# Molecular monitoring of plasmodium falciparum drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaoundé, Cameroon

Sandie Ménard, Isabelle Morlais, Rachida Tahar, Collins Sayang, Pembe Mayengue, Xavier Iriart, Françoise Benoit-Vical, Brigitte Lemen, Jean-François Magnaval, Parfait H Awono-Ambéné, et al.

► To cite this version:

Sandie Ménard, Isabelle Morlais, Rachida Tahar, Collins Sayang, Pembe Mayengue, et al. Molecular monitoring of plasmodium falciparum drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaoundé, Cameroon: Implications for the future. Malaria Journal, 2012, 11 (1), pp.113. 10.1186/1475-2875-11-113. ird-02896995

HAL Id: ird-02896995

https://ird.hal.science/ird-02896995

Submitted on 7 Jun 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Molecular monitoring of *Plasmodium falciparum* drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaoundé, Cameroon: Implications for the future

Sandie Menard¹, Isabelle Morlais²,³, Rachida Tahar³,⁴, Collins Sayang³,⁵, Pembe Issamou Mayengue⁶, Xavier Iriart¹, Françoise Benoit-Vical⁶, Brigitte Lemen⁷, Jean-François Magnaval⁸, Parfait Awono-Ambene³, Leonardo K Basco³,⁹ and Antoine Berry¹*

**Abstract**

**Background:** Regular monitoring of the levels of anti-malarial resistance of *Plasmodium falciparum* is an essential policy to adapt therapy and improve malaria control. This monitoring can be facilitated by using molecular tools, which are easier to implement than the classical determination of the resistance phenotype. In Cameroon, chloroquine (CQ), previously the first-line therapy for uncomplicated malaria was officially withdrawn in 2002 and replaced initially by amodiaquine (AQ) monotherapy. Then, artemisinin-based combination therapy (ACT), notably artesunate-amodiaquine (AS-AQ) or artemether-lumefantrine (AL), was gradually introduced in 2004. This situation raised the question of the evolution of *P. falciparum* resistance molecular markers in Yaoundé, a highly urbanized Cameroonian city.

**Methods:** The genotype of *pfcrt* 72 and 76 and *pfmdr1* 86 alleles and *pfmdr1* copy number were determined using real-time PCR in 447 *P. falciparum* samples collected between 2005 and 2009.

**Results:** This study showed a high prevalence of parasites with mutant *pfcrt* 76 (83%) and *pfmdr1* 86 (93%) codons. On the contrary, no mutations in the *pfcrt* 72 codon and no samples with duplication of the *pfmdr1* gene were observed.

**Conclusion:** The high prevalence of mutant *pfcrt* 76T and *pfmdr1* 86Y alleles might be due to the choice of alternative drugs (AQ and AS-AQ) known to select such genotypes. Mutant *pfcrt* 72 codon was not detected despite the prolonged use of AQ either as monotherapy or combined with artesunate. The absence of *pfmdr1* multicopies suggests that AL would still remain efficient. The limited use of mefloquine or the predominance of mutant *pfmdr1* 86Y codon could explain the lack of *pfmdr1* amplification. Indeed, this mutant codon is rarely associated with duplication of *pfmdr1* gene. In Cameroon, the changes of therapeutic strategies and the simultaneous use of several formulations of ACT or other anti-malarials that are not officially recommended result in a complex selective pressure, rendering the prediction of the evolution of *P. falciparum* resistance difficult. This public health problem should lead to increased vigilance and regular monitoring.

**Keywords:** Malaria, Cameroon, *pfcrt*, *pfmdr1*, *pfmdr1* copy number, Resistance, LNA probes

<footnote>* Correspondence: berry.a@chu-toulouse.fr

¹Service de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Toulouse et UMR152 UPS-IRD, Université de Toulouse, Toulouse, France
Full list of author information is available at the end of the article</footnote>
**Background**

Monitoring the level of *Plasmodium falciparum* resistance against anti-malarial drugs is one of the keys to a successful malaria control. Although controlled clinical trials are the best available tool for assessing the relevance of anti-malarial treatments, molecular monitoring offers some advantages. Studies on single-nucleotide polymorphisms (SNPs) and duplication of genes involved in resistance can be carried out with more ease and are less time-consuming. Furthermore, molecular monitoring may reveal trends, allowing anticipation in the changes of treatment policies.

In Cameroon, the first-line recommended therapy for uncomplicated malaria was chloroquine (CQ) until 2002 and amodiaquine (AQ) monotherapy between 2002 and 2004. In January 2004, the artesunate-amodiaquine (AS-AQ) combination was officially adopted and artemether-lumefantrine (AL) was added as an alternative artemisinin-based combination therapy (ACT) in 2006 [1]. In practice, AS-AQ and AL have been used nationwide since 2007. AS-AQ is widely available in public health care centres while AL is relatively less prescribed because of its low supply in the public sector at a subsidized price [2].

The amplification of *pfmdr1* gene is a common molecular marker of mefloquine (MQ) resistance. An increase in the *pfmdr1* copy number is associated with clinical failures to MQ [3] and *in vitro* resistance to lumefantrine, which is an amino-alcohol, like MQ [4]. The amplification of *pfmdr1* gene has also been demonstrated to decrease the susceptibility to artemisinin derivatives in the field as well as in laboratory-adapted *P. falciparum* strains [4-8]. Furthermore, a recent study in eastern Sudan reported an association between the carriage of parasites with increased *pfmdr1* copy number before treatment and recurrent parasitaemia after AL therapy [9].

*In vitro*, the *pfmdr1* N86 wild-type allele, independently of the *pfmdr1* copy number, is associated with a higher susceptibility to lumefantrine and MQ [3,10,11]. In parallel, in the field, *pfmdr1* N86 and *pfcrt* K76 wild-type alleles were selected by artemether-lumefantrine (AL) treatment whereas they were not selected by artesunate-amodiaquine (AS-AQ) or amodiaquine-sulphadoxine-pyrimethamine (AQ-SP) [12-15]. Conversely, the *pfmdr1* 86Y and *pfcrt* 76T mutant alleles are associated with CQ resistance and also with AQ monotherapy failure [16-19]. Likewise, these haplotypes are selected by the association AS-AQ [20,21].

A *pfcrt* genotype conferring high levels of resistance to AQ, corresponding to SVMNT haplotype of the codons 72-76, has been identified, first in Tanzania and more recently in Angola [22,23]. This haplotype, widely observed in Asia and South America, seems to be strongly selected by the use of AQ [24,25].

The objective of this study was to determine the prevalence of *pfmdr1* multiple copies and mutant *pfcrt* 72

and 76 and *pfmdr1* 86 codons in Yaoundé, Cameroon at the time of the introduction of ACT. It is important to have a “molecular snapshot” of *P. falciparum* isolates at the beginning of this new anti-malarial therapeutic strategy, first, in order to make meaningful comparisons in the future and, secondly, to determine if there is any evidence of molecular mark suggesting a rapid evolution towards resistance.

**Methods**

**Study sites and origin of samples**

The study was carried out between 2005 and 2009, on a total of 447 samples from patients with a microscopy-confirmed diagnosis of uncomplicated *falciparum* malaria. The recruitment sites were in Yaoundé *intra-muros* (3° 52' N, 11° 31' E), including the healthcare centre of Nkoln-dongo (49 patients, median of three years old [0 month to 47 years]), the healthcare centre of Olembe (42 children, median of 2.5 years old [eight months to 12 years]), and the healthcare centre of Nlongkak (125 patients, median of two years old [six months to five years]). Two hundred and thirty-one samples were obtained from asymptomatic children aged from five to 11 years in Mfou (3°43'N, 11° 38'E), 26 km from the centre of Yaoundé. This study was reviewed and approved by the Cameroonian National Ethics Committee and Cameroonian Ministry of Public Health.

Before patients with a positive thick smear have received an ACT treatment, finger-pricked capillary blood sample was collected on different filter papers, Whatman (Whatman plc, Middlesex, UK) or IsoCode STIX (Schleicher & Schuell, Keene, NH, USA). DNA from paper filters was extracted using the chelex-100 boiling method [26], concentrated by ethanol precipitation and frozen at -20°C until amplification.

**Determination of pfmdr1 copy number**

To determine the copy number of *pfmdr1*, a qPCR method described previously was used [27]. All samples were tested in triplicate in 96-well plates on a LightCycler® 480 system (Roche Diagnostics, Neuilly sur Seine, France). Each run included two control DNA samples of reference *P. falciparum* clones, FCM29/Cameroon and Dd2/Indochina, which are known to have one and two-three copies of *pfmdr1* gene, respectively [27].

**Genotyping of pfcrt and pfmdr1**

Genotyping of *pfcrt* 76 and *pfmdr1* 86 codons was performed with a qPCR assay using Fluorescence Resonance Energy Transfer (FRET) hybridization probes and an analysis of the melting curve described previously [28,29]. Each run included two control DNA samples of *P. falciparum*: the CQ-susceptible F32/Tanzania strain with *pfcrt* K76 and *pfmdr1* N86 wild-type alleles and the
CQ-resistant FCM29/Cameroon clone, carrying *pfcrt* 76T and *pfmdr1* 86Y mutant alleles.

The detection of the *pfcrt* 72S mutant allele was
performed with a TaqMan probe-based genotyping assay
the originality of which resides in the presence of Locked
Nucleic Acid (LNA) inside the probes. LNA is a synthetic
RNA analogue which, when integrated into an oligonu-
cleotide, shows a strong affinity for their complementary
targets [30]. Because of their thermal stability when
hybridized to DNA, oligonucleotides containing LNA
have a higher melting temperature (T_m) and could be
used as primers, probes or clamps to improve molecular
detection [31-33]. In general, sequences from *P. falciparum* contain a high percentage of adenine (A) and thymine (T) resulting in a low Tm and complicating molecular analysis. The introduction of LNA bases is a powerful tool to obtain discriminating probes with a moderate length and a probe hybridization that may occur during the annealing step in PCR. Consequently, this technique was particularly well suited for the experimental conditions described here. The *pfcrt* gene was amplified by using primers P.falcA (5'-CAATT TTgTTTAAAgTTCTTTTAgCAA-3') and P.falcF (5'-gTTCTTGgTCTTggTAAATgTgCTCA-3'). To genotype the different alleles, the amplified product was detected with one of the specific TaqMan probes: AF233067 probe, 5'-YAK-AATTgTATTCATT + A + C + ACTT + A + CA-BBQ-3' hybridized with the *pfcrt* 72S mutant allele (SVMNT haplotype) and HM854027 probe, 5'-LC670-AATTgTTTCAATT + A + C + ACATA + CA-BBQ-3' hybridized with the *pfcrt* C72 wild-type allele (CVIET haplotype) (the presence of a LNA nucleotide is preceded by the sign +). The primers and probes were designed in collaboration with Tib MolBiol Syntheselabor (Berlin, Germany). Master mixes contained 1 µl GeneAmp® 10 × PCR Gold Buffer (Applied Biosystems, Branchburg, NJ), 2.5 mM MgCl₂, 200 µM pooled dNTP, 500 nM of forward and reverse primers, 250 nM of each probe, 1 U per reaction of AmpliTaq® Gold DNA Polymerase (Applied Biosystems) and 2 µl of template DNA for a total reaction volume of 10 µl. Each reaction plate was run with control DNA samples of *P. falciparum*, in particular the 7G8/Brazil strain known to harbour the *pfcrt* 72S mutated allele [34], F32/Tanzania and FCM29/ Cameroon as *pfcrt* C72 wild-type control [28], water and DNA of healthy patient, which served as negative external amplification controls. The multiplex TaqMan assay reactions were carried out in a LightCycler® 480 Multiwell Plate 384 (Roche Diagnostics) with the following PCR programme: an initial step at 95°C for 12 minutes followed by 45 cycles of 10 seconds at 95°C and 45 seconds at 60°C. Data analysis for allelic discrimination was performed with the LightCycler® 480 software (Roche Diagnostics).

