High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy
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Evaluation Summary:
The study reports the development of high-throughput droplet digital PCR to detect Plasmodium falciparum parasites carrying pfhrp2 and pfhrp3 gene deletions. These mutations usually cause false-negative RDT results on malaria tests. Although there are several PCR-based detection methods already available, the assay is useful as an alternative, particularly in countries and settings where droplet digital PCR is routinely used. The strength lies in its capability to detect hrp2 and hrp3 deletions in samples with multiclonal (more than one clone) infections. This has the potential to assist in surveillance for pfhrp2/3 deletions programs where RDTs designed to detect HRP2 are the primary test leading to false negative results, particularly in medium to high transmission settings. The study will be of interest to those studying infectious diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Most rapid diagnostic tests for Plasmodium falciparum malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the hrp2 and hrp3 genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of hrp2 / hrp3 deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies hrp2 , hrp3 , and a control gene with very high accuracy. The theoretical limit of detection was 0.33 parasites/µl. The deletion was reliably detected in mixed infections with wild-type and hrp2 -deleted parasites at a density of >100 parasites/reaction. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR hrp2 deletion was observed in 8% of samples. The ddPCR assay was applied to screen 830 samples from Kenya, Zanzibar/Tanzania, Ghana, Ethiopia, Brazil, and Ecuador. Pronounced differences in the prevalence of deletions were observed among sites, with more hrp3 than hrp2 deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e., hrp2 deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.
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Evaluation Summary:
The study reports the development of high-throughput droplet digital PCR to detect Plasmodium falciparum parasites carrying pfhrp2 and pfhrp3 gene deletions. These mutations usually cause false-negative RDT results on malaria tests. Although there are several PCR-based detection methods already available, the assay is useful as an alternative, particularly in countries and settings where droplet digital PCR is routinely used. The strength lies in its capability to detect hrp2 and hrp3 deletions in samples with multiclonal (more than one clone) infections. This has the potential to assist in surveillance for pfhrp2/3 deletions programs where RDTs designed to detect HRP2 are the primary test leading to false negative results, particularly in medium to high transmission settings. The study will be of interest to those …
Evaluation Summary:
The study reports the development of high-throughput droplet digital PCR to detect Plasmodium falciparum parasites carrying pfhrp2 and pfhrp3 gene deletions. These mutations usually cause false-negative RDT results on malaria tests. Although there are several PCR-based detection methods already available, the assay is useful as an alternative, particularly in countries and settings where droplet digital PCR is routinely used. The strength lies in its capability to detect hrp2 and hrp3 deletions in samples with multiclonal (more than one clone) infections. This has the potential to assist in surveillance for pfhrp2/3 deletions programs where RDTs designed to detect HRP2 are the primary test leading to false negative results, particularly in medium to high transmission settings. The study will be of interest to those studying infectious diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study is about the development of a novel assay based on droplet digital PCR (ddPCR) to detect malaria parasites capable of evading rapid diagnostic test (RDT) through the deletion of histidine-rich proteins (HRPs) commonly recognised by the RDTs. The HRP proteins are encoded by hrp2 and hrp3 genes. The ddPCR is probe-based assay targeting four regions in three genes: hrp2 (two regions), hrp3 and tRNA.
The strength of the assay lies on its capability to detect hrp2 and hrp3 deletions in samples with multiclonal (more than one clone) infections. The assay also is capable of measuring the DNA concentration using absolute quantification.
The study has several weaknesses. Firstly, the assay lack internal control to monitor variations caused by external factors such as pipetting and other handling steps such …
Reviewer #1 (Public Review):
This study is about the development of a novel assay based on droplet digital PCR (ddPCR) to detect malaria parasites capable of evading rapid diagnostic test (RDT) through the deletion of histidine-rich proteins (HRPs) commonly recognised by the RDTs. The HRP proteins are encoded by hrp2 and hrp3 genes. The ddPCR is probe-based assay targeting four regions in three genes: hrp2 (two regions), hrp3 and tRNA.
The strength of the assay lies on its capability to detect hrp2 and hrp3 deletions in samples with multiclonal (more than one clone) infections. The assay also is capable of measuring the DNA concentration using absolute quantification.
The study has several weaknesses. Firstly, the assay lack internal control to monitor variations caused by external factors such as pipetting and other handling steps such as during DNA extraction. Secondly, data for validation of the hrp3 assay is missing - HRP3 protein is known to give signal in RDT in the absence of HRP2, particularly when the parasite density if high. Therefore, it is equally important to validate the performance of the assay in detecting hrp3 deletion. Thirdly, the authors used "genome" copy to assess the performance of the assay and it is hard to compare its performance with other assays such as nested PCRs and quantitative PCRs that use parasite density for performance evaluation. Fourthly, the presence of more than one clone in the samples was not verified by other methods, such as by qPCR or genome coverage, and it is difficult to validate the accuracy of the ddPCR. Finally, no data is available to show that both hrp2 and tRNA assays have similar or the same amplification efficiency permitting the use of the assays for calculating the ratio DNA concentration to determine deletions in multiclonal infections.
The assay has the potential to be used as a tool to rapidly detect RDT-evading malaria parasites and contribute to control and elimination programs.
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Reviewer #2 (Public Review):
Vera-Arias et al developed and evaluated a new molecular method using droplet digital PCR (ddPCR) for surveying hrp2/hrp3 deletions in Plasmodium falciparum parasites. These deletions are problematic because they produce false negative results when rapid diagnostic tests (RDT) are used in endemic areas. In this study, they compared the new ddPCR method with existing nested PCR (nPCR) method and found the new ddPCR method has increased sensitivity and accuracy and recommended the use of ddPCR. The conclusions of this paper are well supported by data.
The strength of this paper is that the authors have demonstrated the use of this new assay in mixed culture in experimental setting and extended this evaluation in field isolates as mixed infections are the common occurrence in the endemic areas. An assay with …
Reviewer #2 (Public Review):
Vera-Arias et al developed and evaluated a new molecular method using droplet digital PCR (ddPCR) for surveying hrp2/hrp3 deletions in Plasmodium falciparum parasites. These deletions are problematic because they produce false negative results when rapid diagnostic tests (RDT) are used in endemic areas. In this study, they compared the new ddPCR method with existing nested PCR (nPCR) method and found the new ddPCR method has increased sensitivity and accuracy and recommended the use of ddPCR. The conclusions of this paper are well supported by data.
The strength of this paper is that the authors have demonstrated the use of this new assay in mixed culture in experimental setting and extended this evaluation in field isolates as mixed infections are the common occurrence in the endemic areas. An assay with high sensitivity to detect these key deletions amongst mixed infection is particularly important in monitoring the true prevalence of these deletions in low-density transmission settings.
The weakness of this paper is that the authors did not evaluate against the existing real-time PCR methods. A good and extensively validated real-time PCR assay can sometimes work as effectively as a ddPCR assay with similar sensitivity. Some data from real-time PCR can be useful in term of comparison existing techniques to novel ones.
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