Genetic diversity and population structure of Plasmodium falciparum in Nigeria: insights from microsatellite loci analysis.

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.

Characterization of Plasmodium falciparum structure in Nigeria with malaria SNPs barcode.

BACKGROUND: Plasmodium falciparum malaria remains a major health challenge in Nigeria despite the global decline of its incidence and mortality rates. Although significant progress has been made in preventing the transmission of P. falciparum and controlling the spread of the infection, there is much to be done in the area of proper monitoring, surveillance of the parasite, investigating the population dynamics and drug resistance profiling of the parasite as these are important to its eventual eradication. Polymorphic loci of msp1, msp2 and/or glurp genes or microsatellites have been traditionally used to characterize P. falciparum population structure in various parts of Nigeria. The lack of standardization in the interpretation of results, as well as the inability of these methods to distinguish closely related parasites, remains a limitation of these techniques. Conversely, the recently developed 24 single nucleotide polymorphism (SNP)-based molecular barcode assay has the possibility of differentiating between closely related parasites and offer additional information in determining the population diversity of P. falciparum within and between parasite populations. This study is therefore aimed at defining the population diversity of P. falciparum in and between two localities in Nigeria using the SNPs barcode technique. METHODS: The 24-SNP high-resolution melt (HRM) barcode assay and msp2 genotyping was used to investigate both intra and inter population diversity of the parasite population in two urban cities of Nigeria. RESULTS: Based on SNP barcode analysis, polygenomic malaria infections were observed in 17.9% and 13.5% of population from Enugu and Ibadan, respectively, while msp2 analyses showed 21% and 19.4% polygenomic infections in Enugu and Ibadan, respectively. Low levels of genetic diversity (π) of 0.328 and 0.318 were observed in Enugu and Ibadan parasite populations, respectively, while the FST value of 0.02 (p = 0.055) was obtained when the genetic divergence of both populations was considered. CONCLUSIONS: The 24-SNP barcode assay was effective in analysing P. falciparum population diversity. This study also showed that P. falciparum populations in Enugu and Ibadan had a degree of intra-population diversity, but very low divergence between the population. A low degree of polygenomic infections were also observed in the two parasite populations unlike previous years. This maybe as a result of the effect of artemisinin-based combination therapy (ACT), long-lasting insecticide-treated nets (LLITNs) and intermittent preventive treatments in the study populations.

Early rising asexual parasitaemia in Nigerian children following a first dose of artemisinin-based combination treatments of falciparum malaria.

BACKGROUND: Early rising asexual parasitaemia (ERAP), initially defined as 'an increase in the parasite count over the baseline pre-treatment level during the first 24 h of treatment' of falciparum malaria with artemisinin derivatives is well documented, but there is no characterization of its risk factors, kinetics, molecular features or relationship to late-appearing anaemia (LAA) in acute falciparum malaria in African children following oral artemisinin-based combination therapies (ACTs). METHODS: ERAP was defined as ≥5% increase in pre-treatment parasitaemia within 8 h of initiating treatment. Parasitaemia was quantified pre-treatment and 1-2 hourly for 8 h, and less frequently thereafter for 6 weeks following randomized treatment of acutely malarious children with artesunate-amodiaquine, artemether-lumefantrine or dihydroartemisinin-piperaquine. Risk factors were determined by stepwise multiple logistic regression model. Kinetics of release into and of elimination of asexual parasites and DNA clones from peripheral blood were evaluated by method of residuals and non-compartment model, respectively. Parasite population changes were evaluated morphologically and by molecular genotyping. RESULTS: ERAP occurred in 205 of 416 children. A parasitaemia <100,000/µL and parasitaemia 1 day post-treatment initiation were independent predictors of ERAP. In children with ERAP: mean and peak time of increase in parasitaemia were 105.6% (95% CI 81-130.1) and 2.5 h (95% CI 2.2-2.7), respectively. Mean lag time, half-time and rate constant of release were 0.2 h (95% CI 0.2-0.3), 1 h (95% CI 0.9-1.1), and 0.9 h-1 (95% CI 0.8-1), respectively. Schizonts and young gametocytes were seen only in peripheral blood of few children with ERAP. In age-, gender-, baseline parasitaemia- and treatment-matched children with and without ERAP, parasite DNA clearance time and area under curve of number of DNA clones versus time were significantly higher in children with ERAP indicating peripheral retention of released parasites followed by elimination. DNA clone elimination was monoexponential. CONCLUSION: ERAP is common, occurs rapidly as first order process and may be due to mobilization of parasites from deep tissue following a first dose of ACTs of acute childhood falciparum malaria. TRIALS REGISTRATION: Pan African Clinical Trial Registry PACTR201508001188143 , 3 July 2015; PACTR201510001189370, 3 July 2015; PACTR201508001191898, 7 July 2015 and PACTR201508001193368, 8 July 2015.

