Sysmex Educational Enhancement and Development No

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1 SEED Haematology No Introduction to the basics of CD4 and HIV Viral Load Testing The purpose of this newsletter is to provide an introduction to the basics of the specific laboratory tests that are a core component of HIV management and treatment programmes. It is specifically aimed at those individuals that are involved in conducting these tests but have not necessarily received any formal laboratory based training. Key words: HIV, CD4, CD4%, viral load, HIVVL, resistance, ART, HIV DNA PCR, EID The current global HIV landscape HIV/AIDS remains one of the world s most significant public health challenges, with the vast majority of affected individuals living in low- and middle-income countries, with the continent of Africa carrying the highest burden. As a result of united global efforts, access to antiretroviral therapy (ART) has been majorly expanded and consequently, HIV-positive people now live longer and healthier lives. Expanding access to treatment not only serves to improve the health of the individual, but also prevents onward transmission of HIV. In 2013 it was estimated that 35 million people are living with HIV worldwide, of which 85% are eligible for ART (based on the WHO 2013 consolidated guidelines for the use of antiretroviral drugs). As only 12 million people had access to treatment, reaching the goal of treatment for all remains a major challenge. In December 2013, buoyed by the success of what has already been achieved, UNAIDS issued what they have called an ambitious treatment target to end the AIDS epidemic referred to as the targets: By 2020, 90% of all people living with HIV will know their HIV status By 2020, 90% of all people with diagnosed HIV infection will receive sustained antiretroviral therapy By 2020, 90% of all people receiving antiretroviral therapy will have viral suppression. The World Health Organisation (WHO) 2013 consolidated guidelines highlight the key role that CD4+ T-cell count plays in the evaluation of all patients newly diagnosed with HIV infection. Moreover, viral load testing is now recommended as the preferred approach to monitoring ART success and diagnosing treatment failure, complementing clinical and immunological monitoring of people receiving ART. Previously this was almost exclusively monitored with CD4 counts. It is widely believed that the most feasible means to expanding access to ART is to decentralise testing, hence there is a major drive within industry and research

2 2 organisations to fast track the development of point of care testing platforms for both CD4 and HIV viral load (HIVVL) testing. Several small testing devices, such as the Sysmex- Partec CyFlow minipoc CD4 analyser (see Fig.1), are already commercially available which have contributed significantly and will continue to facilitate the rapid expansion of CD4 testing. A consequence of this expansion of testing beyond the realm of the central laboratory into remote healthcare centres and even non-laboratory based settings is that the operators will most likely not be skilled professionals whose sole responsibility is laboratory testing. Aligned with the push towards the targets, the number of nonlaboratory personnel involved in all aspects of laboratory testing is expected to grow exponentially. The basic pathogenesis of HIV infection The immune system is comprised largely of cells called lymphocytes. The two main types of lymphocytes are B-cells and T- cells. There are two types of T-cells: CD4 cells - these cells have molecules called CD4 on their surface. They start the immune response that protects the body from invasion by bacteria and viruses. CD8 cells - these cells have molecules called CD8 on their surface. They destroy other infected cells and produce antiviral substances that fight off infectious organisms. The pathogenic mechanisms of HIV infection are complex. The human immunodeficiency virus primarily targets CD4+ T-cells, attaching itself to the surface and gaining entry into the cell where it starts to multiply. In the early stages of infection, viral replication is prominent resulting in rapid and extensive depletion of CD4+T-cells. This leads to massive viraemia and widespread dissemination to all lymphoid tissues of the body. By the time an individual has developed antibodies to the virus and tests positive for HIV with conventional serology tests, the infection is already well-established. Fig. 1 Partec CyFlow minipoc analyser The body produces billions of new CD4+ cells to replace those that have been destroyed. If left untreated, infection with HIV will however result in the progressive deterioration of the immune system, breaking down the body s ability to fight off infections and other diseases. AIDS (Acquired CD4 + T Lymphocyte Count (cells/μl) Acute HIV syndrome Primary Wide dissemination of virus Death Infection Seeding of lymphoid organs Opportunistic Diseases Clinical Latency Constitutional Symptoms Weeks Years HIV RNA Copies per ml Plasma Fig. 2 The relationship between CD4 count and HIV viral load as markers of HIV disease progression in untreated individuals (Adapted from Pantaleo, Graziosi and Fauci 1 )

