Expansion of artemisinin partial resistance mutations and lack of histidine rich protein-2 and -3 deletions in Plasmodium falciparum infections from Rukara, Rwanda.

BACKGROUND: Emerging artemisinin partial resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (k13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. k13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015, but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, an assessment was conducted to evaluate recent k13-561H prevalence changes, as well as other key mutations. Prevalence of hrp2/3 deletions was also assessed. METHODS: Samples collected in Rukara in 2021 were genotyped for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. RESULTS: Clinically validated k13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of k13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other anti-malarials were variable, with high levels of multidrug resistance 1 (mdr1) N86 (95.5%) associated with lumefantrine decreased susceptibility and dihydrofolate reductase (dhfr) 164L (24.7%) associated with a high level of antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (crt) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. CONCLUSIONS: Increasing prevalence of artemisinin partial resistance due to k13-561H and the rapid expansion of k13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative RDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin partial resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

Neurocognitive outcomes in Malawian children exposed to malaria during pregnancy: An observational birth cohort study.

BACKGROUND: Annually 125 million pregnancies are at risk of malaria infection. However, the impact of exposure to malaria in pregnancy on neurodevelopment in children is not well understood. We hypothesized that malaria in pregnancy and associated maternal immune activation result in neurodevelopmental delay in exposed offspring. METHODS AND FINDINGS: Between April 2014 and April 2015, we followed 421 Malawian mother-baby dyads (median [IQR] maternal age: 21 [19, 28] years) who were previously enrolled (median [IQR] gestational age at enrollment: 19.7 [17.9, 22.1] weeks) in a randomized controlled malaria prevention trial with 5 or 6 scheduled assessments of antenatal malaria infection by PCR. Children were evaluated at 12, 18, and/or 24 months of age with cognitive tests previously validated in Malawi: the Malawi Developmental Assessment Tool (MDAT) and the MacArthur-Bates Communicative Development Inventories (MCAB-CDI). We assessed the impact of antenatal malaria (n [%] positive: 240 [57.3]), placental malaria (n [%] positive: 112 [29.6]), and maternal immune activation on neurocognitive development in children. Linear mixed-effects analysis showed that children exposed to antenatal malaria between 33 and 37 weeks gestation had delayed language development across the 2-year follow-up, as measured by MCAB-CDI (adjusted beta estimate [95% CI], -7.53 [-13.04, -2.02], p = 0.008). Maternal immune activation, characterized by increased maternal sTNFRII concentration, between 33 and 37 weeks was associated with lower MCAB-CDI language score (adjusted beta estimate [95% CI], -8.57 [-13.09, -4.06], p < 0.001). Main limitations of this study include a relatively short length of follow-up and a potential for residual confounding that is characteristic of observational studies. CONCLUSIONS: This mother-baby cohort presents evidence of a relationship between malaria in pregnancy and neurodevelopmental delay in offspring. Malaria in pregnancy may be a modifiable risk factor for neurodevelopmental injury independent of birth weight or prematurity. Successful interventions to prevent malaria during pregnancy may reduce the risk of neurocognitive delay in children.

Interactions Between Antenatal Sulfadoxine-Pyrimethamine, Drug-Resistant Plasmodium falciparum Parasites, and Delivery Outcomes in Malawi.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is used as intermittent preventive therapy in pregnancy (IPTp) for malaria in sub-Saharan Africa. The resistance marker dhps A581G has been associated with reduced IPTp-SP efficacy and enhanced morbidity in SP recipients. METHODS: We measured SP-resistance allele frequencies in Malawian women participating in a trial comparing IPTp with SP against intermittent screening by rapid diagnostic tests (ISTp). We genotyped polymerase chain reaction-detected parasites using deep sequencing of SP-resistance alleles. RESULTS: Among 125 placental infections, A581G-bearing parasites were associated with reduced birth weight (mean difference [MD], 252 g; 95% confidence interval [CI], 46-457; P = .017). Relative to ISTp, IPTp-SP was associated with higher birth weights in women with wild-type parasites (MD, 116 g; 95% CI, -40 to 272; P = .142) and lower birth weights in women with A581G-bearing parasites (MD, 192 g; 95% CI, -264 to 648; P = .385) (Pinteraction = .033). Similar associations were noted on gestational age (Pinteraction = .075). Amongst only IPTp-SP recipients, relative to women who last received SP > 4 weeks before delivery, recent SP receipt was associated with lower birth weight in women with wild-type parasites (MD, 118 g; 95% CI, -376 to 139; P = .361) and higher birth weight in women with A581G-bearing parasites (MD, 783 g; 95% CI, -20 to 1586; P = .054) (Pinteraction = .005). CONCLUSIONS: The effectiveness in birth weight of IPTp-SP is compromised by A581G-bearing parasites, but there was no evidence that the adverse effects of these parasites are exacerbated by antenatal SP. ISRCTN REGISTRY: www.isrctn.com/ISRCTN69800930.

