Abstract
The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 μg of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.
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 10−6) 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).
When the ratio obtained between the titer in the absence and that in the presence of ACV was ≥10−2, the strain was considered ACV susceptible (ACV 50% inhibitory concentration [IC50] of <1 μg/ml for HSV-1 and ACV IC50 of <2 μg/ml for HSV-2). Ratios lower than 10−1 indicated ACV resistance (ACV IC50 of ≥1 μg/ml for HSV-1 and ACV IC50 of ≥2 μg/ml for HSV-2), and ratios between 10−1 and 10−1.9 were interpreted as indeterminate results.
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.).
The mean OD of the cell control wells was determined, and only wells showing a mean OD ± 10% were considered in further calculations. This mean OD was assigned a value of 100%. The mean OD of the virus control wells was assigned a value of 0%. The titer of virus was expressed as a 50% dye uptake value (DU50) and was calculated by determining the dilution of virus producing a 50% OD reading by linear regression analysis of the data. The percentage of protection of virus-infected cells from HSV-induced destruction due to ACV was calculated for each virus dilution by the formula
 |
 |
where (ODACV)MOCK, (ODACV)HSV, (ODNO ACV)MOCK, and (ODNO ACV)HSV indicate the mean absorbances of the mock-infected control with ACV, test sample with ACV, mock-infected control without ACV, and virus-infected control, respectively.
The percentage of protection values at each dilution of virus were used to carry out a linear regression analysis of the data, and then the susceptibility to ACV (percentage of protection) was calculated at a virus concentration of 100 DU50s.
When the percentage of protection was >50%, the HSV strain was considered susceptible to ACV (ACV IC50 of <1 μg/ml for HSV-1 and ACV IC50 of <2 μg/ml for HSV-2). When this percentage of protection was ≤50%, the strain was considered resistant (ACV IC50 of ≥1 μg/ml for HSV-1 and ACV IC50 of ≥2 μg/ml for HSV-2) (5).
The DUA was carried out according to normal procedures (5). We considered ACV resistance as an IC50 of ≥1 μg/ml for HSV-1 and ≥2 μg/ml for HSV-2, and we defined susceptibility to ACV as an IC50 of <1 μg/ml for HSV-1 and <2 μg/ml for HSV-2 (1, 2, 7).
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 >10−2 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 <10−1, 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).
TABLE 1.
Comparison of mean (± standard deviation) IC50s, titer ratios, and percentages of protection at 100 DU50s for HSV-1 and HSV-2
| IC50 range | Mean IC50 (μg/ml)a | Mean titer ratio (log10 negative)b | Mean % protection at 100 DU50sc |
|---|---|---|---|
| HSV-1 | |||
| <1 μg/ml (ACV susceptible [n = 17]) | 0.55 ± 0.23 | 2.76 ± 0.89 | 81.6 ± 12.1 |
| ≥1 μg/ml (ACV resistant [n = 2]) | 2.53 ± 0.53 | 0.33 ± 0.33 | 10.9 ± 9.9 |
| HSV-2; <2 μg/ml (ACV susceptible [n = 11]) | 1.04 ± 0.49 | 3.14 ± 0.69 | 81.8 ± 11 |
Concentration of ACV producing 50% inhibition of dye uptake (as a measure of the reduction of virus-induced cytopathic alterations) in comparison with virus (0%) and cell control (100%).
Titer in the absence versus that in the presence of ACV determined by Rapid CIA and expressed as log10 negative. When this ratio was ≥−2, the strain was considered ACV susceptible; Ratios <−1 indicated acyclovir resistance; and ratios between −1 and −1.99 were interpreted as indeterminate results.
Percentage of protection of virus-infected cells from HSV-induced destruction due to ACV at 100 DU50s determined by Rapid DUA.
TABLE 2.
