INTRODUCTION — Human immunodeficiency virus type 1 (HIV-1) RNA can be measured using qualitative or quantitative techniques. Qualitative testing (commonly referred to as nucleic acid testing or NAT) is used as a screening test to identify HIV-infected individuals, such as screening possible blood donors. Quantification of HIV-RNA (viral load measurements) can be used as a diagnostic test in certain situations; however, the HIV viral load is primarily used for management/monitoring of HIV-1-infected individuals.
This topic will address the laboratory methods for quantitation of HIV-1 RNA and the use of viral load for clinical management. Additional information on nucleic acid, HIV-2 RNA, and CD-4 cell count testing is found elsewhere. (See "Blood donor screening: Laboratory testing", section on 'HIV-1 and HIV-2' and "Clinical manifestations and diagnosis of HIV-2 infection", section on 'Testing for HIV-2 infection' and "Techniques and interpretation of measurement of the CD4 cell count in people with HIV".)
THE USE OF HIV VIRAL LOAD MEASUREMENTS — Studies have found HIV-1 RNA levels to be a predictor of the time to progression to acquired immunodeficiency syndrome (AIDS) and death that is independent of CD4 cell counts [1-6]. (See "The natural history and clinical features of HIV infection in adults and adolescents".)
Viral load measurements are primarily used for monitoring the response to treatment [7]. The use of HIV-1 RNA measurements in clinical practice are discussed separately. (See "Selecting antiretroviral regimens for treatment-naïve persons with HIV-1: General approach" and "Patient monitoring during HIV antiretroviral therapy".)
In specific situations (neonatal infection and acute infection), HIV-1 RNA levels also may be useful in establishing the diagnosis of HIV infection. However, in most other situations, combination tests that detect HIV p24 antigen and HIV antibodies are primarily used. (See "Screening and diagnostic testing for HIV infection" and "Diagnostic testing for HIV infection in infants and children younger than 18 months" and "Acute and early HIV infection: Clinical manifestations and diagnosis", section on 'Diagnostic test performance in early HIV infection'.)
LABORATORY METHODS FOR QUANTITATION OF HIV-1 RNA
Available tests — There are three real-time reverse transcriptase polymerase chain reaction (RT-PCR) commercial tests that are commonly used to quantify HIV-1 RNA from plasma samples:
●The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2 (Roche Diagnostics) [8]
●The RealTime HIV-1 (Abbott Molecular)
●Aptima HIV-1 Quant Dx Assay (Hologic)
Other viral load tests had been used in the past (Amplicor Monitor, bDNA, and NucliSens). However, these have been replaced by real-time RT-PCR assays, which are more sensitive (they detect 20 to 40 copies/mL of HIV RNA), have a broad linear range (they detect virus to at least 10 million copies/mL), and pose a lower risk of carry over contamination than prior PCR assays. (See 'Quantification and linear range' below.)
Studies of the three real-time RT-PCR tests have shown excellent agreement in viral load values between the COBAS TaqMan and Abbott RealTime tests [9]. There is also very good agreement in viral load values between the Aptima test and the COBAS TaqMan and RealTime tests [10-12]. However, the agreement is decreased at the lower limit of quantification of the tests [9,13]. Thus, it is important to use the same assay when following HIV-1 RNA levels over time. If the viral load assay used to monitor a patient is changed, a new baseline viral load measurement should be obtained, especially when monitoring patients with low or undetectable viral loads.
An important difference between the real-time tests is the gene target; the COBAS TaqMan test targets both the gag gene and the long terminal repeat, the Aptima test targets the pol gene and long terminal repeat, while the Abbott RealTime test targets the integrase gene. With the increasing use of integrase inhibitors, monitoring for resistance mutations in the integrase gene is important to ensure that the primer and probe binding sites are not impacted, which could lead to under quantification of HIV-1 RNA. Active surveillance programs for this purpose have been implemented, and as of 2020, this has not been reported.
Some clinical laboratories test cerebral spinal fluid (CSF) specimens; however, this requires a separate validation process as HIV-1 RNA tests are not FDA approved for use on CSF. The use of CSF viral load testing is discussed elsewhere (see "HIV-associated neurocognitive disorders: Epidemiology, clinical manifestations, and diagnosis"). Viral load testing of other specimens (eg, dried blood spots, cervical fluid, semen) has only been used in the research setting.