Effect of individual and community-level bed net usage on malaria prevalence among under-fives in the Democratic Republic of Congo.

BACKGROUND: Understanding the contribution of community-level long-lasting, insecticidal net (LLIN) coverage to malaria control is critical to planning and assessing intervention campaigns. The Democratic Republic of Congo (DRC), which has one of the highest burdens of malaria cases and deaths and has dramatically scaled up LLIN ownership in recent years thus it is an ideal setting to evaluate the effect of individual versus community-level use to prevent malaria among children under the age of 5. RESULTS: Data were derived from the 2013-2014 DRC Demographic and Health Survey. Community-level LLIN usage was significantly associated with protection against malaria, even when individual-level LLIN usage was included in the model. In stratified analysis, higher levels of community LLIN coverage enhanced the protective effect of individual LLIN usage, resulting in lower malaria prevalence among individuals who used a LLIN. A sub-analysis of individual LLIN usage by insecticide type revealed deltamethrin-treated nets were more protective than permethrin-treated nets, suggesting that mosquitoes in the DRC are more susceptible to deltamethrin. CONCLUSIONS: This study examines the effects of individual and community-level LLIN usage in young children in an area of high ITN usage. Individual and community LLIN usage were significantly associated with protection against malaria in children under 5 in the DRC. Importantly, the protective effect of individual LLIN usage against malaria is enhanced when community LLIN coverage is higher, demonstrating the importance of increasing community-level LLIN usage. LLINs treated with deltamethrin were shown to be more protective against malaria than LLINs treated with permethrin. Demographic and Health Surveys are thus a novel and important means of surveillance for insecticide resistance.

Correction to: Individual and household characteristics of persons with Plasmodium falciparum malaria in sites with varying endemicities in Kinshasa Province, Democratic Republic of the Congo.

Unfortunately after publication of the original article [1], it came to the author's attention that there is an error in the caption of Fig. 2.

Minimal Impact by Antenatal Subpatent Plasmodium falciparum Infections on Delivery Outcomes in Malawian Women: A Cohort Study.

Antenatal malaria screening with a rapid diagnostic test (RDT) and treatment only of women with positive RDT findings may potentially prevent low birth weight resulting from malaria. The consequences of subpatent antenatal infections below the detection limit of RDTs are incompletely understood. In Malawi, pregnant women of any gravidity status were tested at each antenatal visit for Plasmodium falciparum, using an RDT and polymerase chain reaction analysis, and were followed until delivery. Associations between antenatal infections and delivery outcomes were assessed with Poisson regression or analysis of variance. Compared with women with no detected antenatal P. falciparum infection, women with positive RDT findings delivered babies with a lower mean birth weight (2960 vs 2867 g; mean difference, -93 g [95% confidence interval {CI}, -27 to -159]; P = .006); this was not observed among women with only subpatent infections (mean birth weight, 3013 g; mean difference, 54 [95% CI, -33-140]; P = .2268). These differences were apparent early in pregnancy, during the second trimester: compared with uninfected women, women with positive RDT findings delivered babies with a lower mean birth weight (mean difference, -94 g [95% CI, -31 to -156]; P = .003), but women with subpatent infections did not (mean difference, 36 g [95% CI, -49-122]; P = .409). Subpatent antenatal P. falciparum infections were not associated with adverse delivery outcomes. The association of patent infections at enrollment with low birth weight suggests the importance of preventing P. falciparum infection early in pregnancy.

