Types of viral load tests

Viral load assays measure HIV genetic material called RNA from virus particles called virions in the blood plasma. The three most commonly used assays for measuring viral load are:

• HIV-1 RNA polymerase chain reaction (PCR)
• Branched chain DNA (bDNA)
• Nucleic acid sequence-based amplification (NASBA)

Each test has particular characteristics, specificity, and varying costs. Results from different test methodologies may not be strictly comparable.

A fourth assay that is beginning to be used more frequently in resource-limited areas is an ELISA-based method of determining viral load by quantifying reverse transcriptase activity.

Each assay has particular characteristics and results from different test kits may not be strictly comparable.

HIV-1 RNA PCR (polymerase chain reaction) multiplies the amount of HIV RNA in a blood sample through use of an enzyme. The resulting chemical reaction marks virus and the markers are measured to calculate the amount of HIV-1 RNA. This process is called viral nucleic acid amplification.

The Amplicor HIV-1 Monitor Test 1.5 assay has a lower detection limit of 400 copies/ml and the ultra-sensitive assay can detect virus above 50 copies/ml. The upper limit of detection is 10,000,000 copies/ml. If someone’s viral load is below the limit of detection of a particular assay, an 'undetectable' result is reported; this only means that the assay was not sensitive enough to detect virus, not that HIV is not present.

When first introduced, the Amplicor assay was only able to detect HIV clade B accurately; it now can used clades A, B, C, D, E, and G. This test is FDA-approved in the United States.

In a recent study, it was reported that the COBAS AmpliPrep/COBAS Amplicor HIV-1 Monitor Test, version 1.5 (CAP/CA) consolidated and reduced labour while slightly increasing assay throughput over the COBAS Amplicor HIV-1 Monitor Test, version 1.5.¹

The Abbott RealTime HIV assay can quantify virus from 10,000,000 to 40 copies/ml, as can another RT assay, the COBAS AmpliPrep-COBAS TaqMan HIV-1 test. The LCx HIV RNA Quantitative assay by Abbott also shows comparable results.²

A branched DNA (bDNA) assay uses a substance that produces light when it combines with HIV particles. The amount of light is measured and converted to a viral count. The test is marketed as Versant HIV-1 RNA 3.9 Assay by Bayer. The branched-chain DNA (or bDNA for short) test, marketed under the trade name Quantiplex 3.0 in Europe or Versant 3.0 in the United States, has a lower detection limit of 50 copies/ml and an upper limit of 500,000 copies/ml. It is FDA-approved in the US.

This assay can also be used for clades A, B, C, D, E, and G and has the advantage of less risk of contamination than PCR. 

Nucleic acid sequence based amplification (NASBA) is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature. 

The main advantage of this technique is that it works at isothermal conditions, usually at a constant temperature of 41°C. NASBA has been shown to give faster results than PCR and can, in addition, also be more sensitive.

The NucliSens HIV-1 QT by bioMerieux has a cut-off of 400 copies/ml and can be used for clades A, B, C, and D. An advantage of this technique is that it can be used for all biological fluids. In the US, this test is FDA-approved.

The Cavidi ExaVir Load assay is an ELISA-based method of determining viral load by measuring reverse transcriptase (RT) activity. RT is an essential enzyme for viral replication and is present in all HIV-1 and HIV-2 subtypes. As RT is unaffected by mutations in virus, this assay can detect all HIV types and subtypes, including O- and N-group. Because this assay uses standard ELISA equipment with ExaVir Load start-up equipment, it allows for viral load testing in simple laboratory environments.

The ExaVir Load assay, version 2 by Cavidi is a manual RT assay that performs well when compared to commercial viral load assays. Its sensitivity is close to that of RT-PCR. One advantage to this test is that it can be used with smaller sample sizes, a useful quality for paediatric use.³

The ExaVir Load version 3 is relatively affordable and 30% faster than the previous version, capable of 50% greater throughput per week, but equipment for each version is not interchangeable. Although this assay was designed for measuring RT activity and for in vitro diagnostic use, it has been used in research settings to diagnose HIV in infants.⁴ Version 3 has improved sensitivity compared to version 2, according to a study of its use for viral load monitoring.⁵

Performance of the Cavidi ExaVir version 2 RT assay was compared to that of the gold standard Roche RNA PCR and Bayer Versant HIV-1 RNA (bDNA) assays in its ability to assess virological failure in 100 Kenyan patients initiating antiretroviral therapy. At 48 weeks, there was greater than a half log biologic limit of agreement, meaning that the RT assay could not be used interchangeably with the others in one individual. 

The RT assay had 100% sensitivity in detecting viral load equal to or greater than 400 copies/ml. It also had a low positive predictive value (86% compared to RNA PCR and 70% as compared to bDNA) in groups with a low prevalence of virologic failure. Through an agreement between the Clinton Foundation and Cavidi Tech, the RT assay can be done at 15% of the cost of other assays.⁶

An Australian study comparing performance use of the Cavidi ExaVir RT assay to those of the Roche COBAS Amplicor RNA PCR assay in adults and children found that results between the assays were comparable. Further, smaller volumes of blood than the recommended 1ml could be used when doing the RT assay in children.³

Virco has developed a genotypic and phenotypic recombinant virus assay to determine resistance against integrase inhibitors. The assay amplifies HIV-1 integrase, reverse transcriptase, and RNAseH. The overall success of the assays was highest for samples with a viral load above 3 log copies/ml and works for both B and non-B HIV subtypes.⁷