Factors contributing to anaemia after uncomplicated falciparum malaria in under five year-old Nigerian children ten years following adoption of artemisinin-based combination therapies as first-line antimalarials.

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 ].

Clinical illness and outcomes in Nigerian children with late-appearing anaemia after artemisinin-based combination treatments of uncomplicated falciparum malaria.

BACKGROUND: Late-appearing anaemia (LAA) following treatment with artemisinins for severe malaria has been reported and well described, but there are limited clinical and parasitological data on LAA in African children with uncomplicated falciparum malaria following oral artemisinin-based combination therapies (ACTs). METHODS: This was an open label study with the main objectives of evaluating the clinical features, the risk factors for, the temporal changes in haematocrit and the outcomes of a LAA in malarious children treated with artesunate-amodiaquine (AA), artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DHP). The diagnosis of LAA was made using the criteria: clearance of parasitaemia, fever and other symptoms within 1 week of commencing treatment; adequate clinical and parasitological response at 4-6 weeks after treatment began; haematocrit ≥30 % 1 and/or 2 weeks after treatment began; and haematocrit <30 %, parasite negativity by microscopy and polymerase chain reaction and absence of concomitant illness 3-6 weeks after treatment began. RESULTS: LAA occurred in 84 of 609 children, was mild, moderate or severe in 77, 6 or 1 child, respectively and was relatively asymptomatic. Mean time elapsing from commencement of treatment to LAA was 27.1 days (95 % CI 25.3-28.9). In a multivariate analysis, an age <3 years (adjusted odd ratio [AOR] = 2.6, 95 % CI 1.3-5.2, P = 0.005), fever 1 day after treatment began (AOR = 3.8, 95 % CI 1.8-8.2, P < 0.0001), haematocrit <25 % at presentation (AOR = 2.2, 95 % CI 1.3-3.7, P = 0.003), haematocrit <30 % 1 day after treatment began (AOR = 2.1, 95 % CI 1.0-4.3, P = 0.04), parasite reduction ratio >10(4) 2 days after treatment began (AOR = 2.1, 95 % CI 1.1-3.9, P = 0.03) and spleen enlargement at presentation (AOR = 2.0, 95 % CI 1.1-3.9, P < 0.0001) were independent predictors of LAA. During 6 weeks of follow-up, uneventful recovery from anaemia occurred in 56 children [mean recovery time of 11.8 days (95 % CI 10.3-13.3)]. The only independent predictor of failure of recovery was LAA occurring 4 weeks after starting treatment (AOR = 7.5, 95 % CI 2.5-22.9, P < 0.0001). CONCLUSION: A relatively asymptomatic LAA with uneventful recovery can occur in young malarious children following ACTs. Its occurrence may have implications for case and community management of anaemia and for anaemia control efforts in sub-Saharan Africa where ACTs have become first-line antimalarials. TRIALS REGISTRATION: Pan African Clinical Trial Registry PACTR201508001188143, 3 July 2015; PACTR201510001189370, 3 July 2015; PACTR201508001191898, 7 July 2015 and PACTR201508001193368, 8 July 2015 http://www.pactr.org .

Temporal changes in haematocrit following artemisinin-based combination treatments of uncomplicated falciparum malaria in children.

