A new affordable flow cytometry based method to measure HIV‐1 viral load

Abstract

Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5 0 LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes.

Advances in biosensing strategies for HIV-1 detection, diagnosis, and therapeutic monitoring

“…In one example, anti-Digoxigenin (anti-DIG) and anti-Dinitrophenyl (anti-DNP) coated microparticles were used to capture to PCR end-products. By performing flow cytometric analyses, they were able to detect different HIV-1 subtypes, including the subtypes O and N, with a detection range of 50 to 1 million copies [42]. …”

Innovations in cytometry methods and technology

“…The resulting data display a high signal‐to‐noise ratio and yield data comparable to those obtained by traditional but more complex (and expensive) flow cytometers. Such cytometers of low complexity and pricing are dedicated for use in resource limited places, specialized on a limited diagnostic range and underline the efforts taken by cytometrists in this area (1, 2).…”

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

“…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.…”

“…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) low‐cost instrumentation (PCR machines as well as cytometers) and (2) trained technicians who can prepare samples and be 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).…”

“…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.…”