SAMPLE. Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard

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1 February 2004 Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard This document provides a protocol for the performance of the plaque reduction assay for phenotypic antiviral susceptibility testing of herpes simplex virus. A standard for global application developed through the Clinical and Laboratory Standards Institute consensus process.

2 Clinical and Laboratory Standards Institute Setting the standard for quality in clinical laboratory testing around the world. The Clinical and Laboratory Standards Institute (CLSI) is a not-for-profit membership organization that brings together the varied perspectives and expertise of the worldwide laboratory community for the advancement of a common cause: to foster excellence in laboratory medicine by developing and implementing clinical laboratory standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global applicability. Consensus Process Consensus the substantial agreement by materially affected, competent, and interested parties is core to the development of all CLSI documents. It does not always connote unanimous agreement, but does mean that the participants in the development of a consensus document have considered and resolved all relevant objections and accept the resulting agreement. Commenting on Documents CLSI documents undergo periodic evaluation and modification to keep pace with advancements in technologies, procedures, methods, and protocols affecting the laboratory or health care. CLSI s consensus process depends on experts who volunteer to serve as contributing authors and/or as participants in the reviewing and commenting process. At the end of each comment period, the committee that developed the document is obligated to review all comments, respond in writing to all substantive comments, and revise the draft document as appropriate. Comments on published CLSI documents are equally essential, and may be submitted by anyone, at any time, on any document. All comments are addressed according to the consensus process by a committee of experts. Appeals Process If it is believed that an objection has not been adequately addressed, the process for appeals is documented in the CLSI Standards Development Policies and Process document. All comments and responses submitted on draft and published documents are retained on file at CLSI and are available upon request. Get Involved Volunteer! Do you use CLSI documents in your workplace? Do you see room for improvement? Would you like to get involved in the revision process? Or maybe you see a need to develop a new document for an emerging technology? CLSI wants to hear from you. We are always looking for volunteers. By donating your time and talents to improve the standards that affect your own work, you will play an active role in improving public health across the globe. For further information on committee participation or to submit comments, contact CLSI. Clinical and Laboratory Standards Institute 950 West Valley Road, Suite 2500 Wayne, PA USA P: F: standard@clsi.org

3 NOTE: This document is no longer being reviewed as part of the CLSI consensus process. However, because of its usefulness to segments of the health care community, it is available for its informational content. ISBN Vol. 24 No. 7 Replaces M33-P ISSN Vol. 20 No. 22 Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard Volume 24 Number 7 Ella M. Swierkosz, Ph.D., Co-Chairholder Richard L. Hodinka, Ph.D., Co-Chairholder Barbara M. Moore Stephen Sacks, M.D. David R. Scholl, Ph.D. D. Kathleen Wright Abstract Clinical and Laboratory Standards Institute document Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard, the first to address phenotypic antiviral susceptibility testing, standardizes susceptibility testing of herpes simplex virus (HSV). This standard contains information about performance of the plaque reduction assay (PRA) for HSV susceptibility testing, the test method to which other antiviral susceptibility testing methods are usually compared. This standard provides detailed instructions for preparation of the viral inoculum, preparation and dilution of antiviral agents, inoculation of cell cultures, preparation of the overlay medium, incubation of inoculated cell cultures, fixation and staining of cell cultures, counting of plaques, calculation of endpoints, and interpretation of results. A quality control section addresses procedures for evaluation of cell lines, selection of reference strains and quality control ranges, standardization of inocula, and other quality control issues. Clinical and Laboratory Standards Institute (CLSI). Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard. CLSI document (ISBN ). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania USA, The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: ; Fax: ; customerservice@clsi.org; Website:

4 Number 7 Copyright 2004 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of content from a CLSI copyrighted standard, guideline, companion product, or other material requires express written consent from CLSI. All rights reserved. Interested parties may send permission requests to permissions@clsi.org. CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedure manual at a single site. To request permission to use this publication in any other manner, permissions@clsi.org. Suggested Citation CLSI. Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard. CLSI document. Wayne, PA: Clinical and Laboratory Standards Institute; Proposed Standard November 2000 Approved Standard February 2004 ISBN ISSN ii

