HIV resistance testing

# Overview
The emergence of resistance to one or more antiretroviral drugs is one of the more common reasons for therapeutic failure in the treatment of HIV. In addition, the emergence of resistance to one antiretroviral drug sometimes confers a reduction in or a loss of susceptibility to other or all drugs of the same class. The application of laboratory technologies, such as gene amplification, automated nucleic acid sequencing, and nucleic acid hybridization, and the availability of recombinant viruses for testing phenotypic susceptibility have permitted advances in HIV resistance testing. Many clinicians and investigators are currently using these technologies in the clinical management of HIV.

# Rationale for Drug Resistance Testing
Drug resistance remains a crucial issue:
- Centers for Disease Control (CDC) did a survery, covering 2003 to 2006, 10.9% of patients exhibited resistance to anti-HIV drugs, with the majority having resistance to nonnucleoside analogue reverse transcriptase inhibitors (NNRTIs) and 1.9% having resistance to at least 1 drug in numerous drug classes.
- Thousands of individuals who began therapy in the early and mid-1990s already harbor multidrug-resistant viruses. Drug resistance is observed in most countries where ART is prescribed, thus jeopardizing the success of the therapy.
- Resistance testing can improve treatment outcomes for infected individuals.

# Goals of Resistance testing
The goals of resistance testing are to:
- Determine the effect of an antiviral drug on the evolution of the virus.
- Identify the baseline genotypic and phenotypic determinants of virologic success or failure (or clinical success or failure) in the study.

# Indications
Resistance testing is recommended in the following:
- At the time of diagnosis of HIV infection, as a part of initial patient assessment.
While starting therapy for treatment-naive patients.
Person-to-person transmission of drug resistant viruses have known to occur between adults and from mother to child.
First evaluation of chronic HIV-1 infection.
Treatment initiation for chronic HIV-1 infection.
- While starting therapy for treatment-naive patients.
Person-to-person transmission of drug resistant viruses have known to occur between adults and from mother to child.
- Person-to-person transmission of drug resistant viruses have known to occur between adults and from mother to child.
- First evaluation of chronic HIV-1 infection.
- Treatment initiation for chronic HIV-1 infection.
- All cases of virologic failure.
- Special settings like pregnancy.
- Before initiating therapy with chemokine receptor antagonist.

# Advantage
Despite limitations of resistance assays and their interpretation, several randomized controlled studies have demonstrated that virologic outcome, at least over the short term, may be improved when genotypic or phenotypic data are used to guide choice of drug regimens in patients with loss of virologic response to prior regimens.
Resistance testing has also shown to improve survival in HIV patients.
The FDA recommends that characterization of resistance and cross-resistance be a part of antiretroviral drug development so that clinically relevant information is available at the time of approval.

# Limitations
- Performance characteristics (e.g., sensitivity, specificity, and reproducibility) for many of the assays in investigational use have not been fully established.
- The clinical significance of many mutations or mutational patterns has not been defined completely for many antiretroviral drugs.
- The quantitative relationship between reductions of cell culture susceptibility and loss of clinical activity has not been established for most drugs.
- The ability to detect drug-resistance mutations, can vary substantial among different laboratories.

# Types
There are 2 main methods to detect antiretroviral resistance in HIV:
- Genotypic testing.
- Phenotypic testing.
Other resistant testing procedures are under development and may prove to be useful in future. These are as follows:
- Allele specific PCR assay 
- Single-genome assay 
- Ultra-deep sequencing assay 
These assays are used to investigate the role of minority variants (those having drug resistant trait), that are present below the level of detection by other methods.

# Genotypic testing

## Underlying Concept
Drug-resistance mutations have been well known and characterized from patient specimens and in vitro work. Rapid detection of probable resitance mutation to different ART can be detected by amplification and sequencing.

## Process
- Mutations responsible for reductions in susceptibility to a drug can be identified by DNA sequence analysis of the relevant portions of the virus genome.
- The complete coding sequence of the gene for the target protein should be determined in the early stages of characterization of mutations associated with reduced drug susceptibility.
- Once mutations are identified, their ability to confer phenotypic resistance should be evaluated in a recombinant virus system (e.g., by using site-directed mutagenesis or polymerase chain reaction (PCR) amplification of relevant portions of the virus genome to introduce these mutations into a standard laboratory HIV genetic background) or other suitable system, such that the mutations necessary to reproduce the resistant phenotype are identified.
- If site-directed mutagenesis experiments within the target gene fail to recapitulate the resistance phenotype, then the potential effects of mutations elsewhere in the viral genome should be examined.
- In the case of studying mutations in the envelope gene, which is highly variable, a possible option is to introduce mutations into the parental envelope to assess their contribution to the resistance phenotype.
- Recombinant virus should then be tested for drug susceptibility in cell culture. Shifts in drug susceptibility (fold- increases in EC50 value) for recombinant virus relative to WT should be determined.

## Limitations
- Low viral load
It not only decreases the sensitivity of the test but also make this test redundant.
Low-level resistance can be lost in the mixed population of viral pseudospecies, which often exists in HIV infected persons.
- It not only decreases the sensitivity of the test but also make this test redundant.
- Low-level resistance can be lost in the mixed population of viral pseudospecies, which often exists in HIV infected persons.

# Phenotypic testing

## Underlying Concept
It test the ability of Human Immunodeficiency Virus to replicate in the presence of a particular antiretroviral drug i.e. it measures the phenotype (external behavior) of patient's HIV in response to ART. The virus is grown in test-tubes with anti-retroviral drug. It is then evaluated whether it grows (or not grow) under the presence of that drug.

## Limitations
- Low viral load limits the test.
- It is a very labor intensive process.

## Advantages
It is better than genotypic testing at detecting mixed populations of resistant and sensitive pseudospecies.
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# Recommendations

## U.S. Department of Health and Human Services Recommendations
Indications for Antiretroviral Drug Resistance Testing in HIV-Infected Pregnant Women
- HIV drug-resistance testing is recommended for:
All pregnant women with HIV RNA levels above the threshold for resistance testing (e.g., >5001,000 copies/mL) not currently receiving antiretroviral (ARV) drugs, before starting treatment or prophylaxis (AIII).
All pregnant women receiving antenatal ARV drugs who have suboptimal viral suppression or persistant viral rebound to detectable levels after prior viral suppression on an ARV regimen (AII).
- All pregnant women with HIV RNA levels above the threshold for resistance testing (e.g., >5001,000 copies/mL) not currently receiving antiretroviral (ARV) drugs, before starting treatment or prophylaxis (AIII).
- All pregnant women receiving antenatal ARV drugs who have suboptimal viral suppression or persistant viral rebound to detectable levels after prior viral suppression on an ARV regimen (AII).
- Empiric initiation of ARV drugs before results of resistance testing are available may be warranted, with adjustment as needed after the test results are available, for optimal prevention of perinatal transmission (BIII).

## WHO-recommended strategy
The WHO-recommended strategy provides country-level guidance for the following activities:
- National HIVDR working groups.
- HIVDR Early Warning Indicators (EWI).
- Surveys to assess acquired HIV drug resistance.
- Surveillance of transmitted HIV drug resistance.
- Surveillance of initial HIV drug resistance
- National drug resistance database.
- Global drug resistance laboratory network.

# Reference
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