Baseline Ex Vivo and Molecular Responses of Plasmodium falciparum Isolates to Piperaquine before Implementation of Dihydroartemisinin-Piperaquine in Senegal.

Dihydroartemisinin-piperaquine, which was registered in 2017 in Senegal, is not currently used as the first-line treatment against uncomplicated malaria. A total of 6.6% to 17.1% of P. falciparum isolates collected in Dakar in 2013 to 2015 showed ex vivo-reduced susceptibility to piperaquine. Neither the exonuclease E415G mutation nor the copy number variation of the plasmepsin II gene (Pfpm2), associated with piperaquine resistance in Cambodia, was detected in Senegalese parasites.

Low polymorphisms in pfact, pfugt and pfcarl genes in African Plasmodium falciparum isolates and absence of association with susceptibility to common anti-malarial drugs.

BACKGROUND: Resistance to all available anti-malarial drugs has emerged and spread including artemisinin derivatives and their partner drugs. Several genes involved in artemisinin and partner drugs resistance, such as pfcrt, pfmdr1, pfK13 or pfpm2, have been identified. However, these genes do not properly explain anti-malarial drug resistance, and more particularly clinical failures observed in Africa. Mutations in genes encoding for Plasmodium falciparum proteins, such as P. falciparum Acetyl-CoA transporter (PfACT), P. falciparum UDP-galactose transporter (PfUGT) and P. falciparum cyclic amine resistance locus (PfCARL) have recently been associated to resistance to imidazolopiperazines and other unrelated drugs. METHODS: Mutations on pfugt, pfact and pfcarl were characterized on 86 isolates collected in Dakar, Senegal and 173 samples collected from patients hospitalized in France after a travel in African countries from 2015 and 2016 to assess their potential association with ex vivo susceptibility to chloroquine, quinine, lumefantrine, monodesethylamodiaquine, mefloquine, dihydroartemisinin, artesunate, doxycycline, pyronaridine and piperaquine. RESULTS: No mutations were found on the genes pfugt and pfact. None of the pfcarl described mutations were identified in these samples from Africa. The K784N mutation was found in one sample and the K734M mutation was identified on 7.9% of all samples for pfcarl. The only significant differences in ex vivo susceptibility according to the K734M mutation were observed for pyronaridine for African isolates from imported malaria and for doxycycline for Senegalese parasites. CONCLUSION: No evidence was found of involvement of these genes in reduced susceptibility to standard anti-malarial drugs in African P. falciparum isolates.

Association between Polymorphisms in the Pfmdr6 Gene and Ex Vivo Susceptibility to Quinine in Plasmodium falciparum Parasites from Dakar, Senegal.

Polymorphisms and the overexpression of transporter genes, especially of the ATP-binding cassette superfamily, have been involved in antimalarial drug resistance. The objective of this study was to use 77 Senegalese Plasmodium falciparum isolates to evaluate the association between the number of Asn residues in the polymorphic microsatellite region of the Plasmodium falciparum multidrug resistance 6 gene (Pfmdr6) and the ex vivo susceptibility to antimalarials. A significant association was observed between the presence of 7 or 9 Asn repeats and reduced susceptibility to quinine.

Confirmation of Plasmodium falciparum in vitro resistance to monodesethylamodiaquine and chloroquine in Dakar, Senegal, in 2015.

