Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018-2019.

BACKGROUND: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. METHODS: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. RESULTS: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed. CONCLUSION: No molecular markers currently known to be associated with resistance to the first-line ACT in use were detected in isolates of P. falciparum from treatment failure patients. Regular monitoring through in vivo drug efficacy and molecular studies must continue to ensure the effectiveness of malaria treatment in Democratic Republic of Congo.

Comparing the durability of the long-lasting insecticidal nets DawaPlus® 2.0 and DuraNet© in northwest Democratic Republic of Congo.

BACKGROUND: Anecdotal reports from DRC suggest that long-lasting insecticidal nets (LLIN) distributed through mass campaigns in DRC may not last the expected average three years. To provide the National Malaria Control Programme with evidence on physical and insecticidal durability of nets distributed during the 2016 mass campaign, two brands of LLIN, DawaPlus® 2.0 and DuraNet©, were monitored in neighbouring and similar health zones in Sud Ubangi and Mongala Provinces. METHODS: This was a prospective cohort study of representative samples of households from two health zones recruited at baseline, 2 months after the mass campaign. All campaign nets in these households were labelled, and followed up over a period of 31 months. Primary outcome was the "proportion of nets surviving in serviceable condition" based on attrition and integrity measures and the median survival in years. The outcome for insecticidal durability was determined by bio-assay from subsamples of campaign nets. RESULTS: A total of 754 campaign nets (109% of target) from 240 households were included in the study. Definite outcomes could be determined for 67% of the cohort nets in Sud Ubangi and 74% in Mongala. After 31 months all-cause attrition was 57% in Sud Ubangi and 76% in Mongala (p = 0.005) and attrition due to wear and tear was 26% in Sud Ubangi and 48% in Mongala (p = 0.0009). Survival in serviceable condition at the last survey was 37% in Sud Ubangi and 17% in Mongala (p = 0.003). Estimated median survival was 1.6 years for the DawaPlus® 2.0 in Mongala (95% CI 1.3-1.9) and 2.2 years for the DuraNet in Sud Ubangi (95% CI 2.0-2.4). Multivariable Cox proportionate hazard models suggest that the difference between sites was mainly attributable to the LLIN brand. Insecticidal effectiveness was optimal for DuraNet©, but significantly dropped after 24 months for DawaPlus® 2.0. CONCLUSIONS: In the environment of northwest DRC the polyethylene LLIN DuraNet© performed significantly better than the polyester LLIN DawaPlus® 2.0, but both were below a three-year median survival. Improvement of net care behaviours should be able to improve physical durability.

Long-lasting insecticidal net (LLIN) ownership, use and cost of implementation after a mass distribution campaign in Kasaï Occidental Province, Democratic Republic of Congo.

BACKGROUND: Long-lasting insecticidal nets (LLIN) are a highly effective means for preventing malaria infection and reducing associated morbidity and mortality. Mass free distribution campaigns have been shown to rapidly increase LLIN ownership and use. Around 3.5 million LLINs were distributed free of charge in the Kasaï Occidental Province in the Democratic Republic of Congo (DRC) in September-October 2014, using two different approaches, a fixed delivery strategy and a door-to-door strategy including hang-up activities. METHODS: Repeated community-based cross-sectional surveys were conducted 2 months before and six months after the mass distribution. Descriptive statistics were used to measure changes in key malaria household indicators. LLIN ownership and use were compared between delivery strategies. Univariate and multivariate logistic regression analyses were used to identify factors associated with LLIN use before and after the mass distribution. A comparative financial cost analysis between the fixed delivery and door-to-door distribution strategies was carried out from the provider's perspective. RESULTS: Household ownership of at least one LLIN increased from 39.4% pre-campaign to 91.4% post-campaign and LLIN universal coverage, measured as the proportion of households with at least one LLIN for every two people increased from 4.1 to 41.1%. Population access to LLIN within the household increased from 22.2 to 80.7%, while overall LLIN use increased from 18.0 to 68.3%. Higher LLIN ownership was achieved with the fixed delivery strategy compared with the door-to-door (92.5% [95% CI 90.2-94.4%] versus 85.2% [95% CI 78.5-90.0%]), while distribution strategy did not have a significant impact on LLIN use (69.6% [95% CI 63.1-75.5%] versus 65.7% [95% CI 52.7-76.7%]). Malaria prevalence among children aged 6-59 months was 44.8% post-campaign. Living in a household with sufficient numbers of LLIN to cover all members was the strongest determinant of LLIN use. The total financial cost per LLIN distributed was 6.58 USD for the fixed distribution strategy and 6.61 USD for the door-to-door strategy. CONCLUSIONS: The mass distribution campaign was effective for rapidly increasing LLIN ownership and use. These gains need to be sustained for long-term reduction in malaria burden. The fixed delivery strategy achieved a higher LLIN coverage at lower delivery cost compared with the door-to-door strategy and seems to be a better distribution strategy in the context of the present study setting.

Field efficacy and acceptability of PermaNet® 3.0 and OlysetNet® in Kinshasa, Democratic Republic of the Congo.

