Molecular determinants of sulfadoxine-pyrimethamine resistance in
Plasmodium falciparum in Nigeria and the regional emergence of dhps
431V
Mary C. Oguike
a
,
*
,
e
,
1
,
h
,
1
,
a
Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United
Kingdom
b
Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Nigeria
c
Department of Pharmacology and Therapeutics, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Nigeria
d
Department of Child Health, University of Benin Teaching Hospital, Benin City, Nigeria
e
Department of Paediatrics, Specialist Hospital Maiduguri, Borno State, Nigeria
f
Malaria Consortium, Regional Ofce for Africa, Kampala, Uganda
g
Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
h
Malaria Consortium, London, United Kingdom
i
Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United
Kingdom
article info
Article history:
Received 12 March 2016
Accepted 12 August 2016
Available online 29 September 2016
Keywords:
Sulfadoxine-pyrimethamine
dhps
dhfr
mutations
Nigeria
abstract
There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate
reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a
novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To
assess how widespread this mutation is among parasites in different parts of the country and conse-
quently ll the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively
analysed 1000 lter paper blood spots collected in surveys of pregnant women and children with un-
complicated falciparum malaria between 2003 and 2015 from four sites in the south and north.
Genomic DNA was extracted from lter paper blood spots and placental impressions. Point mutations
at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of
the dhps gene were evaluated by nested PCR amplication followed by direct sequencing.
The distribution of the dhps-431V mutation was widespread throughout N igeria with the highest
prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from
0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with
431V, the combination 431V þ 436A þ 437Gþ581Gþ613S was the most common.
All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum
DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could
alter the effects of both S436A/F and A437G, which closely follow the 2nd
b
-strand. Con sequently, it is
now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use
of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy
(IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and
2015.
© 2016 The Authors. Published by Elsevie r Ltd on behalf of Australian Society for Parasitology. This is an
open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
* Corresponding author. Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
E-mail address: mary.oguike@lshtm.ac.uk (M.C. Oguike).
1
Deceased.
Contents lists available at ScienceDirect
International Journal for Parasitology:
Drugs and Drug Resistance
journal homepage: www.elsevier.com/locate/ijpddr
http://dx.doi.org/10.1016/j.ijpddr.2016.08.004
2211-3207/© 2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
International Journal for Parasitology: Drugs and Drug Resistance 6 (2016) 220e229
1. Introduction
Malaria is a major public health challenge in sub-Saharan Africa.
In 2015, there was an estimated 214 million cases and 438,000
deaths due to malaria globally with Nigeria accounting for 25% of
these (WHO, 2015). Malaria poses health risks to both neonate and
mother during pregnancy. It leads to low birth-weight, placental
malaria, severe maternal anaemia (especially in primigravidae),
and perinatal mortality (Shulman et al., 1999; Menendez et al.,
2010; Oyibo and Agomo, 2011). Pregnant women are usually at
higher risk of malaria infection than their non-pregnant counter-
parts due to temporary depression of immunity during foetal
development (Menendez, 1995). The World Health Organization
(WHO) has recommended intermittent preventive treatment dur-
ing pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) as part
of strategies to control malaria in most endemic countries (WHO,
2009). IPTp-SP involves the administration of a supervised cura-
tive treatment dose of SP at each scheduled antenatal care visit
starting as early as possible in second trimester and at an interval
not less than 4 weeks apart and up to the time of delivery (WHO,
2014). WHO recommended IPTp-SP as a strategy for prevention
of malaria in pregnancy in 2001 but IPTp-SP was only adopted in
2005 as national policy in Nigeria (WHO, 2000; FMOH, 2005). The
implementation of this strategy is being faced with challenges such
as timing of SP administration (Onoka et al., 2012), knowledge and
practices of the population (Onwujekwe et al., 2012; Diala et al.,
2013) and rising levels of parasite resistance to SP in the general
malaria chemoprevention (SMC) is another malaria control inter-
vention, which uses SP. It is the administration of a complete
treatment course of amodiaquine plus SP to children aged between
3 and 59 months at monthly intervals, beginning at the start of the
transmission season to a maximum of four doses during the malaria
transmission season (WHO, 2012). SMC is only recommended in
areas with highly seasonal malaria transmission in the Sahel sub-
region of sub-Saharan Africa, where P. falciparum is sensitive to
both antimalarial medicines. SMC has been fully deployed in Kat-
sina and Jigawa states of northern Nigeria.
