Friday, August 19, 2011

Thinking Small To Save Big: new research combines nanotechnology with immunology to develop an inexpensive, accurate diagnostic tool for the developing world


SEM  image of HIV virons (green) infecting a CD4+ T cell (red) from CDC.gov.
Public Interest Note:
            Can you live on an annual salary of less than $2,000?  Even if you could somehow, manage to have some clothes, water, food and shelter, what happens when you get sick? These are the difficulties facing the millions of people living in resource-poor countries.  Resource-poor countries are often plagued by a combination of factors including: dense population, low GDP per capita, economic and political instability. 1  Consequently, these countries often have weak health care systems and lack the finances and proper education in combating these countries’ biggest health challenges. Additionally, these countries are typically the most disease-ridden areas in the world. The most prevalent diseases that affect areas of the world such as Sub-Saharan Africa, Indonesia, and India are not the ones we, in America, see as threat, because despite our current economic woes we can afford valuable education and treatments for these diseases.  Thanks in part to huge scientific and medical advances in vaccination, education, and preventative strategies, the developed world has been able to lower the rate of new cases of HIV/AIDS, tuberculosis, and malaria. 2   
    From Tortora, B. "Sub-Saharan Africans Struggle Financially Even as GDP Grows". Gallup. (Jan. 2011)
Unfortunately, technology is generally expensive. Expensive to produce and subsequently expensive to distribute and purchase. Common diagnostic tests performed in U.S. hospitals can cost upwards of a few hundred dollars each. And that’s the cost for the patient, these prices do not incorporate what it costs the hospital to purchase the test from the drug manufacturing company, the cost in paying trained individuals to administer and interpret the test, or any external equipment needed such as electronics and labware!  Although, there have been recent successes in lowering the cost of antiviral drugs in developing countries, there remains a lack of affordable, by means of the developing world, to receive the same quality of accurate and effective diagnostic testing we receive in America.
The exciting thing is, is that despite this particularly difficult endeavor, the scientific community continues to strive to discover innovative ways to improve health in these areas. The newest published research from Samuel Sia’s laboratory at Columbia University exemplifies the ultimate goals, I hope every scientist seeks: to utilize both creativity and intellect to discover something meaningful that will better the world.    
Sia and colleagues present a novel diagnostic device the size of an index card that has the ability to screen for multiple diseases simultaneously! Besides the awesome science behind it (discussed below), the test, itself occurs on a small chip costing as little as $1 to produce, which then is quickly analyzed by a machine “as inexpensive and simple to use as a cellular phone”, and requires little to no training to interpret the results. Oh, did I mention the whole test requires a single drop of blood and only takes 15-20 minutes to complete? A patient can receive results from a single pinprick to results in hand in less than a half an hour! 3
There is no doubt that this technology will be beneficial to resource-poor countries and will help instigate a movement to develop more diagnostic tests in the future, the only problem is: who will fund this kind of production? Such developments need the public’s constant support by understanding the science, providing funding and enhancing communication between scientists and companies that manufacture and distribute these novel devices. Increased support within the public will likely result in providing these diagnostic tools to resource-poor countries while while keeping the cost as minimal as possible. 
 
