Download frequency distribution of antimalarial drug

Am. J. Trop. Med. Hyg., 74(2), 2006, pp. 205–210
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
FREQUENCY DISTRIBUTION OF ANTIMALARIAL DRUG-RESISTANT ALLELES
AMONG ISOLATES OF PLASMODIUM FALCIPARUM IN BANGUI, CENTRAL
AFRICAN REPUBLIC
DIDIER MENARD,* DJIBRINE DJALLE, FERDINAND YAPOU, ALEXANDRE MANIRAKIZA,
ANTOINE TALARMIN
Pasteur Institute of Bangui, Bangui, Central African Republic
AND
Abstract. We determined the baseline frequency distribution of mutant alleles of genes associated with resistance to
chloroquine and sulfadoxine-pyrimethamine in Plasmodium falciparum isolates in Bangui, Central African Republic.
Mutant alleles of the P. falciparum chloroquine resistance transporter (pfcrt) gene were found in all samples and the
frequency of the deduced CIET pfcrt haplotype was high (45%). The most common allele of the P. falciparum multidrug
resistance 1 (pfmdr1) gene among the field isolates of P. falciparum was 86Y (21.9%). The 1246Y allele was also
common (18.0%). Of the 167 P. falciparum isolates in which the dihydrofolate reductase gene was studied, only 11
carried the wild-type allele (6.6%) whereas many (50.3%) were quadruple mutants (50R, 51I, 59R, 108N). The frequency
of the 436A mutant allele of the dihydropteroate synthase gene was high (74.3%), but the frequencies of the 437G
(18.6%) and 540E (5.2%) mutant alleles were low. Molecular analyses of antimalarial drug-resistant alleles of P.
falciparum isolates in Bangui strongly suggest the widespread distribution of chloroquine and pyrimethamine resistance
and to a lesser extent sulfadoxine resistance.
To complete the description of resistance in Bangui, Central African Republic, we assessed the drug resistance status
of the local malaria parasite population using molecular
markers. Analysis of the molecular basis of antimalarial drug
resistance over the last few decades has showed various mutant alleles of the P. falciparum chloroquine resistance transporter (pfcrt) and the P. falciparum multidrug resistance 1
(pfmdr1) genes that are associated with resistance to chloroquine.6 Similarly, mutant alleles of the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes had
also been implicated in P. falciparum resistance to antifolates
and sulfa drugs, respectively.7 There have been numerous
epidemiologic studies on the frequency distribution of the
alleles in P. falciparum isolates in different geographic regions. These studies have been invaluable in evaluating the
spread of drug resistance in many countries.8–18
The purpose of this study was to determine the baseline
frequency distribution of the mutant alleles of genes associated with resistance to chloroquine and sulfadoxinepyrimethamine in P. falciparum isolates from Bangui, the
capital of the Central African Republic before artemisininbased combination therapies are used as first-line antimalarial
drugs.
INTRODUCTION
Malaria is a major infectious disease worldwide and is a
leading cause of morbidity and mortality, particularly among
children. In Africa, more than 90% of cases and between 1.5
and 3 million deaths occur in children less than five years of
age.1 Chloroquine has been the drug of choice for treating
Plasmodium falciparum malaria for more than 50 years. However, the use of chloroquine as a prophylactic drug and as a
treatment for malaria is being limited because of the spread of
chloroquine-resistant P. falciparum strains throughout most
malaria-endemic areas.
In the Central African Republic, P. falciparum resistance to
chloroquine and sulfadoxine-pyrimethamine has been documented since 19832 and 1987,3 respectively. The most recent
in vivo study based on the World Health Organization standard protocol (2001) was conducted in Bangui in 2002. It
showed that the overall rates of treatment failure were 40.9%
with chloroquine, 20.0% with amodiaquine, 22.8% with sulfadoxine-pyrimethamine, 7.2% with the chloroquine plus sulfadoxine-pyrimethamine combination, and 0% with the amodiaquine plus sulfadoxine-pyrimethamine combination.4 In
accordance with in vivo results, in vitro isotopic drug sensitivity assays in Bangui in 2004 showed that the proportion of
resistant isolates was 37% for chloroquine, 15.9% for amodiaquine, 0% for quinine, 0% for dihydroartemisinin, 1.6% for
mefloquine, 3.8% for halofantrine, 4.0% for atovaquone, and
83% for pyrimethamine. No multi-resistant isolates (showing
resistance to more than three drugs) were found.5 These findings resulted in the Ministry of Health of the Central African
Republic Ministry of Health replacing chloroquine with the
amodiaquine plus sulfadoxine-pyrimethamine combination.