Individual and household characteristics of persons with Plasmodium falciparum malaria in sites with varying endemicities in Kinshasa Province, Democratic Republic of the Congo.

BACKGROUND: The Democratic Republic of the Congo (DRC) bears a large share of global malaria burden despite efforts to control and eliminate the disease. More detailed understanding of individual and household level characteristics associated with malaria are needed, as is an understanding of how these characteristics vary spatiotemporally and across different community-level malaria endemicities. An ongoing study in Kinshasa Province is designed to address gaps in prior malaria surveillance in the DRC by monitoring malaria across seasons, age groups and in high and low malaria sites. Across seven sites, 242 households and 1591 individuals are participating in the study. Results of the enrollment questionnaire, rapid diagnostic tests and PCR testing of dried blood spots are presented. RESULTS: Overall malaria prevalence in the study cohort is high, 27% by rapid diagnostic test and 31% by polymerase chain reaction, and malaria prevalence is highly varied across very small geographic distances. Malaria prevalence is highest in children aged 6-15. While the majority of households own bed nets, bed net usage is less than 50%. CONCLUSIONS: The study cohort will provide an understanding of how malaria persists in populations that have varying environmental exposures, varying community-level malaria, and varying access to malaria control efforts.

Surveillance for sulfadoxine-pyrimethamine resistant malaria parasites in the Lake and Southern Zones, Tanzania, using pooling and next-generation sequencing.

BACKGROUND: Malaria in pregnancy (MiP) remains a major public health challenge in areas of high malaria transmission. Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended to prevent the adverse consequences of MiP. The effectiveness of SP for IPTp may be reduced in areas where the dhps581 mutation (a key marker of high level SP resistance) is found; this mutation was previously reported to be common in the Tanga Region of northern Tanzania, but there are limited data from other areas. The frequency of molecular markers of SP resistance was investigated in malaria parasites from febrile patients at health centres (HC) in seven regions comprising the Lake and Southern Zones of mainland Tanzania as part of the ongoing efforts to generate national-wide data of SP resistance. METHODS: A cross-sectional survey was conducted in the outpatient departments of 14 HCs in seven regions from April to June, 2015. 1750 dried blood spot (DBS) samples were collected (117 to 160 per facility) from consenting patients with positive rapid diagnostic tests for malaria, and no recent (within past 2 months) exposure to SP or related drugs. DNA was extracted from the DBS, pooled by HC, and underwent pooled targeted amplicon deep sequencing to yield estimates of mutated parasite allele frequency at each locus of interest. RESULTS: The dhps540 mutation was common across all 14 sites, ranging from 55 to 98.4% of sequences obtained. Frequency of the dhps581 mutation ranged from 0 to 2.4%, except at Kayanga HC (Kagera Region, Lake Zone) where 24.9% of sequences obtained were mutated. The dhfr164 mutation was detected only at Kanyanga HC (0.06%). CONCLUSION: By pooling DNA extracts, the allele frequency of mutations in 14 sites could be directly determined on a single deep-sequencing run. The dhps540 mutant was very common at all locations. Surprisingly, the dhps581 was common at one health center, but rare in all the others, suggesting that there is geographic micro-heterogeneity in mutant distribution and that accurate surveillance requires inclusion of multiple sites. A better understanding of the effect of the dhps581 mutant on the efficacy of IPTp-SP is needed.

Scheduled Intermittent Screening with Rapid Diagnostic Tests and Treatment with Dihydroartemisinin-Piperaquine versus Intermittent Preventive Therapy with Sulfadoxine-Pyrimethamine for Malaria in Pregnancy in Malawi: An Open-Label Randomized Controlled Trial.

