Limitations of the Procedure

Numerous methodological factors can influence the results of any antiviral susceptibility assay (Drew and Matthews, 1989) including the DNA-DNA hybridization procedure. These include:

1. the strain of virus examined ("wild" versus "laboratory adapted"). Continuous subpassage of clinical isolates of HSV should be avoided prior to performing susceptibility testing so the isolate tested is representative of the wild-type virus initially identified in a clinical specimen.

2. heterogeneity of the virus population. A given isolate of HSV may be a heterogeneous population containing both susceptible and resistant viruses, which may make in vitro results difficult to interpret (Sacks et al., 1989). Also, different body sites from a patient may yield different strains of the same HSV type with dramatically different susceptibility patterns.

3. size of the virus inoculum.

4. type of host-cell system employed, the age and confluency of the cells when used in the assay, and the cell passage level.

5. range of drug concentrations employed.

6. incubation time for virus adsorption and length of incubation of infected cells with drug prior to determination of the ID50 value.

7. the use of ID50 or ID^ drug end points. A consensus has not been reached concerning which value is most appropriate.

8. lack of assay standardization between laboratories, making it difficult to compare results. All assays require the ability of the virus to replicate in vitro in a host cell system, but each laboratory may use different parameters of viral replication (e.g., CPE, plaque formation, virus yield, viral antigens, viral nucleic acid, viral enzyme activity, or cell transformation) to measure the activity of an antiviral agent. Comparisons of in vitro susceptibility data among different laboratories are valid only if the same assay, virus inoculum, and cell type are used. The definition of a sensitive or resistant isolate may differ for each assay system and for different laboratories performin the same assay. When comparing the activity of different antiviral agents against a virus isolate, the same assay system should also be used.

9. absence of well-characterized, drug-resistant, and -sensitive control strains. These strains are not widely available, but may be obtained as gifts from fellow investigators. Currently, they cannot be purchased through commercial sources.

Certain clinical information should be considered when interpreting the results of in vitro antiviral susceptibility assays.

1. Antiviral susceptibility testing appears to be indicated for patients in whom resistance tends to appear the most. These patients normally include immunocompromised individuals, especially transplant recipients and AIDS patients previously treated with anti-HSV agents (Table 3). One report suggests recurrent acyclovir-resistant HSV infection in an immunocompetent individual (Kost et al., 1993) which correlated with clinical failure of antiviral therapy.

2. Clinical situations that favor the development of resistance include long-term suppressive therapy, recurrent intermittent therapy, and the use of less than optimum doses of the antiviral agent.

3. Resistance of HSV isolates to acyclovir is becoming more commonplace, occuring at rates of 2-14% in the immunocompromised host (Wade et al. 1983; Englund et al., 1990). In these patients, resistant HSV can be associated with severe progressive disease that does not respond to antiviral treatement (Bean et al., 1987; Norris et al., 1988; Erlich et al., 1989a; Sacks et al., 1989; Gateley et al., 1990; Ljungman et al., 1990).

4. Data suggest that in vitro resistance may predict treatment failure and lead to the institution of alternative antiviral therapy (Youle et al., 1988; Chatis et al., 1989; Erlich et al., 1989a,b; Engel et al., 1990; Safrin et al., 1990).

5. Antiviral susceptibility testing may become useful in choosing between continuous high-dose acyclovir and foscarnet to treat acyclovir-resistant HSV. As more antiviral agents are developed and utilized, in vitro results may also guide the physician in the choice of the most appropriate therapy.

6. Results of in vitro susceptibility testing are not always predictive of a clinical response. HSV isolates resistant to acyclovir in vitro have responded to antiviral treatment (Straus et al., 1984; Englund et al., 1990) and patients with isolates that remain susceptible to the drug in vitro have failed therapy (Barry et al., 1985; Erlich et al., 1989a).

7. Acyclovir-resistant clinical isolates of HSV have also been obtained from individuals before therapy (Parris and Harrington, 1982) and from healthy individuals on chronic suppressive therapy (Straus et al., 1984), although no correlation was established between these isolates and the clinical outcome.

8. Acyclovir-resistant isolates may become latent and cause recurrent disease (Erlich etal., 1989b; Safrineia/., 1990). In some patients, recurrences are caused by drug-sensitive isolates after the successful treatment of infection with a resistant virus (Larder and Darby, 1984; Collins, 1988).

9. The absolute ID50 value for a single isolate of HSV may be less important than the relative change in ID50 values between paired isolates.

10. In general, HSV-2 strains exhibit higher ID50 values for acyclovir than HSV-1 strains.

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