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One of the most prominent successes in the use of chemotherapy for virus infections is the discovery of an antiherpetic agent, acyclovir (ACV). However, the appearance of ACV-resistant mutants of herpes simplex virus (HSV) has been reported after frequent use or prolonged treatment with ACV (3, 4). The increasing number of effective antiviral therapies for the treatment of several virus infections and the emergence of drug-resistant virus strains underscore the need for rapid methods for evaluating virus susceptibilities to these agents.

In this study, we evaluated the utility of two rapid screening tests developed in our laboratory, Rapid CIA and Rapid DUA, which estimate the susceptibility of an HSV strain to ACV within 3 days after the virus has been isolated.

Thirty clinical isolates from HSV-infected patients (19 with HSV-1 and 11 with HSV-2) were used in the susceptibility studies. Specimens were processed on the day of collection and inoculated into MRC-5 cells for conventional tube culture and shell vial cell culture by standard techniques. When >95% of the cells showed a cytopathic effect (CPE), the supernatant was aspirated and centrifuged to clarity, and the strains of virus were used for the determinations of drug sensitivity. Control strains FP16 and 8 WT (ACV-susceptible) and 8 FCV (ACV-resistant) were kindly provided by "Servicio de Microbiología Diagnóstica" (Instituto de Salud Carlos III, Madrid, Spain). An ACV-resistant strain (FP16ACV) was obtained in our laboratory by sequential passage of strain FP16 seven times in Vero cells in the presence of 10 µg of ACV per ml (6).

Rapid CIA. The rapid CPE inhibition assay (Rapid CIA) was performed as follows. MRC-5 cells, Vero cells, or BGM cells were seeded in a 96-well microtiter plate (Falcon no. 3075; Becton Dickinson, Lincoln Park, N.J.) until a confluent monolayer was obtained. Serial 10-fold dilutions of virus (ranging from 10⁰ to 10⁶) were done, and 100 µl of each dilution of virus per well was inoculated in eight wells of a plate. One row of wells was mock infected with 100 µl of culture medium per well as a cell control. The microtiter tray was centrifuged for 45 min at 700 × g at room temperature, and the medium was then removed from each well without disturbing the cells. Two hundred microliters of culture medium was added to four wells of each viral dilution, and 200 µl of culture medium containing drug (1 µg of ACV per ml for HSV-1 or 2 µg of ACV per ml for HSV-2) was added to the remaining four wells of each viral dilution (1, 2, 7). After an incubation period of 72 h at 37°C in a humidified atmosphere of 5% CO₂, the development of CPE was examined by light microscopy. Subsequently, viral titration and calculation of the 50% tissue culture infective dose (TCID₅₀) by the Kärber method were done in viral cultures both with and without ACV (3a).

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Rapid DUA. For the rapid dye uptake assay (Rapid DUA), virus, cells, and compounds were prepared as described for the Rapid CIA. After an incubation period of 72 h, 50 µl of a 0.15% solution of neutral red in 0.1 M sodium phosphate monobasic buffer (pH 6) was placed in each well. The plates were then incubated for 45 min at 37°C in a humidified atmosphere of 5% CO₂. After that, medium and residual stain were removed, and the wells were rinsed twice with phosphate-buffered saline (pH 6.5). Next, 150 µl of phosphate ethanol buffer (0.1 M sodium phosphate monobasic and 95% ethanol at 1:1 [vol/vol]; pH 4.2) was distributed into each well for elution of the dye incorporated by supposedly viable cells. The optical density of the solutions was read at 550 nm (OD₅₅₀) with an automatic spectrophotometer (Titertek Multiskan; Flow Laboratories, Inc., McLean, Va.).

Results. ACV susceptibility was determined in 30 clinical isolates of HSV (19 HSV-1 and 11 HSV-2) and 4 control strains (2 ACV susceptible and 2 ACV resistant) by Rapid CIA, Rapid DUA, and traditional DUA. In Table 1 are shown the mean ACV IC₅₀s mean percentages of protection at a virus concentration of 100 DU₅₀s, and mean titer ratios of the 30 strains isolated from clinical samples according to whether they were resistant to ACV or not. The ACV-susceptible control strains (8 WT and FP16) with IC₅₀s of <1 µg/ml corresponded to titer ratios of >10² and percentages of protection at 100 DU₅₀s of >50%. The ACV-resistant control strains (8 FCV and FP16ACV) with IC₅₀s >1 µg/ml corresponded to titer ratios of <10¹, and the percentage of protection at 100 DU₅₀ was <50%. Two out of 30 clinical isolates, both HSV-1 strains, were shown to be ACV resistant by all three methods. Three HSV-1 strains showed an indeterminate result by Rapid CIA (titer ratios of between 10^1.5 and 10^1.66) and were shown to be ACV susceptible by Rapid DUA and traditional DUA, and the remaining isolates were ACV susceptible by all three methods. Furthermore, increasing IC₅₀s corresponded with diminishing of titer ratios and disminishing percentages of protection. (Table 2).

Discussion. In the present study, a good correlation was observed between the results with the Rapid CIA and those with the traditional DUA. Strains of known sensibility were tested repeatedly, and in all cases, the susceptibility results determined by both methods agreed. In our method, we consider a strain to be sensitive to ACV when the titer ratio of the virus is at least 10², this equals the reduction of 100 TCID₅₀s. We observe that all of the strains with differences of 10² for Rapid CIA corresponded to strains sensitive to ACV by the traditional method. Furthermore, strains that showed a titer ratio <10¹ were also shown to be resistant to ACV by the traditional method. According to the mathematical model, the limit of susceptibility to ACV corresponds to a titer ratio of 10^1.7. Due to the fact that the determination of CPE is accomplished in a qualitative way, we consider the results with titer ratios of between 10¹ and 10^1.9 to be indeterminate.