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Q&A: University of Alberta Team's Real-Time PCR Method Could Enable POC Blood-Based Malaria Dx

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yanow1.jpgNAME: Stephanie Yanow

POSITION: Program leader, R&D, Alberta Provincial Laboratory for Public Health (ProvLab); Assistant professor, School of Public Health, University of Alberta

Researchers from the University of Alberta School of Public Health and the Alberta Provincial Laboratory for Public Health have developed a protocol for amplifying DNA from malaria-causing Plasmodium species directly from whole blood and filter paper samples.

The work, which is described in a paper published this month in Malaria Journal, is part of a larger effort by the group and by commercial partners Aquila Diagnostics to develop infectious disease and cancer genotyping assays on a microliter-scale gel-based lab-on-a-chip platform called In-Gel, also sprung from laboratories at the University of Alberta (PCR Insider, 9/9/10). In the paper, the researchers describe how they used inhibitor-resistant Taq mutants and an enhancer cocktail within the PCR master mix in order to overcome the quenching effect of hemoglobin on the fluorophores used in direct PCR.

Stephanie Yanow, corresponding author on the paper and a member of the malaria assay-development group, discussed the work recently in an interview with PCR Insider. Following is an edited transcript of the interview.

What is your affiliation and how did you become interested in developing this method for real-time PCR detection of Plasmodium directly from whole blood and filter paper?

I work for Alberta Health Services at the Provincial Lab for Public Health, but I also have an academic appointment at the School of Public Health at the University of Alberta. Through both of those organizations, we collaborate with a group called Team Microfluidics, which is actually an interdisciplinary group that has funding to develop a point-of-care device for diagnosing malaria or other pathogens – essentially developing real-time PCR assays on a lab-on-a-chip.

Through that project we've been trying to develop a test for malaria that we could use directly from blood on a lab-on-a-chip that could be developed into a low-cost device for use in developing countries where malaria is endemic.

One of the challenges we faced when we started to develop this technology was: How do you do real-time PCR for malaria straight from blood? Do we extract the DNA first? This means it would be a much more complicated device than if we could just simply take a drop of blood, put it on the device, and test immediately. That's where this paper stems from — a need to have an assay that can work straight from blood so we can have an assay that works in the field.

Can you tell me a little bit about the need for a blood-based point-of-care test for malaria? How is testing currently done in developing countries?

The most traditional method for diagnosing malaria in the field is by microscopy. You just take a blood sample, make a smear on a slide, and look for parasites. There are challenges with microscopy – you need a functioning microscope, you need staff that are trained to use it properly, to know how to read the slides. And then there are issues with access to power and maintaining the microscopes and that kind of thing.

Fairly recently many countries have introduced rapid diagnostic tests, which are antigen-based tests. It's still from the blood, but it's kind of like a pregnancy test where you're looking for a line to show up. These are being widely implemented because they are very low cost and very easy to use. The challenge with these tests is that sometimes their sensitivity can be poor, though not always. It's better than microscopy, but not as good as a molecular test like PCR. Also, many of these tests are geared toward detecting P. falciparum, which is the most lethal species of malaria, and the most dominant in sub-Saharan Africa, but they're less sensitive in detecting P. vivax, which is more prevalent in Latin American and Southeast Asia.

Here at the Provincial Lab, where we offer reference testing for malaria, we offer real-time PCR, which is the most sensitive and specific method of testing for malaria. So the idea was, can we bring this technology — which obviously in our current setting requires expensive equipment and trained staff — to the field on a lab-on-a-chip device? And by working with our colleagues in Colombia we are also addressing the issue of detection P. vivax and P. falciparum, because they have both of those species in that country. If we can develop this lab-on-a-chip, then Colombia would be one of our sites to pilot this technology, in addition to some colleagues in Africa.

Tell me about these inhibitor-resistant Taq mutants and enhancer cocktail that you tested. Are these commercially available reagents?

They are commercially available [from] a company in the [US] called [DNA Polymerase Technology], which developed these reagents in collaboration with academics from [Washington University School of Medicine in 1998]. They published some of their work using these reagents around the same time they started this company, and they were looking at a number of different specimen types [like] blood, serum, [and] plasma; and were looking at a number of different targets like viral targets and human genomic DNA, for cancer screening.

So we took some of those reagents and tried to see if we could get it to work for our malaria assay. The current assay that we use involves DNA extraction and then TaqMan-based chemistry to detect malaria DNA. With the reagents developed by this company we were using a SYBR Green detection system, so we ported that over for this malaria test.

In the paper, you determined the optimal SYBR Green concentration and blood volume for the test, and you had a range of sensitivities and specificities from 93 percent to 100 percent compared with PCR performed on purified Plasmodium DNA. How close are you to where you want to be in terms of sensitivity and specificity?

