Unravelling the quality of malaria microscopy across Kinshasa, DR Congo.

Introduction: In the current study we assessed clinical laboratories' staff ability across the city of Kinshasa with particular focus on their practices and performance regarding malaria microscopy. Materials and Methods: This was a non-random cross-sectional study included clinical laboratories in Kinshasa and focused on cross-checking of blood slides, a questionnaire and checklist according to standardised analytic malaria microscopy procedures. Regarding the cross-checking of slides, participant responses were considered 'corrects' in cases of complete congruence with the reference; 'acceptable' for malaria-positive slides but no identification of Plasmodium species, stage of development, parasite density and/or reported as P. falciparum instead of 'P. non falciparum'; and 'incorrect' if 'false positive' and 'false negative' cases. Results: Eighty-eight among the 90 targeted clinical laboratories (participation 97.8%) took part in the investigation from February to July 2019. The ability assessment revealed that individuals qualified to perform thick blood films (TBF) according to the national malaria control program (NMCP) procedures ranged from 48.6% to 100.0%. Overall cross-checking performance of 167 eligible routine slides was relatively low: 37.7%; 25.8% and 36.5% of correct, acceptable and incorrect responses, respectively. The first routine slide was correctly and acceptably scored respectively by 35.3% and 28.2% of participating laboratories (n = 85); and the second, by 40.2% and 23.2% respectively (n = 82). The sensitivity and specificity were found to be 79.4% and 53.8%, respectively. However, the relative high scores reported in relation with the ability needed to perform TBF based on NMCP standards contrasted with the poor performance from cross-checking slides. Consecutively, only one-third of the 88 participating laboratories reached a score > 60% in agreement with NMCP procedures and had acceptable responses to cross-checked slides. Conclusions: The study was conducted as part of the activities relating to "Ensuring early diagnosis and prompt malaria treatment" component of the national malaria control strategy with NMCP support. More laboratories must implement clear and standardised malaria microscopy procedures, and need to include more rigorous quality control.

Identification of the PfK13 mutations R561H and P441L in the Democratic Republic of Congo.

OBJECTIVES: Partial artemisinin resistance, mediated by Plasmodium falciparum K13 (PfK13) mutations, has been confirmed in certain areas of East Africa that are historically associated with high-level antimalarial resistance. The Democratic Republic of Congo (DRC) borders these areas in the East. This study aimed to determine the prevalence of resistance markers in six National Malaria Control Program surveillance sites; Boende, Kabondo, Kapolowe, Kimpese, Mikalayi, and Rutshuru. METHODS: The single nucleotide polymorphisms (SNPs) in P. falciparum genes PfK13, Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt were assessed using targeted next-generation sequencing of isolates collected at enrollment in therapeutic efficacy studies. RESULTS: PfK13 SNPs were detected in two samples: in Kabondo (R561H) and in Rutshuru (P441L), both areas near Uganda and Rwanda. The Pfdhps ISGEGA haplotype, associated with reduced sulfadoxine-pyrimethamine chemoprevention efficacy, ranged from 0.8% in Mikalayi (central DRC) to 42.2% in Rutshuru (East DRC). CONCLUSIONS: R561H and P441L observed in eastern DRC are a concern, as they are associated with delayed artemisinin-based combination therapies-clearance and candidate marker of resistance, respectively. This is consistent with previous observations of shared drug resistance profiles in parasites of that region with bordering areas of Rwanda and Uganda. The likely circulation of parasites has important implications for the ongoing surveillance of partial artemisinin-resistant P. falciparum and for future efforts to mitigate its dispersal.

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.

Association between domesticated animal ownership and Plasmodium falciparum parasite prevalence in the Democratic Republic of the Congo: a national cross-sectional study.

