Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR

BackgroundSusceptibility testing of herpes simplex virus type 1 (HSV-1) is traditionally performed by a plaque reduction assay (PRA), but this is labor intensive, time consuming and has a manual read out.ObjectivesThe goal of this study was to develop an internally controlled real time PCR-based phenotypical susceptibility test for HSV-1 that is suitable for use in a clinical diagnostic setting.Study designA DNA reduction assay (DRA) was developed and validated on a test panel of 26 well-characterized isolates of varying susceptibility to aciclovir or foscarnet, including low-level resistant isolates. The DRA consisted of pre-culture of a clinical sample for 48 h and subsequent culture in the presence of antivirals for 24 h. Viral DNA concentration in the culture lysates was measured by an internally controlled quantitative real-time HSV-1 PCR and corrected for cell count and lysis by beta-globin PCR. DRA results were compared to results from PRA and sequence analysis.ResultsDRA results were in accordance with PRA results for both aciclovir and foscarnet susceptibility and appeared to have good discriminative value for low-level resistance due to UL30 gene mutations. Although the direct application of DRA in clinical samples appeared not possible, short pre-culture of 48 h was sufficient and ensured results within a clinically relevant time frame of 5 days.ConclusionsDRA is an accurate, rapid and easy to perform phenotypical susceptibility test for HSV-1.