BACKGROUND: In Africa, most plasmodium infections during pregnancy remain asymptomatic, yet are associated with maternal anemia and low birthweight. WHO recommends intermittent preventive therapy in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP). However, sulfadoxine-pyrimethamine (SP) efficacy is threatened by high-level parasite resistance. We conducted a trial to evaluate the efficacy and safety of scheduled intermittent screening with malaria rapid diagnostic tests (RDTs) and treatment of RDT-positive women with dihydroartemisinin-piperaquine (DP) as an alternative strategy to IPTp-SP. METHODS AND FINDINGS: This was an open-label, two-arm individually randomized superiority trial among HIV-seronegative women at three sites in Malawi with high SP resistance. The intervention consisted of three or four scheduled visits in the second and third trimester, 4 to 6 wk apart. Women in the IPTp-SP arm received SP at each visit. Women in the intermittent screening and treatment in pregnancy with DP (ISTp-DP) arm were screened for malaria at every visit and treated with DP if RDT-positive. The primary outcomes were adverse live birth outcome (composite of small for gestational age, low birthweight [<2,500 g], or preterm birth [<37 wk]) in paucigravidae (first or second pregnancy) and maternal or placental plasmodium infection at delivery in multigravidae (third pregnancy or higher). Analysis was by intention to treat. Between 21 July 2011 and 18 March 2013, 1,873 women were recruited (1,155 paucigravidae and 718 multigravidae). The prevalence of adverse live birth outcome was similar in the ISTp-DP (29.9%) and IPTp-SP (28.8%) arms (risk difference = 1.08% [95% CI -3.25% to 5.41%]; all women: relative risk [RR] = 1.04 [95% CI 0.90-1.20], p = 0.625; paucigravidae: RR = 1.10 [95% CI 0.92-1.31], p = 0.282; multigravidae: RR = 0.92 [95% CI 0.71-1.20], p = 0.543). The prevalence of malaria at delivery was higher in the ISTp-DP arm (48.7% versus 40.8%; risk difference = 7.85%, [95% CI 3.07%-12.63%]; all women: RR = 1.19 [95% CI 1.07-1.33], p = 0.007; paucigravidae: RR = 1.16 [95% CI 1.04-1.31], p = 0.011; multigravidae: RR = 1.29 [95% CI 1.02-1.63], p = 0.037). Fetal loss was more common with ISTp-DP (2.6% versus 1.3%; RR = 2.06 [95% CI 1.01-4.21], p = 0.046) and highest among non-DP-recipients (3.1%) in the ISTp-DP arm. Limitations included the open-label design. CONCLUSIONS: Scheduled screening for malaria parasites with the current generation of RDTs three to four times during pregnancy as part of focused antenatal care was not superior to IPTp-SP in this area with high malaria transmission and high SP resistance and was associated with higher fetal loss and more malaria at delivery. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201103000280319; ISRCTN Registry ISRCTN69800930.

The A581G Mutation in the Gene Encoding Plasmodium falciparum Dihydropteroate Synthetase Reduces the Effectiveness of Sulfadoxine-Pyrimethamine Preventive Therapy in Malawian Pregnant Women.

BACKGROUND: The A581 G: mutation in the gene encoding Plasmodium falciparum dihydropteroate synthase (dhps), in combination with the quintuple mutant involving mutations in both dhps and the gene encoding dihydrofolate reductase (dhfr), the so-called sextuple mutant, has been associated with increased placental inflammation and decreased infant birth weight among women receiving intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) during pregnancy. METHODS: Between 2009 and 2011, delivering women without human immunodeficiency virus infection were enrolled in an observational study of IPTp-SP effectiveness in Malawi. Parasites were detected by polymerase chain reaction (PCR); positive samples were sequenced to genotype the dhfr and dhps loci. The presence of K540 E: in dhps was used as a marker for the quintuple mutant. RESULTS: Samples from 1809 women were analyzed by PCR; 220 (12%) were positive for P. falciparum. A total of 202 specimens were genotyped at codon 581 of dhps; 17 (8.4%) harbored the sextuple mutant. The sextuple mutant was associated with higher risks of patent infection in peripheral blood (adjusted prevalence ratio [aPR], 2.76; 95% confidence interval [CI], 1.82-4.18) and placental blood (aPR 3.28; 95% CI, 1.88-5.78) and higher parasite densities. Recent SP use was not associated with increased parasite densities or placental pathology overall and among women with parasites carrying dhps A581 G: . CONCLUSIONS: IPTp-SP failed to inhibit parasite growth but did not exacerbate pathology among women infected with sextuple-mutant parasites. New interventions to prevent malaria during pregnancy are needed urgently.

