Viral load testing. medical monitoring: viral load testing: 1

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1 medical monitoring: viral load testing: 1 medical monitoring: viral load testing Viral load testing

2 medical monitoring: viral load testing: 2 Slide 1 Viral load The viral load test measures HIV in the blood As the amount of HIV in the blood rises, more CD4 cells are lost Viral load rises very high just before a person becomes sick due to HIV viral load testing: 1 Viral load What is viral load? Viral load refers to the amount (the number of copies) of HIV genetic material (RNA) circulating in the blood plasma (the fluid that carries blood cells). A viral load test is therefore a blood test. A viral load test indicates the number of copies of HIV RNA per millilitre of blood. Viral load test results can range from less than 50 copies to more than 1 million copies. Some experimental tests used in research can detect as little as 5 copies per millilitre.

3 medical monitoring: viral load testing: 3 Slide 2 Natural history of disease progression key to symbols viral load (level in blood) CD4 count (level in blood) set point below the limit of the test viral load testing: 2 Natural history of disease progression Viral load is an indicator of disease progression. It shows how much virus is available to damage the immune system, and it can therefore be used to predict speed with which CD4 cells will be lost. Viral load steadily increases over time if no ART is used. The higher the viral load, the more damage the virus can cause to the immune system, and the more CD4 cells may be lost as a result. The lower someone s viral load, the less likely they are to get sick in the near future.

4 medical monitoring: viral load testing: 4 Slide 3 Predictors of disease progression Imagine HIV is a train heading for Paris. The time the train will arrive depends on both the speed of the train (viral load), and the length of the track left before Paris (CD4 count). viral load testing: 3 Predictors of disease progression Imagine HIV is a train heading for Paris. The time the train will arrive depends on both the speed of the train (viral load), and the length of the track left before Paris (CD4 count).

5 medical monitoring: viral load testing: 5 Slide 4 Key facts about viral load (1) Viral load of 100,000 is high Viral load of 10,000 is low When viral load is high, the risk of HIV transmission is greater viral load testing: 4 Key facts about viral load A viral load greater than 100,000 is considered to be high. A viral load less than 10,000 is considered low. Most people follow a pattern of: high viral load and low CD4 count, or low viral load and high CD4 count, However, a minority of people have discordant results which do not to fit the overall pattern (for example, high CD4 and high viral load). People who become HIV-positive over 50 years of age usually have lower CD4 counts, higher viral loads, and greater risk of disease progression than younger people. The reasons for this are unknown, though it may be due to the presence of other diseases in older people. Viral load and transmission If there are high levels of virus in the blood, there are also likely to be high levels of virus in semen or vaginal fluid. Therefore, people with high viral loads are likely to be more infectious. People with undetectable blood levels of virus might also continue to be infectious; however, because viral load in blood, semen and vaginal fluid does not match exactly. Although the risk of mother-to-baby transmission is very low in women with undetectable viral load, transmission can still occur. Similarly, it is possible to pass HIV to a sexual partner through exposure to semen or vaginal fluid, despite having an undetectable viral load.

6 medical monitoring: viral load testing: 6 Slide 5 Key facts about viral load (2) Viral load may be lower in women, so important to pay attention to CD4 count Viral load provides additional information to CD4 count but CD4 most important laboratory measurement viral load testing: 5 Key facts about viral load Viral load in women HIV viral load stabilises at a lower level in women following seroconversion. Among men, a low viral load following seroconversion is associated with less risk of disease progression. This is not the case for women. Early research into viral load and disease progression looked mainly at male-only cohorts of gay men and haemophiliacs. It was assumed that there was no difference in the way disease progressed in men and women. However, more recent research shows that women s viral loads are significantly lower compared to men with the same CD4 count. The reason for the sex difference in viral load and disease progression risk is unknown. A number of theories have been suggested, including: superior immune response to viral infections in women due to hormonal factors, reduced viral production in women, or exposure to low amounts of HIV due to mode of transmission. Viral load provides additional information Viral load provides additional information in untreated people in itself it is not sufficiently predictive because measurements can change considerably from one test to another, especially if blood is not stored properly or the test is not sufficiently sensitive to the subtype of HIV. The level of viral load is likely to predict the speed at which the CD4 cell count will fall, when considered in combination with the CD4 cell count.

