Sir,

We present a patient who developed Infective crystalline keratopathy (ICK) after penetrating keratoplasty. Scanning electron microscopy revealed coccal microorganisms surrounded by mucopolysaccharides consistent with a biofiolm. Coccal organisms have not previously been reported in association with biofilm and ICK.

Case report

An 81-year-old Caucasian woman with chronic blepharitis underwent a left intracapsular capsular extraction in 1974 and a right extracapsular extraction with an iris clip lens in 1983. Although the surgery was uneventful she subsequently developed pseudophakic bullous keratopathy in her right eye and she underwent a penetrating keratoplasty with secondary anterior chamber intraocular lens exchange in 1993. The postoperative visual acuities were 6/60 OS and 6/60 OD due to retinal pigment epithelium change at the macula, secondary to cystoid macular oedema. The sutures were removed from the corneal graft in December 1998. She subsequently received topical dexamethasone 0.1%, once daily, in the right eye. Four months later she developed an anterior stromal, crystalline, radially branching opacity with an intact overlying epithelium within the donor cornea at the 2-o’clock position (Figure 1). The patient was uncomfortable and there was no evidence of any associated anterior chamber inflammation or hypopyon. Despite 3 months of intensive topical treatment initially with ofloxacin and then fortified penicillin, the lesion did not improve.

Figure 1
figure 1

(a) Branching crystalline appearing corneal lesion. (b) The appearance of the lesion following excision.

Full size image

In September 1999 the lesion was carefully excised. A 2-o’clock partial thickness incision section was made in the graft/host interface and then a crescent blade was used superficial and deep to the lesion so that the greater part of it was excised ’flat’ (Figure 1). We irrigated this slit-like bed thoroughly with cefuroxime. The excised corneal lesion was sent to the histopathologist for scanning electron microscopy.

The tissue was fixed and prepared for scanning electron microscopy by a procedure which involves critical point drying. Initially it was fixed with 2.5% glutaraldehyde in 0.15 M phosphate-buffered saline for 1 h at room temperature. Then it was treated with 1% osmium tetroxide for 1 h, washed with distilled water and treated with 1% uranyl acetate for 1 h and washed again with distilled water. It was then dehydrated with ethanol solutions. The sample was then dried to critical point by using a Polaron critical point drier, coated with gold. It was then reviewed under a scanning electron microscope (SEM) which showed coccal microorganisms surrounded by fine branching processes (Figure 2). These are mucopolysaccharides representing biofilm.

Figure 2
figure 2

Scanning electron microscopy showing numerous coccal microorganisms surrounded by fine branching processes. (a) Low magnification; (b) higher magnification.

Full size image

At follow-up in December 1999 the patient was asymptomatic and the lesion has entirely resolved without signs of recurrence.

Comment

Infective crystalline keratopathy (ICK) is a chronic corneal infection characterised by the development of branching, white crystalline opacities of the cornea and associated with minimum inflammation.1 The crystalline deposits are found mainly in the anterior and less commonly in the midstromal location.1

It is a slowly progressive infection with poor response to antimicrobial treatment.1 Most of the infections occur in corneal grafts patients who also receive topical steroids but may also occur in normal corneas.2 Recent suture manipulations, persistent epithelial defects, loose sutures, contact lenses and topical anaesthetic abuse have been associated with ICK.3,4 Lack of epithelial integrity is presumed to allow for intrastromal inoculation of organisms, whereas the local microenvironment of cell mediated immunodeficiency helps promote bacterial colonization.3 Streptococcus viridans is the most common cause of ICK.5 Other organisms have been reported including Pseudomonas, Haemophilus, and Candida.4,5

The clinicopathological features may be at least partially explained by the formation of a biofilm. Biofilm is difficult to stabilize for visualisation by electron microscopy and is lost during routine fixation techniques unless a specific technique is used at the time of handling the fresh specimen.1 In this case the corneal tissue was fixed and prepared for SEM by a procedure which involved critical point drying, as described above.

A biofilm is defined as an aggregation of microorganisms organised within an extensive exopolymer matrix.6 Elder JM et al were the first to describe a case of ICK due to Candida albicans and showing features of biofilm formation using transmission electron microscopy.1

Excretion of the exopolysaccharide glycocalyx polymer depends on the type of microorganism, its growth phase, the nutrient status and the temperature.6 Microorganisms within this exopolymer divide five to 15 times slower than under planktonic condition due to limited nutrients.1 Also microorganisms are better able to survive attacks by antibodies and other bacteriophages due to limited access.6 In vitro antibiotic concentration has shown that the level must be 20–1000 times greater to achieve adequate growth inhibition in a biofilm compared with the same bacteria in a planktonic state.6 Bacterial growth rate, nutrient limitation and reduced drug penetration into biofilm, all play a role in this resistance to antibiotics.7

This case report is the first reported case of ICK in which scanning electron microscopy shows coccal microorganisms surrounded by biofilm. In cases of ICK a corneal biopsy may help to identify the pathogenesis of the lesion and the nature of any organisms. The tissue should be specifically prepared for examination with a scanning electron microscope. If early aggressive therapy with broad spectrum antibiotics is unsuccessful, Yag laser or penetrating keratoplasty may be warranted.8