Ethics Statements and Study Sites
DBS used for this study were collected as part of a TES based on the standard WHO protocol 19 designed to assess the efficacy of artemether-lumefantrine, artesunate-amodiaquine, and dihydroartemisinin-piperaquine in six sites representing different epidemiologic zones within DRC: Kabondo, a district of the city of Kisangani, in the northern province of Tshopo; Bolenge, located on the Congo River in the province of Equateur that borders the Republic of Congo; Rutshuru, in the mountainous province of North Kivu bordering Uganda and Rwanda; Kimpese, in the Kongo Central province that borders Angola; Mikalayi, in the province of Kasai Central; and Kapolowe, situated on the shore of Lake Tshangalele in the tropical southern province of Haut Katanga bordering Zambia (Figure 1). Based on the 2017-2018 Multiple Indicator Cluster Survey published by the National Institute of Statistics (INS) of DRC 20, the malaria RDT prevalence among children aged 6–59 months was reported to be 38.5% nationally and 52.2% in Tshopo, 45.5% in Kasai Central, 40.0% in Kongo Central, 11.4% in North Kivu, 43.7% in Haut Katanga, and 39.0% in Equateur Province. Based on the national and regional malaria RDT prevalence rates, these study sites should be representative of the nation as a whole for malaria transmission.
Study recruitment took place from March 2017 to January 2018. Ethical clearance for this study was provided by the DRC Ethics Committee of the School of Public Health of the University of Kinshasa, and the protocol was approved as a non-research program evaluation by the Office of the Associate Director for Science, Center for Global Health at Centers for Disease Control and Prevention (CDC; 2018-035). Patient recruitment was performed in accordance with the Declaration of Helsinki. Patients were only enrolled in this study if their parent or guardian chose to give consent. Informed consent forms were made available in French and translated into Lingala, Kikongo, Swahili, and Tshiluba, and read aloud in its entirety to parents and guardians to ensure understanding of the potential risks and benefits of the study. Parents or guardians who were unable to read or write were also given the opportunity to select study-independent co-signers for informed consent forms. All patient information was kept confidential, and an individual identification number was assigned to each sample for data entry. Staff from the CDC provided technical assistance 18; this activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.
Patient Enrollment and Sample Collection
Children aged 6–59 months were recruited at participating health facilities following microscopy confirmed P. falciparum infection with parasite density between 2,000–200,000 trophozoites/µl and temperature of ≥37.5°C. Patients were automatically excluded from the study if they showed the presence of any of the following conditions: mixed infection with other Plasmodium species, other severe illness, malnutrition, hemoglobin level less than 5 g/dL, hematocrit level less than 15%, or weight of less than 5 kg. Informed consent was obtained from the accompanying parent or guardian. Informed consent forms were available in French and translated into the following local languages: Kikongo, Lingala, Swahili, and Tshiluba. Upon enrollment (day 0), medical history was recorded, and capillary blood samples were collected on 903 Whatman filter paper (GE Healthcare, Chicago, IL). Of the 1613 enrolled participants, 1109 DBS samples from day 0 were provided to US CDC in Atlanta for antigen detection assays and subsequent pfhrp2/3 genotyping if applicable.
Bead-Based Antigen Multiplex Assay
A bead-based multiplex antigen detection assay was used to determine the levels of pan-Plasmodium pAldolase and pLDH, and P. falciparum HRP2 antigens as previously described 21-23. A complete description of this assay as performed for this study is included in the Supplemental Methods. Antigen profiles generated by multiplex assay were used to establish samples with atypically low HRP2 levels compared to either pan-Plasmodium antigen. Because the HRP2 signal is obtained from the binding of antibodies to multiple repeat epitopes present in both HRP2 and HRP3 proteins, both antigens contribute to the HRP2 assay signal. Additionally, for wild-type P. falciparum isolates, the assay signal for HRP2 is higher than either pan-Plasmodium antigen since the HRP2 protein is more highly expressed throughout the erythrocytic stage of infection 7. Samples classified as HRP2 low (or HRP2 negative) were selected for further characterization by genetic assays because these samples were much more likely to have pfhrp2 and/or pfhrp3 deletions, as described previously 23,24.
DNA Extraction
For selected samples, genomic DNA was extracted from DBS by column-based purification. A single 6mm hole punch was taken from each selected DBS and processed using the QIAamp Blood Mini Kit (Qiagen Inc.) according to the manufacturer’s protocol for DNA purification from DBS. Extracted DNA was stored at 4°C for immediate use or at -20°C for long-term (> 4 weeks) storage.
Confirmation of P. falciparum infection by photo-induced electron transfer polymerase chain reaction
Photo-induced electron transfer polymerase chain reaction using fluorogenic primers (PET-PCR), was performed to confirm the presence of Plasmodium genus and P. falciparum DNA using the extracted genomic DNA 25. A full description of primers, master mix preparation, and PCR conditions used for this study are included in the Supplemental Methods.
PCR assays for pfmsp1, pfmsp2, pfhrp2, and pfhrp3 genotyping
Following the confirmation of the presence of Plasmodium genus and P. falciparum DNA by PET-PCR, samples were further screened for DNA quality by nested PCR for pfmsp1 and pfmsp2 single-copy genes 26. Samples that failed to amplify by pfmsp1 or pfmsp2 were omitted from further genotyping analysis because DNA quality (or quantity) was assumed to be compromised. A one-step PCR was used to determine the presence of the pfhrp2 gene 27, and two separate nested PCRs were used for the confirmation of the presence of the pfhrp3 gene: a pfhrp3 exon 1-2 PCR and an exon 2 PCR 22,26. Deletion of the pfhrp3 gene was confirmed if the sample was unable to amplify the exon 1-2 PCR product. For all genotyping PCR assays, any samples that produced discordant results after two runs were subjected to a third run to obtain final tie-breaker results. The full description of these genotyping assays as performed here is included in the Supplemental Methods. For all PCRs, P. falciparum DNA controls 3D7, 7G8, Hb3, Dd2, and a water non-template control were used. The presence or absence of the sequence of interest in the final PCR product was confirmed by gel electrophoresis.