Increased pfmdr1 copy number in Plasmodium falciparum isolates from Suriname


  • Mergiory Labadie-Bracho,

    1. “Prof. Dr. Paul C. Flu” Institute for Biomedical Sciences, Paramaribo, Suriname
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  • Malti R. Adhin

    Corresponding author
    1. Department of Biochemistry, Faculty of Medical Sciences, Anton de Kom Universiteit van Suriname, Paramaribo, Suriname
    • “Prof. Dr. Paul C. Flu” Institute for Biomedical Sciences, Paramaribo, Suriname
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Corresponding Author Malti R. Adhin, Faculty of Medical Sciences, Department of Biochemistry, Anton de Kom Universiteit van Suriname, Kernkampweg # 5, PO Box 537, Paramaribo, Suriname, Tel.: +597 441007; Fax: +597 441080; E-mail:


Amplification of the pfmdr1 gene is associated with clinical failures and reduced in vivo and in vitro sensitivity to both mefloquine and artemether–lumefantrine in South-East Asia. Several African countries have reported the absence or very low prevalence of increased copy number, whilst South American reports are limited to Peru without and Venezuela with increased pfmdr1 multiplication. The relative pfmdr1 copy numbers were assessed in 68 isolates from Suriname collected from different endemic villages (2005) and from mining areas (2009). 11% of the isolates harbour multiple copies of the pfmdr1 gene. Isolates originating from mining areas do not yet display a higher tendency for increased copy number and no significant differences could be registered within a time span of 4 years, but the mere presence of increased copy number warrants caution and should be considered as an early warning sign for emerging drug resistance in Suriname and South America.


L'amplification du gène pfmdr1 est associée à des échecs cliniques et à une sensibilité réduite in vivo et in vitro à la fois à la méfloquine et à l'artéméther-luméfantrine, en Asie du sud-est. Plusieurs pays africains ont rapporté l'absence ou la très faible prévalence d'un nombre accru de copies du gène, tandis que les reports d'Amérique du sud sont limités au Pérou et au Venezuela avec une augmentation de la multiplication de pfmdr1. Les nombres relatifs de copies de pfmdr1 ont été évalués sur 68 isolats de Suriname recueillis dans différents villages endémiques (2005) et dans des zones minières (2009). 11% des isolats hébergeaient de multiples copies du gène pfmdr1. Les isolats provenant des zones minières n'affichaient pas encore une tendance plus élevée de nombre accru de copies et aucune différence significative n'a pu être enregistrée sur une période de quatre ans. Mais la simple présence d'une augmentation du nombre de copies justifie une attention et doit être considérée comme un signe d'alerte précoce pour l’émergence de résistance aux médicaments au Suriname et en Amérique du Sud.


La amplificación del gen pfmdr1 está asociado con el fallo clínico y una reducción en la sensibilidad in vivo e in vitro frente a la mefloquina y el artemeter-lumefantrina en el Sudeste Asiático. Varios países africanos han reportado la ausencia o muy baja prevalencia de un aumento en el número de copias, mientras que en Sudamérica los informes se limitan al Perú sin un aumento, y Venezuela con un aumento en la multiplicación de pfmdr1. El número relativo de copias de pfmdr1 se evaluó en 68 aislados de Surinam, recolectados en diferentes poblados endémicos (2005) y en áreas mineras (2009). Un 11% de los aislados contienen copias múltiples del gen pfmdr1. Los aislados cuyo origen son áreas mineras aún no muestran mayor tendencia a un aumento en el número de copias, ni se han registrado diferencias significativas dentro del periodo de cuatro años, pero la sola presencia de un aumento en el número de copias exige proceder con cautela y debería considerarse como una señal de advertencia temprana del posible surgimiento de resistencia a medicamentos en Surinam y Sudamérica.


Malaria control worldwide is severely impeded by the emergence of drug resistance. Plasmodium falciparum (Pf) strains are commonly resistant to chloroquine, antifolates, quinine and mefloquine (Wongsrichanalai et al. 2002). Artemisinin combination therapy (ACT), including artesunate–mefloquine and artemether–lumefantrine (AL), is currently the recommended strategy worldwide for clinical care and also for the prevention of drug resistance (WHO 2001, 2003). In January 2004, Suriname adopted the ACT regimen with AL (Coartem®; Novartis Pharma AG, Basel, Switzerland) as first-line treatment of uncomplicated P. falciparum malaria and mefloquine as prophylaxis for travellers to malaria endemic regions and as treatment for pregnant women in case of P. falciparum malaria.

