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Table 1 PCR assays for detecting pfhrp2 and pfhrp3 genes with advantages and disadvantages

From: Screening strategies and laboratory assays to support Plasmodium falciparum histidine-rich protein deletion surveillance: where we are and what is needed

Advantages

Disadvantages

References

Conventional PCR

 

Numerous, summarized in: [27, 30]

 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

 

Requires high volume of DNA for > 6 PCR reactions

 

Multiplex real-time PCR

 

[37, 38]

 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

 

[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

 

[20, 36, 49, 50]

 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

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