S. Kobayashi (Candidate No: 7273) Centre No: 51537

Fast and Accurate HIV Diagnosis for Use in 3rd World Countries

[1.1] The problem

HIV, or Human Immunodeficiency Virus, is a lentiviral disease which has been estimated to infect at least 0.48% of the world’s population, some 33.3 million people 1a. 68% (22.5 million people) of all HIV sufferers in the world live in sub-Saharan Africa 1b, and the image on the right2 shows the affected areas. The dark red countries in the very south of the continent (South Africa, Botswana, Namibia, Lesotho, Swaziland, Zimbabwe, Zambia and Mozambique) are the most severely affected, with over 15% of the adult population being HIV-positive. Other countries, such as Kenya, Tanzania and Uganda, marked on the map in bright red, have between 5-15% of their adult population infected with HIV. Almost all of these are 3rd world countries with little or no healthcare and a very high proportion of HIV sufferers in these countries have never been tested for the disease, and are ignorant of their condition, presenting a severe obstacle in the introduction of treatments.

There are several diagnosis techniques available today for HIV, the most famous of which is the Western Blot. Also known as a Protein Immunoblot, this technique can be used to diagnose a range of diseases, and employs an electric current passed through a gel medium matrix in order to separate various proteins in the bloodstream according to shape, size, and therefore type. In the case of HIV Western Blotting, the particular protein of note is the anti-HIV antibody, and its presence indicates that the patient is HIV positive. It has proven to be very successful in more developed countries, and its high accuracy has led it to be the current standard against which all other HIV diagnostic methods are compared. However, the Western Blot has severe drawbacks, common to all other current tests: it is very slow, taking 1 or 2 weeks for results, and it is “complex to administer” 3, requiring very sophisticated on-site laboratory equipment and trained medical staff. This makes these types of diagnosis all but impossible to implement successfully in the poverty-stricken 3 rd

world countries where they are most needed.

Therefore, a radically new testing technique is needed for less developed nations, offering a level of reliability on par with the Western Blot, yet at a fraction of the cost and complexity.

[1.2] The Solution: Labs-On-A-Chip

Recent advances in technology have resulted in the development of cheap lab-on-a-chip diagnosis devices. These chips are designed as one-use disposable tests providing a full diagnosis in less than 20 minutes. LOCs work by exploiting the odd movement of liquids at microscopic scales known as

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S. Kobayashi (Candidate No: 7273) Centre No: 51537

microfluidics, allowing techniques such as PCR (Polymerase Chain Reaction) to be conducted in real time.

PCR is used to ‘amplify’ certain strands of DNA through enzymatic replication, and is usually a complicated, multi-step process. The DNA is first “denatured” into separate strands through the application of heat. Specially designed strands known as “primers” are introduced. They contain bases complimentary to the region on the denatured DNA strands to be replicated. DNA polymerase then commences synthesis using free nucleotides, creating two double strands of DNA in the notable region. This process is repeated in a chain reaction, resulting in the exponential amplification of the target DNA4. In the case of HIV, PCR of DNA with primers targeted for HIV-specific viral genomes can be used to isolate and amplify the specific DNA sequence if present. As the reaction can only take place in the presence of HIV genomes, it acts as a diagnostic indicator for HIV positivity. PCR already has an advantage over other diagnosis techniques: HIV antibodies can take weeks or months to reach detectable levels, and the use of PCR can enable diagnosis at an earlier stage of infection than antibody-based methods. If PCR is conducted in premanufactured lab-on-a-chip systems, the complicated steps do not have to be conducted in the field with laboratory setups, significantly reducing the time and labour required for tests.

The major problem that has limited LOC use so far is the fact that they are “hard to manufacture and are consequently too expensive for throwaway systems”5. However, continuing worldwide research into the subject has resulted in a number of innovative new manufacturing techniques for these tiny medical chips.

For example, one of the most interesting of these followed a discovery that ordinary office paper possesses miniscule pores which allow microfluidity. The technique, FLASH (Fast Lithographic Activation of Sheets) has been demonstrated to work (a ‘showcase’ microfluidic device made using the method is shown on the left5), and is very simple: soaking the paper in a chemical called Photoresist which hardens under

UV light. A transparent cover is placed on top, and the chip’s channels are printed on the cover with opaque ink, after which it is exposed to UV light. The paper not under the ink is hardened and sealed, leaving the same microfluidic channels as the printed pattern6.

