Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon

Abstract

Background: Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing.

Methods: Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (Pf-SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach.

Results: We evaluated Pf-SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf-SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf-SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed.

Conclusion: Overall, Pf-SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible.

Keywords: Plasmodium falciparum; amplicon sequencing; antimalarial resistance; genetic relatedness; malaria.

Figure 1

Sample flow. One hundred samples from parasitemic participants were used in the study. These samples were PCRed and sequenced with replicates (R1 and R2) and processed in SeekDeep. To ensure high quality field sample data, we examined the replicates for each field sample. Replicates that differed in the haplotypes reported or that showed >10% variation in haplotype frequency between replicates (N=35) were re-amplified and re-sequenced, with one exception. In addition, a subset of 50 samples also underwent MIP sequencing to allow for comparison of relatedness and antimalarial resistance frequency. Lastly, the 100 samples were used to generate non-overlapping pools of N=10, N=20, N=50, and N=100 to evaluate the utility of the assays for pooled sequencing. All final sample (and pool) genotyping calls are based upon haplotypes that occurred in both replicates (R1 and R2) and within sample haplotype frequency was averaged between replicates.

Figure 2

Estimated complexity of infection for each sample. Red text denotes the mean (as a central dot) and standard deviation (as arms above and below the mean). Black dots represent COI values for individual samples. (A) Depicts the COI for Pf-SMARRT based on the amplicon that provided the highest number of haplotypes. (B) Shows the estimated COI determined by THE REAL McCOIL for Pf-SMARRT and MIPs. Significance determined by t-test. ns = not significant.

Figure 3

Principal component analysis of 100 Cameroonian samples collected from three hospitals in Dschang, Cameroon. Samples are color coded by the patient's village/town/city of origin.

Figure 4

Correlation of antimalarial resistance SNP frequency in pools compared to individual samples. Weighted mean allele frequency for key resistance mutations was estimated across replicates for each pool group and compared with allele frequencies across replicates for the individual samples included in each pool. Allele frequencies in the pool were weighted by parasite density of each individual sample included in the pool. Pearson’s correlation values were high across all pool groups. Each frame is labeled as ##pool#, where ## is the pool size and # is the pool number.