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.

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.

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.

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.

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.

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.

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.

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.

Arboviruses as an unappreciated cause of non-malarial acute febrile illness in the Dschang Health District of western Cameroon.

Acute febrile illness is a common problem managed by clinicians and health systems globally, particularly in the Tropics. In many regions, malaria is a leading and potentially deadly cause of fever; however, myriad alternative etiologies exist. Identifying the cause of fever allows optimal management, but this depends on many factors including thorough knowledge of circulating infections. Arboviruses such as dengue (DENV) cause fever and may be underdiagnosed in sub-Saharan Africa where malaria is a major focus. We examined cases of fever in western Cameroon that tested negative for malaria and found 13.5% (13/96) were due to DENV, with 75% (9/12) of these being DENV serotype 2 infections. Two complete DENV2 genomes were obtained and clustered closely to recent isolates from Senegal and Burkina Faso. The seroprevalence of DENV in this region was 24.8% (96/387). Neutralizing antibodies to DENV2 were detected in all (15/15) seropositive samples tested. Chikungunya (CHIKV) is an arthritogenic alphavirus that is transmitted by Aedes mosquitoes, the same principal vector as DENV. The seroprevalence for CHIKV was 15.7% (67/427); however, CHIKV did not cause a single case of fever in the 96 subjects tested. Of note, being seropositive for one arbovirus was associated with being seropositive for the other (Χ2 = 16.8, p<0.001). Taken together, these data indicate that Aedes-transmitted arboviruses are endemic in western Cameroon and are likely a common but underappreciated cause of febrile illness. This work supports the need for additional study of arboviruses in sub-Saharan Africa and efforts to improve diagnostic capacity, surveillance systems, and arbovirus prevention strategies.

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.

The epidemiology of Plasmodium vivax among adults in the Democratic Republic of the Congo.

Reports of P. vivax infections among Duffy-negative hosts have accumulated throughout sub-Saharan Africa. Despite this growing body of evidence, no nationally representative epidemiological surveys of P. vivax in sub-Saharan Africa have been performed. To overcome this gap in knowledge, we screened over 17,000 adults in the Democratic Republic of the Congo (DRC) for P. vivax using samples from the 2013-2014 Demographic Health Survey. Overall, we found a 2.97% (95% CI: 2.28%, 3.65%) prevalence of P. vivax infections across the DRC. Infections were associated with few risk-factors and demonstrated a relatively flat distribution of prevalence across space with focal regions of relatively higher prevalence in the north and northeast. Mitochondrial genomes suggested that DRC P. vivax were distinct from circulating non-human ape strains and an ancestral European P. vivax strain, and instead may be part of a separate contemporary clade. Our findings suggest P. vivax is diffusely spread across the DRC at a low prevalence, which may be associated with long-term carriage of low parasitemia, frequent relapses, or a general pool of infections with limited forward propagation.

A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping.

BACKGROUND: CRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection. METHODS: We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance. FINDINGS: The suite of Plasmodium assays achieved analytical sensitivities ranging from 2•5-18•8 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities. INTERPRETATION: These novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance. FUNDING: National Institutes of Health (T32GM007092, R21AI148579, K24AI134990, R01AI121558, UL1TR002489, P30CA016086).

Individual, household and neighborhood risk factors for malaria in the Democratic Republic of the Congo support new approaches to programmatic intervention.

BACKGROUND: The Democratic Republic of the Congo (DRC) remains one of the countries most impacted by malaria despite decades of control efforts, including multiple mass insecticide treated net (ITN) distribution campaigns. The multi-scalar and complex nature of malaria necessitates an understanding of malaria risk factors over time and at multiple levels (e.g., individual, household, community). Surveillance of households in both rural and urban settings over time, coupled with detailed behavioral and geographic data, enables the detection of seasonal trends in malaria prevalence and malaria-associated behaviors as well as the assessment of how the local environments within and surrounding an individual's household impact malaria outcomes. METHODS: Participants from seven sites in Kinshasa Province, DRC were followed for over two years. Demographic, behavioral, and spatial information was gathered from enrolled households. Malaria was assessed using both rapid diagnostic tests (RDT) and polymerase chain reaction (PCR) and seasonal trends were assessed. Hierarchical regression modeling tested associations between behavioral and environmental factors and positive RDT and PCR outcomes at individual, household and neighborhood scales. RESULTS: Among 1591 enrolled participants, malaria prevalence did not consistently vary seasonally across the sites but did vary by age and ITN usage. Malaria was highest and ITN usage lowest in children ages 6-15 years across study visits and seasons. Having another member of the household test positive for malaria significantly increased the risk of an individual having malaria [RDT: OR = 4.158 (2.86-6.05); PCR: OR = 3.37 (2.41-4.71)], as did higher malaria prevalence in the 250 m neighborhood around the household [RDT: OR = 2.711 (1.42-5.17); PCR: OR = 4.056 (2.3-7.16)]. Presence of water within close proximity to the household was also associated with malaria outcomes. CONCLUSIONS: Taken together, these findings suggest that targeting non-traditional age groups, children >5 years old and teenagers, and deploying household- and neighborhood-focused interventions may be effective strategies for improving malaria outcomes in high-burden countries like the DRC.