Developing New Diagnostic Tools: Why This Research Matters
            Sia and his research team set out to design a new diagnostic tool that would be able to accurately screen people for two of the most common infectious diseases in developing countries: HIV and syphilis. The authors of the published paper explain they “chose an HIV-syphilis combination test because HIV and syphilis are treatable in diagnosed pregnant mothers, for whom short-course antiretroviral prophylaxis reduces transmission of HIV, and treatment with penicillin reduces congenital syphilis, which can be fatal for the newborn”.  3 By focusing on diseases that are passively transmitted from mother to baby, the scientists hope to increase the rate of treating infected individuals and subsequently improving the overall healthcare in infectious disease ridden countries.  Sounds like a pretty amicable quest right? Not only would there be hurdles designing the world’s first-ever multi-diagnostic test for these diseases, but the goal was to achieve all of this with quickest results return rate as possible. 
            How do scientists think of ways to do this? They think small: small devices, small patient samples, small amount of reagents needed, etc.  All of this adds up, theoretically, to a small cost to the patient.  The most common testing technology that fits these criteria is categorized as point-of-care testing (POCT).  The purpose of POCT is to provide on-site immediate medical diagnoses that are portable for patients-no matter if they live in a big city, in the country, or in the most isolated, resource-poor habitats in the world. Commonly used POCTs include: individual blood-glucose tests, pregnancy test strips, and rapid agglutination tests for blood-typing. All of these examples are extremely valuable tools because not only are the tests reliable, but they are also inexpensive, easy to use, easy to interpret, and require the minimal amount of external equipment, thus keeping the cost low. All of the mentioned POCTs test for harmless molecules, but how do you keep the convenience of POCTs in mind when designing a tool that is going to detect highly infectious agents? 
            For a long time, it was believed that microscopy was the only way to detect pathogens in an infected person’s blood.  Because you can literally see the presence of a particular bacteria, virus, or parasite, thanks to a variety of dyes and light maneuvering microscopy techniques, you can confidently determine if someone was in fact infected with a deadly microbe. However, microscopes are expensive (as a point of reference, the cheaper student-microscopes used in the General Microbiology Labs I teach, are worth hundreds of dollars each and don’t offer any fancy optics!). Notably, microscopy done right is not as easy as one might believe, it requires extensive training which requires more money in addition to thousands of dollars spent for the microscope, lens, slides, dyes and reagents needed.  Furthermore, microscopes are generally not the most accessible devices, it seems quite challenging to lug a microscope into the middle of the Congo, for example, without somehow breaking one of the objective lens. According to a recent review article on the impact of POCTs on global health, “over a century of poor microscopy performance has contributed to the culture of mistrusts and undervalue of diagnostic test results by health care providers in low-resource settings”.  4
            For these reasons, health care providers in developing countries have turned to immunoassays, which rather than seeing whole microbes, can quantify how much of pathogenic protein is present in a patient’s blood sample.

Screening for Infections in Developing Countries: Challenges and Advances
            POCTs represent a fantastic example of cooperation between scientists, engineers, businesses and public health personnel. There are the biological research laboratories that focus on identifying antigens, molecules that stimulate the immune response, associated with certain infectious diseases, the engineers who then figure out a way to detect these antigens in biological samples, and finally there is the mass-production and distribution of these diagnostic devices by businesses and global health agencies. 
            One of the biggest challenges in designing the most effective POCTs, that is the ones that can realistically be used in the field no matter the location or finances available, are ones that can operate with as little equipment as possible.  This is particularly important for resource-poor countries such as ones located in Sub-Saharan Africa.  Biosaftey and waste disposal tend to be quite poor in developing country clinical facilities, which is especially a concern for areas plagued by high infectious disease rates.  Improper handling of needles, culture plates, tubes, pipet tips, and any other equipment that stores biological samples, increases the spread of disease and further hinder a country’s overall health care. 4    For this reason, the most commonly used POCT to detect pathogenic infection currently is the lateral flow test.  
Lateral flow tests are also called immunochromatic strip (ICS) tests: “immuno” meaning antibodies are used to detect an antigen present in a sample and “chromatic” indicating that some sort of chromatography will be used to visualize the result of the test.  Lateral flow tests are very simple and consist of a small strip of nitrocellulose that has some antibody bound to it at one end of the strip. On the opposite end of the antibody spot, a small blood sample, obtained from a pinprick, is mixed with a buffer that will help carry the blood through the entire nitrocellulose strip simply by capillary action. If a person has the antigen present in their blood that the bound antibody recognizes, it will stick to the strip. As more antigen accumulates on the antibody strip, the strip will produce a visible line to indicate the presence of that antigen. This is the most common method to currently detect malaria and HIV in developing countries. As perhaps, a more easily understood example of lateral flow tests in action, this is the mechanism behind how take-home pregnancy tests work too!  So why invent something new? How could these screening for infectious diseases be improved upon for resource-poor nations?

