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The aetiology of Thygeson’s superficial punctate keratitis (TSPK) remains elusive. A viral aetiology has been suggested by the absence of bacterial infection and clinical resemblance to other viral keratopathies. We report the results of polymerase chain reaction analysis for the detection of herpes simplex virus (HSV) 1 and 2, herpes zoster virus, varicella zoster virus (VZV) and adenovirus from corneal epithelial samples from patients with active signs and symptoms of TSPK.Schirmer strip impressions were taken from the epithelium of eight patients with a known history of TSPK and symptoms and signs of active disease. Three patients were recruited as positive controls (two with herpes simplex keratitis and one with herpes zoster ophthalmicus). Samples from a further three patients acted as negative controls. All 14 samples underwent polymerase chain reaction testing for HSV 1, HSV 2, VZV and adenovirus.DNA corresponding to the expected viral DNA was amplified from all three positive control samples. The three negative control samples showed no evidence of viral DNA. Similarly, all samples from patients with TSPK showed no evidence of the presence of HSV 1, HSV 2, VZV or adenovirus.We conclude that HSV, VZV and adenovirus are not present in the epithelium of patients with TSPK. These results are considered in light of existing theories regarding the aetiology and treatment of this condition.