Souleymane Dama

No Evidence of Delayed Parasite Clearance after Oral Artesunate Treatment of Uncomplicated Falciparum Malaria in Mali

Plasmodium falciparum resistance to artemisinins by delayed parasite clearance is present in Southeast Asia. Scant data on parasite clearance after artemisinins are available from Africa, where transmission is high, burden is greatest, and artemisinin use is being scaled up. Children 1-10 years of age with uncomplicated malaria were treated with 7 days of artesunate and followed for 28 days. Blood smears were done every 8 hours until negative by light microscopy. Results were compared with a similar study conducted in the same village in 2002-2004. The polymerase chain reaction-corrected cure rate was 100%, identical to 2002-2004. By 24 hours after treatment initiation, 37.0% of participants had cleared parasitemia, compared with 31.9% in 2002-2004 (P = 0.5). The median parasite clearance time was 32 hours. Only one participant still had parasites at 48 hours and no participant presented parasitemia at 72 hours. Artesunate was highly efficacious, with no evidence of delayed parasite clearance. We provide baseline surveillance data for the emergence or dissemination of P. falciparum resistance in sub-Saharan Africa.

Molecular markers of resistance to sulphadoxine-pyrimethamine one year after implementation of intermittent preventive treatment of malaria in infants in Mali.

BackgroundIntermittent preventive treatment in infants (IPTi) with sulphadoxine-pyrimethamine (SP) given during routine vaccinations is efficacious in preventing malaria disease and shows no interaction with the vaccines. However, there is a fear that IPTi may result in a rapid increase of parasite resistance to SP.MethodsTo evaluate the impact of IPTi on SP-resistance point mutations, the 22 health sub-districts in the district of Kolokani, Mali, were randomized in a 1:1 ratio and starting in December 2006, IPTi with SP was implemented in 11 health sub-districts (intervention zone), while the other 11 health sub-districts served as the control (non-intervention zone). Blood smears and blood dots on filter paper were obtained from children aged 0-5 years, randomly selected in each of heath sub-districts during two cross-sectional surveys. The first survey was conducted in May 2007 before the start of the transmission season to collect baseline prevalence of the molecular markers of resistance to SP and the second in December 2007 after the end of the transmission season and one year after implementation of IPTi. A total of 427 and 923 randomly selected blood samples from the first and second surveys respectively were analysed by PCR for dhfr and dhps mutations.ResultsEach of the three dhfr mutations at codons 51, 59 and 108 was present in 35% and 57% of the samples during the two surveys with no significant differences between the two zones. Dhps mutations at codons 437 and 540 were present respectively in about 20% and 1% of the children during the two surveys in both zones at similar proportion. The prevalence of quadruple mutants (triple dhfr-mutants + dhps-437G) associated with in-vivo resistance to SP in Mali after one year implementation of IPTi was also similar between the two zones (11.6% versus 11.2%, p = 0.90) and to those obtained at baseline survey (10.3% versus 8.1%).ConclusionThis study shows no increase in the frequency of molecular markers of SP resistance in areas where IPTi with SP was implemented for one year.

Efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria: revisiting molecular markers in an area of emerging AQ and SP resistance in Mali

BackgroundTo update the National Malaria Control Programme of Mali on the efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine in the treatment of uncomplicated falciparum malaria.MethodsDuring the malaria transmission seasons of 2002 and 2003, 455 children – between six and 59 months of age, with uncomplicated malaria in Kolle, Mali, were randomly assigned to one of three treatment arms. In vivo outcomes were assessed using WHO standard protocols. Genotyping of msp1, msp2 and CA1 polymorphisms were used to distinguish reinfection from recrudescent parasites (molecular correction).ResultsDay 28 adequate clinical and parasitological responses (ACPR) were 14.1%, 62.3% and 88.9% in 2002 and 18.2%, 60% and 85.2% in 2003 for chloroquine, amodiaquine and sulphadoxine-pyrimethamine, respectively. After molecular correction, ACPRs (cACPR) were 63.2%, 88.5% and 98.0% in 2002 and 75.5%, 85.2% and 96.6% in 2003 for CQ, AQ and SP, respectively. Amodiaquine was the most effective on fever. Amodiaquine therapy selected molecular markers for chloroquine resistance, while in the sulphadoxine-pyrimethamine arm the level of dhfr triple mutant and dhfr/dhps quadruple mutant increased from 31.5% and 3.8% in 2002 to 42.9% and 8.9% in 2003, respectively. No infection with dhps 540E was found.ConclusionIn this study, treatment with sulphadoxine-pyrimethamine emerged as the most efficacious on uncomplicated falciparum malaria followed by amodiaquine. The study demonstrated that sulphadoxine-pyrimethamine and amodiaquine were appropriate partner drugs that could be associated with artemisinin derivatives in an artemisinin-based combination therapy.