BACKGROUND: Artemisinin-based combination treatments (ACTs) or intravenous artesunate are used in over 100 countries for uncomplicated or severe falciparum malaria. Although intravenous artesunate may cause delayed haemolytic anaemia, there is little evaluation of the temporal changes in haematocrit following ACTs. METHODS: Clinical and parasitological parameters were measured before and following treatment of uncomplicated falciparum malaria in children with artesunate-amodiaquine (AA) or artemether-lumefantrine (AL) over 6-weeks. Changes in haematocrit were characterized in individual patients based on a haematocrit <30 % or ≥30 % before and following treatment. Kinetics of the deficit in haematocrit from <30 % until attainment of ≥30 % were estimated by a non-compartment model. RESULTS: In 248 of 1180 children eligible for evaluation, common temporal patterns were: no change or increase in haematocrit from ≥ 30 % [50 % of patients], haematocrit >30 % at presentation declining to <30 % within 2 weeks (early monophasic fall) [19 % of patients], and haematocrit <30 % at presentation increasing to ≥ 30 % [23 % of patients]. Haematocrit >30 % at presentation declining to <30 %, 3-5 weeks later (late monophasic fall) occurred in 7 children (3 %). Fall in haematocrit ≥5 units following treatment occurred in 57 children [23 %] between 14 and 28 days after treatment began. Baseline parasitaemia and proportion with > 100,000µL(-1) asexual forms were significantly higher in children with ≥5 units compared to <5 units fall in haematocrit 21 or 28 days after treatment began. Irrespective of pattern, declines in haematocrit deficit from <30 % were mono-exponential, with similar half-times for AA- and AL-treated children (1.32 d versus 1.14 d). Anaemia half-time correlated significantly positively with anaemia recovery time in the same patients (r = 0.55, P < 0.0001). Bland-Altman analysis of 9 or 10 multiples of anaemia half-time and anaemia recovery times showed narrow limit of agreement with insignificant biases (P = 0.19 or 0.63, respectively). CONCLUSIONS: In uncomplicated falciparum malaria, increases or falls in haematocrit are common following ACTs. Falls in haematocrit ≥ 5 units are common and may or may not result in early or late anaemia. In children who recovered from acute falciparum malaria-associated anaemia following ACTs, decline in haematocrit deficit is mono-exponential. TRIALS REGISTRATION: Pan African Clinical Trial Registry PACTR201508001188143 , 3 July 2015; PACTR201508001191898 , 7 July 2015 http://www.pactr.org .

Interaction between rifampicin, amodiaquine and artemether in mice infected with chloroquine resistant Plasmodium berghei.

BACKGROUND: Artemisinin-based combination therapy (ACT) remains the most effective chemotherapeutic strategy in the management of malaria. However, reports of reduced susceptibility of Plasmodium falciparum to the ACT justify the need for continued search for alternative anti-malarial drugs. The use of antibiotics with anti-malarial properties represents a potentially valuable chemotherapeutic option for the management of drug resistant infections. Thus, the intrinsic anti-malarial activity of the combination of clinical doses of rifampicin with amodiaquine and artemether was evaluated in an animal model using Plasmodium berghei. METHODS: A modification of the suppressive tests in vivo was employed. The anti-malarial activity of standard doses of amodiaquine (AQ) with or without artemether (ART) and combined with varying doses of rifampicin (RIF 15 mg/kg or RIF 30 mg/kg body weight) was evaluated in 40 mice sub-divided into eight groups and inoculated intraperitoneally with 1 × 10(7) red blood cells infected with chloroquine-resistant P. berghei ANKA strain. There were two control groups of animals, one group received amodiaquine alone while the other group received saline. Parasiticidal activity and survival of the animals were assessed over 21 days. RESULTS: Parasitaemia in the control animals peaked at 38% on day 9 and all animals died by day 10. The combination of amodiaquine with rifampicin 15 mg/kg body weight was the most effective of all the combinations and more efficacious than amodiaquine alone. The order of superiority of anti-malarial efficacy of the combinations was as follows; AQ + RIF 15 > AQ > AQ + ART + RIF 30 > AQ + ART + RIF 15 > AQ +RIF 30. CONCLUSION: The combination of the clinical dose of rifampicin (15 mg/kg) with amodiaquine represents a potentially valuable treatment option in management of drug resistant malaria. In addition, the role of pharmacokinetic interaction in multiple drug therapy cannot be over-emphasized.

Fall in hematocrit per 1000 parasites cleared from peripheral blood: a simple method for estimating drug-related fall in hematocrit after treatment of malaria infections.