[1.3] Effectiveness and Relevance of Solution

The theory behind LOCs may be sound, but are they appropriate for use in 3rd world countries? Equipment for LOC devices usually comes in a ‘reader-and-disposable-chip’ combination, like the one shown in the picture on the right7. Each disposable chip costs around $1.50 to manufacture, a price that will decrease with bulk orders. The main capital investment comes with the “shoebox-sized” chip reader, which currently costs $3200. Although a hefty sum, the process of “miniaturization” of the device has already begun

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S. Kobayashi (Candidate No: 7273) Centre No: 51537

in order to “reduce manufacturing costs”8. It must also be remembered that only one reader is required for hundreds of diagnoses to be performed, and due to the low cost of the chips themselves, each test will be many times cheaper than a standard Western Blot.

As mentioned before, LOCs work through the phenomenon of microfluidity, the odd movement of liquids at microscopic scales. Exploiting microfluidic physics, accurate results can be gained from very small volumes of testing body fluid and reagents. This allows all reagent chemicals to be stored inside the chip itself, and therefore no complicated laboratory setup and work is required.. Furthermore, the low cost and simplicity of the chips’ user-end design allows them to be used by almost anyone. The chip reader setup itself requires very little technical or medical expertise meaning that LOCs are perfectly suited to ‘field-diagnoses’ in sub-Saharan 3rd world countries where HIV diagnosis has been restricted due to the lack of medical staff and equipment, not only improving the health of the local populace, but also significantly aiding the global battle against HIV/AIDS as easier diagnosis will lead to a wealth of new data, accelerating the research and development of potential cures and treatments. LOC technology can also be easily adapted to test for other viral or bacterial diseases, meaning that it has far more scope for use than just HIV diagnosis.

The two graphs on the left9 are both from Yolé Développement, a well-known Market Research and Consultancy company which specialises in new areas of medical engineering. Taken from the 2010 report on Point of Care applications of microfluidic devices (such as Labs-on-a-chip), the growth of the microfluidic POC market can be seen very clearly in the first graph (the dark green sections of the bars, and the data in the red box), with 257.2 million units projected to be sold in 2014 compared to just 29.1 million in 2006. This huge growth is further emphasised in the second graph, in which it has been estimated that microfluidic POC diagnostics (again in dark green) will

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S. Kobayashi (Candidate No: 7273) Centre No: 51537

experience 39% growth between 2009 and 2014, the highest in any field applying microfluidic principles.

This is evidence for the extensive research and development being carried out in LOCs and microfluidic POC diagnosis devices by many of the world’s largest pharmaceutical companies, and a sure sign that in this hugely competitive and growing field, LOC manufacturers will have to improve reliability and lower costs simultaneously, providing benefits for the consumers.

[2.1a] Implications of LOC Technology: Economic

However, there is another potential implication for LOC technology that is less optimistic. Since the manufacture of the chips themselves is quite a complex process, they will have to be made in more developed countries, most likely by large pharmaceutical corporations. This means that the future of this technology may depend on profit-motivated companies who may not see any personal monetary benefits in investing in these chips.

However, it is equally, if not more, likely that the ease of LOC mass-manufacture, as well as their large demand will promote the technology as a viable investment in the pharmaceutical industry. Indeed, the market analysis graphs shown in the previous section indicate that the LOC market is increasing rapidly, driving down costs due to increasing competition.

[2.1b] Implications of LOC Technology: Social

Furthermore, in certain areas of the world, especially in countries which would see the heaviest use of labs-on-a-chip, HIV infection “carries a stigma” and has even become a taboo subject. HIV-positive people can “be shunned or suffer violence after being diagnosed”10. This means that having such an easy diagnosis device for HIV as LOC may not benefit the social environment of these countries as much as might be hoped. However, there is an possible optimistic implication of LOCs on this situation. The ease with which HIV diagnoses can be conducted may help raise awareness in the local populace, perhaps leading to the breaking of this social barrier which segregates those who are infected with HIV.

[2.2a] Benefits of LOC Technology

There are a large number of benefits associated with the widespread use of LOC technology, not least of which is that the quick diagnosis allows the sufferer to start undergoing HIV-combating treatments earlier. Whereas in the past, only a few people could undergo diagnosis with Western Blot, a process that took one or two weeks, now, with LOCs, the same standard of diagnosis can be conducted on a huge scale in a matter of minutes. For example treatments such as HAART (Highly active antiretroviral therapy) are highly effective in preventing HIV transmission from pregnant mothers to their babies. However, due to the delicate nature of a pregnancy, this treatment must be started at the earliest opportunity, and a diagnosis one or two weeks earlier can make all the difference.

[2.2b] Risks of LOC Technology

LOC is a relatively new form of diagnosis, albeit one in which a very large amount of research has been conducted. It has not been thoroughly field tested, and therefore unforeseen problems may

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S. Kobayashi (Candidate No: 7273) Centre No: 51537

arise in the future. However, it must be remembered that they are a diagnostic device and not a treatment, cure or drug. Due to this, problems would most likely be of a technical nature, such as errors in the manufacturing or calibration stages of the process, and are highly unlikely to cause direct harm to people, as a drug might. However, these miscalculations may cause indirect harm through misdiagnosis. False negatives, if they occur, could lead to further spread of HIV through individuals who falsely believe they are uninfected. False positives, on the other hand, could result in uninfected people having to undergo drug treatments which are unnecessary and potentially harmful, as well as perhaps being subjected to the social stigma of being HIV-positive.

