Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

The ability to identify mixed-species infections and track the origin of Plasmodium parasites can further enhance the development of treatment and prevention recommendations as well as outbreak investigations. Here, we explore the utility of using the full Plasmodium mitochondrial genome to classify Plasmodium species, detect mixed infections, and infer the geographical origin of imported P. falciparum parasites to the United States (U.S.). Using the recently developed standardized, high-throughput Malaria Resistance Surveillance (MaRS) protocol, the full Plasmodium mitochondrial genomes of 265 malaria cases imported to the U.S. from 2014-2017 were sequenced and analyzed. P. falciparum infections were found in 94.7% (251/265) of samples. Five percent (14/265) of samples were identified as mixed- Plasmodium species or non-P. falciparum, including P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri. P. falciparum mitochondrial haplotypes analysis revealed greater than eighteen percent of samples to have at least two P. falciparum mitochondrial genome haplotypes, indicating either heteroplasmy or multi-clonal infections. Maximum-likelihood phylogenies of 912 P. falciparum mitochondrial genomes with known country origin were used to infer the geographical origin of thirteen samples from persons with unknown travel histories as: Africa (country unspecified) (n = 10), Ghana (n = 1), Southeast Asia (n = 1), and the Philippines (n = 1). We demonstrate the utility and current limitations of using the Plasmodium mitochondrial genome to classify samples with mixed-infections and infer the geographical origin of imported P. falciparum malaria cases to the U.S. with unknown travel history.

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon 1 PCR product must be confirmed by sequencing. Here we describe a new one-step PCR method for detection of pfhrp2. To determine sensitivity and specificity, all PCRs were performed in triplicate. Using photo-induced electron transfer (PET) PCR detecting 18srRNA as true positive, one-step had comparable sensitivity of 95.0% (88.7-98.4%) to nested exon 1, 99.0% (94.6-99.9%) and nested exon 2, 98.0% (93.0-99.8%), and comparable specificity 93.8% (69.8-99.8%) to nested exon 1 100.0% (79.4-100.0%) and nested exon 2, 100.0% (74.4-100.0%). Sequencing revealed that one step PCR does not amplify pfhrp3. Logistic regression models applied to measure the 95% level of detection of the one-step PCR in clinical isolates provided estimates of 133p/μL (95% confidence interval (CI): 3-793p/μL) for whole blood (WB) samples and 385p/μL (95% CI: 31-2133 p/μL) for dried blood spots (DBSs). When considering protocol attributes, the one-step PCR is less expensive, faster and more suitable for high throughput. In summary, we have developed a more accurate PCR method that may be ideal for the application of the WHO protocol for investigating pfhrp2 deletions in symptomatic individuals presenting to health care facilities.

Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated Plasmodium falciparum infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers. Samples from treatment failures were genotyped for the pfk13, pfcrt, and pfmdr1 genes. Day 3 clearance rates were ≥95% in all arms. Uncorrected day 28 Kaplan-Meier efficacy estimates ranged from 84.2 to 90.1% for the AL arms and 84.7 to 100% for the ASAQ arms. Corrected day 28 estimates were 87.6% (95% confidence interval [CI], 81 to 95%) for the AL arm in Lunda Sul, 92.2% (95% CI, 87 to 98%) for AL in Zaire, 95.6% (95% CI, 91 to 100%) for ASAQ in Zaire, 98.4% (95% CI, 96 to 100%) for AL in Benguela, and 100% for ASAQ in Benguela and Lunda Sul. All 103 analyzed samples had wild-type pfk13 sequences. The 76T pfcrt allele was found in most (92%; 11/12) ASAQ late-failure samples but in only 16% (4/25) of AL failure samples. The N86 pfmdr1 allele was found in 97% (34/35) of treatment failures. The AL efficacy in Lunda Sul was below the 90% World Health Organization threshold, the third time in four rounds that this threshold was crossed for an AL arm in Angola. In contrast, the observed ASAQ efficacy has not been below 95% to date in Angola, including this latest round.

Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites.