Acyclovir (ACV) is the most commonly used drug for herpes simplex virus (HSV) infection therapy. Prolonged antiviral therapy or prophylaxis in immunocompromised patients may promote the development of drug-resistant strains. Due to the high polymorphism in genes involved in drug resistance, phenotypic methods, although work-intensive, are still required to test drug susceptibility. Real-time cell analysis (RTCA) based methods could offer a rapid and less labor-intensive alternative for phenotypic testing of ACV resistance.
To investigate the utility of a new RTCA based assay (RTCAA) to test acyclovir susceptibility of HSV clinical isolates.
Four reference strains and 93 clinical isolates (60 HSV-1 and 33 HSV-2) were tested by RTCAA. In the presence of ACV concentrations from 2.2 to 140.8 μM, Vero cells were infected with different virus dilutions. IC values were calculated by dose-response curve (DRC) with area-under-curve (AUC) method. The reference strains and 22 clinical isolates were additionally tested by dye-uptake assay, and IC values of both methods were compared.
IC values from RTCAA and dye-uptake assays were positively correlated (Spearman’s rho = 0.897, p < 0.001) and quantitatively agreed (Bland-Altman plot). Based on a cut-off of 4 μM for HSV-1 and 13 μM for HSV-2, 87 isolates were classified as ACV-sensitive and 6 isolates as ACV-resistant. The reference strains showed the expected results of ACV susceptibility.
RTCAA agrees well with the dye-uptake assay. Compared with other phenotypic methods, RTCAA requires less manipulation, reduces the workload and the turnaround time, and appears to be an objective and reliable method to test ACV susceptibility.

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