I think they're definitely in the right range. Ideally we would want around 95 percent. Depending on which test – whole blood or filter papers – we ranged, as you said, from 93 to 100 percent. We were quite pleased with the results compared to our gold standard testing. It's definitely within the range we were looking for, and the results were very clean and easy to interpret.

With the lab-on-a-chip, we'll be dealing with much smaller volumes of blood, because everything is nanoscale. So the next step is to see whether we can get that same level of specificity and sensitivity compared to reference testing, but with smaller volumes. There is the risk that we'll lose some of our sensitivity because we'd be dealing with sub-microliter volumes of blood.

The reaction volumes in our tests were around 20 microliters, and the lab-on-a-chip will be about 0.6 microliters. So there may be some further optimization required, but at least, in principle, we've shown that we can amplify the DNA; that we can do it from different species; and that we can even do it from filter papers, which is a different application from lab-on-a-chip, but we still got good specificity and sensitivity from that.

Would the ability to do the test from filter papers even come into play if you are able to successfully develop the point-of-care test for blood samples?

We're thinking as well about how to use filter papers on the chip, but we haven't really done much with that. The thing with filter papers is that currently that's how blood is collected in the field for epidemiological studies and for vaccine efficacy studies, because many labs can't run real-time PCR in the field. What they tend to do is make blood spots and send them to the research lab. And there, too, you need to extract DNA from the filters, which can be very expensive if you're testing thousands of samples. The second part of this paper explores whether we can just put the filter papers straight into a tube without having to go through the extraction steps. That also was very successful, and that shows us that it's a way to improve the throughput for field studies on malaria. It may not be that important for diagnostics, but it's a research tool.

Are there any other caveats or major hurdles to move this to the next phase into a chip-based field test?

The only other caveat … is that primer design is really important for these assays, because you're dealing with a sample that has a lot of inhibition and is obviously not the optimal sample for molecular testing. These reagents have allowed us to overcome the inhibition, but one of the issues is the potential formation of primer-dimers. In some of our speciation panels, we could see a little primer-dimer curve creeping up. And if we want these assays to be quantitative, then that needs to be addressed, because with SYBR Green it will skew your quantification.

With our consensus reaction, where we detect all the species of Plasmodium, we don't have any problem with primer-dimers, and I think we only saw that in a test for one of the species. But we know that by simply improving the primer design or further optimizing the assay we can get rid of those. I'd say it's a minor caveat, but it's something that's inherent to this type of assay where you're dealing with raw specimen.

Would even a broad, non-species-specific test be valuable for the field compared to what is done currently?

It does, depending on the geographical region. If you're looking at certain parts of Africa where they only see P. falciparum … of course you want to identify and treat it immediately, and can probably safely make the assumption that it's P. falciparum. But in other parts of the world, like Papua New Guinea, they have all four species of Plasmodium, and you need to know which one is causing the infection so you can prescribe the appropriate treatment. And in Latin America where they have P. vivax and P. falciparum, you need to know which species is present because P. vivax needs another treatment to kill the liver-stage parasites that can cause relapses. The quantification may or may not be as important for diagnosis.

What steps is Team Microfluidics taking to commercialize this assay once it's fully developed?

We do have a company that is commercializing this technology called Aquila Diagnostics. They are looking at a number of different areas for the [gel-based PCR array] technology, such as other infectious diseases and cancer genotyping. My group is heading the malaria application, so essentially we needed a way to use that gel-based PCR technology but with whole blood.

Is your academic group and Aquila just collaborating informally at this point?

Yes, Aquila is fairly new, and they're looking at the commercial aspects – manufacturing, how to make these chips for a low cost; and the academic side is really developing and validating the assays.

Is there an agreement for Aquila to license assays and other technologies from your academic group and from the University of Alberta?

Yes, for certain applications. They have a license for blood-borne pathogens, and malaria falls into that.

Do you plan to move forward with the inhibitor-resistant Taq mutants and PCR enhancer cocktail from DNA Polymerase Technology for your malaria test?

Yes, at the moment. We already have preliminary data showing that we can take the same clinical samples we used, along with the TaqMan and [Applied Biosystems 7500 Fast system] and put them on these gel post chips, and we can get very good amplification. So we've already moved into the stage of taking those reagents and moving them onto the lab-on-a-chip.

Now we are trying to adapt the consensus reaction and all the speciation reactions onto the chip and to make sure that we're not having primer-dimer formation; and we're looking at the sensitivity on the chip. Ultimately we'll be comparing the clinical sensitivity and specificity with results from the reference lab, similar to what we did with this paper, but putting the sample right on the gel post chip.

And this is something that you believe will have utility for other blood-borne pathogens besides malaria?

I do think so, and in one of the original papers from DNA Polymerase Technologies, they had shown that they can take blood samples and spike virus and detect it straight from blood. Although we haven't explored other pathogens in blood, it seems perfectly feasible to me that assays could be developed for them … which would reduce costs even for a reference lab, to be able to eliminate that extraction step.

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