Comparison of a DNA probe assay with the plaque reduction assay for measuring the sensitivity of herpes simplex virus and varicella-zoster virus to penciclovir and acyclovir

The commonly used antiviral drugs acyclovir (ACV) and penciclovir (PCV) possess similarly potent antiviral activities in vivo against herpes simplex virus (HSV). Assay methods for sensitivity to ACV are not necessarily transferable to PCV, even though the two drugs have similar in vivo potencies and mechanisms of action. We determined by plaque reduction assay the relative activities of ACV and PCV against five laboratory-adapted strains of HSV types 1 and 2 (including sensitive and resistant strains) in seven human cell lines and one nonhuman primate cell line. Seven characteristics were used to evaluate the cell lines. All cell lines were similar in their plating efficiencies and abilities to discriminate between sensitive and resistant HSV isolates. Vero and MRC-5 cells yielded the most discordant 50% inhibitory concentrations (IC 50 s) for the two HSV types, while Vero and WI-38 VA-13 cells yielded large differences in the IC 50 s of ACV and PCV. The limited life spans and poor plaque morphologies of the fibroblast lines were undesirable characteristics. Among the transformed cell lines producing well-defined plaques, A549 cells provided the best concordance between IC 50 s for the two HSV types and two antiherpes drugs. Comparison experiments with a yield reduction format indicated that the use of assays of this type might allow some of the cell-specific properties observed in plaque reduction assays to be avoided. Antiviral susceptibility testing of herpes simplex virus (HSV)clinical isolates is important because of the frequency of infection of immunocompromised hosts with resistant HSV strains and the increasing worldwide use of antiviral analog therapy. The National Committee for Clinical Laboratory Standards (NCCLS) has proposed a standard method (18) for in vitro determination of HSV susceptibility to two antivirals, acyclovir (ACV) and foscarnet (phosphonoformic acid [FOS]). For other prescribed antiviral drugs, such as penciclovir (PCV) and its prodrug famciclovir, the standard may need to be modified. The plaque reduction assay (PRA) provided in the NCCLS standard is the most commonly reported method for evaluations of the susceptibilities of clinical isolates to ACV and the technique to which new methods are compared (16,18,22,24,26,28,30). ACV resistance of HSV measured by PRA correlates with isolates from clinical infections unresponsive to ACV therapy; thus, the PRA is biologically relevant (25).Testing of the susceptibilities of HSV isolates to ACV has typically been performed in Vero (African green monkey kidney), MRC-5, or HEL (diploid human embryonic lung) cells. A priori, lines derived from human cells would be preferred for a standard assay, since current and future drugs are developed and optimized for activity in the human system. Vero cells do represent a continuous cell line that readily forms distinct plaques upon infection with HSV, but they are nonhuman in origin. Conversely, MRC-5 and HEL cells, which are human in origin and which have the appropriate metabolism (11, 32), represen...

Section: Antiviral Susceptibility Testing Of Herpes Simplex Virus (Hsv)mentioning

confidence: 99%

Susceptibilities of Herpes Simplex Viruses to Penciclovir and Acyclovir in Eight Cell Lines

Leary

Wittrock

Sarisky

et al. 2002

“…The latter has been shown to correlate well with PRA. Other currently used antiviral susceptibility assays involve the use of DNA hybridization (9,30,31), flow cytometric analysis (20) and transgenic HSV inducible reporter cells (32).With the increasing numbers of immunocompromised individuals, there is a need for the widespread routine availability of antiviral drug susceptibility assays, which would be rapid, reproducible and clinically relevant. Currently used methods, except for the MISE-test, suffer from certain pitfalls, which preclude their routine use.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Application of Real-Time PCR for Determination of Antiviral Drug Susceptibility of Herpes Simplex Virus

Stránská

Loon

Polman

et al. 2002

A quantitative real-time PCR (TaqMan) assay was developed for determination of antiviral drug susceptibility of herpes simplex virus (HSV). After short-time culture of the virus, the antiviral drug susceptibility of HSV isolates for acyclovir (ACV) was determined by measuring the reduction of the HSV type 1 (HSV-1) DNA levels in culture supernatants using real-time PCR. The 50% inhibitory concentration was reported as the concentration of antiviral drug that reduced the number of HSV-1 DNA copies by 50%. A total of 15 wellcharacterized ACV-sensitive or -resistant strains and clinical isolates were used for assay evaluation. The new assay with real-time PCR readout permitted rapid (3 days), objective, and reproducible determination of HSV-1 drug susceptibilities with no need for stringent control of initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (r ‫؍‬ 0.86) with those for the plaque reduction assay. In conclusion, the real-time PCR assay described here is a suitable quantitative method for determination of antiviral susceptibility of HSV-1, amenable for use in the routine diagnostic virology laboratory.Extensive use of acyclovir and other antiviral drugs for prophylaxis and treatment of herpes simplex virus (HSV) infections exerts a continuous selection pressure on the HSV virus population. HSV antiviral drug resistance occurs relatively frequently especially in immunocompromised patients such as those undergoing bone marrow (6 to 12%) or solid organ transplantation (ϳ4%) or AIDS patients (ϳ6%) Several phenotypic assays have been described, and some of them are used in clinical practice, with the plaque reduction assay (PRA) as the most frequently used drug susceptibility assay. Although this technique is laborious and time-consuming, it still remains the "gold standard" method by which other tests are evaluated (24). The majority of alternative susceptibility assays is based on reduction in cytopathic effect (CPE), which is either microscopically evaluated or colorimetrically detected (7,13,15,19,23,29). Assays based on enzyme-linked immunosorbent assay (ELISA) include the sandwich ELISA (33) and the microplate in-situ ELISA (MISE-test) (16,21,25). The latter has been shown to correlate well with PRA. Other currently used antiviral susceptibility assays involve the use of DNA hybridization (9,30,31), flow cytometric analysis (20) and transgenic HSV inducible reporter cells (32).With the increasing numbers of immunocompromised individuals, there is a need for the widespread routine availability of antiviral drug susceptibility assays, which would be rapid, reproducible and clinically relevant. Currently used methods, except for the MISE-test, suffer from certain pitfalls, which preclude their routine use. Most of the assays are time-consuming and labor-intensive; some may have subjective endpoints, require special equipment or trained laboratory personnel. Therefore we set out to develop an assay, which would overcome most of the aforementioned restrictio...

“…Rapid assays for the detection of resistant VZV or HSV have been reported (1,11,13,38,41,44), and all these assays depend on cell culture methods to obtain the initial virus sample for analysis. This may be limiting and time-consuming, in particular, for VZV.…”

Section: Discussionmentioning

confidence: 99%

A Rapid Phenotypic Assay for Detection of Acyclovir-Resistant Varicella-Zoster Virus with Mutations in the Thymidine Kinase Open Reading Frame

Sahli¹,

Andrei

Estrade³

et al. 2000

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl ␤-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.