BACKGROUND: Domesticated animal ownership is an understudied aspect of the human environment that influences mosquito biting behaviour and malaria transmission, and is a key part of national economies and livelihoods in malaria-endemic regions. In this study, we aimed to understand differences in Plasmodium falciparum prevalence by ownership status of common domesticated animals in DR Congo, where 12% of the world's malaria cases occur and anthropophilic Anopheles gambiae vectors predominate. METHODS: In this cross-sectional study, we used survey data from individuals aged 15-59 years in the most recent (2013-14) DR Congo Demographic and Health Survey and previously performed Plasmodium quantitative real-time PCR (qPCR) to estimate P falciparum prevalence differences by household ownership of cattle; chickens; donkeys, horses, or mules; ducks; goats; sheep; and pigs. We used directed acyclic graphs to consider confounding by age, gender, wealth, modern housing, treated bednet use, agricultural land ownership, province, and rural location. FINDINGS: Of 17 701 participants who had qPCR results and covariate data, 8917 (50·4%) of whom owned a domesticated animal, we observed large differences in malaria prevalence across types of animals owned in both crude and adjusted models. Household chicken ownership was associated with 3·9 (95% CI 0·6 to 7·1) more P falciparum infections per 100 people, whereas cattle ownership was associated with 9·6 (-15·8 to -3·5) fewer P falciparum infections per 100 people, even after accounting for bednet use, wealth, and housing structure. INTERPRETATION: Our finding of a protective association conferred by cattle ownership suggests that zooprophylaxis interventions might have a role in DR Congo, possibly by drawing An gambiae feeding away from humans. Studies of animal husbandry practices and associated mosquito behaviours could reveal opportunities for new malaria interventions. FUNDING: The National Institutes of Health and the Bill & Melinda Gates Foundation. TRANSLATIONS: For the French and Lingala translations of the abstract see Supplementary Materials section.

Digitalized long-lasting insecticidal nets mass distribution campaign in the context of Covid-19 pandemic in Kongo Central, Democratic Republic of Congo: challenges and lessons learned.

BACKGROUND: The Democratic Republic of the Congo (DRC) organized a first mass distribution campaign of long-lasting insecticidal nets (LLINs) with digitalized data management with coordinated support from the Ministry of Health (MOH) and Santé Pour Tous En Milieu Rural-an 'Association sans but lucratif' (SANRU Asbl), in the context of the Covid-19 pandemic in Kongo Central province. This article describes the planning and implementation process of this campaign as well as the challenges and lessons learned. METHODS: The planning and implementation process was performed in line with the standard guidance issued by the National Malaria Control Programme (NMCP) following the start of Covid-19. The changes and adaptations put in place as well as the challenges encountered are described. RESULTS: A total of 5,629,211 people were registered (7.7% above projection) in 1,065,537 households (6.2% below projection) giving an average of 5.3 people per household. Of a total of 3,062,850 LLINs ordered, 2,886,096 were distributed to households (94%). Out of 11,070 villages and 3,947 teams planned, 91.7% of villages were reached and 93% of teams were established. CONCLUSION: The revision of standards of campaign implementation during Covid-19, as well as effective coordination supported by real-time decision-making through digital data management, have been factors in the success of this campaign. Maintaining this momentum is essential to ensure the continuity of malaria prevention services for the population.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018.

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

Therapeutic Efficacy of Artemisinin-Based Combination Therapies in Democratic Republic of the Congo and Investigation of Molecular Markers of Antimalarial Resistance.

Routine assessment of the efficacy of artemisinin-based combination therapies (ACTs) is critical for the early detection of antimalarial resistance. We evaluated the efficacy of ACTs recommended for treatment of uncomplicated malaria in five sites in Democratic Republic of the Congo (DRC): artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Children aged 6-59 months with confirmed Plasmodium falciparum malaria were treated with one of the three ACTs and monitored. The primary endpoints were uncorrected and polymerase chain reaction (PCR)-corrected 28-day (AL and ASAQ) or 42-day (DP) cumulative efficacy. Molecular markers of resistance were investigated. Across the sites, uncorrected efficacy estimates ranged from 63% to 88% for AL, 73% to 100% for ASAQ, and 56% to 91% for DP. PCR-corrected efficacy estimates ranged from 86% to 98% for AL, 91% to 100% for ASAQ, and 84% to 100% for DP. No pfk13 mutations previously found to be associated with ACT resistance were observed. Statistically significant associations were found between certain pfmdr1 and pfcrt genotypes and treatment outcome. There is evidence of efficacy below the 90% cutoff recommended by WHO to consider a change in first-line treatment recommendations of two ACTs in one site not far from a monitoring site in Angola that has shown similar reduced efficacy for AL. Confirmation of these findings in future therapeutic efficacy monitoring in DRC is warranted.

