Under the Radar: Epidemiology of Plasmodium ovale in the Democratic Republic of the Congo.

BACKGROUND: Plasmodium ovale is an understudied malaria species prevalent throughout much of sub-Saharan Africa. Little is known about the distribution of ovale malaria and risk factors for infection in areas of high malaria endemicity. METHODS: Using the 2013 Democratic Republic of the Congo (DRC) Demographic and Health Survey, we conducted a risk factor analysis for P. ovale infections. We evaluated geographic clustering of infections and speciated to P. ovale curtisi and P. ovale wallikeri through deep sequencing. RESULTS: Of 18 149 adults tested, we detected 143 prevalent P. ovale infections (prevalence estimate 0.8%; 95% confidence interval [CI], .59%-.98%). Prevalence ratios (PR) for significant risk factors were: male sex PR = 2.12 (95% CI, 1.38-3.26), coprevalent P. falciparum PR = 3.52 (95% CI, 2.06-5.99), and rural residence PR = 2.19 (95% CI, 1.31-3.66). P. ovale was broadly distributed throughout the DRC; an elevated cluster of infections was detected in the south-central region. Speciation revealed P. ovale curtisi and P. ovale wallikeri circulating throughout the country. CONCLUSIONS: P. ovale persists broadly in the DRC, a high malaria burden country. For successful elimination of all malaria species, P. ovale needs to be on the radar of malaria control programs.

Drug-Resistance and Population Structure of Plasmodium falciparum Across the Democratic Republic of Congo Using High-Throughput Molecular Inversion Probes.

A better understanding of the drivers of the spread of malaria parasites and drug resistance across space and time is needed. These drivers can be elucidated using genetic tools. Here, a novel molecular inversion probe (MIP) panel targeting all major drug-resistance mutations and a set of microsatellites was used to genotype Plasmodium falciparum infections of 552 children from the 2013-2014 Demographic and Health Survey conducted in the Democratic Republic of the Congo (DRC). Microsatellite-based analysis of population structure suggests that parasites within the DRC form a homogeneous population. In contrast, sulfadoxine-resistance markers in dihydropteroate synthase show marked spatial structure with ongoing spread of double and triple mutants compared with 2007. These findings suggest that parasites in the DRC remain panmictic despite rapidly spreading antimalarial-resistance mutations. Moreover, highly multiplexed targeted sequencing using MIPs emerges as a cost-effective method for elucidating pathogen genetics in complex infections in large cohorts.

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.

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.

Low prevalence of Plasmodium malariae and Plasmodium ovale mono-infections among children in the Democratic Republic of the Congo: a population-based, cross-sectional study.

BACKGROUND: In an effort to improve surveillance for epidemiological and clinical outcomes, rapid diagnostic tests (RDTs) have become increasingly widespread as cost-effective and field-ready methods of malaria diagnosis. However, there are concerns that using RDTs specific to Plasmodium falciparum may lead to missed detection of other malaria species such as Plasmodium malariae and Plasmodium ovale. METHODS: Four hundred and sixty six samples were selected from children under 5 years old in the Democratic Republic of the Congo (DRC) who took part in a Demographic and Health Survey (DHS) in 2013-14. These samples were first tested for all Plasmodium species using an 18S ribosomal RNA-targeted real-time PCR; malaria-positive samples were then tested for P. falciparum, P. malariae and P. ovale using a highly sensitive nested PCR. RESULTS: The prevalence of P. falciparum, P. malariae and P. ovale were 46.6, 12.9 and 8.3 %, respectively. Most P. malariae and P. ovale infections were co-infected with P. falciparum-the prevalence of mono-infections of these species were only 1.0 and 0.6 %, respectively. Six out of these eight mono-infections were negative by RDT. The prevalence of P. falciparum by the more sensitive nested PCR was higher than that found previously by real-time PCR. CONCLUSIONS: Plasmodium malariae and P. ovale remain endemic at a low rate in the DRC, but the risk of missing malarial infections of these species due to falciparum-specific RDT use is low. The observed prevalence of P. falciparum is higher with a more sensitive PCR method.

Participant perspectives to improve tenofovir adherence in the prevention of mother-to-child transmission of hepatitis B virus in Kinshasa, DRC.