Surveillance of SP resistance levels must be achieved by moni-
toring of molecular markers (WHO, 2004). SP resistance is linked
with substitutions of amino acids in the enzymes dihydropteroate
synthetase (DHPS) and dihydrofolate reductase (DHFR) in the folate
1994; Brooks et al., 1994). Pyrimethamine targets the enzyme
DHFR disrupting catalysis of the NADPH-dependent reduction of 7,
8-dihydrofolate to 5,6,7,8-tetrahydrofolate (Blakeley, 1984) while
sulfadoxine blocks the folate biosynthetic pathway at the DHPS
level by disrupting the coupling of 7,8,-dihydro-6-
hydroxymethylpterin pyrophosphate with para-amino benzoic
acid (pABA) to yield 7, 8- dihydropteroate (Walter, 1991).
Resistance to SP has evolved worldwide, and is caused by point
mutations that accumulate at multiple sites in both the dhfr and
dhps genes (Wang et al., 1997). In both genes, each successive
mutation has been shown to incrementally increase the parasite's
tolerance to the drug in vitro (Triglia et al., 1997, 1998). An aspara-
gine substitution at codon 108 of dhfr
followed by substitution at
codons 51 and 59 seem to be necessary for pyrimethamine resis-
tance while an additional mutation at codon 164 (I164L) has been
associated with high grade pyrimethamine resistance (Plowe et al.,
1997). Mutations at codons 437 and 540 of dhps play the most
signicant role in sulfadoxine resistance among African parasites.
In East and South Africa, mutations at the 437 and 540 codons are
found together while in West and Central Africa the 437 is found on
its own (Pearce et al., 2009). Laboratory studies show that the
A437G and K540E substitutions in combination raise sulfadoxine
tolerance of sensitive DHPS by 200 fold, compared to just 10 fold for
the A437G substitution alone (Triglia et al., 1997). Hence East Af-
rican parasites are predicted to withstand higher doses of SP than
West African parasites. The efcacy of IPTp-SP is being further
compromised in east Africa by the additional emergence of dhps
mutation at codon 581 in northern Tanzania (Gesase et al., 2009)
which has been shown to reduce the efcacy of IPTp-SP (Minja
et al., 2013) termed super resistance (Naidoo and Roper, 2013).
WHO recommended that prior to implementation of IPTp-SP in any
region with moderate to high malaria transmission, the prevalence
of K540E and A581G should be determined. IPTp-SP should be used
in regions with a prevalence rate K540E less than 50% and A581G
less than 10% (WHO, 2013a).
Hitherto the dhps K540E and A581G mutations have been rare in
West and Central Africa and this is consistent with evidence of
IPTp-SP efcacy during the same period (Falade et al., 2007; Aziken
Reports of novel dhps mutations at codon 431(I431V) from UK
imported malaria infections originating from Nigeria (Sutherland
et al., 2009) and pregnant women from Cameroon (Chauvin et al.,
2015) suggest this mutation is emerging. In Nigeria, there has
been a dearth of molecular surveillance data (Naidoo and Roper,
2011; Drug resistance maps, http://www.drugresistancemaps.org/
ipti/) which makes this difcult to substantiate. Crucially this
needs to be addressed to underpin the continuing use of SP for IPTp
and seasonal malaria chemoprevention (SMC).
In order to ll the gap in SP resistance surveillance data in
Nigeria, we analysed retrospectively 1000 lter paper blood spots
collected from malaria-infected pregnant women (with or without
IPTp-SP intervention) and children with uncomplicated falciparum
malaria from four geopolitical zones in Nigeria. We also evaluated
the patterns of genetic changes in the parasite between 2003 and
2015. This study is the rst of its kind providing patterns of SP
resistance in different regions of Nigeria.