BACKGROUND: In response to increasing resistance to anti-malarial drugs, Senegal adopted artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria in 2006. However, resistance of Plasmodium falciparum parasites to artemisinin derivatives, characterized by delayed parasite clearance after treatment with ACT or artesunate monotherapy, has recently emerged and rapidly spread in Southeast Asia. After 10 years of stability with rates ranging from 5.6 to 11.8%, the prevalence of parasites with reduced susceptibility in vitro to monodesethylamodiaquine, the active metabolite of an ACT partner drug, increased to 30.6% in 2014 in Dakar. Additionally, after a decrease of the in vitro chloroquine resistance in Dakar in 2009-2011, the prevalence of parasites that showed in vitro chloroquine resistance increased again to approximately 50% in Dakar since 2013. The aim of this study was to follow the evolution of the susceptibility to ACT partners and other anti-malarial drugs in 2015 in Dakar. An in vitro test is the only method currently available to provide an early indication of resistance to ACT partners. RESULTS: Thirty-two P. falciparum isolates collected in 2015 in Dakar were analysed using a standard ex vivo assay based on an HRP2 ELISA. The prevalence of P. falciparum parasites with reduced susceptibility in vitro to monodesethylamodiaquine, chloroquine, mefloquine, doxycycline and quinine was 28.1, 46.9, 45.2, 31.2 and 9.7%, respectively. None of the parasites were resistant to lumefantrine, piperaquine, pyronaridine, dihydroartemisinin and artesunate. These results confirm an increase in the reduced susceptibility to monodesethylamodiaquine observed in 2014 in Dakar and the chloroquine resistance observed in 2013. The in vitro resistance seems to be established in Dakar. Additionally, the prevalence of parasites with reduced susceptibility to doxycycline has increased two-fold compared to 2014. CONCLUSIONS: The establishment of a reduced susceptibility to monodesethylamodiaquine as well as chloroquine resistance, and the emergence of a reduced susceptibility to doxycycline are disturbing. The in vitro and in vivo surveillance of anti-malarial drugs must be implemented in Senegal.

Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014.

We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August-December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted.

Emergence of Mutations in the K13 Propeller Gene of Plasmodium falciparum Isolates from Dakar, Senegal, in 2013-2014.

The kelch 13 (K13) propeller gene is associated with artemisinin resistance. In a previous work, there were no mutations found in 138 Plasmodium falciparum isolates collected in 2012 and 2013 from patients residing in Dakar, Senegal (M. Torrentino-Madamet et al., Malar J 13:472, 2014, http://dx.doi.org/10.1186/1475-2875-13-472). However, the N554H, Q613H, and V637I mutations were identified in the propeller region of K13 in 92 (5.5%) isolates in 2013 and 2014. There were five polymorphisms identified in the Plasmodium/Apicomplexa-specific domain (K123R, N137S, N142NN/NNN, T149S, and K189T/N).

Absence of Association between Polymorphisms in the RING E3 Ubiquitin Protein Ligase Gene and Ex Vivo Susceptibility to Conventional Antimalarial Drugs in Plasmodium falciparum Isolates from Dakar, Senegal.

The RING E3 ubiquitin protein ligase is crucial for facilitating the transfer of ubiquitin. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation (62.5%) but was not significantly associated with the 50% inhibitory concentration (IC50) of conventional antimalarial drugs. However, some mutated isolates (D113N) present a trend of reduced susceptibility to piperaquine (P = 0.0938). To evaluate the association of D113N polymorphism with susceptibility to antimalarials, more isolates are necessary.

Prevalence of anti-malarial resistance genes in Dakar, Senegal from 2013 to 2014.

BACKGROUND: To determine the impact of the introduction of artemisinin-based combination therapy (ACT) on parasite susceptibility, a molecular surveillance for antimalarial drug resistance was conducted on local isolates from the Hôpital Principal de Dakar between November 2013 and January 2014 and between August 2014 and December 2014. METHODS: The prevalence of genetic polymorphisms in antimalarial resistance genes (pfcrt, pfmdr1, pfdhfr and pfdhps) was evaluated in 103 isolates. RESULTS: The chloroquine-resistant haplotypes CVIET and CVMET were identified in 31.4 and 3.9 % of the isolates, respectively. The frequency of the pfcrt K76T mutation was increased from 29.3 % in 2013-2014 to 43.2 % in 2014. The pfmdr1 N86Y and Y184F mutations were identified in 6.1 and 53.5 % of the isolates, respectively. The pfdhfr triple mutant (S108N, N51I and C59R) was detected in the majority of the isolates (82.3 %). The prevalence of quadruple mutants (pfdhfr S108N, N51I, C59R and pfdhps A437G) was 40.4 %. One isolate (1.1 %) harboured the pfdhps mutations A437G and K540E and the pfdhfr mutations S108N, N51I and C59R. CONCLUSIONS: Despite a decline in the prevalence of chloroquine resistance due to the official withdrawal of the drug and to the introduction of ACT, the spread of resistance to chloroquine has continued. Furthermore, susceptibility to amodiaquine may be decreased as a result of cross-resistance. The frequency of the pfmdr1 mutation N86Y declined while the Y184F mutation increased in prevalence, suggesting that selective pressure is acting on pfmdr1, leading to a high prevalence of mutations in these isolates and the lack of specific mutations. The 50.5 % prevalence of the pfmdr1 polymorphisms N86Y and Y184F suggests a decrease in lumefantrine susceptibility. Based on these results, intensive surveillance of ACT partner drugs must be conducted regularly in Senegal.

