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HAART resistance genotyping

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Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Antiretroviral, antiretroviral resistance genotyping, GeneSeqTM, GenoSure (Plus)TM, genotypic assay, genotype testing, HAART, highly active antiretroviral therapy, highly active antiretroviral therapy resistance genotyping, HIV-1 TrueGeneT, human immunodeficiency virus, mutation, opportunistic infections, PCR, phenotype testing, polymerase chain reaction, PR region, retrovirus, RT region, sequencing assay, ViroSeqTM.

Background
  • Human immunodeficiency virus (HIV): HIV is a retrovirus that causes AIDS (acquired immune deficiency syndrome), a disease of the human immune system. Retroviruses are a group of viruses that contain ribonucleic acid (RNA) and replicate by reverse transcription. Reverse transcription is the process of producing a double-stranded deoxyribonucleic acid (DNA) from a single-stranded RNA.
  • The retrovirus primarily attacks the body's immune system, making the body extremely vulnerable to opportunistic infections. Opportunistic infections are infections that occur in people with weak immune systems (and generally do not occur in healthy people). Examples of such infections include toxoplasmosis, cytomegalovirus, and Pneumocystis jiroveci pneumonia (formerly called Pneumocystis carinii or PCP).
  • HIV-1 and HIV-2 are the two known strains of HIV. The HIV-1 virus is highly infectious (virulent), relatively easily transmitted, and is the cause of most HIV infections globally. HIV-2 is less easily transmitted and is largely confined to West Africa.
  • Highly active antiretroviral therapy (HAART): HAART refers to the combination of multiple antiretroviral drugs used to treat HIV-positive patients. Antiretroviral drugs inhibit the ability of HIV or other types of retroviruses to multiply in the body. Some of the classes of antiretroviral drugs include reverse transcriptase inhibitors, protease inhibitors, and integrase inhibitors. Reverse transcriptase inhibitors are drugs that inhibit the enzyme reverse transcriptase, which is required for the reverse transcription process. Reverse transcriptase inhibitors include zidovudine, tenofovir, and efavirenz. Protease inhibitors are drugs that inhibit the viral protease, an enzyme that HIV uses to produce infectious viral particles. When protease is blocked, new copies of HIV are not formed properly, and they are unable to infect new cells. Examples of protease inhibitors include saquinavir, ritonavir, and indinavir.
  • Resistance to HAART: Resistance to HAART is an important cause of treatment failure, and it is estimated to be as high as 50% among those receiving treatment in the United States. Resistance is the capacity of disease-causing microorganisms to withstand exposure to drugs previously toxic to them. It is acquired either through spontaneous mutation or by gradual selection of a relatively resistant virus after drug exposure.
  • Determination of resistance has implications not only in the treatment of the individual patient, but also among the general population because they may be exposed to drug-resistant viruses. Another critical problem is the possibility that a resistant strain may have resistance to other drugs that have not yet been prescribed in that particular patient (cross-resistance).
