Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

The estimated burden of fungal diseases in Mali.

Mali is a developing country facing several health challenges with a high rate of tuberculosis (TB) and a moderate HIV infection burden. Little is known or done about fungal diseases, yet they represent a significant public health problem in certain populations. The aim of this study was to estimate the national burden of fungal disease, and summarize data, diagnostic and treatment gaps. We used national demographics and PubMed searches to retrieve articles on published data on these infections and at-risk populations (pulmonary TB, HIV/AIDS patients, patients receiving critical care etc.) in Mali. The estimated Malian population was 21,251,000 in 2020 (UN), of which 45% were children <14 years. Among HIV patients, we estimate an annual incidence of 611 cryptococcosis, 1393 Pneumocystis pneumonia, 180 histoplasmosis and >5,700 esophageal candidiasis and some microsporidiosis cases. Our prevalence estimates for tinea capitis are 2.3 million, for recurrent vulvovaginal candidiasis 272,460, ∼60,000 fungal asthma and 7,290 cases of chronic pulmonary aspergillosis (often mistaken for TB). Less common acute fungal infections are probably invasive aspergillosis (n=1230), fungal keratitis (n=2820), candidaemia (>1,060) and mucormycosis (n=43). Histoplasmin was found in 6% in general population. A few cases of mycetoma are described in Mali. Many WHO Essential medicines and Diagnostics are not available in Mali. This shows a marked disparity in documented and estimated cases of fungal diseases in Mali. These infections are underestimated due to the lack of accurate diagnosis tools and lack of support for fungal diseases diagnosis and management.

A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Persistent Submicroscopic Plasmodium falciparum Parasitemia 72 Hours after Treatment with Artemether-Lumefantrine Predicts 42-Day Treatment Failure in Mali and Burkina Faso.

A recent randomized controlled trial, the WANECAM (West African Network for Clinical Trials of Antimalarial Drugs) trial, conducted at seven centers in West Africa, found that artemether-lumefantrine, artesunate-amodiaquine, pyronaridine-artesunate, and dihydroartemisinin-piperaquine all displayed good efficacy. However, artemether-lumefantrine was associated with a shorter interval between clinical episodes than the other regimens. In a further comparison of these therapies, we identified cases of persisting submicroscopic parasitemia by quantitative PCR (qPCR) at 72 h posttreatment among WANECAM participants from 5 sites in Mali and Burkina Faso, and we compared treatment outcomes for this group to those with complete parasite clearance by 72 h. Among 552 evaluable patients, 17.7% had qPCR-detectable parasitemia at 72 h during their first treatment episode. This proportion varied among sites, reflecting differences in malaria transmission intensity, but did not differ among pooled drug treatment groups. However, patients who received artemether-lumefantrine and were qPCR positive at 72 h were significantly more likely to have microscopically detectable recurrent Plasmodium falciparum parasitemia by day 42 than those receiving other regimens and experienced, on average, a shorter interval before the next clinical episode. Haplotypes of pfcrt and pfmdr1 were also evaluated in persisting parasites. These data identify a possible threat to the parasitological efficacy of artemether-lumefantrine in West Africa, over a decade since it was first introduced on a large scale.

Ex-vivo Sensitivity of Plasmodium falciparum to Common Anti-malarial Drugs: The Case of Kéniéroba, a Malaria Endemic Village in Mali.