Evaluation of Malaria Rapid Diagnostic Test Performance and pfhrp2 Deletion in Tanzania School Surveys, 2017.

As part of malaria nationwide monitoring and evaluation initiatives, there is an increasing trend of incorporating malaria rapid diagnostic tests (mRDTs) in surveys conducted within primary schools to detect malaria parasites. However, mRDTs based on the detection of histidine-rich protein 2 (HRP2) are known to yield false-positive results due to persistent antigenemia, and false-negative results may result from low parasitemia or Plasmodium falciparum hrp2/3 gene deletion. We evaluated diagnostic performance of an HRP2 and pan-parasite lactate dehydrogenase (HRP2/pLDH) mRDT against polymerase chain reaction (PCR) for detection of P. falciparum among 17,051 primary school-age children from eight regions of Tanzania in 2017. According to PCR, the prevalence of P. falciparum was 19.2% (95% CI: 18.6-19.8). Using PCR as reference, the sensitivity and specificity of mRDT was 76.2% (95% CI: 74.7-77.7) and 93.9% (95% CI: 93.5-94.3), respectively. Test agreement was lowest in low transmission areas, where true-positive mRDTs were outnumbered by false-negatives due to low parasitemia. Discordant samples (mRDT-negative but PCR-positive) were screened for pfhrp2/3 deletion by real-time PCR. Among those with a parasite density sufficient for analysis, pfhrp2 deletion was confirmed in 60 samples, whereas pfhrp3 deletion was confirmed in two samples; one sample had both pfhrp2 and pfhrp3 deletions. The majority of samples with gene deletions were detected in the high-transmission Kagera region. Compared with mRDTs, PCR and other molecular methods offer increased sensitivity and are not affected by pfhrp2/3 deletions, making them a useful supplement to mRDTs in schools and other epidemiological surveys.

Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale.

BACKGROUND: The parasite species Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), formerly known as Plasmodium ovale, are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species. METHODS: Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC. RESULTS: The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/µl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/µl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/µl for P. ovalecurtisi and P. ovalewallikeri, respectively. CONCLUSIONS: We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing.

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.

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: P lasmodium f alciparum 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.

Prevalence of Falciparum and non-Falciparum Malaria in the 2014-15 Rwanda Demographic Health Survey.

Background: Malaria remains a major cause of morbidity in sub-Saharan Africa. Undetected asymptomatic falciparum malaria results in a large transmission reservoir and there is evidence of increasing non-falciparum malaria as malaria is controlled in Africa, both resulting in challenges for malaria control programs. Methods: We performed quantitative real time PCR for 4 malaria species in 4,596 individuals from the 2014-2015 Rwanda Demographic Health Survey. Bivariate models were used to determine species-specific associations with risk factors. Results: Asymptomatic falciparum malaria, P. ovale spp., and P. malariae infection had broad spatial distribution across Rwanda. P. vivax infection was rare. Overall infection prevalence was 23.6% (95%CI [21.7%, 26.0%]), with falciparum and non-falciparum at 17.6% [15.9%, 19.0%] and 8.3% [7.0%, 10.0%], respectively. Parasitemias tended to be low and mixed species infections were common, especially where malaria transmission was the highest. Falciparum infection was associated with socio-econiomic status, rural residence and low altitude. Few risk factors were associated with non-falciparum malaria. Conclusions: Asymptomatic falciparum malaria and non-falciparum malaria are common and widely distributed across Rwanda. Continued molecular monitoring of Plasmodium spp. is needed to monitor these threats to malaria control in Africa.

A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria.

