NAME: Edwin Kamau
POSITION: Chief, Molecular Diagnostics, Malaria Vaccine Development at Walter Reed Army Institute of Research
As is the case with many infectious diseases, quantitative real-time PCR is quickly moving toward becoming the gold standard for diagnosing malaria, ostensibly replacing the current gold standard of microscopy. The advantages of qPCR are many: it is faster, equally or more sensitive, and enables identification, speciation, and quantitation of the several species of Plasmodium parasites that can cause the disease.
As the number of assays for detecting Plasmodium increase in the literature, so too has arisen a need to reliably compare and evaluate the performance of the tests — a difficult task considering the fact that most of the qPCR assays for malaria that have been reported have been evaluated using different methods, reagents, standards, and instrument platforms, just to name a few variables.
A group led by Edwin Kamau of the Military Malaria Research Program of the Malaria Vaccine Branch of Walter Reed Army Institute of Research recently sought to address these discrepancies. In a paper published in August in the Malaria Journal, Kamau et al. compared the performance of seven published qPCR assays that detect Plasmodium spp. or Plasmodium falciparum (the most common malaria-causing parasite), using standard DNA and samples from a clinical trial.
Besides evaluating the assays using the same study parameters, the researchers also made sure to follow the minimum information for publication of quantitative real-time PCR experiments, or MIQE, guidelines — a set of qPCR standards published by concerned members of the research community that were intended to serve as a checklist for authors to ensure their qPCR-based experiments could be reproduced by others (PCR Insider 6/3/2010 and 12/31/2009).
Kamau and colleagues found that assays with high PCR efficiencies outperformed those with low efficiencies in all categories including sensitivity, precision, and consistency regardless of the assay format and background. They also found that, with the exception of one assay, all of the evaluated tests showed lower sensitivity compared to what had been previously published.
PCR Insider recently interviewed Kamau via email to discuss his group's study and the further development of its malaria assay, which was one of the tests that they evaluated. Following is an edited version of the interview.
What was the primary motivation for conducting this work?
The motive was to initiate awareness — to address the need for qPCR to be in harmony and to have a standardized method of conducting experiments and reporting results. That way, data established using qPCR assays from different labs can be evaluated by other researchers with greater confidence.
How do the assays compared in your study generally differ from one another? Is it simply a difference in the primers used? Are there other factors?
There are several differences. First, the primers and probe sequences are designed to target different areas within the malaria genome. The most common target is the 18S rRNA gene. Second, the assays are performed in different types of master mixes, which we have found can greatly affect the sensitivity of the assay. Third, the platforms on which the assays are performed are different. In this effort, we assessed primer/probe sets as previously published, with the goal of trying to level the playing field. The only thing different was the primer/probe chemistry — everything else was kept constant.
What specific aspects of the assay did your group make uniform for the purposes of your study?
We modified the probe reporter to make it uniform for all the assays. To level the playing field, all assays were analyzed using the same master mixes background and the same platform. Further, where possible, all assays were performed on the same plate, on the same day, and by the same technician to eliminate the possibility of any bias. Several master mixes were tested to try and capture different environments in which these assays were initially performed. It would be interesting if the work that we have done here could be repeated in other labs, especially if several labs can participate in such an effort — even more so if more [and] different master mixes can be used. In such a scenario, participating laboratories would be supplied with the same aliquot of samples to test.
An assay that you and your colleagues previously developed was found to be one of the highest performing assays. Why do you think that is?
This was the first question that the reviewers asked and fussed about. When we initially designed the assay, we followed good molecular assay guidelines [such as MIQE] to ensure good performance of the assay. Also, although not captured in the original publication, we tested about 15 different primer/probe assays, which included several combinations of primers and probes, until we found chemistry that we felt was most optimal. In addition, since we published the assay it has been cited several times; and I have talked to many labs that are now using our assay exclusively. Two of those labs that I spoke with stopped using their own assays in favor of our assay based purely on performance of the assay. We are currently taking the assay through the [US Food and Drug Administration] validation process, which will be a first for a malaria PCR [assay]. This will allow further analysis of the assay. There is also the possibility [that] the assay performed very well because it was designed on the same platform [that was used to compare it] with other assays. However, we have argued that the focus of the PCR assays should be the chemistry regardless of the background or platform. As we continue performing the assay on different platforms —which we are now: the assay is being used on an [Agilent] Stratagene, [Qiagen] RotorGene, and Bio-Rad [platform] — we will continue collecting important data. But we strongly believe, as pointed out in the publication, that if the chemistry is right, the results will be right.
On the other hand, what are the main reasons for the poor performance of the lower-performing assays, especially the FAL assay?
There could be many reasons, but as clearly shown, the assay did not meet some of the key characteristics. The chemistry of the primers [and] probe might not have been optimal. P. falciparum being such an AT-rich genome makes it challenging to obtain optimal chemistries for primers/probes. Therefore, if someone had assessed the assay conceptually when they were designing it, they could have realized the assay performance might not stand the test of time especially since the P. falciparum genome is so diverse.
Are any of these assays the basis for commercial malaria assay products?
No, they are not. There is only one commercial assay that we know of by Qiagen, which has expressed interest in adapting our assay to replace what it currently has. The assay commercialized by Qiagen, [the Artus RealArt Malaria LC Assay], was [evaluated] by Farcas et al. in the Journal of Clinical Microbiology in Feb. 2004.
Is your group following up on this work at all?
Yes, very much so. We are currently part of a working group that is trying to harmonize PCR assays for laboratories across the world, with a special focus on a controlled human malaria infection model for clinical trials. The working group involves many organizations including the European Vaccine Initiative, University of Washington Medical Center, and Sanaria, to mention a few. Also, as mentioned, the assay is going through the FDA validation process, [an] effort which started two years ago. This effort will include filling for 510k [clearance].