Specimen collection — The real-time RT-PCR tests have been designed to measure HIV-1 RNA from plasma specimens. Finger prick samples have also been tested in the Abbott RealTime test, and due to reduced sample volume, the test had a lower limit of quantification of approximately 500 copies/mL [14].
For routine clinical use, the real-time PCR tests require between 0.5 and 1.0 mL of plasma. The blood should ideally be anticoagulated in EDTA (purple top tube) since the virus is most stable in this anticoagulant. Acid citrate dextrose (ACD) can also be used as an anticoagulant, but there may be a 15 percent decrease in the viral load measurement due to the volume of the anticoagulant [15,16]. Blood anticoagulated in heparin is unacceptable for the RT-PCR assays. It is important to follow the recommendations for specimen collection outlined in the package insert since they may differ for each test.
Due to the instability of HIV-1 RNA in whole blood, it is critical to handle clinical specimens properly to minimize the risk of RNA degradation. Plasma should be separated within six hours of collection and ideally stored at -20ºC, although plasma viral RNA is stable at 4ºC for several days. Failure to separate plasma promptly may lead to degradation of viral RNA and a falsely decreased viral load measurement [15,16]. In addition, viral load measurements may be reduced by as much as 50 percent if serum samples are used, probably due to trapping of the viral particles in the clot [16]. For this reason, plasma specimens should be used when viral load measurements are followed over time.
Plasma preparation tubes (PPTs) contain a gel barrier that physically separates plasma from the cellular components, and have an advantage in that whole blood can be collected in these tubes and held at room temperature for up to six hours prior to centrifugation and shipped in their original tube at ambient temperature without affecting the viral load values [17]. However, after centrifugation, PPTs should either be stored refrigerated or plasma should be removed from PPTs prior to freezing, as freezing the plasma samples in PPTs can falsely elevate the viral load values [18,19].
Quantification and linear range — The available assays differ in their limit of quantification and linear range. The lower limit of quantification is a key characteristic of viral load assays, and how this parameter is defined is equally important. The limit of detection is best defined as the amount of nucleic acid that can be verified in 95 percent of replicate samples. Using this definition, the limit of quantification and linear range of the assays is as follows:
●The COBAS AmpliPrep/COBAS TaqMan version 2 HIV-1 test – 20 to 10 million copies/mL
●The RealTime HIV-1 – 40 to 10 million copies/mL
●Aptima HIV-1 Quant Dx test – 30 to 10 million copies/mL
INTERPRETATION — There are several issues that must be considered when interpreting the results of HIV-1 RNA measurements. These include the clinical significance of changes in viral load, the relationship of HIV-1 RNA to the CD4 cell count, and the possible presence of HIV-1 non-B subtypes.
Clinically significant changes in viral load — To determine if changes in viral load represent clinically significant changes in viral replication both intra-assay and biologic variation need to be considered. Each of the viral load assays has a low intra-assay variation, between 0.12 and 0.2 log10, on repeated testing of a single specimen [8,11,20-22]. In general there is greater variability in the tests near the lower limit of detection. In clinically stable patients, HIV-1 RNA levels are relatively stable from week to week and month to month, provided antiretroviral therapy has not been initiated or changed. The biologic variability of viral RNA measurements is about 0.3 log10 [23].
These data taken together have led to the recommendation that changes in HIV-1 RNA levels must exceed at least 0.5 log10 or threefold in magnitude to represent biologically relevant changes in viral replication [23,24]. It is important not to over interpret small increases or decreases in viral RNA.
Exogenous factors that may affect HIV RNA levels — Viral RNA levels can transiently rise during acute illness [25], an outbreak of herpes simplex infection [26], or vaccination against a variety of pathogens including influenza, pneumococcus, and tetanus [27,28]. The increases may be quite dramatic, 1 log10 (tenfold) or greater; however, values usually return to baseline within one month. Thus, plasma HIV-1 RNA levels should not be measured within one month of any of these events.
Relationship of HIV-1 RNA to CD4 cell count — CD4 cell counts are correlated with viral RNA measurements, although the association is weak. In general, the higher the CD4 cell count, the lower the viral RNA. However, for any given CD4 cell count, there may be a 3 log10 (1000-fold) range in viral RNA measurements [1,24]. As an example, patients with a CD4 count of 200 cells/microL may have HIV-1 RNA levels ranging from 1000 to 100,000 copies/mL.
As a result, viral RNA measurements should not replace CD4 cell counts in the management of HIV-1-infected patients; rather, the two parameters should be used in parallel. CD4 cell counts are useful in determining when to initiate prophylactic therapy for opportunistic infections. (See "Techniques and interpretation of measurement of the CD4 cell count in people with HIV" and "Overview of prevention of opportunistic infections in patients with HIV".)
