Molecular surveillance of drug-resistance associated mutations of Plasmodium falciparum in south-west Tanzania.

Abstract

In Tanzania, drug-resistant malaria parasites are an increasing public health concern. Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2001. Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes.
In samples from 86 patients with uncomplicated Plasmodium falciparum malaria from Mbeya and Matema, Mbeya region, south-western Tanzania, the occurrence of mutations was investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr and pfdhps, conferring SP resistance, as well in cytb which is linked to resistance to atovaquone.
Pfcrt T76 occurs in 50% and pfmdr1 Y86 in 51.7%. Pfdhfr triple mutations coexisting with pfdhps double mutations were detected in 64.3% of the P. falciparum isolates. This quintuple mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of the cytb gene were not detected.
These findings of a high prevalence of mutations conferring SP resistance correspond to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation of changing first-line treatment to artemisinin-based combination therapy.

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BioMed Central
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Malaria Journal
Open Access
Research
Molecular surveillance of drug-resistance associated mutations of
Plasmodium falciparum in south-west Tanzania
Mirjam Schönfeld1, Isabel Barreto Miranda1, Mirjam Schunk1,
Ibrahim Maduhu2, Leonard Maboko3, Michael Hoelscher1,3, Nicole Berens-
Riha1, Andrew Kitua4 and Thomas Löscher*1
Address: 1Department of Infectious Diseases and Tropical Medicine, Ludwig-Maximilians- University (LMU), Munich, Germany, 2Department of
Paediatrics and Child Health, Mbeya Referral Hospital, Tanzania, 3Mbeya Medical Research Programme (MMRP), Mbeya, Tanzania and 4National
Institute for Medical Research (NIMR), Dar es Salaam, Tanzania
Email: Mirjam Schönfeld - mimischoen@web.de; Isabel Barreto Miranda - barretomiranda@lrz.uni-muenchen.de;
Mirjam Schunk - mschunk@mmrp.org; Ibrahim Maduhu - nyasilu55@yahoo.com; Leonard Maboko - lmaboko@mmrp.org;
Michael Hoelscher - hoelscher@lrz.uni-muenchen.de; Nicole Berens-Riha - berens@lrz.uni-muenchen.de; Andrew Kitua - akitua@nimr.or.tz;
Thomas Löscher* - loescher@lrz.uni-muenchen.de
* Corresponding author
Abstract
Background: In Tanzania, drug-resistant malaria parasites are an increasing public health concern.
Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment
recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2001.
Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the
dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes.
Methods: In samples from 86 patients with uncomplicated Plasmodium falciparum malaria from
Mbeya and Matema, Mbeya region, south-western Tanzania, the occurrence of mutations was
investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr
and pfdhps, conferring SP resistance, as well in cytb which is linked to resistance to atovaquone.
Results: Pfcrt T76 occurs in 50% and pfmdr1 Y86 in 51.7%. Pfdhfr triple mutations coexisting with
pfdhps double mutations were detected in 64.3% of the P. falciparum isolates. This quintuple
mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of
the cytb gene were not detected.
Conclusion: These findings of a high prevalence of mutations conferring SP resistance correspond
to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation
of changing first-line treatment to artemisinin-based combination therapy.
Background
In Sub-Saharan Africa malaria is a leading cause of mor-
bidity and mortality, especially in children under five
years [1]. Despite intensive campaigns, over 10 Million
malaria cases occurred in Tanzania in 2003, about 30%
more compared to the previous year [2]. Most of these
cases were caused by Plasmodium falciparum. This may be
partly due to better surveillance systems, but raising drug
Published: 15 January 2007
Malaria Journal 2007, 6:2 doi:10.1186/1475-2875-6-2
Received: 05 October 2006
Accepted: 15 January 2007
This article is available from: http://www.malariajournal.com/content/6/1/2
© 2007 Schönfeld 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.