2. Materials and methods
We identied molecular markers of SP resistance by nested PCR
and direct sequencing in 1000 malaria positive blood spots
collected from pregnant women and children attending hospitals
across Southwest, Southeast, South-south and Northeast Nigeria
(
Fig. 1). Southern Nigeria comprises of the tropical rain forest with
perennial malaria transmission occurring in rural and urban areas
while the northern part is mostly characterized as arid savannah
with less annual rainfall and more seasonal transmission (Ekanem
et al., 1990). In the past, chloroquine and sulfadoxine-
pyrimethamine were used but later abandoned in 2005 due to
increased threat of drug resistance. The antimalarial drug regimens
for all parts of Nigeria is currently artemether-lumefantrine and
amodiaquine-artesunate. Filter paper blood spots and placental
impressions were collected from pregnant women attending St
Mary's Catholic Hospital Eleta Ibadan between May 2003 and
October 2004 (Falade et al., 2007), Damboa General hospital be-
tween 2010 and 2012 (Damboa LGA, Borno State), Polyclinic (an
extension of Park Lane hospital) and Balm of Gilead Specialist
hospital between 2010 and 2012 (both in Enugu, Enugu State). Filter
paper blood spots were also collected from children with uncom-
plicated malaria presenting at General Outpatient Department of
the University College Hospital (UCH), Ibadan, and the Primary
Health Care Center (PHC), Idi-Ayunre, Oluyole Local Government
Area (both in Oyo State) between August 2007 and May 2008
(Falade et al., 2014); University of Benin Teaching Hospital (UBTH)
and Sickle-cell centre (both in Edo State) between September 2014
M.C. Oguike et al. / International Journal for Parasitology: Drugs and Drug Resistance 6 (2016) 220e229 221
and April, 2015 (details of this study is being prepared for a separate
publication). Oyo state is located in Southwest, Borno state in
Northeast, Enugu state in Southeast and Edo state in South-south
geopolitical zones of Nigeria. Informed consent was obtained
from all subjects before enrolment. All lter paper blood samples
were shipped to London School of Hygiene and Tropical Medicine
for molecular analysis. Ethical approval for the 2003 study was
obtained from the University of Ibadan/University College Hospital
Institutional Review Committee and the Boston University Insti-
tutional Review Board. The 2007 study was approved by the Uni-
versity of Ibadan/UCH Institutional Review Committee and Oyo
State ministry of health ethical review committee. The 2010 study
was approved by the Health Research Ethics Committee of the
University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu while
the 2015 study was approved by the Ethics and Research commu-
nity, University of Benin Teaching hospital and Hospital Manage-
ment Board.
2.1. DNA preparation, PCR diagnosis, PCR genotyping and
sequencing
2.1.1. DNA preparation
DNA extraction from blood spots was carried out in 96-well
plate format using the Chelex extraction method as described
elsewhere (Plowe et al., 1995).
2.1.2. Description of study samples
DNA was extracted from a total of 1000 lter paper blood spots.
In Ibadan, samples from two different time points (2003 and 2007)
were used. In 2003, fty (50) matched maternal and placental
blood spots (100 individual samples) from pregnant women were
evaluated. In 2007e2008, two hundred (200) lter paper blood
spots obtained at enrolment from children with uncomplicated
falciparum malaria were evaluated. In 2010e2012, samples
obtained from an in vivo efcacy study of IPTp-SP in Maiduguri and
Enugu (Component A) were evaluated. A further subset from a
cross-sectional study of pregnant women in Enugu (Component B)
were also evaluated. In Maiduguri, a total of one hundred and forty-
two (142) lter paper blood spots were evaluated. These consisted
of day0, 7, 14, 21, 28, 42, maternal blood and placental samples from
each sample ID. Likewise in Enugu (Component A), a total of two
hundred and thirty-three (233) samples were evaluated from day0,
7, 14, 21, 28, 42, maternal and placental blood spots. For Enugu
(Component B), two hundred and twenty-ve (225) samples were
evaluated. In 2014e2015, one hundred (100) samples from a cross-
sectional survey amongst sickle cell and normal children were
evaluated. Components A and B were the followed up (FU) and non-
followed up (Non-FU) pregnant women, respectively.
2.1.3. PCR diagnosis, PCR genotyping and sequencing
One thousand samples were screened for infection with
P. falciparum using nested PCR (Snounou et al., 1993). Point muta-
tions at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and
codons 431, 436, 437, 540, 581 and 613 of the dhps gene were
evaluated by nested PCR amplication as earlier described (Pearce
et al., 2003). 594 bp and 711 bp products of dhfr and dhps genes
respectively were sized against 100 bp molecular weight marker on
1.2% agarose gel stained with ethidium bromide. PCR products were
enzymatically puried using Exonuclease I-Fast Alkaline Phospha-
tase according to the manufacturer's instructions followed by direct
sequencing of products. Sequences were analysed using Lasergene
analysis software (DNAStar, Madison, WI).
2.2. P. falciparum DHPS modelling
2.2.1. Preparing the intensive homology model of PfDHPS
A modied Phyre2 model (Kelley et al., 2015) was developed for
the P. falciparum IT (wild type enzyme) using the intensive option.
