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Accurate assessment and monitoring of the Plasmodium falciparum Kelch 13 (pfk13) gene associated with artemisinin resistance is critical to understand the emergence and spread of drug-resistant parasites in malaria-endemic regions. In this study, we evaluated the genomic profile of the pfk13 gene associated with artemisinin resistance in P. falciparum in Nigerian children by targeted sequencing of the pfk13 gene. Genomic DNA was extracted from 332 dried blood (DBS) spot filter paper samples from three Nigerian States. The pfk13 gene was amplified by nested polymerase chain reaction (PCR), and amplicons were sequenced to detect known and novel polymorphisms across the gene. Consensus sequences of samples were mapped to the reference gene sequence obtained from the National Center for Biotechnology Information (NCBI). Out of the 13 single nucleotide polymorphisms (SNPs) detected in the pfk13 gene, five (F451L, N664I, V487E, V692G and Q661H) have not been reported in other endemic countries to the best of our knowledge. Three of these SNPs (V692G, N664I and Q661H) and a non-novel SNP, C469C, were consistent with late parasitological failure (LPF) in two States (Enugu and Plateau States). There was no validated mutation associated with artemisinin resistance in this study. However, a correlation of our study with in vivo and in vitro phenotypes is needed to establish the functional role of detected mutations as markers of artemisinin resistance in Nigeria. This baseline information will be essential in tracking and monitoring P. falciparum resistance to artemisinin in Nigeria., Competing Interests: The authors have declared that no competing interests exist.

Background: Malaria remains a public health burden especially in Nigeria. To develop new malaria control and elimination strategies or refine existing ones, understanding parasite population diversity and transmission patterns is crucial., Methods: In this study, characterization of the parasite diversity and structure of Plasmodium falciparum isolates from 633 dried blood spot samples in Nigeria was carried out using 12 microsatellite loci of P. falciparum. These microsatellite loci were amplified via semi-nested polymerase chain reaction (PCR) and fragments were analysed using population genetic tools., Results: Estimates of parasite genetic diversity, such as mean number of different alleles (13.52), effective alleles (7.13), allelic richness (11.15) and expected heterozygosity (0.804), were high. Overall linkage disequilibrium was weak (0.006, P < 0.001). Parasite population structure was low (Fst: 0.008-0.105, AMOVA: 0.039)., Conclusion: The high level of parasite genetic diversity and low population structuring in this study suggests that parasite populations circulating in Nigeria are homogenous. However, higher resolution methods, such as the 24 SNP barcode and whole genome sequencing, may capture more specific parasite genetic signatures circulating in the country. The results obtained can be used as a baseline for parasite genetic diversity and structure, aiding in the formulation of appropriate therapeutic and control strategies in Nigeria.

Background: The development and spread of artemisinin-resistant Plasmodium falciparum malaria in Greater Mekong Subregion has created impetus for continuing global monitoring of efficacy of artemisinin-based combination therapies (ACTs). This post analyses is aimed to evaluate changes in early treatment response markers 10 years after the adoption of ACTs as first-line treatments of uncomplicated falciparum malaria in Nigeria., Methods: At 14 sentinel sites in six geographical areas of Nigeria, we evaluated treatment responses in 1341 children under 5 years and in additional 360 children under 16 years with uncomplicated malaria enrolled in randomized trials of artemether-lumefantrine versus artesunate-amodiaquine at 5-year interval in 2009-2010 and 2014-2015 and at 2-year interval in 2009-2010 and 2012-2015, respectively after deployment in 2005., Results: Asexual parasite positivity 1 day after treatment initiation (APPD1) rose from 54 to 62% and 2 days after treatment initiation from 5 to 26% in 2009-2010 to 2014-2015 (P = 0.002 and P <  0.0001, respectively). Parasite clearance time increased significantly from 1.6 days (95% confidence interval [CI]: 1.55-1.64) to 1.9 days (95% CI, 1.9-2.0) and geometric mean parasite reduction ratio 2 days after treatment initiation decreased significantly from 11 000 to 4700 within the same time period (P <  0.0001 for each). Enrolment parasitaemia > 75 000 μl - 1 , haematocrit > 27% 1 day post-treatment initiation, treatment with artemether-lumefantrine and enrolment in 2014-2015 independently predicted APPD1. In parallel, Kaplan-Meier estimated risk of recurrent infections by day 28 rose from 8 to 14% (P = 0.005) and from 9 to 15% (P = 0.02) with artemether-lumefantrine and artesunate-amodiaquine, respectively. Mean asexual parasitaemia half-life increased significantly from 1.1 h to 1.3 h within 2 years (P <  0.0001)., Conclusions: These data indicate declining parasitological responses through time to the two ACTs may be due to emergence of parasites with reduced susceptibility or decrease in immunity to the infections in these children., Trial Registration: Pan African Clinical Trial Registration PACTR201508001188143 , 3 July 2015; PACTR201508001191898 , 7 July 2015 and PACTR201508001193368 , 8 July 2015 PACTR201510001189370 , 3 July 2015; PACTR201709002064150 , 1 March 2017; https://www.pactr.samrca.ac.za.

