Skip to content

Advertisement

Open Access

Ex vivo susceptibility and genotyping of Plasmodium falciparum isolates from Pikine, Senegal

  • Aminata Mbaye1Email authorView ORCID ID profile,
  • Amy Gaye1,
  • Baba Dieye1,
  • Yaye D. Ndiaye1,
  • Amy K. Bei1, 2,
  • Muna Affara4,
  • Awa B. Deme1,
  • Mamadou S. Yade1,
  • Khadim Diongue1,
  • Ibrahima M. Ndiaye1,
  • Tolla Ndiaye1,
  • Mouhamed Sy1,
  • Ngayo Sy1,
  • Ousmane Koita5,
  • Donald J. Krogstad3,
  • Sarah Volkman2,
  • Davis Nwakanma4 and
  • Daouda Ndiaye1, 2
Malaria Journal201716:250

https://doi.org/10.1186/s12936-017-1897-6

Received: 23 January 2017

Accepted: 6 June 2017

Published: 14 June 2017

Abstract

Background

The monitoring of Plasmodium falciparum sensitivity to anti-malarial drugs is a necessity for effective case management of malaria. This species is characterized by a strong resistance to anti-malarial drugs. In Senegal, the first cases of chloroquine resistance were reported in the Dakar region in 1988 with nearly 7% population prevalence, reaching 47% by 1990. It is in this context that sulfadoxine–pyrimethamine temporarily replaced chloroquine as first line treatment in 2003, pending the introduction of artemisinin-based combination therapy in 2006. The purpose of this study is to assess the ex vivo sensitivity to different anti-malarial drugs of the P. falciparum population from Pikine.

Methods

Fifty-four samples were collected from patients with non-complicated malaria and aged between 2 and 20 years in the Deggo health centre in Pikine in 2014. An assay in which parasites are stained with 4′, 6-di-amidino-2-phenylindole (DAPI), was used to study the ex vivo sensitivity of isolates to chloroquine, amodiaquine, piperaquine, pyrimethamine, and dihydroartemisinin. High resolution melting was used for genotyping of pfdhps, pfdhfr, pfmdr1, and pfcrt genes.

Results

The mean IC50s of chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine were, respectively, 39.44, 54.02, 15.28, 2.23, and 64.70 nM. Resistance mutations in pfdhfr gene, in codon 437 of pfdhps gene, and an absence of mutation at position 540 of pfdhps were observed. Mutations in codons K76T of pfcrt and N86Y of pfmdr1 were observed at 51 and 11% population prevalence, respectively. A relationship was found between the K76T and N86Y mutations and ex vivo resistance to chloroquine.

Conclusion

An increase in sensitivity of isolates to chloroquine was observed. A high sensitivity to dihydroartemisinin was observed; whereas, a decrease in sensitivity to pyrimethamine was observed in the parasite population from Pikine.

Keywords

ChemosensitivityGenotyping Plasmodium falciparum Pikine

Background

Malaria is a parasitic disease which was responsible for nearly 429,000 deaths worldwide in 2015. More than 92% of these deaths occur in Africa in children [1]. Children under 5 years and pregnant women are among those most vulnerable to malaria. This can be explained by the fact that in malaria-endemic areas, after years of exposure, non-sterilizing immunity develops that is protective against severe malaria. Pregnant women tend to lose this acquired immunity due to the immune-suppression which occurs during pregnancy, and studies have shown that placental parasitaemia in these individuals is higher than those of peripheral blood [2], putting both mother and child at high risk. Many tools are currently available to fight malaria in these vulnerable groups such as intermittent preventive treatment of pregnant women with sulfadoxine–pyrimethamine (SP) [3, 4] and seasonal malaria chemoprevention (SMC) for children under 5 years of age in areas with seasonal transmission of malaria [5]. Currently in Senegal, for SMC in children, the drug regimen of choice is SP-amodiaquine. Amodiaquine is also used in combination with artesunate for treatment of uncomplicated malaria. Cross-resistance has been observed between amodiaquine and chloroquine. Thus, it would be important to monitor the sensitivity of parasites to SP to ensure the effectiveness of these preventive combination treatments. A good correlation between in vivo resistance to SP and in vitro resistance has been characterized as well as a strong association between in vivo resistance and single nucleotide polymorphisms in pfdhps (Plasmodium falciparum dihydropteroate synthetase) and pfdhfr (P. falciparum dihydrofolate reductase) genes [610]. In vitro resistance to pyrimethamine is associated with the mutation at codon S108N of pfdhfr gene, whereas the resistance to sulfadoxine is associated with the mutation K540E of the pfdhps gene. In West Africa, triple mutations at codons N51I, C59R and S108N of the pfdhfr gene and the mutation G437A/T in the pfdhps gene are frequently observed. In Senegal, the quadruple mutation representing the triple mutation of the pfdhfr gene plus the mutation G437A/T in the pfdhps is also frequently observed [1113]. In East Africa, a further mutation in pfdhps at codon K540E has been described [9]. This quintuple mutation is highly associated with a therapeutic failure to SP. WHO recommends that in areas where the quintuple mutation reaches greater than 50% population prevalence that SP use should be abandoned for chemoprevention of malaria [2].

