Abstract
BackgroundIn 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. Since the introduction of ACT, there have been very few reports on the level of resistance of P. falciparum to anti-malarial drugs. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility and drug resistance molecular marker study was conducted on local isolates obtained from the Centre de santé Elizabeth Diouf (Médina, Dakar, Senegal).MethodsThe prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., pfcrt, pfdhfr, pfdhps and pfmdr1, were evaluated for a panel of 165 isolates collected from patients recruited from 17 August 2010 to 6 January 2011. The malaria isolates were assessed for susceptibility to chloroquine (CQ); quinine (QN); monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine; mefloquine (MQ); lumefantrine (LMF); dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives; and doxycycline (DOX) using the Plasmodium lactate dehydrogenase (pLDH) ELISA.ResultsThe prevalence of the in vitro resistant isolates, or isolates with reduced susceptibility, was 62.1% for MQ, 24.2% for CQ, 10.3% for DOX, 11.8% MDAQ, 9.7% for QN, 2.9% for LMF and 0% for DHA. The Pfcrt 76T mutation was identified in 43.6% of the samples. The pfmdr1 86Y, 184F and 1246Y mutations were found in 16.2%, 50.0% and 1.6% of the samples, respectively. The pfdhfr 108N, 51I and 59R mutations were identified in 81.9%, 77.4% and 79.4% of the samples, respectively. The double mutant (108N and 51I) was detected in 75.5% of the isolates, and the triple mutant (108N, 51I and 59R) was detected in 73.6% of the isolates. The pfdhps 437G, 436A and 613S mutations were found in 54.4%, 38.6% and 1.2% of the samples, respectively. There was only one double mutant, 437G and 540E, and one quintuple mutant, pfdhfr 108N, 51I and 59R and pfdhps 437G and 540E. The prevalence of the quadruple mutant (pfdhfr 108N, 51I and 59R and pfdhps 437G) was 36.7%.ConclusionsThe results of this study indicate that an intensive surveillance of the in vitro P. falciparum susceptibility to anti-malarial drugs must be conducted in Senegal.
Highlights
In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria
To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility study was conducted on local isolates from Dakar obtained from the Centre de santé Elizabeth Diouf (Médina, Dakar, Senegal)
The malaria isolates were assessed for susceptibility to chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine, mefloquine (MQ), lumefantrine (LMF), dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives and doxycycline (DOX)
Summary
Plasmodium falciparum isolates In total, 165 patients (63 females, 3 to 70 years old and 102 males, 2 to 67 years old) with malaria were recruited from 17 August 2010 to 6 January 2011 at the Centre de santé Elizabeth Diouf. The reaction mixture for PCR amplifications included 2.5 μl of genomic DNA, 2.5 μl of 10X reaction buffer (Eurogentec), 0.5 μM of each primer, 200 μM of a deoxynucleoside triphosphate mixture (dGTP, dATP, dTTP and dCTP) (Euromedex, Souffelweyersheim, France), 2.5 mM MgCl2 and 1 unit of RedGoldStarW DNA polymerase (Eurogentec) in a final volume of 25 μl. The reaction mixture contained 2.5 μl of genomic DNA, 2.5 μl of 10X reaction buffer (Eurogentec), 0.5 μM each primer, 2.5 mM MgCl2, 200 μM deoxynucleoside triphosphate mixture (dGTP, dATP, dTTP and dCTP) (Euromedex, Souffelweyersheim, France) and 1 U of RedGoldStarW DNA polymerase (Eurogentec) in a final volume of 25 μl. Using the Plasmodium lactate dehydrogenase (pLDH) ELISA under Genbag conditions, the cut-off values for in vitro resistance, or reduced susceptibility, were 77 nM, 61 nM, 115 nM, 12 nM, 611 nM, 30 nM and 37 μM for CQ, MDAQ, LMF, DHA, QN, MQ and DOX, respectively [40]
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