Interpretive Handbook

HIV is an RNA virus that infects human host cells and is then converted to complementary DNA (cDNA) by the action of viral reverse transcriptase. HIV is the causative agent of AIDS, a severe, life-threatening condition. 

Currently, there are 2 types of HIV, HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV-1 has been isolated from patients with AIDS, AIDS-related complex, and asymptomatic infected individuals at high-risk for AIDS. Accounting for >99% of HIV infection in the world, HIV-1 is transmitted by sexual contact, by exposure to infected blood or blood products, from an infected pregnant woman to fetus in utero or during birth, or from an infected mother to infant via breast feeding. HIV-2 has been isolated from infected patients in West Africa and it appears to be endemic only in that region. However, HIV-2 also has been identified in individuals who have lived in West Africa or had sexual relations with individuals from that geographic region. HIV-2 is similar to HIV-1 in its morphology, overall genomic structure, and ability to cause AIDS.

Multiple clinical studies of plasma HIV-1 viral load (expressed as HIV-1 RNA copies/mL of plasma) have shown a clear relationship of HIV-1 RNA copy number to stage of HIV-1 disease and efficacy of anti-HIV-1 therapy. Quantitative HIV-1 RNA level in plasma (ie, HIV-1 viral load) is an important surrogate marker in assessing the risk of disease progression and monitoring response to anti-HIV-1 drug therapy in the routine medical care of HIV-1-infected patients.

Studies have identified a number of mutations associated with antiviral resistance. Genotypic analysis allows identification of nucleotide changes associated with HIV drug resistance. When combination therapy fails, genotyping for drug resistance mutations may help direct appropriate changes in antiretroviral therapy and may result in at least a short-term benefit, as evidenced by viral load reduction.

Quantifying plasma HIV-1 RNA levels (viral load) in HIV-1-infected patients

In patients with HIV-1 RNA levels >1,000 copies/mL, genotypic determination and prediction of phenotypic viral resistance to anti-HIV drugs is performed to guide therapy

Guiding initiation or change of antiretroviral regimens

This assay has a plasma HIV-1 RNA quantification result range from 20 to 10,000,000 copies/mL (1.30-7.00 log copies/mL).

An "Undetected" result indicates that the assay was unable to detect HIV-1 RNA within the plasma specimen.

A "Detected" result with the comment, "HIV-1 RNA level is <20 copies/mL (<1.30 log copies/mL). This assay cannot accurately quantify HIV-1 RNA below this level" indicates that HIV-1 RNA is detected, but the level present is less than the lower quantification limit of this assay.

A "Detected" result with the comment, "HIV-1 RNA level is >10,000,000 copies/mL (>7.00 log copies/mL). This assay cannot accurately quantify HIV-1 RNA above this level" indicates that HIV-1 RNA is detected, but the level present is above the upper quantification limit of this assay.

Due to the increased sensitivity of this assay, patients with previously low or undetectable HIV-1 viral load may show increased or detectable viral load with this assay. However, the clinical implications of a viral load below 50 copies/mL remain unclear. Possible causes of such a result include very low plasma HIV-1 viral load present (eg, in the range of 1-19 copies/mL), very early HIV-1 infection (ie, <3 weeks from time of infection), or absence of HIV-1 infection (ie, false-positive).

For the purpose of patient monitoring, the United States Department of Health and Human Services Panel on Antiretroviral Guidelines for Adults and Adolescents defines virologic failure as a confirmed viral load of >200 copies/mL, which eliminates most cases of viremia resulting from isolated blips or assay variability. Confirmed viral load rebound (ie, >200 copies/mL) on 2 separate tests obtained at least 2 to 4 weeks apart should prompt a careful evaluation of patient's tolerance of current drug therapy, drug-drug interactions, and patient adherence.

If the viral load is > or =1,000 copies/mL, genotypic anti-HIV-1 drug resistance mutation analysis is performed automatically at an additional charge. Sequence data of the patient's viral strain is compared with those in a database of known drug resistance mutations. Results are provided that highlight those codon changes associated with specific drug resistance. These mutations are categorized and reported.

Genotypic drug resistance:

-"Susceptible" indicates that the genotypic mutations present in patient's HIV-1 strain have not been associated with resistance to the specific drug in question.

-"Resistant" indicates that genotypic mutations (see specific list in corresponding result comment) detected have been associated with maximum reduction in susceptibility to the specific drug.