A simple method to estimate antimalarial drug-related fall in hematocrit (FIH) after treatment of Plasmodium falciparum infections in the field is described. The method involves numeric estimation of the relative difference in hematocrit at baseline (pretreatment) and the first 1 or 2 days after treatment begun as numerator and the corresponding relative difference in parasitemia as the denominator, and expressing it per 1000 parasites cleared from peripheral blood. Using the method showed that FIH/1000 parasites cleared from peripheral blood (cpb) at 24 or 48 hours were similar in artemether-lumefantrine and artesunate-amodiaquine-treated children (0.09; 95% confidence interval, 0.052-0.138 vs 0.10; 95% confidence interval, 0.069-0.139%; P = 0.75) FIH/1000 parasites cpb in patients with higher parasitemias were significantly (P < 0.0001) and five- to 10-fold lower than in patients with lower parasitemias suggesting conservation of hematocrit or red cells in patients with higher parasitemias treated with artesunate-amodiaquine or artemether-lumefantrine. FIH/1000 parasites cpb were similar in anemic and nonanemic children. Estimation of FIH/1000 parasites cpb is simple, allows estimation of relatively conserved hematocrit during treatment, and can be used in both observational studies and clinical trials involving antimalarial drugs.

Therapeutic efficacy of artesunate-amodiaquine combinations and the plasma and saliva concentrations of desethylamodiaquine in children with acute uncomplicated Plasmodium falciparum malaria.

The treatment efficacy of artesunate-amodiaquine (AQ) coformulated or copackaged, and the plasma and saliva concentrations of desethylamodiaquine (DEAQ), the active metabolite of AQ, were evaluated in 120 and 7 children, respectively, with uncomplicated Plasmodium falciparum malaria treated with oral daily doses of the 2 formulations for 3 days. All children recovered clinically. Fever clearance (1.1 ± 0.2 vs 1.0 ± 0 days) and parasite clearance times (21.1 ± 10.2 vs 19.0 ± 7.0 hours) in artesunate-AQ coformulated and artesunate-AQ copackaged treated children, respectively, were similar. All children remained aparasitemic for at least 28 days. Blood and saliva samples were collected over 35 days and DEAQ in plasma and saliva was determined by high-performance liquid chromatography. DEAQ was detectable in plasma and saliva within 40 minutes of oral administration of artesunate-AQ. DEAQ concentrations 7 days after the start of therapy were 247.8 and 125.1 ng/mL in plasma and saliva, respectively. The concentration-time curves of plasma and saliva in declining phases were approximately parallel giving a similar half-life of 169.1 ± 16.4 and 142.8 ± 6.5 hours in plasma and saliva, respectively. Clearance from plasma and saliva was also similar (335.6 and 443.4 mL·h·kg, respectively). Area under concentration-time curves (AUC0-35d) for plasma and saliva were 94,744.9 and 74,004.2 ng·mL·h, respectively. In general, Saliva-plasma concentration ratio was 0.25-0.4. DEAQ concentrations in saliva may be useful for monitoring therapy and for the evaluation of the disposition of AQ in children with falciparum malaria treated with AQ-based combination.

In vitro-reduced susceptibility to artemether in P. falciparum and its association with polymorphisms on transporter genes.

Plasmodium falciparum with reduced sensitivity to artemisinin derivatives has been observed in endemic areas, but the molecular mechanisms for this reduced sensitivity remain unclear. We evaluated the association between in vitro susceptibility of P. falciparum isolates obtained from southwest Nigeria and polymorphisms in selected putative transporter genes (PFE0775C, PF13_0271, pfmrp1, pfcrt, and pfmdr1). Modified schizont inhibition assay was used to determine the in vitro parasite susceptibility to artemether (ATH). Polymorphisms in selected genes were detected by polymerase chain reaction followed by direct DNA sequencing. The half-maximal inhibitory concentration (IC(50)) geometric mean (GM) for all P. falciparum isolates was 1.78 nM (range, 0.03-10.43 nM). Polymorphisms at codons 241, 86, and 76 of PFE0775C, pfmdr1, and pfcrt genes, respectively, were associated with reduced susceptibility to ATH. A new S263P single-nucleotide polymorphism on the PFE0775C gene was also detected in 27% of the isolates. Patient isolates harboring V241L or S263P polymorphisms on the PFE0775C gene showed increased IC(50) (GM: 3.08 nM and 1.79 nM, respectively). Plasmodium falciparum isolates harboring mutant Y86 pfmdr1 and P263 PFE0775C alleles showed a 2.5-5.5-fold increase in ATH IC(50.) This study shows that polymorphisms on the PFE0775C and pfmdr1 genes are associated with reduced sensitivity to ATH in fresh isolates of P. falciparum from Nigeria.

