This article was originally published on April 7.
Scientists from the US Centers for Disease Control have for the first time directly compared the ability of three types of PCR assays to detect DNA from various species of Plasmodium, the parasite that causes malaria.
According to the results of their work, the so-called "nested" PCR assay — currently considered the gold standard despite being more time- and labor-intensive — is still more effective than easier-to-perform semi-nested or single-tube multiplex PCR assays for detecting Plasmodium.
Their findings, published last week in the online edition of BMC Research Notes, support the belief that development of a fast and easy assay that retains the same level of sensitivity as nested PCR will likely require the identification and incorporation of new molecular targets for the parasite, the researchers told PCR Insider this week.
Because of the prevalence of malaria in the developing world, healthcare providers have long desired a sensitive, inexpensive, and easy-to-administer test for detecting and identifying one of the four Plasmodium species known to cause malaria in humans: P. falciparum, P. vivax, P. malariae, and P. ovale.
It is generally accepted that molecular tests trump other methods of detection, such as microscopy, which requires specialized training and often leads to mischaracterization of species; and blood-based rapid-diagnosis tests, which, despite being fast and easy to administer, are not able to identify all species or quantify parasitemia — important factors in determining treatment regimens.
PCR-based amplification of the 18S ribosomal DNA gene is currently one of the most commonly used molecular tests to detect mixed Plasmodium species infections. Of the various PCR tests, the nested PCR method, developed in the early 1990s by researchers at London's National Institute for Medical Research, has been widely used in laboratory studies and clinical diagnosis.
However, because the nested reaction requires five separate PCR reactions — one for each species plus one genus-specific reaction — it is time-consuming, expensive, and labor-intensive, and thus not always conducive to laboratories in developing nations.
"When we first started this research, the nested … method was the gold standard for malaria diagnosis because it was very sensitive and specific," Tonya Mixson-Hayden, first author on the BMC Research Notes paper and a senior service fellow with the CDC, told PCR Insider.
"But it's five different sets of reactions, and if you're doing 100 samples, it can become quite tedious," Mixson-Hayden added. "We wanted to look through the literature and find some other methods that were hopefully just as sensitive but were less tedious."
They identified "semi-nested" and single-tube multiplex PCR assays, both of which target a region of the 18S gene, as alternatives. The semi-nested procedure comprises two PCR reactions: one "outer" reaction specific to the Plasmodium genus, and one "inner" reaction specific to all mammalian 18S rDNA.
Meantime, in the single-tube method, a single multiplexed PCR reaction combines a genus-specific reverse primer with four forward species-specific primers to detect all four Plasmodium species. "It's not nested, therefore you are going to lose a little sensitivity," Mixson-Hayden said — although the authors of this method, researchers from the National Institute for Biological Standards and Control in the UK, have claimed that its sensitivity is comparable to the nested reaction.
Mixson-Hayden and colleagues set out to evaluate the three assays in the hopes that either the semi-nested or single-tube assay could distinguish between the four Plasmodium species as effectively as the nested reaction.
To do this, they compared the methods using known quantities of laboratory-derived Plasmodium DNA alone and in experimentally mixed cocktails containing all four species. They found that the nested PCR assay was able to detect single Plasmodium species infections in concentrations as low as 0.4 parasites/µl.
In comparison, the semi-nested assay was as sensitive as the nested assay at detecting P. falciparum and P. vivax; and more sensitive at detecting P. malariae; but was only able to detect P. ovale down to 4 parasites/µl. Meantime, the multiplex single-tune assay had a similar limit of detection as the other two assays for P. falciparum; but was only able to detect down to 4 parasites/µl for P. ovale and 40 parasites/µl for P. malariae and P. vivax.
In addition, over the course of seven replicate experiments, the nested reaction was the only assay that consistently detected all four species in the cocktail at sensitivity levels reported for singly infected samples.
"Clearly the nested PCR method is the best PCR-based assay for the diagnosis of mixed infections and of subclinical infections among the three methods tested in this study," the researchers wrote in the paper.
They also wrote that "further optimization of primers and reaction conditions could possibly improve the semi-nested and multiplex methods." However, Mixson-Hayden told PCR Insider that identifying genes other than the 18S rDNA gene will likely be a more fruitful endeavor.
Researchers "are trying to work on a different kind of assay," Mixson-Hayden said. The 18S gene "is still pretty much the most sensitive gene to detect these species, and we hope that we can find different molecular markers that would allow us to improve our sensitivity."
In addition, Mixson-Hayden's co-author Venkachalam Udhayakumar, chief of the genetics and immunology lab in CDC's malaria branch, is attempting to develop a modified assay based on loop-mediated isothermal amplification, or LAMP.
This assay still uses the 18S gene, but "uses more sensitive approaches and simple molecular devices" that may be employable "even in remote places," Udhayakumar told PCR Insider. Udhayakumar's team is currently compiling data from experiments using the LAMP method, and expects to submit a manuscript for publication in the near future, he said.