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Keywords:

  • malaria;
  • Plasmodium falciparum;
  • pfmdr1;
  • amplification;
  • Kenya
  • Malaria;
  • Plasmodium falciparum;
  • pfmdr1;
  • amplification;
  • Kenya
  • malaria;
  • Plasmodium falciparum;
  • pfmdr1;
  • amplificación;
  • Kenia

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objectives  Many countries are now adopting artemisinin-based combination therapy (ACT) for treatment of Plasmodium falciparum malaria. In multi-drug resistant areas in South East Asia amplifications of the pfmdr1 gene are frequent and tentatively associated with reduced susceptibility to the common quinoline partner drugs mefloquine and lumefantrine. In Africa where amodiaquine is one of the favoured quinoline partner drugs in ACT, studies on multi-drug resistance associated pfmdr1 gene amplifications are urgent. Our aim was to determine the current prevalence of pfmdr1 gene amplifications and a possible association between pfmdr1 gene copy number and amodiaquine treatment outcome in Kenya.

Methods  Seventy-two children with Plasmodium falciparum infection in Kenya were treated with amodiaquine monotherapy and followed for 21 days. Possible amplification of the pfmdr1 gene was assessed from blood-spotted filterpaper by TaqMan® probe based real-time polymerase chain reaction.

Results  The recrudescent rate was 14 of 72 (19%). All children had single pfmdr1 copy infections, with the exception of one child who had an infection with two pfmdr1 copies. This child had an adequate treatment response.

Conclusion Pfmdr1 amplifications do exist in Kenya but at a very low frequency. Yet, the substantial number of children with recrudescent infections implies that amodiaquine resistance is not related to pfmdr1 gene amplifications in Kenya.

Objectifs  De nombreux pays adoptent maintenant le traitement combinéà base d'artemisinine pour la malaria àPlasmodium falciparum. Dans les régions à résistance multiple aux médicaments en Asie du sud-est, des amplifications du gène pfmdr1 sont fréquentes et ont tendance àêtre associées avec une susceptibilité réduite pour les médicaments tels que la méfloquine et la luméfantrine, accompagnant couramment les quinolines. En Afrique où l'amodiaquine est l'accompagnant de choix dans les combinaisons avec l'artemisinine, des études sur la résistance multiple et son association avec l'amplification de pfmdr1 sont urgemment nécessaires. Notre but était de déterminer la prévalence actuelle des amplifications de pfmdr1 et l’éventuelle association entre le nombre de copies de pfmdr1 et le résultat du traitement à l'amodiaquine au kenya.

Méthodes  72 enfants avec une infection àP. falciparum ont été traités avec l'amodiaquine en monothé rapie et ont été suivis pendant 21 jours. L'amplification éventuelle de pfmdr1 a étéévaluée partir d’échantillons de sang collectés sur papier filtre et testés par real time PCR utilisant des sondes TaqMan®.

Résultats  Le taux de recrudescence était de 19% (14/72). Tous les enfants avaient des infections avec une copie unique de pfmdr1à l'exception d'un seul enfant avec une infection à double copie de pfmdr1. Cet enfant a répondu adéquatement au traitement.

Conclusion  Des amplifications de pfmdr1 existent au Kenya mais à très basse fréquence. Cependant le nombre important d'enfants avec une recrudescente des infections suggère que la résistance à l'amodiaquine n'est pas liée à l'amplification du gène pfmdr1 au Kenya.

Objetivos  Muchos países están adoptando la terapia de combinación con artemisininas (TCA) para el tratamiento de la malaria por Plasmodium falciparum. En áreas del Sureste Asiático con multiresistencia, la amplificación del gen pfmdr1 gene es frecuente y podría estar asociada a una reducción de la susceptibilidad a las quinolinas más comúnmente utilizadas en terapia de combinación: la mefloquina y la lumefantrina. En África, en donde la amodiaquina es una de las quinolinas usadas con preferencia en la TCA, son urgentes los estudios sobre multiresistencia asociados a la amplificación del gen pfmdr1. Nuestro objetivo era determinar la prevalencia actual de las amplificaciones del gen pfmdr1 y la posible asociación entre el número de copias de este gen y el resultado frente al tratamiento con amodiquina en Kenia.

Métodos  72 niños con infección por Plasmodium falciparum en Kenia fueron tratados con amodiaquina en monoterapia, y seguidos durante 21 días. La posible amplificación del gen pfmdr1 se evaluó mediante PCR a tiempo real a partir de muestras de sangre recogidas en papel de filtro TaqMan®.

Resultados  La tasa de recrudescencia fue de 14/72 (19%). Todos los niños tenían infecciones con una sola copia de pfmdr1, con excepción de un niño que presentaba una infección con dos copias de pfmdr1. Este niño respondió adecuadamente al tratamiento.

