HIV diagnostics pipeline
The effectiveness of antiretroviral treatment delivery was traditionally thought to depend on the use of sophisticated diagnostic tests.
Earlier this year, important findings from the Development of AntiRetroviral Therapy in Africa (DART) trial, conducted in Zimbabwe and Uganda were published, showing impressive survival rates in people receiving routine clinical monitoring alone. This trial randomised patients to receive either clinical monitoring or laboratory (hematology, biochemistry and CD4) plus clinical monitoring. At five years, 87% and 90% of patients were alive in the clinical and laboratory arms respectively. 
These results were even more impressive as patients in this trial had a median baseline CD4 count of 86 cells/mm3.
The DART investigators concluded that ART could be delivered safely with good quality clinical care, allowing treatment delivery to be decentralised. Despite DART results being occasionally misinterpreted to suggest that this argues for no monitoring at all, the investigators also recommended that there is a role for CD4 testing from the second year on ART to guide the switch to a second-line regimenand clearly to start treatment. They added that this should encourage accelerated development of simpler, cheaper, point-of-care CD4 tests.
Furthermore, in an accompanying editorial, Andrew Phillips and Joep van Oosterhout argue that, although the authors suggest that the advantage of measuring viral load on survival may have been modest in this cohort, the reduction of transmission of drug-resistant virus, which will undermine the effectiveness of antiretroviral therapy over the longer term may be another reason to measure it. Therefore development of cheap robust assays for viral load that can easily be used in rural and urban settings is of the highest priority for researchers.
There are many arguments for the development of affordable point of point-of-care assays. CD4 measurements are essential for knowing when to initiate ART. CD4 counts also guide the use of opportunistic infection prophylaxis.
Viral load testing is essential to diagnose HIV-infected infants (see next chapter) and to monitor virological suppression in pregnant women to reduce the risk of mother to child transmission. These tests can be used to monitor adherence early on, and better inform decisions about treatment modifications or switches. [3, 4]
The next edition of the Pipeline Report will include a more detailed survey of diagnostics appropriate to low resource settings. Here we look at the qualities required from a point-of-care test to be useful and two initiatives that appear to be close to emerging from the pipeline.
Requirements for point-of-care tests
Point-of-care refers to a test that can be conducted in the same facility where a patient receives his/her treatment and other care. In rural areas many patients have to travel vast distances to reach testing facilities and then return to get their results. There is considerable loss to follow up from people who do not return for their test results.
These technologies need to be quick and easy to use and interpret, that work with a finger-prick blood sample or other non-venous sample and have a simple read out. They need to be appropriate to settings without sophisticated laboratories, where electricity and running water cannot be guaranteed. They need to perform in hot, humid or dusty environments and have a long shelf life. They need to be used and maintained by health workers without advanced technical skills.
The CD4 initiative point-of-care CD4 test
The CD4 Initiative, at Imperial College, was established in 2005. The Initiative came about through a substantial activist push from Gregg Gonsalves, and a generous grant from the Gates Foundation. 
Led by principal investigator Dr Hans-Georg Batz and project manager Dr Steven Reid, their objective is to develop new, low-cost, rapid point-of-care tests to measure CD4 counts, which are suitable for use in rural areas of low-income countries.
The initiative set out to produce tangible results in four years, which is a very ambitious timescale to develop this type of product.
The project began by establishing a set of predetermined specifications:
- Simple and robust
- Semi-quantitative, minimum cut off of 250 cells/mm3
- Stable at 40ºC for 12 months
- Quality assurance material to check correct functioning of test
- Use of finger-prick blood/other non-venous blood sample
- Simple to perform, few steps and <2 hours training required
- <30 minutes from patient to result
- Simple read out
- All-in-one kit
- 25 tests performed/person/day
- Target price around $2 per test
- Customer capital outlay (if any) <$1,000
- Safe solution for infectious waste materials
Phase I of the project was proof of concept, in which new tests were developed, that can reliably measure CD4 counts in blood. Phase II involved developing prototypes, scale-up and validation. That is, to establish whether the new tests met most, if not all, of the specifications and provided reliable and reproducible results in medium-scale production.
In Phase III the tests will be trialled in resource-poor settings to make sure that specifications are achievable in field conditions. The costs will also be evaluated. The group has completed two rounds of independent verification using samples from clinics in London. Field-testing will start later this year in Malawi and Uganda.
Three prototypes (Beckman Coulter, Burnet Institute and Zyomyx Inc.) were assessed against the gold standard method of measuring CD4 counts called flow cytometry. Flow cytometry is a technique for counting CD4 cells by suspending them in a stream of liquid and passing them by an electronic detector. The machines used to perform these tests are big and expensive and require an uninterrupted supply of electricity and highly trained technicians.
This verification trial used around 150 blood samples from HIV-positive patients in London to see if the same results could be obtained with the prototype tests as with flow cytometry. The results showed that only the test from Zyomyx, Inc compared favorably with flow cytometry. The other two tests failed to correctly identify samples from patients with low CD4 counts with sufficient sensitivity, thus the initiative is proceeding with the Zyomyx Inc., point-of-care CD4 test.
Zyomyx is based in San Francisco. The device is a CD4-gauge much like a thermometer and this simple test will be able to provide a quantitative CD4 count from a finger-prick of bloodexceeding the original specif ication of semi quantitative with a minimum cut-off.
After the field-testing, phase IV of the project will be technology transfer (ideally in the developing world), large-scale production and distribution.
This test can be used for both adults and children with a different measuring range for younger children and infants.
Point-of-care viral load test
The Diagnostics Development Unit based at the Department of Haematology, University of Cambridge, and led by Dr Helen Lee, are developing a simple amplification based assay (SAMBA). This is a nucleic test based on visual detection of viral nucleic acid by dipstick designed to detect a broad range of HIV-1 subtypes typical to sub-Saharan Africa. 
Like the CD4 Initiative, the group conducted a survey of potential users in resource-limited settings. Again, respondents need tests that are quick and simple and can be performed while patients are at the clinic. The SAMBA needs to be, simple, robust, inexpensive, stable and self contained with predispensed unit-dose reagents and disposables included and with a design and procedure that comply with biosafety standards.
Recent data published in the April 2010 supplement of the Journal of Infectious Diseases showed the sensitivity and subtype coverage of the SAMBA test, when tested with 69 samples provided by The Royal London Hospital, to be at least as good as those of commercially available diagnostic tests.
The investigators suggested that the SAMBA system is not restricted to HIV and could also be adapted to detect other infections.
- The DART trial team. Routine versus clinically driven laboratory monitoring of HIV antiretroviral therapy in Africa (DART): a randomised non-inferiority trial. The Lancet, Volume 375, Issue 9709, Pages 123-131, 9 January 2010.
- Phillips A and van Oosterhout J. DART points the way for HIV treatment programmes. The Lancet, Volume 375, Issue 9709, Pages 96-98, 9 January 2010
- Calmy A et al. HIV viral load monitoring in resource limited regions: optional or necessary/ Clin Infect Dis 2007; 44(1):128-134
- Usdin M et al. Patient needs and Point-of-Care Requirements for HIV Load Testing in Resource Limited Settings. J Infect Dis 2010 (suppl 1) 573-577
- The CD4 Initiative. Accessed July 5, 2010 from: http://www1.imperia l.ac.uk/medicine/about/divisions/departmentofmedicine/infectiousdiseases/infectious_diseases/cd4_initiative/
- Lee H H et al. Simple Amplification-Based Assay: A Nucleic Acid-Based Point-of-Care Platform for HIV-1 Testing. Jour Infect Dis 2010:201 (Supp 1) S65-72.