Results for strains tested by the three susceptibility assays
| Strain | Type of HSV | IC50 (μg/ml)a | % Protection at 100 DU50sb | Titer ratio (log10 negative)c | ACV susceptibilityd |
|---|---|---|---|---|---|
| 94/2902 | 2 | 1 | 83.1 | 3.5 | S |
| 94/3221 | 2 | 0.54 | 81.7 | 3.5 | S |
| 94/3232 | 1 | 0.76 | 80.4 | 3.25 | S |
| 94/3676 | 2 | 1.30 | 73.5 | 4 | S |
| 94/3710 | 2 | 0.95 | 79.1 | 3 | S |
| 94/4073 | 1 | 0.84 | 90.2 | 3 | S |
| 94/4620 | 2 | 1.43 | 77.8 | 3.33 | S |
| 94/4842 | 2 | 0.86 | 99.9 | 4.25 | S |
| 94/4968 | 1 | 0.49 | 99.9 | 4.75 | S |
| 95/848 | 1 | 0.23 | 99.9 | 3.5 | S |
| 95/966 | 1 | 0.45 | 67.6 | 2 | S |
| 95/1014 | 1 | 0.66 | 60.2 | 2.33 | S |
| 95/1065 | 2 | 0.16 | 99.9 | 4 | S |
| 95/1242 | 1 | 0.34 | 88.5 | 2.5 | S |
| 95/1447 | 1 | 0.83 | 78.5 | 1.5 | S |
| 95/1616 | 1 | 0.29 | 96.9 | 2.67 | S |
| 95/2970 | 1 | 0.56 | 82.4 | 1.66 | S |
| 95/3013 | 1 | 0.42 | 91 | 3 | S |
| 95/3730 | 1 | 0.25 | 71.7 | 4 | S |
| 95/3783 | 2 | 0.44 | 92 | 3 | S |
| 95/3838 | 1 | 0.25 | 91.4 | 4 | S |
| 95/4052 | 2 | 1.46 | 62.7 | 2.33 | S |
| 95/4179 | 1 | 0.92 | 64.5 | 2 | S |
| 95/4414 | 1 | 0.65 | 73.6 | 2.75 | S |
| 95/6108 | 1 | 2 | 1 | 0 | R |
| 96/615 | 2 | 1.43 | 71.9 | 2.33 | S |
| 96/667 | 1 | 0.71 | 82.2 | 1.5 | S |
| 96/668 | 1 | 0.85 | 68.2 | 2.66 | S |
| 96/876 | 2 | 1.86 | 78.6 | 2.33 | S |
| 96/3369 | 1 | 3.07 | 20.8 | 0.67 | R |
| 8 WT | 1 | 0.25 | 99.9 | 3.83 | S |
| FP16 | 1 | 0.41 | 88.8 | 3.25 | S |
| FCV | 1 | 16 | 3.6 | 0 | R |
| F16ACV | 1 | 8 | 1 | 0 | R |
Concentration of ACV producing 50% inhibition of dye uptake (as a measure of the reduction of virus-induced cytopathic alterations) in comparison with virus (0%) and cell control (100%).
Percentage of protection of virus-infected cells from HSV-induced destruction due to ACV at 100 DU50s determined by Rapid DUA.
Titer in the absence versus that in the presence of ACV determined by Rapid CIA and expressed as log10 negative. When this ratio was ≥−2, the strain was considered ACV susceptible; ratios <−1 indicated acyclovir resistance; and ratios between −1 and −1.99 were interpreted as indeterminate results.
Determined by the traditional methods. S, ACV susceptible; R, ACV resistant.
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 10−2, this equals the reduction of 100 TCID50s. We observe that all of the strains with differences of ≥10−2 for Rapid CIA corresponded to strains sensitive to ACV by the traditional method. Furthermore, strains that showed a titer ratio <10−1 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 10−1 and 10−1.9 to be indeterminate.
There was also complete correlation between Rapid DUA and traditional methods: strains with a percentage of protection >50% corresponded to strains sensitive to ACV, and strains with a percentage of protection <50% corresponded to resistant ones. The three strains which gave an indeterminate result with Rapid CIA and were susceptible by the traditional method were also shown to be susceptible by Rapid DUA.
In summary, rapid CIA is a fast and repetitive method for the rapid determination of the susceptibility of HSV-1 and HSV-2 to ACV, employing a single dose of the drug which is related to the limits of susceptibility of the virus (1 μg of ACV per ml for HSV-1 or 2 μg of ACV per ml for HSV-2). This method, combined with the Rapid DUA method, allowed the determination of susceptibility in 100% of the HSV isolates.
These methods could be employed to determine the susceptibility of HSV to other antiviral agents and could also be used in studies of susceptibility of other cytopathic viruses (cytomegalovirus and varicella-zoster virus).
Acknowledgments
We thank the Hospital of Asturias for providing clinical samples and data and Guillermo Viejo, Ana Martinez, and Flor Hidalgo for editorial assistance.
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