Associating the scale-up of insecticide-treated nets and use with the decline in all-cause child mortality in the Democratic Republic of Congo from 2005 to 2014.

BACKGROUND: To reduce the malaria burden and improve the socioeconomic status of its citizens, the Democratic Republic of Congo scaled up key malaria control interventions, especially insecticide-treated nets (ITNs), between 2005 and 2014. Since then, the effects of these interventions on malaria mortality and morbidity have not been assessed. This study aimed to measure the impact of the National Malaria Control Programme's efforts and to inform future control strategies. METHODS: The authors used data from the Demographic and Health Surveys 2007 and 2013-2014 to assess trends in all-cause childhood mortality (ACCM) against trends in coverage of malaria interventions at national and subnational levels. The authors used the plausibility argument to assess the impact of the malaria control interventions and used Kaplan-Meier survival probability and Cox proportional hazard models to examine the effect of ITN ownership on child survival. Contextual factor trends affecting child survival were also considered. RESULTS: Countrywide, household ownership of at least one ITN increased, from 9% in 2007 to 70% in 2013-2014. All provinces experienced similar increases, with some greater than the national level. ITN use increased between 2007 and 2013-2014 among children under five (6% to 55%). Severe anaemia (haemoglobin concentration < 8 g/dl) prevalence among children aged 6-59 months significantly decreased, from 11% (95% confidence interval [CI] 9-13%) in 2007 to 6% (95% CI 5-7%) in 2013-2014. During the same period, ACCM declined, from 148 (95% CI 132-163) to 104 (95% CI 97-112) deaths per 1000 live births. The decline in ACCM was greater among children aged 6-23 months (relative reduction of 36%), compared to children aged 24-59 months (relative reduction of 12%). Cox regression indicated that household ownership of at least one ITN reduced the risk of mortality by 24% among children under five (risk ratio = 0.76, 95% CI 0.64-0.90). Contextual factor analysis revealed marginal improvements in socioeconomic indicators and other health interventions. CONCLUSIONS: Given the patterns of the coverage of malaria control interventions, patterns in ACCM by province, and marginal improvements in contextual factors, the authors conclude that the malaria control interventions have plausibly contributed to the decrease in ACCM in the Democratic Republic of Congo from 2005 to 2014.

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.

Household Prevalence of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale in the Democratic Republic of the Congo, 2013-2014.

In a cross-sectional molecular study in the Democratic Republic of the Congo, 78% of households had ≥1 member infected with Plasmodium falciparum, Plasmodium vivax, and/or Plasmodium ovale spp.; 47% of children and 33% of adults tested positive for ≥1 species. Risk factors varied by species and age group.

Highly targeted cholera vaccination campaigns in urban setting are feasible: The experience in Kalemie, Democratic Republic of Congo.