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resistance is the most likely reason for this tremendous
increase.
Chloroquine (CQ) was the antimalarial treatment of
choice during the second half of the 20th century. But
increasing rates of CQ resistance led Tanzania to change
its first line treatment of uncomplicated malaria to sulf-
adoxine-pyrimethamine (SP) in 2001 [3]. This antifolate
combination seemed to be an effective and reasonable
alternative, but resistance to SP was rapidly gaining
ground, facilitated by the slow elimination from the body.
New data show a high level (45%) of SP treatment failures
in Muheza, northeast Tanzania [4]. Other effective drugs,
where resistance is as yet not a frequent problem, such as
atovaquone-proguanil or mefloquine, are of limited value
due to their high current costs [5]. A useful alternative is
artemisinin-based combination therapy (ACT), e.g., arte-
mether-lumefantrine which is introduced as the new first
line drug in Tanzania in mid 2006. At the moment the
availability of ACT is limited; while artemisinin mono-
therapy formulations and SP are commonly used, espe-
cially in the private sector. Some drug shops still sell the
relegated CQ.
CQ resistance is associated with an amino acid change
from lysine to threonine in codon 76 of the P. falciparum
chloroquine resistance transporter gene (pfcrt) [6], and a
mutation from asparagine to tyrosine in codon 86 of the
multidrug resistant gene (pfmdr) [7-10]. Besides there are
indices that pfmdr1 N86 is associated with resistance to
lumefantrine that is widely used in combination with
artemether [11], and also with decreased sensitivity to
artemisinins [12].
SP resistance is associated with mutations in the dihydro-
folate reductase (pfdhfr) and dihydropteroate synthase
(pfdhps) genes. Pyrimethamine is a selective, competitive
inhibitor of dihydrofolate reductase and earlier in the
folate pathway sulfa drugs inhibit dihydropteroate syn-
thase [13,14]. Several point mutations are connected to
antifolate drug resistance. The quintuple mutation (triple
pfdhfr: I51, R59, N108 and double pfdhps: G437, E540) is
discussed as a relevant molecular marker of SP treatment
failure [15,16].
Atovaquone-proguanil is a relatively new antimalarial
drug that inhibits mitochondrial electron transport. Point
mutations in the cytb codon 268 are associated with resist-
ance to this combination [17-19].
Methods
Overall 86 blood samples were collected from patients
with clinically diagnosed uncomplicated P. falciparum
malaria (fever ≥ 38.0°C, parasitaemia ≥ 2 000/μl) from
August to October in 2004 and from June to July in 2005
in Mbeya region of South-western Tanzania. Patients were
enrolled at two locations: Matema Health Care Center
located in a poor rural area at the shores of Lake Malawi
with holoendemic malaria transmission and the Mbeya
Referral Hospital, a tertiary hospital at an altitude of 1 700
meters that admits complicated cases from surrounding
mesoendemic areas or people that have travelled within
Tanzania. Written informed consent was obtained from
each patient or the parental guide. The study was reviewed
and approved by the local IRB at the Mbeya Referral Hos-
pital and the Tanzanian National Ethics Board of the
National Institute for Medical Research, Dar es Salaam.
From each patient a finger prick blood sample was taken
for thick and thin blood film and another for filter paper
blood sample. Giemsa-stained blood films were exam-
ined for malaria parasites (per 200 white blood cells) and
densities were assessed based on a assumed mean WBC
count of 8 000/μl. DNA extraction from filter paper
bloodspots was done using Chelex® (Bio-Rad, Germany)
as described elsewhere [20] Nested PCR assays were used
to verify the parasite species [21]. The DNA was amplified
by nested PCR and digested by the RFLP-method to detect
the mutations of P. falciparum pfcrt 76, pfmdr1 86, dhfr 16,
51, 59, 108, 164, dhps 436, 437, 540, 581, 613 and cytb
268 [17,22,23]. Mixed alleles (wild type and mutant)
were assessed as mutant. The median age of the 86
patients with P. falciparum infection (45 female, 41 male)
was 21 years (range 8 month to 55 years). All 86 isolates
showed P. falciparum mono-infection, there was no P.
vivax, P. ovale, or P. malariae infection. The geometric
mean parasite density was 29 992/μl (range 3 320/μl to
127 440/μl).
Results
Table 1 displays the prevalence of pfcrt, pfmdr1, pfdhfr,
pfdhps and cytb alleles in Mbeya & Matema. Pfcrt T76
mutation is expressed by 58.8% in Mbeya and 47.8% in
Matema. 70.6% of the isolates from Mbeya and 47.1%
from Matema showed the pfmdr1 Y86 mutation. Almost
100% of both settings exhibited the pfdhfr N108 muta-
tion. Likewise, nearly all samples showed the I51 muta-
tion. All but one displayed the R59 mutation in Mbeya.
Amino acid changes from alanine to glycine at codon 437
and from lysine to glutamine at codon 540 of the pfdhps
gene were detected in 81% and 86.9%, respectively. Only
one specimen in Matema displayed the G581 variant. No
pfdhfr L164 mutation was seen. T108 and V16 variants
which are linked with cycloguanil resistance were not
present as well. In line with previous reports no evidence
for cytb codon 268 mutations were found in south-west-
ern Tanzania [24].