Comparison of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular biology techniques for identification of Culicoides (Diptera: ceratopogonidae) biting midges in senegal.

Biting midges of the genus Culicoides are implicated as vectors for a wide variety of pathogens. The morphological identification of these arthropods may be difficult because of a lack of detailed investigation of taxonomy for this species in Africa. However, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiling is efficient for arthropod identification at the species level. This study established a spectrum database of Culicoides spp. from Senegal using MALDI-TOF. Identification of Culicoides insects to the species level before mass spectrometry was performed on the basis of morphological characters. MALDI-TOF MS reference spectra were determined for 437 field-caught Culicoides of 10 species. The protein profiles of all tested Culicoides revealed several peaks with mass ranges of 2 to 20 kDa. In a validation study, 72 Culicoides specimens in the target species were correctly identified at the species level with a similarity of 95 to 99.9%. Four Culicoides protein profiles were misidentified. Nevertheless, six SuperSpectra (C. imicola, C. enderleini, C. oxystoma, C. kingi, C. magnus, and C. fulvithorax) were created. Abdomens of midges were used to amplify and sequence a portion of the mitochondrial cytochrome oxidase I gene (COI). The results obtained using the MALDI-TOF MS method were consistent with the morphological identification and similar to the genetic identification. Protein profiling using MALDI-TOF is an efficient approach for the identification of Culicoides spp., and it is economically advantageous for approaches that require detailed and quantitative information of vector species that are collected in field. The database of African Culicoides MS spectra created is the first database in Africa. The COI sequences of five Culicoides species that were previously noncharacterized using molecular methods were deposited in GenBank.

Plasmodium falciparum susceptibility to standard and potential anti-malarial drugs in Dakar, Senegal, during the 2013-2014 malaria season.

BACKGROUND: In 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 Plasmodium falciparum resistance to anti-malarial drugs. An ex vivo susceptibility study was conducted on local isolates obtained from the Hôpital Principal de Dakar (Dakar, Senegal) from November 2013 to January 2014. METHODS: Eighteen P. falciparum isolates were sussessfully assessed for ex vivo susceptibility to chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine, mefloquine (MQ), lumefantrine (LMF), artesunate (AS), dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives, pyronaridine (PND), piperaquine (PPQ), and, Proveblue (PVB), a methylene blue preparation, using the HRP2-based ELISA test. RESULTS: The prevalence of isolates with reduced susceptibility was 55.6% for MQ, 50% for CQ, 5.6% for QN and MDAQ, and 0% for DHA, AS and LMF. The mean IC50 for PND, PPQ and PVB were 5.8 nM, 32.2 nM and 5.3 nM, respectively. CONCLUSIONS: The prevalence of isolates with a reduced susceptibility to MQ remains high and stable in Dakar. Since 2004, the prevalence of CQ resistance decreased, but rebounded in 2013 in Dakar. PND, PPQ and PVB showed high in vitro activity in P. falciparum parasites from Dakar.

Usefulness of MALDI-TOF Mass Spectrometry for Routine Identification of Candida Species in a Resource-Poor Setting.