Background: P. ovale spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, P. ovale curtisi (Poc) and P. ovale wallikeri (Pow). These species are thought to differ in clinical symptomatology and latency, but existing diagnostic assays have limited ability to detect and distinguish them. In this study, we developed a new duplex assay for the detection and differentiation of Poc and Pow that can be used to improve our understanding of these parasites. Methods: Repetitive sequence motifs were identified in available Poc and Pow genomes and used for assay development and validation. We evaluated the analytical sensitivity and specificity of the best-performing assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), then validated its performance using 55 P. ovale spp. samples and 40 non-ovale Plasmodium samples from the DRC. Poc and Pow prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated. Results: The best-performing Poc and Pow targets had 9 and 8 copies within the reference genomes, respectively. Our duplex assay had 100% specificity and 95% confidence lower limits of detection of 4.2 and 41.2 parasite genome equivalents/µl for Poc and Pow, respectively. Species was determined in 80% of all P. ovale spp.-positive field samples and 100% of those with >10 parasites/µl. Most P. ovale spp. field samples from the DRC were found to be Poc infections. Conclusions: We identified promising multi-copy targets for molecular detection and differentiation of Poc and Pow and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density Pow infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of P. ovale spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.

Expansion of Artemisinin Partial Resistance Mutations and Lack of Histidine Rich Protein-2 and -3 Deletions in Plasmodium falciparum infections from Rukara, Rwanda.

Background: Emerging artemisinin resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (K13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. K13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015 but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, we sought to assess recent K13-561H prevalence changes, as well as for other key mutations. Prevalence of hrp2/3 deletions was also assessed. Methods: We genotyped samples collected in Rukara in 2021 for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. Results: Clinically validated K13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of artemisinin resistance mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of K13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other antimalarials were variable, with high levels of multidrug resistance 1 (MDR1) N86 (95.5%) associated with lumefantrine resistance and dihydrofolate reductase (DHFR) 164L (24.7%) associated with antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (CRT ) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. Conclusions: Increasing prevalence of artemisinin partial resistance due to K13-561H and the rapid expansion of K13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative mRDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in Kinshasa Province, Democratic Republic of Congo.

Reports suggest non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa but their epidemiology is ill-defined, particularly in highly malaria-endemic regions. We estimated incidence and prevalence of PCR-confirmed non-falciparum and Plasmodium falciparum malaria infections within a longitudinal study conducted in Kinshasa, Democratic Republic of Congo (DRC) between 2015-2017. Children and adults were sampled at biannual household surveys and routine clinic visits. Among 9,089 samples from 1,565 participants, incidences of P. malariae, P. ovale spp., and P. falciparum infections by 1-year were 7.8% (95% CI: 6.4%-9.1%), 4.8% (95% CI: 3.7%-5.9%) and 57.5% (95% CI: 54.4%-60.5%), respectively. Non-falciparum prevalences were higher in school-age children, rural and peri-urban sites, and P. falciparum co-infections. P. falciparum remains the primary driver of malaria in the DRC, though non-falciparum species also pose an infection risk. As P. falciparum interventions gain traction in high-burden settings, continued surveillance and improved understanding of non-falciparum infections are warranted.

Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in a highly malaria-endemic country: a longitudinal cohort study in Kinshasa Province, Democratic Republic of Congo.

Background: Increasing reports suggest that non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa, but their epidemiology is not well-defined. This is particularly true in regions of high P. falciparum endemicity such as the Democratic Republic of Congo (DRC), where 12% of the world's malaria cases and 13% of deaths occur. Methods and Findings: The cumulative incidence and prevalence of P. malariae and P. ovale spp. infection detected by real-time PCR were estimated among children and adults within a longitudinal study conducted in seven rural, peri-urban, and urban sites from 2015-2017 in Kinshasa Province, DRC. Participants were sampled at biannual household survey visits (asymptomatic) and during routine health facility visits (symptomatic). Participant-level characteristics associated with non-falciparum infections were estimated for single- and mixed-species infections. Among 9,089 samples collected from 1,565 participants over a 3-year period, the incidence of P. malariae and P. ovale spp. infection was 11% (95% CI: 9%-12%) and 7% (95% CI: 5%-8%) by one year, respectively, compared to a 67% (95% CI: 64%-70%) one-year cumulative incidence of P. falciparum infection. Incidence continued to rise in the second year of follow-up, reaching 26% and 15% in school-age children (5-14yo) for P. malariae and P. ovale spp., respectively. Prevalence of P. malariae, P. ovale spp., and P. falciparum infections during household visits were 3% (95% CI: 3%-4%), 1% (95% CI: 1%-2%), and 35% (95% CI: 33%-36%), respectively. Non-falciparum malaria was more prevalent in rural and peri-urban vs. urban sites, in school-age children, and among those with P. falciparum co-infection. A crude association was detected between P. malariae and any anemia in the symptomatic clinic population, although this association did not hold when stratified by anemia severity. No crude associations were detected between non-falciparum infection and fever prevalence. Conclusions: P. falciparum remains the primary driver of malaria morbidity and mortality in the DRC. However, non-falciparum species also pose an infection risk across sites of varying urbanicity and malaria endemicity within Kinshasa, DRC, particularly among children under 15 years of age. As P. falciparum interventions gain traction in high-burden settings like the DRC, continued surveillance and improved understanding of non-falciparum infections are warranted.