Recent reports from Cambodia have indicated that resistance to the artesunate–mefloquine combination may be emerging (Dondorp et al. 2009; Rogers et al. 2009) and this might not be confined to Cambodia, as attested by a study from Thailand (Phyo et al. 2012).

Drug resistance has been associated with a variety of mutations in the P. falciparum multidrug resistance gene 1 (pfmdr1), although no association could be demonstrated between pfmdr1 loci and parasite clearance time (Takala-Harrison et al. 2013). The parasites circulating in Suriname correspond with the fully mefloquine sensitive isolates found in neighbouring Brazil, as was demonstrated in a recent molecular characterisation of pfmdr1 at codons 86, 184, 1034, 1042 & 1242 (Adhin MR, Labadie-Bracho M & Bretas G, submitted). However, an increased pfmdr1 copy number is also positively associated with decreased sensitivity to mefloquine and artemisinin derivatives in vitro (Wilson et al. 1993 Price et al. 1999; Price et al. 2004) and with AL failure (Price et al. 2006).

In this retrospective study, we investigated the pfmdr1 copy number to assess the potential threat of emerging drug resistance in Suriname. To observe differences in time, isolates were collected in two time periods, 2005 and 2009. The impact of the mobile mining population, which is assumed to be responsible for maintaining and disseminating the disease across the country (PAHO 2010), was assessed through comparison of copy number results from isolates collected in villages or mining areas.


Suriname is a small tropical country situated in the South American continent. The country is inhabited by nearly half a million people living predominantly in or around Paramaribo, the capital city (ABS 2005). The interior is occupied mostly by Amerindians and Maroons and more recently by a mobile population, mainly Brazilian immigrants, dedicated to small-scale gold mining.

We analysed pfmdr1 copy numbers in parasite DNA, derived from blood-spotted filters (3MM, Whatman) from patients with a febrile clinical infection collected during two time periods 2005 (n = 41) and 2009 (n = 27). The samples from 2005 from different endemic areas, obtained from villagers not involved in gold mining activities, were provided by Dr. S. Vreden; the samples from 2009 originated from gold mining areas and were retrieved from the National Malaria Gene Bank. All 68 patients [63% males (n = 43) and 37% females (n = 25)] had a P. falciparum mono-infection as confirmed with a positive Giemsa-stained thick blood smear. All patients had provided informed consent for molecular malaria research, and the ethics committee of the institute approved the study.

Parasite DNA was extracted from blood stored on filter paper with a modified Chelex extraction (Fischer et al. 2004).

The relative pfmdr1 copy number (CN) was assessed as described by Price et al. (2004) with minor modifications. Briefly, the pfmdr1 copy number was quantified by TaqMan real-time PCR (StepOnePlus™ Real-Time PCR system; Applied Biosystems, Foster City, CA) under the conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s and 58 °C for 60 s. Reference DNA samples (3D7, W2-mef and Dd2 with 1, 2 and 3–4 copies, respectively) were run in quadruplicates in each experiment. DNA from the reference strains was kindly provided by Dr. Venkatachelam Udhayakumar. The 12.5 μl multiplex reaction mix contained Platinum Quantitative PCR SuperMix-UGD (Invitrogen/Life Technologies, NY, USA), 300 nm of both forward and reverse primer, 150 nm of each target probe, 500 nm of ROX dye as passive reference signal and 2 μl of template DNA. The single-copy β-tubulin gene was used as endogenous control. At least two independent experiments were performed per sample, and amplifications were run in triplicates. Individual replicates were rejected if the variance was >50% of the mean estimate within the experiment. A third experiment was performed if the standard deviation in any of the two experiments was >0.3 or the average CN was between 1.3 and 1.7. Copy numbers were rounded to the nearest integer, and isolates with a mean CN estimate >1.5 copies in each experiment were considered multicopy.

Estimates were regarded as invalid if the SD in the third experiment remained >0.3. Assays were repeated with 5 times concentrated DNA if the average cycle threshold number (Ct) for P. falciparum β-tubulin was > 34. CN estimates were considered invalid if the Ct remained >34 after the third repeat with concentrated DNA.

Statistical analyses were performed using the chi-square (χ2) test.