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Discussion
More relevant for predicting treatment failure or emerging
resistance are combinations of the point mutations
described above (Table 2). In Matema, the rural setting,
the Pfdhfr quintuple mutation was more common than in
the Mbeya Referral Hospital (67.2% vs 52.9%), while the
Pfdhfr triple mutation, suggested to be an early molecular
marker for SP resistance in Tanzania [25], was more fre-
quent in Mbeya (79.7% vs 94.1%). Although the predic-
tive value of these markers for SP treatment failure has not
been established in the study regions, these results are in
line with the high level of treatment failure (42.3%) in a
multi-site survey in Tanzania [26].
Even so this study investigated a limited number of
patients and therefore differences may not reach signifi-
cance, differences in mutation rates might reflect differ-
ences of access to health care between the two locations.
While individuals in the rural area receive their malaria
treatment, currently SP, almost exclusively through the
Matema Health Care Center, patients from Mbeya can
choose upon a wide variety of health care facilities and
antimalarial drugs. Ongoing usage of CQ may be assumed
there.
Although considerably higher resistance rates before 2001
are probable, the still relatively high rate of pfcrt and
pfmdr1 mutations is contrary to other reports that have
demonstrated a complete regression of pfcrt mutations
several years after leaving CQ as first – line antimalarial
drug [27,28].
In vivo selection of pfmdr1 86N allele by artemether-lume-
fantrine has been found in Tanzania [11] and pfmdr copy
numbers seem to influence susceptibility to lumefantrine
and artemisinin [29]. There is no prediction possible due
to our results but continuing surveillance would be inter-
esting concerning pfmdr1 polymorphisms.
Table 2: Prevalence of pfcrt, pfmdr1, dhfr and dhps genotype combinations conferring chloroquine and sulfadoxine-pyrimethamine
resistance in Mbeya & Matema, together and each seperated. The risk ratio is the prevalence ratio of the combinations between
Mbeya and Matema.
Grouped alleles
Mbeya & Matema (%)Mbeya (%) Matema (%) Risk ratio
Pfcrt+pfmdr1
Dhfr
T76+Y86**
I51+N108*
I51+R59*
I51+R59+ N108*
G437+E540***
29 (34.1)
80 (93)
71 (82.6)
71 (82.6)
68 (81)
54 (64.3)
9 (52.9)
17 (100)
16 (94.1)
16 (94.1)
10 (58.8)
9 (52.9)
20 (29.4)
63 (91.3)
55 (79.9)
55 (79.7)
58 (86.6)
45 (67.2)
0.5556
0.9130
0.8469
0.8469
1.4716
1.2687
Dhfr triple
Dhps double
Dhfr/dhps quintuple (triple dhfr + double dhps) ***
* N = 86 ** N = 85 *** N = 84
Table 1: Prevalence of mutations conferring resistance to chloroquine, sulfadoxine-pyrimethamine and atovaquone-proguanil in
Plasmodium falciparum isolates from Mbeya & Matema, southern Tanzania
GeneMutationNMutation (%)Mixed type (%)
Pfcrt
Pfmdr1
T76
Y86
86
85
37 (43)
38 (44.7)
6 (7)
6 (7.1)
Dhfr
I51
N108
T108
R59
V16
L164
86
86
86
86
86
86
80 (93)
84 (97.7)
0 (0)
69 (80.2)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
6 (7)
0 (0)
0 (0)
Dhps
A436
G437
E540
G581
N613
84
84
84
81
81
4 (4.8)
66 (78.6)
65 (77.4)
1 (1.2)
0 (0)
3 (3.6)
2 (2.4)
8 (9.5)
0 (0)
0 (0)
Cytb
N268850 (0)0 (0)

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Conclusion
This study confirms the high prevalence of point muta-
tions in the pfdhfr and pfdhps genes in Tanzania which are
associated with SP treatment failure [25,26]. The rate of
quintuple pfdhfr/pfdhps mutations in the Mbeya region,
south-western Tanzania, is in the upper range of frequen-
cies reported in East Africa. Data from Malawi, Kenya,
Tanzania and Ethiopia range from 10 to 78% [25,30-32].
The absence of cytb codon 268 supports atovaquone-pro-
guanil as a possible second- or third-line drug for treat-
ment of uncomplicated malaria.
The study data might be used as a basis for surveillance of
resistance markers after introduction of ACT and might
later indicate the possibility for reintroduction of one of
the other drugs.
Authors' contributions
LM, AK, MH and TL designed the study. MS, IM and LM
were responsible for patient recruitment and parasitolog-
ical examinations. MS, IBM and MS did the PCR assays.
MH did the data processing. MS, AK, IBM, MS, NBR and
TL wrote the paper with major contributions of the other
authors.
Conflict of interest
The authors do not have a commercial or other associa-
tion that might pose a conflict of interest.
Acknowledgements
We thank MMRP for making their facilities available for this project. We
acknowledge the contributions of NIMR, Dr Eleuter Samky, Diretor of
Mbeya Referal Hospital and Dr. Donnan Mmbando, Regional Medical
Officer for Mbeya Region and Heike Schimanowski, Head of the Matema
Health Care Center, for the permission to use their respective facilities and
facilitating ethical clearance. Vera Kleinfeldt was responsible for training of
all laboratory technicians that participated in the study. This study is part of
the thesis work of Mirjam Schönfeld at the Ludwig-Maximilians-University
(LMU) Munich, Germany.
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