This combination was to be used as an interim first-line antimalarial treatment until better, alternative treatments, such
as artemisinin-based combination therapies, became available
at low prices in the Central African Republic.
MATERIALS AND METHODS
Study site. The location of Bangui is shown in Figure 1. The
Central African Republic is a subtropical country bordered
by Sudan to the east, Cameroon to the west, Chad to the
north, and the Republic of Congo to the south. It has an area
of 632,000 km2 and an estimated population of 3,823,929.
Bangui is located near the Oubangui River (7⬘N, 21⬘E). The
climate is tropical and rainfall is highest from April to November. The average temperature varies from 19°C to 32°C.
Malaria transmission occurs throughout the year, with peaks
at the beginning and the end of the rainy season. Malaria is
hyperendemic in this region and P. falciparum is the predominant malaria species. The prevalence of this parasite in children less than five years of age is 31.8%.19
Sample collection. This study was conducted between
March and July 2004. Clinical isolates of P. falciparum were
* Address correspondence to Didier Menard, Institut Pasteur de
Madagascar, BP 1274, Antananarivo 101, Madagascar. E-mail:
[email protected]
205
206
MENARD AND OTHERS
FIGURE 1.
Central African Republic showing the location of Bangui.
obtained from symptomatic Central African patients before
they were treated. These patients attended several health centers (in the northern districts of Boy Rabe and Gobongo and
the southern districts of La Kouanga and Ouango). Blood
samples, collected in a tube coated with EDTA (Vaicutainer威
tubes; Becton Dickinson, Rutherford, NJ), were obtained
from patients who provided informed consent during routine
malaria diagnosis. Giemsa-stained thin and thick blood
smears were examined to check for mono-infection with P.
falciparum and to determine parasite density. The patients
were treated with the amodiaquine-sulfadoxine-pyrimethamine combination or quinine, as recommended by the
National Malaria Control Program in the Central African
Republic.4
Ethical approval. Since there is no National Ethics Committee in the Central African Republic, study protocols were
reviewed and approved by the expert committee for antimalarial drug policy and the Central African Republic Ministry
of Health.
Extraction of DNA. The DNA template for a polymerase
chain reaction (PCR) and detection of mutant alleles was
prepared from the whole blood sample. The blood was centrifuged and the erythrocytes were frozen at −20°C until extraction. Parasite DNA was extracted from 100 ␮L of thawed
red blood cell pellets by treatment with 0.1 M NaOH for three
minutes at 100°C. After centrifugation, the supernatant was
collected, treated with 250 ␮L of lysing solution (150 mM
Tris-HCl pH 7.5, 1% [v/v] Triton 100X, 150 mM NaCl, 1%
sodium dodecyl sulfate, 1 mM EDTA) and proteinase K (20
mg/mL) for one hour at 37°C, and extracted twice with phenol:chloroform (1:1). The DNA was then precipitated with
ethanol, resuspended in 100 ␮L of distilled water, and stored
at −20°C.
Amplification by PCR and detection of mutant alleles. The
PCR and restriction fragment length polymorphism (RFLP)
analysis were conducted for four genes (dhfr, dhps, pfcrt, and
pfmdr1) to identify the presence of mutant alleles. A detailed
description of these methods is available from http://
medschool.umaryland.edu/CVD/plowe.html.
Statistical analysis. Data were analyzed using Epi-Info 2000
softeware (Centers for Disease Control and Prevention, Atlanta, GA) and MedCalc version 8.0.0.1 software (MedCalc
Software, Mariakerke, Belgium). Fisher’s exact test was used
to compare two variables with two categories. Spearman’s
correlation coefficients were calculated to assess associations
between codons in wild-type and mutant alleles.
RESULTS
A total of 386 blood samples from P. falciparum-infected
individuals were examined for the presence of mutations in
the pfcrt, pfmdr1, dhfr, and dhps genes: only 267 (69/2%) gave
PCR results. There were 135 samples (50.5%) that contained
mixed infections of both wild-type and mutant alleles. Allele
frequencies of mutations in pfcrt and pfmdr1 are shown in
Table 1 and in dhfr and dhps in Table 2.
Mutant alleles of the pfmdr1 and pfcrt genes. Analysis of
pfmdr1 gene PCR products indicated that mutant alleles of
this gene were present in 55 (31.8%) of 173 samples. The most
common allele of the pfmdr1 gene in the field isolates of P.
falciparum was 86Y (21.9%). The 1246Y allele was also observed at a lower frequency (18.0%).