The monitoring of amodiaquine, chloroquine, piperaquine, and dihydroartemisinin sensitivity is a clear priority in the fight against malaria. As amodiaquine is used for prevention and treatment of malaria in Senegal, it is important to determine if there is a decrease of sensitivity of P. falciparum population to this drug and if this decrease is related to the past (cross-resistance between amodiaquine and chloroquine) or current amodiaquine use. The overall objective of this study is to assess the ex vivo sensitivity of P. falciparum isolates from Pikine to SP, amodiaquine, chloroquine, piperaquine, and dihydroartemisinin.

Methods

Sample collection

In 54 children aged between 5 and 20 years of age who came for consultation at the Deggo health centre in Pikine in 2014, both venous blood and filter paper were collected. These children suffered from non-complicated malaria with confirmation by drop thick and thin smears. Informed consent by the child and or guardian was requested before any samples were taken. The study protocol was validated by the Human Subjects Committee of Tulane University and the Ethics Committee of the Ministry of Health of Senegal. The work is funded by the International Centres of Excellence for Malaria Research, (ICEMR) West Africa (U19AI089696).

Ex vivo assays

Drug preparation

Pyrimethamine (Sigma), chloroquine diphosphate salt, amodiaquine hydrochloride, dihydroartemisinin, and piperaquine were reconstituted with dimethyl sulfoxide (DMSO). The dilution was performed with the non-supplemented Roswell Park Memorial Institute Medium (RPMI). Twofold serial dilutions were performed with the non-supplemented Roswell Park Memorial Institute Medium (RPMI). The highest drug concentrations plated were 750 nM for chloroquine, 500 nM for piperaquine, 100 nM for amodiaquine, 50 nM for dihydroartemisinin, and 295,056 nM for pyrimethamine. Each drug concentration was plated in duplicate. Plates were frozen at −20 °C until required.

Culture and CI50 determination

The tubes of venous blood collected for DAPI test were transported to Aristide Le Dantec Hospital within 6 h of blood draw. The plasma was removed by centrifugation (2500g for 10 min). The pellet was then washed twice with unsupplemented RPMI by centrifugation at 2500g for 5 min. Parasitaemia was adjusted to between 0.4 and 1% and haematocrit was adjusted to 2%. The parasitaemia and haematocrit adjusted parasite mixture was distributed on the previously dosed 96-well drug plates and incubated in the presence of gas (94% N2, 5% CO2, 1% O2) [14] at 37 °C. After 48 h of incubation, the growth of parasite in positive control wells specifically plated for microscopic evaluation was checked. The assays was determined to be complete when parasites had re-invaded as new rings. Plates were frozen at −20 °C until reading and reading was performed for all plates at once.

For staining and reading the DAPI assay, 100 µl of membrane lysis buffer containing the molecule DAPI was distributed to each well. After a 30 min incubation, plates were centrifuged 4000g for 10 min, washed with PBS, and fluorescence was measured using a Fluoroskant Ascent. IC50 values were calculated using graph Pad Prism software version 5. Reference clone 3D7, sensitive to all anti-malarial drugs tested, was used for each batch of drug plates as a positive control.