3 3 Immune Deficiency Syndrome) refers to the most advanced stages of HIV infection, defined by the occurrence of any of several opportunistic infections or related cancers. The treatment of HIV infection As ongoing viral replication is directly linked to progressive loss of CD4 cells in both function and number, the mainstay of treatment is to disrupt the viral lifecycle. A vast body of research data has identified that effective viral suppression requires the use of a cocktail of drugs, collectively referred to as antiretrovirals (ARVs), the name reflecting the family Retroviridae to which HIV belongs. As these drugs and the tests needed to monitor patients on ART were initially prohibitively expensive for wide scale implementation, treatment was initially reserved for individuals with advanced immunosuppression. The WHO has development comprehensive guidelines on how to determine who is eligible for enrollment into ART programmes. Although other clinical factors are considered, the mainstay of such selection is the CD4 count. The initial recommended threshold was a CD4 count of 200 cells/µl but this has been progressively raised, first to 350 and now 500 in the 2013 WHO guidelines. A combination of a reduction in costs of both drugs and laboratory tests, based on economies of scale of mass production, and an increased global commitment to fighting HIV with concomitant increase in funding has made this possible. The CD4 count The CD4 count is the most important laboratory indicator of immune function in HIV infected individuals. Once an individual is confirmed to be HIV positive, a CD4 test is performed to assess whether the person is eligible for ART. It also serves to identify those patients with more advanced immunosuppression who are at risk of developing opportunistic infections and need to be initiated on prophylactic antibiotic treatment. The CD4 count can be expressed as an absolute CD4 count or as a CD4 percentage (CD4%) count. What is the absolute CD4 count? The absolute CD4 count is a measurement of how many functional CD4 T-cells are circulating in the blood. The lower the absolute CD4 count, the weaker the immune system. The absolute CD4 count is measured by a simple blood test and is reported as the number of CD4 cells per µl of blood. HIV negative people typically have absolute CD4 counts of between 600 and 1200 CD4 cells/µl. Lymphocytes, including CD4 T-cells are typically higher in children than in adults. Neonates can have CD4 counts as high as 5000 cells/ µl. These high values gradually decline and approximate adult levels by the age of 5 years. The CD4 count in HIV infected individuals is highly variable and would depend on the degree of immunosuppression. Values will mostly be less than 500 cells/µl and less than 200 cells/µl in those that have AIDS. The absolute CD4 count is a calculated value based on the total white blood cell count (WBC) and the percentages of CD4 T-cells. When performing an absolute CD4 test, typically, the total WBC and the percentage of CD4 cells of the total WBC (not to be confused with CD4% count described below) are measured. The absolute CD4 count is then calculated from these two values. The absolute CD4 count may fluctuate in individuals or may be influenced by factors that may affect the total WBC count and lymphocyte percentages, which for example, may occur in the presence of infections. What is the CD4 percentage count? CD4% represents the portion of total lymphocytes that are CD4 cells. The CD4% is measured using the same blood test as the absolute CD4 count. Typically, HIVnegative people will have a CD4% of about 40% (with a wide normal range of about 25-55%). The CD4% of HIV infected individuals is usually less than 30%. Table 1 below shows an approximate relationship between CD4% and absolute CD4 count values.

4 4 Table 1 The approximate relationship between CD4% and absolute CD4 counts Absolute CD4 count ~200 cells/µl ~12 15% ~350 cells/ µl ~22% ~500 cells/ µl ~25 30% CD4 percentage count As more and more countries adopt the 2013 guidelines, the role of the CD4% count is likely to diminish. Those individuals that have a CD4 count above the treatment threshold should have their CD4 counts monitored regularly with the most widely adopted time interval being 6 months. Whilst the absolute CD4 count can be quite variable, the CD4% tends to be much more stable. An unexpected drop in the absolute CD4 count without any change in the CD4% count is not clinically significant. Absolute CD4 versus CD4% count when does one use which test? Initiation of ART Absolute CD4 counts are used to assess the baseline immune function of HIV positive individuals at the initial clinic visit. The actual value at which treatment will be started will depend on individual country guidelines, not all of which have the resources to adopt the latest WHO recommendation of <500 cells/µl. This is applicable for adults and children over the age of 5 years. Because of the very high absolute CD4 counts in children, and their inherent fluctuation, CD4% counts are preferred for children. As the 2013 WHO guidelines recommend treatment for all children under the age of 5 years, irrespective of CD4 count, they do not refer to a specific CD4% value that would be equivalent to the absolute CD4 cut-off value of 500 cells/ µl. The 2010 guidelines state that all children less than 1 year should be treated, irrespective of CD4 count and for those from 2 to 5 years, at a CD4% of <25% or an absolute CD4 count of <750 cells/µl. Monitoring of CD4 counts Once ART has been commenced, it is important to monitor the response to treatment. The frequency of testing may differ based on individual country guidelines. Because depletion of CD4 T cells is the hallmark of HIV infection and the apparent source of the central immune defect of HIV disease, determination of the CD4 count (or CD4% for children under the age of 5 years) has been the most important laboratory marker of disease progression. With treatment the CD4 count is expected to rise. For most patients on therapy an adequate response is generally defined as a rise of about 50 to 150 cells/µl during the first year of treatment. Thereafter a 50 to 100 cell/µl increase per year may be observed until a steady state level is attained. Patients that have a very low CD4 count when they start ART may have a blunted increase in their cell counts despite viral suppression. HIV Viral Load HIV viral load (HIVVL) is a measure of how many virus particles are present in blood. Testing is most commonly performed on the plasma component of blood. There are several commercial kits available for HIVVL testing all based on molecular techniques. Whilst they all differ slightly in which part of the virus is targeted, they all follow the same broad principle of amplification, detection and quantification steps.