The efficacies of 3-day regimens of artemether-lumefantrine (AL), artesunate-amodiaquine (AA), and dihydroartemisinin-piperaquine (DHP) were evaluated in 910 children < 5 years old with uncomplicated malaria from six geographical areas of Nigeria. Parasite positivity 1 day and Kaplan-Meier estimated risk of persistent parasitemia 3 days after therapy initiation were both significantly higher, and geometric mean parasite reduction ratio 1 day after treatment initiation (PRRD1) was significantly lower in AL-treated children than in AA- and DHP-treated children. No history of fever, temperature > 38°C, enrollment parasitemia > 75,000 μL -1 , and PRRD1 < 5,000 independently predicted persistent parasitemia 1 day after treatment initiation. Parasite clearance was significantly faster and risk of reappearance of asexual parasitemia after initial clearance was significantly lower in DHP-treated children. Overall, day 42 polymerase chain reaction-corrected efficacy was 98.3% (95% confidence interval [CI]: 96.1-100) and was similar for all treatments. In a non-compartment model, declines of parasitemias were monoexponential with mean terminal elimination half-life of 1.3 hours and unimodal frequency distribution of half-lives. All treatments were well tolerated. In summary, all three treatments evaluated remain efficacious treatments of uncomplicated malaria in young Nigerian children, but DHP appears more efficacious than AL or AA.

Background: Plasmodium falciparum , the most dangerous malaria parasite species to humans remains an important public health concern in Okelele, a rural community in Ilorin, Kwara State, Nigeria. There is however little information about the genetic diversity of Plasmodium falciparum in Nigeria., Objective: To determine the population genomic diversity of Plasmodium falciparum in malaria patients attending Okelele Community Healthcare Centre, Okelele, Ilorin, Kwara State., Methods: In this study, 50 Plasmodium falciparum strains Merozoite Surface Protein 1, Merozoite Surface Protein 2 and Glutamate Rich Protein were analysed from Okelele Health Centre, Okelele, Ilorin, Nigeria. Genetic diversity of P. falciparum isolates were analysed from nested polymerase chain reactions (PCR) of the MSP-1 (K1, MAD 20 and RO33), MSP-2 (FC27 and 3D7) and Glutamate Rich Protein allelic families respectively., Results: Polyclonal infections were more in majority of the patients for MSP-1 allelic families while monoclonal infections were more for MSP-2 allelic families. Multiplicity of infection for MSP-1, MSP-2 and GLURP were 1.7, 1.8 and 2.05 respectively., Conclusion: There is high genetic diversity in MSP - 2 and GLURP allelic families of Plasmodium falciparum isolates from Okelele Health Centre, Ilorin, Nigeria.