A simple dose regimen of artesunate and amodiaquine based on age or body weight range for uncomplicated falciparum malaria in children: comparison of therapeutic efficacy with standard dose regimen of artesunate and amodiaquine and artemether-lumefantrine.

A new dose regimen of artesunate and amodiaquine (NDRAA) based on age or body weight range was compared with standard dose regimen of artesunate and amodiaquine (SDRAA) calculated according to body weight and with fixed-dose artesunate-amodiaquine (FDAA) and artemether-lumefantrine (AL) in 304 children afflicted by malaria aged 15 years or younger. In initial comparison (n = 208), children on NDRAA received 1-3 times amodiaquine per kilogram of body weight and 1-1.5 times of artesunate per kilogram of body weight compared with those receiving SDRAA. Parasite but not fever clearance was significantly faster in children who received NDRAA (19.4 ± 8.4 hours vs. 24.6 ± 15.5 hours, P = 0.003). Polymerase chain reaction-uncorrected cure rates on days 28-42 were also significantly higher in children who received NDRAA (P < 0.02 in all cases). Therapeutic responses in children younger than 5 years (n = 96) treated with NDRAA, FDAA, and AL were similar. Changes in hematocrit values and reported adverse events after commencing therapy were similar in those who received NDRAA and SDRAA. All drug regimens were well tolerated. NDRAA based on age or body weight range is simple, is therapeutically superior to SDRAA calculated according to body weight, and is as efficacious as AL in children younger than 5 years.

Oral artesunate-amodiaquine and artemether-lumefantrine in the treatment of uncomplicated hyperparasitaemic Plasmodium falciparum malaria in children.

The therapeutic efficacy, changes in haematocrit and declines in parasitaemias were evaluated in 56 children with uncomplicated falciparum hyperparasitaemia after oral artesunate-amodiaquine or artemether-lumefantrine. All children recovered clinically within 2 days and without progression to severe malaria. Falls in haematocrit in the first 3 days after treatment began were similar and <5%. Declines in parasitaemias were monoexponential with both treatments with an estimated half-life of 1 h.

A simple dose regimen of artesunate and amodiaquine based on arm span- or age range for childhood falciparum malaria: a preliminary evaluation.

A dose regimen of artesunate and amodiaquine based on arm span- or age range (DRAAAS), derived from a study of 1674 children, was compared with standard dose regimen of the same drugs calculated according to body weight (SDRAA) in 68 malarious children. Children on DRAAAS received 0.8-1.0 of artesunate/kg and 0.9-1.2 times amodiaquine/kg compared with those receiving SDRAA. Parasite and fever clearance and fall in hematocrit in the first 3 days were similar; both regimens were well tolerated. DRAAAS is simple and is efficacious.

Risk factors for Plasmodium falciparum hyperparasitaemia in malarious children.

BACKGROUND: Hyperparasitaemia is a feature of childhood severe malaria but there is little information on the risk factors for hyperparasitaemia in malarious children METHODS: The risk factors associated with Plasmodium falciparum hyperparasitaemia, defined as asexual parasitaemia > 250,000/µl, at presentation were evaluated in 3338 malarious children enrolled prospectively between 2008 and 2010 in an endemic area of southwestern Nigeria. RESULTS: At enrolment, 97 (3%) of 3338 malarious children had hyperparasitaemia. In a multiple regression model, 3 factors were found to be independent risk factors for the presence of hyperparasitaemia at enrolment: an age ≤ 11 years (Adjusted odds ratio [AOR] = 2.85, 95% confidence interval [CI] 1.23-6.61, P = 0.014), fever (AOR = 2.02, 95% CI 1.23-3.29, P = 0.005), and enrolment after year 2008 (AOR = 0.42, 95% CI 0.24-0.73, P = 0.002). Duration of illness ≤ 3 d was associated with increased risk of hyperparasitaemia. There was no association between season and hyperparasitaemia. Compared to non-hyperparasitaemia, hyperparasitaemia was associated with an increased risk of progression to cerebral malaria (P < 0.0001). The risk of progression in hyperparasitaemic children was higher in < 5-year olds (P = 0.02). CONCLUSION: Young age and presence of fever are independent risk factors for hyperparasitaemia which is associated with an increased risk of progression to cerebral malaria. The findings have implications for case and community management of childhood hyperparasitaemia and for malaria control efforts in sub-Saharan Africa where severe malaria is relatively common.