DNA extraction and single nucleotide polymorphism typing

Parasitic DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen) according to manufacturer instructions. Codons 51, 59–108 of the pfdhfr gene, 436, 437, 540, and 613 of pfdhps, 76 of pfcrt and 86 of pfmdr1 were genotyped by HRM [12]. Glass capillaries were used with a 10 µl final volume. All PCR were performed using 2.5X LightScanner master mix (Biofire), with forward primers at a final concentration of 0.05 µM, reverse primers at a final concentration of 0.2 µM (asymmetric PCR), allele specific probes at a final concentration of 0.2 µM, and 1 µl of genomic DNA, as previously described [12]. Standard software included with the instruments was used for unlabelled probe analysis to visualize melting peaks based on different melting temperatures, indicative of different base pairs, and compared with controls to call alleles for a given assay.

Statistical analysis

The Graph-pad Prism Software version 5 was used to calculate the IC50 value for all drugs for each parasite isolate tested. For each codon position, the distribution of IC50s were compared using the Mann–Whitney U test. The test is significant if the P value is less than 0.05.

Results

Ex vivo sensitivity to chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine

Good sensitivity of the 3D7 reference strain to chloroquine (22.05 nM) was observed. The geometric mean IC50 for all isolates for chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine were 35.44, 54.02, 15.28, 2.23, and 64.70 nM, respectively (Fig. 1).
Figure 1
Fig. 1

Distribution of IC50 value among parasites collected in Pikine in 2014 and tested again Chloroquine (a), Piperaquine (b), Amodiaquine (c), Dihydroartemisinin (d) and Pyrimethamine (e). Horizontal lines indicate the geometric mean of IC50 value in red with the 95% CI. For all panels IC50 high value is not represented

Prevalence of point mutations of pfdhps, pfdhfr, pfcrt, and pfmdr1 genes

Mutations at codons 51, 59 and 108 of dhfr were highly prevalent with 100, 95 and 96% observed, respectively. For codon 437, a proportion of 44% with mixed wild type and mutant (2%) was observed. No mutation was observed at the 540 position of pfdhps gene. For codon 76 of the pfcrt gene 51% of mutations and 6% of mixed was found. About the codon 86 of pfmdr1, the mutation rate amounted to 11% (Table 1).
Table 1

Prevalence of point mutation of pfdhps, pfdhfr, pfcrt, and pfmdr1 genes at positions 51, 59, 108, 437, 540, 76, and 86

Gene

Codon

Allele

Prevalence

Pfdhfr

N51I

I

100% (51/51)

C59R

R

96% (49/51)

S108N

N

98% (50/51)

Mixte

2% (1/51)

Pfdhps

A437G

G

44% (24/54)

G/A

2 (1/54)

K540E

K

100% (54/54)

E

0%

Pfcrt

K76T

T

51% (28/540)

Mixte

6% (3/54)

Pfmdr1

N86Y

Y

11%

Mixte

0%

All strains that contained the mutation N86Y of pfmdr1 gene were mutant or mixed at codon K76T of pfcrt. For pfdhps and pfdhfr triple, quadruple and quintuple mutation were observed at 95, 39 and 0% prevalence, respectively (Table 2).
Table 2

Prevalence of mutant haplotype in pfdhps and pfdhfr

Haplotype

Prevalence

Triple mutation

95% (40/42)

Quadruple mutation

39% (15/38)

Quintuple mutation

0% (0/44)

Association between genotype and phenotype

The ex vivo resistance to chloroquine has been found to be linked to the mutation at codon K76T of pfcrt gene and N86Y of pfmdr1 (Table 3). For amodiaquine, the observation was that the values found for the mutant strains on these codons were higher but not significantly different. In our study, no relationship was found between the mutation K76T and N86Y and the decrease in ex vivo sensitivity to piperaquine, dihydroartemisinin and pyrimethamine. High prevalent of mutation on codon 51, 59 and 108 of the pfdhfr gene was found, probably related to the decrease in sensitivity to pyrimethamine.
Table 3

Correlation between mutation at Pfcrt codon K76T and Pfmdr1 codon N86Y and sensitivity to chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine

Compound

Codon

GM of IC50 (nM) for WA

GM of IC50 (nM) for MA

P value

Chloroquine

K76T

13.86

74.15

0.0195

N86Y

33.90

391.2

0.0279

Amodiaquine

K76T

9.924

10.47

0.0539

N86Y

9.417

20.69

0.5290

Piperaquine

K76T

56.16

46.45

0.9370

N86Y

27.15

58.00

0.8155

Dihydroartemisinin

K76T

2.139

1.773

0.4614

N86Y

2.165

1.906

0.7727

Pyrimethamine

K76T

11,596

4981

0.8366

N86Y

2.165

3.377

0.4605

P value is significant when less than 0.05

GM: geometric mean, IC 50 : half maximal inhibitory concentration, WA: wild allele, MA: mutant allele

