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

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.

Molecular Profile of Malaria Drug Resistance Markers of Plasmodium falciparum in Suriname.

In Suriname, an artesunate monotherapy therapeutic efficacy trial was recently conducted to evaluate partial artemisinin resistance emerging in Plasmodium falciparum We genotyped the PfK13 propeller domain of P. falciparum in 40 samples as well as other mutations proposed to be associated with artemisinin-resistant mutants. We did not find any mutations previously associated with artemisinin resistance in Southeast Asia, but we found fixed resistance mutations for chloroquine (CQ) and sulfadoxine-pyrimethamine. Additionally, the PfCRT C350R mutation, associated with reversal of CQ resistance and piperaquine-selective pressure, was present in 62% of the samples. Our results from neutral microsatellite data also confirmed a high parasite gene flow in the Guiana Shield. Although recruiting participants for therapeutic efficacy studies is challenging in areas where malaria endemicity is very low due to the low number of malaria cases reported, conducting these studies along with molecular surveillance remains essential for the monitoring of artemisinin-resistant alleles and for the characterization of the population structure of P. falciparum in areas targeted for malaria elimination.