Analysis of false-negative rapid diagnostic tests for symptomatic malaria in the Democratic Republic of the Congo.

The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen's kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

Spatial and epidemiological drivers of Plasmodium falciparum malaria among adults in the Democratic Republic of the Congo.

BACKGROUND: Adults are frequently infected with malaria and may serve as a reservoir for further transmission, yet we know relatively little about risk factors for adult infections. In this study, we assessed malaria risk factors among adults using samples from the nationally representative, cross-sectional 2013-2014 Demographic and Health Survey (DHS) conducted in the Democratic Republic of the Congo (DRC). We further explored differences in risk factors by urbanicity. METHODS: Plasmodium falciparum infection was determined by PCR. Covariates were drawn from the DHS to model individual, community and environmental-level risk factors for infection. Additionally, we used deep sequencing data to estimate the community-level proportions of drug-resistant infections and included these estimates as potential risk factors. All identified factors were assessed for differences in associations by urbanicity. RESULTS: A total of 16 126 adults were included. Overall prevalence of malaria was 30.3% (SE=1.1) by PCR; province-level prevalence ranged from 6.7% to 58.3%. Only 17% of individuals lived in households with at least one bed-net for every two people, as recommended by the WHO. Protective factors included increasing within-household bed-net coverage (Prevalence Ratio=0.85, 95% CI=0.76-0.95) and modern housing (PR=0.58, 95% CI=0.49-0.69). Community-level protective factors included increased median wealth (PR=0.87, 95% CI=0.83-0.92). Education, wealth, and modern housing showed protective associations in cities but not in rural areas. CONCLUSIONS: The DRC continues to suffer from a high burden of malaria; interventions that target high-risk groups and sustained investment in malaria control are sorely needed. Areas of high prevalence should be prioritised for interventions to target the largest reservoirs for further transmission.

The impact of antimalarial resistance on the genetic structure of Plasmodium falciparum in the DRC.

The Democratic Republic of the Congo (DRC) harbors 11% of global malaria cases, yet little is known about the spatial and genetic structure of the parasite population in that country. We sequence 2537 Plasmodium falciparum infections, including a nationally representative population sample from DRC and samples from surrounding countries, using molecular inversion probes - a high-throughput genotyping tool. We identify an east-west divide in haplotypes known to confer resistance to chloroquine and sulfadoxine-pyrimethamine. Furthermore, we identify highly related parasites over large geographic distances, indicative of gene flow and migration. Our results are consistent with a background of isolation by distance combined with the effects of selection for antimalarial drug resistance. This study provides a high-resolution view of parasite genetic structure across a large country in Africa and provides a baseline to study how implementation programs may impact parasite populations.

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.

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.

Matched Placental and Circulating Plasmodium falciparum Parasites are Genetically Homologous at the var2csa ID1-DBL2X Locus by Deep Sequencing.

In pregnancy-associated malaria, infected erythrocytes accumulate in the placenta. It is unclear if in polyclonal infections this results in distinct peripheral and placental parasite populations. We used long amplicon deep sequencing of Plasmodium falciparum var2csa ID1-DBL2X from 15 matched peripheral and placental samples collected at delivery from a high transmission area to determine genetic homology. Despite substantial sequence variation and detecting 23 haplotypes, the matched pairs mostly contained the same genetic variants, with 11 pairs sharing 100% of their variants, whereas others showed some heterogeneity. Thus, at delivery, peripheral and placental parasites appear to intermix and placental genotypes can be inferred through peripheral sampling.