Discussion

It is essential to have effective anti-malarial drugs to fight malaria. Artemisinin combination therapies were introduced as first-line therapy in this context. However, growing resistance to ACT has been observed in Southeast Asia: in Cambodia in 2006, Myanmar and Thailand in 2008, and Vietnam in 2009, and Laos in 2013 [15]. The rationale for monitoring resistance phenotypically by the in vitro method is that several anti-malarial drugs can be tested at the same time, and the evolution of the sensitivity or resistance of parasite populations to drugs either in use or no longer in use can be studied. The study of molecular markers of resistance informs the level of resistance of the Plasmodium population to drugs at the genetic level. This will result in better understanding of which drugs to monitor in vivo, which combinations to avoid, and those that can be used effectively for the management of malaria. In Senegal, pyrimethamine combined with sulfadoxine is used for intermittent preventive treatment for pregnant women. Further SP, plus amodiaquine is used for preventive seasonal treatment for children under 5 years old in areas with high transmission of malaria [16]. Chloroquine was eliminated in Senegal in 2003 following cases of resistance in vivo [17]. Piperaquine combined with dihydroartemisinin is used for the third-line treatment of non-complicated malaria. For molecular markers of resistance, the mutation on codons K76T of pfcrt gene and N86Y of pfmdr1 has been demonstrated to be associated with resistance to chloroquine [1822]. Resistance to amodiaquine is associated with the N86Y mutation and cases of cross-resistance between amodiaquine and chloroquine have been observed. For pyrimethamine, the mutation on codon S108N is strongly associated with resistance [23]. In vivo resistance of P. falciparum to chloroquine has been confirmed in Pikine, Moulomp (Casamance) and Fatick [24]. Indeed, the emergence of resistance to chloroquine in Senegal were reported in 1988 in Dakar with 5.7% therapeutic failure [25]. These cases then increased to 47.5% in 1990 and 25–30% in 1992 in Pikine [26], leading to the withdrawal of chloroquine for treatment of non-complicated malaria in Senegal in 2003. However, amodiaquine, which has some cross-resistance with chloroquine, is always used in combination for the treatment or prevention of malaria. In Dakar in 2010 a geometric mean of 41.63 nM for chloroquine and 19.4 nM for amodiaquine was found with another ex vivo technique [13]. At Pikine, an in vitro sensitivity study conducted in 2000 showed 31% of resistance to chloroquine with a geometric mean of 272 nM [27]. In 2001, a geometric mean of 135 nM was registered [28]. Prevalence of mutation of 51% on codon K76T of pfcrt and 11% on the N86Y of pfmdr1 gene was recorded in 2014.

The prevalence of the 76T allele in isolates from Pikine was 72.4% when chloroquine was used (2000–2003), 47.16% during the period of the use of amodiaquine-SP for first-line treatment (2004–2005) and 59.46% with ACT used between 2006 and 2009. N86Y mutation had decreased between 2005 and 2009 and it was about 20% in 2009 [29]. A selection of N86 and K76 alleles were noted in Thiès, another region in Senegal, in 2013 [30]. The results of this study have shown that the mutation on codon N86Y was related to the decrease in sensitivity to chloroquine. For amodiaquine, the geometric mean of the isolates with the mutation N86Y was higher compared to isolates with wild-type allele, but the difference was not significant. For piperaquine, no relationship between genotype and IC50 was observed. An association was found between the presence of the 76T allele and the decrease in sensitivity to chloroquine (p = 0.0195) but not to amodiaquine (0.0539) and piperaquine (0.9370). A decrease in ex vivo sensitivity and an increase in the prevalence of the N86Y mutation relationship was not significantly found with amodiaquine (p = 0.5290) and the geometric mean for piperaquine was very low compared to that found in other countries [3034]. These compounds are currently used in combination with dihydroartemisinin for piperaquine, SP and artesunate for amodiaquine. Good ex vivo sensitivity of isolates to dihydroartemisinin was found, implying continued effectiveness of one of the partner drugs of ACT used in the treatment of non-complicated malaria in Pikine.

