Sulfadoxine-pyrimethamine (SP) is the first line antimalarial treatment in the Democratic Republic of Congo. Using polymerase chain reaction, we assessed the prevalence of mutations in the dihydrofolate reductase (dhfr) (codons 108, 51, 59) and dihydropteroate synthase (dhps) (codons 437, 540) genes of Plasmodium falciparum, which have been associated with resistance to pyrimethamine and sulfadoxine, respectively. Four hundred seventy-four patients were sampled in Kilwa (N = 138), Kisangani (N = 112), Boende (N = 106), and Basankusu (N = 118). The proportion of triple mutations dhfr varied between sites but was always > 50%. The proportion of dhps double mutations was < 20%, with some sites as low as 0.9%. A quintuple mutation was present in 12.8% (16/125) samples in Kilwa; 11.9% (13/109) in Kisangani, 2.9% (3/102) in Boende, and 0.9% (1/112) in Basankusu. These results suggest high resistance to pyrimethamine alone or combined with sulfadoxine. Adding artesunate to SP does not seem a valid alternative to the current monotherapy

Bonnet, M

Cohuet, S

D'Alessandro, U

Guthmann, J P

Van Herp, M

Van Overmeir, C

English

MSF Field Research

SHORT REPORT: MOLECULAR MARKERS ASSOCIATED WITH PLASMODIUM
FALCIPARUM RESISTANCE TO SULFADOXINE-PYRIMETHAMINE IN THE
DEMOCRATIC REPUBLIC OF CONGO
SANDRA COHUET, MARYLINE BONNET, MICHEL VAN HERP, CHANTAL VAN OVERMEIR,
UMBERTO D’ALESSANDRO, AND JEAN-PAUL GUTHMANN*
Epicentre, Paris, France; Médecins Sans Frontières, Brussels, Belgium; Prince Leopold Institute of Tropical Medicine,
Antwerp, Belgium
Abstract. Sulfadoxine-pyrimethamine (SP) is the first line antimalarial treatment in the Democratic Republic of
Congo. Using polymerase chain reaction, we assessed the prevalence of mutations in the dihydrofolate reductase (dhfr)
(codons 108, 51, 59) and dihydropteroate synthase (dhps) (codons 437, 540) genes of Plasmodium falciparum, which have
been associated with resistance to pyrimethamine and sulfadoxine, respectively. Four hundred seventy-four patients
were sampled in Kilwa (N  138), Kisangani (N  112), Boende (N  106), and Basankusu (N  118). The proportion
of triple mutations dhfr varied between sites but was always > 50%. The proportion of dhps double mutations was <
20%, with some sites as low as 0.9%. A quintuple mutation was present in 12.8% (16/125) samples in Kilwa; 11.9%
(13/109) in Kisangani, 2.9% (3/102) in Boende, and 0.9% (1/112) in Basankusu. These results suggest high resistance to
pyrimethamine alone or combined with sulfadoxine. Adding artesunate to SP does not seem a valid alternative to the
current monotherapy.
Since 1997, the Democratic Republic of Congo (DRC) has
been affected by continuous civil war, despite a 2003 cease-
fire. With ongoing poor access to health care, the health status
of the civilian population has continued to deteriorate. Since
2002, Médecins Sans Frontières (MSF) has supported health
structures in areas neighboring the ceasefire line (Kisangani
area in Oriental province, Boende and Basankusu areas in
Equator province, and Kilwa area in Katanga; Figure 1),
where access to the most vulnerable population is difficult.
These are areas of perennial seasonal malaria affecting
mainly small children. Malaria is one of the most significant
health problems in these areas, accounting for 35% of all
health center attendances in 2002 (Ministry of Health, unpub-
lished data). The national protocol, adopted in October 2001,
recommends sulfadoxine-pyrimethamine (SP) as the first-line
treatment of uncomplicated malaria. However, resistance to
this drug is expanding in many African countries.
1,2 Despite
variable levels of SP resistance recently measured in vivo,
3,4
no data (in vivo, in vitro) were available for our intervention
sites. In Kisangani and Basankusu, MSF used SP as the first-
line treatment, whereas in Kilwa and Boende, a combination
of SP and amodiaquine was used.
A good correlation has been shown between mutations in
the dihydrofolate reductase (dhfr) and dihydropteroate syn-
thase (dhps) genes of Plasmodium falciparum (Pf) and resis-
tance to pyrimethamine and sulfadoxine, respectively.
5,6 As-
sessing the prevalence of dhfr and dhps mutations has been
suggested as an alternative measure of SP resistance when in
vivo studies are difficult to implement,
7 which was the case in
our sites. We therefore determined the prevalence of the mu-
tations in codons 51, 59, and 108 of the dhfr gene and in
codons 437 and 540 of the dhps in each of our intervention
sites. Triple mutations of the dhfr gene, double mutations of
