Corneal infections still keep me awake at night and I am sure I am not alone.

While most infections can be successfully treated using a topical antibiotic and close monitoring, some require culturing for pathogen identification and treatment specificity. Standard culturing techniques remain the standard of care but new techniques allow for faster identification.

Lesions located in the center of the cornea, involving the deeper stromal layers, or are large (> 2 mm) should be cultured to reduce the risk of vision loss. Risk increases with a history of contact lens over-wear or use of contaminated solutions, immunocompromised states (such as HIV or active chemotherapy), exposure to lakes, swimming pools, or hot tubs prior to infection, or vegetative material in the eye. Ulcers with feathery edges, opaque or gray color, severe anterior chamber reactions, multiple (satellite) lesions, or those associated with descemetocele should be considered highly suspicious. Culturing identifies pathogens as bacterial (Gram-positive, negative), fungal, parasitic, and viral, and aids in specific treatment.

It is common for corneal specialists to keep several supplies on hand for culturing corneal infections. Gram stains are performed for the earliest identification for pathogen type, and are typically the first response obtained from the lab when culturing. Blood agar plates are used to detect aerobic bacteria such as Staph and Strep. Chocolate agar plates are used to isolate gram-negative bacteria such as Haemophilus and Neisseria. Thioglycolate broth tube testing is used to identify anaerobic bacteria, and tryptan soy broth can be used to identify aerobic bacteria. Less commonly used are potato dextrose for fungi pathogens, Lowenstein-Jensen for mycobacteria, and pink viral media to test for Herpes Simplex virus.

While culturing remains the standard of care, there are some issues that complicate these techniques. Sampling, testing, and reporting takes time. You cannot wait for the results to initiate treatment, so many start with fortified Tobramycin, Vancomycin and/or Cephalosporins after culturing. Commonly used fortified anti-infectives are listed in Table 1. Patient requiring culturing may have already been treated using an antibiotic and may require cessation of treatment for 12-24 hours prior to culturing.

Once cultured, the patient is typically seen frequently to determine if initial treatment choice is effective. Gram stain results may be received within 24-36 hours while final pathogen identification and sensitivity results may take one week. If the patient responds and the isolate is not sensitive to current medication, a second medication determined by sensitivity testing may be required. If the patient worsens, additional medication may be added until the sensitivity testing is returned. If the lesion progresses when there are no organisms present in the culture, culturing may need to be repeated with an expanded scope, or biopsy may be required.

New molecular techniques such as Polymerase Chain Reaction (PCR) and Surface plasmon resonance (SPR) methods offer high diagnostic specificity and sensitivity. PCR was developed by the Nobel Prize winner Kary Mullis and associates in 1985. Trace amounts of DNA (or RNA) are amplified and then used to determine with a high probability the identity of the pathogen. After the DNA or RNA is amplified, it is compared to other nucleotide segments from a known source using electrophoresis. Advantages include simplified collection using a single vial, 24-48 reporting times, specimen viability up to 5 days after collection and ability to request additional testing up to 30 days after collection. Blood and mucus do not affect results, and no refrigeration of supplies is required. PCR has been found particularly advantageous in Herpetic infections, where it provides better sensitivity that diagnosis based on clinical presentation.

Panda et al investigated the utility of PCR in bacterial corneal ulcers and compared sensitivity and specificity of PCR to conventional laboratory methods in 122 eyes of presumed bacterial keratitis. Samples were collected for bacterial and fungal culture, Gram stain smear and a separate sample for PCR. Gram-stained smear revealed presence of bacteria in 23.9% of specimens and PCR positivity was evident in 45.5%. In eyes previously treated with antibiotics, culture was positive in 30%, Gram stain in 18%, and PCR in 36%. In untreated eyes, positivity of culture, as well as PCR, was noted in 52.7% while only 27.7% was found with Gram stain. Sensitivity of Gram stain was 45.28% while PCR was 88.68%. Specificity was 92.75% for Gram stain and 86.96% for PCR. The average time taken for PCR reaction was 4-8 h while culture reporting took at least 24-48 hours.

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique used to simultaneously monitor interactions of many biomolecules immobilized on a thin gold film without the use of fluorescent, enzymatic, or radioactive tags. SPR has been used to perform, real-time discrimination of S. aureus, P. aeruginosa, C. tetani and C. perfringens in mixed bacterial infections. A novel hand-held unit using this technology to identify biomarkers in corneal infection and measure osmolarity is also in the pipeline. (

My office currently employs both standard culturing methods with a swab for PCR analysis. I look forward to using advanced diagnostics testing to reduce risk of vision-threatening infections in 2018.

Table-1: Commonly Used Fortified Anti-infective Medications6, 7



Tobramycin (Gram positive/negative bacilli and pseudomonas bacterial keratitis) 14mg/ml(1.4%)
Gentamicin Eye Drops (Gram positive/negative bacilli bacterial keratitis) 14mg/ml(1.4%)
Amikacin Eye Drops (Gram-negative bacilli, mycobacteria post-corneal penetration such as after LASIK Bacterial keratitis) 2.5%
Cefazolin Eye Drops (Gram-positive cocci cacterial keratitis) 50mg/ml( 5%)
Ceftazidime eye drops (Gram-negative cocci Bacterial keratitis) 50mg/ml( 5%)
Vancomycin Eye Drops (Gram-positive bacterial keratitis) 50mg/ml(5%)
Linezolid (Bacterial keratitis) 2 mg/ml (0.2%)
Colistin (Colistimethate sodium powder) 0.19%
Imipenem–Cilastin eye drops 1%
Amphotericin B (antifungal) 0.15%
Voriconazole Eye Drops 1%


²TN Azher, X Yin, D Tajfirouz, AJW Huang, PM Stuart.  Herpes simplex keratitis: challenges in diagnosis and clinical management.   Clin Ophthalmol. 2017; 11: 185–191.

³A Panda, ST Pal, G Satpathy, M Wadhwani, M Monika.  Comparison of polymerase chain reaction and standard microbiological techniques in presumed bacterial corneal ulcers.  Int Ophthalmol. 2015 Apr;35(2):159-65.

4EA Smith, MG Erickson, AT Ulijasz, B Weisblum, RM Corn.  Surface Plasmon Resonance Imaging of Transcription Factor Proteins: Interactions of Bacterial Response Regulators with DNA Arrays on Gold Films.  Langmuir 2003, 19, 1486-1492.

5Wang, Jue & Luo, Yang & Zhang, Bo & Chen, Ming & Huang, Junfu & Zhang, Kejun & Gao, Weiyin & Fu, Weiling & Jiang, Tianlun & Liao, Pu. (2011). Rapid label-free identification of mixed bacterial infections by surface plasmon resonance. Journal of translational medicine. 9. 85. 10.1186/1479-5876-9-85.

6SG Shah, NS Gokhale.Instruction manual for preparation of fortified antimicrobial eye drops.  Mumbai Cornea Club.  Accessed 12/28/2017.

7PM Chachadi.  Fortified Antibiotics for Corneal Ulcers.  Accessed 12/28/2017.