Used since 2003 in Senegal, first as temporary replacement of chloroquine for the treatment of non-complicated malaria, SP is now used for preventive treatment of malaria. The results showed low ex vivo sensitivity of isolates to pyrimethamine. This was accompanied by a high prevalence of mutations in codons N51I, C59R and S108N of the pfdhfr gene. A strong presence of mutation on codon S108N (67 and 24%) and 51/59 (40 and 20%) were recorded, respectively, for Thiès in 2003 and Pikine in 2002. At Pikine, 65% (N51I), 61% (C59R) and 78% (S108N) of mutation was found [35]. These results suggest that resistance to pyrimethamine emerged before the introduction of the SP association. The double mutation 437/540 of the pfdhps gene has been demonstrated as being related to resistance to sulfadoxine [10]. Ex vivo sensitivity to sulfadoxine of isolates has not been studied. However, an absence of mutation on codon K540E and high prevalence of mutation at codon G437A/T has been recorded, which confirms the efficacy of sulfadoxine. On the other hand, studies revealed that triple mutation 108–59–51 is strongly associated with resistance to SP in African isolates [36] and that the presence of the double mutation 437/540 indicates a high risk of treatment failure in SP [9]. An increase of triple mutation and quadruple mutation in Pikine was noted [35], but the quintuple mutation was absent. However, mutation of 2.12% at codon 540 in pfdhfr was found in Dakar [36].

Conclusion

Monitoring the sensitivity of P. falciparum populations to anti-malarial drugs is a necessity for effective malaria case management. An increase in the sensitivity of isolates to chloroquine Good efficacy of dihydroartemisinin amodiaquine and piperaquine and decrease in sensitivity to pyrimethamine were observed.

Abbreviations

SP: 

sulfadoxine–pyrimethamine

ACT: 

artemisinin-based combination therapy

Pfdhps: 

Plasmodium falciparum dihydropteroate synthetase

Pfdhfr: 

Plasmodium falciparum dihydrofolate reductase

WHO: 

World Health Organization

ICEMR: 

International Centres of Excellence for Malaria Research

DMSO: 

dimethyl sulfoxide

RPMI medium: 

Roswell Park Memorial Institute medium

HRM: 

high resolution melting

DNA: 

deoxy ribo nucleic acid

PCR: 

polymerase chain reaction

Pfcrt: 

Plasmodium falciparum chloroquine resistance transporter

Pfmdr1: 

Plasmodium falciparum multidrug resistance protein 1

CQ: 

chloroquine

AMQ: 

amodiaquine

PQ: 

piperaquine

DHA: 

dihydroartemisinin

Declarations

Authors’ contributions

AM, BD, AG, YDN, ABD, MSY, IMN, TN, AKB carried out the experiments and collected data. DN, OK, DK, SV, DN conceived and designed the study. AM and AKB analysed the data. AM, BD and KD wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We would like to acknowledge the International Centre for Excellence in Malaria Research (ICEMR) project and the Parasitology and Mycology Laboratory Le Dantec Hospital. We thank Cyrille Diedhiou, Nasserdine Papa Nze, Dior Diop, Younouss Diedhiou, Lamine Ndiaye, Amadou Mactar Mbaye, the Degoo patients and staff for their contribution to this study.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

The participants in this study consented publication.

Ethics approval and consent to participate

The Human Subjects Committee of Tulane University and the Ethics Committee of the Senegal Ministry of Health in Dakar both approved the protocols used in these studies.

Funding

The work was supported by the International Centers of Excellence for Malaria Research, (ICEMR) West Africa (U19AI089696).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Laboratory of Parasitology/Mycology HALD, Cheikh Anta Diop University of Dakar, Dakar, Senegal
(2)
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, USA
(3)
Tulane University, New Orleans, USA
(4)
Medical Research Council Unit, The Gambia, Fajara, Gambia
(5)
University of Bamako, Bamako, Mali

