Malaria Journal BioMed Central

Research Open Access

Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium
falciparum isolates by FRET and in vivo response to chloroquine
among children from Osogbo, Nigeria
Olusola Ojurongbe1,2,4, Titus O Ogungbamigbe3, Adetayo F Fagbenro-
Beyioku4, Rolf Fendel1, Peter G Kremsner1 and Jürgen FJ Kun*1

Address: 1Department of Parasitology, Institute of Tropical Medicine, University of Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany,
2Department of Medical Microbiology & Parasitology College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria,
3Department of Pharmacology & Therapeutics College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria and
4Department of Medical Microbiology and Parasitology College of Medicine, University of Lagos, Nigeria

Email: Olusola Ojurongbe - stojurongbe@yahoo.com; Titus O Ogungbamigbe - bisiogun1562@yahoo.com; Adetayo F Fagbenro-

Beyioku - afbeyioku@yahoo.com; Rolf Fendel - rolf_fendel@yahoo.de; Peter G Kremsner - peter.kremsner@uni-tuebingen.de;
Jürgen FJ Kun* - juergen.kun@uni-tuebingen.de
* Corresponding author

Published: 11 April 2007 Received: 20 December 2006

Accepted: 11 April 2007
Malaria Journal 2007, 6:41 doi:10.1186/1475-2875-6-41
This article is available from: http://www.malariajournal.com/content/6/1/41
© 2007 Ojurongbe et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Chloroquine (CQ) has been in use in Africa for a long time. Because of misuse, this drug
has now lost its efficacy due to the emergence of resistance strains in most parts of Africa. Recently, it was
shown that after chloroquine has been withdrawn from the market, chloroquine-sensitive Plasmodium
falciparum re-emerged and chloroquine could again be used successfully as an antimalarial. Surveillance of
parasite populations is, therefore, important to decide whether chloroquine could be re-introduced.
Methods: To estimate the prevalence of the most pivotal polymorphisms, including Pfcrt K76T, Pfmdr1
N86Y and Pfmdr1 Y184F mutations, and their contributions to the outcome of CQ treatment, isolates
from Osogbo Western Nigeria were tested using the Fluorescence Resonance Energy Transfer (FRET)
method on a real-time PCR instrument.
Results: 116 children with acute uncomplicated P. falciparum malaria infections were treated with the
standard dosage of CQ and followed-up for 28 days. Blood samples were collected on filter paper at
enrollment and during follow-up for identification of parasite carrying the chloroquine resistant
transporter (pfcrt) and P. falciparum-multi drug resistance (pfmdr1) gene mutations. Parasitological
assessment of response to treatment showed that 62% of the patients were cured and 38% failed the CQ
treatment. The presence of single mutant pfcrt (T76) alleles (P = 0.003) and in combination with mutant
pfmdr1 Y86 (P = 0.028) was significantly associated with in vivo CQR. No other mutation on its own or in
combinations was significantly associated with treatment outcome. Mutant pfcrt was more prevalent in
both pre- and post-treatment isolates. No association was observed between age or initial level of
parasitaemia and chloroquine treatment outcome.
Conclusion: The result established the usefulness and accuracy of real time PCR in pfcrt and pfmdr1
mutation detection and also give further evidence to the reliability of the pfcrt T76 point mutation as a
molecular marker for CQ resistance.

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Background many other malaria endemic regions the therapeutic effi-