The sequence ran from just before the start of the DHPS sequence at
residue Ile 366 to the C-terminus at Val 706. 11 May 13.33 2016
(ref:0347ffe1a64d64b1). The program chose 6 template crystal
structures for the nal modelling process as follows:
1 AD1 (a) Staphylococcus aureus
1 AJZ (a) Escherichia coli
3 TR9 (a) Coxiella burnetti
1 EYE (a) Mycobacterium tuberculosis
2 BMB (a) Saccharomyces cerevisiae
1 TX2 (a) Bacillus anthracis
Eighty-nine percent (89%) of residues were modelled at high
(>90%) condence but condence in the second P. falciparum insert
towards the end of the sequence 624e666 was low. The QMEAN
test (Benkert et al., 2009) was used to determine the reliability of
the modelled protein. The QMEAN value was 0.567 with a Z-score
of 2.43 (estimated reliability between 0 and 1).
2.2.2. Introduction of single mutations using the site directed
mutator DUET (Pires et al., 2014)
DUET can be used on homology-modelled or crystal structures
to check the impact of individual drug-resistance mutations, in
addition to allowing examination of structures in the modied
*.pdb le in pdb viewers.
The Program works by measurement of Gibbs energy change
(delta delta Gibbs,
dd
G) (in kilocalories/mol) involved in folding and
unfolding a wild type or a mutated structure, taking the overall
energy involved for the wild type as zero, and comparing the in-
crease or decrease of energy seen in the same process for the
mutant. This gives a positive (stabilizing) value or a negative
Fig. 1. Prevalence of dhps 431 mutation haplotypes between 2003 and 2014 in Nigeria.
A map of Nigeria showing the study locations and the prevalence of the various
combinations of the dhps-431V haplotypes between 2003 and 2014/2015. The preva-
lence of dhps-431V haplotypes in Yaounde, Cameroon (2015) is also shown.
M.C. Oguike et al. / International Journal for Parasitology: Drugs and Drug Resistance 6 (2016) 220 e229222
(destabilizing) value for the new residue. Mutations having very
positive or very negative
dd
G values are likely to render a protein
less t than where
dd
G is moderate, so the values expected for most
common non-damaging mutations are of the order of þ or - 1.0. It
follows that under natural conditions the advantage gained by the
organism, after residue change, will depend very much on
balancing the intrinsic loss of tness with the intensity of drug-
pressure.
3. Results
Point mutations in dhps and dhfr genes were evaluated in 1000
malaria-positive (PCR-conrmed) lter paper blood spots obtained
from pregnant women and children in the southern and northern
parts of Nigeria between 2003 and 2015. In Ibadan (2003), of the
fty (50) matched maternal and placental blood spots evaluated,
thirty-eight (38) were successfully amplied and sequenced. In
2007e2008, of the two hundred (200) lter paper blood spots
evaluated, 198 were successfully amplied and sequenced for dhps
while 191 were successfully sequenced for dhfr mutations. In
Maiduguri (2010e2012), of the one hundred and forty-two (142)
lter paper blood spots evaluated, 53 and 48 samples were used for
dhps and dhfr analyses respectively. A sample ID with similar
haplotypes on day0, 7, 14, 21, 28, 42, maternal or placental were
counted as one haplotype. Likewise, in Enugu (Component A), of
the 233 samples evaluated from day0, 7,14, 21, 28, 42, maternal and
placental blood spots, 145 samples were left for dhps and 139 for
dhfr analyses after ltering off the duplicates by sample IDs. For
Enugu (Component B), of the two hundred and twenty ve (225)
evaluated only 60 (maternal or placental e duplicate haplotypes
were dropped) blood spots were successfully sequenced for dhps
and 51(maternal or placental) blood spots for dhfr.In2014e2015, of
the one hundred samples evaluated, ninety-ve (95) and seventy-
nine (79) were successfully sequenced for dhps and dhfr muta-
tions respectively. In all, a total of 589 and 546 analysable data were
obtained for dhps and dhfr mutations respectively. It is noteworthy
that the nested Snonou protocol for malaria diagnosis is highly
sensitive compared to the nested PCR protocols for dhps and dhfr
gene amplication and as such fewer successful sequence data
were obtained for the pregnant women.