Use of area under the curve to evaluate the effects of antimalarial drugs on malaria-associated anemia after treatment.

To evaluate the effects of antimalarial drugs on Plasmodium falciparum malaria-associated anemia, we use the area under the curve (AUC) of anemia levels after treatment as an approach to combine their duration and magnitude. The method involves numeric estimation, by trapezoidal rule, of AUC from a plot of deficit in hematocrit levels from 30% (the lower threshold of normal) versus time in anemic children. Using the method, we evaluated, in randomized trials, the effects of artesunate-mefloquine versus mefloquine alone and artemether-lumefantrine versus amodiaquine-artesunate on the time course of recovery from malaria-associated anemia in 109 children. Anemia resolution times were similar (10.9 ± 6.2 [standard deviation] versus 13.3 ± 8.9 days, P = 0.2), but mean AUC was significantly lower in artesunate-mefloquine- compared with mefloquine-treated children (35.5 ± 7.1 [standard error of mean] versus 49.8 ± 11.3 %·h, P = 0.02) indicating larger exposure to anemia in mefloquine-treated children. In artemether-lumefantrine- and amodiaquine-artesunate-treated children, both anemia resolution times (8.6 ± 5.3 [standard deviation] versus 8.6 ± 4.8 days, P = 0.98) and mean AUC (57.1 ± 12.9 [standard error of mean] versus 46.3 ± 8.7 %·h, P = 0.74) were similar. Estimation of AUC appears more robust than estimation of anemia resolution time in evaluating antimalarial drug effects and can be used in both observational studies and clinical trials assessing the effects of therapies on malaria-associated anemia.

Risk factors for gametocyte carriage in uncomplicated falciparum malaria in children before and after artemisinin-based combination treatments.

BACKGROUND: Artemisinin-based combination treatments (ACTs) are the recommended first-line antimalarials globally, but their influence on the risk factors associated with gametocyte carriage has had little evaluation in endemic areas. METHODS: The risk factors associated with gametocytaemia at presentation and after ACTs were evaluated in 835 children assigned to artesunate, artesunate-amodiaquine, artesunate-mefloquine or artemether-lumefantrine. RESULTS: Gametocyte carriage at enrolment was 8.4%. During follow-up, 24 patients (2.8%) developed gametocytaemia, which in 83% (20 patients) had developed by day 7 following treatment. In a multiple regression model, 2 factors were independent risk factors for the presence of gametocytaemia at enrolment, namely age <3 years (adjusted odds ratio 2.03, 95% confidence interval 1.01-4.05; p = 0.04) and enrolment before 2009 (adjusted odds ratio 4.2, 95% confidence interval 2.09-8.44; p < 0.001). Haematocrit <25% and parasitaemia <50,000/µl blood were associated with an increased risk of gametocytaemia. Following treatment, 3 factors were independent risk factors for gametocytaemia, namely gametocytaemia at enrolment (adjusted odds ratio 46.39, 95% confidence interval 22.3-96.46; p < 0.0001) and treatment with artesunate (adjusted odds ratio 6.74, 95% confidence interval 1.79-25.27; p = 0.005) or artesunate-mefloquine (adjusted odds ratio 9.66, 95% confidence interval 2.87-32.46; p < 0.0.0001) relative to other ACTs. CONCLUSION: ACTs modified the risk factors associated with gametocyte carriage after use.

Plasmodium falciparum gametocyte carriage, sex ratios and asexual parasite rates in Nigerian children before and after a treatment protocol policy change instituting the use of artemisinin-based combination therapies.