BACKGROUND: In 2006, the National Malaria Control Program in Mali recommended artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. Since the introduction of artemisinin-based combination therapy, few reports are available on the level of resistance of Plasmodium falciparum to the most common anti-malarial drugs in Mali. METHODS: From 2016 to 2017, we assessed the ex-vivo drug sensitivity of P. falciparum isolates in Kéniéroba, a village located in a rural area of southern Mali. We collected P. falciparum isolates from malaria-infected children living in Kéniéroba. The isolates were tested for ex-vivo sensitivity to commonly used anti-malarial drugs, namely chloroquine, quinine, amodiaquine, mefloquine, lumefantrine, dihydroartermisinin, and piperaquine. We used the 50% inhibitory concentration determination method, which is based on the incorporation of SYBR® Green into the parasite's genetic material. RESULTS: Plasmodium falciparum isolates were found to have a reduced ex-vivo sensitivity to quinine (25.7%), chloroquine (12.2%), amodiaquine (2.7%), and mefloquine (1.3%). In contrast, the isolates were 100% sensitive to lumefantrine, dihydroartermisinin, and piperaquine. A statistically significant correlation was found between 50% inhibitory concentration values of quinine and amodiaquine (r = 0.80; p < 0.0001). CONCLUSIONS: Plasmodium falciparum isolates were highly sensitive to dihydroartermisinin, lumefantrine, and piperaquine and less sensitive to amodiaquine (n = 2), mefloquine (n = 1), and quinine (n = 19). Therefore, our data support the previously reported increasing trend in chloroquine sensitivity in Mali.

A randomized trial of dihydroartemisinin-piperaquine versus artemether-lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Mali.

BACKGROUND: Artemether-lumefantrine (AL) and artesunate-amodiaquine are first-line treatment for uncomplicated malaria in many endemic countries, including Mali. Dihydroartemisinin-piperaquine (DHA-PQ) is also an alternative first-line artemisinin-based combination therapy, but only few data are available on DHA-PQ efficacy in sub-Saharan Africa. The main aim of this study was to compare clinical efficacy of DHA-PQ versus AL, using the World Health Organization (WHO) 42-day in vivo protocol. METHODS: The efficacy of three-dose regimens of DHA-PQ was compared to AL combination in a randomized, comparative open label trial using the WHO 42-day follow-up protocol from 2013 to 2015 in Doneguebougou and Torodo, Mali. The primary endpoint was to access the PCR-corrected Adequate Clinical and Parasitological Responses at day 28. RESULTS: A total of 317 uncomplicated malaria patients were enrolled, with 159 in DHA-PQ arm and 158 in AL arm. The parasite positivity rate decreased from 68.4% (95% CI 60.5-75.5) on day 1 to 3.8% (95% CI 1.4-8.1) on day 2 for DHA-PQ and 79.8% (95% CI 72.3-85.7) on day 1 to 9.5% (95% CI 5.4-15.2) on day 2 for AL, (p = 0.04). There was a significant difference in the uncorrected ACPR between DHA-PQ and AL, both at 28-day and 42-day follow-up with 97.4% (95% CI 93.5-99.3) in DHA-PQ vs 84.5% (95% CI 77.8-89.8) in AL (p < 0.001) and 94.2% (95% CI 89.3-97.3) in DHA-PQ vs 73.4% (95% CI 65.7-80.2) in AL, respectively (p < 0.001). After molecular correction, there was no significant difference in ACPRc between DHA-PQ and AL, both at the 28-day and 42-day follow-up with 99.4% (95% CI 96.5-100) in DHA-PQ versus 98.1% (95% CI 94.5-99.6) in AL (p = 0.3) and 99.3% (95% CI 96.5-100) in DHA-PQ vs 97.4% (95% CI 93.5-99.3) in AL (p = 0.2). There was no significant difference between DHA-PQ and AL in QTc prolongation 12.1% vs 7%, respectively (p = 0.4). CONCLUSION: The results showed that dihydroartemisinin-piperaquine and artemether-lumefantrine were clinically efficacious on Plasmodium falciparum parasites in Mali.

Efficacy of artesunate-amodiaquine, dihydroartemisinin-piperaquine and artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Maradi, Niger.

