Commentary
Diagnosing HIV Infection Using Flow Cytometry: From Antigenic Analyses to a Specifically Dedicated Bead-Based Assay to Measure Viral Load Gerard Lizard* Key terms bead-based assay; HIV; viral load; flow cytometry
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
Centre de Recherche Inserm 866 (Equipe Biochimie Metabolique et Nutritionnelle), Faculte des Sciences Gabriel, Universite de Bourgogne, 21000 Dijon, France Received 28 October 2008; Accepted 13 November 2008 *Correspondence to: Gerard Lizard, Centre de Recherche Inserm 866 (Equipe Biochimie Metabolique et Nutritionnelle), Universite de Bourgogne, Faculte des Sciences Gabriel, 6 Bd Gabriel, 21000 Dijon, France.
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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-
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[email protected] Published online 12 December 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/cyto.a.20694 © 2008 International Society for Advancement of Cytometry
COMMENTARY
Figure 1. The figures shown illustrate a global view of HIV infection and of the estimated number of people in need of antiretroviral therapy in low and middle income countries (World Health Organization (WHO), HIV/AIDS department). The author thanks WHO to permit the reproduction of this document (http://www.who.int/hiv/data/en/index.html).
ginal achievement, not only marking major progress in the fight against HIV infection, but also providing applications for other infectious diseases (26). Furthermore, this method is significant in that it could rapidly and accurately identify infectious agents in newborns or in premature newborns when biological samples are available only in small amounts. In addition, the low cost per test (around US $12) makes it possible to regularly conduct a complete monitoring program of HIV infection using only flow cytometry (CD4 cell count and viral load), most particularly in resource-limited countries. However, the successful implementation of the bead-based test described by Greve et al. (12) will also depend upon (1) lowcost instrumentation (PCR machines as well as cytometers) and (2) trained technicians who can prepare samples and be Cytometry Part A • 75A: 172174, 2009
responsible for quality control at different levels (blood collection conditions, sample and reagent storage, sample preparation, flow cytometry analysis conditions, and data interpretation). In fact, ongoing efforts by the ISAC past-president (Prof. J. Paul Robinson) and his colleagues at Purdue University have focused on the development of an ultra-low-cost flow cytometer with point-of-care utility for CD4 testing (http://www.cytometryforlife.org). If the same low-cost cytometer could be adapted to HIV viral load as well as CD4 levels, two important problems in HIV diagnosis could be overcome. However, the trained staff required to prepare samples and the need for a PCR machine do not lend themselves to low-cost solutions. Nevertheless, in the near future, it is tempting to speculate that the development of easy to use 173
COMMENTARY bead-based microfluidic immunoassays resulting from progress in nanotechnologies (27) as well as the ability to identify nucleotide sequences without PCR (28) will find applications in the diagnosis and follow-up of various diseases, including HIV infection. As the development of accurate and low-cost methods for HIV diagnosis is crucial to efficiently fight AIDS, I believe that it is essential to underscore the importance of the original work carried out by Greve et al. (12), which will undoubtedly have consequences on the management of HIV infection worldwide, but which could also have several other applications in a variety of human diseases. In addition, this investigation clearly illustrates the significant potential of bead-based assays, as yet underestimated, even though they open many avenues for fundamental and clinical research.
ACKNOWLEDGMENT The author is indebted to Ms Linda Northrup for reviewing the English version of the manuscript.
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Diagnosing HIV Infection—From Antigenic Analyses to Bead-Based Assay for Viral Load