the dhps gene, and especially quintuple mutations of dhfr and
dhps genes are strongly correlated in vivo with SP treatment
failure.
8,9
Authorization to conduct these analyses was given by the
national health authorities. After obtaining written informed
consent, a blood sample was collected from each patient pre-
senting with uncomplicated malaria at Kisangani, Basankusu,
Kilwa, and Boende health centers. All patients with Pf ma-
laria confirmed both by a rapid test (Paracheck-Pf, Orchid,
Goa, India) and a thick/thin smear had a second capillary
blood smear collected on an Isocode stix (Schleicher &
Schuell, Dassel, Germany) for genomic analysis. A minimum
of 100 samples per health center (i.e., a total of 400 samples)
was considered logistically manageable and sufficient to esti-
mate the prevalence of the mutations. Samples were air dried,
stored adequately, and transported to the Institute of Tropi-
cal Medicine (Antwerp, Belgium) where parasite DNA ex-
traction and analysis of dhfr and dhps genes were performed.
Mutation-specific nested polymerase chain reaction (PCR)
and/or restriction digestions were used to analyze dhfr and
dhps mutations as described elsewhere.
10,11 A detailed de-
scription of these methods is available at http://www
.medschool.umaryland.edu/CVD/plowe.html.
The classification of samples was based on a published
methodology.
9 In brief, each dhfr and dhps codon was char-
acterized as wild-type (no mutation present), mixed (both
wild and mutant genotypes clearly present in the same infec-
tion), or pure mutant (only mutant genotypes detected).
Then, dhfr and dhps genotypes for each infection were cat-
egorized as follows: wild-type, no mutation detected; single,
infection involving parasites with a single mutation; double,
infection involving parasites with a double mutation; triple,
infection involving parasites with all three mutations de-
tected. Finally, infections involving parasites both with triple
dhfr mutations and double dhps mutations were categorized
as quintuple mutations.
Between September 2003 and March 2004, > 100 samples
were collected in each site, and most of them could be geno-
typed (Table 1). The proportion of triple mutations dhfr var-
ied between sites but was always > 50%. The proportion of
dhps double mutations was much lower, < 20%, with some
sites as low as 0.9%. Overall, a quintuple mutation was
present in 12.8% (16/125) of the samples in Kilwa; 11.9%
* Address correspondence to Jean-Paul Guthmann, Epicentre, 8 rue
Saint Sabin, 75011 Paris, France. E-mail: jguthmann@epicentre.msf.org
Am. J. Trop. Med. Hyg., 75(1), 2006, pp. 152–154
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
152(13/109) in Kisangani; 2.9% (3/102) in Boende; and 0.9%
(1/112) in Basankusu.
This survey provided an estimation of the SP resistance in
different areas where in vivo test data were not available. Our
sample was relatively small in size, because of the logistic
difficulties. However, the confidence intervals are relatively
narrow, allowing a fair estimate of the situation in the study
sites In our sites, the high frequency of dhfr triple mutations
and the rather high frequency of dhps double mutations in
two sites suggest that SP resistance is already well established.
These findings are not surprising considering that SP resis-
tance has been shown to increase quickly when used as mono-
therapy.
12–14 This situation could rapidly lead to low SP effi-
cacy because of selection of resistant parasites by widespread
use of SP. Previous studies have suggested that they may be a
stepwise accumulation of mutations in response to increasing
drug pressure.
15 This could be also the case in our sites. How-
ever, the data presented here cannot confirm this hypothesis.
It is important to note that differences between regions may
also reflect variation in the duration and magnitude of SP
use,
16 although these differences were not significant. This
study on molecular markers associated with SP resistance
should contribute to inform health authorities on anti-
malarial efficacy in these sites and guide treatment policy.
Indeed, in situations such as those we have described here,
where in vivo tests cannot be carried out, the estimation of the
prevalence of mutations offers an easy and rapid alternative.
The prevalence of quintuple mutations varied from 12% to
1%; however, the information available (especially concern-
ing the frequency and adequacy of antimalarial use in each
site) does not provide a clear explanation for these differ-
ences between sites.
