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Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility
Robert T Sarisky1, Paul Crosson, Rachel Cano
1Department of Host Defense, The Antimicrobial and Host Defense Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, UP 1450, Collegeville, PA 19426-0989, USA. robert_t_sarisky@gsk.com
Background:
A number of in vitro assays are used to determine susceptibility of HSV to antiviral agents, but results from these in vitro assays do not necessarily correlate with treatment outcome.
Objectives:
A method with improved capability for identifying an isolate as acyclovir (ACV) or penciclovir (PCV) resistant when resistance is borderline could greatly improve the management of HSV disease.
Study Design:
A comparative evaluation of four in vitro assays, plaque reduction (PRA), DNA hybridization, plating efficiency (PEA) and plaque autoradiography (PAR) was performed to accurately identify and measure resistance of a TK-altered clinical HSV isolate (HSV-1 N4) from a patient who was non-responsive to ACV treatment. Two established criteria for the prediction of antiviral resistance, IC(50)> or =2.0 microg/ml or an IC(50) greater than 10x above a sensitive virus IC(50), as well as testing in human (MRC-5) and nonhuman (Vero and CV-1 monkey kidney) cell lines were evaluated.
Results:
The PRA and DNA hybridization assays accurately identified HSV-1 N4 as ACV(r) in human cells when using the 10x above sensitive virus IC(50) resistance criterion. Moreover, the PEA and PAR assays failed to classify HSV-1 N4 as drug resistant and indicate that these technologies alone are inadequate for identifying resistant virus.
Conclusions:
The data presented herein indicate that the PRA and DNA hybridization assays most accurately identified an otherwise borderline-resistant isolate as drug resistant: (i) when a sensitive virus is used within each individual assay as a control, (ii) when ACV and PCV susceptibility is evaluated in human cells, and (iii) when the 10x above sensitive IC(50) criterion is used to classify a virus as drug-resistant. Testing of additional clinical samples is warranted to further confirm these findings.