7 medical monitoring: viral load testing: 7 Slide 6 MACS study Multi Center AIDS Cohort Study (MACS) Viral Load CD4 Count Below Above 750 Below 1500 ** ** ** ,501-7,000 ** ** ,001-20,000 ** ,001-55, above 55, Percentage of people who develop AIDS within three years (assuming no treatment). Viral load levels relate to results using RT-PCR test method, such as Roche Amplicor test. ** Indicates lack of data viral load testing: 6 MACS study The US Multicenter AIDS Cohort study (MACS) is the largest study which has explored the relationship between viral load and the natural progression of HIV infection if HIV remains untreated. The study enrolled over 1,600 symptom-free HIV-positive patients between 1984 and Blood samples were taken and frozen when they joined the trial and at six monthly intervals. Ten years later, when viral load testing became available, researchers went back to these samples and measured the viral load, comparing each individual s test results with his medical history. In this way, the researchers were able to build a picture of how future risk of illness varies according to a range of viral load and CD4 levels. This is shown in the graph which accompanies this section. The MACS study found that two people with the same CD4 count but different viral loads, might have a very different prognosis. The person with the higher viral load would be more likely to progress to AIDS faster. For example, at a viral load above 55,000 copies and a CD4 count above 750, there was a 32.6% chance of disease progression within three years. At the same viral load level but with a CD4 count below 200, the risk was 85.5%. Conversely, at viral loads below 7,000 copies, there was a very low risk of developing symptomatic disease (less than 3.7% at any CD4 level). Information from this study is still used in treatment centres to help with decision-making about ART. However, the results represents general trends and they are not a rule book which can be applied to all people with HIV. It is also important to remember that the study looked at the risk of disease progression in untreated people, and its findings cannot be applied to people whose test results have been influenced by the use of ART. The graph shows the percentage of people who might be expected to develop AIDS within three years (assuming they took no anti-hiv treatment), based on given viral loads and CD4 levels. Viral load measurements are related to results using the RT-PCR testing method, such as the Roche Amplicor test.

8 medical monitoring: viral load testing: 8 Slide 7 Viral load testing during treatment (1) May be measured just before starting, to get baseline' measurement Viral load should be `undetectable` after six months of treatment Undetectable = virus levels below the limit of detection of test Undetectable does not mean no virus viral load testing: 7 Viral load testing during treatment Where the test is available, viral load may be tested immediately before treatment is started, to provide a baseline measurement. If viral load is undetectable after six months, this is a sign that treatment is successfully controlling virus activity. The normal limit of detection is 50 copies or 400 copies. A viral load test with a limit of detection of 50 copies is more sensitive. However undetectable viral load does not mean that virus activity has stopped. It means that the test cannot detect virus levels of les than 50 copies.

9 medical monitoring: viral load testing: 9 Slide 8 Viral load testing during treatment (2) If virus levels rise above limit of detection, treatment may be failing Small increase (not above 1,000 copies) may be a temporary blip due poor adherence or minor infection Test again If viral load continues to rise, treatment is failing viral load testing: 8 Viral load testing during treatment If viral load subsequently rises above the limit of detection, the viral load test should be carried out again, since viral load can sometimes rise and fall for no important reason. If the second result is below the limit of detection, treatment does not need to be switched. If viral load has risen again, it is best to switch treatment as quickly as possible in order to reduce the risk of developing drug resistance. A rising viral load will usually be the first warning that treatment is failing. The sooner that treatment can be changed, the smaller the risk that the patient will develop drug resistance. If the CD4 count falls during treatment, it may be useful to test viral load to check whether this fall is caused by the failure of treatment or not. Factors affecting viral load With viral load tests (and with CD4 tests) it is the trend of results over time that matters, rather than the results of a single test. A temporary rise in viral load can be caused by various factors, especially things that stimulate the immune system, such as vaccinations. In a person not receiving ARV treatment, an opportunistic infection will cause a temporary tenfold increase in viral load, which will fall again when the infection has been successfully treated. The viral load (and CD4 count) will be different at different times of the day. It should therefore be measured each time at a similar time of day. It is also possible that viral load changes throughout the menstrual cycle in women.

10 medical monitoring: viral load testing: 10 Natural variations Researchers have investigated viral load changes in people who are not receiving ART. They have found that when levels are low (under 30,000 copies per ml) results from two separate tests on the same sample of blood can differ by a factor of five. For example, one sample might give a value of 5,000 and another sample a value of 25,000. In people receiving ARV treatment, viral load may increase temporarily above the limits of detection of the test if a person has an infection, or misses a dose or two of medication immediately before the test. The viral load may return below the limits of detection of its own accord within a few days without the need to change to treatment. However, if the viral load continues to rise, this is a sign that treatment is failing, and it should be changed as soon as possible in order to reduce the risk of developing resistance.