While there is an active search for new antimalarial drug cacy of CQ has decreased considerably. This, therefore,
combinations that could prevent or delay further spread has led to the change in the first line drug for the treat-
of resistance, there is a need to understand the basis of ment of malaria to artemisinin-based combination,
parasites resistance to chloroquine (CQ) and other anti- although, CQ is still widely used in the country. In order
malarial drugs and explore potentials to use the data in to explore the roles of pfcrt and pfmdr1 polymorphisms in
improving the potency and rational for selecting compo- CQR, the Fluorescence Resonance Energy Transfer (FRET)
nents for effective drug combination. Constant observa- method was used to determine these polymorphisms and
tion of the existing parasite population concerning their their in vivo sensitivity to chloroquine in P. falciparum iso-
genetic makeup determining the resistance to CQ became lates from Osogbo Western Nigeria. The use of a Real
even more important since it was shown that after CQ Time PCR assay for a rapid, sensitive, and specific detec-
withdrawal for therapy CQ-sensitive parasite re-occurred tion of these mutations was also assessed.
[1]. So written-off drugs may come into focus again.
Materials and methods
The molecular basis of CQ resistance in Plasmodium falci- Study site and patients
parum is still unclear, and the association of point muta- The study was undertaken between July 2004 and January
tions in different genes with chloroquine-resistance has 2005 in the town of Osogbo located in the western part of
been largely studied in the last decade. In 2000, pfcrt gene Nigeria. Osogbo is the state capital of Osun state Nigeria
was identified [2]. This gene consisting of 13 exons and it represents a typical urban setting in Nigeria. Malaria
showed 6–8 point mutations including one that appears is present throughout the year with a marked increase dur-
to play a crucial role in CQR [3]. A lysine to threonine ing the raining season (i.e. April – September). Febrile
change at position 76 (K76T) which was subsequently children (1–12 years old) attending the Osun state Hospi-
found in every in vitro CQR parasite from around the tal and Ladoke Akintola University Teaching hospital
world [4,5] was identified as an important mutation asso- were screened for P. falciparum parasitaemia. Blood films
ciated with CQR. The resistance was associated with a were stained with 10% Giemsa and examined microscop-
reduced accumulation of CQ in the parasite digestive vac- ically. Criteria for recruitment in this study were: (1) asex-
uole but how the pfcrt gene exerts such an effect on the ual parasitaemia between 2,000/μl and 200,000/μl, (2)
digestive vacuole is still unclear. Many studies have shown no signs of severity or severe malaria (including severe
that the pfcrt play a crucial on CQR, but this mutation was anaemia defined by haemoglobin <5 g/dl), (3) no intake
not the sole requirement, suggesting that other factors of antimalarial drugs during the preceding four weeks, (4)
including host factors are responsible for the clearance of informed consent from the patient parent or guardian.
CQR parasites [6]. The detected parasitaemic cases were treated with 25 mg/
kg chloroquine in divided doses for three days at 10 mg/
Polymorphisms in pfmdr1, a gene located on chromo- kg daily for D0 and D1 and 5 mg/kg for D2. Subsequent
some 5 which encodes the P. falciparum P-glycoprotein follow up appointments were scheduled for days 3, 7, 14,
homologue-1 is also thought to modulate CQR. It is a typ- 21 and 28.
ical member of the ATP-binding cassette transporter
superfamily localized in the parasite vacuole, where it Classification of responses to treatment was done accord-
may regulate intracellular drug concentrations [7]. Muta- ing to the WHO criteria [14]. The cure rate on day 28 of
tions were observed at the amino acids 86, 184, 1034, the follow-up was defined as the percentage of children
1042, and 1246, which were strongly linked to the CQR who remained free of parasites. Two drops of blood were
in laboratory clones obtained from various regions [8]. also blotted onto 3 MM Whatman filter paper on days 0
However, the link between pfmdr1 and CQR still remains before treatment and during following up when there was
unclear and controversial [6,9]. While some field studies re-occurrence of clinical symptoms for extraction and
had indicated that there is positive association between analysis of parasites DNA. Treatment failure rates were
CQR and mutation (asparagine to tyrosine change) at corrected by msp-2 genotyping of parasites at enrollment
position 86 (N86Y) [10,11], several others had doubts and recrudescence of infections [15]. The study received
about this association [12,13]. Currently, pfmdr1 muta- ethical approval from the ethical review committee
tions are said to assist the CQR parasites by augmenting boards of the joint College of Health Sciences/Ladoke
the level of resistance. A combination of pfcrt and pfmdr1 Akintola University Teaching Hospital and Osun State
polymorphisms is believed to result in higher levels of Hospitals Management Board.
CQR [4,7].
Detection of Pfcrt and Pfmdr1 mutations by Real time PCR
In Nigeria, CQ has been used for many years as the first- Parasite genomic DNA was extracted from blood samples
line treatment for uncomplicated malaria. However, like collected on filter paper using a QIAamp DNA blood kit

