Diagnosis of Fusarium keratitis in an animal model using the polymerase chain reaction

Abstract

Aims/background: The purpose of this study was apply the polymerase chain reaction (PCR) to develop a sensitive, specific, and rapid test to diagnose Fusarium keratitis. Fusarium is the most common cause of fungal corneal infection in some parts of the world. It is often difficult to establish that a keratitis is due to fungal infection.

Methods: Fusarium solani keratitis was induced in three eyes of three rabbits by injection of a suspension of the fungus into the anterior corneal stroma. In one rabbit the contralateral eye served as a control. From four to 28 days after inoculation, the corneas were scraped for culture, then scraped and swabbed for PCR analysis. The PCR was performed with primers directed against a portion of the Fusarium cutinase gene, and the presence or absence of this amplified target sequence was determined by agarose gel.

Results: The amplified DNA sequence was detected in 25 of 28 samples from the corneas infected with Fusarium, for a sensitivity of 89%. Only three of the 14 samples from these eyes with Fusarium keratitis were positive by culture, for a sensitivity of 21%. Seven of eight control samples were negative by the PCR based test, for a specificity of 88%.

Conclusion: This PCR based test holds promise of being an effective method of diagnosing Fusarium keratitis as well as Fusarium infections at other sites.

Figure 1

A rabbit cornea 5 days after inoculation with Fusarium solani, showing a focal infiltrate and diffuse corneal oedema.

Figure 2

A rabbit cornea 12 days after inoculation with Fusarium solani, showing vascularisation of the infiltrate.

Figure 3

A rabbit cornea 4 days after inoculation with Candida albicans, showing a focal infiltrate and diffuse corneal oedema.

Figure 4

Agarose gel showing PCR results of samples collected by scraping and swabbing Fusarium infected and control corneas. Many of the positive results from the Fusarium infected eyes consist of only faint bands of the target fragment of the cutinase gene, which are lost on reproduction of the photograph. However, the faint bands were confirmed by Southern blot analysis. As expected, the PCR products from the control eye contained no detectable target DNA. A separate negative control was from the PCR performed at the same time with sterile water substituted for the sample. The positive control was PCR product from purified DNA extracted from fresh Fusarium mycelium.

Figure 5

Agarose gel of restriction endonuclease digests of the PCR product. Fragments of the expected sizes were produced (Sty1: 122 base pair (bp) and 67 bp; BsaJ1: 87 bp, 67 bp, and 35 bp; EcoO109I: 174 bp and 15 bp), confirming that the product was the target fragment of the Fusarium cutinase gene.

Figure 6

Agarose gel of PCR products from a serial dilution of Fusarium solani spores, demonstrating that this PCR based test can detect from 10 to 10 000 organisms in a sample. Positive and negative controls are as described in Figure 4.