The effects of artemisinin-based combination therapies (ACTs) on transmission of Plasmodium falciparum were evaluated after a policy change instituting the use of ACTs in an endemic area. P. falciparum gametocyte carriage, sex ratios and inbreeding rates were examined in 2,585 children at presentation with acute falciparum malaria during a 10-year period from 2001-2010. Asexual parasite rates were also evaluated from 2003-2010 in 10,615 children before and after the policy change. Gametocyte carriage declined significantly from 12.4% in 2001 to 3.6% in 2010 (χ2 for trend = 44.3, p < 0.0001), but sex ratios and inbreeding rates remained unchanged. Additionally, overall parasite rates remained unchanged before and after the policy change (47.2% vs. 45.4%), but these rates declined significantly from 2003-2010 (χ2 for trend 35.4, p < 0.0001). Chloroquine (CQ) and artemether-lumefantrine (AL) were used as prototype drugs before and after the policy change, respectively. AL significantly shortened the duration of male gametocyte carriage in individual patients after treatment began compared with CQ (log rank statistic = 7.92, p = 0.005). ACTs reduced the rate of gametocyte carriage in children with acute falciparum infections at presentation and shortened the duration of male gametocyte carriage after treatment. However, parasite population sex ratios, inbreeding rates and overall parasite rate were unaffected.

Plasmodium falciparum gametocyte carriage, emergence, clearance and population sex ratios in anaemic and non-anaemic malarious children.

Anaemia in falciparum malaria is associated with an increased risk of gametocyte carriage, but its effects on transmission have not been extensively evaluated in malarious children. Plasmodium falciparum gametocyte carriage, emergence, clearance, population sex ratios (SR) (defined as the proportion of gametocytes that are male), inbreeding rates and temporal changes in SR were evaluated in 840 malarious children. Gametocyte carriage pre-treatment was at a level of 8.1%. Anaemia at enrolment was an independent risk factor for gametocyte carriage post-treatment. The emergence of gametocytes seven days post-treatment was significantly more frequent in anaemic children (7/106 vs. 10/696, p = 0.002). In the initially detected gametocytes, the proportion of children with a male-biased SR (MBSR) (> 0.5) was significantly higher in anaemic children (6/7 vs. 3/10, p = 0.027). Pre-treatment SR and estimated inbreeding rates (proportion of a mother's daughters fertilised by her sons) were similar in anaemic and non-anaemic children. Pre-treatment SR became more female-biased in non-anaemic children following treatment. However, in anaemic children, SR became male-biased. Anaemia was shown to significantly increase gametocyte emergence and may significantly alter the SR of emerging gametocytes. If MBSR is more infective to mosquitoes at low gametocytaemia, then these findings may have significant implications for malaria control efforts in endemic settings where malaria-associated anaemia is common.

In vitro antiplasmodial activity and toxicity assessment of some plants from Nigerian ethnomedicine.

CONTEXT: The emergence and spread of Plasmodium falciparum-resistant parasites to nearly all available antimalarial drugs pose a threat to malaria control and necessitates the need to continue the search for new effective and affordable drugs. Ethnomedicine has been shown to be a potential source of antimalarial compounds or source of template for the synthesis of novel antimalarial molecules. OBJECTIVE: The antiplasmodial activity and toxicity assessment of 30 plant extracts from eight medicinal plants identified in Nigerian ethnomedicine for the treatment of febrile illnesses were evaluated. MATERIALS AND METHODS: In vitro antimalarial activity was evaluated using Plasmodium falciparum NF54 (sensitive to all antimalarial drugs) and K1 (chloroquine/pyrimethamine resistant) strains in the [(3)H]-hypoxanthine incorporation assay. Toxicity was determined against mammalian L6 cells using Alamar blue assay. RESULTS: The ethyl acetate extract of leaves of Ocimum gratissimum Linn. (Labiatae) and hexane extract of stem bark of Trema orientalis (L.) Blume (Ulmaceae) showed the highest antiplasmodial activity (IC(50) 1.8-1.93 µg/mL) against P. falciparum K1 strain but elicited low cytotoxicity (selective index >10). However, hexane, ethyl acetate or methanol extracts of leaves of Terminalia catappa Linn. (Combretaceae), Jatropha curcas Linn. (Euphorbiaceae), Vitex doniana Sweet. (Verbenaceae) and stem bark of Vitex doniana displayed antiplasmodial activity (IC(50) 2.3-16.9 µg/mL) with good selectivity (21-120) for malaria parasites. DISCUSSION AND CONCLUSION: The antiplasmodial activity of Terminalia catappa and Vitex doniana against P. falciparum K1 is being reported for the first time in Nigerian ethnomedicine and these plants could be potential source of antimalarial agents.

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique.

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.