Acta Tropica 149 (2015) 5963

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Survey of chloroquine-resistant mutations in the Plasmodium

falciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen
Omar A.A. Bamaga a , Mohammed A.K. Mahdy a,b,c, , Yvonne A.L. Lim a,
a
b
c

Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Tropical Disease Research Center, University of Science and Technology, Sanaa, Yemen
Department of Parasitology, Faculty of Medicine, Sanaa University, Yemen

a r t i c l e

i n f o

Article history:
Received 7 February 2015
Received in revised form 14 May 2015
Accepted 16 May 2015
Available online 19 May 2015
Keywords:
Malaria
Drug resistance
Molecular marker
Hadhramout-Yemen

a b s t r a c t
Malaria is still a major public health problem in Yemen. More than 95% of the malaria cases are due to
Plasmodium falciparum . Recently in Yemen, the antimalarial treatment policy was changed from chloroquine (CQ) to artemisinin combination therapy (ACTs). However, CQ is still available and prescribed in
the Yemeni market. The persistence of CQ resistance will be prolonged if the shift to ACT and the simultaneous withdrawal of CQ are not rigorously implemented. The aim of the current survey is to detect
chloroquine-resistant mutations in P. falciparum chloroquine-resistance transporter (pfcrt) and P. falciparum multi-drug resistance-1 (pfmdr1) genes. These data will be important for future monitoring and
assessment of antimalarial drug policy in Yemen. Blood specimens were collected from 735 individuals
from different districts of the Hadhramout province, Yemen by house-to-house visit. Mutation-specic
nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCRRFLP) methods were used to investigate the mutations in the pfmdr1(codons 86 and 1246) and pfcrt (codons 76, 271,
326, 356 and 371) genes. The overall prevalence of pfcrt mutations at codons 76, 271, 326 and 371 were
50.4%, 58.7%, 54.3% and 44.9%, respectively. All isolates had wild-type pfcrt 356 allele. The majority of
pfmdr1 86 alleles (83.3%) and all pfmdr1 1246 alleles were wild type. There was no association between
pfcrt mutations and symptomatology, gender and age groups. In conclusion, point mutations in codons
76, 271, 326 and 371 of pfcrt of P. falciparum are high suggesting a sustained high CQ resistance even
after 4 years of shifting to ACTs. These ndings warrant complete withdrawal of CQ use from the Yemeni
market for P. falciparum and careful usage of CQ for treating Plasmodium vivax.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Malaria, especially falciparum malaria is one of the main causes
of mortality and morbidity worldwide. In Yemen, malaria is classied as an afro-tropical type and 99% of malaria cases are due to
Plasmodium falciparum as the predominant species with Anopheles arabiensis and Anopheles sergenti as the main mosquito vectors
(WHO, 2013). Socotra Island and the eastern governorate of AlMaharah belong to the oriental type of malaria with Anopheles
culicifacies as the predominant vector (NMCP, 2011). About 62%
of the population are at risk of malaria with approximately 43%
at high risk (WHO, 2012). Mortality rates ranged from 2.1 to 4.7%

Corresponding author at: Tropical Disease Research Center, University of Science

and Technology, Sanaa, Yemen. Tel.: +967 711177711.
Corresponding author.
E-mail addresses: alsharaby9@yahoo.com (M.A.K. Mahdy),
limailian@um.edu.my (Y.A.L. Lim).
http://dx.doi.org/10.1016/j.actatropica.2015.05.013
0001-706X/ 2015 Elsevier B.V. All rights reserved.