3.1. Prevalence of dhps haplotypes
Table 1 shows the prevalence of dhps haplotypes from the
various geographical regions between 2003 and 2015. Of the 38
successfully sequenced isolates from Ibadan in 2003, seventy-eight
percent (78%) were of the single mutant dhps haplotype ISG
KAA,
10% were of the IAG
KAA, 5% IAGKAS and another 5% comprised of
mixed haplotypes. No dhps 431V haplotype was observed in 2003.
In Ibadan (2007), of the 198 successfully sequenced isolates, sixty-
one percent (61%) harboured the single mutant ISG
KAA, 4% IAG-
KAA, 5.1% IAGKAS, 16.2% mixed haplotypes, 5.6% IAAKAA, 0.5% with
double mutant dhps ISGE
AA and 0.5% of other minor haplotypes.
There was an observed increase of 5% 431V haplotype e 3% VAG-
KAA, 1% VAGKAS and 1% VAGKGS. In Maiduguri (2010), of the 53
isolates analysed, 20.8% harboured the single mutant ISG
KAA, 3.8%
IAG
KAA, 3.8% mixed haplotypes, 39.6% IAAKAA, 3.8% IFAKAS, 1.9%
ISAKAA (wild-type), 1.9% IA
AKGS and 1.9% ICAKAA. A prevalence of
22.6% was observed for the 431V haplotypes e 11.3% VAG
KAA and
11.3% VAG
KGS. In Enugu (2010- Component A), of the 145 analys-
able samples, 25.5% harboured the ISG
KAA, 6.2% IAGKAA, 9.6%
mixed haplotypes, 1.4% IA
AKAA, 0.7% IAAKGA and 2.1% IFAKAS.A
total prevalence of 54.5% was observed for the 431V haplotypes -
6.9% VAG
KAA,1.4% VAGKAS and 46.2% VAGKGS. In the component B
study e Enugu 2010, of the 60 successfully analysed samples, 40%
harboured the ISG
KAA, 1.7% IAGKAA, 3.3% IAGKAS, 6.7% mixed
haplotypes, 6.7% IA
AKAA and 1.7% ISAKAA. The 431V haplotype was
observed in 40% of the isolates and these were all VAG
KGS. In Benin
City (2015), of the 95 successfully sequenced isolates, 30.5% har-
boured ISG
KAA, 4.2% IAGKAA, 2.1% IAGKAS, 1.1% mixed haplotypes,
3.2% IA
AKAA and 1.1% of other haplotypes. The 431V haplotype
occurred in more diverse forms with a total prevalence of 53.8%e
2.1% VAG
KAA, 3.2% VAGKAS, 7.4% VAGKGA, 1.1% VSGKAA, 1.1%
V
SGKGA, 2.1% VSGKGS and 36.8% VAGKGS.
3.2. Prevalence of dhfr haplotypes
The prevalence of dhfr haplotypes in the various regions be-
tween 2003 and 2015 are shown in Table 2 with no signicant
change observed across the sites. In Ibadan (2003), all of the 38
successfully sequenced isolates harboured the triple mutant dhfr
ACIRN
VI. In 2007, 96.9% of the 191 isolates harboured the triple
mutant ACIRN
VI, 0.5% ACICNVI, 0.5% ACNCSVI (wild type), 1.6%
ACNRN
VI and 0.5% mixed haplotypes. In Maiduguri (2010), of the
48 isolates, 81.3% harboured ACIRN
VI, 4.2% ACNCSVI, 10.4%
ACNRN
VI and 4.2% mixed haplotypes. In Enugu (Component A-
2010), of the 139 isolates, 94.2% harboured ACIRN
VI, 0.7% ACICNVI,
0.7% ACNRN
VI, 2.9% mixed haplotypes and 1.4% ACNCNVI. In Enugu
(Component B), of the 51 isolates, 90.2% harboured ACIRN
VI, 2%
ACI
CNVI, 3.9% ACNCSVI and 3.9% mixed haplotypes. In Benin City,
98.7% of the 79 isolates harboured ACIRN
VI while 1.3% had ACICNVI.
The triple mutant dhfr is predominantly high across all years with
100 percent frequency observed in 2003. No mutations were
observed at codons 16, 50, 140 and 164 of the dhfr gene.
