Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

PLoS One. 2022 Jan 14;17(1):e0262616. doi: 10.1371/journal.pone.0262616. eCollection 2022.

Abstract

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antimalarials / pharmacology
  • Dried Blood Spot Testing / methods
  • Drug Resistance / genetics*
  • Epidemiological Monitoring
  • Haiti
  • High-Throughput Nucleotide Sequencing / methods
  • High-Throughput Screening Assays / methods*
  • Humans
  • Malaria / epidemiology
  • Malaria, Falciparum / parasitology
  • Nucleic Acid Amplification Techniques / methods
  • Parasites / genetics
  • Plasmodium falciparum / drug effects
  • Plasmodium falciparum / genetics*
  • Plasmodium falciparum / pathogenicity
  • Polymerase Chain Reaction / methods
  • Sequence Analysis, DNA

Substances

  • Antimalarials

Grants and funding

We acknowledge support from the Advanced Molecular Detection (https://www.cdc.gov/amd/index.html) Initiative at the CDC. SL and JM is supported by The Oak Ridge Institute for Science and Education (https://orise.orau.gov) and funded by the CDC OAMD program. DP is employed by Williams Consulting LLC (https://www.williamsconsultingllc.com), which provided support in the form of salary for DP. CH was supported by the CDC Foundation (https://www.cdcfoundation.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.