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(Qiagen, Hilden, Germany), according to the manufac- sensor probes. For pfmdr1 codons 86 and 184, the PCR
turer's instructions. The oligonucleotide probes and program was as follows: 5 min at 95°C, 40 cycles of 94°C
primer used to detect the polymorphisms in pfcrt and for 10 s, 52°C for 30 s, and 72°C for 40 s. The melting
pfmdr1 are shown in Table 1. program consisted of one cycle of 95°C for 15 s and heat-
ing from 36 to 85°C rising by 1°C.
For pfcrt analysis the sensor probe labeled with fluorescein
at the 3' end is designed to be perfectly complementary to Statistical analysis
the mutation site. An amplification primer iLC labeled For analysis purposes, each isolate was classified based on
with Cy5 on the third base from the 3'end is used as a the presence or absence of a resistance-associated allele
reverse primer which is extended during amplification. and infections with mixed wild-type/mutant alleles of
During FRET, fluorescein which is excited by the light pfcrt or pfmdr1 were treated as mutants. Data were ana-
source of the Rotor Gene instrument transfers its energy to lysed using the statistical programs JMP for Windows. For
the Cy5 incorporated into the PCR product working as univariate analysis, frequencies were compared using the
anchor probe [16,17]. A specific melting temperature is Fisher's exact tests. Two sided P values < 0.05 indicated
then obtained for each genotype: a sensor probe spanning statistical significance. McNemar's test was used to com-
one mismatch could still hybridize to the target sequence pare the samples before and after treatment.
but will melt off at lower temperature than a sensor probe
with a perfect match. Primers and probes for pfcrt were Results
designed and synthesized by TIB MOLBIOL (DNA synthe- Patient treatment outcome
sis service, Berlin, Germany) The potential for detection of a point mutation in pfcrt
and pfmdr1 genes using hybridization probes on Rotor
For pfmdr1 mutations, hybridization probes consisted of Gene technology has been evaluated. DNA from 116 sam-
two different oligonucleotides that bind to an internal ples of patients treated with chloroquine and successfully
sequence amplified by forward and reverse primers Table followed up for 28 days were evaluated for pfcrt and
1. The sensor probe, labeled at the 3'end with FAM, is Pfmdr1 (86 and 184) mutations.
designed to match the mutation sites. The anchor probe,
labeled at the 5' end with Cy5 and phosphorylated at the Of these, 69 (59%) were males and 47 (41%) were
3' end to prevent extension by Taq polymerase, is females. The mean age of the children was 46 ± 36 months
designed to conserved sequences adjacent to the mutation (6–144 months). Geometric mean parasite density was
sites. Both probes, localised on the same DNA strand, 9,061 parasites/μl of blood on the day of enrollment.
could hybridize in a head-to tail arrangement, bringing While infections in 72 (62%) of the patients were cured
the two fluorescent dyes into close proximity. During with a standard dosage of CQ, 44 (38%) failed to respond
FRET, FAM was excited by the light source of the Rotor to treatment. The clinical data and therapeutic responses
Gene instrument. The excitation energy was transferred to of the patients stratified by age is shown in Table 2.
the acceptor fluorophore, Cy5, and the emitted fluores-
cence was measured on the Rotor Gene channel in contin- Real-time PCR for Pfcrt and Pfmdr1 gene detection in
uous during the melting phase. A specific melting blood samples
temperature is then obtained for each genotype as Wild-type and mutant alleles were rapidly determined by
described above. Pfmdr1 primers and probes were synthe- comparing the melting temperature for the alleles with
sized by Operon Biotechnology (Cologne, Germany). the melting temperature of the reference alleles obtained
by the FRET assay. The reference strains used for this study
PCR amplification were 3D7, FCR, S007 and K1 laboratory strains which
Amplification was performed with Rotor Gene 3000 were all sequenced to determine the mutations that were
(Corbbett, Sydney, Australia). For pfcrt forward primer present. A confirmed field sample of Plasmodium malariae
was added at a final concentration of 0.4 μM, iLC primer was used as control along with no template. P. falciparum
0.5 μM and probe at 0.2 μM. The amplification program strain K1 parasites were kindly provided by Katja Becker
consisted of an initial step at 95°C for 10 min, amplifica- (Justus-Liebig-University Giessen). Both 3D7 and FCR
tion was performed with 40 cycles of denaturation (95°C showed the wild type sequence for pfcrt (K76) and Pfmdr1
for 10 s), annealing (50°C for 15 s), and extension (65°C (N86 and Y184). K1 showed mutant nucleotide at posi-
for 15 s). The melting curve program consisted of one tion 76 (T76) for Pfcrt and position 86 (Y86) for pfmdr1
cycle of 95°C for 15 s, and heating at 36°C to 75°C rising while S007 showed mutation at positions 76 for pfcrt, 86
by 1°C. and 184 for pfmdr1. DNA yielded a specific melting tem-
perature of 46.5 ± 0.3°C for the pfcrt mutant alleles and
For pfmdr1, the master mix contains a final concentration 65.3 ± 0.4 for the wild alleles. The melting temperatures
of 0.4 μM of both primers and 0.2 μM of both Anchor and