The “mChip”: The Immunology Behind the Technology


Those were the questions that motivated Sia and his research team to create the “mobile Microfluidic chip for immunoassay on protein markers” or the "mChip" for short (a bit more catchy too right?!). Essentially, the mChip is a full laboratory-based assay shrunk down to fit on a plastic chip not much bigger than a credit card.  The strongest advantage the mChip has over lateral flow tests is that you can screen for multiple antigens or diseases at the same time!
            So how does the test work? Well, part of the name is immunoassay, so again, this test utilizes antibodies specific for certain antigens associated with disease. The laboratory-based test that is miniaturized is the Enzyme-linked Immunosorbant Assay (ELISA).  The ELISA, is perhaps the most commonly used immunological test in the world and is used everywhere from hospitals to academic research labs to undergraduate immunology courses. ELISAs are an extremely valuable test because: 1) you can test multiple samples simultaneously, 2) is very good for detecting even small amounts of antigen and 3) the results are typically colormetric, making the interpretation of results simpler.  You can detect very small amounts of antigen that usually is below the threshold of detection in other screening strategies, because of the Enzyme-linked detection antibodies used in the assay. 
Diagram illustrating an ELISA done commonly in labs in hospitals to test for the presence of disease-related antibodies and pathogens. The mChip miniturized this whole assay onto a polystyrene chip and used metal nanoparticles in place of enyzmes to simplify the technique. From signosisinc.com
          An ELISA begins by coating a 96-well polystyrene plastic plate with a capture antibody (that’s right you can test 96 samples at once!). The capture antibody is an antibody that is specific for a particular antigen, say gp41- a well-known HIV antigen.  Polystyrene plastic is used because it binds proteins extremely tightly, which is important when you need to wash the plate to remove unbound antigen.  By removing anything that doesn’t stick to the capture antibody, the level of background interference decreases dramatically.  So if a person has HIV, the gp41 will stick to the capture antibody on the plate, but how will you visualize this?  That’s where the enzyme-linked antibody comes into play.  In an ELISA, a second antibody is used to detect the bound antigen.  Think of this as a sandwich: where on a plate there’s an antigen trapped between two antibodies-both antibodies detect the same antigen. This second, detection antibody is covalently linked to an enzyme, so you can add a particular substrate that if the enzyme-linked antibody is stuck to the plate, will cleave the substrate revealing a color. Therefore in a typical ELISA, the darker the color means the more enzyme there is, thus the more antigen there is detected in the sample!
            By greatly reducing the volumes of reagents used, and shrinking the plate to a polystyrene chip with seven channels, Sia and his team effectively minimized the size of a typical laboratory-based ELISA. This sounds great, but based on personal experience, ELISAs take a long time to complete. Each step has to be incubated for about an hour and involves a lot of pipetting and waste, so this wouldn’t really work for a resource-poor environment. To circumvent these concerns, the mChip utilizes microfluidics and multistep reactions to significantly reduce the amount of time and labware needed to do the test!  The chips come pre-coated with specific capture antibodies so the only part that would have to be done in the field is running the sample.  All the pipetting and experimental techniques are replaced with a small tube that a field technician can easily prepare by simple syringe-induced vacuum so no reagents will contact the syringe itself.
             Another innovative difference between the classic ELISA and the mChip is that instead of using enzyme-linked detection antibodies, the mChip uses gold nanoparticles attached to secondary antibodies. After the secondary, gold-linked antibodies are loaded into the chip, a flush of silver nanoparticles flow through the chip.  The silver nanoparticles will react with any bound gold nanoparticles, resulting in the release of light-emitting silver ions.   
           These ions can then be exposed to an inexpensive, simple optical beam such as light-emitting diodes (LEDs) and generate a visible signal within-get this- 10-15 minutes!! Because the compact LED device has an imaging screen, a technician can quickly and easily see the results. 
What else makes the mChip advantageous over lateral flow tests? Another caveat to currently available screen tests, is the subjectivity of interpreting results. This is especially true for developing countries where efficient training is lacking. For example, it can be difficult for one to distinguish between a negative result and a positive result if the dye on the strip is not super strong.  When there is no clear threshold or value placed on an observation, it is up to the experience of the available technician to conclude the results of the test. To correct for this, the Columbia University bioengineering team developed a simple compact device costing less than $1 per unit hat converts the light emitted to an electronic signal. No calculations are required of the clinic-so by simply seeing a signal greater than that of background (a sample run without any blood), indicates a positive result.   
Pretty cool, right? But, does this new technology work in reality, in the field-say, for example in Rwanda?  That’s exactly where the team started to test their mChip, where nearly 3% of the nation is infected with HIV and turnaround times for on-site ELISA tests can take several weeks to obtain. By using only a pinprick of unprocessed whole blood, the mChip was used to screen the presence of HIV (gp41) and syphilis (TpN17).  According tothe study, the assay took less than 15 minutes to complete.  Out of a total of 70 specimens with known HIV status, only one tested false, resulting in overall sensitivity of 100% (98.9-100) and specificity of 96% (88.7-100), rivaling the accuracy of lab-based HIV testing”.  Similar results were obtained for syphilis detection with 94% sensitivity and 76% specificity with samples collected in Project Ubuzima in Rwanda. Importantly, the lab also tested the stability of reagents used for the mChip, and noted that they remain stable for at least 6 months at room temperature. This is especially important for it to be used in areas where refrigeration is hard to find.