11 medical monitoring: viral load testing: 11 Slide 9 Log scale 1.0 logs logs logs logs 316 viral load testing: 9 Log scale Log changes Viral load test results are often written in a form known as the logarithmic scale, or logs. The logarithmic scale is used because it is a useful way for researchers to present trial data which include a wide range of values, from very high ones to very low ones. A one log change is a ten-fold change. For example, with a baseline load of 10 copies, a ten-fold increase would mean that the viral load rises to 100 copies (a one log change), a 2 log increase (a hundred-fold change) would see the viral load rise to 1,000 copies, a 3 log increase (a thousand-fold change) would see the viral load rise to 10,000 copies. Log values can be computed with a calculator, but here are a few examples: 1.0 logs = logs = logs = logs = logs = 1, logs = 3, logs = 10, logs = 31, logs = 100, logs = 316,228

12 medical monitoring: viral load testing: 12 Slide 10 HIV sub-types and viral load testing Numerous sub-types, or families, of HIV Viral load tests developed to recognise sub-type B in Europe, N America Not as sensitive to other subtypes? HIV-2 in West Africa: some viral load tests do not recognise viral load testing: 10 HIV sub-types and viral load testing Globally, HIV has evolved over time into a number of sub-types, or virus families. People who were infected with HIV in North America and Europe usually have HIV-1 sub-type B. People with HIV from other parts of the world (the majority of infected people worldwide) carry other sub-types. This has caused problems in the past with monitoring viral loads and tests for drug resistance,. Kits developed for use in North American and European markets have not always been sensitive to HIV sub-types found in other parts of the world. Fortunately this problem is gradually being resolved, and people with non-b HIV-1 sub-types are more likely now to receive accurate blood test results. However, this problem might reappear when new tests are developed in the future. Tests for HIV-2, which occurs in West Africa, have not been commercially available until recently. Some are now able to detect all subtypes of both HIV-1 and HIV-2, but others are still limited in the ranges of subtypes which they can detect.

13 medical monitoring: viral load testing: 13 Slide 11 Types of viral load test kit Roche PCR Amplicor HIV-1 Monitor Lower Limit of Detection: 50 copies. Bayer bdna Versant 3.0 Lower Limit of Detection: 50 copies. Organon Technika Nuclisens Lower Limit of Detection: 40 copies. viral load testing: 11 Types of viral load test kit There are two main types of viral load test: those that employ molecular techniques and those that use viral culture. The commercially available tests are molecular techniques, and most of these use quantitative PCR, or branchedchain DNA. Each test works in a slightly different way, but all tests look for HIV s genetic material. Because the results for different types of test may not be strictly comparable, it is best to ensure that the same type of test kit is always used to measure a patient s viral load using the same type of test kit. It is important to remember, and to tell the patient, that when a viral load result is classified as undetectable, it only means that the viral load is below the limit of detection of the testing kit. It does not mean that there is no virus in the patient s blood; The Roche PCR test is called the Amplicor HIV-1 Monitor. This detects HIV sub-types B, C and D,.but there is some doubt about its ability to detect other sub-types There have been two versions of this kit: the standard kit, with a lower detection limit of 400 copies and an upper limit of 750,000 copies the ultrasensitive kit, with a lower detection limit of 50 copies and an upper limit of 100,000; this version is used to monitor people with HIV in well-resourced settings such as the UK. A branched-chain DNA (bdna) test is marketed by Bayer under the name Versant 3.0. This is an ultrasensitive kit with a lower limit of 50 copies, and an upper limit of 500,000 copies. It has been validated for accurate detection of viral load in people infected with all sub-types of HIV. The Versant 3.0 is cheaper and easier to carry out and there is some evidence that it is better at detecting very low levels of virus. The Organon Technika Nuclisens test has a lower detection limit of 40 copies. It has been suggested that this test is less sensitive than both the Roche and Bayer tests and that it is less accurate at measuring non-b sub-types.

14 medical monitoring: viral load testing: 14 Comparing results Results from the older, less sensitive viral load kits, such as the standard Amplicor HIV-1 Monitor (lower detection limit 400 copies) and the Quantiplex 2.0 (lower detection limit 500 copies), could not be compared. A reading on the Quantiplex 2.0 needs to be doubled to get a comparable reading on the Roche kit, or divided to convert in the opposite direction.

