BackgroundAn accurate diagnosis is essential for the rapid and appropriate treatment of malaria. The accuracy of the histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT) Palutop+4® was assessed here. One possible factor contributing to the failure to detect malaria by this test is the diversity of the parasite PfHRP2 antigens.MethodsPfHRP2 detection with the Palutop+4® RDT was carried out. The pfhrp2 and pfhrp3 genes were amplified and sequenced from 136 isolates of Plasmodium falciparum that were collected in Dakar, Senegal from 2009 to 2011. The DNA sequences were determined and statistical analyses of the variation observed between these two genes were conducted. The potential impact of PfHRP2 and PfHRP3 sequence variation on malaria diagnosis was examined.ResultsSeven P. falciparum isolates (5.9% of the total isolates, regardless of the parasitaemia; 10.7% of the isolates with parasitaemia ≤0.005% or ≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT. Low parasite density is not sufficient to explain the PfHRP2 detection failure. Three of these seven samples showed pfhrp2 deletion (2.4%). The pfhrp3 gene was deleted in 12.8%. Of the 122 PfHRP2 sequences, 120 unique sequences were identified. Of the 109 PfHRP3 sequences, 64 unique sequences were identified. Using the Baker’s regression model, at least 7.4% of the P. falciparum isolates in Dakar were likely to be undetected by PfHRP2 at a parasite density of ≤250 parasites/μl (slightly lower than the evaluated prevalence of 10.7%). This predictive prevalence increased significantly between 2009 and 2011 (P = 0.0046).ConclusionIn the present work, 10.7% of the isolates with a parasitaemia ≤0.005% (≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT (7.4% by the predictive Baker’model). In addition, all of the parasites with pfhrp2 deletion (2.4% of the total samples) and 2.1% of the parasites with parasitaemia >0.005% and presence of pfhrp2 were not detected by PfHRP2 RDT. PfHRP2 is highly polymorphic in Senegal. Efforts should be made to more accurately determine the prevalence of non-sensitive parasites to pfHRP2.

The genetic variability and population structure of Plasmodium falciparum are key factors in malaria control strategies. Studies have suggested no P. falciparum population structure although linkage disequilibrium was observed in some African areas. We have assessed length polymorphism at 6-22 microsatellites in four urban and rural sites (Djibouti, Dakar, Niamey, and Zouan-Hounien, n = 240 blood samples). Results have shown a P. falciparum population structure in Africa (Fst = 0.17-0.24), lower genetic diversity in Djibouti (He = 0.53) than in the other sites (He = 0.73-0.76), and 3) significant linkage disequilibrium in Djibouti. These results could be related to geographic isolation and low flow of parasites between sites. They also suggest a potential effect of rural suburbs to generate genetic diversity in towns. This could affect the dispersal of selected drug resistance and should be considered when adapting urban malaria control strategies.

The chemosusceptibility and genetic polymorphism of Plasmodium falciparum populations from 48 patients hospitalized for malaria at the Hospital Principal in Dakar, Senegal were investigated during the 2002 malaria transmission season. Sixty-two percent of the isolates collected were from patients with severe malaria and 38% were from patients with mild malaria. In vitro activities of chloroquine, quinine, cycloguanil, atovaquone, mefloquine, halofantrine, and artesunate were evaluated. The prevalence of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) and dihyropteroate synthetase (dhps) genes and the P. falciparum chloroquine resistance transporter (Pfcrt) gene associated with cycloguanil, pyrimethamine, sulfadoxine, and chloroquine resistance were estimated. The genetic polymorphism of the parasite populations was evaluated by analysis of the highly polymorphic regions of merozoite surface protein 1 (msp1) block 2 and msp2. Seventy percent of the isolates were assessed by an in vitro assay. Fifty-two percent of the isolates were chloroquine resistant, 45% were cycloguanil resistant, and 24% were atovaquone resistant. Four percent had low susceptibility to quinine. The Pfcrt and dhfr mutations were associated with in vitro chloroquine- and antimetabolic drug-resistant isolates, respectively. Approximately 70% of the isolates contained two or more clones. Genetic diversity of P. falciparum was high. The prevalence of allelic family K1 of msp1 was 68%. Isolates of P. falciparum were highly resistant to chloroquine, cycloguanil and atovaquone. The transmission rate of malaria in Dakar is low but a high degree of genetic polymorphism can increase severe malaria, as shown by persons coming to Dakar from areas highly endemic for malaria. Areas with urban malaria should use vector control measures and efficient chemoprophylaxis for non-immune populations.

