1/22/2017. Indication to Culture. Indication to Culture. Indication to Culture

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1 Fusarium Scar Christine W. Sindt OD FAAO Clinical Professor Tressa Larson OD FAAO Assistant Professor HSV Neurotrophic/Lagophthalmos Indication to Culture Syphilitic Interstitial keratitis Serratia Severe or sight threatening Involving the visual axis (within 3 mm) Affecting > 25% of the cornea or scleral extension > 50% corneal thinning Hypopyon Gentamicin Toxicity Acanthamoeba Indication to Culture Indication to Culture History Suggesting of Unusual Pathogen Trauma with vegetable matter Immune supression Ocular surface disease Use of certain contact lens solutions Findings of Unusual Pathogen Raised, gray ulcer Satellite or multiple lesions Feather edge Keratoneuritis Failure to respond to initial therapy 1

2 Culture if: Culture Results 1) > 1+ cells 2) > 2mm in diameter 3) Within 3mm of visual axis Factors with NO correlation to positive culture size depth stromal edema sex antibiotic at presentation Factors with correlation to positive culture age (more with younger patients) anterior chamber cells Do CL Wearers Need Culture? CL WEARERS Contact lens wear as only risk factor NO 0/75 patients treated empirically in general clinic required medication adjustment VS 10/82 in corneal referral clinic General patients had smaller, peripheral ulcers, shorter duration of symptoms and minimal risk factor except contact lenses Annual incidence of contact lens associated microbial keratitis in the US 0.04% DW SCL 0.21% EW SCL Positively correlated with the number of consecutive days of lens wear Martins etal CLAO 28(3): ewscl wearers with presumed microbial keratitis 29% bandage lenses / 71% cosmetic lenses Concordance between corneal and contact lens / case cultures Fungal 100% Amoebic 80% Bacterial 75% (Pseudomonas most common) Rodman, et al. Ophthalmology 104: , 1997 Inform the patient about the procedure s risks and benefits, and obtain proper consent before proceeding. Instill topical anesthetic. Proparacaine is preferred over other anesthetics, as it is less bactericidal.4 A nonpreserved anesthetic should provide the best results.3 Use a sterile spatula, spud or swab. Cotton or wood swabs are not recommended as natural fibers have bacteriocidal properties. Most swabs are made of Rayon or Dacyron. 2

3 Scrape the ulcer at its base and at the leading edge of the infiltrate- greatest microbial yield Use enough pressure to indent the cornea slightly. Deep scrape into ulcer if suspicious for fungus Avoid base and apply less pressure if there is thinning, so as to decrease the likelihood of a perforation. obtaining only purulent material, as it is unlikely to yield a positive result. Short/Easy Version: If you do not keep plates on hand/do not culture frequently Open an account with a local lab that does culturing such as Quest Diagnostics or Lab Corp. When patient needs to be cultured go to lab and pick up Amie s (aerobic) gel or thioglycollate broth collection tube. Can also obtain plates etc from the lab to plate yourself per upcoming directions Use synthetic fiber swab or corneal spatula to innoculate collection tube. Return to lab for culturing and incubation. Larger labs have online results. Transfer the specimen to the plate, spreading the material throughout the agar. If you are also preparing slides, place the specimen on the slide first and then the plate. Rescrape the ulcer for each different plate or agar, using a sterilized tool each time. A platinum spatula can be beneficial, as it can be heat-sterilized between specimens, and cools quickly. When preparing plates, avoid breaking the surface of the solid agars. It is convention that a corneal specimen be drawn on the plate in the shape of a C, however this is not necessary as long as the plate is clearly labeled as a corneal culture. The specific patterns on the plates become more important if you are also collecting cultures from the lids or conjunctiva. 3

4 able 1. Nutrient Agar Plates Media Blood agar Chocolate agar Sabouraud dextrose agar MacConkey IMA with gentamicin Thioglycollate broth Löwenstein-Jensen medium Non-nutrient agar with Escherichia coli Brain heart infusion Cooked meat broth Growth Supported Most bacteria and fungi, except Neisseria, Haemophilus, and Moraxella Haemophilus, Moraxella and Neisseria Fungi Gram negative bacteria only, differentiate lactose positive and negative, which is helpful in identifying Pseudomonas Fungi Wide range of bacteria, including anaerobic, and fungi Mycobacteria and Nocardia Acanthamoeba Streptococci, meningococci, yeast and fungi Anaerobic and fastidious bacteria Measure the defect after culturing, as there will be greater epithelial disruption following the procedure. Label the plates, vials and slides with information identifying the patient and locations cultured. Fill out the laboratory request form, including information about the site of the ulcer, what plates you are sending for testing and the tests you wish to have performed. You will also want to request sensitivities for medications, including the specific medications that the patient is using or will be starting. Immediately start the patient on empirical treatment Amie s Gel Non-nutrient inorganic buffer to limit indiscriminate overgrowth, and semi-solid gel to reduce oxygen diffusion. Sterile swab kits have a long shelf life and can be stored at ambient temperatures. use of Amies transport medium without charcoal was shown to be as good as direct patient side processing of corneal scrapings for culture of bacteria and fungi after storage for 24 h at room temperature. 4