**Statistical analysis**

The proportions were compared using $\chi^2$ test thanks to SigmaStat® software. The significance level (*P*) was fixed at 0.05.

**Results**

*Pfmdr1* copy number

The copy number of *pfmdr1* was determined for only 215 isolates from healthcare centres of central Yaoundé because of the limited amount of DNA samples from Mfou. Regardless of where the tested isolates were collected, none of them were identified with *pfmdr1* gene amplification (Table 1). The estimated gene copy number from all analysed isolates was close to 1, with an average copy number of 1.05 and a standard deviation of 0.20 (data not shown).

Pfmdr1 and pfcrt genotypes

The prevalence of *pfmdr1* and *pfcrt* alleles in blood sam-
ples obtained from different sites in Yaoundé is presented
in Table 1. The frequencies of the *pfmdr1* 86Y mutant
genotype were 76% (153/201) and 84% (175/209) in
Yaoundé and Mfou, respectively. Wild-type *pfmdr1* N86
genotype was observed in 10% (20/201) and 4% (9/209) of
isolates, and 14% (28/201) and 12% (25/209) of isolates
presented a mixed genotype in Yaoundé and Mfou,
respectively. No significant differences were observed
between Yaoundé and Mfou.

The frequencies of *pfcrt* 76T mutant genotype were
71% (145/203) and 55% (125/229), the *pfcrt* K76 wild-
type allele was present in 19% (38/203) and 15% (35/229)
and mixed *pfcrt* alleles occurred in 10% (20/203) and 30%
(69/229) of the isolates in Yaoundé and Mfou, respec-
tively, with a significant difference (p < 0.001).

Contrary to *pfcrt* 76, a significant difference (p =
0.042) of the distribution of the alleles was observed
between Yaoundé and Mfou when all samples with
mixed *pfmdr1* 86 genotype are classified in mutant
group.

No significant differences were observed either
between the different healthcare centres of Yaoundé or
between the different times of sample collection (data
not shown).

None of the 414 samples tested for the codon 72 of *pfcrt*
gene was found with the mutant 72S allele (SVMNT
haplotype).

**Discussion**

As elsewhere in the world, a very rapid development of
resistance to anti-malarial drugs in Africa requires a reg-
ular monitoring in multiple and strategic points. With
53% of the population living in cities against 38% WHO
African region, Cameroon is a highly urbanized African
country [35]. This demonstrates the importance of
<table><caption>Table 1 Frequency of mutations and/or gene amplification in anti-malarial resistance markers *pfmdr1* and *pfcrt* in *Plasmodium falciparum* isolates in Yaoundé and Mfou, Cameroon</caption><thead><tr><th rowspan="2">Genes and alleles</th><th colspan="3">Number of samples (%)</th><th colspan="2">P</th></tr><tr><th>Both sites</th><th>Yaoundé</th><th>Mfou<br>(Suburb of Yaoundé)</th><th></th><th></th></tr></thead><tbody><tr><td><strong>pfmdr1 amplification (n<sup>a</sup> = 215, n<sup>b</sup> = NA)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1 <i>pfmdr1</i> copy number</td><td>215</td><td>(100%)</td><td>215</td><td>(100%)</td><td>NA</td></tr><tr><td>&gt; 1 <i>pfmdr1</i> copy number</td><td>0</td><td>(0%)</td><td>0</td><td>(0%)</td><td>NA</td></tr><tr><td><strong>pfmdr1 codon 86 (n<sup>a</sup> = 201, n<sup>b</sup> = 209)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mutant 86Y haplotype only</td><td>328</td><td>(80%)</td><td>153</td><td>(76%)</td><td>175 (84%)</td></tr><tr><td>Mixed N86 and 86Y haplotypes</td><td>53</td><td>(13%)</td><td>28</td><td>(14%)</td><td>25 (12%)</td></tr><tr><td>Wild-type N86 haplotype only</td><td>29</td><td>(7%)</td><td>20</td><td>(10%)</td><td>9 (4%)</td></tr><tr><td><strong>Total mutant 86Y haplotype<sup>c</sup></strong></td><td><strong>381</strong></td><td><strong>(93%)</strong></td><td><strong>181</strong></td><td><strong>(90%)</strong></td><td><strong>200</strong> (96%)</td></tr><tr><td><strong>pfcrt codon 76 (n<sup>a</sup> = 203, n<sup>b</sup> = 229)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mutant 76T haplotype only</td><td>270</td><td>(62%)</td><td>145</td><td>(71%)</td><td>125 (55%)</td></tr><tr><td>Mixed K76 and 76T haplotypes</td><td>89</td><td>(21%)</td><td>20</td><td>(10%)</td><td>69 (30%)</td></tr><tr><td>Wild-type K76 haplotype only</td><td>73</td><td>(17%)</td><td>38</td><td>(19%)</td><td>35 (15%)</td></tr><tr><td><strong>Total mutant 76T haplotype<sup>c</sup></strong></td><td><strong>359</strong></td><td><strong>(83%)</strong></td><td><strong>165</strong></td><td><strong>(81%)</strong></td><td><strong>194</strong> (85%)</td></tr><tr><td><strong>pfcrt codon 72 (n<sup>a</sup> = 202, n<sup>b</sup> = 212)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mutant 72S haplotype</td><td>0</td><td>(0%)</td><td>0</td><td>(0%)</td><td>0 (0%)</td></tr><tr><td>Wild-type C72 haplotype</td><td>414</td><td>(100%)</td><td>202</td><td>(100%)</td><td>212 (100%)</td></tr></tbody></table>

NA: not analysable because of the limited amount of DNA samples

NS: non significant

a: Number of total samples tested in the centre of Yaoundé

b: Number of total samples tested in the suburb of Yaoundé

c: Total mutant haplotype regroups single mutant haplotype and mixed haplotype, all samples with mixed genotype for the considered allele are classified in mutant group.

d: comparison of total mutant haplotypes versus wild-type haplotypes

e: comparison of mutant, wild-type and mixed haplotypes

epidemiological studies in large cities such as Yaoundé, which currently has a population of over 1,800,000 inhabitants.