BackgroundAs a result of widespread chloroquine and sulphadoxine-pyrimethamine resistance, artemisinin-based combination therapy (ACT) (which includes artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Since then, there have been very few reports on the ex vivo susceptibility of Plasmodium falciparum to anti-malarial drugs. To examine whether parasite susceptibility has been affected by the widespread use of ACT, the ex vivo susceptibility of local isolates was assessed at the military hospital of Dakar.MethodsThe ex vivo susceptibility of 93 P. falciparum isolates from Dakar was successfully determined using the Plasmodium lactate dehydrogenase (pLDH) ELISA for the following drugs: chloroquine (CQ), quinine (QN), mefloquine (MQ), monodesethylamodiaquine (MDAQ), lumefantrine (LMF), dihydroartemisinin (DHA) and doxycycline (DOX).ResultsAfter transformation of the isolate IC50 in ratio of IC50 according to the susceptibility of the 3D7 reference strain (isolate IC50/3D7 IC50), the prevalence of the in vitro resistant isolates with reduced susceptibility was 50% for MQ, 22% for CQ, 12% for DOX, 6% for both QN and MDAQ and 1% for the drugs LMF and DHA. The highest significant positive correlations were shown between responses to CQ and MDAQ (r = 0.569; P < 0.0001), LMF and QN (r = 0.511; P < 0.0001), LMF and DHA (r = 0.428; P = 0.0001), LMF and MQ (r = 0.413; P = 0.0002), QN and DHA (r = 0.402; P = 0.0003) and QN and MQ (r = 0.421; P = 0.0001).ConclusionsThe introduction of ACT in 2002 has not induced a decrease in P. falciparum susceptibility to the drugs DHA, MDAQ and LMF, which are common ACT components. However, the prevalence of P. falciparum isolates with reduced susceptibility has increased for both MQ and DOX. Taken together, these data suggest that intensive surveillance of the P. falciparum in vitro susceptibility to anti-malarial drugs in Senegal is required.

BackgroundThe emergence of Plasmodium falciparum resistance to artemisinin and its derivatives, manifested as delayed parasite clearance following the treatment, has developed in Southeast Asia. The spread of resistance to artemisinin from Asia to Africa may be catastrophic for malaria control and elimination worldwide. Recently, mutations in the propeller domain of the Kelch 13 (k13) gene (PF3D71343700) were associated with in vitro resistance to artemisinin and with delayed clearance after artemisinin treatment in southern Asia. The aim of the study was to characterize the genetic variability of k13 and to evaluate the molecular resistance to artemisinin for the first time in Senegal.MethodsPlasmodium falciparum isolates were collected from 138 malaria patients in Dakar and its districts during the rainy season of October 2012 to January 2013 at the Hôpital Principal de Dakar. The k13 gene was amplified using nested PCR and sequenced.ResultsA very limited variability within the k13 gene in Senegalese P. falciparum isolates was identified. No polymorphism was detected in the six k13-propeller blades. Only two mutations, T149S (6.3%) and K189T (42.2%), and one (N) or two (NN) asparagine insertion at the codon 142 (4.7 and 6.3%, respectively) were detected in the Plasmodium/Apicomplexa-specific domain. None of the polymorphisms associated with artemisinin resistance in Southeast Asia was detected in the 138 P. falciparum from Dakar.DiscussionThe present data do not suggest widespread artemisinin resistance in Dakar in 2012–2013. Notably, the C580Y, R539T or Y493H substitutions that were associated with in vitro resistance or delayed parasite clearance in Southeast Asia were not observed in Dakar, nor were any of the polymorphisms observed in parasites from Southeast Asia, nor the M476I mutation that was selected in vitro with artemisinin pressure in a African parasite line.