  • Mutations causing resistance: A point mutation involves a single nucleotide; it may consist of the loss of a nucleotide, substitution of one nucleotide for another, or the insertion of an additional nucleotide. A region in the HIV-1 gene that codes for the protease enzyme is called PR, and the region that codes for the reverse transcriptase enzyme is called RT. Some of the point mutations that confer antiretroviral resistance include RT M184V (amino acid methionine is replaced by amino acid valine at position 184 of RT), PR V82A (amino acid valine is replaced by amino acid alanine at position 82 of PR), and PR I84V (amino acid isoleucine is replaced by amino acid valine at position 84 of PR). Amino acids are the building blocks of proteins. Proteins are any of a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of one or more chains of amino acids.
  • Causes of resistance: Causes of resistance include reduced compliance with treatment; reduced absorption and metabolism of a drug in individual patients; initiation of antiretroviral therapy at an advanced stage of HIV infection; host genetic variability to drug efficacy and HIV susceptibility; drug interactions; poor follow-up; and prescription of ineffective regimens. Drug metabolism is the enzymatic degradation or modification that a drug undergoes in the body. Host genetic variability refers to the variations in the genetic makeup of individuals, which may result in differences in response to a drug. These factors lead to selection of pre-existing or newly developed resistant strains. Identification of the presence of drug resistance in a patient helps the healthcare provider choose the right combination of antiretrovirals to suppress the viral replication in the patient.
  • Drug resistance testing: Drug resistance testing is used to determine whether an HIV patient has a mutated form of the virus that does not respond to antiretroviral therapy. There are two main types of drug resistance tests: genotypic and phenotypic. Genotypic tests examine the genetic structure of a patient's HIV, while phenotypic tests examine the sensitivity of a patient's HIV to a specific drug. Genotype is the genetic constitution of an organism. Phenotype is the manifestation of a genotype or the combined manifestations of several different genotypes. Genotype assays are tests that detect viral mutations that may cause changes in viral susceptibility to particular drugs or classes of drugs. The assay makes use of polymerase chain reaction (PCR), which amplifies (copies) the HIV-1 gene of interest from the patient's blood samples. PCR is an enzymatic method for the repeated copying of the two strands of DNA of a particular gene sequence. It is widely used to amplify minute quantities of biologic material so as to provide adequate specimens for laboratory study. Phenotypic assays first insert the patient's PR and RT genes into a standard reference virus, then test the amount of drug required to inhibit the virus replication by 50%. The test is done in vitro, that is, under laboratory conditions and not in the patient.
  • Types of genotype assays: Sequencing assays and point-mutation assays are the two types of genotypic assays.Sequencing assays are used to detect mutations in either the reverse transcriptase or protease genes. Both of these genes are involved in the replication of HIV. Reverse transcriptase is an enzyme that allows HIV and similar viruses to insert their genetic information into the DNA of a human cell, causing the cell to produce more viruses. Proteases are a class of enzymes that degrade proteins by breaking the peptide bonds that join the amino acids in a protein. The HIV virus makes use of proteases to split proteins during the assembly of virions (virus particles). Point-mutation assays are used to detect mutations that are known to cause drug resistance in these genes.
  • Current genotypic resistance tests include Bayer Health Diagnostics' HIV-1 TrueGeneT, Celera Diagnostics/Abbott Laboratories' ViroSeqT, LabCorp's GenoSure (Plus)T, and Monogram Biosciences' GeneSeqT. Among these, HIV-1 TrueGeneT is the only U.S. Food and Drug Administration (FDA)-approved genotyping assay kit.

