Global health diagnostics for HIV

 

Human immune-deficiency virus (HIV), tuberculosis, and malaria are the most prevalent fatal infectious diseases in global health. HIV alone affects an estimated 33 million people.  Many live with HIV worldwide with appropriate treatment but 1.4 million die annually from acquired immune deficiency syndrome (AIDS), which develops from untreated advanced HIV. The good news is that effective anti-retroviral and anti-protease drugs are available to slow down the reproduction of HIV in the body.  The bad news is that only one in eight infected persons are tested for HIV and AIDS. There is a serious lack of testing to direct the use of effective anti-retroviral drugs that can prolong life in those infected with HIV. Targeted treatment of HIV can convert what is a lethal disease to a chronic one that adds many years of life to so many victims.  The very worst news is that the vast majority of those who are not tested live in the least developed countries of the world. The huge challenge is to provide a technology to test for the presence of HIV and its severity in these least developed countries at a cost and format that is feasible and affordable.  Read More

Components of the HIV virus

Components of the HIV virus

What is HIV and a CD4+ T-cell

HIV is a single-stranded RNA retrovirus that contains only an mRNA genome.  Once bound to a host cell and transported into its cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its own viral mRNA genome.  This process is the reverse of the usual pattern, thus retro (backwards).  This new viral DNA is then incorporated into the host DNA genome.  Subsequently, the host cell transcribes the viral DNA along with its own genomic DNA to produce both its own proteins as well as retroviral proteins required to reproduce the HIV retrovirus.

Binding of the HIV virus to the CD4+ T-cell host

Binding of the HIV virus to the CD4+ T-cell host

In humans, the host cell for the HIV retrovirus is the CD4+ T-cells, which are mature Th cells (helper T-cells), that express the CD4 cell surface protein. HIV attaches to the CD4 receptor and gp 120 protein to enter the host cell.  These CD4+ T-cells are critical in the immune system because for example, when an antigen-presenting cell expresses an antigen on a MHC class II cell, a CD4+ T-cell will aid those cells to create a specific immune response to the antigen. This process is critical to mount effective specific responses to pathogens (harmful microorganisms).

Latent versus active infection of HIV in CD4+ T-cell host

Latent versus active infection of HIV in CD4+ T-cell host

HIV is a particularly diabolical pathogen because of two most unfortunate characteristics. First, it attacks the very cells (CD4+ T-cells) that would normally be responsible to help eliminate it and thus renders the infected CD4+ cells ineffective against HIV. This becomes particularly important towards the end of an HIV infection, in which the number of functional CD4+ T cells seriously decreases. This CD4+ T-cell deficiency leads to the symptomatic stage of infection known as AIDS.  Making matters worse, HIV has a tremendous capacity to undergo genomic mutation, which continually changes the retroviral nucleic acid and protein structures over time. Knowing the status of the retroviral mutations is also an important feature that is used to select the most effective drug treatments.

Why count CD4+ T-cells in austere environments at low cost

Global prevalence of HIV/AIDS

Global prevalence of HIV/AIDS

There have been tremendous advances in the use of combinations of anti-retroviral and anti-protease drugs to transform the previously fatal HIV infections into a condition in which patients can live for many years in coexistence with HIV infection. Selection of the anti-retroviral and anti-protease drug treatments is driven by the current status of the infection that includes multiple parameters: the numbers of CD4+ T-cells and retroviral copies that are in the patient’s circulation as well as the mutational status of the retrovirus in the patient’s blood.

The basic problem is that many, if not most, of those infected with HIV live in least developed countries. In these austere environments, although the anti-retroviral and anti-protease drugs are available, their rational and effective use is limited by the availability of sophisticated monitoring of the current status of the infection. The most serious challenge is the need to provide sophisticated testing to determine the current status of the infection so that proper ongoing treatment of the HIV retrovirus can be implemented in a truly austere environment at extremely low costs.