5 Volume 24 Contents Abstract... i Committee Membership... iii Foreword... vii 1 Introduction and Scope Antiviral Agents for Treatment of HSV Acyclovir Valacyclovir Famciclovir/Penciclovir Foscarnet Idoxuridine and Trifluridine Vidarabine Definitions Variables of Antiviral Susceptibility Testing Host Cell Line, Viral Inoculum, Incubation Time, and Range of Concentration of the Antiviral Agent Heterogeneity of Virus Population and Strain of Virus Used ( Wild versus Laboratory Adapted ) Reference Strains Assay Method Endpoint Criteria Calculation of Endpoints Interpretation of Endpoints Plaque Reduction Assay (PRA) Cells, Media, and Reagents Supplies Equipment Storage Instructions Preparation of Cell Culture Plates, HSV Isolates, and Antiviral Agents Performance of the Plaque Reduction Assay Quality Control Procedures Purpose Quality Control Responsibilities Reference Strains for Quality Control Storing Quality Control Strains Medium Quality Control Cell Line Quality Control Antiviral Drug Quality Control Antiviral Cytotoxicity Control Inoculum Control Quality Control Ranges Frequency of Quality Control Testing v

6 Number 7 Contents (Continued) 7 Results: Interpretation and Reporting Data Analysis Interpretation and Reporting Limitations of Procedure Safety References Summary of Comments and Subcommittee Responses Summary of Delegate Comments and Subcommittee Responses The Quality System Approach Related NCCLS Publications vi

7 Volume 24 Foreword The NCCLS Subcommittee on Antiviral Susceptibility Testing was formed in 1997 and is composed of representatives from the professions, government, and industry, including virology and microbiology laboratories, healthcare providers and educators, and diagnostic microbiology and pharmaceutical industries. Our ultimate goal is to develop standards that provide specific details for the materials, methods, and practices necessary for the correct performance and appropriate reporting of phenotypic antiviral susceptibility tests. Although antiviral agents have been used in clinical practice for two decades, in vitro testing methods have never been standardized. This has hampered comparison of susceptibility testing results from different laboratories as well as development of definitive interpretive breakpoints denoting susceptibility and resistance of viral isolates to tested drugs. As more patients fail to respond to appropriate therapy and additional antiviral agents are produced, it has become increasingly important for diagnostic laboratories to provide antiviral susceptibility testing to assist physicians in defining drug resistance and choosing alternative therapies. Because many variables can affect antiviral susceptibility testing, including the cell line, virus inoculum, and assay method, this subcommittee has chosen to restrict the focus of this document to testing of herpes simplex virus (HSV). There are three major reasons for our decision. First, many antiviral agents have been approved for treatment of HSV, and a large body of literature exists regarding emergence of drug-resistant HSV and management of patients with resistant virus. Second, many of the variables that affect antiviral susceptibility testing have been defined for HSV. Third, because of the relative ease of culturing HSV, it should be possible to complete validation studies of this standard in a timely manner. The plaque reduction assay (PRA) was the first antiviral susceptibility testing method performed to determine the susceptibility of cytopathic viruses to antiviral agents and traditionally has been the standard to which newer methods are compared. 1 For this reason, this NCCLS approved standard will limit its discussion to the performance of the PRA. This is necessary as a first step to standardizing other methodologies, interpretive criteria, and quality control parameters so that results from different assays can be correlated with PRA. The methods described herein are generic reference procedures that can be used for routine antiviral susceptibility testing by clinical laboratories, or that can be used by clinical laboratories to evaluate commercial devices for possible routine use. Results generated by the NCCLS reference methods may be used by the United States Food and Drug Administration to evaluate the performance of commercial systems before clearance is given for marketing in the United States. Clearance by the FDA indicates that the agency concludes that commercial devices provide antiviral susceptibility results that are substantially equivalent to results generated using the NCCLS reference methods for the viruses and antiviral agents described in the manufacturer s approved package insert. Key Words Antiviral agents, plaque reduction assay, susceptibility testing vii