The implementation of artemisinin-based combinations (or
ACT) recommended by WHO
17 needs careful consideration
of the partner drug to be associated with the artemisinin com-
ponent. The choice of new protocols should be seriously dis-
cussed among relevant authorities and malaria experts in
DRC. The information provided here should be added and
discussed considering other data available for the country.
The high SP resistance suggested by our studies indicate that
combining artesunate to SP would not increase the efficacy of
the first-line treatment, at least not in the long term. Arte-
mether + lumefantrine (Coartem, Novartis Pharma, Basel,
Switzerland) has been shown to be very efficacious even in
unsupervised conditions,
18 but one major limitation is the
cost. At the current cost of 2.4 US$/adult treatment, many
African countries are unable to afford Coartem for public
sector use without the support of international donors (e.g.,
the Global Fund). Artesunate + amodiaquine is another in-
teresting alternative, less expensive than Coartem, and should
TABLE 1
Prevalence of mutations at codons 108, 51, and 59 in dhfr and of mutations at codons 437 and 540 of dhps, by site
Dhfr*
Kilwa (n  128) Kisangani (n  109) Boende (n  102) Basankusu (n  112)
n % 95% CI n % 95% CI n % 95% CI n % 95% CI
Wild 5 3.9 1.4–9.3 3 2.8 0.7–8.4 3 2.9 0.6–8.3 1 1 0–5.6
Single mutation 4 3.1 1–8.3 0 0 – 2 2.0 0.2–6.9 5 4.5 1.6–10.6
Double mutation 19 14.8 9.4–22.5 43 39.4 30.3–49.3 35 34.3 25.2–44.4 33 29.4 21.4–38.9
Triple mutation 100 78.2 69.8–84.7 63 57.8 47.9–67.0 62 60.8 50.6–70.3 73 65.1 55.5–73.7
Mixte 49 38.4 29.9–47.3 35 32.1 23.7–41.8 44 43.1 33.4–53.3 64 57.1 47.4–66.3
Pure 51 39.8 34.4–48.9 28 25.7 18.0–35.1 18 17.6 10.8–26.4 9 8.0 3.9–15.1
Dhps†
Kilwa (n  135) Kisangani (n  109) Boende (n  102) Basankusu (n  112)
n % 95% CI n %9 5 C I n % 95% CI n % 95% CI
Wild 102 75.6 62.3–82.4 62 56.9 47–66.2 30 29.4 20.8–39.2 38 34 25.4–43.5
Single mutation 15 11.1 6.6–17.9 26 23.8 16.4–33.1 68 66.7 56.6–75.7 73 65 55.5–73.8
Double mutation 18 13.3 8.3–20.5 21 19.3 12.6–28.2 4 3.9 1.1–9.7 1 0.9 0.0–5.6
Mixte 10 7.4 3.8–13.5 8 7.4 3.4–14.4 1 1.0 0.0–5.3 0 0 –
Pure 8 5.9 2.8–11.7 13 11.9 6.7–19.9 3 2.9 0.6–8.3 1 1 0.0–5.6
Dhfr + dhps
Kilwa (n  125) Kisangani (n  109) Boende (n  102) Basankusu (n  112)
n % 95% CI n % 95% CI n % 95% CI n % 95% CI
Quintuple mutation 16 12.8 7.7–20.2 8 7.3 3.4–14.3 3 2.9 0.6–8.3 1 0.9 0.0–5.6
Mixte 11 8.8 4.7–15.5 5 4.6 1.5–10.4 2 2 0.2–6.9 1 0.9 0.0–5.6
Pure 5 4 1.5–9.5 3 2.7 0.6–7.8 1 0.9 0.0–5.3 0 0 –
* Dhfr, single  108, 51, or 59, double  108 and 51 or 59, triple  108-51-59.
† Dhps: simple  437 or 540, double  437 and 450.
FIGURE 1. Location () of health structures where the survey was
carried out.
MOLECULAR MARKERS OF PF SP RESISTANCE IN DRC 153be soon available in blister packs as a fixed combination.
Information available for amodiaquine
19 indicates that this
drug is safe. Other promising alternatives (such as chlor-
proguanil-dapsone-artesunate, piperaquine-dihydroartemisinin,
or pyronaridne-artesunate) are currently under development
and should soon become available, increasing considerably
the options for the National Malaria Control Programs.
Received October 3, 2005. Accepted for publication February 9,
2006.
Acknowledgments: The authors thank Dr. Nsibu Ndosimao, General
Director of the PNLP, for authorizing this study, Dr. Alphonse
Swana-Nimy for representing PNLP on the field, and Dr. Kristina
Persson and Dr. Anja Huefner who supervised the data collection on
the field. We also thank Rebecca Freeman Grais at Epicentre head-
quarters for reviewing an earlier draft of the manuscript.
Financial support: This study was funded by Médecins sans Fron-
tières. The American Society of Tropical Medicine and Hygiene
(ASTMH) assisted with publication expenses.
Authors’ addresses: Sandra Cohuet, Maryline Bonnet, and Jean-
Paul Guthmann, Epicentre, 8 rue Saint Sabin, 75011 Paris, France.
E-mails: sandra.cohuet@lshtm.ac.uk, maryline.bonnet@
geneva.msf.org, and jguthmann@epicentre.msf.org; Michel van Herp,
Médecins sans Frontières, 94 rue Dupré, 1090 Brussels, Belgium,
E-mail: michel.vanherp@msf.be; Chantal van Overmeir and Umberto
D’Alessandro, Prince Léopold Institute of Tropical Medicine, 155
Nationalestraat, B-2000 Antwerp, Belgium, E-mails: cvoverm@itg.be
and udalessandro@itg.be.
Reprints requests: Jean-Paul Guthmann, Epicentre, 8 rue Saint
Sabin, 75011 Paris, France. E-mail: jguthmann@epicentre.msf.org.
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COHUET AND OTHERS 154