INTRODUCTION: Oral cholera vaccines are primarily recommended by the World Health Organization for cholera control in endemic countries. However, the number of cholera vaccines currently produced is very limited and examples of OCV use in endemic countries, and especially in urban settings, are scarce. A vaccination campaign was organized by Médecins Sans Frontières and the Ministry of Health in a highly endemic area in the Democratic Republic of Congo. This study aims to describe the vaccine coverage achieved with this highly targeted vaccination campaign and the acceptability among the vaccinated communities. METHODS AND FINDINGS: We performed a cross-sectional survey using random spatial sampling. The study population included individuals one year old and above, eligible for vaccination, and residing in the areas targeted for vaccination in the city of Kalemie. Data sources were household interviews with verification by vaccination card. In total 2,488 people were included in the survey. Overall, 81.9% (95%CI: 77.9-85.3) of the target population received at least one dose of vaccine. The vaccine coverage with two doses was 67.2% (95%CI: 61.9-72.0) among the target population. The vaccine coverage was higher during the first round (74.0, 95%CI: 69.3-78.3) than during the second round of vaccination (69.1%, 95%CI: 63.9-74.0). Vaccination coverage was lower in male adults. The main reason for non-vaccination was to be absent during the campaign. No severe adverse events were notified during the interviews. CONCLUSIONS: Cholera vaccination campaigns using highly targeted strategies are feasible in urban settings. High vaccination coverage can be obtained using door to door vaccination. However, alternative strategies should be considered to reach non-vaccinated populations like male adults and also in order to improve the efficiency of the interventions.

[Asymptomatic Parasitemia in under five, school age children and households self-medication, Lubumbashi, Democratic Republic of Congo]. / Parasitémie asymptomatique chez les enfants de moins de 5 ans, enfants en âge scolaire et prise en charge des épisodes fébriles dans les ménages de Lubumbashi, République Démocratique du Congo.

INTRODUCTION: Long neglected, asymptomatic malaria is currently recognized as a potential threat and obstacle to malaria control. In DR Congo, the prevalence of this parasite is poorly documented. This study aims to determine the prevalence of asymptomatic parasitaemia in children less than 5 years of age as well as in those aged over five years for what concerns ongoing mass control interventions (LLINs). METHODS: This is a cross-sectional study conducted among school age children, children less than 5 years of age living in the household of Lubumbashi. Schools, students and children less than 5 years of age were selected randomly. Thick and thin blood smears and rapid tests were performed and read. RESULTS: Out of 350 examined students, 43 (12, 3%), IC 95% (9, 14-16, 04) had positive thick smear. Only plasmodium falciparum was identified in all the 43 cases. 314 households (90.5%) declared that they had administered anti-malarial drugs to their children to treat fever at home. More than one-third of households (39.9%) declared that they had administered antipyretics to their children to relieve fever, 19.7% administered quinine and only less than 2% artemether-lumefantrine. Considering the use of the TDR technique, the prevalence of asymptomatic parasitaemia was 3%, IC 95% (from 2.075 to 4.44), but if we consider microscopy as the gold standard, the prevalence was 1.9%, IC 95% (from 1.13 to 3.01). CONCLUSION: Asymptomatic malaria is not without health consequences, so it is important to conduct such investigations to detect new malaria device programmes.

Adherence to Artemisinin Combination Therapy for the treatment of uncomplicated malaria in the Democratic Republic of the Congo.

Between 2011 and 2013 the number of recorded malaria cases had more than doubled, and between 2009 and 2013 had increased almost 4-fold in MSF-OCA (Médecins sans Frontières - Operational Centre Amsterdam) programmes in the Democratic Republic of the Congo (DRC). The reasons for this rise are unclear. Incorrect intake of Artemisinin Combination Therapy (ACT) could result in failure to treat the infection and potential recurrence. An adherence study was carried out to assess whether patients were completing the full course of ACT. One hundred and eight malaria patients in Shamwana, Katanga province, DRC were visited in their households the day after ACT was supposed to be completed. They were asked a series of questions about ACT administration and the blister pack was observed (if available). Sixty seven (62.0%) patients were considered probably adherent. This did not take into account the patients that vomited or spat their pills or took them at the incorrect time of day, in which case adherence dropped to 46 (42.6%). The most common reason that patients gave for incomplete/incorrect intake was that they were vomiting or felt unwell (10 patients (24.4%), although the reasons were not recorded for 22 (53.7%) patients). This indicates that there may be poor understanding of the importance of completing the treatment or that the side effects of ACT were significant enough to over-ride the pharmacy instructions. Adherence to ACT was poor in this setting. Health education messages emphasising the need to complete ACT even if patients vomit doses, feel unwell or their health conditions improve should be promoted.