Prevention of mother-to-child transmission (PMTCT) programs for hepatitis B virus (HBV) are critical to reach the World Health Organization's 2030 HBV elimination goals. Despite demonstrated feasibility utilizing HIV infrastructure, HBV PMTCT programs are not implemented in many African settings, including in the Democratic Republic of Congo (DRC). In a previous pilot of HBV PMTCT implementation in DRC's capital, Kinshasa, we observed low TDF metabolite levels at delivery among women with high-risk HBV who were given tenofovir disoproxil fumarate (TDF) antiviral therapy. As such, we conducted qualitative interviews with women who received TDF to understand facilitators and barriers of medication adherence. We used a modified Information-Motivation-Behavioral Skills model (IMB+) as a framework for thematic content analysis. We found that trust in healthcare workers, familial support, and improved awareness of the disease and treatment options were important facilitators of TDF adherence; pill size, social stigma, and low HBV knowledge were barriers to adherence. While overall acceptance of TDF was high in this pilot, improved TDF adherence is needed in order to reach efficacious levels for preventing transmission from mothers to newborns. We suggest ongoing HBV sensitization within existing maternity and HIV care infrastructure would address gaps in knowledge and stigma identified here. Additionally, given the trust women have towards maternity center staff and volunteers, scaled HBV PMTCT interventions should include specific sensitization and education for healthcare affiliates, who currently receive no HBV prevention or information in DRC. This study is timely as TDF, particularly future long-acting formulations, could be considered as an alternate rather than adjuvant to birth-dose vaccination for HBV PMTCT in sub-Saharan Africa.

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.

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.

Plasmodium vivax Infections in Duffy-Negative Individuals in the Democratic Republic of the Congo.

Although Plasmodium vivax has been assumed to be absent from sub-Saharan Africa because of the protective mutation conferring the Duffy-negative phenotype, recent evidence has suggested that P. vivax cases are prevalent in these regions. We selected 292 dried blood spots from children who participated in the 2013-2014 Demographic and Health Survey of the Democratic Republic of the Congo (DRC), to assess for P. vivax infection. Four P. vivax infections were identified by polymerase chain reaction, each in a geographically different survey cluster. Using these as index cases, we tested the remaining 73 samples from the four clusters. With this approach, 10 confirmed cases, three probable cases, and one possible case of P. vivax were identified. Among the 14 P. vivax cases, nine were coinfected with Plasmodium falciparum. All 14 individuals were confirmed to be Duffy-negative by sequencing for the single point mutation in the GATA motif that represses the expression of the Duffy antigen. This finding is consistent with a growing body of literature that suggests that P. vivax can infect Duffy-negative individuals in Africa. Future molecular and sequencing work is needed to understand the relationship of these isolates with other P. vivax samples from Asia and South America and discover variants linked to P. vivax virulence and erythrocyte invasion.

The links between agriculture, Anopheles mosquitoes, and malaria risk in children younger than 5 years in the Democratic Republic of the Congo: a population-based, cross-sectional, spatial study.

