There are two main types of viral load tests: molecular techniques and viral culture.

Molecular techniques, known as polymerase chain reaction (PCR) assays, are most commonly used in United Kingdom clinics. PCR assays measure HIV genetic material called RNA from virus particles called virions in the blood plasma. There are two main assays: the Roche PCR assay and the Chiron / Bayer bDNA assay. Each test has particular characteristics, and results from different test kits may not be strictly comparable.

Amplicor HIV-1 Monitor

The Roche PCR test is called the Amplicor HIV-1 Monitor. The standard Amplicor assay has a lower detection limit of 400 copies/ml, whereas the ultra-sensitive assay can detect virus above 50 copies/ml. The upper limit of detection is 75,000 copies/ml. If a person's viral load is below the limit of detection of a particular assay, they will receive an 'undetectable' result; this does not mean they have no virus in their blood, but that the assay could not detect any virus. The ultra-sensitive test is increasingly used in clinics in the United Kingdom, and the British HIV Association (BHIVA) treatment guidelines recommend that the test be used routinely.

When first introduced, the Amplicor assay was only able to detect HIV sub-type B accurately. People infected with other sub-types and tested with the Amplicor assay in the past may have shown inaccurately low levels of viral load. However, the Amplicor assay has now been adjusted, and now detects subtypes B, C and D. There is still some doubt about its ability to accurately detect other subtypes. Preliminary research carried out by the Public Health Laboratory Service suggests that all viral load tests may be less sensitive to sub-type A than to other HIV sub-types.

Quantiplex / Versant 3.0 bDNA assay

The branched-chain DNA (or bDNA for short) test, marketed under the tradename 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. The standard version of this test is the Quantiplex 2.0 bDNA assay that has a lower limit of detection of 500 copies/ml. This assay was developed by Chiron Diagnostics which is now owned by Bayer.

The Quantiplex 3.0 assay was previously considered less accurate at detecting low levels of HIV than the Amplicor assay. However, there is evidence that the ultra-sensitive Quantiplex 3.0 assay may be more sensitive than the Amplicor ultra-sensitive test for measuring low levels of HIV. A study of 130 blood samples from people on treatment found that the Quantiplex 3.0 test detected virus in 82% samples, compared with Amplicor assay which found virus in only 58%. Thirty-four samples which were below 50 by the Amplicor test were above 50 copies/ml on the bDNA test (Erice 1999).

A comparison of the Quantiplex and Amplicor 1.5 assays found that across all sub-types, the bDNA assay was more reliable than the PCR assay. Much less variation was seen when multiple samples of the same sub-type were tested with bDNA than with PCR. The study also found significant variation between laboratories in the reading given by the Amplicor assay. This did not occur with the bDNA assay (Elbeik 2002).

The Quantiplex 3.0 test is easier to carry out, cheaper and has been validated for the accurate quantification of viral load in people infected with all sub-types of HIV. For these reasons the Quantiplex 3.0 test has been adopted by a number of clinics which previously used the Amplicor assay.

Other assays

The Organon-Teknika (NASBA) assay has a lower detection limit of 500 copies/ml. The lower limit of detection for Organon-Teknika's Nuclisens assay is 40 copies/ml. Comparing the three tests, a team from British Columbia found that sensitivity was greater with the Quantiplex 3.0 and Amplicor assays than the Organon-Teknika test (Sherlock 1999).

The Amplicor and Quantiplex (bDNA) assays appear to be equally accurate in measuring non-B subtypes of HIV, which are more likely to be found in people infected outside Europe or North America. However, the NASBA assay is less accurate when measuring non-B subtypes.

In July 2004, the development of a new technique that detects HIV viral load as low as two copies/ml was announced. Called real-time immuno-PCR, this technique looks for an inner protein of HIV called p24, and can detect HIV infection as soon as one week after infection. However, the test is not yet commercially available and needs to be validated in clinical trials (Barletta 2004).

Ultra-sensitive tests

It is now widely accepted that ultra-sensitive viral load tests are more useful in assessing the effects of treatment than standard assays. In a substantial proportion of people whose viral load is below 400 copies/ml, the true level of virus is between 50 and 400 copies/ml i.e. it is detectable with a more sensitive test. Several studies have found that people who achieve viral suppression below 50 copies/ml tend to sustain viral suppression for much longer than people whose viral load remains in the 50 to 400 copies/ml range. The long-term health benefits of sustaining viral load below 50 copies/ml have not been proven.

The accuracy of undetectable results is limited. There is a five-fold variability in viral load measurements at values lower than 30,000 copies/ml. This means that a person's real viral load may be five times higher, or five times lower, than indicated by a test result. This explains why viral load may appear to rebound temporarily above the limit of detection; no real increase in virus production may have occurred, and what is being seen may be the normal margin of error of the test.

See Viral load blips in Anti-HIV therapy: Changing treatment for further discussion of temporary increases in viral load above the limits of detection.

Viral cultures

The other type of viral load test involves HIV being grown or cultured in the laboratory. To do an HIV viral culture, blood from a person with HIV infection is separated into blood cells (with lymphocytes containing HIV) and the non-cellular part of blood (plasma containing virions of HIV). Both parts can be introduced into donor human cells to grow HIV artificially in the laboratory and the quantity of new HIV produced after (usually) two weeks can be estimated with fair accuracy. Both cell and plasma cultures produce measurements with roughly the same significance when used as prognostic markers.

References

Barletta JM et al. Lowering the detection limits of HIV-1 viral load using Real-Time Immuno-PCR for HIV-1 p24 antigen. Am J Clin Pathol 122: 20-27, 2004.

Elbeik T et al. Comparative analysis of HIV-1 viral load assays on subtype quantification: Bayer Versant HIV-1 RNA 3.0 versus Roche Amplicor HIV-1 Monitor version 1.5. J Acquir Immune Defic Syndr 29(4): 330-9, 2002.

Erice A et al. Comparison of ultrasensitive assays for quantification of HIV RNA in plasma samples. Sixth Conference on Retroviruses and Opportunistic Infections, Chicago, abstract 149, 1999.

Sherlock CH et al. Comparison of copy numbers generated by 3 commercial highly sensitive HIV viral load measurement systems. Sixth Conference on Retroviruses and Opportunistic Infections, Chicago, abstract 145, 1999.