Advantages | Disadvantages | References |
---|---|---|
Conventional PCR | Â | |
 Can be performed in most labs that can perform molecular diagnosis of malaria | Time-intensive— > 2 h per reaction |  |
 Can readily check presence of flanking genes | Requires multiple distinct PCR reactions; ≥ 6 reactions per sample for control and pfhrp2/3 genes |  |
 Can detect single and dual exon deletions | Need to visualize PCR products on agarose gels |  |
Do not detect gene deletions when gene deleted parasites are mixed with wild-type parasites in the same sample Nested assays are prone to contamination | Â | |
Requires high volume of DNA for > 6 PCR reactions |  | |
Multiplex real-time PCR | Â | |
 Streamlined workflow | Requires multichannel real-time PCR machine |  |
 Quantitative read-out | Proper interpretation of results requires training | |
 Can detect mixed infections involving pfhrp2/3-deleted and intact strains | Careful optimization required for individual laboratories | |
 Only one PCR reaction and requires less volume of DNA | May not detect some partial gene deletions involving one exon as most assays targeting one exon only | |
 Includes internal control |  |  |
Digital droplet PCR | Â | [42] |
 Higher confidence deletion calls than other PCR methods  Can detect mixed infections involving pfhrp2/3-deleted and intact strains | Specialized equipment that is not widely available Requires advanced laboratory and analysis expertise More expensive than conventional approaches |  |
Sequencing approaches | Â | |
 Enable identification of mutations that affect HRP2 expression (e.g., coding changes)  Mapping and comparison of deletion breakpoints is possible using next-generation sequencing approaches (amplicon-based and whole-genome)  Enables analysis of parasite relatedness, transmission, and evolution | Current approaches are not well-suited for initial deletion identification, especially in lower parasite density samples Specialized equipment that is not widely available Requires advanced laboratory and analysis expertise More expensive than conventional approaches |  |