In the present study, a high prevalence of mutations associated with drug resistance was found in Yaoundé and its suburbs in both codon 76 of the *pfcrt* gene (83%) and codon 86 of the *pfmdr1* gene (93%) when all samples with mixed genotype were classified as mutant (Table 1). These results are in agreement with several other studies. Previous works of Basco *et al* carried out in Yaoundé showed that 70% of 157 *P. falciparum* clinical isolates had the mutant *pfcrt* 76T codon in 2001 [36], and a large majority of isolates (88% of 64) carried the *pfmdr1* 86Y mutant allele between 1997 and 2000 [37]. Similarly, Mbacham *et al* reported 77% and 76% prevalence of mutant *pfcrt* 76T and *pfmdr1* 86Y codons, respectively, in samples collected during the period 2004-2006 in Yaoundé [38]. Despite different classification of double populations and techniques with different sensitivity, the prevalence of mutations appears to increase (*pfcrt*) or remains at a high and relatively stable level (*pfmdr1*) until 2009 in spite of the official withdrawal of CQ from Cameroon in 2002. In some endemic areas, stopping the

widespread use of CQ resulted in a return of chloroquine-sensitivity associated with the reappearance of wild-type genotypes. In the absence of drug pressure, *P. falciparum* wild-type haplotypes have a selective advantage over mutants. For example, in 1993, Malawi was the first sub-Saharan African country to replace CQ with SP nationwide in response to the high rates of CQ-resistant *falciparum* malaria. This change resulted in a decrease in the prevalence of the mutant *pfcrt* haplotype associated with CQ resistance from 85% in 1992 to 13% in 2000. For *pfmdr1* 86Y mutant codon, the same study showed similar results but with lower amplitude (from about 60% in 1993 to around 20% in 2000) [39]. The recovery of CQ-sensitivity phenotype and genotype was also observed elsewhere in Malawi [40], Kenya [41] and China [42].

This stability of mutant *pfcrt* 76T and *pfmdr1* 86Y genotypes observed in Yaoundé and suburb may be the result of many factors. First of all, the choice of the replacement treatment logically influences the type of selected isolates. The use of SP, which has no influence on the selection of mutant *pfcrt* and *pfmdr1* genotypes, has been shown to favour, by a phenomenon of selective
advantage, the reappearance of CQ-sensitive isolates
harbouring wild-type *pfcrt* K76 and *pfmdr1* N86 geno-
types [39-41]. The use of AL or artesunate-mefloquine
(AS-MQ) seems to favour the return to the predomi-
nance of wild-type *pfmdr1* N86 genotype and, to a lesser
extent, to the wild-type *pfcrt* K76 genotype by an active
selection [14,43-45]. Inversely, AQ, a close Mannich
base analogue of CQ, or AS-AQ promotes the mainte-
nance of CQ-resistant isolates with the mutant *pfcrt* and
*pfmdr1* genotypes by an active selective pressure [20,46],
as observed in the present study. Whereas in East Afri-
can countries like Malawi or Kenya, SP or AL had lar-
gely replaced CQ [47], in Yaoundé, in 2005, AQ was
still prescribed as a first-line anti-malarial drug in 20%
and 63% of adults and children under five years old,
respectively [2], and AS-AQ in 4.5% and 1.5%. AL was
used only in 8.3% and 2.4%, AS-MQ in 1.5% and 0.8%,
and SP in 5.8% and 0% of adults and children less than
five years old, respectively [2].

Secondly, the changes of *P. falciparum* resistance phe-
notype and genotype after the withdrawal of CQ depend
on the rapidity of drug replacement. For example, in
Malawi where a profound and rapid return to CQ sensi-
bility was observed, the change in drug policy from CQ
to SP was swift and efficient, so that SP became the only
available anti-malarial drug in less than one year after the
implementation of the new drug policy. In contrast, these
changes were progressive and lasted several years in
many areas as in Cameroon. In fact, in Yaoundé,
although the National Malaria Control Programme of
Cameroon had replaced CQ by AQ in 2002 and then AQ
monotherapy by AS-AQ since January 2004, CQ was still
largely accessible through the informal outlets (e.g. food
market) in August 2005 [2].

Finally, in a more general way, fitness loss of mutant
*P. falciparum* might be associated with the development
of compensatory mechanisms able to maintain mutant
parasites even in the absence of drug pressure [48]. This
feature might explain, at least in part, the persistence of
mutant *pfcrt* codon in Southeast Asia and South America
[49-51] and also in Cameroon, as described here.

In Mfou, a higher frequency of mixed *pfcrt* haplotypes was observed at the expense of mutant *pfcrt* population. This observation was not done for *pfmdr1* haplotypes. A possible reason for this observation is a drug pressure selection different from that existing in Yaoundé.

Since the probes used to detect the mutation in codon
76 of *pfcrt* gene were not able to detect that of codon 72, a
new technique using LNA probes was developed in the
present study to discriminate the mutant SVMNT haplo-
type (72S mutation) from the wild-type CVIET haplotype
(C72 wild-type). Previous studies and data collected from
countries like Bolivia or India suggested that AQ has an
early and prominent role in the selection of parasites

carrying SVMNT haplotype associated with drug resistance [24]. These parasites are highly resistant to AQ, but only moderately resistant to CQ. Contrary to CVIET haplotype, once the SVMNT haplotype emerges in a given parasite population and CQ and AQ are removed, the repopulation of sensitive strains may be very slow to occur [24]. As the SVMNT haplotype was recently described in Tanzania and Angola [22,23], it was important to verify whether this haplotype existed in Yaoundé. None of the samples tested for the codon 72 of *pfcrt* was found to carry the SVMNT haplotype. These results are contrary to what was observed in nearby African countries, such as in Ghana [52], Tanzania [22] and Angola [23] where the prevalence of this haplotype was between 3.9% and over 50%. It is possible that the observed predominance of SVMNT haplotype in Angola is the result of frequent travels of Brazilian and Angolan citizens between the two countries [23], which is not the case in Cameroon. However, the monitoring of *pfcrt* codons 72-76 should be pursued because AQ has long been prescribed in Cameroon before and since the cessation of the use of CQ (2002) and until 2005 [2] and seems to have an important role in the selection of the SVMNT haplotype [22].

