Jacob M Sadler ¹ Alfred Simkin ² Valery P K Tchuenkam ³ Isabela Gerdes Gyuricza ¹ Abebe Fola ² Kevin Wamae ⁴ Ashenafi Assefa ¹ Karamoko Niaré ² Kyaw Thwai ¹ Samuel J White ¹ William Moss ⁵ Rhoel Dinglasan ⁶ Sandrine Eveline Nsango ⁷ Christopher B Tume ³ Jonathan B Parr ¹ Innocent Mbulli Ali ³ Jeffrey A Bailey ² Jonathan J Juliano ^1*

• ¹ University of North Carolina at Chapel Hill, Chapel Hill, United States
• ² Brown University, Providence, Rhode Island, United States
• ³ University of Dschang, Dschang, Cameroon
• ⁴ KEMRI Wellcome Trust Research Programme, Kilifi, Kenya
• ⁵ Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States
• ⁶ University of Florida, Gainesville, Florida, United States
• ⁷ Centre Pasteur du Cameroun, Yaoundé, Cameroon

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.