BACKGROUND: Malaria endemic countries need to assess efficacy of anti-malarial treatments on a regular basis. Moreover, resistance to artemisinin that is established across mainland South-East Asia represents today a major threat to global health. Monitoring the efficacy of artemisinin-based combination therapies is of paramount importance to detect as early as possible the emergence of resistance in African countries that toll the highest burden of malaria morbidity and mortality. METHODS: A WHO standard protocol was used to assess efficacy of the combinations artesunate-amodiaquine (AS-AQ Winthrop®), dihydroartemisinin-piperaquine (DHA-PPQ, Eurartesim®) and artemether-lumefantrine (AM-LM, Coartem®) taken under supervision and respecting pharmaceutical recommendations. The study enrolled for each treatment arm 212 children aged 6-59 months living in Maradi (Niger) and suffering with uncomplicated falciparum malaria. The Kaplan-Meier 42-day PCR-adjusted cure rate was the primary outcome. A standardized parasite clearance estimator was used to assess delayed parasite clearance as surrogate maker of suspected artemisinin resistance. RESULTS: No early treatment failures were found in any of the study treatment arms. The day-42 PCR-adjusted cure rate estimates were 99.5, 98.4 and 99.0% in the AS-AQ, DHA-PPQ and AM-LM arms, respectively. The reinfection rate (expressed also as Kaplan-Meier estimates) was higher in the AM-LM arm (32.4%) than in the AS-AQ (13.8%) and the DHA-PPQ arm (24.9%). The parasite clearance rate constant was 0.27, 0.26 and 0.25 per hour for AS-AQ, DHA-PPQ and AM-LM, respectively. CONCLUSIONS: All the three treatments evaluated largely meet WHO criteria (at least 95% efficacy). AS-AQ and AL-LM may continue to be used and DHA-PPQ may be also recommended as first-line treatment for uncomplicated falciparum malaria in Maradi. The parasite clearance rate were consistent with reference values indicating no suspected artemisinin resistance. Nevertheless, the monitoring of anti-malarial drug efficacy should continue. Trial registration details Registry number at ClinicalTrial.gov: NCT01755559.

Reduced ex vivo susceptibility of Plasmodium falciparum after oral artemether-lumefantrine treatment in Mali.

BACKGROUND: Artemisinin-based combination therapy is the recommended first-line treatment for uncomplicated falciparum malaria worldwide. However, recent studies conducted in Mali showed an increased frequency of recurrent parasitaemia following artemether-lumefantrine (AL) treatment. METHODS: Study samples were collected during a large WANECAM study. Ex-vivo Plasmodium falciparum sensitivity to artemether and lumefantrine was assessed using the tritiated hypoxanthine-based assay. The prevalence of molecular markers of anti-malarial drug resistance (pfcrt K76T, pfmdr1 N86Y and K13-propeller) were measured by PCR and/or sequencing. RESULTS: Overall 61 samples were successfully analysed in ex vivo studies. Mean IC50s increased significantly between baseline and recurrent parasites for both artemether (1.6 nM vs 3.2 nM, p < 0.001) and lumefantrine (1.4 nM vs 3.4 nM, p = 0.004). Wild type Pfmdr1 N86 allele was selected after treatment (71 vs 91%, 112 of 158 vs 95 of 105, p < 0.001) but not the wild type pfcrt K76 variant (23.5 vs 24.8%, 40 of 170 vs 26 of 105, p = 0.9). Three non-synonymous K13-propeller SNPs (A522C, A578S, and G638R) were found with allele frequencies <2%. CONCLUSION: Malian post-AL P. falciparum isolates were less susceptible to artemether and lumefantrine than baseline isolates.

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.

Efficacy of sulphadoxine-pyrimethamine + artesunate, sulphadoxine-pyrimethamine + amodiaquine, and sulphadoxine-pyrimethamine alone in uncomplicated falciparum malaria in Mali.