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Table 1: Sequence of primers and probes used for Pfcrt and Pfmdr1 amplification and melting temperatures of the sensor probes of
each allele

Melting temperatures (°C) of the sensor probes

Sequence 5' to 3' Wild Mutant

Pfcrt
Forward Primer: CTTGTCTTGGTAAATGTGCTCA
iLC Primer: GTTACCAATTTTGTTTAAAGTTCT
Sensor Probe: TGTGTAATTGAAACAATTTTTGCTAA 46.5 ± 0.2 65.3 ± 0.4

Pfmdr1
Forward Primer: TGTATTATCAGGAGGAACATTACC
Reverse Primer: ACCACCAAACATAAATTAACGGA
Sensor Probe 86: ATTAATATCATCATAAATACATG 51.8 ± 0.3 56.5 ± 0.2
Anchor Probe 86: TCTTTAATATTACACCAAACACAGATAT
Sensor Probe 184: TAAAAAATGCACGTTTGACTTTATGTATTA 53.0 ± 0.2 58.7 ± 0.3
Anchor Probe 184: CCTTTTTAGGTTTATTTATTTGGTCAT

for all the alleles are shown in Table 1. The melting anal- associated with CQ treatment failure (P = 0. 003). Both
ysis is shown in Figures 1&2 pfmdr1 N86Y and Y184F did not show any significant
association. The combination of pfcrt 76 and pfmdr1 86
Association between Pfcrt and Pfmdr1 mutations and also produces weak significant association (p = 0.028)
chloroquine treatment failure (Table 3).
The prevalence of Pfcrt T76 and Pfmdr1 (Y86 and F184)
mutations known to be involved in CQR resistance in For analysis of parasites strains before and after treatment,
West Africa was evaluated. By in-vivo testing, 38% of the P. 44 pairs was collected; each consisting of the parasite
falciparum isolates were chloroquine resistant, although strain before treatment and that from the recrudescence
by molecular analysis 74% of the pre-treatment isolates infection. All the 44 samples had either mutant or mixed
carried the mutant pfcrt T76 and 14% had mixed T76 allele in their pre-treatment sample and 43/44 (98%)
(T76+K76) allele that has been associated with chloro- had the mutant pfcrt allele after treatment. Only one iso-
quine resistance in other studies [6,18]. The pfmdr1 Y86 late showed the wild pfcrt allele and genotyping by MSP2
and F184 alleles were present in 29% and 64% of the pre- confirmed the isolate to be a new infection. For Pfmdr1
treatment isolates, respectively (Table 3). Y86 and F184 the prevalence of the alleles at pre-treat-
ment isolates were 17% and 33%, respectively, and at
The hypothesis whether allelic variations in pfcrt and recrudescence the prevalence of Y86 was 18% while that
pfmdr1 of P. falciparum isolates are associated with CQ of F184 was 27%. The analysis of nonresponding and
treatment outcome was tested in the study area. The pres- recrudescent parasites showed a pronounced significant
ence of pfcrt or pfmdr1 mutations or the combination of difference between the samples collected before and after
these mutations in samples collected before chloroquine chemotherapy for the isolates having pfmdr1 F184 muta-
treatment was examined for their association with patient tion (p = 0.031) while for other alleles tested there was no
treatment outcome. The pfcrt K76T allele was significantly