3.3. Prevalence of dhps alleles
Table 3 summarizes the prevalence of the individual dhps alleles
(codons 431, 436, 437, 540, 581 and 613). The allelic frequencies for
studies in pregnant women were split into maternal (M) and
placental (P) samples. The prevalence of placental parasitaemia was
very low in 2003 and 2010 except for Component B (Enugu). This is
expected since Component B was a non-intervention arm with
most women not actively taking IPTp-SP and as a result the level of
protection from placental parasitaemia was low. Fig. 1 shows a
descriptive map of Nigeria with the emergence of dhps-VAG
KGS
haplotype and a complete absence of 431V-haplotype in 2003
(Ibadan). It also highlights the prevalence of 431V-haplotype in
Yaounde, Cameroon previously described by Chauvin et al. (2015).
3.4. A homology model of P. falciparum DHPS
Fig. 2 shows the full model PfDHPS showing H-bonded parallel
b
-strands 1 and 2. At the C-terminus of
b
-2 is loop 2 containing
substrate-binding mutable residues Ser436 and Ala437. The ho-
mology model of PfDHPS suggests the probable effect of the I431 to
431V change. Supplementary Fig. 1 describes
b
-1 and
b
-2 strands of
PfDHPS in the wild type I431 and mutant type 431V respectively. In
our homology model for the wild-type PfDHPS component, the side
chain of Ile 431 in
b
-2 shows hydrophobic interaction (2.763 Å)
with that of Leu 395 in
b
-1. The Ile 431 Val change is expected to
prevent this inter-strand stabilizing hydrophobic interaction since
the closest side-chain approach is 4.157 Å. This may render the
substrate-binding residues in loop 2 marginally less stable,
enhancing resistance to drug inhibition.
Fig. 3 shows the clustal alignment of DHPS sequences to locate
structural features. This alignment is largely from Pemble et al.,
(2010) but sequences used, apart from P. falciparum, are from
FASTA texts published with the crystal structures. The reactive
enzymatic parts of the DHPS (dihydropteroate synthase) structure
M.C. Oguike et al. / International Journal for Parasitology: Drugs and Drug Resistance 6 (2016) 220e229 223
are enclosed in a supporting TIM Barrel (Triose phosphate IsoM-
erase Barrel structure) which is a conserved protein fold consisting
of eight
a
-helices (
heavy-underlining
) in the alignment. Inside are
eight
b
-strands (shaded) (arranged in 4, backbone H-bonded,
parallel pairs) that alternate with the
a
-helices along the peptide
backbone. The drug-binding DHPS residues associated with sul-
phonamide resistance (*) are seen almost exclusively in exible
loops (underlined
), which are located between the relatively-rigid
Table 1
Prevalence of dhps haplotypes in Nigeria (2003e2015).
dhps haplotype Ibadan 2003 (%) Ibadan 2007/8 (%) Maiduguri 2010 (%) Enugu FU 2010 (%) Enugu non-FU 2010 (%) Benin city 2014/15 (%)
ISGKAA 30 (78.9) 121 (61.1) 11 (20.8) 37 (25.5) 24 (40) 29 (30.5)
IAGKAA 4 (10.5) 8 (4) 2 (3.8) 9 (6.2) 1 (1.7) 4 (4.2)
IAGKAS 2 (5.3) 10 (5.1) 2 (3.3) 2 (2.1)
MIXED 2 (5.3) 32 (16.2) 2 (3.8) 14 (9.6) 4 (6.7) 1 (1.1)
IAAKAA 11 (5.6) 21 (39.6) 2 (1.4) 4 (6.7) 3 (3.2)
IAAKGA 1 (0.5) 1 (0.7)
IFAKAS 1 (0.5) 2 (3.8) 3 (2.1)
ISAKAA 1 (0.5) 1(1.9) 1 (1.7)
ISGEAA 1 (0.5)
ISGKAS 1 (0.5) 1 (1.1)
IYAKAS 1 (0.5)
IAAKGS 1 (1.9)
ICAKAA 1 (1.9)
IAGKGA 1 (1.1)
IAAKGS
IAGKGS 1 (1.1)
ISGKGA 1 (1.1)
ISGKGS 1 (1.1)
VAGKAA 6 (3) 6 (11.3) 10 (6.9) 2 (2.1)
VAGKAS 2 (1) 2 (1.4) 3 (3.2)
VAGKGS 2 (1) 6 (11.3) 67 (46.2) 24 (40) 35 (36.8)
VAGKGA 7 (7.4)
VSGKAA 1 (1.1)
VSGKGA 1 (1.1)
VSGKGS 2 (2.1)
VAAKAA
TOTAL 38 198 53 145 60 95
FU e followed up; nonFU e non follow up.
Bold means actual gures while normal text indicates percentages.