TABLE 1
Allele frequencies of mutations in pfcrt and pfmdr1 in isolates of
symptomatic individuals from Bangui, Central African Republic
with uncomplicated Plasmodium falciparum malaria in 2004*
Frequency
Genes
Codons
pfcrt
72
74
75
76
pfmdr1
86
1246
Amino acids
C
C/S
S
M
M/I
I
N
N/E
E
L
K/T
T
N
N/Y
Y
D
D/Y
Y
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
Counts
%
95% CI
168
1
7
79
18
80
3
4
172
72
33
75
175
25
24
146
18
14
95.4
0.6
4.0
44.6
10.2
45.2
1.7
2.2
96.1
40.0
18.3
41.7
78.1
11.2
10.7
82.0
10.1
7.9
91.2–98.0
0–3.1
1.6–8.1
37.2–52.3
6.1–15.6
37.7–52.8
0.3–4.8
0.6–5.6
92.1–98.4
32.8–47.6
13.0–24.8
34.4–49.2
72.1–83.4
7.4–16.0
7.0–15.5
75.6–87.4
6.1–15.5
4.4–12.8
* pfcrt ⳱ P. falciparum chloroquine resistance transporter; pfmdr1 ⳱ P. falciparum multidrug resistance gene 1; CI ⳱ confidence interval; W ⳱ wild-type allele; M ⳱ mutant allele.
207
ANTIMALARIAL DRUG-RESISTANT ALLELES IN BANGUI
TABLE 2
Allele frequencies of mutations in dhfr and dhps in isolates of symptomatic individuals from Bangui, Central African Republic, with
uncomplicated Plasmodium falciparum malaria in 2004*
Deduced haplotypes
in dhfr gene
Frequency
Genes
Codons
16
dhfr
50
51
59
108
164
dhps
436
437
540
581
613
Amino acids
A
C
C/R
R
N
N/I
I
C
C/R
R
S
S/N
N
T
I
I/L
L
S
S/A
A
A
A/G
G
K
K/E
E
A
A
W
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
Mixed
M
W
W
TABLE 4
Deduced haplotype profiles for the dhfr gene of Plasmodium falciparum isolates from Bangui, Central African Republic in 2004*
Counts
%
95% CI
181
37
15
119
46
33
94
39
28
108
43
9
125
1
176
1
1
37
37
113
149
13
21
91
2
3
84
82
100
21.6
8.8
69.6
26.6
19.1
54.3
22.3
16.0
61.7
24.2
5.1
70.2
0.6
98.8
0.6
0.6
18.9
20.0
61.1
81.4
7.1
11.5
94.8
2.1
3.1
100
100
98.0–100
15.7–28.6
5.0–14.1
62.1–76.4
20.2–33.8
13.5–25.7
46.6–61.9
16.4–29.2
10.9–22.3
54.1–68.9
18.1–31.1
2.3–9.4
62.9–76.8
0–3.1
96.0–99.9
0.0–3.1
0.0–3.1
13.5–25.3
13.0–26.5
53.7–68.1
75.0–86.8
3.8–11.8
7.2–17.0
88.3–98.3
0.3–7.3
0.6–8.9
95.7–100
95.6–100
* dhfr ⳱ dihydrofolate reductase; dhps ⳱ dihydropteroate synthase. For definitions of
other abbreviations, see Table 1.
Mutant alleles of the pfcrt gene were found in all samples
(171 of 171). Deduced haplotype frequencies in the pfcrt gene
are shown in Table 3. There was a significant correlation in
wild-type and mutant alleles between codons 74 and 76 (P <
10−6). Two mutant alleles (86Y and 1246Y) of pfmdr1 gene
were paired with the 76T mutant allele of pfcrt gene in only 16
(9.2%) of 173 isolates.
Mutant alleles of the dhfr and dhps genes. Mutant alleles of
the dhfr gene were found in all but 11 samples. Deduced
haplotype frequencies for the dhfr gene are shown in Table 4.
The most prevalent haplotype carried four mutant alleles:
50R, 51I, 59R, and 108N. The mutant allele with a mutation at
ARIRNI
ARIRSI
ACIRNI
ARNRNI
ACNCSI
ARNCNI
ACNCNI
ACICNI
ARNRSI
ARICNI
ACIRSI
ARICSI
ACICSI
ARIRNL
ARIRSL
ACNRNI
ACNCTI
ACNRSI
ARNCSI
No. of
mutant alleles
Counts
%
4
3
3
3
0
2
1
2
2
3
2
2
1
5
4
2
1
1
1
84
14
12
12
11
10
4
3
3
2
2
2
2
1
1
1
1
1
1
50.3
8.4
7.2
7.2
6.6
6.0
2.4
1.8
1.8
1.2
1.2
1.2
1.2
0.6
0.6
0.6
0.6
0.6
0.6
* dhfr ⳱ dihdydrofolate reductase. Aminio acids conferring resistance are shown in bold.