Background: Artemisinin resistance in Plasmodium falciparum manifests as slow parasite clearance but this measure is also influenced by host immunity, initial parasite biomass and partner drug efficacy. This study collated data from clinical trials of artemisinin derivatives in falciparum malaria with frequent parasite counts to provide reference parasite clearance estimates stratified by location, treatment and time, to examine host factors affecting parasite clearance, and to assess the relationships between parasite clearance and risk of recrudescence during follow-up., Methods: Data from 24 studies, conducted from 1996 to 2013, with frequent parasite counts were pooled. Parasite clearance half-life (PC1/2) was estimated using the WWARN Parasite Clearance Estimator. Random effects regression models accounting for study and site heterogeneity were used to explore factors affecting PC1/2 and risk of recrudescence within areas with reported delayed parasite clearance (western Cambodia, western Thailand after 2000, southern Vietnam, southern Myanmar) and in all other areas where parasite populations are artemisinin sensitive., Results: PC1/2 was estimated in 6975 patients, 3288 of whom also had treatment outcomes evaluate d during 28-63 days follow-up, with 93 (2.8 %) PCR-confirmed recrudescences. In areas with artemisinin-sensitive parasites, the median PC1/2 following three-day artesunate treatment (4 mg/kg/day) ranged from 1.8 to 3.0 h and the proportion of patients with PC1/2 >5 h from 0 to 10 %. Artesunate doses of 4 mg/kg/day decreased PC1/2 by 8.1 % (95 % CI 3.2-12.6) compared to 2 mg/kg/day, except in populations with delayed parasite clearance. PC1/2 was longer in children and in patients with fever or anaemia at enrolment. Long PC1/2 (HR = 2.91, 95 % CI 1.95-4.34 for twofold increase, p < 0.001) and high initial parasitaemia (HR = 2.23, 95 % CI 1.44-3.45 for tenfold increase, p < 0.001) were associated independently with an increased risk of recrudescence. In western Cambodia, the region with the highest prevalence of artemisinin resistance, there was no evidence for increasing PC1/2 since 2007., Conclusions: Several factors affect PC1/2. As substantial heterogeneity in parasite clearance exists between locations, early detection of artemisinin resistance requires reference PC1/2 data. Studies with frequent parasite count measurements to characterize PC1/2 should be encouraged. In western Cambodia, where PC1/2 values are longest, there is no evidence for recent emergence of higher levels of artemisinin resistance.

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7&#95;1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

Background: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies., Methods: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined., Results: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days., Conclusions: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).

Background: Artemisinin-based combination therapies (ACTs) are the recommended treatment for uncomplicated Plasmodium falciparum malaria in all malaria endemic countries. Artemisinin resistance, partner drug resistance, and subsequent ACT failure are widespread in Southeast Asia. The more recent independent emergence of artemisinin resistance in Africa is alarming. In response, triple artemisinin-based combination therapies (TACTs) are being developed to mitigate the risks associated with increasing drug resistance. Since ACTs are still effective in Africa, where malaria is mainly a paediatric disease, the potential deployment of TACTs raises important ethical questions. This paper presents an analysis of stakeholders' perspectives regarding key ethical considerations to be considered in the deployment of TACTs in Africa provided they are found to be safe, well-tolerated and effective for the treatment of uncomplicated malaria. Methods: We conducted a qualitative study in Burkina Faso and Nigeria assessing stakeholders' (policy makers, suppliers and end-users) perspectives on ethical issues regarding the potential future deployment of TACTs through 68 in-depth interviews and 11 focus group discussions. Findings: Some respondents suggested that there should be evidence of local artemisinin resistance before they consider deploying TACTs, while others suggested that TACTs should be deployed to protect the efficacy of current ACTs. Respondents suggested that additional side effects of TACTs compared to ACTs should be minimal and the cost of TACTs to end-users should not be higher than the cost of current ACTs. There was some disagreement among respondents regarding whether patients should have a choice of treatment options between ACTs and TACTs or only have TACTs available, while ACTs are still effective. The study also suggests that community, public and stakeholder engagement activities are essential to support the introduction and effective uptake of TACTs. Conclusion: Addressing ethical issues regarding TACTs and engaging early with stakeholders will be important for their potential deployment in Africa. [ABSTRACT FROM AUTHOR]