Baseline in vitro efficacy of ACT component drugs on Plasmodium falciparum clinical isolates from Mali.

In vitro susceptibility to antimalarial drugs of Malian Plasmodium falciparum isolates collected between 2004 and 2006 was studied. Susceptibility to chloroquine and to three artemisinin-based combination therapy (ACT) component drugs was assessed as a first, to our knowledge, in vitro susceptibility study in Mali. Overall 96 Malian isolates (51 from around Bamako and 45 collected from French travellers returning from Mali) were cultivated in a CO(2) incubator. Fifty percent inhibitory concentrations (IC(50)s) were measured by either hypoxanthine incorporation or Plasmodium lactate dehydrogenase (pLDH) ELISA. Although the two sets of data were generated with different methods, the global IC(50) distributions showed parallel trends. A good concordance of resistance phenotype with pfcrt 76T mutant genotype was found within the sets of clinical isolates tested. We confirm a high prevalence of P. falciparum in vitro resistance to chloroquine in Mali (60-69%). While some isolates showed IC(50)s close to the cut-off for resistance to monodesethylamodiaquine, no decreased susceptibility to dihydroartemisinin or lumefantrine was detected. This study provides baseline data for P. falciparum in vitro susceptibility to ACT component drugs in Mali.

A molecular map of chloroquine resistance in Mali

Plasmodium falciparum chloroquine resistance (CQR) transporter point mutation (PfCRT 76T) is known to be the key determinant of CQR. Molecular detection of PfCRT 76T in field samples may be used for the surveillance of CQR in malaria-endemic countries. The genotype-resistance index (GRI), which is obtained as the ratio of the prevalence of PfCRT 76T to the incidence of CQR in a clinical trial, was proposed as a simple and practical molecular-based addition to the tools currently available for monitoring CQR in the field. In order to validate the GRI model across populations, time, and resistance patterns, we compiled data from the literature and generated new data from 12 sites across Mali. We found a mean PfCRT 76T mutation prevalence of 84.5% (range 60.9-95.1%) across all sites. CQR rates predicted from the GRI model were extrapolated onto a map of Mali to show the patterns of resistance throughout the participating regions. We present a comprehensive map of CQR in Mali, which strongly supports recent changes in drug policy away from chloroquine.

Gametocyte clearance dynamics following oral artesunate treatment of uncomplicated falciparum malaria in Malian children

Artemisinin-based combination therapies decrease Plasmodium gametocyte carriage. However, the role of artesunate in monotherapy in vivo, the mechanisms involved, and the utility of gametocyte carriage as a potential tool for the surveillance of antimalarial resistance are poorly understood. In 2010–2011, we conducted an open-label, prospective efficacy study of artesunate as monotherapy in children 1–10 years of age with uncomplicated falciparum malaria in Bougoula-Hameau, Mali. Standard oral doses of artesunate were administered for 7 days and patients were followed up for 28 days. The data were compared to a similar study conducted in 2002–2004. Of 100 children enrolled in the 2010–2011 study, 92 were analyzed and compared to 217 children enrolled in the 2002–2004 study. The proportion of gametocyte carriers was unchanged at the end of treatment (23% at baseline vs. 24% on day 7, p = 1.0) and did not significantly decline until day 21 of follow-up (23% vs. 6%, p = 0.003). The mean gametocyte density at inclusion remained unchanged at the end of treatment (12 gametocytes/μL vs. 16 gametocytes/μL, p = 0.6). Overall, 46% of the 71 initial non-carriers had gametocytes detected by day 7. Similar results were found in the 2002–2004 study. In both studies, although gametocyte carriage significantly decreased by the end of the 28-day follow-up, artesunate did not clear mature gametocytes during treatment and did not prevent the appearance of new stage V gametocytes as assessed by light microscopy. Baseline gametocyte carriage was significantly higher 6 years after the deployment of artemisinin-based combination therapies in this setting.