Table 2: Enrollment clinical data and therapeutic responses of patients with acute uncomplicated Plasmodium falciparum treated with
chloroquine stratified by age

Age Freq Sex Temp Mean Mean Mean T76 Y86 F184 CQ Treatment
(Yrs) M F (SD) PD FC PC (%) (%) (%) Cured Failed

0<4 72 39 33 37.7 (± 1.13) 8261.066 2.7 2.6 67 (93%) 27 (38%) 49 (68%) 46 (58%) 26 (36%)
4<8 27 20 7 38.0 (± 1.3) 11632.6 1.2 2.4 22 (81%) 9 (33%) 20 (74%) 17 (63%) 10 (37%)
8–12 17 10 7 37.8 (± 0.84) 9015.282 1.3 2.5 16 (94%) 6 (35%) 12 (71%) 9 (53%) 8 (47%)
Total 116 69 47 37.8 (± 1.3) 9061.375 1.7 2.5 102 (88%) 42 (36%) 81 (70%) 72 (62%) 44 (38%)

Key: Freq = Frequency

SD = Standard deviation
PD = Parasite density on Day 0
FC = Fever clearance time in days
PC = Parasite clearance time in days

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K76

T76

Mixed K76 + T76

Figure 1of a typical real time rum showing melting curve analysis of Pfcrt position 76
Example
Example of a typical real time rum showing melting curve analysis of Pfcrt position 76.

significant difference between the pre and post treatment attractive strategy that could easily be adapted to large-
samples Table 4. scale studies of drug resistance.

Discussion Point mutations in the pfcrt gene and to a lesser extent, in

In this study, a Real Time PCR assay for the detection of the pfmdr1 gene are thought to be associated with CQR
pfcrt and pfmdr1 alleles thought to be associated with CQ [2,6]. The goal of this study was to evaluate the utility of
susceptibility and resistance was described. This Real- these molecular markers as indicators of chloroquine
Time PCR assay was demonstrated to be rapid, sensitive, resistance in isolates of P. falciparum obtained from
and specific for the detection and characterization of P. Osogbo western Nigeria using a hybridization probe
falciparum genetic marker of CQR. The assay detected method on a Real Time PCR technology platform. The
mixed alleles infections and clearly discriminated result of this study showed a high prevalence of pfcrt T76
between CQ-susceptible and CQ-resistant isolates. Its (74%). This observation is consistent with the previous
speed (up to 72 samples can be assayed in a 2-h experi- reports from various malaria endemic regions where chlo-
ment) and performance characteristics may eliminate the roquine has been widely used. A significant association
need for more complicated approaches and make it an was also found between the overall in vivo rate of treat-

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Y86
N86

Mixed N86 + Y86

Figure
Example2of a typical melting curve analysis generated by real time PCR of the Pfmdr position 86
Example of a typical melting curve analysis generated by real time PCR of the Pfmdr position 86.

