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Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum
Jonathan B Parr1, Olivia Anderson2, Jonathan J Juliano3,2,4
1Division of Infectious Diseases, Department of Medicine, University of North Carolina, 130 Mason Farm Rd, Chapel Hill, NC, 27599, USA. jonathan_parr@med.unc.edu.
Malaria Journal
|April 4, 2018
Summary
Detecting Plasmodium falciparum malaria parasites that lack the pfhrp2 and pfhrp3 genes requires careful laboratory work. Optimized PCR methods improve detection limits and accuracy for these resistant malaria strains.
Area of Science:
- Molecular biology
- Parasitology
- Genetics
Background:
- Rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria rely on detecting histidine-rich protein 2 (PfHRP2).
- Emerging P. falciparum strains lacking pfhrp2 and/or pfhrp3 genes are reported globally, challenging malaria diagnosis.
- Existing PCR assays for detecting gene deletions vary, necessitating standardization.
Purpose of the Study:
- To characterize and compare published PCR assays for detecting pfhrp2- and pfhrp3-negative P. falciparum.
- To optimize PCR conditions for improved detection limits and specificity.
- To propose a streamlined testing algorithm for identifying these resistant malaria parasites.
Main Methods:
- Evaluation of six distinct PCR assays targeting pfhrp2 and pfhrp3 genes.
- Optimization of PCR by adjusting elongation temperature to 60°C.
- Assessment of assay sensitivity, specificity, and limit of detection using P. falciparum 3D7 strain DNA.
Main Results:
- The lower limit of detection for tested PCR assays ranged from 0.01 pg/µL to 0.1 ng/µL (approx. 0.4-4000 parasite genomes/µL).
- Lowering elongation temperature to 60°C improved detection limits tenfold for exon 1 targets.
- Assays targeting exon 1 showed spurious amplification of paralogous genes, indicating potential specificity issues.
Conclusions:
- Accurate surveillance of pfhrp2- and pfhrp3-negative P. falciparum necessitates meticulous laboratory protocols.
- PCR-based methods, combined with microscopy or antigen testing, are crucial for informing malaria control policies.
- Standardized PCR approaches are vital for assessing the epidemiological impact of these resistant malaria parasites.
