium: thioglycollate and Amies medium without charcoal. Swabbings were then subcultured onto BAP at two time points: 2 and 24 hours after collection in transport medium. Plates were evaluated 48 hours later. Organism recovery rates were measured in terms of the number of positive culture plates observed and the bacterial colony counts on each plate.. The rate of positive cultures overall was 69%. The recovery rates were similar for direct inoculation, inoculation via Amies held for 2 hours, and inoculation via Amies held for 24 hours. Direct inoculation yielded fewer colonies than indirect inoculation via Amies held for 24 hours (p = 0.008). Direct inoculation yielded a higher rate of positive cultures than did thioglycollate held for 2 hours (p = 0.004) or 24 hours (p < 0.001). The rate of nonpneumococcal contaminants ranged from 6% of BAP subcultured from thioglycollate held for 24 hours to 28% of directly inoculated BAP.. Amies medium without charcoal may be used as a transport medium for up to 24 hours in the recovery of S. pneumoniae from corneal ulcers in this rabbit model. Thioglycollate appears to be less effective as a transport medium. Results of this study may justify studies of other transport media and/or other corneal pathogens. Altogether, such studies may provide justification for human clinical trials.

Topics: Agar; Animals; Bacteriological Techniques; Culture Media; Disease Models, Animal; Eye Infections, Bacterial; Keratitis; Rabbits; Specimen Handling; Streptococcal Infections; Streptococcus pneumoniae

Annellated conidiogenous cells of both the mononematous (Scedosporium state) and synnematous (Graphium state) conidiophores develop in the conidial state of Petriellidium (= Allescheria) boydii, Scedosporium (= Monosporium) apiospermum. Conidiogenesis in this species is characterized by percurrent proliferation of the conidiogenous cells and the conidia are holoblastic and annellidic.

Topics: Adult; Agar; Ascomycota; Cell Wall; Humans; Keratitis; Lung Diseases, Fungal; Male; Mycoses; Spores, Fungal