Methods
  • General: HIV-1 TrueGeneT genotyping kit is a sequencing assay. Sequencing assays are used to detect mutations in either the reverse transcriptase or protease genes. Both of these genes are involved in the replication of human immunodeficiency virus (HIV). Reverse transcriptase is an enzyme that allows HIV and similar viruses to insert their genetic information into the deoxyribonucleic acid (DNA) of a human cell, causing the cell to produce more viruses. DNA is the long thread-like molecule made up of large numbers of nucleotides. Proteases are a class of enzymes that degrade proteins by breaking the peptide bonds that join the amino acids in a protein. The HIV virus makes use of proteases to split proteins during the assembly of virions (virus particles). The assay is used in combination with the patient's clinical history and other laboratory tests, such as CD4 count and number of viral copies per milliliter, in the management of HIV-1, subtype B infection. RNA is a polymeric constituent of all living cells and many viruses, consisting of a long, usually single-stranded chain of alternating phosphate and ribose units with the bases adenine, guanine, cytosine, and uracil bonded to the ribose.
  • There are two major types of HIV, types 1 and 2. HIV type 1 is further classified into groups M, N, and O. Subtype B is a further classification under group M. There are two regions on the HIV-1 genome which code for resistance against antiretroviral drugs: the protease region (codons 1 to 99) codes against protease inhibitors, and the RT region (codons 40 to 247) codes against the reverse transcriptase inhibitors. A codon is a set of three consecutive nucleotides in a strand of DNA or RNA that provides the genetic information to code for a specific amino acid that will be incorporated into a protein chain or serve as a termination signal.
  • The protease region (codons 1 to 99) and RT region (codons 40 to 247) of the HIV-1 genome, where mutations that code for resistance against the antiretrovirals protease inhibitors and the reverse transcriptase inhibitors are found, are the targets of this assay.
  • The kit consists of three components: chemistry, hardware, and software. The chemistry kit consists of the reagents (chemicals) necessary to perform reverse transcription and amplification of the extracted RNA virion. A virion is the complete virus particle that is structurally intact and infectious. Reverse transcription refers to the production of a double-stranded DNA from a single-stranded RNA, and amplification (replication/multiplication) is the process of production of multiple copies of a sequence of DNA or RNA. Reagents for CLIP sequencing reactions of the protease I and reverse transcriptase portions of the HIV-1 genome are also present. CLIP reaction refers to amplification (replication/multiplication) by polymerase chain reaction (PCR) simultaneously with nucleic acid sequencing.
  • The hardware component consists of the Long-Read tower Automated DNA sequencer, MicroCelTM cassettes, and a polymerization unit, the GelToasterTM. The Long-Read tower can read a sequence of 1,000 bases within four hours when combined with the MicroCelTM cassette. The MicroCelTM cassette is a polyacrylamide, electrophoretic gel cassette, which acts as a medium for the Long-Read DNA sequencer. Elelctrophoresis is the movement of charged colloidal particles through the polyacrylamide medium in which the particles are dispersed as a result of changes in electrical potential. It is used in the analysis and separation of proteins. Proteins are any group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of one or more chains of amino acids. The GelToasterTM is a device that polymerizes the acrylamide gel medium through which the DNA is separated. Polymerization is the process in which many small molecules called monomers are joined to form giant molecules called polymers.
  • The software assembles the data and determines the DNA sequences by reading the order of bases. It compares the DNA sequence of the patient's sample with that of an HIV-1 database and identifies the incompatible bases and produces the final report. The interpretation makes use of a set of criteria developed by an international panel of HIV-1 clinical and research experts and coded by the Visible Genetics Research & Development group, based on the GuideLinesTM Rules interpretation algorithm.
  • Interpretation of test results: The results may be listed as one of the follows: no resistance, possible resistance, resistance, and insufficient evidence. When no mutations are detected or the resistance is not associated with the mutations detected by this assay, the result is ''no resistance.'' If the mutations detected by this assay are associated with a definite resistance, the result will read ''resistance.'' If the mutations detected by the assay have been associated with resistance in some patients or an in vitro reduction in viral susceptibility to a certain extent, the result is ''possible resistance.'' If no definite conclusion is drawn from the assay, then the result is given as ''insufficient evidence.'' The results help the clinician choose a suitable antiretroviral drug regimen to which the patient's HIV is not resistant.