The amplification of *pfmdr1* gene has been more closely
linked to MQ and halofantrine (HAL) resistance [53-55].
In this study, *pfmdr1* amplification was not observed in
Yaoundé between 2005 and 2009. Elsewhere in Africa, the
situation seems to be contrasted. In various studies con-
ducted in East Africa, only four samples were found with
*pfmdr1* gene duplication, one in Kenya and three in Sudan
(near the Ethiopian border) among 475 isolates tested (57
in Sudan [9], 72 in Kenya [46], 186 in Zanzibar [53] and
160 in Malawi [54]). In West Africa, on the one hand,
none of 580 samples tested in Liberia and Guinea-Bissau
between 1981 and 2005 was found to be duplicated [55];
on the other hand, two studies had identified in Burkina
Faso, Ivory Coast, Togo and Madagascar, six *pfmdr1*
duplications among 112 samples tested [27,56]. In Central
Africa, data are limited since only 32 samples were
screened and all of them had a single copy of *pfmdr1* gene
[27]. In this region, an exception is the study of Uhlemann
et al who found the duplication of *pfmdr1* gene in five of
62 clinical isolates tested (8%) in 1995 in Lambaréné,
Gabon [57]. Four of these five patients harboured the
wild-type N86 *pfmdr1* codon even though during this per-
iod 90% of isolates carried the mutant *pfmdr1* codon 86
around Lambaréné [58]. However, in 2002 at the same
study site, none of 37 samples tested had *pfmdr1* gene
duplication. These observations on *pfmdr1* gene amplifica-
tion in Lambaréné are difficult to explain outside of the
possible selection of such a clone by previous clinical trials
on the same site, using low dose of mefloquine [58-60].
Nevertheless, these data showed that *P. falciparum* isolates
from Central Africa can have *pfmdr1* gene duplication.
The lack of *pfmdr1* gene duplication in Yaoundé may
possibly be due to the very low use of MQ or HAL,
which represented only 1.5% of first-line treatments
against malaria in 2005 [2], but also partly to the high
prevalence of the *pfmdr1* Y86 mutant allele. Indeed, in
Southeast Asia, *pfmdr1* amplification has been suggested
to be incompatible in the presence of the mutant
*pfmdr1* 86Y allele [61]. However, the conclusion of that
Asian study has not been confirmed in Africa, where
the existence of parasites harbouring a duplicated
*pfmdr1* gene with mutant 86Y codon has been reported
from Sudan [9], Gabon [57] and Ivory Coast [27,56].

The molecular analysis performed in the present study
did not find any *pfcrt* 72S mutation, which may be a
good sign for the continued use of AQ in combination
with AS. A regular evaluation of AS-AQ efficacy, in par-
allel with molecular surveillance, is required to ensure
the utility of AS-AQ in Cameroon. This ACT contri-
butes to the maintenance of a high prevalence of mutant
*pfcrt* 76T and *pfmdr1* 86Y alleles. The pressing question
is to predict how these parasites will evolve in the pre-
sence of AL pressure. Several scenarios can be envi-
sioned. Firstly, they could behave like Southeast Asian
isolates and will not progress to the duplication of
*pfmdr1* gene in the absence of wild-type *pfmdr1* N86
allele. Secondly, as already observed in some cases in
Africa [9,27,56,57], the parasites may acquire multico-
pies of *pfmdr1* despite the *pfmdr1* 86Y mutation. Only
regular and exhaustive molecular monitoring of *P. falcى-
parum* clinical isolates can provide the answer. How-
ever, the relevance of these results would be improved if
they were associated with information on different anti-
malarial drugs that are really taken by the patients
because these data often differ from the current national
recommendation.

Abbreviations

ACT: Artemisinin-based combination therapy; AL: Artemether-lumefantrine;
AQ: Amodiaquine; AQ-SP: Amodiaquine-sulphadoxine-pyrimethamine; AS:
Artesunate; AS-AQ: Artesunate-amodiaquine; AS-MQ: Artesunate-mefloquine;
CQ: Chloroquine; FRET: Fluorescence resonance energy transfer; HAL:
Halofantrine; LNA: Locked nucleic acid; MQ: Mefloquine; *pfcrt*: *Plasmodium*
*falciparum* chloroquine resistance transporter; *pfmdr1*: *Plasmodium falciparum*
multidrug resistance gene 1; qPCR: Quantitative polymerase chain reaction;
SNP: Single-nucleotide polymorphism; SP: Sulphadoxine-pyrimethamine; Tm:
Melting temperature.

Acknowledgements

The authors thank the patients and health staff of different health care
centres in Yaoundé and Mfou for their participation in the study. The
authors also thank Odile Mercereau-Puijalon for generously providing 7G8/
Brazil DNA. This work was supported by the Service de Coopération et
d'Action Culturelle Français (SCAC) au Cameroun/Ministère des Affaires
Etrangères Français (MAE), the European Union (Redox Antimalarial Drug
Discovery, READ-UP, FP6-2004-LSH-2004-2.3.3-7, STREP no. 018602), and
French Agence Nationale de la Recherche (project RES-ATQ, ANR-08-MIE-
024). CS was supported by a PhD grant of the Agence Universitaire de la
Francophonie.

Author details

¹Service de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Toulouse et UMR152 UPS-IRD, Université de Toulouse, Toulouse, France.

²Institut de Recherche pour le Développement, UMR MIVEGEC, Montpellier, France. ³Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), Yaoundé, Cameroon. ⁴Unité Mixte de Recherche 216, Institut de Recherche pour le Développement (IRD), Université Paris Descartes, Laboratoire de Parasitologie, Faculté de Pharmacie, Paris, France. ⁵Centre de Formation et de Recherche en Médecine et Santé Tropicale, Faculté de Médecine Nord, Université de la Méditerranée, Marseille, France. ⁶CNRS, LCC (Laboratoire de Chimie de Coordination), et Université de Toulouse Paul Sabatier, UPS, INPT, LCC, Toulouse, France. ⁷Centre d'Animation Sociale et Sanitaire (CASS), Yaoundé, Cameroon. ⁸CNRS UMR 5288 Anthropobiology, Université de Toulouse, Toulouse, France. ⁹Unité Mixte de Recherche 198, Institut de Recherche pour le Développement (IRD), Faculté de Médecine La Timone, Université de la Méditerranée, Marseille, France.