The six-letter codes show amino acid residues at positions 16, 50, 51, 59, 108, and 164.
codon 16 was not detected in any of our samples examined. In
one isolate, the 108T mutation was found as a single mutation.
Significant correlations were found in wild-type and mutant
alleles between codons 50 and 51 (P ⳱ 0.002), codons 50 and
59 (P < 10−6), codons 50 and 108 (P ⳱ 0.002), codons 51 and
59 (P < 10−6), codons 51 and 108 (P ⳱ 0.005) and codons 59
and 108 (P ⳱ 0.0005).
Deduced haplotype frequencies in the dhps gene are shown
in Table 5. The most common haplotype was 436A as single
mutation (74.3%). It was paired with the 437G allele in five
samples (6.8%). Mutant alleles at codons 581 and 613 were
not detected in any of the samples tested. A significant correlation was found in wild-type and mutant alleles between
codons 436 and 437 (P < 10−6).
The triple dhfr variant (51I, 59R, and 108N) was found in
combination with the dhps variant 436A in parasites from
41.6% of the individuals, with dhps variant 437G in parasites
from 3.4% of the individuals, and with the dhps variant 540E
in parasites from 2.2% of the individuals. No quintuple variant (combined triple mutations in dhfr and double mutations
in dhps) was observed.
DISCUSSION
TABLE 3
Deduced haplotype profiles for the pfcrt gene of Plasmodium falciparum isolates from Bangui Central African Republic in 2004
Deduced haplotypes
in pfcrt genet
No. of
mutant alleles
Counts
%
CIET
CMEK
CMET
CIEK
SIET
CMNT
SIEK
CINT
SMET
3
1
2
2
4
1
3
2
3
77
57
19
8
5
2
2
1
1
45.0
33.3
11.1
4.7
2.9
1.2
1.2
0.6
0.6
* pfcrt ⳱ P. falciparum chloroquine resistance transporter. Amino acids conferring resistance are shown in bold. The four-letter codes show amino acid residues at positions 72, 74,
75, and 76.
Until recently, our knowledge of the epidemiology of drugresistant malaria was based on the collection of in vivo data
TABLE 5
Deduced haplotype profiles for the dhps gene of Plasmodium falciparum isolates from Bangui Central African Republic in 2004*
Deduced haplotypes
in dhps gene
No. of
mutant alleles
Counts
%
AAKAA
SAKAA
SGKAA
AGKAA
SGEAA
1
0
1
2
2
55
7
6
5
1
74.3
9.5
8.1
6.8
1.4
* dhps ⳱ dihydropteroate synthase. Amino acids conferring resistance are shown in bold.
The five-letter codes show amino acid residues at positions 436, 437, 540, 581, and 613.
208
MENARD AND OTHERS
from symptomatic patients to whom different antimalarial
drugs were administered and, to a lesser extent, on in vitro
drug sensitivity assays. The limitations of these methods for
studying drug-resistant malaria and elucidating molecular
mechanisms of resistance to some antimalarial drugs have
stimulated the use of a third approach based on molecular
markers for resistance.20 Thus, to complete recent in vivo4
and in vitro5 data on drug-resistant malaria in Bangui, Central
African Republic, we determined the baseline frequency distribution of the mutant alleles of genes associated with resistance to chloroquine and sulfadoxine-pyrimethamine in the
P. falciparum population. Samples collected in 2004 for in
vitro drug sensitivity assays were used for testing for markers
of drug resistance.5
There is still no single, universally accepted PCR protocol
to amplify the target genes. Consequently, we used a PCRRFLP technique. Compared with other techniques used to
determine DNA sequences, for example hybridization with
DNA probes or direct sequencing of amplified fragments, the
PCR-RFLP technique has been found to be robust21 and is
widely used in other African countries. It was also the easiest
technique for us to use because our laboratory was already
equipped with the necessary material.
We found mixed infections of both wild-type and mutant
alleles in more than half of the samples. This is not surprising
and is consistent with our previous study on the genetic diversity and clone multiplicity of P. falciparum infections in
symptomatic individuals living in Bangui. In this study, we
showed a high percentage of multiclonal infections (42.7%
with merozoite surface protein 1 [msp 1] gene and 76.7% with
the merozoite surface protein 2 [msp 2] gene), with a mean
of 1.7 genotype with msp 1 and 2.8 genotypes with msp 2,
which probably reflects a high rate of transmission (unpublished data).