Efficacy of fixed-dose combination artesunate-amodiaquine versus artemether-lumefantrine for uncomplicated childhood Plasmodium falciparum malaria in Democratic Republic of Congo: a randomized non-inferiority trial.

BACKGROUND: In 2005, the Democratic Republic of Congo (DRC) adopted artesunate and amodiaquine (ASAQ) as first-line anti-malarial treatment. In order to compare the efficacy of the fixed-dose formulation ASAQ versus artemether-lumefantrine (AL), a randomized, non-inferiority open-label trial was conducted in Katanga. METHODS: Children aged six and 59 months with uncomplicated Plasmodium falciparum malaria were enrolled and randomly allocated into one of the two regimens. The risk of recurrent parasitaemia by day 42, both unadjusted and adjusted by PCR genotyping to distinguish recrudescence from new infection, was analysed. RESULTS: Between April 2008 and March 2009, 301 children were included: 156 with ASAQ and 145 with AL. No early treatment failures were reported. Among the 256 patients followed-up at day 42, 32 patients developed late clinical or parasitological failure (9.9% (13/131) in the ASAQ group and 15.2% (19/125) in the AL group). After PCR correction, cure rates were 98.3% (95%CI, 94.1-99.8) in the ASAQ group and 99.1% (95%CI, 94.9-99.9) in the AL group (difference -0.7%, one sided 95% CI -3.1). Kaplan-Meier PCR-adjusted cure rates were similar. Both treatment regimens were generally well tolerated. CONCLUSION: Both ASAQ and AL are highly effective and currently adequate as the first-line treatment of uncomplicated falciparum malaria in this area of Katanga, DRC. However, in a very large country, such as DRC, and because of possible emergence of resistance from other endemic regions, surveillance of efficacy of artemisinin-based combination treatments, including other evaluations of the resistance of ASAQ, need to be done in other provinces. TRIAL REGISTRATION: The protocol was registered with the clinicaltrials.gov, open clinical trial registry under the identifier number NCT01567423.

Ready-to-use therapeutic food for catch-up growth in children after an episode of Plasmodium falciparum malaria: an open randomised controlled trial.

BACKGROUND: Catch-up growth after an infection is essential for children to maintain good nutritional status. To prevent malnutrition, WHO recommends that children are given one additional healthy meal per day during the 2 weeks after onset of illness. We investigated to what extent ready-to-use therapeutic food (RUTF) promotes catch-up growth in children after an acute, uncomplicated episode of Plasmodium falciparum malaria. METHODS: We did an open randomised trial of children aged 6-59 months with confirmed malaria who attended a Médecins Sans Frontières-supported outpatient clinic in Katanga Province, Democratic Republic of Congo. All children received a clinical examination and malaria treatment. Patients were then randomly assigned to either an RUTF group, who received daily supplemental RUTF (a high-protein peanut-based paste) for 14 days, or to a control group, who received no supplemental food. Children were weighed at baseline and on days 14 and 28. The primary outcome was mean weight change after 14 days' RUTF. Analysis was by intention-to-treat. RESULTS: 93 children received RUTF and 87 received no food supplementation. At day 14, the RUTF group had a mean weight gain of 353 g compared with 189 g in the control group (difference 164 [95%CI 52-277], p = 0.005). However, at day 28 there was no statistically significant difference between the groups (539 g versus 414 g, respectively [p = 0.053]). Similarly, rate of weight gain per kg bodyweight per day was significantly higher at day 14 in the RUTF group (2.4 g/kg per day versus 1.3 g/kg per day, p = 0.005) but at day 28 was 1.9 g/kg per day in the RUTF group versus 1.5 g/kg per day in the control group (p = 0.076). CONCLUSIONS: Children receiving RUTF for 14 days after effective treatment of an uncomplicated malaria episode had a faster weight gain than children not given supplementation, reducing the period that children were at risk of malnutrition. TRIAL REGISTRATION: ClinicalTrials.gov NCT00819858.