Authors' contributions

SM, PIM, carried out the molecular genetic studies. SM, AB, XI conceived and designed the study protocol and analysed the results. SM, AB, FBV, LKB drafted the manuscript. JFM, FBV, XI participated in its design and helped to draft the manuscript. RT, CS, LKB and BL participated in study design, supervised clinical and laboratory diagnosis in the health care centres, and collected blood samples in Nlongkak, Olembe, and Nkolndongo, IM and PAA had the same role for the blood samples collected in Mfou. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 24 January 2012 Accepted: 12 April 2012

Published: 12 April 2012

References

1. WHO: World Malaria Report 2008. 2008 [http://www.who.int/malaria/publications/atoz/9789241563697/en/index.html].

2. Sayang C, Gausseres M, Vernazza-Licht N, Malvy D, Bley D, Millet P:
Treatment of malaria from monotherapy to artemisinin-based
combination therapy by health professionals in urban health facilities in
Yaounde, central province, Cameroon. *Malar J* 2009, 8:176.

3. Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, Patel R, Laing K, Looareesuwan S, White NJ, Nosten F, Krishna S: Mefloquine resistance in *Plasmodium falciparum* and increased *pfmdr1* gene copy number. *Lancet* 2004, 364:438-447.

4. Sidhu AB, Uhlemann 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, 194:528-535.

5. Lim P, Alker AP, Khim N, Shah NK, Incardona S, Doung S, Yi P, Bouth DM, Bouchier C, Puijalon OM, Meshnick SR, Wongsrichanalai C, Fandeur T, Le Bras J, Ringwald P, Ariey F: *Pfmdr1* copy number and artemisinin derivatives combination therapy failure in *falciparum* malaria in Cambodia. *Malar J* 2009, 8:11.

6. Carrara VI, Zwang J, Ashley EA, Price RN, Stepniewska K, Barends M, Brockman A, Anderson T, McGready R, Phaiphun L, Poux S, Van Vugt M, Hutagalung R, Maung Lwin K, Pyae Phyo A, Preechapornkul P, Imwong M, Pukrittayakamee S, Singhasivanon P, White NJ, Nosten F: *Changes in the treatment responses to artesunate-mefloquine on the northwestern border of Thailand during 13 years of continuous deployment*. *PLoS One* 2009, 4:e4551.

7. Begum K, Kim HS, Okuda Y, Wataya Y, Kimura M, Huruta T: *Genomic analysis of mefloquine-resistant *Plasmodium falciparum*. Nucleic Acids Res Suppl* 2002, 2:223-224.

8. Teuscher F, Gatton ML, Chen N, Peters J, Kyle DE, Cheng Q: *Artemisinin-induced dormancy in *Plasmodium falciparum*: duration, recovery rates, and implications in treatment failure. *J Infect Dis* 2010, 202:1362-1368.

9. Gadalla NB, Adam I, Elzaki SE, Bashir S, Mukhtar I, Oguke M, Gadalla A, Mansour F, Warhurst D, El-Sayed BB, Sutherland CJ: Increased *pfmdr1* Copy Number and Sequence Polymorphisms in *Plasmodium falciparum*
Isolates from Sudanese Malaria Patients Treated with Artemether-Lumefantrine. *Antimicrob Agents Chemother* 2011, **55**:5408-5411.

10. Sidhu AB, Valderramos SG, Fidock DA: *pfmdr1* mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in *Plasmodium falciparum*. *Mol Microbiol* 2005, **57**:913-926.

11. Duraimingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC: The tyrosine-86 allele of the *pfmdr1* gene of *Plasmodium falciparum* is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. *Mol Biochem Parasitol* 2000, **108**:13-23.

12. Martensson A, Stromberg J, Sisowath C, Msellem MI, Gil JP, Montgomery SM, Olliaro P, Ali AS, Bjorkman A: Efficacy of artesunate plus amodiaquine versus that of artemether-lumefantrine for the treatment of uncomplicated childhood *Plasmodium falciparum* malaria in Zanzibar, Tanzania. *Clin Infect Dis* 2005, **41**:1079-1086.

13. Baliraine FN, Rosenthal PJ: Prolonged selection of *pfmdr1* polymorphisms after treatment of *falciparum* malaria with artemether-lumefantrine in Uganda. *J Infect Dis* 2011, **204**:1120-1124.

14. Sisowath C, Stromberg J, Martensson A, Msellem M, Obondo C, Bjorkman A, Gil JP: *In vivo* selection of *Plasmodium falciparum* *pfmdr1* 86 N coding alleles by artemether-lumefantrine (Coartem). *J Infect Dis* 2005, **191**:1014-1017.

15. Some AF, Sere YY, Dokomajilar C, Zongo I, Rouamba N, Greenhouse B, Ouedraogo JB, Rosenthal PJ: Selection of known *Plasmodium falciparum* resistance-mediating polymorphisms by artemether-lumefantrine and amodiaquine-sulfadoxine-pyrimethamine but not dihydroartemisinin-piperazine in Burkina Faso. *Antimicrob Agents Chemother* 2010, **54**:1949-1954.

16. Ochong EO, van den Broek IV, Keus K, Nzila A: Short report: association between chloroquine and amodiaquine resistance and allelic variation in the *Plasmodium falciparum* multiple drug resistance 1 gene and the chloroquine resistance transporter gene in isolates from the upper Nile in southern Sudan. *Am J Trop Med Hyg* 2003, **69**:184-187.

17. Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A: *Amodiaquine resistant Plasmodium falciparum* malaria *in vivo* is associated with selection of *pfcrt* 76T and *pfmdr1* 86Y. *Infect Genet Evol* 2006, **6**:309-314.

