Rapid malaria tests have become a vital tool in controlling malaria outbreaks, requiring small drops of blood, providing results in just 15 minutes and costing less than $1 each. These tests typically work by detecting the Histidine-Rich Proteins (HRP) 2 and 3 in malaria parasites, but some parasites have mutated to lack the genes for HRP2 and HRP3, leading to potential false negative results when using a rapid test. An international team led by researchers at the University of Notre Dame have now proposed a method that could help determine when an alternate rapid test is needed to detect malaria variants, using digital droplet PCR (ddPCR) to accurately quantify the rates of HRP2 and HRP3 gene deletion across geographic areas.
The World Health Organization (WHO) recommends that malaria control programs only select an alternate diagnostic test if more than 5% of malaria parasites in an area lack the HRP2 and HRP3 genes, as alternative rapid tests relying on other proteins may be less sensitive. While there are several methods that can test for HRP2 and HRP3 gene deletions in malaria parasites, these methods often need to be repeated and may not produce the most accurate results, said lead author Claudia A. Vera-Arias. On the other hand, ddPCR separates blood samples into approximately 15,000 microdroplets and runs PRC on each droplet simultaneously; furthermore, it allows up to 96 samples to be run in parallel on one plate. Compared with more traditional PCR techniques, ddPCR is more efficient and sensitive, and can detect deletions in the genes for HRP2 and HRP3 in individuals who are infected with multiple types of malaria parasites – both those that do and do not have the genes.
The researchers compared the performance of ddPCR with conventional nested PCR (nPCR) by testing 248 samples from people with asymptomatic infections in Kenya. The nPCR approach incorrectly determined that 8% lacked HRP2 and HPR3 genes, meaning an alternative test would have to be used according to WHO’s guidance. However, the more sensitive ddPCR technique determined none of the samples had deletions, allowing the preferred HRP2 and HRP3-based tests to be used.
The team further demonstrated how ddPCR could be used to detect deletions in different geographic areas by testing 830 patient blood samples from Brazil, Ecuador, Ethiopia, Ghana, Kenya and Zanzibar. In Kenya and Zanzibar, the method found no deletions of either gene, and only one sample from Ghana lacked the genes. In Ethiopia, about 2% of samples had an HRP2 gene deletion, and almost 75% had HRP3 deletions. In Brazil, 62% of samples lacked the gene for HRP3, and 46% lacked both the genes for HRP2 and HRP3. In Ecuador, no samples had deletions of the gene for HRP2, but about 54% had deletions of the gene for HRP3. Fewer than 3% of the ddPCR tests had to be repeated because of unclear results. This research was published in eLife.
“Our new ddPCR technique yielded highly accurate results in countries with vastly different rates of deletions in the genes encoding HRP2 and HRP3 and reduced the risk of inaccurate results,” Vera-Arias said.
Senior author Cristian Koepfli added: “Using the more efficient and sensitive ddPCR testing to monitor malaria parasites for deletions of these genes could lead to much better selection of diagnostic tests.”
The results of the study show that traditional PCR approaches may overestimate how common the HRP2 and HRP3 gene deletions are, which may lead some malaria control programs to needlessly abandon the use of HRP-based rapid tests. The researchers are now working to establish a lab for deletion typing in Ethiopia to screen samples from across the region and inform malaria control programs, according to Koepfli. The team also hopes to build more similar reference laboratories in other malaria-endemic regions in the future, Koepfli added.