Conclusión  Las amplificaciones de pfmdr1 existen en Kenia pero con una frecuencia muy baja. Sin embargo, el número sustancial de niños con infecciones recrudescentes implica que la resistencia a la amodiquina no está relacionada con la amplificación de pfmdr1 en Kenia.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The burden of Plasmodium falciparum mortality and morbidity has increased significantly in Africa the last decades due to expansion of resistance to the mainstay antimalarials chloroquine (CQ) and sulphadoxine–pyrimethamine (SP). Artemisinin-based combination therapy (ACT) is a part of the new global disease control strategy and is under implementation on the African continent (WHO 2001). The differences in pharmacokinetic characteristics between the potent short half-life artemisinin derivative (ART) and the long half-life partner drug in ACT implies that in areas of high malaria transmission the recurrent parasites will be exposed to the latter alone. This can potentially lead to progressively more tolerant parasites to the partner drug, through sub-therapeutic drug exposure (Hastings & Ward 2005; Sisowath et al. 2005). In Africa, amodiaquine (AQ) with its long half-life metabolite desethylamodiaquine (DEAQ) is presently one main partner drug option either for first or second line ACT.

Decreased susceptibility to DEAQ in vitro has been correlated with polymorphisms in the P. falciparum CQ resistance transporter gene (pfcrt) with decreased mean hydrophobicity of the amino acids 72–76 (Warhurst 2003). Resistance to AQ/DEAQ in vivo has been associated with pfcrt 76T and an 86Y SNP in the P. falciparum multiple drug resistance 1 gene (pfmdr1) (Ochong et al. 2003; Holmgren et al. 2006). However, these alterations cannot fully explain the treatment failures in this study, suggesting that AQ/DEAQ resistance must also involve other yet to be identified and putatively more important genetic alterations (Holmgren et al. 2006).

The pfmdr1 gene codes for an ABC transporter homologue of the human drug resistance associated P-glycoprotein 1 (Pgh1). Pfmdr1 gene amplification, resulting in an over-expression of Pgh1, has been consistently associated with decreased parasite sensitivity in vitro and/or in vivo to the aryl-methanol quinolines mefloquine (MQ), halofantrine (HF), quinine (QN) and also tentatively to the new related drug lumefantrine (LF), as well as to the endoperoxide artemisinin derivatives (ART) (Barnes et al. 1992; Price et al. 1999; Duraisingh et al. 2000; Price et al. 2004; Ashley & White 2005; Duraisingh & Cowman 2005; Nelson et al. 2005). In contrast, de-amplification of the pfmdr1 gene has been associated to increased parasite resistance in vitro to the 4-aminoquinoline CQ (Barnes et al. 1992). No information is available for AQ/DEAQ.

In some parts of Thailand the prevalence of pfmdr1 amplifications is as high as 30–40% (Price et al. 2004). Information on the prevalence of pfmdr1 amplifications in Africa is scarce with only one recent study from Gabon, where the prevalence was as low as 5% in 1995 and none of the samples collected 7 years later had pfmdr1 amplifications (Uhlemann et al. 2005). At a point when most African countries are adopting ACT, studies on the prevalence of multi-resistance associated pfmdr1 gene amplifications and the possible relation to potential ACT partner drugs response are urgent.

We revisited our in vivo study on AQ monotherapy in Kenya (Holmgren et al. 2006) to determine the current prevalence of pfmdr1 gene amplifications in this East African setting, and to identify a possible association between pfmdr1 gene copy number and AQ treatment outcome, especially in the context of AQ as a potential ACT partner drug.

Material and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Samples

Samples for molecular analysis were derived from our clinical trial on AQ in monotherapy conducted between November and December 2004 in Western Kenya (Holmgren et al. 2006). Plasmodium falciparum clones Dd2 and 3D7 were generously supplied by Prof. D. Walliker (Institute for Animal and Population Genetics, Edinburgh University, Edinburgh, UK)

Molecular analysis

Blood samples on filterpapers (3MM®; Whatman) from all patients were available for extraction. Possible amplification of the pfmdr1 gene was assessed by TaqMan® probe based real-time PCR on an ABI Prism® 7000 sequence detector (Applied Biosystems) as described elsewhere (Price et al. 2004). In summary, PCR amplifications of the pfmdr1 and β-tubulin genes were done in multiplex in Thermo-Fast® 96-well PCR plates (Abgene; Advanced Biotechnologies Ltd). All samples were made in triplicate of 25 μl containing 12.5 μl TaqMan® Universal Mix, 300 nm of each forward and reverse primers and 100 nm of each FAM-labelled pfmdr1 and VIC-labelled β-tubulin probes and 5 μl of DNA template. Each PCR plate contained two samples known to have one pfmdr1 copy (clone 3D7) and one sample known to have circa three pfmdr1 copies (clone Dd2) (Uhlemann et al. 2005) as calibrators and one negative control. After 50 PCR cycles of 95 °C for 15 s and 58 °C for 1 min, the fluorescent data was analysed with the comparative ΔCT (cycle threshold) method. The results qualified if CT < 35 and ΔCT spread <1.5 or CT 35–40, SD CT < 0.5 and ΔCT spread <1.5. Numbers of copies were rounded to the nearest integer unless otherwise stated. The pfmdr1 N86Y genotyping analysis is described in a previous report (Holmgren et al. 2006).