Immunoaffinity capture of CD4+ T-cells

Label-free immunoaffinity capture of CD4+ T-cells

Label-free immunoaffinity capture of CD4+ T-cells

To count the number of CD4+ T-cells, the cells must be captured and then counted in a simple, accurate, and cost-effective manner.  A simple cost-effective CD4+ cell counting device was urgently needed to stage and monitor HIV-infected patients in austere environments. To address the limitations of the currently expensive and cost-prohibitive approaches, a simple and label-free CD4 cell counting device was designed using microfluidic technology. The fabrication of this microfluidic system for high-efficiency isolation of pure populations of CD4+ T-cells has been based on cell affinity chromatography operated under controlled flow conditions. The performance of a microfluidic CD4 cell counting device was compared against standard flow cytometry in 49 HIV-positive subjects over a wide range of absolute CD4 cell counts. We observed a close correlation between CD4 cell counts from the microchip device and measurements by flow cytometry. Sensitivity for distinguishing clinically-relevant thresholds of 200, 350, and 500 cells/µL and specificity were excellent. This device now serves as a functional cartridge for fast, accurate, affordable, and simple CD4 cell counting in austere environments.

Quantifying CD4+ T-cells using electrolysis

Validation studies performed at MGH

Validation studies performed at MGH

The microfluidic CD4+ T-cell biochip that enumerates CD4+ T lymphocytes from leukocyte populations using cell affinity chromatography has been combined with a fabricated device that uses electrical impedance sensing. Using the electrical impedance, T cell counts are measured by obtaining the difference between the number of leukocytes before and after depleting CD4+ T-cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of about 9 cells per µL. In addition, the biochip provides T cell counts, which correlated closely with an optical control for CD4 T-cells concentrations ranging from approximately 100 – 700 cells per µL.  These combined technologies have been further explored for commercialization by Daktari Diagnostics, Inc.

Daktari Diagnostics, Inc. 

Daktari CD4 cartridge

Daktari CD4 cartridge

Daktari Diagnostics, Inc. was formed to commercialize these two basic technologies of immunoaffinity capture of CD4+ T-cells and quantifying CD4+ T-cells using electrolysis.  The Daktari CD4 system is a portable and robust platform that can be used anywhere from a doctor’s office to the most remote areas of the world. The Daktari CD4 system combines the innovations in microscale technologies with a simplicity in design and use. The Daktari CD4 system has been successfully tested in a four-center clinical trial in Sub-Sahara Africa to quantify CD4+ T-cells in an austere environment with little or no access to sophisticated diagnostics.

The Daktari technology brings this essential blood test to parts of the world where millions of people can have access to quality diagnostics as well as life-saving drugs.

Relevant publications

Watkins NN, Sridhar S, Cheng X, Chen GD, Toner M, Rodriguez W, et al. A microfabricated electrical differential counter for the selective enumberation of CD4+ T lymphocytes. Lab Chip. 2011 Apr 21;11(8):1437-47. PubMed PMID: 21283908; PubMed Central PMCID: PMC3141315

Watkins N, Venkatesan BM, Toner M, Rodriguez W, Bashir R. A robust electrical microcytometer with 3-dimensional hydrofocusing. Lab Chip 2009 Nov 21;9(22):3177-84. PubMed PMID: 19865723; PubMed Central PMCID: PMC4142320

Cheng X, Gupta A, Chen C, Tompkins RG, Rodriguez W, Toner M. Enhancing the performance of a point-of-care CD4+ T-cell counting microchip through monocyte depletion for HIV/AIDS diagnostics. Lab Chip 2009 May 21;9(10):1357-1357-64. PubMed PMID: 19417901; PubMed Central PMCID: PMC4064257

Cheng X, Liu YS, Irimia D, Demirci U, Yang L, Zamir L, et al. Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices. Lab Chip 2007 Jun;7(6):746-55. PubMed PMID: 17538717

Cheng X, Irimia D, Dixon M, Ziperstein JC, Demirci U, Zamir L, Tompkins RG, Toner M, Rodriguez WR. A microchip approach for practical label-free CD4+ T-cell counting of HIV-infected subjects in resource-poor settings. J Acquir Immune Defic Syndr 2007 Jul 1;45(3):257-61. PubMed PMID: 17414933

Cheng X, Irimia D, Dixon M, Sekine K, Demirci U, Zamir L, et al. A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects.   Lab chip. 2007 Feb;7(2):170-8. PubMed PMID:17268618; PubMed Central PMCID: PMC4028372

Contact

Mehmet Toner, Ph.D. William Rodriguez, M.D. Rashid Bashir, Ph.D.
mtoner@hms.harvard.edu
617-724-5336 617-724-5336 617-724-5336

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Grace McDonald-SmithGlobal health diagnostics for HIV