Diagnosing HIV infection using flow cytometry: From antigenic analyses to a specifically dedicated bead‐based assay to measure viral load

Abstract

SINCE the discovery of HIV in 1984 (1,2), millions have been infected and have died of AIDS. In 2006, the World Health Organization identified 40 million HIV-infected subjects, noting that antiretroviral therapy was required throughout the world (Fig. 1; http://www.who.int/hiv/data/en/index.html). Today, the situation is very similar and the HIV pandemic remains a major world health problem (3). The treatment of AIDS with highly active antiretroviral therapy (HAART) involves regular monitoring of different blood parameters, requiring the development of convenient and accurate methods that can evaluate HIV infection even in resource-poor countries (4,5). In addition to CD4 cell count, which is described as the best surrogate marker (6–8), blood serum virus concentration (viral load) is the most meaningful parameter to evaluate treatment success and to decide on initiating HAART (9). Virus detection is also important for evaluation and prevention of mother-to-child transmission (10) and in persons with an atypical disease course (11). Viral load tests are usually performed in specialized laboratories because they require specifically dedicated materials and are based on highly specific real-time PCR, to determine viral serum concentration over a wide scale. Therefore, these tests can be difficult to conduct in developing countries. I have, therefore, read with great interest the paper by Greve et al. (12), published in the current issue of Cytometry Part A, describing a new affordable flow cytometric bead-based assay to measure HIV-1 viral load. This test, combining PCR, a novel and original bead-based assay, and flow cytometric analysis, clearly shows that various HIV1 subtypes can be identified and that the sensitivity of the method makes it possible to quantify as few as 50 copies of virus. Interestingly, the copreparation and amplification of an internal standard provides quality control of all the steps from RNA purification over PCR-reaction to the quantification of the amplified HIV-1 product. Currently, flow cytometry bead-based assays (13–15) can be carried out either on specifically dedicated machines (Luminex Technology) or on various conventional flow cytometers (16,17). They are frequently used to simultaneously identify and quantify cytokines, adipokines, growth factors, and auto-antibodies as well as cancer, cardiac, and neurological markers in a small volume (10–50 ll) of different body fluids (plasma, serum, tears, cerebrospinal fluid, synovial fluid, and bronchoalveolar lavage fluid). These methods can also be used on culture media or on cellular extracts (18). In the latter, bead-based assays are promising tools to study fusion proteins in leukemic cells (19) or protein phosphorylation on culture cells or tissue samples to investigate cell signaling under various conditions (20). The ability to combine PCR methods with cytometric bead assays was previously reported by various authors (21–23), and the method was successfully used to investigate DNA polymorphism (24,25). In virological diagnosis, detecting HIV with the combined use of PCR on a conventional flow cytometer is an ori-