Preliminary Preparations

1. Drug Dilutions

1. The dilutions and volumes of drug needed to perform a susceptibility test are dependent on the range of drug concentrations employed and the total number of HSV isolates to be tested. Figures 3 and 4 illustrate the typical ranges of acyclovir and foscarnet, respectively, employed in the HSV hybridization assay and the configurations of the 24-well plates when one clinical HSV isolate and two control virus strains are examined.

2. Sufficient volumes of individual drug dilutions should be prepared

Sensitive Control

Sensitive Control

Patient Isolate
Figure 3 Illustration of the typical ranges of acyclovir (/ng/ml) employed in the HSV hybridization assay, and the configuration of the 24-well plates when two control virus strains (A) and one clinical HSV isolate (B) are examined.

Sensitive Control

Resistant Control

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Patient Isolate

Patient Isolate

Figure 4 Illustration of the typical ranges of foscarnet (/ng/ml)employed in the HSV hybridization assay and the configuration of the 24-well plates when two control virus strains (A) and one clinical HSV isolate (B) are examined.

in sterile EMEM containing 5% FBS and 2 m M L-glutamine (replacement medium) so 1.0 ml of each drug concentration can be added to the appropriate wells of a 24-well plate.

3. All drug dilutions should be prepared immediately before use and each dilution should be mixed thoroughly before making the next dilution.

2. Dilution of Clinical HSV Isolates and Control Strains

The optimum virus inoculum for the susceptibility assay can range from 100 to 10,000 PFU per well. This level can be achieved by growing a clinical HSV isolate or control virus strain to 50-100% CPE in a single 16 x

125-mm tissue culture tube of CV-1 cells. Virus isolates are usually harvested when approximately 75% of the monolayer is infected.

1. Scrape the infected cell monolayer from the tube surface into 2.0 ml culture medium using the tip of a sterile disposable 1.0-ml pipette.

2. Disperse the cells by gently pipetting them up and down enough times to break apart cell aggregates and produce an even suspension.

3. Dilute this cell suspension 1000-fold with EMEM containing 10% FBS and 2 m M L-glutamine. Inoculate 0.2 ml into each well of 24-well plate.

Using this dilution scheme, an inoculum containing approximately 2,000-3,000 PFU is obtained that is suitable for optimum results.

3. 24-Weil Cell Culture Plates

1. Determine the number of 24-well plates of CV-1 cells needed to perform a given susceptibility assay and order the appropriate number of test kits to be used.

2. As a general guide, for each antiviral agent employed, a single 24-well plate is needed for each clinical HSV isolate and an additional plate is required for the control strains (Figs. 3 and 4). Therefore, a total of two clinical isolates and two controls can be tested using the described format and a single 72-assay Hybriwix™ kit.

3. The kits may be purchased at a lower cost without cultured CV-1 cells, but the cells must then be maintained and propagated in the laboratory for preparation of 24-well plates.

4. The cell monolayers of the plates should be examined by microscopy before use to determine the quality of the cells. At the time of virus inoculation, the cell monolayers should be freshly confluent and not overgrown.

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