Antiviral Therapy, Susceptibility Testing, and Prevention

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Antiviral Therapy, Susceptibility Testing, and Prevention

Antiviral Therapy

Antiviral therapy has expanded over the past several years as a treatment for a number of viral infections. Although most of the population is susceptible to such treatments, overuse of these agents has led to the emergence of drug-resistant strains, especially in immunocompromised patients. Resistance is known to develop to all agents and may be detected in vitro by using antiviral susceptibility testing. Virology laboratories are increasingly being asked to perform in vitro testing of antiviral agents when a patient’s infection fails to respond clinically to antiviral therapy, but testing for antiviral resistance is not currently available in many clinical settings. This chapter provides an overview of the viral diseases in which antiviral resistance has emerged, the need for in vitro susceptibility testing, and the phenotypic and genotypic susceptibility testing methods currently available.

Antiviral Resistance

Antiviral resistance means that a virus has changed in such a way that the antiviral drug is less effective in preventing illness. Antiviral resistance is indicated if a patient is taking an antiviral drug that has been proven in vitro to be effective against a virus, but the patient shows no improvement and continues to deteriorate clinically. Drug resistance must be distinguished from clinical resistance. With clinical resistance, the viral infection fails to respond to the antiviral therapy because of factors other than a change in the virus; such factors may include the patient’s immunologic status, the pharmacokinetics of the antiviral drug in the individual patient, and, if a combination of drugs is administrated, potential antagonism and interference with the absorption of one or more drugs. Other patient factors that also affect the success of drug therapy include nonadherence to or intolerance of a specific drug and prescriptive errors, such as inappropriate doses or route of administration. In addition, infections in immunocompromised patients may fail to respond to therapy that has proven effective in immunocompetent individuals.

Very few standards have been established for antiviral susceptibility testing. The development of such protocols began in 2004 with the establishment of an approved standard for susceptibility testing for herpes simplex virus (HSV) by the Clinical and Laboratory Standards Institute (CLSI).

The final result of antiviral susceptibility testing is determined by many variables, and these variables also hinder the standardization of antiviral susceptibility testing. Some of these variables include the following:

Each of these categories in turn has variables that affect the final results. For example, if the inoculum quantity is too large, a susceptible isolate may appear resistant; if the inoculum quantity is too small, the isolate may appear susceptible. The complexity of all these variables makes it imperative that established control strains also be tested when antiviral susceptibility testing is performed. Controls should include both drug-resistant and drug-susceptible isolates that have been well characterized. Several research laboratories across the nation can provide reference and drug-resistant strains of a virus for susceptibility testing; they include the National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Program (niaid.nih.gov) and the American Type Culture Collection (atcc.org). Pharmaceutical companies also are a source.

Methods of Antiviral Susceptibility Testing

The purpose of antiviral susceptibility testing is to evaluate new antiviral chemoprophylaxis, to test for cross resistance or cross reactivity to alternate agents, and to determine how frequently drug-resistance viral mutations occur.

The two general types of antiviral susceptibility testing are phenotypic testing and genotypic testing. Phenotypic susceptibility assays measure viral replication in the presence of antiviral agents; they measure the inhibitory effect of an antiviral agent on the entire virus population in a clinical isolate. Genotypic susceptibility assays use polymerase chain reaction (PCR) to detect genes known to be responsible for resistance, coupled with molecular sequencing to determine whether genome alterations associated with resistance have occurred. These assays use the virus’s nucleic acid to determine whether the virus has mutations capable of causing viral drug resistance. Alternately, a combination of the two general types of antiviral susceptibility, known as a virtual phenotype resistance assay, may be performed. This assay is a characterization of the patient’s virus genotype compared to a data base that includes paired genotypic and phenotypic information. This information is then used to estimate the most likely phenotype of the patient’s virus. The success of this approach varies with the virus type and antiviral drugs examined.