However, as the PCR/Immunoassay used in chips have been based on previously successful and accurate tests such as ELISA or the Western Blot, large issues are unlikely to occur, and any that do will be swiftly corrected.

Additional disadvantages include harm to the environment caused by irresponsible disposal of used chips.

[2.3a] Alternate Solution: Lateral Flow

Lateral flow immunochromatographic assays, to give LF testing its full name, are more commonly, and famously, used in the commercially available point-of-care ‘blue strip’ pregnancy tests. However, the principle can easily be applied to detect, for example, HIV-linked antigens. When the test serum is deposited a one end of the Lateral Flow apparatus, capillary action causes it to move into a pad containing unreactive coloured particles, “usually colloidal gold particles or latex microspheres”. The coloured serum then moves up a nitrocellulose membrane on which various “anti-target analyte antibodies are immobilised” in lines. If HIV-linked antigens are present in the serum, they will be stopped by the corresponding antibody, forming a coloured line11. As the technique has been tried, tested and improved over the years of pregnancy test development, lateral flow testing has become quite reliable, with a wide variety of different point-of-care products available today. An example, developed by Orasure Technologies Inc., able to produce results in “< 30 min”, is shown on the right12. Furthermore, when compared to the Western Blot, such tests can be done in less than half the number of steps. However, such point of care devices currently act as primary stage pre-diagnosis indicators, and cannot provide a full diagnosis on their own; something that labs-on-a-chip will be able to when fully developed. LF works by detecting antigens, and can therefore only be performed at later stages of infection compared to LOCs performing polymerase chain reactions.

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S. Kobayashi (Candidate No: 7273) Centre No: 51537

[2.3b] Alternate Solution: Particle Agglutination

Particle agglutination (clumping) tests are a fairly old and well established technique commonly used to determine blood types through ‘bedside cards’ such as Serafol. However, like the Lateral Flow described above, PA testing can also be applied to detect HIV-linked antigens. A particularly good example of this method is the Capillus HIV-1/HIV-2 latex agglutination test, which has been around for some years. This test can be employed with blood, plasma or serum samples, and uses HIV antibodies as its analyte. The addition of a solution containing latex based “analyte-specific detector molecule”13 results in a reaction which bonds the HIV antibody analyte and the detector molecule if the former is present. As shown in the image below14, a positive result is shown by the clear presence of “white clumping”. Although fairly reliable and well-established, particle agglutination tests have begun falling out of favour with the medicals community, and popularity has increased for more modern forms of Lateral Flow due to the difficulty of determining agglutination in cases of low analyte concentration, such as in the early stages of HIV infection.

It is necessary to point out that both alternate solutions outlined above are examples of currently existing Point-Of-Care products and are therefore in a completely different category from labs-on-a-chip. LOC technology is the cutting edge of diagnosis through laboratory miniaturisation, providing a western-blot style full diagnosis on credit-card-sized equipment, whereas current POC devices are meant as quick confirmatory checks. This is what makes LOCs completely revolutionary- they work on principles common to nothing else available today, and can, and probably will, change the medical world completely in the next decade or so.

BIBLIOGRAPHY

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1 “UNAIDS Report on The Global AIDS Epidemic”By the Joint United Nations Programme on HIV/AIDS (2010)Executive Director: Michel Sidibé ISBN 978-92-9173-871-7a p.21 b p.25

Evaluation of source 1: Gathering reliable worldwide data on HIV/AIDS is understandably very difficult, not least because it is a taboo subject in many areas of the world. The Joint United Nations Programme on HIV/AIDS (UNAIDS) exists to research the global epidemic, and to provide reliable data on it. I believe that all UNAIDS reports are accurate and valid, and constant research and revision done by the organisation (which published updates almost every year) ensures that all figures are up to date. UNAIDS data is gathered in partnership with the World Health Organisation (WHO), further ensuring the data’s reliability and validity. The figures given by the UNAIDS report of “33.3 million” in the world affected by HIV, with “68%” in sub-Saharan Africa is confirmed by the United States HIV/AIDS resource USAID (http://www.usaid.gov/our_work/global_health/aids/News/aidsfaq.html) which says that “In 2009, 33.3 million

people around the world were living with HIV/AIDS” and “Sub-Saharan Africa….is home to 67 percent of all people living with HIV

worldwide”, and also by Avert (http://www.avert.org/worldstats.htm - “People living with HIV/AIDS in2009: 33 Million”) and AIDSmap (http://www.aidsmap.com/hiv-basics/HIV-AIDS/page/1412437/ - “at the end of 2008 there were 33.4 million

people living with HIV”). However, it should be remembered that the initial source of all this information is almost certain to have been the UNAIDS report, although this widespread trust and use of the report means that it is all the more reliable.