Table 2
Prevalence of dhfr haplotypes in Nigeria (2003e2015).
dhfr haplotype Ibadan 2003 (%) Ibadan 2007/8 (%) Maiduguri 2010 (%) Enugu FU 2010 (%) Enugu non-FU 2010 (%) Benin city 2014/15 (%)
ACIRNVI 38 (100) 185 (96.9) 39 (81.3) 131 (94.2) 46 (90.2) 78 (98.7)
ACICNVI 1 (0.5) 1 (0.7) 1 (2) 1 (1.3)
ACNCSVI 1 (0.5) 2 (4.2) 2 (3.9)
ACNRNVI 3 (1.6) 5 (10.4) 1 (0.7)
MIXED 1 (0.5) 2 (4.2) 4 (2.9) 2 (3.9)
ACNCNVI 2 (1.4)
TOTAL 38 191 48 139 51 79
Table 3
Summary of dhps alleles in 4 different regions of Nigeria (2003e2015).
dhps allele Ibadan 2003
n ¼ 38
Ibadan 2007/8 n ¼ 198 Maiduguri
2010 n ¼ 53
Enugu FU
2010 n ¼ 145
Enugu non-
FU 2010
n ¼ 60
Benin city 2014/15 n ¼ 95
MP MPM PMP
I431 36 2 188 38 3 60 5 20 15 48
V431 13 11 2 89 2 18 8 52
S436 31 1 153 12 1 43 5 16 11 39
A436 7 1 70 34 4 108 3 19 16 58
F436 1 2 7 2
C436 1
Y436 1
A437 24 26 2 11 2 6 1 3
G437 36 2 183 23 3 133 6 32 23 92
K540 36 2 197 48 5 139 6 38 22 95
E540 2
A581 36 2 195 43 4 73 5 20 16 48
G581 5 6 1 74 2 18 8 50
A613 35 1 179 41 4 66 3 23 11 50
S613 2 1 24 8 1 82 2 17 10 49
M e Mother; P e Placenta.
M.C. Oguike et al. / International Journal for Parasitology: Drugs and Drug Resistance 6 (2016) 220 e229224
b
-strands and the
a
-helices. Substrate-binding sites (þ) are found
in the loops and in
b
-strands.
Although the genus Plasmodium is atypical in having a bifunc-
tional HPPK/DHPS where, as seen in the yeasts Francisella (Pemble
et al., 2010) and Saccharomyces (Lawrence et al., 2005) DHPS is
fused to the preceding enzyme in the folate synthetic pathway, 6-
hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) (de
Beer et al., 2006). The 3D structure in both HPPK-fused and sepa-
rate DHPS is quite typical.
In the homology model of PfDHPS the
b
-strands t readily into
the typical picture but where earlier resistance-associated residue
changes have only been seen in exible loops, Ile431Val is seen in a
beta-strand,
b
-2, preceded by the completely conserved Asp 430
residue, a substrate-binding site and followed by the completely-
conserved Gly 432.
b
-1 and
b
-2 are structurally adjacent parallel
b
-strands, H-bonded together by backbone atoms into a rigid
b
-
sheet. In
b
-1 of our homology model for the wild-type PfDHPS
component, the bulky side chain of Ile 431 in
b
-2 hydrophobically
interacts with the side chain of Leu 395 in
b
Fig. 1). This strengthens the link between C-terminal ends of
these
b
-strands, participating in stabilizing the position of the
mutable substrate-binding residues Ser 436 and Ala 437 in exible
Loop-2.
In the PfDHPS sequence, the residues subject to mutation are
highlighted in red (Fig. 3). Additionally, in
b
-1, completely
conserved Asn 396, follows hydrophobically interacting Leu 395
directly, and this interaction is lost in I431V. The mutation may very
likely affect N 396 in its role as a substrate-binding site, as well as
making the positions of drug-interacting and substrate-binding
residues in the attached loop less xed. There is only one
resistance-associated residue change in a
b
-strand in the DHPS
alignment of Pemble et al., (among 12 noted overall), which is in
b
-
4, the equivalent WT residue in
b
-4 in the PfDHPS structure being
Leu 501, next to completely conserved Asn 502 and Asp 503, the
rst of which is a substrate binding site.
Table 4 shows the Gibbs energy change (delta delta Gibbs,
dd
G)
(in kilocalories/mol) involved in folding and unfolding a wild type
or a mutated structure. The values reect the degree of stabilizing
or destabilizing effect of the changes. The I431V has a
dd
G value
of 1.622 which is similar to that of A581G (1.64) and A613S
(1.626) with destabilizing effects.