Each of these types of susceptibility testing has unique properties that can be used to complement each other. Phenotypic assays are better used to assess the combined effect of multiple-resistance mutations on drug susceptibility, but they are labor intensive, expensive, and have lengthy end result times. Genotypic assays have a shorter turnaround time and are less expensive than phenotypic assays, but they can detect only defined viral mutations.

Phenotypic Assays

Phenotypic assays use a variety of end-point measurements to determine whether a virus is inhibited by an antiviral drug or demonstrates drug resistance. Some of these end-point measurements include a reduction in the number of plaques, inhibition of viral DNA synthesis, a reduction in the yield of a viral structural protein, or a reduction of the enzymatic activity of a functional protein. As mentioned, an advantage of phenotypic assays is that they are much better for assessing the combined effect of multiple-resistance mutations on drug susceptibility. This is useful for assaying viruses such as hepatitis B virus (HBV), human immunodeficiency virus type 1 (HIV-1), and human cytomegalovirus (HCMV), which acquire resistance mutations in multiple genes. The disadvantages of phenotypic assays are that they are labor intensive, expensive, and require weeks to perform.

Genotypic Susceptibility Assays

Genotypic susceptibility assays use PCR to detect genes known to be responsible for resistance, coupled with genetic sequencing to determine whether genome alterations associated with resistance have occurred. Genotypic assays use DNA sequencing by automated sequencers, PCR amplification and restriction enzyme digestion of the products, and hybridization to microarrays containing multiple oligonucleotide probes. These assays are rapid, because isolation of the virus in culture is not necessary for testing.

The response to an antiviral agent is also measured by quantitative monitoring of the viral load (by means of the nucleic acid concentration) in the patient’s blood. Such testing is common in patients infected with HBV, hepatitis C virus (HCV), and cytomegalovirus (CMV). The viral load should diminish significantly after addition of an antiviral agent to which the virus is susceptible. Using molecular testing (e.g., quantitative PCR) to measure the amount of virus in serum is a surrogate test for resistance to antiviral agents. The viral load rises quickly when resistance appears.

Pyrosequencing

DNA sequencing is among the most important testing methods for the study of biologic entities. Pyrosequencing, which is relatively new, is a sequence-based detection method that allows rapid, accurate quantification of sequence variation. It allows rapid acquisition of short reads (100 to 200 bp) of genomic sequence to identify known mutations. It is based on the technology of detection of released pyrophosphate (PPi) during DNA synthesis. In a sequence of enzymatic reactions, a enzyme (polymerase) catalyzes the addition of nucleotides into a nucleic acid chain. As a result of this addition, a PPi molecule is released and converted to adenosine triphosphate (ATP) by the ATP enzyme sulfurylase. Visible light is produced when a luciferin molecule is oxidized during the luciferase reaction. The visible light or signal strength generated is proportional to the number of nucleotides incorporated into the final product.

The two types of pyrosequencing methods currently available are solid-phase pyrosequencing and liquid-phase pyrosequencing. Solid-phase pyrosequencing involves a three-enzyme system that uses immobilized DNA, and a washing step is performed to remove excess substrate after each nucleotide addition. In liquid-phase pyrosequencing, a fourth nucleotide-degrading enzyme (made from potato) is added. The advantage of the liquid-phase system is that it eliminates the need for solid support and the intermediate washing step, allowing the reaction to be performed in a single tube.

Because of its rapid, accurate quantification of sequence variation, pyrosequencing is an adaptable tool that can be used for a wide range of applications. Automation with pyrosequencing is made possible by the liquid-phase methodology. Pyrosequencing signals are quantitative, which allows a large number of people to be screened through examination of the allelic frequency in a population. This technique also is used taxonomically to group different organisms into strains or subtypes, and it can be applied to bacteria, yeasts, and viruses. It is currently the fastest method for sequencing a PCR product and can be applied to the resequencing of PCR-amplified disease genes for mutation screening. It also is used to screen clinical isolates for the genes that confer resistance to antiviral therapy, such as for analysis of influenza specimens for the adamantine resistance mutation.

Human Immunodeficiency Virus

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