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Volume 19, Number 5—May 2013

Letter

Plasmodium falciparum with Multidrug Resistance 1 Gene Duplications, Senegal

Suggested citation for this article

To the Editor: Amplification and overexpression of the Plasmodium falciparum multidrug resistance 1 gene (Pfmdr1) have been associated with mefloquine resistance in P. falciparum malaria in Asia (1). Amplification of Pfmdr1 in Africa has occurred rarely. Only 12 isolates with >2 copies of Pfmdr1 were identified in Africa during 1993–2012: 3 in Côte d’Ivoire (2,3), 1 in Burkina Faso (3), 1 in Togo (3), 3 in eastern Sudan (4), 2 in Kenya (5,6), and 1 Senegal (7). Another isolate was obtained in a patient from Benin who did not respond clinically to mefloquine treatment (8). Pfmdr1 amplification has not been found in samples collected either before or after treatment for recurring P. falciparum infection in Africa in many studies.

In Dakar, Senegal, and its surrounding suburbs, malaria is transmitted with spatial heterogeneity to the human mosquito bite rate, which ranged from 0.1 to 250 bites per person per night during the rainy seasons of 2007–2010. P. falciparum isolates from patients with malaria who lived in Dakar (>80%) and the surrounding area and did not travel during the previous month were obtained during the rainy seasons of October 2009–January 2010 (172 patients, 42% female) and August 2010–January 2011 (129 patients, 38% female). Informed verbal consent from the patients and/or their parents/guardians was obtained before blood collection; the study was approved by the ethical committee of the Hôpital Principal de Dakar.

Of the 301 patients, 54% were recruited from the emergency department during each of the 2 seasons; other patients were recruited from the intensive care unit (18% during October 2009–January 2010 and 20% during August 2010–January 2011), pediatric department (9% and 5%), and other units (19% and 21%). No significant differences were found between the 2 seasons for parasitemia (p = 0.160), sex ratio (p = 0.446), living area (p = 0.651), or hospital admission status (p = 0.567). Information on antimalarial treatment before admission was not available.

We analyzed the Pfmdr1 copy number for the 301 isolates by using TaqMan real-time PCR as described (7); 167 isolates were successfully evaluated by using the 72-h histidine-rich protein 2 test as described (9). P. falciparum clones 3D7 (1 Pfmdr copy) and W2 (3 Pfmdr1 copies) were used as controls for the determination of the Pfmdr1 copy number and for the validation of the batches of plates used in the susceptibility tests.

A total of 9 isolates with 2 Pfmdr1 copies were identified, 1 collected during the 2009–2010 season and 8 collected during the 2010–2011 season. This finding reflects a statistically significant 10-fold increase in frequency of isolates from 1 season to the next (p = 0.0057 by Fisher exact test). All of the isolates with duplicated copies had 1 allelic family for each of the 3 genes (msp1, msp2, and glurp), confirming that these infections were single and not mixed.

We did not find an association between Pfmdr1 copy number and the 50% inhibitory concentration values for mefloquine (p = 0.345), monodesethylamodiaquine (p = 0.729), lumefantrine (p = 0.314) and chloroquine (p = 0.579), but a significant association was found for dihydroartemisinin (p = 0.003). Odds ratio for in vitro reduced susceptibility to dihydroartemisinin associated with 2 Pfmdr1 copies was 1.4. Although the number of isolates with 2 Pfmdr1 copies we obtained is small, these data are consistent with previous reports on Pfmdr1 copies and in vitro responses to artemisinin derivatives (10).

The role of the amplification of Pfmdr1 in P. falciparum resistance to antimalarial drugs in Africa is debated. In our study, we identified as many samples with multiple Pfmdr1 copies (9) as had been identified during the previous 19 years. The 9 patients from whom these isolates were collected were successfully treated with quinine. One study found clinical treatment failure for mefloquine in Africa was associated with in vitro resistance and amplification of Pfmdr1 (8). In addition, amodiaquine resistance is not related to the amplification of Pfmdr1 (6). In Sudan, 3 isolates with 2 Pfmdr1 copies were identified in patients before and after treatment with artemether-lumefantrine during the 28-day follow-up period, but these patients were classified as having adequate clinical and parasitologic responses (4).

