HIV diagnosis changes course

 CD4+ T cell levels dictate when an HIV patient begins drug therapy, but the diagnostic test presently requires full laboratory facilities. In the last year, the emergence of disposable, affordable, point-of-care tests has resulted in a method for determining CD4 levels in HIV patients that can be used in even the most remote geographical locations

HIV is a major global health challenge with an estimated 31 out of 34 million HIV­infected people worldwide living in developing countries and five million new cases reported every year. Current treatment recommendations are based on potent antiretroviral therapy (ART) regimens which, when administered before the onset of severe immunodeficiency, suppress the HIV virus and slow the progression of the disease. Since the discovery of the relevant drugs, access to ART has significantly improved quality of life and slowed HIV transmission, considerably reducing death rates in the developed world, and to a growing extent in developing countries, over recent years.

At present, WHO guidelines recommend that ART starts when an individual’s CD4+ T cells fall below 350 cells/µl blood, with levels monitored every three to six months after diagnosis of HIV infection. However, CD4+ T cell levels have traditionally been measured using flow cytometry, which requires full laboratory facilities, expensive and sophisticated equipment, and trained personnel. Consequently, access to CD4 testing has been a logistical, geographical and financial barrier to treating HIV in resource-limited developing countries, resulting in more patients going on to develop full-blown AIDS with huge economic and social consequences.

Seven years ago, two groups of researchers based at the Burnet Institute in Melbourne, Australia, working separately on diagnostics for virus infections and on fundamental research into HIV pathogenesis and the role of CD4+ T cells, turned their attention to the critical clinical need for an affordable point-of-care (POC) test that didn’t need a laboratory, an instrument or trained scientists. Together with an inter-disciplinary team of colleagues from within the Burnet Institute and the US, they began to look at measuring CD4+ T cell counts in terms of the quantity of CD4 protein rather than the more traditional method of counting the actual number of CD4+ T cells present. The new approach relied on the fact that the amount of CD4 protein in each T cell is relatively constant and thus, in theory, the total quantity of CD4 and the CD4+ T cell count can be highly correlated. However, CD4 occurs in three forms in blood; soluble CD4, macrophage CD4, and T cell CD4. Only the T cell CD4 protein is relevant with regard to monitoring HIV, and so an essential step and huge challenge in the test development was to find a way to discriminate between the cell-associated T cell CD4, and the soluble and monocyte forms.

At the same time, and throughout the development process, the team had to keep costs and practicality at the forefront of their product design concept, as their main focus was a solution for resource-poor developing countries. Another key consideration was that the test should be amenable to large-scale manufacture in order to produce sufficient numbers to meet the potential demand; if no other CD4 test was available to outlying remote communities, this could mean over 30 million tests annually. Ideally, not only the manufacturing process and facilities, but also assay components, would need to be readily available and easily affordable.

[caption id="attachment_33695" align="aligncenter" width="300" caption="2CD42: The VISITECT CD4 test can determine CD4 T-cell levels from a finger-prick blood sample"][/caption]

The answer to all of these challenges, far from being a completely novel approach, was an assay based on well-established lateral flow POC assays similar to standard off-the-shelf pregnancy tests. Once the specific biological reagents had been sourced and developed by the team, the concept of measuring protein levels was essentially the same as that used in these over-the-counter tests. Components such as membranes and pads, as well as the manufacturing processes, were readily available for industrial-scale production when required, and the method would be simple, relatively cheap and, most importantly, a practical and familiar solution for remote and basic medical facilities anywhere.

The end result is a semi-quantitative POC laminar flow assay, where the blood sample taken from a finger prick is drawn by capillary action into the sample pad and interacts with reagents that, in three minutes, remove macrophages and red blood cells. After two additions of running buffer, approximately 40 minutes later a gold conjugate gives a visual signal – the test line – and, depending on the intensity of this compared to the reference line for 350 cells/µl blood, decisions are taken about whether or not to commence ART. If the test line is equal to or weaker than the reference line, ART should begin; if it is stronger, no treatment is required.

At the time of the July 2011 International AIDS Society (IAS) meeting in Rome, the POC CD4+ test was a working prototype, and the team at the Burnet Institute was actively looking for a commission partner for the scale up, manufacture and marketing of the final product. Discussions were held with Omega Diagnostics Group PLC and, with the Company’s knowledge and experience of the diagnostic market, it was clear that Burnet’s CD4 test and Omega Diagnostics were a perfect fit. The new test was launched at the IAS meeting in Washington, DC in 2012, and the reaction and excitement shown by healthcare professionals working at grassroots level was overwhelmingly positive. The assay clearly fulfils a long-standing clinical need for a real POC CD4+ T cell test suitable for developing countries and aid agencies. It offers the familiarity of other widely-used lateral flow assays, such as the standard off-the-shelf pregnancy tests and most widely used tests for diagnosing HIV infection. The first clinical trials of the product are scheduled to begin in August 2013 in Mozambique and soon after in Zimbabwe, South Africa, Kenya and India. The assay is expected to make a real difference to treatment decisions in developing countries.

Authors: Associate Professor David Anderson, Deputy Director, the Burnet Institute, Melbourne, Australia and Andrew Shepherd, Chief Executive, Omega Diagnostics Group PLC, UK