The baseline frequency distribution of the mutant alleles of
genes associated with resistance to chloroquine was consistent
with previous studies in Africa. We found mutant alleles of
the pfcrt gene in all samples and high frequencies of the pfcrt
alleles 74I, 75E and 76T, as has been described in numerous
African countries, such as Cameroon,22 Gabon,23 Democratic
Republic of Congo, 24 Sudan, 25 Nigeria, 26 Senegal, 27
Uganda,28 and Mauritania,29 where chloroquine has been
widely used. We also found close relationships between the
overall in vivo rate of treatment failure (40.9%), the proportion of resistant isolates estimated by in vitro isotopic drug
sensitivity assays (37%), and the frequency of the deduced
CIET pfcrt haplotype (45%), as already shown in Lambaréné
in Gabon.23 Mutant alleles 86Y and 1246Y in pfmdr1 gene,
which are often cited as potential contributors to resistance to
chloroquine, were also analyzed. Unlike previous reports in
Africa,30,31 we found only a low prevalence of mutant alleles
86Y and 1246Y in isolates from Bangui and these two alleles
were found in only 16 (9.2%) of 173 isolates. The low prevalence of the pfmdr1 Y1246 allele is consistent with other data
from Africa.32,33 This polymorphism has been found mostly in
isolates originating from South America.34,35 However, the
presence of the Y1246 mutant allele in a few isolates from
Bangui may indicate the introduction of new strains into this
region.
Of the 167 P. falciparum isolates examined in the dhfr gene,
only 11 carried the wild-type allele (6.6%). There was a high
frequency (50.3%) of quadruple mutants (50R, 51I, 59R,
108N). Thus, as suggested by Basco and others,20 there
seemed to be a drastic change from a wild-type allele to quadruple mutants, rather than a gradual accumulation of mutations in the dhfr gene. These results are consistent with other
studies in neighboring countries20,36 and imply a high degree
of resistance to pyrimethamine.37 It was more surprising to
find a high 436A mutant allele frequency, but low frequencies
of the 437G mutant allele (18.6%) and 540E mutant allele
(5.2%) in the dhps gene. However, this is consistent with the
overall in vivo treatment failure rate of 22.8% with sulfadoxine-pyrimethamine in 2002. Regular evaluation of these two
molecular markers in the dhps gene would be valuable in
monitoring the therapeutic efficacy of sulfadoxinepyrimethamine. It is unclear whether these results reflect
drug pressure due to the use of sulfadoxine-pyrimethamine as
the second-line antimalarial treatment or of antibiotics containing antifolates (e.g., cotrimoxale). Cross-resistance between sulfadoxine-pyrimethamine and trimethoprimsulfamethoxazole has been described.37 Strains with mutant
alleles N108, N108-I51, and N108-R59 in the dhfr gene are
less susceptible to both pyrimethamine and trimethoprim
than wild-type isolates,38 and a high rate of bacterial uropathogen resistance to trimethoprim-sulfamethoxazole has
been observed in Bangui.39 Intermittent use of these drugs
could contribute to pyrimethamine resistance. Moreover, the
extensive use of trimethoprim-sulfamethoxazole as prophylaxis against human immunodeficiency virus (HIV) infectionassociated opportunistic infections probably makes a large
contribution to pyrimethamine resistance because HIV seropositivity has now reached 15% in Bangui.40
In conclusion, molecular analyses of antimalarial drugresistance alleles of P. falciparum isolates in Bangui clearly
show the widespread distribution of chloroquine- and pyrimethamine-resistant isolates, and to a lesser extent, sulfadoxine-resistant isolates. The molecular makers used in this study
are valuable tools for describing the epidemiology of drugresistant P. falciparum. Regular monitoring of molecular
makers both in Bangui and the rest of the Central African
Republic would help the National Malaria Control Program
to identify and recommend the best available treatment of
malaria in this country.
Received May 21, 2005. Accepted for publication September 22,
2005.
Acknowledgments: We thank the patients for participating in the
study.
Financial support: This work was supported by the French Government through the FSP/RAI 2001-168 project (French Ministry of Foreign Affairs).
Authors’ addresses: Didier Menard, Institut Pasteur de Madagascar,
BP 1274, Antananarivo 101, Madagascar, Telephone: 261-20-22-41272, Fax: 261-20-22-415-34, E-mail: [email protected]. Djibrine
Djalle, Ferdinand Yapou, Alexandre Manirakiza, and Antoine Talarmin, Institut Pasteur de Bangui, BP 923, Bangui, Central African
Republic.
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