2 Image: “Map of HIV Prevalence in Africa” from Wikipedia http://en.wikipedia.org/wiki/File:Map-of-HIV-Prevalance-in-Africa.png Accessed 08/02/2011

3 “Avert” HIV and AIDS prevention charity http://www.avert.org/testing.htm Accessed 01/02/2011

4 “PCR Animation” By the Dolan DNA Learning Center http://www.dnalc.org/resources/animations/pcr.html Accessed 09/04/2011

5 Article: “Labs-on-a-chip that you can shrink to fit”, New Scientist Magazine Issue 2730 (October 2009), p.24 Author: Paul Marks

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Article: “FLASH: A Rapid Method for Prototyping Paper-based Microfluidic Devices”, Lab On A Chip Journal (RSC) DOI: 10.1039/B811135A Issue 12 (2008), 8, pp.2146-2150 Authors: Andres W. Martinez, Scott T. Phillips, Benjamin J. Wiley, Malancha Gupta and George M. Whitesides

Evaluation of source 6: This academic paper is presented in Lab On A Chip, a peer-reviewed scientific journal. Published by the Royal Society of Chemistry, the journal is widely considered to be the definitive source of information on microfluidic innovation. Martinez, Phillips and Whitesides are leading experts in the field of microfluidics, and have published papers which have appeared in other respected journals such as Analytical Chemistry (2010, 82 (1), pp 3–10 DOI: 10.1021/ac9013989, American Chemical Society). The article on FLASH microfluidics itself has been peer reviewed for scientific accuracy before publication as with most journals, and has since been cited in a large number of papers in other well-known journals, including “Laser-treated hydrophobic paper: an inexpensive microfluidic platform” (Girish Chitnis et al., Lab

Chip, 2011 DOI: 10.1039/C0LC00512F), “Progress in patterned paper sizing for fabrication of paper-based microfluidic sensors” (Xu Li et al., Cellulose, 2010, 17, 649 DOI: 10.1007/s10570-010-9401-2) and

“Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva” (Scott A. Klasner et al., Analytical and Bioanalytical Chemistry, 2010, 397, 1821 

DOI: 10.1007/s00216-010-3718-4). These papers develop the FLASH method into more sophisticated techniques of microfluidic device manufacture. It is obvious that in this process of improvement, the original method has come under intense scrutiny, because any flaws in it would be reflected onto the new version. Due to the fact that the technique has stood up to not one but several of these examinations, I believe that the paper “FLASH: A Rapid Method for Prototyping Paper-based Microfluidic Devices” is a very reliable source, and one which presents valid data and results suitable for this issue report.

7 Image: “Lab On A Chip” from MTB Europe http://www.mtbeurope.info/news/2005/506024.htm Accessed 14/02/2011

8 Article: “URI engineering team invents lab-on-a-chip for fast, inexpensive blood tests”, University of Rhode Island

http://www.uri.edu/news/releases/?id=5657 Author: Todd McLeish Accessed 14/02/2011

9 Graphs: ” Microfluidic Devices market in MUnits” and “Compound Annual Growth Rates – CAG” by Yolé Développement From the 2010 report on “Point of Care Testing : Applications of Microfluidic Technologies”http://www.i-micronews.com/upload/Rapports/POC2010-SAMPLE.pdf

Accessed 20/02/1011

10 Article: “Editorial: Implications of Routine HIV Testing”, New Scientist Magazine Issue 2561 (July 2006), p.5 Author: Editor (Jeremy Webb)11

Article: “Lateral Flow Immunoassays” – by Rapid Test Methods Ltd. http://www.rapidmicrobiology.com/test-methods/Lateral-flow-assay.php12 Accessed 10/04/2011

Article: “Development of a Rapid HIV-1 Confirmatory Test” –Orasure Technologies, Inc.http://www.hivtestingconference.org/hivtesting2005/Posters/45_Rapid%20Confirmatory%20Test.pdfAuthors: Keith Kardos, Vijaya K Mokkapati, Geraldine Guillon and Bonnie MartinezAccessed 20/02/2011

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“Agglutination: How It Works” infographic by Rapid Diagnostics Technology Info (RDTInfo) http://www.rapid-diagnostics.org/tech-agglut-howitworks.htm14 Accessed 10/04/2011

Image: “Capillus HIV Rapid Test Results” –World Health Organisation (WHO) http://www.who.int/diagnostics_laboratory/documents/guidance/capillus.pdf Accessed 20/02/2011