References

  1. WHO. World Malaria Report. Geneva: World Health Organization; 2016.Google Scholar
  2. Bricaire F, Danis M, Gentilini M. Paludisme et grossesse. Cahiers. Santé. 2008;63:289–92.Google Scholar
  3. Bamba S, Séré A, Nikiéma R, Halidou T, Thiéba B, Dao B, et al. Traitement préventif intermittent à la sulfadoxine–pyriméthamine du paludisme chez les femmes enceintes: efficacité et observance dans deux hôpitaux urbains du Burkina Faso. Pan Afr Med J. 2013;14:105.PubMedPubMed CentralGoogle Scholar
  4. Rupérez M, González R, Mombo-Ngoma G, Kabanywanyi AM, Sevene E, Ouédraogo S, et al. Mortality, morbidity, and developmental outcomes in infants born to women who received either mefloquine or sulfadoxine–pyrimethamine as intermittent preventive treatment of malaria in pregnancy: a cohort study. PLoS Med. 2016;13:e1001964.View ArticlePubMedPubMed CentralGoogle Scholar
  5. OMS. Chimioprévention Du Paludisme Saisonnier. 2013. p. 1–45. Available from: http://www.who.int/malaria/areas/preventive_therapies/children/fr/. Accessed 9 June 2017.
  6. Happi CT, Gbotosho GO, Folarin OA, Akinboye DO, Yusuf BO, Ebong OO, et al. Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine–pyrimethamine resistance in malaria-infected patients from Nigeria. Acta Trop. 2005;95:183–93.View ArticlePubMedGoogle Scholar
  7. Hailemeskel E, Kassa M, Taddesse G, Mohammed H, Woyessa A, Tasew G, et al. Prevalence of sulfadoxine-pyrimethamine resistance-associated mutations in dhfr and dhps genes of Plasmodium falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia. Acta Trop. 2013;128:636–41.View ArticlePubMedGoogle Scholar
  8. Nzila AM, Mberu EK, Sulo J, Dayo H, Winstanley PA, Sibley CH, et al. Towards an understanding of the mechanism of pyrimethamine–sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Antimicrob Agents Chemother. 2000;44:991–6.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Naidoo I, Roper C. Mapping, “partially resistant”, “fully resistant”, and “super resistant” malaria. Trends Parasitol. 2013;29:505–15.View ArticlePubMedGoogle Scholar
  10. Abdul-Ghani R, Farag HF, Allam AF. Sulfadoxine-pyrimethamine resistance in Plasmodium falciparum: a zoomed image at the molecular level within a geographic context. Acta Trop. 2013;125:163–90.View ArticlePubMedGoogle Scholar
  11. Ndiaye D, Dieye B, Ndiaye YD, Van Tyne D, Daniels R, Bei AK, et al. Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal. Int J Parasitol Drugs Drug Resist. 2013;3:135–42.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Daniels R, Ndiaye D, Wall M, McKinney J, Séne PD, Sabeti PC, et al. Rapid, field-deployable method for genotyping and discovery of single-nucleotide polymorphisms associated with drug resistance in Plasmodium falciparum. Antimicrob Agents Chemother. 2012;56:2976–86.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Fall B, Pascual A, Sarr FD, Wurtz N, Richard V, Baret E, et al. Plasmodium falciparum susceptibility to anti-malarial drugs in Dakar, Senegal, in 2010: an ex vivo and drug resistance molecular markers study. Malar J. 2013;12:107.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Bei AK, Patel SD, Volkman SK, Ahouidi AD, Ndiaye D, Mboup S, et al. An adjustable gas-mixing device to increase feasibility of in vitro culture of Plasmodium falciparum parasites in the field. PLoS ONE. 2014;9:e90928.View ArticlePubMedPubMed CentralGoogle Scholar
  15. WHO. Status report on artemisinin resistance [Internet]. 2014. p. 1–8. Available from: http://www.who.int/malaria/publications/atoz/status-rep-artemisinin-resistance-sep2014.pdf. Accessed 9 June 2017.
  16. PNLP. Plan Strategique National 2011-2015. 2010. p. 129. Available from: http://www.pnlp.sn/wp-content/uploads/2016/08/PNLP_PSN_VFF_03-02-2016.pdf. Accessed 9 June 2017.
  17. Mouzin E, Thior PM, Diouf MB, Samd B. Focus sur le Senegal. 2010. Available from: http://www.rollbackmalaria.org/files/files/resources/report4-fr.pdf. Accessed 9 June 2017.
  18. Payne D. Spread of chloroquine resistance in Plasmodium falciparum. Parasit Today. 1987;3:241–6.View ArticleGoogle Scholar