HIV-1 subtypes — HIV-1 is a genetically diverse virus that has been classified into three groups based upon significant differences in the sequence of the gag and envelope genes:
●Group M refers to the major group
●Group O refers to the outliers or outgroup
●Group N refers to non-M, non-O
Group M is further divided into nine subtypes, referred to as subtypes A through D, F through H, J, and K. The geographic distribution of the subtypes varies. The most common subtype in North America and Europe is subtype B; in the United States, strains other than subtype B are rare [29], although there are reports of increasing genetic diversity of HIV-1 within the United States [30,31]. Other subtypes, in particular A, C, D, and E, are commonly found in Africa and Asia [29]. A more detailed discussion on the epidemiology of HIV is found elsewhere. (See "Global epidemiology of HIV infection".)
Most commercial assays were initially developed and evaluated for samples containing subtype B. The real-time HIV-1 RNA assays accurately quantify a wide range of HIV-1 subtypes:
●The COBAS AmpliPrep/COBAS TaqMan version 2 HIV-1 test accurately quantifies all group M subtypes, group O, and many recombinants
●The RealTime HIV-1 test accurately quantifies all group M subtypes, group N, group O, and recombinants [32,33]
●The Aptima HIV-1 test accurately quantifies group M, N, and O, and many recombinants
One study that compared various commercial assays with a second generation long terminal repeat-based PCR test demonstrated a high degree of correlation using samples that were genomically and geographically diverse [34]. Any degree of discrepancy that was noted between assays was not associated with a specific subtype, but rather with the degree of genomic diversity within the subtype. A study evaluating the Aptima HIV-1 test using well characterized subtype samples demonstrated very good agreement in viral load values; 95 and 87 percent of the Aptima results where within 0.5 log10 of the COBAS TaqMan and RealTime results, respectively [35].
AVAILABILITY AND COST OF VIRAL LOAD TESTS — HIV-1 RNA measurements are widely available in clinical microbiology and reference laboratories. The cost of the assay varies from $80 to $240 per test. Cost should not be the only factor in determining where to have the testing performed; viral load assays are complex and should be performed by experienced laboratories. There are a variety of proficiency tests available to assist in assessing the performance of laboratories.
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: HIV screening and diagnostic testing" and "Society guideline links: HIV treatment in nonpregnant adults and adolescents".)
SUMMARY AND RECOMMENDATIONS
●Human immunodeficiency virus type 1 (HIV-1) RNA quantification (eg, viral load testing) is routinely used in the management of persons infected with HIV-1. (See 'Introduction' above.)
●Some studies have shown HIV-1 RNA levels to be a predictor of the time to progression to AIDS and death, independent of CD4 cell counts. Viral load measurements are also useful in determining the response to antiretroviral therapy. (See 'The use of HIV viral load measurements' above.)
●There are three real-time polymerase chain reaction tests that are widely used for viral load testing. For routine clinical use, viral load measurements are performed on plasma specimens. (See 'Available tests' above and 'Specimen collection' above.)
●Some clinical laboratories test cerebral spinal fluid (CSF) specimens; however, this requires a separate validation process as HIV-1 RNA tests are not FDA approved for use on CSF. (See 'Available tests' above.)
●In patients who are not taking antiretroviral therapy, there are small fluctuations of viral load that may be seen which are clinically insignificant. Changes in HIV-1 RNA levels must exceed at least 0.5 log10 (or a threefold change in magnitude) to represent biologically relevant changes in viral replication. (See 'Clinically significant changes in viral load' above.)
●Viral RNA levels can transiently rise during acute illness, an outbreak of herpes simplex infection, or after immunization. Thus, plasma HIV-1 RNA levels should not be measured within one month of any of these events. (See 'Clinically significant changes in viral load' above.)
●CD4 cell counts are correlated with viral RNA measurements, although the association is weak. In general, the higher the CD4 cell count, the lower the viral RNA level. (See 'Relationship of HIV-1 RNA to CD4 cell count' above.)
ACKNOWLEDGMENT — We are saddened by the death of John G Bartlett, MD, who passed away in January 2021. UpToDate gratefully acknowledges Dr. Bartlett's role as section editor on this topic, his tenure as the founding Editor-in-Chief for UpToDate in Infectious Diseases, and his dedicated and longstanding involvement with the UpToDate program.