BACKGROUND: Identification of fungal clinical isolates is essential for therapeutic management. In resource-limited settings, identification mostly relies on biochemical tests whose sensitivity and specificity are known to be insufficient for identification of closely related or newly described species. MALDI-TOF has been shown in favored countries to be a reliable and powerful tool for microorganism identification, including yeasts. The aim of this study was to compare MALDI-TOF with routine identification procedures in a resource-poor context. METHODS: A total of 734 clinical specimens (502 vaginal swabs, 147 oral swabs, 61 bronchoalveolar lavage fluids and 24 stool samples) have been tested in the mycology unit of Fann Hospital, Dakar, Senegal. Strains isolated from culture were identified by both conventional phenotypic methods (germ tube formation and biochemical panels) and MALDI-TOF Saramis/VITEK MS, bioMérieux, France. In addition to comparing the final identification, we determined the time of obtaining the results and the cost for both approaches. RESULTS: Overall, 218 (29.7 %) samples were positive for Candida. MALDI-TOF MS enabled the identification of 214 of the 218 strains isolated (98.1 %) at species level. Phenotypic approach yielded identification for 208 strains (95.4 %). Congruence between the tests was observed for 203 isolates. A discrepancy was observed for one isolate identified as Candida krusei with the phenotypic approach and Candida tropicalis with the MALDI-TOF. In addition, ten isolates identified at genus level by phenotypic methods were identified as C. glabrata (n = 8), C. tropicalis (n = 1) and C. parapsilosis (n = 1) by MALDI-TOF. The turnaround time for identification was <1 h using the MALDI-TOF compared to our routine procedures (48 h). The overall cost (reagents + expendables) per isolate was at 1.35 for the MALDI-TOF MS. CONCLUSION: MALDI-TOF clearly outperformed the diagnosis capacities of phenotypic methods by reducing the delay of results and giving accurate identification at species level. Moreover, this approach appears to be cost-effective and should be implemented especially in resource-poor context.

Role of Pfmdr1 in in vitro Plasmodium falciparum susceptibility to chloroquine, quinine, monodesethylamodiaquine, mefloquine, lumefantrine, and dihydroartemisinin.

The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites.

Limited polymorphisms in k13 gene in Plasmodium falciparum isolates from Dakar, Senegal in 2012-2013.

BACKGROUND: The 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. METHODS: Plasmodium 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. RESULTS: A 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. DISCUSSION: The 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.

[Contribution of qPCR to the diagnosis of cervico-vaginal infections at the Hôpital Principal de Dakar, Senegal]. / Apport de la qPCR dans le diagnostic des infections cervicovaginales à l'Hôpital Principal de Dakar, Sénégal.

Objective: To determine the etiology of cervico-vaginal infections by cytobacteriology and the efficacy of qPCR for the diagnosis of sensitive strains such as Streptococcus agalactiae, Borrelia crocidurae, Chlamydia trachomatis, Neisseria gonorrhoeae and Treponema pallidum. Methodology: This prospective cross-sectional study was performed between January and September 2021 in 346 women who were examined for cervico-vaginal infection at the Hôpital Principal de Dakar (HPD). Cytobacteriological (direct examination, agar culture) and molecular analyses were performed. Results: Vaginal flora imbalances predominated, with a rate of 72.3%. The proportion of type IV vaginal flora was 46.5%. Of the 199 germs isolated, Candida albicans (25.1%), Ureaplasma urealyticum (17.6%), S. agalactiae (7.8%), Gardnerella vaginalis (6.6%) and nonalbicans Candida (5.5%) were the main pathogens responsible for cervico-vaginal infections in patients. Among women tested for mycoplasma, U. urealyticum was identified in 43.3% of patients. Among those tested for C. trachomatis, the proportion of infected women was low (4%). The prevalence of C. albicans was higher in pregnant women (38.3%) than in nonpregnant women (19.2%). S. agalactiae strains showed high resistance to certain beta-lactam antibiotics (pristinamycin 100%, gentamycin 100%, ampicillin 92.5% and cefalotin 85.2%) and to a glycopeptide antibiotic (vancomycin 100%). The Staphylococcus aureus strain had good sensitivity to antibiotics except gentamycin (100%) and kanamycin (100%). The enterobacteria tested were all sensitive to phenicols, carbapenems, cephalosporins and aminoglycosides. However, E. coli showed high resistance to tetracycline. The different methods showed low prevalences of C. trachomatis and N. gonorrhoeae, so comparisons Test RapidChlamydia/qPCR for C. trachomatis and culture/qPCR for N. gonorrhoeae were not possible. For S. agalactiae, on the other hand, qPCR was more advantageous than culture. The χ2 test showed a significant difference (Yates χ2 = 33.77 and p = 1-7) for the diagnosis of S. agalactiae. S. agalactiae qPCR had a sensitivity of 40.7%, a specificity of 94%, and positive and negative predictive values of 36.7% and 94.9% respectively, as well as a kappa = 0.33. Conclusion: The methods applied enabled us to identify the pathogens that cause cervicovaginal infections. The results suggest that qPCR may be an alternative, at least for the diagnosis of S. agalactiae. However, culture remains indispensable for studying antibiotic sensitivity. In order to improve patient care, molecular techniques need to be integrated into the HPD testing toolbox. To broaden the repertoire of pathogens to be diagnosed by qPCR, targeted comparison studies will be needed to increase the probability of encountering infected individuals.