18. Happi CT, Gbotosho GO, Folarin OA, Bolaji OM, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AM: Association between mutations in *Plasmodium falciparum* chloroquine resistance transporter and *P. falciparum* multidrug resistance 1 genes and *in vivo* amodiaquine resistance in *P. falciparum* malaria-infected children in Nigeria. *Am J Trop Med Hyg* 2006, **75**:155-161.

19. Tinto H, Guekoun L, Zongo I, Guiguemde RT, D'Alessandro U, Ouedraogo JB: Chloroquine-resistance molecular markers (*Pfcrt* T76 and *Pfmdr-1* Y86) and amodiaquine resistance in Burkina Faso. *Trop Med Int Health* 2008, **13**:238-240.

20. Djimde AA, Fofana B, Sagara I, Sidibe B, Toure S, Dembele D, Dama S, Ouologuem D, Dicko A, Doumbo OK: Efficacy, safety, and selection of molecular markers of drug resistance by two ACTs in Mali. *Am J Trop Med Hyg* 2008, **78**:455-461.

21. Nawaz F, Nsobya SL, Kiggundu M, Joloba M, Rosenthal PJ: Selection of parasites with diminished drug susceptibility by amodiaquine-containing antimalarial regimens in Uganda. *J Infect Dis* 2009, **200**:1650-1657.

22. Alifrangi M, Dalgaard MB, Lusingu JP, Vestergaard LS, Staalsøe T, Jensen AT, Enevold A, Rønn AM, Khalil IF, Warhurst DC, Lemnge MM, Theander TG, Bygbjerg IC: Occurrence of the Southeast Asian/South American SVMNT haplotype of the chloroquine-resistance transporter gene in *Plasmodium falciparum* in Tanzania. *J Infect Dis* 2006, **193**:1738-1741.

23. Gama BE, Pereira-Carvalho GA, Lutucuta Kosi FJ, Almeida NKde Oliveira, Fortes F, Rosenthal PJ, Daniel-Ribeiro CT, de Fatima Ferreira-da-Cruz M: *Plasmodium falciparum* isolates from Angola show the StctVMNT haplotype in the *pfcrt* gene. *Malar J* 2010, **9**:174.

24. Sa JM, Twu O: Protecting the malaria drug arsenal: halting the rise and spread of amodiaquine resistance by monitoring the *pfcrt* SVMNT type. *Malar J* 2010, **9**:374.

25. Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE: Geographic patterns of *Plasmodium falciparum* drug resistance distinguished by differential responses to amodiaquine and chloroquine. *Proc Natl Acad Sci USA* 2009, **106**:18883-18889.

26. Plowe CV, Wellems TE: Molecular approaches to the spreading problem of drug resistant malaria. *Adv Exp Med Biol* 1995, **390**:197-209.

27. Witkowski B, Nicolau ML, Soh PN, Iriart X, Menard S, Alvarez M, Marchou B, Magnaval JF, Benoit-Vical F, Berry A: *Plasmodium falciparum* isolates with increased *pfmdr1* copy number circulate in West Africa. *Antimicrob Agents Chemother* 2010, **54**:3049-3051.

28. Vessiere A, Berry A, Fabre R, Benoit-Vical F, Magnaval JF: Detection by real-time PCR of the *pfcrt* T76 mutation, a molecular marker of chloroquine-resistant *Plasmodium falciparum* strains. *Parasitol Res* 2004, **93**:5-7.

29. Prugnolle F, Ollomo B, Durand P, Yalcindag E, Arnathau C, Elguero E, Berry A, Pourrut X, Gonzalez JP, Nkoghe D, Akiana J, Verrier D, Leroy E, Ayala FJ, Renaud F: African monkeys are infected by *Plasmodium falciparum* nonhuman primate-specific strains. *Proc Natl Acad Sci USA* 2011, **108**:11948-11953.

30. Kumar R, Singh SK, Koshkin AA, Rajwanshi VK, Meldgaard M, Wengel J: The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2'-thio-LNA. *Bioorg Med Chem Lett* 1998, **8**:2219-2222.

31. Fratczak A, Kierzek R, Kierzek E: LNA-modified primers drastically improve hybridization to target RNA and reverse transcription. *Biochemistry* 2009, **48**:514-516.

32. Ugozzoli LA, Latorra D, Puckett R, Arar K, Hamby K: Real-time genotyping with oligonucleotide probes containing locked nucleic acids. *Anal Biochem* 2004, **324**:143-152.

33. Senescau A, Berry A, Benoit-Vical F, Landt O, Fabre R, Lelievre J, Cassaing S, Magnaval JF: Use of a locked-nucleic-acid oligomer in the clamped-probe assay for detection of a minority *pfcrt* K76T mutant population of *Plasmodium falciparum*. *J Clin Microbiol* 2005, **43**:3304-3308.

34. Valderramos SG, Valderramos JC, Musset L, Purcell LA, Mercereau-Puijalon O, Legrand E, Fidock DA: Identification of a mutant *pfcrt*-mediated chloroquine tolerance phenotype in *Plasmodium falciparum*. *PLoS Pathog* 2010, **6**:e1000887.

35. WHO: Country Health System Fact Sheet 2006, Cameroon. 2006 [http://www.afr WHO.who.int/en/cameroon/country-health-profile.html].

36. Basco LK: Molecular epidemiology of malaria in Cameroon. XIII. Analysis of *pfcrt* mutations and *in vitro* chloroquine resistance. *Am J Trop Med Hyg* 2002, **67**:388-391.

37. Basco LK, Ringwald P: Molecular epidemiology of malaria in Cameroon. X. Evaluation of *pfmdr1* mutations as genetic markers for resistance to amino alcohols and artemisinin derivatives. *Am J Trop Med Hyg* 2002, **66**:667-671.

38. Mbacham WF, Evehe MS, Netongo PM, Ateh IA, Mimche PN, Ajua A, Nji AM, Irenee D, Echouffo-Tcheugui JB, Tawe B, Hallett R, Roper C, Targett G, Greenwood B: Efficacy of amodiaquine, sulphadoxine-pyrimethamine and their combination for the treatment of uncomplicated *Plasmodium falciparum* malaria in children in Cameroon at the time of policy change to artemisinin-based combination therapy. *Malar J* 2010, **9**:34.

39. Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimde AA, Kouriba B, Taylor TE, Plowe CV: Reemergence of chloroquine-sensitive *Plasmodium falciparum* malaria after cessation of chloroquine use in Malawi. *J Infect Dis* 2003, **187**:1870-1875.

40. Mita T, Kaneko A, Lum JK, Bwijo B, Takechi M, Zungu IL, Tsukahara T, Tanabe K, Kobayakawa T, Bjorkman A: Recovery of chloroquine sensitivity and low prevalence of the *Plasmodium falciparum* chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. *Am J Trop Med Hyg* 2003, **68**:413-415.

41. Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G, Sasi P, Marsh K, Borrmann S, Mackinnon M, Nzila A: Chloroquine resistance before and after its withdrawal in Kenya. *Malar J* 2009, **8**:106.

42. Wang X, Mu J, Li G, Chen P, Guo X, Fu L, Chen L, Su X, Wellems TE: Decreased prevalence of the *Plasmodium falciparum* chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against *P. falciparum* malaria in Hainan, People's Republic of China. *Am J Trop Med Hyg* 2005, **72**:410-414.

43. Sisowath C, Petersen I, Veiga MI, Premji Z, Bjorkman A, Fidock DA, Gil JP: *In vivo* selection of *Plasmodium falciparum* parasites carrying the chloroquine-susceptible *pfcrt* K76 allele after treatment with artemether-lumefantrine in Africa. *J Infect Dis* 2009, **199**:750-757.

44. Yang Z, Zhang Z, Sun X, Wan W, Cui L, Zhang X, Zhong D, Yan G, Cui L: Molecular analysis of chloroquine resistance in *Plasmodium falciparum* in Yunnan Province, China. *Trop Med Int Health* 2007, **12**:1051-1060.
45. Lekana-Douki JB, Dinzouna Boutamba SD, Zatra R, Zang Edou SE, Ekomy H, Bisvigou U, Toure-Ndouo FS: Increased prevalence of the *Plasmodium falciparum* pfmdr1 86N genotype among field isolates from Franceville, Gabon after replacement of chloroquine by artemether-lumefantrine and artesunate-mefloquine. *Infect Genet Evol* 2011, 11:512-517.

46. 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, 11:1808-1812.

47. Laufer MK, Plowe CV: Withdrawing antimalarial drugs: impact on parasite resistance and implications for malaria treatment policies. *Drug Resist Updat* 2004, 7:279-288.

48. Walliker D, Hunt P, Babiker H: Fitness of drug-resistant malaria parasites. *Acta Trop* 2005, 94:251-259.

49. Cortese JF, Caraballo A, Contreras CE, Plowe CV: Origin and dissemination of *Plasmodium falciparum* drug-resistance mutations in South America. *J Infect Dis* 2002, 186:999-1006.

50. Vieira PP, Das Gracas Alecrim M, Da Silva LH, Gonzalez-Jimenez I, Zalis MG: Analysis of the *pfcrt* K76T mutation in *Plasmodium falciparum* isolates from the Amazon region of Brazil. *J Infect Dis* 2001, 183:1832-1833.

51. Zalis MG, Pang L, Silveira MS, Milhous WK, Wirth DF: Characterization of *Plasmodium falciparum* isolated from the Amazon region of Brazil: evidence for quinine resistance. *Am J Trop Med Hyg* 1998, 58:630-637.

52. Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA: Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite *Plasmodium falciparum*. *Antimicrob Agents Chemother* 2008, 52:2212-2222.

53. Sisowath C, Ferreira PE, Bustamante LY, Dahlstrom S, Martensson A, Bjorkman A, Krishna S, Gil JP: The role of *pfmdr1* in *Plasmodium falciparum* tolerance to artemether-lumefantrine in Africa. *Trop Med Int Health* 2007, 12:736-742.

54. Nkhoma S, Nair S, Mukaka M, Molyneux ME, Ward SA, Anderson TJ: Parasites bearing a single copy of the multi-drug resistance gene (*pfmdr-1*) with wild-type SNPs predominate amongst *Plasmodium falciparum* isolates from Malawi. *Acta Trop* 2009, 111:78-81.

55. Ursing J, Kofoed PE, Rombo L, Gil JP: No *pfmdr1* amplifications in samples from Guinea-Bissau and Liberia collected between 1981 and 2004. *J Infect Dis* 2006, 194:716-718, author reply 718-719.

56. 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, 74:157-166.

57. Uhlemann AC, Ramharter M, Lell B, Kremsner PG, Krishna S: Amplification of *Plasmodium falciparum* multidrug resistance gene 1 in isolates from Gabon. *J Infect Dis* 2005, 192:1830-1835.

58. Mawili-Mboumba DP, Kun JF, Lell B, Kremsner PG, Ntoumi F: *Pfmdr1* alleles and response to ultralow-dose mefloquine treatment in Gabonese patients. *Antimicrob Agents Chemother* 2002, 46:166-170.

59. Radloff PD, Philipps J, Nkeyi M, Sturchler D, Mittelholzer ML, Kremsner PG: Arteflene compared with mefloquine for treating *Plasmodium falciparum* malaria in children. *Am J Trop Med Hyg* 1996, 55:259-262.

60. Lell B, Lehman LG, Schmidt-Ott JR, Sturchler D, Handschin J, Kremsner PG: Malaria chemotherapy trial at a minimal effective dose of mefloquine/ sulfadoxine/pyrimethamine compared with equivalent doses of sulfadoxine/pyrimethamine or mefloquine alone. *Am J Trop Med Hyg* 1998, 58:619-624.

61. Woodrow CJ, Krishna S: Antimalarial drugs: recent advances in molecular determinants of resistance and their clinical significance. *Cell Mol Life Sci* 2006, 63:1586-1596.

doi:10.1186/1475-2875-11-113

Cite this article as: Menard et al: Molecular monitoring of *Plasmodium falciparum* drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaoundé, Cameroon: Implications for the future. *Malaria Journal* 2012 11:113.

Submit your next manuscript to BioMed Central and take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central