5 5 What does an HIVVL tell us? The goal of ART is to suppress viral replication long-term. Viral load is a marker of response to ART. A patient s pre-treatment viral load level and how much and how quickly it falls post initiation of ART will provide an indication of the likelihood of disease progression. If viral load does not decline as expected, and non-adherence to treatment has been ruled out, then it is an indication that the treatment is not effective and a switch to an alternate drug regimen should be considered. If left untreated or if treatment is ineffective, uncontrolled viral replication can produce over a billion viral copies per day with ongoing destruction of the immune system. Where it is available, viral load testing, in conjunction with CD4 testing, is a standard tool to monitor response to ART. However, due to the complexity and cost of the test, most countries have not been able to adopt this strategy. This is however expected to change as the 2013 WHO guidelines now recommend routine viral load testing as the preferred approach to monitoring patients for ART failure. The WHO recommends that HIVVL be done 6 months after commencing treatment, and every 12 months thereafter. In response to this, there has been a massive scale-up in the research and development efforts to fast track the market entry of less complex viral load testing platforms that are suitable for use outside of central laboratories, the almost exclusive domain of currently available HIVVL testing. In recognition of the reality that access to viral load testing will remain limited in the short-term, the WHO recommends selective viral load testing to confirm treatment failure where this is suspected based on immunological (CD4 count) and clinical indicators. Generally CD4 counts and HIVVL values tend to move in opposite directions successful treatment will cause a rise in CD4 and drop in viral load whereas dropping CD4 counts and high HIVVL is observed in untreated individuals, in the event of non-adherence to effective drugs or the development of drug resistance. However, in some people viral load may be undetectable, even if the CD4 count remains low. HIVVL monitoring is therefore important to avoid unnecessary switching to second-line ART regimens. As second-line ART is more costly and may be associated with a higher side effect profile it should be reserved for those that truly need it. How are HIVVL results interpreted? HIVVL test results are expressed as the number of copies of HIV RNA per millilitre of plasma. A value of 100,000 to 1 million copies is considered to be high. For an HIV infected individual not on ART, values less than 10,000 copies are considered to be low. The aim of ART is for a person to have an undetectable viral load. An undetectable viral load is indicative of very slow viral replication, which, besides halting the destruction of the immune system, means that drug resistance is less likely to develop and the risk of transmission of the virus to an uninfected person is significantly reduced. The question however is, what does undetectable mean? In essence this would refer to the sensitivity of the viral load assay in use with earlier versions having between 400 and 500 copies/ml as their limit of detection. The commercial assays that are currently widely used

6 6 are ultra-sensitive so unless otherwise stated, an undetectable viral load referred to in current treatment guidelines is generally understood to mean <50 copies/ml. The WHO 2013 guidelines have defined virological failure as persistent viral load readings above 1000 copies/ml, based on 2 consecutive readings 3 months apart, whilst making sure that the individual is actually taking the drugs as prescribed. This is a reduction from the 2010 WHO guideline threshold of 5000 copies/ml. The values described refer to plasma based viral load testing. Facilities using dry blood spot (DBS) technology should consider retaining a higher threshold because of the possibility of reduced sensitivity for viral load measurement at 1000 copies/ml of DBS samples. HIV DNA Polymerase Chain Reaction A diagnosis of HIV can be made in several ways. This includes HIV antibody detection, HIV p24 antigen detection as well as HIV DNA polymerase chain reaction (PCR). Of these tests, blood based HIV antibody detection are by far the commonest tests used for routine diagnosis. They are however not suitable for use in children under the age of 18 months because of the presence of circulating maternally derived antibodies. HIV DNA PCR, which detects the presence of the actual virus, has been adopted for early infant diagnosis (EID).This, like HIVVL, is a molecular based test and is currently restricted to laboratory based testing. Take home message The CD4 count is the most important test that is used to determine eligibility for initiation of ART. Absolute CD4 counts are used to assess immunological function in HIV infected adults. CD4 percentage counts are used to assess immunological function in HIV infected children less than 5 years of age. HIVVL is the most important indicator to assess response to ART. HIV DNA PCR is used for EID as antibody based HIV tests are not accurate in infants less than 18 months due to the persistence of circulating maternal antibodies. References 1. WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection, June Pantaleo G, Graziosi C, Fauci AS. (1993): New concepts in the immunopathogenesis of human immunodeficiency virus infection. N Engl J Med 328: UNITAID 2014 HIV/AIDS Diagnostics Technology Landscape 4 th edition, June Compiled by Dr Marion Münster HIV drug resistance assays Because of the very high rate of viral replication, mutations occur. When such a mutation affects the area targeted by a specific drug, the result may be the development of drug resistance. This means that viral replication is no longer controlled by that drug. As described above, this is generally considered to be the case if the viral load starts to rise, or remains high in an individual that is diligently adhering to ART. Various assays have been developed to confirm such drug resistance but are not routinely used because of limited availability and high costs. They are mostly used for research at this stage. Sysmex South Africa (Pty) Ltd. Fernridge Office Park Block 2, 5 Hunter Avenue, Ferndale, Randburg 2194, South Africa Phone Fax info@sysmex.co.za