5 Table 2. Slides for Microscopy Stains Slides Organism Identified Glass Slides Gram stain Bacteria, fungi and Microsporidia Giemsa stain Calcofluor white Fungi, Acanthamoeba and Microsporidia Bacteria, fungi, Microsporidia and Acanthamoeba Gram and fungal stains Sharpie marker to demarcate the area of smear Acid-fast stain Mycobacterium and Nocardia Grocott-Gömöri mehenamine-silver Fungi, Acanthamoeba and Microsporidia Periodic acid-schiff (PAS) Fungi and Acanthamoeba Red Blood Agar Aerobic bacteria: Staph, Strep, Pseudomonas Fungi associated with eye infections are usually fast-growing saprophytic fungi that can grow on media, such as blood and chocolate agar, traditionally meant for bacteria. bib1717 Chocolate Agar Variant of the blood agar plate, containing red blood cells that have been lysed by slowly heating to 80 C. Lysing the RBC is important for fastidious bacteria. They need access to the nutrients found inside the cells. Heat also inactivates some of the enzymes that break nutrients down. Hemophilus and Neisseria Thioglycate media Anaerobic bacteria Most frequently used broth in diagnostic bacteriology. Supports growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms. Contains many nutrient factors: casein, yeast, and beef extracts, and vitamins to enhance the growth of most medically important bacteria. Other nutrient supplements includes; oxidationreduction indictor (resazurin), dextrose, vitamin K1, and hemin. Also contains 0.075% agar to prevent convection currents from carrying atmospheric oxygen throughout the broth. Thioglycollate Broth Growth Characteristics of various bacteria in tholgycollate broth A. Gram-negative, facultatively anaerobic bacilli (i.e., those that can grow in the presence or absence of oxygen) generally produce diffuse, even growth throughout the broth. B. Gram positive cocci frequently grow as discrete puffballs. C. Strict aerobic bacteria (i.e., require oxygen for growth), such as Pseudomonas spp., tend to grow toward the surface of the broth, D. Strictly anaerobic bacteria (i.e., those that cannot grow in the presence of oxygen) grow at the bottom of the broth. 5

6 picture can't be displayed. Thioglycollate Broth Primary purpose of ingredients used Cystine and casein: They supply carbon and nitrogenous compounds, Dextrose: It is added as another energy source Yeast extract or papaic digest of soybean meal are added as growth enhancers. Sodium chloride: It maintains osmotic equilibrium. Sodium Thioglycollate: Sodium Thioglycollate is a reducing agent which maintains a low oxygen tension by removing molecular oxygen from the environment i.e., it creates anaerobic conditions when it reduces molecular oxygen to water. Peroxides, which may be lethal to many anaerobic organisms, are not formed under this condition. Resazurin is an oxidation-reduction indicator that turns pink when increased oxidation has occurred, it is colorless when reduced. Agar: The addition of a small amount of agar in Thioglycollate Medium aids in the initiation and growth of small inocula and anaerobes by impeding the diffusion of oxygen into the medium. It also retards the dispersion of CO2 and the reducing substance from the microenvironment surrounding the inoculum. Tryptic soy broth (TSB) Aerobic bacteria Staph, strep, corynebacteria Contains Digests of soybean meal and casein- amino acids and other nitrogenous substances as nutrients for bacteria Sodium chloride- maintain the osmotic equilibrium. Dextrose- energy source. Dipotassium phosphate- buffer to maintain the ph. Slide tubes Potato dextran Fungal culture Lowenstein-Jensen medium OR Middlebrook 7H10 Mycobacterium tuberculosis Viral Cultures Hank's balanced salt solution or 2 sucrose phosphate broth may be used. (ie. Copan Diagnostics s UTM-RT) Viral molecular diagnostic methods for demonstration of viral DNA in clinical samples have taken over the virus isolation. Molecular methods are ideal for viral diagnosis as virus isolation is time consuming, technically demanding and requires special and expensive virology laboratory set up. References Fairbanks A, Provencher L, Greiner M, Sindt C. A Guide to the Corneal Culture. EyeRounds.org. posted September 26, 2016; Available from: Mandell, Douglas, and Bennett (2015). Principles and Practice of Infectious Diseases. Elsevier. Philadelphia, PA. John E. Bennett, Raphael Dolin, Martin J. BlaserMcLeod SD, Kumar A, Cevallos V, Srinivasan M, Whitcher JP, (2005). Reliability of transport medium in the laboratory evaluation of corneal ulcers. Am J Ophthalmol Dec;140(6): Rijal N (2016) Thioglycollate broth: Principle, Composition, Preparation and Uses. Epub Oct 6, Sharma S (2012). Diagnosis of infectious diseases of the eye. Eye (2012) 26, ; doi: /eye ; Epub 18 Nov