Background: Artemisinin-based combination therapies (ACTs) have remained efficacious treatments of acute falciparum malaria in many endemic areas but there is little evaluation of factors contributing to the anaemia of acute falciparum malaria following long term adoption of ACTs as first-line antimalarials in African children.Methods: Malarious <5 year-olds randomized to artemether-lumefantrine, artesunate-amodiaquine or dihydroartemisinin-piperaquine treatments were followed up clinically for 6 weeks. Anaemia was defined as haematocrit <30%; Malaria-attributable fall in haematocrit (MAFH) as the difference between haematocrit 28-42 days post- and pre-treatment; Total MAFH (TMAFH) as the difference between days 28-42 haematocrit and the lowest haematocrit recorded in the first week post-treatment initiation; Drug-attributable fall in haematocrit (DAFH) as the difference between MAFH and TMAFH; Early appearing anaemia (EAA) as haematocrit <30% occurring within 1 week in children with normal haematocrit pre-treatment. Predictors of anaemia pre-treatment, EAA, MAFH or DAFH >4% were evaluated by stepwise multiple logistic regression models. Survival analysis and kinetics of DAFH were evaluated by Kaplan-Meier estimator and non-compartment model, respectively.Results: Pre-treatment, 355 of 959 children were anaemic. Duration of illness >2 days and parasitaemia ≤10,000 μL-1 were independent predictors of anaemia pre-treatment. EAA occurred in 301 of 604 children. Predictors of EAA were age ≤ 15 months, history of fever pre-treatment and enrolment haematocrit ≤35%. The probabilities of progression from normal haematocrit to EAA were similar for all treatments. MAFH >4% occurred in 446 of 694 children; its predictors were anaemia pre-treatment, enrolment parasitaemia ≤50,000 μL-1, parasitaemia one day post-treatment initiation and gametocytaemia. DAFH >4% occurred in 334 of 719 children; its predictors were history of fever pre-and fever 1 day post-treatment initiation, haematocrit ≥37%, and parasitaemia >100,000 μL-1. In 432 children, declines in DAFH deficits were monoexponential with overall estimated half-time of 2.2d (95% CI 1.9-2.6). Area under curve of deficits in DAFH versus time and estimated half-time were significantly higher in non-anaemic children indicating greater loss of haematocrit in these children.Conclusion: After ten years of adoption of ACTs, anaemia is common pre-and early post-treatment, falls in haematocrit attributable to a single infection is high, and DAFH >4% is common and significantly lower in anaemic compared to non-anaemic Nigerian children.Trial Registration: Pan African Clinical Trial Registry (PACTR) [ PACTR201709002064150, 1 March 2017 ]. [ABSTRACT FROM AUTHOR]

Since congenital malaria had previously been thought to be rare, blood film examination for malaria parasites is still sometimes not routinely performed in ill neonates in malaria-endemic regions. Because of increasing published reports of congenital malaria in Nigeria, there is a need to characterize the clinical and laboratory manifestations associated with malaria parasitemia within the first few hours of life. In a 12-month (April 2003-March 2004), multicenter study in Nigeria, thin and thick blood smears made from maternal (finger prick), placental aspirates, cord blood and neonate (heel prick taken within 4 hours of life) were Giemsa-stained and examined by light microscopy for asexual stages of Plasmodium. Parasitemic neonates were closely monitored for clinical and laboratory features of symptomatic malaria. Plasmodium falciparum was found in 5.1% (95/1875) of neonatal heel pricks; mean parasite density was low (mean = 48/μL, range 8–200/μL). Antepartum maternal and placental parasitemia were the most important risk factors for congenital parasitemia (P < 0.001 and P < 0.001). Prolonged labor and prolonged rupture of membranes were also significant factors in the symptomatic neonates. Sixty-one percent (58/95) of parasitemic babies were asymptomatic, while 38.9% (37/95) of them exhibited signs of possible infection. The presence of any symptom was significantly related to parasitemia (P < 0.001). Among the symptomatic parasitemic babies the most common symptoms were, fever (temperature >37.5&degic;) within the first 24 hours of life (100%) and refusal to suck (10.8%). Anemia at birth (hematocrit <42%) was found in 15.7% (15/95) of parasitemic babies as compared to 9.2% in the non-parasitemic ones. (P = 0.03, OR = 1.84). The mean hematocrit of parasitemic neonates within 4 hours of life was 49.5 ± 6.4 as compared to 52.6 ± 8.2 in non-parasitemic babies (P = 0.001). Furthermore, the mean hematocrit was 44.0 ± 5.5% in the symptomatic parasitemic babies. All symptomatic babies were treated with oral chloroquine with a cure rate of 89.1%. Treatment failures subsequently received oral sulfadoxine-pyrimethamine with good outcome. The febrile newborn should be evaluated for malaria especially if there is a history of prolonged labor or in the presence of maternal malaria infection. Efforts should be intensified to reduce the burden of maternal, placental malaria and therefore congenital malaria. [ABSTRACT FROM AUTHOR]