A digital polymerase chain reaction (dPCR) technique for screening malaria parasite genomes will help identify when they have mutations that allow them to dodge detection by some rapid tests, shows a study published in eLife.

The results could help malaria control programs identify which rapid diagnostic tests will work best in their areas. Matching the right test with an area based on the parasite Plasmodium falciparum’s genes reduces false negative malaria tests that hamper efforts to control the disease by rapidly detecting and treating people with the infection.

Rapid Testing for Malaria

Rapid diagnostic tests that detect Histidine-Rich Proteins (HRP) 2 and 3 from malaria parasites in human blood droplets have become vital in controlling malaria. These tests cost less than $1 and provide results in about 15 minutes in almost any setting. But in areas where malaria parasites lack the genes for HRP2 or HRP3, the tests may produce false negative results. As a result, the World Health Organization (WHO) recommends that malaria control programs test malaria parasites for the genes for HRP2 and HRP3 and select an alternate diagnostic test if more than 5% of malaria parasites in an area lack these genes. Some diagnostic tests detect other proteins, but they are less sensitive.

“There are several methods to test for hrp2 and hrp3 gene deletions in malaria parasites,” says lead author Claudia A. Vera-Arias, a PhD candidate at the University of Notre Dame in South Bend, Indiana, US. “But these tests often have to be repeated and may produce inaccurate results.”

Vera-Arias and colleagues developed a new method for detecting the presence of genes for HRP2 and HRP3 in malaria parasites based on a technique called droplet digital polymerase chain reaction (ddPCR). This technique separates blood samples into approximately 15,000 microdroplets and runs PCR on each of them simultaneously. 96 samples can be run in parallel on one plate.

Compared with more traditional PCR techniques, ddPCR is more efficient and sensitive, and can detect deletions of the genes for HRP2 or HRP3 in individuals infected with malaria parasites both with and without these genes.

Details of the Study

The team compared the performance of the ddPCR technique with the conventional nested PCR (nPCR) method by testing 248 samples from people with asymptomatic infections in Kenya. The traditional nPCR approach incorrectly determined that 8% lacked genes for HRP2 or HRP3, which would rule out the use of HRP2- or HRP3-based diagnostic tests based on the WHO’s guidance. However, the ddPCR technique determined that none of the samples had these deletions.

Next, they used ddPCR on 830 patient blood samples from Brazil, Ecuador, Ethiopia, Ghana, Kenya and Zanzibar. In Kenya and Zanzibar, ddPCR found no deletions of the genes for HRP2 or HRP3; only one sample from Ghana lacked these genes. In Ethiopia, about 2% of samples had an HRP2 gene deletion, and almost 75% had HRP3 gene 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. 

“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 says.

She adds that more traditional PCR testing approaches may overestimate how common deletions of the genes for HRP2 and HRP3 are, leading malaria control programs to unnecessarily abandon HRP2-based rapid tests altogether.

“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,” says senior author Cristian Koepfli, assistant professor at the Department of Biological Sciences and affiliate of the Eck Institute for Global Health, University of Notre Dame. “We are already putting the new method to use where it is needed most. With support from the Bill and Melinda Gates Foundation, we are establishing a lab for deletion typing in Ethiopia and screening samples from across the region to inform the malaria control programs whether they should stop using diagnostic tests relying on HRP2 detection. We hope to build similar reference laboratories in other malaria-endemic regions and work with multiple control programs. Our novel molecular assay will thus directly support countries in their selection of the best tests for malaria diagnosis, and thus aid to make further gains towards malaria elimination.”