The increased prevalence of Pfmdr1 duplication in P. falciparum isolates from patients in Dakar within a 2-year period is cause for concern and vigilance. The presence of these duplicated Pfmdr1 copies could be associated with rare clinical failures of P. falciparum infections to respond to mefloquine treatment or artemisinin-based combination therapy. However, our findings highlight the need for active surveillance of the prevalence of Pfmdr1 duplication in P. falciparum isolates and for ex vivo and in vivo studies in Senegal and in other parts of Africa.

Aurélie Pascual, Bécaye Fall, Nathalie Wurtz, Mansour Fall, Cheikhou Camara, Aminata Nakoulima, Eric Baret, Bakary Diatta, Boubacar Wade, Sébastien Briolant, and Bruno PradinesComments to Author 
Author affiliations: Institut de Recherche Biomédicale des Armées, Marseille, France (A. Pascual, N. Wurtz, E. Baret, S. Briolant, B. Pradines); Aix Marseille Université, Marseille (A. Pascual, N. Wurtz, E. Baret, S. Briolant, B. Pradines); Centre National de Référence du Paludisme, Marseille (A. Pascual, B. Pradines); Hôpital Principal de Dakar, Dakar, Sénégal (B. Fall, M. Fall, C. Camara, A. Nakoulima, B Diatta, B. Wade, B. Pradines)

References

  1. Price RN, Uhlemann AC, Brockman A, McReady R, Ashley E, Phaipun L, Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet. 2004;364:43847. DOIPubMed
  2. Basco LK, Le Bras J, Rhoades Z, Wilson CM. Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from Subsaharan Africa. Mol Biochem Parasitol. 1995;74:15766. DOIPubMed
  3. Witkowski B, Nicolau ML, Soh PN, Iriart X, Menard S, Alvarez M, Plasmodium falciparum isolates with increased pfmdr1 copy number circulate in West Africa. Antimicrob Agents Chemother. 2010;54:304951. DOIPubMed
  4. Gadalla NB, Adam I, Elzaki SE, Bashir S, Mukhtar I, Oguike M, Increased pfmdr1 copy number and sequence polymorphisms in Plasmodium falciparum isolates from Sudanese malaria patients treated with artemether-lumefantrine. Antimicrob Agents Chemother. 2011;55:540811. DOIPubMed
  5. Akala HM, Eyase FL, Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, Antimalarial drug sensitivity profile of Western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay and molecular analysis. Am J Trop Med Hyg. 2011;85:3441 . DOIPubMed
  6. Holmgren G, Bjorkman A, Gil JP. Amodiaquine resistance is not related to rare findings of Pfmdr1 gene amplifications in Kenya. Trop Med Int Health. 2006;11:180812. DOIPubMed
  7. Wurtz N, Fall B, Pascual A, Diawara S, Sow K, Baret E, Prevalence of molecular markers of Plasmodium falciparum drug resistance in Dakar, Senegal. Malar J. 2012;11:197. DOIPubMed
  8. Witkowski B, Iriart X, Soh PN, Menard S, Alvarez M, Naneix-Laroche V, Pfmdr1 amplification associated with clinical resistance to mefloquine in West Africa: implications for efficacy of artemisinin combination therapies. J Clin Microbiol. 2010;48:37979. DOIPubMed
  9. Fall B, Diawara S, Sow K, Baret E, Diatta B, Fall KB, Ex vivo susceptibility of Plasmodium isolates from Dakar, Senegal, to seven standard anti-malarial drugs. Malar J. 2011;10:310. DOIPubMed
  10. Sidhu ABS, Uhlmann AC, Valderramos SG, Valderramos JC, Krishna S, Fidock DA. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis. 2006;194:52835. DOIPubMed

Suggested citation for this article: Pascual A, Fall B, Wurtz N, Fall M, Camara C, Nakoulima A, et al. Plasmodium falciparum with multidrug resistance 1 gene duplications, Senegal [letter]. Emerg Infect Dis [Internet]. 2013 May [date cited]. http://dx.doi.org/10.3201/eid1905.121603

DOI: 10.3201/eid1905.121603

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Table of Contents – Volume 19, Number 5—May 2013

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Bruno Pradines, Unité de Parasitologie, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Institut de Recherche Biomédicale des Armées, GSBdD Marseille-Aubagne, 111 Av de le Corse, BP 40026, 13568 Marseille Cedex 02, France





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