a Servicio de Microbiología Clínica y Enfermedades Infecciosas-VIH, Hospital General Universitario Gregorio Marañón', Madrid 28007; b Centro Nacional de Microbiología, Virología e Inmunología Sanitarias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
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Abstract |
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Introduction |
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In-vitro susceptibility of viruses to antiviral agents is normally measured as the inhibitory concentration 50% (IC50), that is the concentration of antiviral that lowers the virus-induced cytopathic effect (CPE) and the number of plaques formed by a given inoculum, by 50%.8 Antiviral methods for the determination of susceptibility in HSV include the plaque reduction assay (PRA), dye uptake (DU), CPE inhibition, virus yield reduction, inhibition of specific immunofluorescence and DNA hybridization. PRA has classically been considered the reference method of choice.9 However, all these methods are tedious and time consuming.
In this study we evaluate a rapid method for the determination of HSV susceptibility to antivirals, in comparison with the PRA.
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Materials and methods |
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We studied a total of 84 HSV clinical isolates collected at the Virology Laboratory of our hospital between September 1991 and December 1995. Forty-three were HSV-1 and 41 were HSV-2.
A wild-type HSV isolate, an acyclovir-resistant laboratory mutant (presumptive thymidine kinase mutant) and an acyclovir- and foscarnet-resistant laboratory mutant (presumptive DNA polymerase mutant) obtained from the Centro Nacional de Microbiología, Virología e Immunología Sanitarias, Instituto de Salud Carlos III were used as control strains.
Isolates were maintained in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS) at 70°C prior to the study.
Once thawed, isolates were inoculated on to Vero cell monolayers until 75% or more of the monolayer showed a CPE. They were then centrifuged and the supernatant used as sample for the susceptibility study; an aliquot of the supernatant was stored in single use sterile tubes at 70°C.
Antiviral agents tested
Acyclovir was provided by GlaxoWellcome, Madrid, Spain and foscarnet by Sigma Chemicals, Madrid, Spain.
Reference assay
The reference method was the PRA, performed according to the American Society for
Microbiology guidelines,8 with adaptation for use with
96-well flat-bottomed sterile plates and lids. Titration of the isolates was performed in order to
obtain inocula concentrations of 200400 pfu/mL. An HSV isolate was considered
susceptible to acyclovir when the IC50 was <2 mg/L and resistant when the IC50 was 2 mg/L. In the case of foscarnet, an HSV isolate was considered
susceptible when the IC50 was <50 mg/L, intermediate when it was between 50
and 100 mg/L, and resistant when the IC50 was
100 mg/L.8
New rapid method
The method evaluated was a qualitative method based on the PRA. Eight serial 1:10 dilutions of the viral isolates were inoculated on to Vero cell monolayers growing in 96-well plates, containing EMEM with 2% FCS as diluent, 1% glutamine and antimicrobials (penicillin, streptomycin and amphotericin B). For every HSV isolate, three sets of 1:10 serial dilutions were used, the first without any antiviral agent, the second with acyclovir (2 mg/L) and the third with foscarnet (100 mg/L). Antiviral agents were omitted in the first well of every set in order to act as growth controls. The lack of toxicity of growth medium on the Vero cells was confirmed by the inclusion of control wells.
Plates were then incubated at 37°C with 5% CO2 for 72 h. After this incubation period, formation of a CPE was observed using an inverted light microscope.
Interpretation of results was performed by comparing the titre obtained in the sets without antiviral agent with those obtained in the sets containing each antiviral agent. The titre of an HSV isolate was the highest dilution of the isolate at which HSV growth was detected. If the titre obtained in the set without antiviral agents was at least one log10 greater than that obtained in the wells containing antiviral agent, then the isolate was considered susceptible to that particular antiviral agent. If titres obtained with and without antiviral were equal, meaning that the isolate was not inhibited by the antiviral, then the isolate was considered resistant to that agent (Figure). Since this method is based on serial dilutions of the initial inoculum, susceptibility results should not be affected by the viral load.
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Results |
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Discussion |
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The rapid method we have described is simpler to perform than the PRA and requires no titration of inoculum. It also does not require the use of radioactive material. The rapid method takes only 72 h to perform. This is compared with the 57 days needed for the PRA. Another advantage of the rapid method is that since it is a qualitative method, it is more reproducible than the PRA. In addition, if results obtained using the rapid method are to be confirmed using any other method (i.e. PRA, DU or others), inocula titration will have already been performed, as the set of wells without antivirals serves this purpose.
Data on sensitivity, specificity and predictive values of resistant and susceptible result' obtained for our assay of acyclovir in this study might be modified when applied to other populations. The predictive value of a resistant result' would be reduced in populations where the prevalence of resistance was lower than in our sample. However, high predictive values of a sensitive result' makes our assay particularly useful in screening large series of strains.
Since acyclovir is currently the first choice antiviral agent to treat infections caused by HSV,2 a feasible rapid method for detection of acyclovir-resistant HSV isolates such as the one we have described, would be a very important tool in clinical virology laboratories.
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Acknowledgments |
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Notes |
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References |
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2 . Hirsch, M. S. (1995). Herpes simplex virus. In Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases, 4th edn, (Mandell, G. L., Bennett, J. E. & Dolin, R., Eds), pp. 133645. Churchill Livingstone, New York.
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4 . Englund, J. A., Zimmerman, M. E., Swierosz, E. M., Goodman, J. L., Scholl, D. R. & Balfour, H. H. (1990). Herpes simplex virus resistant to acyclovir: a study in a tertiary care center. Annals of Internal Medicine 112, 41622.[ISI][Medline]
5 . Erlich, K. S., Mills, J., Chatis, P., Mertz, G. J., Busch, D. F., Follansbee, S. E. et al. (1989). Acyclovir resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. New England Journal of Medicine 320, 2936.[ISI][Medline]
6 . Field, A. K. & Biron, K. K. (1994). The end of innocence' revisited: resistance of herpesviruses to antiviral drugs. Clinical Microbiology Reviews 7, 113.[Abstract]
7 . Safrin, S., Elbeik, T., Phan, L., Robinson, D., Rush, J., Elbaggari, A. et al. (1994). Correlation between response to acyclovir and foscarnet therapy and in-vitro susceptibility result for isolates of herpes simplex virus from human immunodeficiency virus-infected patients. Antimicrobial Agents and Chemotherapy 38,124650.[Abstract]
8 . Sweirkosz, E. M. & Biron, K. K. (1994). Antiviral susceptibility testing. In Clinical Microbiology Procedures Handbook, Supplement 1, (Isenberg, H. D., Ed.), pp. 8.26.121. American Society for Microbiology, Washington, DC.
9 . Sweirkosz, E. M. & Biron, K. K. (1995). Antiviral agents and susceptibility testing. In Manual of Clinical Microbiology, 6th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 141523. American Society for Microbiology, Washington, DC.
Received 2 February 1999; returned 10 May 1999; revised 7 June 1999; accepted 27 July 1999