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SIMPLseq: a high-sensitivity Plasmodium falciparum genotyping and PCR contamination tracking tool.

Philipp Schwabl1,2,3, Jorge-Eduardo Amaya-Romero1,2, Katrina A Kelley1,2

  • 1Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA.

Malaria Journal
|February 3, 2026
PubMed
Summary

A new Plasmodium falciparum amplicon sequencing (AmpSeq) miniplex, SIMPLseq, offers high-sensitivity malaria parasite genotyping and integrated contamination detection. This tool enhances genomic epidemiology for drug resistance tracking and intervention trials.

Keywords:
Plasmodium falciparumAmplicon sequencingContamination detectionEpidemiologyGenotypingHigh-sensitivityMalaria

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Area of Science:

  • Genomic epidemiology
  • Molecular diagnostics
  • Parasitology

Background:

  • Polymerase chain reaction (PCR) amplicon sequencing (AmpSeq) is crucial for pathogen genotyping and disease surveillance.
  • Existing large AmpSeq panels struggle with low-parasitemia samples (<10 parasites/μl), limiting their use in malaria drug resistance tracking.
  • Managing PCR contamination is a significant challenge in AmpSeq methodology.

Purpose of the Study:

  • To develop a high-sensitivity Plasmodium falciparum AmpSeq miniplex (SIMPLseq) for low-parasitemia samples.
  • To integrate a contamination detection system into the miniplex using inline barcodes.
  • To evaluate SIMPLseq's sensitivity, precision, and contamination detection capabilities.

Main Methods:

  • Developed a 6-locus Plasmodium falciparum AmpSeq miniplex (SIMPLseq) with inline barcodes for first-round PCR (PCR1) contamination detection.
  • Assessed in silico panel diversity using whole-genome sequencing (WGS) data.
  • Used mock samples to compare SIMPLseq's sensitivity and precision against the 4CAST miniplex.
  • Deliberately introduced contamination events to assess detection rigor and estimated unintentional contamination rates in samples from Mali.

Main Results:

  • SIMPLseq demonstrated high in silico haplotypic diversity, distinguishing 96.0% of sample pairs.
  • Achieved 100% average locus detection at ≥0.5 parasites/μl and ≥50% at 0.125 parasites/μl, outperforming 4CAST.
  • Inline barcoding did not significantly impact yield and correctly identified all 24 deliberately introduced PCR1 contaminations.
  • Identified 39 unintentional contaminations among 1420 samples from Mali.

Conclusions:

  • SIMPLseq enhances malaria genomic epidemiology by providing high-sensitivity P. falciparum genotyping and PCR contamination detection.
  • The protocol is simple, uses open-source reagents, and avoids costly pre-amplification.
  • SIMPLseq is valuable for classifying recurrent infections, estimating polyclonality, and applying genotypic infection endpoints in trials.