15 medical monitoring: viral load testing: 15 Slide 12 Disadvantages Still costly Requires well-equipped laboratory, reliable electricity supply Requires considerable training of lab staff Some tests may not capture virus subtypes common in Africa and Asia very accurately Blood samples must be refrigerated and spun to separate plasma Accuracy of test will fall quickly if any delays in refrigeration Secure and reliable transport of blood samples viral load testing: 12 Disadvantages Although the Clinton Foundation has negotiated a reduction in the cost of two viral load tests, the tests are still costly. They also require a well-equipped laboratory, reliable electricity supply and highly trained staff who can handle a high volume of samples. It is not worth establishing a viral load testing laboratory unless it will be well used. Some tests may not capture virus subtypes common in Africa and Asia very accurately, particularly at low viral load levels (which are precisely the levels at which the tests need to be accurate if their main use is for monitoring treatment). In clinics where viral load might be used, a number of conditions would need to be met before the current generation of tests could be used in resource-limited settings. Blood samples must be refrigerated soon after they are drawn. Accuracy of the viral load test will fall quickly if any delays in refrigeration occur, or if there are any irregularities in the way that the blood is treated between leaving the patient s body and being used in the test. Secure and reliable transport of blood samples is also essential, so that blood samples do not go missing or become subjected to high temperatures.

16 medical monitoring: viral load testing: 16 Slide 13 Alternatives to viral load Cheaper options now being explored: P24 antigen Reverse transcriptase Dried whole blood spots viral load testing: 13 Alternatives of viral loads Viral load tests - reducing the cost One approach, as with CD4 tests, is to make viral load tests more affordable, through developing new technologies. However, all PCR-based tests depend on a high level of skill and may remain expensive. The other approach is to look at alternative ways of measuring the amount of the virus activity in blood samples. p24 antigen as an alternative to viral load The p24 protein is the main HIV core protein. Infected cells make it in larger quantities than other HIV proteins and therefore it is the easiest to detect. However, wwhen antibodies appear in the blood, they mask the p24 and make it harder to detect. There is now a commercial test kit for p24, from PerkinElmer Life Sciences, based on using heat treatment to separate antibodies that would otherwise mask p24 in the blood of people with HIV. In principle, it is possible to measure the production of the p24 as an alternative to viral load. However, even though the results of p24 tests might be as consistent as, or even more consistent than, viral load tests; they are not measuring exactly the same thing. Clinical studies are required to find out which of the tests is more relevant when a person is receiving ART. The two main advantages of the p24 test are its lower cost and the fact that samples can survive longer transport times, in more difficult conditions, without affecting the reliability of the test. Research continues, in order to validate the test in populations with non-subtype-b viruses that are commonly found in Africa and Asia. Measuring reverse transcriptase activity ExaVir LOAD is a test system from Cavidi Tech (Sweden) that measures levels of reverse transcriptase, the enzyme that defines retroviruses including HIV-1 (Braun).This test can measure the number of retrovirus particles in blood plasma, providing a closer equivalent to viral load testing than p24. It is much cheaper than RNA viral load testing, costing $15 per test, which the company hopes to reduce to $10. The test works equally well for HIV-1, regardless of subtype, and for HIV-2, but it cannot distinguish between them.

17 medical monitoring: viral load testing: 17 The biggest limitation is that it has a lower limit of detection between 350 and 700 copies per mm3 (the company claims 2,000). It requires a large sample - 1ml of plasma - which is a potential problem when diagnosing HIV in newborn babies. It also requires an overnight incubation stage, which requires a well equipped laboratory, and the kit must be stored in a freezer before use. Viral load test by dried blood spot The Retina Rainbow test has been developed by Primagen, a Dutch company. It uses dried blood spots or dried breast milk spots. This system is very convenient and cheap, because it does not need any refrigeration, special storage or transportation. The test is able to detect all HIV-1 subtypes, although accuracy seems to be higher for HIV-1 subtype B.

18 medical monitoring: viral load testing: 18 Slide 15 Questions 1. What does the viral load test measure? 2. When does it provide the most useful information? 3. What is undetectable viral load? 4. Can HIV be transmitted by someone with undetectable viral load? 5. At what level of viral load is treatment likely to be failing? 6. What should be the response to possible treatment failure? viral load testing: 14 Questions 1. What does the viral load test measure? The amount of HIV in the blood. High levels of HIV show that CD4 cells are likely to be lost more quickly, increasing the risk of illness. 2. When does it provide the most useful information? When the CD4 cell count is falling rapidly, and once a person has begun antiretroviral treatment. 3. What is undetectable viral load? Undetectable viral load means that levels of HIV in the blood are too low for the test to detect I.e below copies. 4. Can HIV be transmitted by someone with undetectable viral load? Yes, most likely to happen if they have a sexually transmitted infection that will increase levels of HIV in the genital fluids and blood. 5. At what level of viral load is treatment likely to be failing? If viral load rises above the limits of detection of the test, especially if it rises above 500 1,000 copies 6. What should be the response to possible treatment failure? Test viral load again if it is below copies, if above, refer immediately for treatment change.

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