Ethical approval

Ethical approval was obtained from The Research Ethics Committee at Karolinska Institute in Stockholm, Sweden (KI Dnr 03-545) and The Ethical Research Committée, Nyanza Province, Kenya (GN 153, vol. 1/50).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A total of 78 children with uncomplicated P. falciparum malaria were included and treated with AQ under supervision for 3 days. Six were lost to follow-up due to migration. The remaining 72 patients fulfilled the 21 days follow up. A total of 26 (36.1%, 95% CI 25.1–48.3%) children were found with recurrent parasitaemias during follow-up days 7–21, out of which eight were recrudescent infections, 12 re-infections and six mixed recrudescenses/re-infections, i.e. including the mixed infections a total of 14 (19%, 95%CI 11–30%) were recrudescent infections.

Pfmdr1 amplification analysis was performed on all 72 samples from day 0, of which copy number was successfully ascertained from 58 (80.6%) samples. Of the 26 samples from children with recurrent parasitaemia, copy number was ascertained from 23 (88.5%) samples. Pfmdr1 amplification results are illustrated in Figure 1. When rounded to the nearest integer, all successfully amplified samples were found to have one pfmdr1 copy, with the exception of one day 0 sample (1.4%, 95% CI 0.03–7.5%), which had two pfmdr1 copies. This child had an adequate treatment response with clearance of parasites already by day 2.

image

Figure 1. Pfmdr1 gene copy number distribution.

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The pfmdr1 N86Y polymorphism distribution among the samples was 15N, 40Y and 17 mixed N/Y genotypes on day 0 and 4N, 18Y and 4 mixed N/Y genotypes among the samples with recurrent parasitaemia day 7–21 (Holmgren et al. 2006). The sample with two pfmdr1 copies had an 86N genotype.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We found that pfmdr1 amplifications do exist but at a very low frequency in Kenya. This is coherent with previous published data from Gabon where the most recent study in 2002 did not detect any pfmdr1 amplifications (Uhlemann et al. 2005).

The only child with an infection with a multicopy pfmdr1 gene responded well to AQ treatment, suggesting that the amplification may not interfere with treatment response. However, 14 (19%) patients had recrudescent parasitaemias during the study follow up, implying that AQ resistance is not related to these rare findings of pfmdr1 gene amplifications in Kenya.

In Thailand, pfmdr1 amplifications have only occurred in combination with the genotype pfmdr1 86N (Price et al. 1999, 2004) suggesting that the long use of MQ has selected both for pfmdr1 86N and its amplification (Ashley & White 2005). In our study, the only sample with an amplified pfmdr1 gene also had a pfmdr1 86N genotype. This correlation between pfmdr1 amplifications and the pfmdr1 86N genotype was also observed in Gabon, but at least one strain had an amplified pfmdr1 gene despite the pfmdr1 86Y genotype (Uhlemann et al. 2005). This suggests that parasites with a pfmdr1 86Y genotype can amplify its gene, but may be parasites with a pfmdr1 86N genotype benefits more of its amplification and depending on the epidemiological drug pressure the parasites have to choose which gene alteration is most optimal to survive. This would explain why in Africa it appears that the long use of CQ has selected for the genotype pfmdr1 86Y but not for its amplification, which is reassuring with the massive introduction of the aryl-methanol quinoline ACT (Coartem®, Novartis, Basel, Switzerland). However, this scenario might change since decreased susceptibility to both ART and LF are associated with pfmdr1 86N and potentially also its amplification (Price et al. 1999, 2004; Duraisingh et al. 2000; Hastings & Ward 2005; Sisowath et al. 2005).

Despite the pharmacodynamic advantages with ACT there is a risk for rapid selection for resistance if a partner drug to ART would have an overlapping molecular mechanism of resistance. AQ/DEAQ resistance has been associated with pfcrt 72–76 polymorphisms (Ochong et al. 2003; Warhurst 2003; Holmgren et al. 2006) as well as with pfmdr1 86Y (Holmgren et al. 2006), generally not present in pfmdr1 amplified genes. ART has been associated with pfATP6 S769N (Krishna et al. 2006) as well as with pfmdr1 86N and its amplification (Price et al. 1999, 2004; Duraisingh et al. 2000; Ashley & White 2005; Duraisingh & Cowman 2005). This would suggest that AQ and ART might protect each other against development of resistance because of different mechanism of resistance and considering pfmdr1 polymorphisms and amplifications also a potential opposite genetic selection pattern.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to Mrs Görel Day-Wilson, Coordinator of The Scandinavian Doctor Bank in Kenya, Dr SJ Bongo, District Medical Officer of Health and Mr Chrisantus Makomere, Primary Health Care Officer at Siaya Hospital and the local research team in Kenya for great cooperation and support in the fieldwork. The study was funded by Karolinska University Hospital Research and Development Grants no. 015/04 and European Union 5th Framework Program Ref. No. QLK2-CT-2002-01503.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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