BACKGROUND: Plasmodium falciparum resistance to artemisinin has been reported in South-East Asia. Long half-life drugs are increasingly being used for malaria prevention. The potential spread of parasite resistance to these regimens is real and makes regular efficacy surveillance a priority. METHODS: From August to December 2004 and July to December 2005, a randomized open label trial of sulphadoxine-pyrimethamine (SP) + artesunate (AS) versus SP + amodiaquine (AQ), and SP alone, was conducted in two villages of Mali. PCR was used to distinguish new infections from recrudescent P. falciparum infections. Patients were followed for 28 days to assess treatment efficacy. RESULTS: Overall 912 children aged between six to 59 months, with uncomplicated P. falciparum malaria were recruited. Baseline characteristics were similar in the three treatment arms. Crude ACPRs were 94.9%; 98.6% and 93.5% for SP + AS; SP + AQ and SP alone arms respectively (SP + AS versus SP + AQ, p = 0.01; SP + AS versus SP, p = 0.5; SP + AQ versus SP, p = 0.001). After PCR adjustment, cACPRs were 99%; 100% and 97.2% for SP + AS; SP + AQ and SP alone arms, respectively (SP + AS versus SP + AQ, p = 0.25; SP + AS versus SP, p = 0.12; SP + AQ versus SP, p = 0.007). CONCLUSION: Sulphadoxine-pyrimethamine + amodiaquine therapy was as efficacious as sulphadoxine-pyrimethamine + artesunate, but more efficacious than sulphadoxine-pyrimethamine alone in the treatment of uncomplicated P. falciparum malaria in Mali.

Quinine treatment selects the pfnhe-1 ms4760-1 polymorphism in Malian patients with Falciparum malaria.

BACKGROUND: The mechanism of Plasmodium falciparum resistance to quinine is not known. In vitro quantitative trait loci mapping suggests involvement of a predicted P. falciparum sodium-hydrogen exchanger (pfnhe-1) on chromosome 13. METHODS: We conducted prospective quinine efficacy studies in 2 villages, Kollé and Faladié, Mali. Cases of clinical malaria requiring intravenous therapy were treated with standard doses of quinine and followed for 28 days. Treatment outcomes were classified using modified World Health Organization protocols. Molecular markers of parasite polymorphisms were used to distinguish recrudescent parasites from new infections. The prevalence of pfnhe-1 ms4760-1 among parasites before versus after quinine treatment was determined by direct sequencing. RESULTS: Overall, 163 patients were enrolled and successfully followed. Without molecular correction, the mean adequate clinical and parasitological response (ACPR) was 50.3% (n = 163). After polymerase chain reaction correction to account for new infections, the corrected ACPR was 100%. The prevalence of ms4760-1 increased significantly, from 26.2% (n = 107) before quinine treatment to 46.3% (n = 54) after therapy (P = .01). In a control sulfadoxine-pyrimethamine study, the prevalence of ms4760-1 was similar before and after treatment. CONCLUSIONS: This study supports a role for pfnhe-1 in decreased susceptibility of P. falciparum to quinine in the field.

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.

BACKGROUND: Intermittent 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. METHODS: To 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. RESULTS: Each 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%). CONCLUSION: This study shows no increase in the frequency of molecular markers of SP resistance in areas where IPTi with SP was implemented for one year.

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.

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.

BACKGROUND: To update the National Malaria Control Programme of Mali on the efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine in the treatment of uncomplicated falciparum malaria. METHODS: During 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). RESULTS: Day 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. CONCLUSION: In 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.

Efficacy, safety, and selection of molecular markers of drug resistance by two ACTs in Mali.

We conducted a randomized single-blinded trial comparing the efficacy and safety of artesunate (AS) + amodiaquine (AQ, 3 days) versus AS (3 days) + sulfadoxine-pyrimethamine (SP, single dose) versus AS monotherapy (5 days) in Southern Mali. Uncomplicated malaria cases were followed for 28 days. Molecular markers of drug resistance were determined. After identification of recrudescences by genotyping, both artemisinin-based combination therapies (ACTs) reached nearly 100% efficacy at Day 14 and Day 28 versus 98.3% and 96.5% for AS, respectively (P > 0.05). AS + SP significantly selected DHFR and DHPS mutations associated with sulfadoxine and pyrimethamine resistance (P < 0.001), and AS + AQ equally selected PfCRT and PfMDR1 point mutations associated with chloroquine and AQ resistance (P < 0.001). No significant adverse event attributable to any of the study drugs was found. The ACTs were efficacious and safe, but the selection of markers for resistance to the partner drugs raises concerns over their lifespan in areas of intense malaria transmission.

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.