What’s Next: From Experimental Research to Distribution
A prototype to model the mChip and detector that would like to be distributed and used in the future. 
From popsci.com


            By incorporating physics and microfluidics, the mChip has the potential to deliver advanced immunological diagnostic tests to resource-poor countries. Currently, the mChip is still in the early design phases, due to limited resources for such academic-based studies.  It will take more funding and people to actually design the mChip that Sia envisions that will actually be distributed throughout the world. It will take more funding and public support to cover production and distribution of the mChip. This research was supported largely by the National Institutions of Health and is a contender for a $14 million dollar grant sponsored by USAID, the Gates Foundation and other agencies as part of the Saving Lives at Birth” challenge. Sia’s research team explains in the paper, that the “ultimate goal of this research is to develop a device for infectious-disease screening of pregnant women located in remote areas to prompt early treatment”. This technology has great potential in reducing infectious-disease prevalence in the world, but needs further support to develop this potential into reality.

ResearchBlogging.org Chin CD, Laksanasopin T, Cheung YK, Steinmiller D, Linder V, Parsa H, Wang J, Moore H, Rouse R, Umviligihozo G, Karita E, Mwambarangwe L, Braunstein SL, van de Wijgert J, Sahabo R, Justman JE, El-Sadr W, & Sia SK (2011). Microfluidics-based diagnostics of infectious diseases in the developing world. Nature medicine, 17 (8), 1015-9 PMID: 21804541






References and Further Reading: 
1: Quet, F., et al. "Challenges of epidemiological research on epilepsy in resource-poor countries". Neuroepidemiology. 30: 3-5. (2008) 
2: IB Times Staff Reporter. "Lab on Chip to Offer, Cheaper, Faster HIV Test". (Aug. 2011)
3:Chin, C., et al. "Microfluidics-based diagnostics of infectious diseases in the developing world". Nature Medicine. 17:1015-1019. (2011). 
4: Yager, P., et al. "Point-of-care Diagnostics for Global Health". Annu. Rev. Biomed. 10:107–44 (2008).

14 comments:

  1. good immunology posts
    could you please provide an email or contact form?
    thank you

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  2. Thank you for reading OX40! I will work on setting up a contact form, until then if you have any questions or would like to further discuss any posts, please feel free to leave more comments and I will be happy to discuss them!

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