Plasmodium falciparum susceptibility to anti-malarial drugs in Dakar, Senegal, in 2010: an ex vivo and drug resistance molecular markers study.

BACKGROUND: In 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). METHODS: The 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. RESULTS: The 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%. CONCLUSIONS: The 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.

Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests.

BACKGROUND: An 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. METHODS: PfHRP2 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. RESULTS: Seven 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). CONCLUSION: In 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.

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger transporter (Pfnhe-1) gene in 393 isolates from Dakar, Senegal.

BACKGROUND: Although the World Health Organization recommends replacing quinine (QN) by artesunate due to its increased efficacy and the higher tolerance to the drug in both adults and children, QN remains a first-line treatment for severe malaria, especially in Africa. Investigations of microsatellite Pfnhe-1 ms4760 polymorphisms in culture-adapted isolates from around the world have revealed that an increase in the number of DNNND amino acid motifs was associated with decreased QN susceptibility, whereas an increase in the number of DDNHNDNHNND motifs was associated with increased QN susceptibility. METHODS: In this context, to further analyse associations between Pfnhe-1 ms4760 polymorphisms and QN susceptibility, 393 isolates freshly collected between October 2009 and January 2010 and July 2010 and February 2011, respectively, at the Hôpital Principal de Dakar, Senegal were assessed ex vivo for QN susceptibility, and their genes were amplified and sequenced. RESULTS: Of the 393 Plasmodium falciparum clinical isolates collected, 145 were successfully cultured. The 145 QN IC50s ranged from 2.1 to 1291 nM, and 17 isolates (11.7%) exceed the QN reduced susceptibility threshold of 611 nM. Among the 393 P. falciparum clinical isolates, 47 different alleles were observed. The three most prevalent profiles were ms4760-1 (no = 72; 18.3%), ms4760-3 (no = 65; 16.5%) and ms4760-7 (no = 40; 10.2%). There were no significant associations observed between QN IC50 values and i) the number of repeats of DNNND in block II (p = 0.0955, Kruskal-Wallis test); ii) the number of repeats of DDNHNDNHNND in block V (p = 0.1455, Kruskal-Wallis test); or iii) ms4760 profiles (p = 0.1809, Kruskal-Wallis test). CONCLUSIONS: Pfnhe-1 ms4760 was highly diverse in parasite isolates from Dakar (47 different profiles). Three profiles (ms4760-1, ms4760-3 and ms4760-7) were predominant. The number of repeats for block II (DNNND) or block V (DDNHNDNHNND) was not significantly associated with QN susceptibility. New studies, and especially in vivo studies, are necessary to confirm the role of Pfnhe-1 ms4760 as a marker of QN resistance.

Prevalence of molecular markers of Plasmodium falciparum drug resistance in Dakar, Senegal.