Research
  • The VIRADAPT study aimed at guiding treatment in patients who had treatment failure after receiving at least three months of a protease inhibitor-containing regimen. Protease inhibitors are drugs that inhibit the viral protease, an enzyme that the human immunodeficiency virus (HIV) uses to produce infectious viral particles. When protease is blocked, new copies of HIV are not formed properly, and they are unable to infect new cells. The study sought to evaluate if genotype testing resulted in antiretroviral treatment success in such patients. Genotypic tests examine the genetic structure of a patient's HIV. The study concluded that patients who underwent genotype testing were more likely to achieve HIV-1 ribonucleic acid (RNA) levels of <200 copies per milliliter at six months. About 32% of patients in the genotyped arm achieved HIV-1 RNA levels of <200 copies per milliliter at six months compared to only 14% in the control arm. Also, the results persisted at week 48 in the genotyped arm. RNA is a polymeric constituent of all living cells and many viruses, consisting of a long, usually single-stranded chain of alternating phosphate and ribose units with the bases adenine, guanine, cytosine, and uracil bonded to the ribose.

Implications
  • Genotype testing for human immunodeficiency virus (HIV) is recommended in recent HIV infections; before starting antiretroviral therapy in known cases of HIV; and in pregnant patients with detectable plasma HIV-1 RNA levels.
  • Genotypic resistance testing examines the genetic structure (genotype) of a patient's HIV. In recently acquired HIV infections, the test helps to identify the transmission of a drug-resistant virus and to identify the drugs to which the virus is resistant.
  • In known cases of HIV, the test helps to list the drugs to which the patient's virus is resistant, especially in patients in whom the first regimen itself failed. In patients in whom multiple regimens failed, a regimen to which the virus is susceptible can be chosen; genotype testing helps in determining this and ruling out all the ineffective regimens.
  • Pregnant women may be tested before initiation of antiretroviral therapy during pregnancy or to change a regimen. It is especially useful if the woman has been exposed to many antiretroviral drugs prior to pregnancy. The test aids the clinician in choosing the right regimen to treat the mother as well as to prevent transmission of the virus to the fetus.
  • Inability to obtain results or obtaining an inaccurate result by the use of genotype testing may result in patients receiving inappropriate therapy, thus making the patient susceptible to unnecessary adverse effects and increasing the cost of treatment.

Limitations
  • The present genotypic assays require plasma HIV-1 ribonucleic acid (RNA) levels to be greater than 500-1,000 RNA copies per milliliter of blood.
  • Due to the high cost of the genotyping kits, they are not used regularly in developing countries. Efforts are being made to develop cheaper kits with effectiveness similar to the standard commercial kits for use in these regions, where the disease burden is higher.
  • Occasionally genotype testing may yield inaccurate results, and this may result in patients receiving inappropriate therapy.

Safety




Future research
  • In addition to monitoring the patient's therapy by reviewing side effects and CD4 counts, monitoring drug levels may be of use. Therapeutic drug monitoring (TDM) is an emerging tool in the treatment of human immunodeficiency virus (HIV) patients. TDM refers to the measurement of drug levels in the blood. Usually the blood is drawn just before the next dose is due. It is especially useful in patients who are receiving drugs that interact with one another as the test helps to determine if the required plasma level of the drug is being achieved. Further research is ongoing, and the present data shows that TDM is useful when used along with resistance testing.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography
  1. Centers for Disease Control and Prevention.
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  4. Hanna GJ, D'Aquila RT. Clinical use of genotypic and phenotypic drug resistance testing to monitor antiretroviral chemotherapy. Clin Infect Dis. 2001;32:774-782.
  5. Hirsch MS, Günthard HF, Schapiro JM, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 recommendations of an International AIDS Society-USA panel. Clin Infect Dis. 2008 Jul 15;47(2):266-85.
  6. Natural Standard: The Authority on Integrative Medicine.
  7. Sukasem C, Churdboonchart V, Chasombat S,et al. Surveillance of genotypic resistance mutations in chronic HIV-1 treated individuals after completion of the National Access to Antiretroviral Program in Thailand. Infection. 2007 Apr;35(2):81-8.
  8. Van Laethem K, Vandamme AM. Interpreting resistance data for HIV-1 therapy management--know the limitations. AIDS Rev. 2006 Jan-Mar;8(1):37-43.
  9. Van Vaerenbergh K. Study of the impact of HIV genotypic drug resistance testing on therapy efficacy. Verh K Acad Geneeskd Belg. 2001;63(5):447-73.
  10. World Health Organization.
  11. Wilson JW, Bean P. A physician's primer to antiretroviral drug resistance testing. AIDS Reader. 2000;10:469-478.

Copyright © 2011 Natural Standard (www.naturalstandard.com)


The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.

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