ment failure and the frequency of mutated pfcrt gene in whether this association existed in parasite isolates
the population (P = 0.004) as already shown previously in obtained directly from the field [9,20]. Their analysis
western Nigeria [19] and other malaria endemic areas showed that African isolates predominantly possess poly-
[18]. 93% of the pre-treatment isolates carried the pfcrt morphism at two alleles, codon-86 and codon-184, with
T76 and mixed allele while among the post treatment iso- a positive association, although incomplete, between
late the prevalence was 98%. The facts that this polymor- mutation of codon-86 and CQR. A similar association has
phism was present in all the recrudescence isolates been found in several studies [4,11,21,22]. Nevertheless,
emphasised again the fact that this mutation is important much confusion has surrounded the association of differ-
in CQR. ent pfmdr1 alleles to chloroquine resistance because
numerous studies have had contradictory results. Trans-
The point mutation of asparagine to tyrosine at codon 86 fection studies as well as carefully controlled molecular
of pfmdr1 has been associated with CQR in some studies epidemiologic studies have shown that there are strong
[8,9] but not in others [12]. In the present study, both the associations between pfmdr1 polymorphisms and antima-
pfmdr1 Y86 and F184 mutations showed no correlation larial resistance [7]. However, like many other studies [12]
with resistance to chloroquine. Both the wild type and the the present findings have failed to find such associations
mutant alleles for each locus were present in both sensi- because the presence of both wild-type and mutant-type
tive and resistance isolates. Previously, an association was alleles in our samples were largely independent of their in
established between chloroquine-resistance and alleles of vivo responses. Also the current belief that the combina-
the pfmdr1 gene in laboratory isolates obtained from dif- tion of pfcrt and pfmdr1 polymorphisms result in higher
ferent parts of the world [8]. Others had considered levels of CQR [4] was not observed in this study. Although

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Table 3: Prevalence of pfcrt and pfmdr1 alleles in post-treatment samples of Nigerian children with acute uncomplicated malaria.

Allele Frequency Treatment outcome (%)

Cured N = 72 Failed N = 44 P. value

N = 116
Pfcrt T 76 86 (74%) 51 (71%) 41 (93%)
Pfcrt K 76 11 (12%) 11 (15%) 0 (0%) 0.003*
T76+K76 19 (14%) 10 (14%) 9 (20%)

N = 116
Pfmdr1 Y86 34 (29%) 22 (31%) 12 (27%)
Pfmdr1 N86 74 (64%) 47 (65%) 27 (61%) n.s.
Y86+N86 8 (7%) 3 (4%) 5 (11%)

N = 116
Pfmdr1 F184 74 (64%) 45 (63%) 29(65%)
Pfmdr1 Y 184 35 (30%) 24 (33%) 11 (25%) n.s.
F184+Y184 7 (6%) 3 (4%) 4 (9%)

N = 116
T76 + Y86 37 (32%) 21 (29%) 16 (36%)
K76 + N86 7 (6%) 7 (10%) 0 (0%) 0.028*
Mixed 72 (62%) 44 (61%) 28 (64%)

N = 116
T76 + F186 73 (63%) 40 (56%) 33 (75%)
K76 + Y184 2 (2%) 2 (3%) 0 (0%) n.s.
Mixed 41 (35%) 30 (42%) 11 (25%)

N = 116
T76+Y86+F184 35 (30%) 19 (26%) 16 (36%)
K76+N86+Y184 3 (3%) 3 (4%) 0 (0%) n.s.
Mixed 78 (67%) 50 (70%) 28 (64%)

n.s. is not significant

a significant association was observed (p = 0.028), it was A recent report from Ibadan Nigeria a neighbouring town
not in any way stronger than the one observed with pfcrt to Osogbo had suggested an association and linkage dise-
alone. Analysis of altered gene expression and other quilibrium between the pfcrt T76 and pfmdr1 Y86 alleles
mechanisms that may contribute to a resistant phenotype in chloroquine-resistant isolates [19]. On the contrary this
is needed before the role of pfmdr1 can be excluded. study suggested no possible association between these
two polymorphic alleles and in vivo chloroquine resist-

Table 4: Prevalence of mutant pfmdr1/pfcrt genes in Day 0 and recrudescence samples of patients that failed chloroquine treatment

Prevalence of alleles

Alleles Day 0 Recrudescence Mc Nemar P value

T76 44/44 (100%) 43/44 (98%) 1

Y86 17/44 (39%) 18/44 (41%) 1
F184 33/44 (75%) 27/44 (61%) 0.031*
T76+Y86 44/44 (100%) 43/44 (98%) 1
T76+F184 44/44 (100%) 43/44 (98%) 1
T76+Y86+F184 44/44 (100%) 44/44 (100%) 1