  19. Lim P, Chy S, Ariey F, Incardona S, Chim P, Sem R, et al. pfcrt Polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia. Antimicrob Agents Chemother. 2003;47:87–94.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Sidhu AB, Verdier-Pinard D, Fidock DA. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science. 2002;298:210–3.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Eyase FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, et al. The role of pfmdr1 and pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in Western-Kenya P. falciparum samples during 2008–2011. PLoS ONE. 2013;8:e64299.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Pradines B, Dormoi J, Briolant S, Bogreau H, Rogier C. La résistance aux antipaludiques. Revue Francophone des Laboratoires. 2010;422:51–62.View ArticleGoogle Scholar
  23. Basco LK, Ringwald P. Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil. Am J Trop Med Hyg. 2000;62:271–6.View ArticlePubMedGoogle Scholar
  24. Gaye O, Bah IB, Diallo S, Victorius A, Bengua E, Faye OFO. The emergence of chloroquine-resistant malaria in Dakar. Senegal. Ann Soc Belg Med Trop. 1990;70:33–7.PubMedGoogle Scholar
  25. Trape JF, Legros F, Ndiaye P, Konate L, Bah IB, Diallo S, et al. Chloroquine-resistant Plasmodium falciparum malaria in Senegal. Trans R Soc Trop Med Hyg. 1989;83:761–3.View ArticlePubMedGoogle Scholar
  26. Sokhna CS, Molez JF, Ndiaye P, Sane BTJ. In vivo chemosensitivity tests of Plasmodium falciparum to chloroquine in Senegal: the development of resistance and the assessment of therapeutic efficacy. Bull Soc Pathol Exot. 1997;90:83–9.PubMedGoogle Scholar
  27. Penh P, Thomas SM, Ndir O, Dieng T, Mboup S, Wypij D, et al. In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal. Am J Trop Med Hyg. 2002;66:474–80.View ArticleGoogle Scholar
  28. Dieng T, Bah IB, Ndiaye PM, Diallo I, Diop BM, Brasseur P, et al. Evaluation de la sensibilité in vitro de Plasmodium falciparum à la chloroquine par le deli-microtest dans la région de Dakar (Sénégal). Med Trop (Mars). 2005;65:580–3.PubMedGoogle Scholar
  29. Ly O, Gueye PEO, Deme AB, Dieng T, Badiane AS, Ahouidi AD, et al. Evolution of the pfcrt T76 and pfmdr1 Y86 markers and chloroquine susceptibility 8 years after cessation of chloroquine use in Pikine, Senegal. Parasitol Res. 2012;111:1541–6.View ArticlePubMedGoogle Scholar
  30. Mbaye A, Dieye B, Ndiaye YD, Bei AK, Muna A, Deme AB, et al. Selection of N86F184D1246 haplotype of Pfmrd1 gene by artemether–lumefantrine drug pressure on Plasmodium falciparum populations in Senegal. Malar J. 2016;15:433.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Basco LK, Ringwald P. In vitro activities of piperaquine and other 4-aminoquinolines against clinical isolates of Plasmodium falciparum in Cameroon. Antimicrob Agents Chemother. 2003;47:1391–4.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Nsobya SL, Kiggundu M, Nanyunja S, Joloba M, Greenhouse B, Rosenthal PJ. In vitro sensitivities of Plasmodium falciparum to different antimalarial drugs in Uganda. Antimicrob Agents Chemother. 2010;54:1200–6.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Barends M, Jaidee A, Khaohirun N, Singhasivanon P, Nosten F. In vitro activity of ferroquine (SSR 97193) against Plasmodium falciparum isolates from the Thai-Burmese border. Malar J. 2007;6:81.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Mwai L, Kiara SM, Abdirahman A, Pole L, Rippert A, Diriye A, et al. In vitro activities of piperaquine, lumefantrine, and dihydroartemisinin in Kenyan Plasmodium falciparum isolates and polymorphisms in pfcrt and pfmdr1. Antimicrob Agents Chemother. 2009;53:5069–73.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Ndiaye D, Daily JP, Sarr O, Ndir O, Gaye O, Mboup S, et al. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Senegal. Trop Med Int Health. 2005;4:1176–9.View ArticleGoogle Scholar
  36. Boussaroque A, Fall B, Madamet M, Wade KA, Fall M, Nakoulima A, et al. Prevalence of anti-malarial resistance genes in Dakar, Senegal from 2013 to 2014. Malar J. 2016;15:347.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s) 2017

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Please note that comments may be removed without notice if they are flagged by another user or do not comply with our community guidelines.

Advertisement