PURPOSE: To evaluate the contribution of molecular methods for the diagnosis of an acyclovir-resistant HSV-1 bilateral keratitis in an AIDS patient and to report a new point mutation in the nucleotide sequence of the thymidine kinase (tk) gene involved. METHODS: A 31 year old HIV-positive female presented with severe, active, bilateral and sight-threatening keratitis of 6 months duration, which was treated unsuccessfully with acyclovir. After corneal biopsy, samples were analysed by standard virological procedures, in situ hybridization, and PCR. The tk gene was cloned and subsequently sequenced. RESULTS: Conventional virological methods remained inconclusive. However, in situ hybridization and PCR rapidly confirmed the diagnosis of HSV-1 keratitis. The tk gene sequence revealed the presence of five variations previously described in two reference strains, but also a new point mutation at nucleotide position 431 which leads to an amino-acid change at position 144 that supported the hypothesis of a putatively altered functional form of the enzyme. Intravenous foscarnet treatment in an induction regimen was effective and cicatrization occurred within 3 weeks. CONCLUSIONS: PCR and in situ hybridization are effective and powerful techniques when other virological procedures are non-contributive, particularly in immunocompromised patients previously treated with antiviral drugs. The new point mutation identified in the tk gene may be associated with resistance to acyclovir. Copyright 2000 Academic Press.
PURPOSE: To evaluate the contribution of molecular methods for the diagnosis of an acyclovir-resistant HSV-1 bilateral keratitis in an AIDSpatient and to report a new point mutation in the nucleotide sequence of the thymidine kinase (tk) gene involved. METHODS: A 31 year old HIV-positive female presented with severe, active, bilateral and sight-threatening keratitis of 6 months duration, which was treated unsuccessfully with acyclovir. After corneal biopsy, samples were analysed by standard virological procedures, in situ hybridization, and PCR. The tk gene was cloned and subsequently sequenced. RESULTS: Conventional virological methods remained inconclusive. However, in situ hybridization and PCR rapidly confirmed the diagnosis of HSV-1 keratitis. The tk gene sequence revealed the presence of five variations previously described in two reference strains, but also a new point mutation at nucleotide position 431 which leads to an amino-acid change at position 144 that supported the hypothesis of a putatively altered functional form of the enzyme. Intravenous foscarnet treatment in an induction regimen was effective and cicatrization occurred within 3 weeks. CONCLUSIONS: PCR and in situ hybridization are effective and powerful techniques when other virological procedures are non-contributive, particularly in immunocompromised patients previously treated with antiviral drugs. The new point mutation identified in the tk gene may be associated with resistance to acyclovir. Copyright 2000 Academic Press.