4. Discussion
In this study of SP resistance markers in Nigeria during
2003e2015, we observed an emergence of the dhps I431V mutation
in Ibadan during 2003e2008. The I431V mutation was rst
described in a study by Sutherland and others (2009) in isolates
from malaria patients returning to the UK from Nigeria. It has since
been reported in neighbouring Cameroon (Chauvin et al., 2015)ata
prevalence of 9.8% but not among the numerous dhps sequencing
studies conducted on parasite populations in the rest of Africa
(Naidoo and Roper, 2013). Published reports of the mutation are
currently conned to Nigeria and Cameroon although one appears
in a Ghanaian isolate among publicly available whole genome se-
quences (Plasmoview, http://pathogenseq.lshtm.ac.uk/
plasmoview). Early sequencing surveillance studies of dhps in
Cameroon (Tahar and Basco, 2007) did not detect the 431V among
355 samples collected in multiple sites (Yaounde, Djoum, Manjo,
Bertoua and Garoua) during 1999e2003 which suggests that the
appearance of this mutation is comparatively recent. Reports of the
prevalence of the 431V are mapped in Fig. 1.
The I431V was seen in combination with various other dhps
mutations but interestingly was most abundant in combination
with 437G, 581G and 613S in the VAG
KGS haplotype. The haplotype
prevalences summarised in Fig. 1 show the VAG
KGS haplotype was
more prevalent in Enugu (in 2010) than Maiduguri (in 2010) and
was found at high prevalence in Benin City (2014). The increase in
mutant dhps-VAG
KGS over 12 years seems to indicate that it con-
fers a selective advantage in the presence of SP drug pressure and is
displacing more sensitive haplotypes. As well as IPTp, SP is still used
for the treatment of malaria and is readily available in the Nigerian
market (Ugwu et al., 2013) so ongoing SP drug pressure is strong.
Importantly, the dhps-VAG
KGS and VAGKAA were also widely
dispersed being found in Maiduguri which is far from the other
sites and this indicates that the haplotype may be spreading
throughout Nigeria.
The dhps-540E mutation was observed at low prevalence (0.5%)
in Ibadan in 2007/2008, it was otherwise absent. Contrastingly, we
observed a signicant increase in the prevalence of 581G and 613S
over the same time period. Prevalence increased from 0% in 2003 to
52.6% in 2014 for dhps -581G and 8%e51.6% in 2014 for dhps-613S. A
signicant part of this expansion could be accounted for by the
increase in the VAG
KGS haplotype.
The triple mutant dhfr-IRN was almost xed across the years
with prevalence above 90%. Although the quintuple dhfr/dhps
mutation was almost absent in this study, the combination of IRN
with alternative dhps resistance haplotypes to the east African GEA
and GEG (437 þ 540 þ 581 mutant haplotypes) may be conferring
increased SP tolerance levels.
4.1. What is the signicance of dhps 431V mutations for efcacy of
SP?
Models of the 3-dimensional structure of the DHPS molecule
can be used to explore how the I431V substitution might affect
resistance. The mutation involves a change from isoleucine to
valine in a highly conserved region of the DHPS molecule, close to
mutable residues S436 and A437. The preceding conserved residue
D430 is recognised as a substrate-binding site. Valine is less hy-
drophobic than isoleucine and its side-chain is smaller than that of
isoleucine.
Although we do not yet have conclusive evidence that there is
an effect of the
b
-2 I431V change on drug response for PfDHPS, the
comparison with the effective mutation signalled by Pemble et al.
in
b
-4 is suggestive. In crystal 3MCNa, of F. tularensis HPPK/DHPS,
the side chain of Ile 276 in
b
-4 hydrophobically interacts (3.59 Å)
with that of Ile 291 in
a
-4 (this interaction is lost on mutation to
Val) and with side chain of Met262 (2.97 Å)in
a
-3, which is
retained after the Ile 276 Val mutation. Comparing the calculated
Fig. 2. Full model PfDHPS showing H-bonded parallel
b
-strands 1 and 2. At the C-
terminus of
b
-2 is loop 2 containing substrate-binding mutable residues Ser436 and
Ala437.
M.C. Oguike et al. / International Journal for Parasitology: Drugs and Drug Resistance 6 (2016) 220e229 225