Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus

Swierkosz, Ella M.; Scholl, David R.; Brown, Jerry L.; Jollick, J. D.; Gleaves, Curt A.

doi:10.1128/aac.31.10.1465

Cited by 59 publications

(20 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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%

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

Stránská

Loon

Polman

et al. 2002

Antimicrob Agents Chemother

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 99%

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

Stránská

Loon

Polman

et al. 2002

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…These resistant strains are detected in vitro by phenotypic tests which determine the antiviral concentration inhibiting viral replication by 50%. Several methods have been used to evaluate the sensitivity of the HSV strains to ACV, including techniques to detect the intensity of the cytopathic effect, such as the plaque reduction (17,31) and colorimetric (11,22,26) techniques, but also the detection of DNA replication by hybridization (39) or antigen production by flow cytometry (30).…”

mentioning

confidence: 99%

Surveillance Network for Herpes Simplex Virus Resistance to Antiviral Drugs: 3-Year Follow-Up

et al. 2004

View full text Add to dashboard Cite

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains . We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3,900 isolated strains among 3,357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.Herpes simplex virus (HSV) infections are very common; they are localized on the face and torso in the case of HSV type 1 (HSV-1) and in the genital region in the case of HSV-2. HSV-1 infections in the genital region are on the increase (40). Ocular herpes is less frequent, and neonatal herpes and herpetic meningoencephalitis are very rare but have a severe functional and vital prognosis (37).Since acyclovir (ACV) {9-[(2-hydroxyethoxy)methyl)guanine]} was introduced to the market in 1983, it has been used primarily in the prevention and treatment of HSV infections. ACV-resistant HSV strains have been observed in vivo since the first large therapeutic trials (5, 10, 36). These resistant strains are detected in vitro by phenotypic tests which determine the antiviral concentration inhibiting viral replication by 50%. Several methods have been used to evaluate the sensitivity of the HSV strains to ACV, including techniques to detect the intensity of the cytopathic effect, such as the plaque reduction (17, 31) and colorimetric (11,22,26) techniques, but also the detection of DNA replication by hybridization (39) or antigen production by flow cytometry (30).Previous surveys among immunocompetent patients have shown a prevalence of resistance to ACV varying between 0 and 0.6%, whereas among immunocompromised patients, the prevalence varied between 3 and 6% (9,16,29). The use of ACV is co...

show abstract

“…One method of nucleic acid hybridization for HSV susceptibility has been made commercially available in kit form. It compares well with the plaque reduction assay and involves wicking of lysed, virusinfected cells onto membranes followed by hybridization with a radioiodinated DNA probe (465). Although …”

Section: Latencymentioning

confidence: 99%

Antiviral therapy: current concepts and practices

Bean

1992

Clin Microbiol Rev

View full text Add to dashboard Cite

Drugs capable of inhibiting viruses in vitro were described in the 1950s, but real progress was not made until the 1970s, when agents capable of inhibiting virus-specific enzymes were first identified. The last decade has seen rapid progress in both our understanding of antiviral therapy and the number of antiviral agents on the market. Amantadine and ribavirin are available for treatment of viral respiratory infections. Vidarabine, acyclovir, ganciclovir, and foscarnet are used for systemic treatment of herpesvirus infections, while ophthalmic preparations of idoxuridine, trifluorothymidine, and vidarabine are available for herpes keratitis. For treatment of human immunodeficiency virus infections, zidovudine and didanosine are used. Immunomodulators, such as interferons and colony-stimulating factors, and immunoglobulins are being used increasingly for viral illnesses. While resistance to antiviral drugs has been seen, especially among AIDS patients, it has not become widespread and is being intensely studied. Increasingly, combinations of agents are being used: to achieve synergistic inhibition of viruses, to delay or prevent resistance, and to decrease dosages of toxic drugs. New approaches, such as liposomes carrying antiviral drugs and computer-aided drug design, are exciting and promising prospects for the future.

show abstract

Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus

Cited by 59 publications

References 24 publications

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

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

Surveillance Network for Herpes Simplex Virus Resistance to Antiviral Drugs: 3-Year Follow-Up

Antiviral therapy: current concepts and practices

Contact Info

Product

Resources

About