Mixed infection is considered as mutant

*Significant at P < 0.05

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ance and that these molecular markers by themselves may 5. Vinayak S, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD: Prev-
alence of the K76T mutation in the pfcrt gene of Plasmo-
not predict in vivo chloroquine resistance. dium falciparum among chloroquine responders in India.
Acta Trop 2003, 87:287-293.
Conclusion 6. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte
Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV,
In summary, the results of this study give further evidence Coulibaly D: A molecular marker for chloroquine-resistant
to the reliability of the 76T pfcrt point mutation as a falciparum malaria. N Engl J Med 2001, 344:257-263.
molecular marker for CQ resistance. Conversely, the role 7. Duraisingh MT, Cowman AF: Contribution of the pfmdr1 gene
to antimalarial drug-resistance. Acta Trop 2005, 94:181-190.
of the Y86 and F184 point mutation of pfmdr1 gene in CQ 8. Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cow-
resistance remains elusive. The analysis of the relevant man AF: Several alleles of the multidrug-resistance gene are
closely linked to chloroquine resistance in Plasmodium falci-
mutations by RT-PCR provides a rapid and reliable parum. Nature 1990, 345:255-258.
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Authors' contributions 10. Gomez-Saladin E, Fryauff DJ, Taylor WR, Laksana BS, Susanti AI,
Purnomo, Subianto B, Richie TL: Plasmodium falciparum mdr1
OO performed the in vivo testing and the molecular anal- mutations and in vivo chloroquine resistance in Indonesia.
ysis, and drafted the manuscript. Am J Trop Med Hyg 1999, 61:240-244.
11. Nagesha HS, Din S, Casey GJ, Susanti AI, Fryauff DJ, Reeder JC, Cow-
man AF: Mutations in the pfmdr1, dhfr and dhps genes of Plas-
TOO performed the in vivo testing. modium falciparum are associated with in-vivo drug
resistance in West Papua, Indonesia. Trans R Soc Trop Med Hyg
FR participated in the molecular study. 2001, 95:43-49.
12. Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S,
Inkathone S, Zhong K, Kain KC: Plasmodium falciparum malaria
AFF-B supervised the design of the study. in Laos: chloroquine treatment outcome and predictive
value of molecular markers. J Infect Dis 2001, 183:789-795.
13. Thomas SM, Ndir O, Dieng T, Mboup S, Wypij D, Maguire JH, Wirth
PGK and JFJK supervised the molecular work and the DF: In vitro chloroquine susceptibility and PCR analysis of
interpretation of the data and helped to draft the manu- pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum
isolates from Senegal. Am J Trop Med Hyg 2002, 66:474-480.
script. 14. WHO: Assessment of therapeutic efficacy of antimalarial
drugs for uncomplicated malaria in areas with intense trans-
All authors read and agreed to the content of the manu- mission. Geneva: World Health Organistion WHO/MAL/ 1996:96-1077.
15. Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, Vir-
script. iyakosol S: Biased distribution of msp1 and msp2 allelic vari-
ants in Plasmodium falciparum populations in Thailand.
Trans R Soc Trop Med Hyg 1999, 93:369-374.
Acknowledgements 16. Kearns AM, Guiver M, James V, King J: Development and evalua-
The authors are grateful to all parents and guardian who volunteered to tion of a real-time quantitative PCR for the detection of
participate in the study. We thank Titi Okewumi, Adeola Ayileka and human cytomegalovirus. J Virol Methods 2001, 95:121-131.
Kumbi Akinwale for their assistance in the laboratory. We also thank Eric 17. de Monbrison F, Raynaud D, Latour-Fondanaiche C, Staal A, Favre S,
Kaiser K, Peyron F, Picot S: Real-time PCR for chloroquine sen-
Kenjo for helping with the statistical analysis. We thank Ladoke Akintola sitivity assay and for pfmdr1-pfcrt single nucleotide polymor-
University of Technology for their contribution to malaria research clinic phisms in Plasmodium falciparum. J Microbiol Methods 2003,
and laboratory where the work was carried out. OO is a recipient of a 54:391-401.
Deutscher Akademischer Austausch Dienst (DAAD) Fellowship. 18. Binder RK, Borrmann S, Adegnika AA, Missinou MA, Kremsner PG,
Kun JF: Polymorphisms in the parasite genes for pfcrt and
pfmdr-1 as molecular markers for chloroquine resistance in
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