Assessing the efficacy of chloroquine and sulfadoxine-pyrimethamine for treatment of uncomplicated Plasmodium falciparum malaria in the Democratic Republic of Congo.

Efficacy of mefloquine and sulfadoxine-pyrimethamine for the treatment of uncomplicated Plasmodium falciparum infection in Machinga District,

Efficacy of sulfadoxine/pyrimethamine in the treatment of uncomplicated Plasmodium falciparum malaria in Republic of Congo.

High level of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine in children in Tanzania. Trans R Soc Trop Med Hyg 90:

In vivo selection for a specific genotype of dihydropteroate synthetase of Plasmodium falciparum by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment.

Molecular assays for surveillance of antifolate-resistant malaria.

Molecular epidemiology of malaria in Cameroon. XVI. Longitudinal surveillance of in vitro pyrimethamine resistance in Plasmodium falciparum.

Molecular markers for failure of sulfadoxinepyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance.

Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in-vivo drug resistance in West Papua,

Organization. Position of WHO’s Roll Back Malaria Department on Malaria Treatment Policy. Geneva: World Health Organization,

Prediction of Plasmodium falciparum resistance to sulfadoxine/ pyrimethamine in vivo by mutations in the dihydrofolate reductase and dihydropteroate synthetase genes: a comparative study between sites of differing endemicity.

Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa.

Sulfadoxine-pyrimethamine in treatment of malaria in Western Kenya: increasing resistance and underdosing.

Supervised versus unsupervised intake of six-dose artemether-lumefantrine for treatment of acute, uncomplicated Plasmodium falciparum malaria in Mbarara, Uganda: a randomised trial.

Systematic review of amodiaquine treatment in uncomplicated malaria.

Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites.

Two mutations in dihydrofolate reductase combined with one in the dihydropteroate synthase gene predict sulphadoxine-pyrimethamine parasitological failure in Ugandan children with uncomplicated falciparum malaria.

Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy.

Short report: molecular markers associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine in the Democratic Republic of Congo.

Guthmann, J P P

Short Report: Molecular Markers Associated with Plasmodium Falciparum Resistance to Sulfadoxine-Pyrimethamine in the Democratic Republic of Congo.

Short report: molecular markers associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine in the Democratic Republic of Congo.

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