8 Volume 24 Antiviral Susceptibility Testing: Herpes Simplex Virus by Plaque Reduction Assay; Approved Standard 1 Introduction and Scope During the past decade, safe and effective antiviral therapy has been developed for treatment of a number of viral infections. While the overwhelming majority of clinical viral isolates from drug-naive patients are susceptible to antiviral agents, widespread use of some antiviral agents has led to the emergence of drugresistant strains Most drug resistance has been observed in the immunocompromised host, especially transplant recipients and AIDS patients who fail to respond to therapy for herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV) infections. 4,10,11 This standard, the first to address antiviral susceptibility testing, will limit its scope to a description of antiviral agents, the FDA-approved uses of these drugs for the treatment and prophylaxis of HSV infections, and antiviral susceptibility testing for HSV. Detailed discussions of antiviral susceptibility testing for other viruses, including HSV, have been recently published We have restricted our focus to HSV for three reasons. First, effective antiviral therapy for HSV has been available for over a decade, and there is widespread experience regarding development of resistance and management of patients with resistant virus. Second, there is also abundant laboratory experience with in vitro susceptibility testing of HSV. Many of the variables that affect antiviral susceptibility testing have been defined for HSV so that a standard can be developed. Third, because HSV is relatively easy to culture, validation of this standard can proceed in a straightforward and timely manner. A number of antiviral agents have been developed for the management of HSV infections, including acyclovir, valacyclovir, famciclovir, foscarnet, idoxuridine, trifluridine, and vidarabine (Table 1). Of these drugs, acyclovir has been widely used as an effective treatment for and prophylactic against HSV infections. However, resistance of HSV clinical isolates to acyclovir has emerged with the occurrence of chronic, progressive, debilitating disease in immunocompromised patients receiving prolonged courses of continuous or intermittent suppressive therapy. 16,17 The development of viral resistance to acyclovir and the morbidity and mortality associated with these virus strains is a problem of concern. Foscarnet has been employed successfully as an alternative antiviral agent for treating acyclovir-resistant HSV. 18 Resistance to this drug, however, has also been documented and clinical isolates resistant to both acyclovir and foscarnet have been reported. 19,20 With the rapid advance in the development and use of additional antiviral agents and the continued emergence of drug resistance, there is a definite need for diagnostic laboratories to provide rapid and practical antiviral susceptibility testing. This document provides a brief discussion of the major antiviral agents with activity against HSV, their mechanisms of action and development of resistance, the clinical use of these drugs, and detailed instructions for the performance of the plaque reduction assay as the standard for antiviral susceptibility testing of HSV. Clinical and Laboratory Standards Institute. All rights reserved. 1

9 Number 7 The Quality System Approach NCCLS subscribes to a quality system approach in the development of standards and guidelines, which facilitates project management; defines a document structure via a template; and provides a process to identify needed documents through a gap analysis. The approach is based on the model presented in the most current edition of NCCLS HS1 A Quality System Model for Health Care. The quality system approach applies a core set of quality system essentials (QSEs), basic to any organization, to all operations in any healthcare service s path of workflow. The QSEs provide the framework for delivery of any type of product or service, serving as a manager s guide. The quality system essentials (QSEs) are: Documents & Records Equipment Information Management Process Improvement Organization Purchasing & Inventory Occurrence Management Service & Satisfaction Personnel Process Control Assessment Facilities & Safety addresses the quality system essentials (QSEs) indicated by an X. For a description of the other NCCLS documents listed in the grid, please refer to the Related NCCLS Publications section. Documents & Records Organization Personnel Equipment Purchasing & Inventory Process Control Information Management Occurrence Management X M7 M23 Adapted from NCCLS document HS1 A Quality System Model for Health Care. Path of Workflow A path of workflow is the description of the necessary steps to deliver the particular product or service that the organization or entity provides. For example, GP26-A2 defines a clinical laboratory path of workflow which consists of three sequential processes: preanalytical, analytical, and postanalytical. All clinical laboratories follow these processes to deliver the laboratory s services, namely quality laboratory information. addresses the clinical laboratory path of workflow steps indicated by an X. For a description of the other NCCLS documents listed in the grid, please refer to the Related NCCLS Publications section. Patient Assessment Test Request Assessment Process Improvement Service & Satisfaction Preanalytic Analytic Postanalytic Specimen Collection Specimen Transport Specimen Receipt Testing Review Laboratory Interpretation X X M7 M7 Adapted from NCCLS document HS1 A Quality System Model for Health Care. Results Report X M7 Post-test Specimen Management Facilities & Safety X M29 38 Clinical and Laboratory Standards Institute. All rights reserved.

10 Volume 24 Related NCCLS Publications * M7-A6 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard Sixth Edition (2003). This newly revised standard provides updated reference methods for the determination of minimal inhibitory concentrations (MICs) for aerobic bacteria by broth macrodilution, broth microdilution, and agar dilution. This document contains MIC interpretive criteria and quality control parameters tables updated for M23-A2 Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved Guideline Second Edition (2001). This document addresses the required and recommended data needed for the selection of appropriate interpretative standards and quality control guidelines for new antimicrobial agents. M29-A2 Protection of Laboratory Workers from Occupationally Acquired Infections Second Edition; Approved Guideline (2001). Based on U.S. regulations, this document provides guidance on the risk of transmission of hepatitis viruses and human immunodeficiency viruses in any laboratory setting; specific precautions for preventing the laboratory transmission of blood-borne infection from laboratory instruments and materials; and recommendations for the management of blood-borne exposure. * Proposed- and tentative-level documents are being advanced through the NCCLS consensus process; therefore, readers should refer editions. to the most recent Clinical and Laboratory Standards Institute. All rights reserved. 39

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