BACKGROUND: The relationship between agriculture, Anopheles mosquitoes, and malaria in Africa is not fully understood, but it is important for malaria control as countries consider expanding agricultural projects to address population growth and food demand. Therefore, we aimed to assess the effect of agriculture on Anopheles biting behaviour and malaria risk in children in rural areas of the Democratic Republic of the Congo (DR Congo). METHODS: We did a population-based, cross-sectional, spatial study of rural children (<5 years) in the DR Congo. We used information about the presence of malaria parasites in each child, as determined by PCR analysis of dried-blood spots from the 2013-14 DR Congo Demographic and Health Survey (DHS). We also used data from the DHS, a longitudinal entomological study, and available land cover and climate data to evaluate the relationships between agriculture, Anopheles biting behaviour, and malaria prevalence. Satellite imagery was used to measure the percentage of agricultural land cover around DHS villages and Anopheles sites. Anopheles biting behaviour was assessed by Human Landing Catch. We used probit regression to assess the relationship between agriculture and the probability of malaria infection, as well as the relationship between agriculture and the probability that a mosquito was caught biting indoors. FINDINGS: Between Aug 13, 2013, and Feb 13, 2014, a total of 9790 dried-blood spots were obtained from the DHS, of which 4612 participants were included in this study. Falciparum malaria infection prevalence in rural children was 38·7% (95% uncertainty interval [UI] 37·3-40·0). Increasing exposure to agriculture was associated with increasing malaria risk with a high posterior probability (estimate 0·07, 95% UI -0·04 to 0·17; posterior probability [estimate >0]=0·89), with the probability of malaria infection increased between 0·2% (95% UI -0·1 to 3·4) and 2·6% (-1·5 to 6·6) given a 15% increase in agricultural cover, depending on other risk factors. The models predicted that large increases in agricultural cover (from 0% to 75%) increase the probability of infection by as much as 13·1% (95% UI -7·3 to 28·9). Increased risk might be due to Anopheles gambiae sensu lato, whose probability of biting indoors increased between 11·3% (95% UI -15·3 to 25·6) and 19·7% (-12·1 to 35·9) with a 15% increase in agriculture. INTERPRETATION: Malaria control programmes must consider the possibility of increased risk due to expanding agriculture. Governments considering initiating large-scale agricultural projects should therefore also consider accompanying additional malaria control measures. FUNDING: National Institutes of Health, National Science Foundation, Bill & Melinda Gates Foundation, President's Malaria Initiative, and Royster Society of Fellows at the University of North Carolina at Chapel Hill.

Changing spatial patterns and increasing rurality of HIV prevalence in the Democratic Republic of the Congo between 2007 and 2013.

The Democratic Republic of the Congo (DRC) has one of the lowest HIV prevalence in sub-Saharan Africa, estimated at 1.1% [0.9-1.3] of adults aged 15-49 in 2013 (UNAIDS). Within the 2 million km(2) country, however, there exists spatial variation in HIV prevalence, with the highest HIV prevalence observed in the large cities of Kinshasa and Lubumbashi. Globally, HIV is an increasingly rural disease, diffusing outwards from urban centers of high HIV prevalence to places where HIV was previously absent or present at very low levels. Utilizing data collected during Demographic and Health Surveillance (DHS) in 2007 and 2013 in the DRC, we sought to update the map of HIV prevalence in the DRC as well as to explore whether HIV in the DRC is an increasingly rural disease or remains confined to urban areas. Bayesian kriging and regression indicate that HIV prevalence in rural areas of the DRC is higher in 2013 than in 2007 and that increased distance to an urban area is no longer protective against HIV as it was in 2007. These findings suggest that HIV education, testing and prevention efforts need to diffuse from urban to rural areas just as HIV is doing.

The geography of malaria genetics in the Democratic Republic of Congo: A complex and fragmented landscape.

Understanding how malaria parasites move between populations is important, particularly given the potential for malaria to be reintroduced into areas where it was previously eliminated. We examine the distribution of malaria genetics across seven sites within the Democratic Republic of Congo (DRC) and two nearby countries, Ghana and Kenya, in order to understand how the relatedness of malaria parasites varies across space, and whether there are barriers to the flow of malaria parasites within the DRC or across borders. Parasite DNA was retrieved from dried blood spots from 7 Demographic and Health Survey sample clusters in the DRC. Malaria genetic characteristics of parasites from Ghana and Kenya were also obtained. For each of 9 geographic sites (7 DRC, 1 Ghana and 1 Kenya), a pair-wise RST statistic was calculated, indicating the genetic distance between malaria parasites found in those locations. Mapping genetics across the spatial extent of the study area indicates a complex genetic landscape, where relatedness between two proximal sites may be relatively high (RST > 0.64) or low (RST < 0.05), and where distal sites also exhibit both high and low genetic similarity. Mantel's tests suggest that malaria genetics differ as geographic distances increase. Principal Coordinate Analysis suggests that genetically related samples are not co-located. Barrier analysis reveals no significant barriers to gene flow between locations. Malaria genetics in the DRC have a complex and fragmented landscape. Limited exchange of genes across space is reflected in greater genetic distance between malaria parasites isolated at greater geographic distances. There is, however, evidence for close genetic ties between distally located sample locations, indicating that movement of malaria parasites and flow of genes is being driven by factors other than distance decay. This research demonstrates the contributions that spatial disease ecology and landscape genetics can make to understanding the evolutionary dynamics of infectious diseases.