BACKGROUND & OBJECTIVES: Insecticide resistance in mosquitoes at Kinshasa may jeopardize the efficacy and usage of long-lasting insecticidal nets (LLINs). Entomological impact, user acceptance and bioefficacy of a combination LLIN (PermaNet® 3.0) and a standard LLIN (OlysetNet®) were evaluated at two sites in Kinshasa characterized by high densities of either Anopheles gambiae s.s. (Kindele) or Culex spp (Kimbangu). METHODS: Insecticide susceptibility (permethrin, deltamethrin, bendiocarb, propoxur and DDT) was determined via tube tests and bottle assays. Entomological impact of unwashed and washed LLINs and untreated nets was assessed via Latin square, based on rotation of nets and their users through selected houses at each site. User acceptability was evaluated through interviews using a questionnaire and net bioefficacy was measured via cone bioassays with field-derived An. gambiae s.s. RESULTS: The An. gambiae s.s. population from Kindele was resistant to DDT and permethrin with mortality rate of 27.3 and 75.8%, respectively, and kdr mutations (L1014F) plus suspected metabolic resistance. The Culex spp population was resistant to all five insecticides tested. No differences in entomological indices were observed for the five net treatments, but bioefficacy against An. gambiae was significantly higher for unwashed and washed PermaNet 3.0 (100 and 71% mortality) than for OlysetNet (56 and 36%). Householders reported a good sleep most often when using unwashed and washed PermaNet (94 and 88%) and least often with unwashed OlysetNet (46%). INTERPRETATION & CONCLUSION: High bioefficacy via cone bioassays against an An. gambiae s.s. population with kdr and suspected metabolic resistance was observed with PermaNet 3.0. Lower biting rates and a higher chance of a good night of sleep were reported when using PermaNet 3.0 compared to OlysetNet.

Molecular detection and species identification of Plasmodium spp. infection in adults in the Democratic Republic of Congo: A population-based study.

BACKGROUND: In efforts to control malaria infection, the Democratic Republic of Congo has implemented several strategies. Studies assessing their efficiency mainly involved at-risk groups, especially children under five years of age. This study aimed to determine the prevalence and identify the risk factors associated with Plasmodium spp. infection. METHODS: From October 2014 to March 2015, individuals aged at least 15 years were selected randomly and enrolled in a cross-sectional study conducted throughout the country. Microscopy and polymerase chain reaction (PCR) analysis were used for the detection of Plasmodium ssp. RESULTS: From 2286 individuals recruited, 1870 with valid laboratory results were included in the study for further analysis. The prevalence of Plasmodium spp. infection assessed by microscopy (355/ 1870 (19%) was lower than that estimated by PCR (580/1870 (31%). In addition, the difference between the two results was statistically significant (P < 0.0001). The most prevalent Plasmodium species was P. falciparum, either as mono-infection (96.3%; 95% C.I. 93.9-98.1) or combined with P. malariae (3.7%; 95% C.I. 2.8-5.9). The mean parasite density was 3272739 trophozoites/µL of blood. Women had higher risks of being infected than men (OR 2.03, 95% C.I.: 1.96. 2.62, P = 0.041)]. CONCLUSION: In this study, the molecular detection and species identification of Plasmodium spp. showed that, despite all efforts for malaria control, malaria remains a public health problem in the Democratic Republic of Congo. The high prevalence and parasite density of Plasmodium spp. in adults make this age group a potential parasitic infectious reservoir for the at-risk groups and supports the need to include this age group in further programs for malaria control.

Presence of species within the Anopheles gambiae complex in the Democratic Republic of Congo.

BACKGROUND: Anopheles gambiae s.l. is the primary vector of malaria in the Democratic Republic of Congo, however, there is little data on the species from this complex present in the country. METHODS: This paper presents the species collected (as determined by PCR) between 2004 and 2011 in 16 locations across the country. RESULTS AND CONCLUSIONS: The two species from the An. gambiae complex that were detected were An. coluzzii and An. gambiae s.s. An. gambiae s.s. was predominant in eastern DRC, whereas An. coluzzii was the main species found in several locations in Bandundu. The species were also found in sympatry in several locations (Kinshasa, Kisangani, Lodja). These results provide a basis for future work, which is needed to accurately describe the distribution of the An. gambiae complex species in DRC.

High Prevalence of Plasmodium falciparum Infection in Asymptomatic Individuals from the Democratic Republic of the Congo.

Malaria remains a major public health problem in the Democratic Republic of Congo (DRC) with 14 million cases reported by the WHO Malaria Report in 2014. Asymptomatic malaria cases are known to be prevalent in endemic areas and are generally untreated, resulting in a significant source of gametocytes that may serve as reservoir of disease transmission. Considering that microscopy certainly underestimates the prevalence of Plasmodium infections within asymptomatic carriers and that PCR assays are currently recognized as the most sensitive methods for Plasmodium identification, this study was conducted to weigh the asymptomatic carriage in DRC by a molecular method. Six provinces were randomly selected for blood collection in which 80 to 100 individuals were included in the study. Five hundred and eighty blood samples were collected and molecular diagnosis was performed. Globally, almost half of the samples collected from asymptomatic individuals (280/580; 48.2%) had Plasmodium infections and the most species identified was P. falciparum alone in combination with P. malariae. The high prevalence reported here should interpellate the bodies involved in malaria control in DR Congo to take into account asymptomatic carriers in actions taken and consider asymptomatic malaria as a major hurdle for malaria elimination.

Investigation on possible transmission of monkeys' Plasmodium to human in a populations living in the equatorial rainforest of the Democratic republic of Congo.

Plasmodiums are protozoa that may infect various hosts. Only five species are now recognized as naturally parasitizing humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. This fifth species, P. knowlesi, previously identified as naturally parasitizing the monkey Macaca fascicularis, has been microscopically confused for a long time with P. malariae or P. falciparum and it was not possible to correctly differentiate them until the advent of molecular biology. To date, natural human infections with P. knowlesi only occur in Southeast Asia and a similar phenomenon of natural transmission of simian plasmodium to humans has not been reported elsewhere. This study was conducted to investigate a possible transmission of African small monkey's plasmodium to humans in populations living near the rainforest of the Democratic Republic of Congo (DRC) where several species of non-human primates are living. Two successive real-time PCRs were identified in the literature and used in combination for purpose. Only P. falciparum was found in this study. However, studies with larger samples and with more advanced techniques should be conducted.