24.2 Assisting with Conjunctival & Corneal Specimen Collection

When specimens are taken

Not every red eye is cultured. Indications for microbiological sampling include:

• Corneal ulcer that is central, large (commonly cited as over 1 to 2 mm), deep, sight-threatening, or associated with a hypopyon
• Any ulcer not responding to empirical treatment
• Suspected fungal or Acanthamoeba keratitis
• Hyperacute purulent conjunctivitis — suspected gonococcal
• Neonatal conjunctivitis (ophthalmia neonatorum)
• Chronic or recurrent conjunctivitis that has failed treatment
• Suspected endophthalmitis — vitreous and aqueous taps, taken by the clinician
• Infection in a corneal graft or after refractive surgery

The timing rule that determines the result: collect before starting antibiotics. Even a short course substantially reduces culture yield, and a patient already on drops should ideally stop for a period at the clinician's direction before sampling.

Preparing the setting

The technologist's role is usually to prepare and to assist, and the preparation determines whether the sample is usable.

Set out before the clinician starts:

Standard media:

Media handling: allow refrigerated plates to reach room temperature before inoculation, check expiry dates, and do not use plates that are dry, cracked or contaminated.

The collection sequence

1. Anaesthetise with preservative-free anaesthetic.
2. Conjunctival swab first if both conjunctival and corneal samples are needed and the conjunctiva is being sampled for comparison — although in practice the clinician may prefer the corneal sample first to avoid contamination.
3. Corneal scraping: the clinician scrapes at the advancing edge and the base of the ulcer, not the necrotic slough in the centre, because the slough contains dead material and few viable organisms.
4. Inoculate each plate directly at the slit lamp, in a row of C-shaped streaks — the "C" marks which part of the plate holds the specimen so that growth on the C can be distinguished from airborne contamination elsewhere on the plate.
5. Do not stab into the agar; streak on the surface.
6. Prepare slides by spreading the material thinly in a defined area, air drying and labelling.
7. Repeat the scrape with a fresh sterile instrument for each medium and each slide, to avoid carrying organisms between plates.
8. Label everything at the point of collection — patient identifiers, eye, site, date, time and the medium.
9. Transport promptly; note that plates for Acanthamoeba and fungi have different incubation requirements and must be clearly flagged.

Conjunctival swabs and scrapings

Swab technique: moisten the swab with preservative-free sterile saline (a dry swab yields less), then roll it firmly along the inferior fornix from the nasal to the temporal side. Avoid touching the lid margin and lashes, which carry commensal flora that confuse the result. Use a separate swab for each eye and label them individually — comparing the two eyes is often the point.

Conjunctival scraping (for Giemsa cytology and for Chlamydia): after anaesthesia, the clinician scrapes the palpebral conjunctiva, usually of the everted upper lid, because chlamydial inclusions are found in epithelial cells rather than in discharge. Sampling discharge alone is the classic reason a chlamydial test comes back falsely negative.

Cytology is informative:

Special situations

Contact lens wearers. Culture the lens, the case and the solution in addition to the cornea. In Acanthamoeba and Pseudomonas keratitis the case is frequently the richest source, and a positive case culture can establish the organism when the corneal scrape is negative. Ask the patient to bring the case and solution in.

Suspected fungal keratitis. A deeper scrape is needed because hyphae are in the stroma. Sabouraud agar is held for up to four weeks, so a negative report at one week means nothing. A corneal biopsy may be needed if scrapings are repeatedly negative.

Suspected Acanthamoeba. Non-nutrient agar with an E. coli overlay; confocal microscopy may show cysts non-invasively and is faster than culture.

Molecular testing. Polymerase chain reaction is increasingly used for herpes simplex, varicella zoster, adenovirus, Chlamydia, Acanthamoeba and bacterial 16S sequencing. It is faster and more sensitive but does not give antibiotic susceptibilities, so culture remains important in severe keratitis.

Handling, labelling and safety

Labelling errors are the commonest cause of a wasted specimen. Every container and slide needs the patient's full identifiers, the eye, the site sampled, the date and time, and the collector. Label at the point of collection, never afterwards from memory, and never pre-label before the sample is taken.

The request form must carry the clinical question — "suspected fungal keratitis, contact lens wearer, on moxifloxacin for four days" tells the laboratory to hold the Sabouraud plate and to interpret a negative bacterial culture appropriately. A form that says only "red eye" wastes the specimen.

Safety: treat all specimens as infectious; wear gloves; transport in a sealed, leak-proof container inside a specimen bag; never carry uncontained plates through public areas; dispose of used spatulas and blades immediately into a sharps container; and clean the slit lamp and work surface afterwards.

Documentation: record what was taken, from which eye, which media were inoculated, which slides were made, the time, who collected the sample and where it was sent.