BACKGROUND: As a result of the widespread resistance to chloroquine and sulphadoxine-pyrimethamine, artemisinin-based combination therapy (ACT) (including artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Intermittent preventive treatments with anti-malarial drugs based on sulphadoxine-pyrimethamine are also given to children or pregnant women once per month during the transmission season. Since 2006, there have been very few reports on the susceptibility of Plasmodium falciparum to anti-malarial drugs. To estimate the prevalence of resistance to several anti-malarial drugs since the introduction of the widespread use of ACT, the presence of molecular markers associated with resistance to chloroquine and sulphadoxine-pyrimethamine was assessed in local isolates at the military hospital of Dakar. METHODS: The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., Pfcrt, Pfdhfr, Pfdhps and Pfmdr1, and the copy number of Pfmdr1 were evaluated for a panel of 174 isolates collected from patients recruited at the military hospital of Dakar from 14 October 2009 to 19 January 2010. RESULTS: The Pfcrt 76T mutation was identified in 37.2% of the samples. The Pfmdr1 86Y and 184F mutations were found in 16.6% and 67.6% of the tested samples, respectively. Twenty-eight of the 29 isolates with the 86Y mutation were also mutated at codon 184. Only one isolate (0.6%) had two copies of Pfmdr1. The Pfdhfr 108N/T, 51I and 59R mutations were identified in 82.4%, 83.5% and 74.1% of the samples, respectively. The double mutant (108N and 51I) was detected in 83.5% of the isolates, and the triple mutant (108N, 51I and 59R) was detected in 75.3%. The Pfdhps 437G, 436F/A and 613S mutations were found in 40.2%, 35.1% and 1.8% of the samples, respectively. There was no double mutant (437G and 540E) or no 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 36.5%. CONCLUSIONS: Since 2004, the prevalence of chloroquine resistance had decreased. The prevalence of isolates with high-level pyrimethamine resistance is 83.5%. The prevalence of isolates resistant to sulphadoxine is 40.2%. However, no quintuple mutant (Pfdhfr 108N, 51I and 59R and Pfdhps 437G and 540E), which is associated with a high level of sulphadoxine-pyrimethamine resistance, has been identified to date. The resistance to amodiaquine remains moderate.

Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs.

BACKGROUND: As 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. METHODS: The 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). RESULTS: After 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). CONCLUSIONS: The 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.

Absence of association between polymorphisms in the pfcoronin and pfk13 genes and the presence of Plasmodium falciparum parasites after treatment with artemisinin derivatives in Senegal.

Due to resistance to chloroquine and sulfadoxine/pyrimethamine, treatment for uncomplicated Plasmodium falciparum malaria switched to artemisinin-based combination therapy (ACT) in 2006 in Senegal. Several mutations in the gene encoding the kelch13 helix (pfk13-propeller) have been identified as associated with in vitro and in vivo artemisinin resistance in Southeast Asia. Additionally, three mutations in the pfcoronin gene (G50E, R100K and E107V) have been identified in two culture-adapted Senegalese field isolates that became resistant in vitro to artemisinin after 4 years of intermittent selection with dihydroartemisinin. The aims of this study were to assess the prevalence of pfcoronin and pfk13 mutations in Senegalese field isolates from Dakar and to investigate their association with artemisinin derivative clinical failures. A total of 348 samples of P. falciparum from 327 patients, collected from 2015-2019 in Dakar, were successfully analysed. All sequences had wild-type pfk13 allele. The three mutations (G50E, R100K and E107V), previously identified in parasites with reduced susceptibility to artemisinin, were not found in this study, but a new mutation (P76S) was detected (mean prevalence 16.2%). The P76S mutation was identified in 5 (31.3%) of 16 isolates collected from patients still parasitaemic on Day 3 after ACT treatment and in 31 samples (15.3%) among 203 patients considered successfully cured. There was no significant association between in vivo reduced efficacy to artemisinin derivatives and the P76S mutation (P = 0.151, Fisher's exact test). These data suggest that polymorphisms in pfk13 and pfcoronin are not the best predictive markers for artemisinin resistance in Senegal.