The median age of the study population was 29.0 years (range of 4–72), with a median age of 33.0 years (range of 4–50) for the miners and 24.5 years (range of 4–72) for the villagers, respectively. No statistical significance was observed for age between both subpopulations.

The relative pfmdr1 copy number was assessed in 68 P. falciparum-positive patients. From the samples with a valid CN estimate (n = 63), 89% carried a single copy of pfmdr1, whilst multiple copies of the gene were found in 11% of the isolates (Table 1). None of the multicopy isolates exceeded two copies of the pfmdr1 gene. The success rate of quantification for samples collected in 2005 was 97.6% [40/41], attesting to the quality of the stored patient samples. 87.5% of these isolates were characterised as pfmdr1 single copy, whilst 12.5% were multicopy (Table 1). From the samples collected in the mining areas in 2009, the copy number determination had a success rate of 85.2% [23/27] with 91.3% carrying a single copy and 8.7% harbouring multiple copies of the gene. No statistical significance was observed between either population in reference to the amount of multicopy isolates nor between age and copy number.

Table 1. pfmdr1 copy number results from two time periods from malaria endemic villages and mining areas in Suriname
Study participantsSingle copyMulticopyTotal
  1. a

    Five CN estimates were invalid.

Villagers (2005)87.5% (35/40)12.5% (5/40)40
Miners (2009)91.3% (21/23)8.7% (2/23)23
Total study participants89% (56/63)11% (7/63)63a


The presence of a multiplicated pfmdr1 gene has been linked with several aspects of resistance to a variety of antimalarial drugs. Absence of or extremely low prevalence of an increased pfmdr1 copy number was found in several African countries as Papua New Guinea (Hodel et al. 2008), Malawi (Nkhoma et al. 2009), Uganda (Baliraine & Rosenthal 2011), Kenya (Holmgren et al. 2007) and Tanzania (Humphreys et al. 2007). In South America, reports on pfmdr1 copy numbers have been limited to Peru (Bacon et al. 2009), where only single-copy isolates were detected and to Venezuela, revealing 12% of multicopy isolates (Griffing et al. 2010). The observed increased pfmdr1 copy number in 11% of isolates from Suriname is to our knowledge the second study to report that the pfmdr1 copy number is also increasing in South America.

The revealed combination of increased copy number with the fixed presence of the pfmdr1 N86 allele is in contrast to results obtained from Malawi (Nkhoma et al. 2009), where a high prevalence of pfmdr1 N86 was accompanied by a virtual absence of pfmdr1 amplification, and with results from Sudan (Gadalla et al. 2011), where the isolates with a multiplicated pfmdr1 gene displayed the mutated pfmdr1 N86Y allele. Our results, however, are in accordance with the haplotypes circulating in Cambodia (Lim et al. 2009).

Although gold miners are believed to be reservoirs of malaria resistance, we did not yet detect a higher tendency for increased copy number in isolates originating from mining areas. Also, no significant increase in copy number was registered over 4 years and none of the isolates exceeded two copies. We should, however, note that the sample size is small, the time span is limited and the comparisons of non-overlapping years and mutually exclusive populations can only be justified because of the absence of significant changes.

These copy number results provide more differentiation between isolates from Suriname because earlier studies on single-nucleotide polymorphisms in the pfATP6 gene (Adhin et al. 2012), the pfmdr1 gene and the pfcrt gene (Adhin MR, Labadie-Bracho M & Bretas G, submitted) revealed the existence of a widely distributed monomorphic genotype in Suriname. Because this molecular profile resembles the haplotypes circulating in neighbouring Brazil (Zalis et al. 1998) and Guyana (Plummer et al. 2004), these copy number results not only serve as an early warning sign for the malaria management in Suriname, but may also signal emerging resistance in South America.

The current finding of increased copy number in conjunction with the recently observed increased incidence of day 3 parasitaemia in Suriname after AL treatment (Vreden SGS, Jitan JK, Bansie RD & Adhin MR, submitted) and the steady presence of the pfmdr1 N86 wild-type allelic form, which is believed to be the first in a series of mutations leading to artemisinin resistance (Hastings & Ward 2005), highlights the importance of a vigilant surveillance in Suriname to monitor emerging of antimalarial resistance.


This work was supported by PAHO's Amazon Network for the Surveillance of Antimalarial Drug Resistance in cooperation with the United States Agency for International Development's Amazon Malaria Initiative. The views expressed herein are those of the authors and do not necessarily reflect the opinions of the PAHO, USAID and RAVREDA.