LUMEFANTRINE DISPOSITION AFTER REPETITIVE TREATMENT OF UNCOMPLICATED MALARIA PATIENTS WITH ARTEMETHER-LUMEFANTRINE IN MALI

Background Since 2006 the national malaria control program in Mali recommended artemether-lumefantrine (AL) as the first-line treatment of uncomplicated malaria. The role of lumefantrine in this combination is to eliminate remaining parasites after the action of artemether and to protect the patient against a new blood infection. Some studies showed a correlation between lumefantrines day 7 concentration and the efficacy of AL after treatment of a single episode of malaria. The objective of this work is to validate this observation after repetitive treatment of uncomplicated malaria patients with AL. Methods During a phase IIIb/IV comparative, randomised, multicentre, clinical study of artemisinin-based combination therapies, we collected plasma on Day 7 from patients treated with standard dose of AL in Sotuba, Bougoula Hameau, and Kolle (Mali). The age of the patients enrolled in this study was from 6 months old. The plasma samples were kept at – 80°C until lumefantrine analysis using high performance liquid chromatography was performed. Results We included 1076 subjects, of which 595 were females and a mean age of 12 years old in this analysis. The median concentration was 66% higher (p<0.0001) in patients without recurrent parasite on day 28 compared to patients with recurrent parasitaemia: 509.1 ng/ml (inter quartile range: 329.6–723.2; n=919) vs 372.5 (255.7–538.4; n=157). Day 7 concentrations increased with age; the difference between age group was statistically significant: 305.9 (207.3–491.5, n=140), 447 (290.7–622.9, n=399), 544.7 (383.9–738.5, n=254) and 571.1 (378.8–850.9), n=283) in patients under 5 years old, 5–9 years old, 10–14 years old and 15 years old and older, respectively. Girls under 5 years old had a lower lumefantrine concentration at day 7 compared to other age groups of 223.3 ng/ml (159.7–425.6, n=37). Conclusions We found a lower concentration of lumefantrine in patients with recurrent parasitaemia at day 28.

EFFECT OF ARTESUNATE MONOTHERAPY ON PLASMODIUM FALCIPARUM IN VIVO GENOMIC EXPRESSION

Background Artemisinin-based combination therapies (ACTs) are the main treatment for malaria in endemic countries. Plasmodium falciparum resistance to artemisinins is described as delayed parasite clearance, which is associated with mutations on the parasite K13 propeller gene. Both the mechanisms of action and mechanisms of resistance to artemisinins are poorly understood. Transcriptomic studies can help in improving our understanding of these processes. Here we explore P. falciparum in vivo RNA expression profile after a curative dose of artesunate monotherapy. Methods During a prospective study of the efficacy of artesunate in monotherapy in children aged 1–10 years and presenting uncomplicated P. falciparum malaria in Bougoula-Hameau, Mali, venous blood was collected on PAXgen blood RNA tubes before treatment (H0) and one (H1), two (H2) and three hours (H3) after treatment. RNA was extracted from these respective blood samples and used for microarray experiments with Plasmodium/Anopheles GeneChips and the Affymetrix® platform. Results A total of 23 samples from 6 patients were included in the final analysis after quality control using Affimetrix® and Qlucore® softwares. With a 2-groups comparison of H0/H after treatment, 236 genes were identified as differentially expressed. Overall 42 genes were up-regulated including a knob-associated histidine-rich protein, rifins (pf.12.409.0, pf.13_399.0), stevors (pf.3.184.0), RESA-like proteins with DNAJ domain and thioredoxins. Heat shock protein (Pf.5.258.0), a number of AP2 domain-containing genes (Pf.6.27.0, Pf.11.99.0), an ABC transporter (Pf.12.250.0), genes involved in cell cycle regulation and many exported protein genes with unknown function and membrane proteins genes were among the 194 down-regulated genes. Conclusions Our data support a role for these genes in the in vivo response of P. falciparum to artesunate administration.