Plasmodium falciparum with pfhrp2/3 Deletion Not Detected in a 2018-2021 Malaria Longitudinal Cohort Study in Kinshasa Province, Democratic Republic of the Congo.

Histidine-rich protein 2- (HRP2-) based rapid diagnostic tests (RDTs) are widely used to detect Plasmodium falciparum in sub-Saharan Africa. Reports of parasites with pfhrp2 and/or pfhrp3 (pfhrp2/3) gene deletions in Africa raise concerns about the long-term viability of HRP2-based RDTs. We evaluated changes in pfhrp2/3 deletion prevalence over time using a 2018-2021 longitudinal study of 1,635 enrolled individuals in Kinshasa Province, Democratic Republic of the Congo (DRC). Samples collected during biannual household visits with ≥ 100 parasites/µL by quantitative real-time polymerase chain reaction were genotyped using a multiplex real-time PCR assay. Among 2,726 P. falciparum PCR-positive samples collected from 993 participants during the study period, 1,267 (46.5%) were genotyped. No pfhrp2/3 deletions or mixed pfhrp2/3-intact and -deleted infections were identified in our study. Pfhrp2/3-deleted parasites were not detected in Kinshasa Province; ongoing use of HRP2-based RDTs is appropriate.

Impact of malaria diagnostic choice on monitoring of Plasmodium falciparum prevalence estimates in the Democratic Republic of the Congo and relevance to control programs in high-burden countries.

Malaria programs rely upon a variety of diagnostic assays, including rapid diagnostic tests (RDTs), microscopy, polymerase chain reaction (PCR), and bead-based immunoassays (BBA), to monitor malaria prevalence and support control and elimination efforts. Data comparing these assays are limited, especially from high-burden countries like the Democratic Republic of the Congo (DRC). Using cross-sectional and routine data, we compared diagnostic performance and Plasmodium falciparum prevalence estimates across health areas of varying transmission intensity to illustrate the relevance of assay performance to malaria control programs. Data and samples were collected between March-June 2018 during a cross-sectional household survey across three health areas with low, moderate, and high transmission intensities within Kinshasa Province, DRC. Samples from 1,431 participants were evaluated using RDT, microscopy, PCR, and BBA. P. falciparum parasite prevalence varied between diagnostic methods across all health areas, with the highest prevalence estimates observed in Bu (57.4-72.4% across assays), followed by Kimpoko (32.6-53.2%), and Voix du Peuple (3.1-8.4%). Using latent class analysis to compare these diagnostic methods against an "alloyed gold standard," the most sensitive diagnostic method was BBA in Bu (high prevalence) and Voix du Peuple (low prevalence), while PCR diagnosis was most sensitive in Kimpoko (moderate prevalence). RDTs were consistently the most specific diagnostic method in all health areas. Among 9.0 million people residing in Kinshasa Province in 2018, the estimated P. falciparum prevalence by microscopy, PCR, and BBA were nearly double that of RDT. Comparison of malaria RDT, microscopy, PCR, and BBA results confirmed differences in sensitivity and specificity that varied by endemicity, with PCR and BBA performing best for detecting any P. falciparum infection. Prevalence estimates varied widely depending on assay type for parasite detection. Inherent differences in assay performance should be carefully considered when using community survey and surveillance data to guide policy decisions.