-"Possibly resistant" indicates that genotypic mutations detected have been associated with 1 or both of the following outcomes:

 - Diminished virologic response in some, but not all, patients having virus with these mutations

 - Intermediate decrease in susceptibility of the virus to the specific drug

-"Insufficient evidence" indicates that there is inadequate direct or indirect evidence to determine susceptibility of the virus to the specific drug on the basis of the genotypic mutations present, according to the opinion of the consensus panel of leading experts in the field of HIV resistance

-"Unable to genotype" indicates that the sequence data obtained are of poor quality to determine the presence or absence of genotypic resistant mutations in the patient's HIV strain. Possible causes of such poor sequence data include low HIV viral load (ie, <1,000 copies/mL) and polymorphism in the region of the sequencing primers interfering with primer binding and subsequent sequencing reaction.

If HIV type 1 genotypic drug resistance mutation analysis detects drug resistance mutations, then VPHIV/88781 HIV-1 Phenotypic Drug Resistance Prediction (Add-On) will be performed at an additional charge.

Phenotypic drug resistance prediction:

-For each drug, fold change (FC) is predicted by statistical comparison of the IC50 from all of the clinical virus specimens matching the patient's genotypic mutation data to that of the wild-type HIV-1 strain (susceptible reference virus). For example, an FC value of 3.0 for a specific drug indicates that the IC50 of that drug for the patient's virus is predicted to be 3 times higher than that of the susceptible reference virus.

-Predicted FC values should be interpreted with reference to clinical cutoff (CCO) or biological cutoff (BCO) values. CCO values are based on clinical observations of virologic response (change in viral load) in treated patients, indicating how that response is affected by viral resistance. BCO values are derived from laboratory observations indicating the normal range of in vitro susceptibility of wild-type viruses. CCO1 is the baseline FC associated with a 20% loss of the wild-type virologic response due to drug resistance, whereas CCO2 is the baseline FC associated with an 80% loss of the wild-type virologic response due to drug resistance.

Resistance analysis for a given drug is based on the magnitude of the predicted FC in IC50 relative to the CCO or BCO values:

-"Susceptible" indicates that the predicted FC in IC50 is < or =BCO value for the given drug.

-"Resistant" indicates that the predicted FC in IC50 is >BCO value for the given drug.

-"Maximal response" indicates that the predicted FC in IC50 is < or =CCO1 value for the specific drug.

-"Reduced response" indicates that the predicted FC in IC50 is >CCO1 but < or =CCO2 values for the specific drug.

-"Minimal response" indicates that the predicted FC in IC50 is >CCO2 value for the specific drug.

This test is not licensed by the FDA as a screening test for HIV-1 infections in a post-exposure setting. Although this quantitative HIV-1 RNA test is not FDA-approved for diagnostic purposes, the United States Working Group on Antiretroviral Therapy and Medical Management of HIV-Infected Children recommends the use of molecular-based assays to detect HIV-1 RNA or proviral DNA for the diagnosis of HIV infection in infants of <18 months of age and born to HIV-infected mothers.

A single HIV-1 viral load test result should not be used as the sole criterion in guiding therapeutic decisions and intervention in the clinical care of HIV-1-infected patients. Viral load results should be correlated with patient symptoms, clinical presentation, and CD4 cell count. Due to the inherent variability in the assay, physiologic variation and concurrent illnesses in the infected patients, <100-fold (<2 log) changes in plasma HIV-1 viral load should not be considered to be significant changes.

Viral load results of <20 copies/mL do not necessarily indicate absence of HIV-1 viral replication. Inhibitory substances may be present in the plasma specimen, leading to negative or falsely low HIV-1 RNA results. Improper specimen collection or storage may lead to negative or falsely lower plasma viral load results.

Although this commercial HIV-1 viral load assay is optimized for quantification of plasma viral load in HIV-1 infection due to HIV-1 groups M (subtypes A to H) and O strains, results generated from HIV-1 group O strains may be discordant (> or =0.5 log copies/mL) with those obtained from other commercially available HIV-1 viral load assays. The assay is not reliable for quantifying plasma viral loads in infection caused by HIV-1 group N and HIV-2 strains.

ACD-plasma specimens are not optimal for HIV-1 viral load testing because such plasma specimens show quantitative HIV-1 RNA levels that are approximately 15% lower than those collected in tubes containing EDTA.

Undetected

1. Relucio K, Holodniy M: HIV-1 RNA and viral load. Clin Lab Med 2002;22:593-610

2. Braun P, Ehret R, Wiesmann F, et al: Comparison of four commercial quantitative HIV-1 assays for viral load monitoring in clinical daily routine. Clin Chem Lab Med 2007;45(1):93-99

3. DHHS Panel on Antiretroviral Guidelines for Adults and Adolescents: Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Available from URL: http://aidsinfo.nih.gov/Guidelines January 29, 2008