in children (Al-Taiar et al., 2006). Although Hadhramout, a Yemeni

province in the east, has been suggested to be in the pre-elimination
stage, the west provinces of Yemen are still in the control phase. In
contrast, the neighbouring countries, such as Saudi Arabia is in the
elimination phase and Oman is in the prevention of re-introduction
phase (WHO, 2013; Bamaga et al., 2014). The national strategy of
malaria control involves early diagnosis and proper treatment to
prevent mortality and to reduce morbidity, indoor residual spraying, distribution of insecticide-treated mosquito nets, and reactive
and proactive case surveillance (WHO, 2013).
For years, CQ had been the rst line treatment. The rst detection of the indigenous chloroquine resistance (CQR) cases in Yemen
was reported in 1989 in Taiz (Mamser, 1989; Alkadi et al., 2006),
and then in Hodeidah (Al-Shamahy et al., 2006). In addition, recent
studies have shown high prevelance of CQR marker pfcrt 76T in
Hodeidah, Dhammar, Rymah and Taiz (Al-Mekhla et al., 2011;
Abdul-Ghani et al., 2013; Al-Hamidhi et al., 2013). In November
2005, antimalaria drug policy shifted from CQ to a combina-

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O.A.A. Bamaga et al. / Acta Tropica 149 (2015) 5963

tion of artesunatesulphadoxine/pyrimethamine as the rst-line

therapy and artemetherlumefantrine as a second line treatment
for uncomplicated malaria. However, this new policy was only
implemented four years later in 2009 after proper training and
distribution of the national guideline for antimalaria drugs were
carried out (WHO, 2012). Despite these efforts, previous studies
conducted in Hadhramout province reported that CQ is still commonly prescribed (18 out of 42 prescriptions) and some clinicians
were not aware and had poor knowledge about the new national
drug policy (Bashrahil et al., 2010; Ghouth, 2013). Continued use
of CQ sustains the selection of CQ resistant mutations leading to
persistence of mutant parasite. The complete withdrawal of CQ use
may enhance the emergence of CQ sensitive parasite over time and
make CQ possible to be re-introduced for malaria treatment (Kublin
et al., 2003; Laufer et al., 2006). However, the persistence of CQ
resistance will be prolonged if the shift to ACT and the simultaneous
withdrawal of CQ are not rigorously implemented. The prevalence
of CQ resistant mutations in Hadhramout is not at present known.
The aim of the current survey is to obtain such data, since they will
be important for future monitoring and assessment of antimalarial
drug policy in Yemen.
2. Methods
2.1. Survey area/subjects and ethical consideration
This survey was carried out in Hadhramout province, which represent about 36% of the total area of Yemen and located in the
southeast corner of the country with a population estimated at
1,028,556 (CSO, 2013). Hadhramout has coastal plain region and
mountainous area. The coastal plain region (with an altitude of
0600 m) is characterized by a hot climate throughout the year
with little rainfall ranging between 50 and 100 mm per annum.
The coastal region is endemic for malaria (only in valleys) with a
seasonal transmission occurring from November to April (NMCP,
2011). The sources of income of the population include funds
transferred from natives working in neighbouring Gulf countries,
agriculture, shing, livestock or handicraft. A total of 735 participants of all ages and both genders were enrolled in this survey;
nger prick blood samples were collected from participants in three
villages in Hajer and four villages in Al-RaydahQusyer districts
(Fig. 1). These villages were selected because they are endemic for
malaria and houses were selected by random sampling. The survey
was conducted between July 2011 and May 2012. Informed consent
was obtained from each participant, and for children, consent was
obtained from their parents after a clear explanation of the survey
objectives was given. Prior to sample collection, the survey protocol
was approved by the Faculty of Medicine, Hadhramout University
for Science and Technology, the Ministry of Health and Population,
Yemen, and NMCP division in Hadhramout governorate. All positive cases were treated by NMCP following the recommendations
of the national drug policy.
2.2. Samples and microscopy
Blood was collected by nger prick and thin and thick blood
lms were prepared and left to air-dry and thin lm was xed
with methanol. Blood smears were brought back from the eld
and stained as soon as possible with 10% diluted Giemsa stain and
screened under a microscope for the presence of malaria in the laboratory. A negative result was recorded after screening at least 200
elds under the oil immersion lens of the light microscope. The
positive specimens for P. falciparum were used for molecular studies. Filter paper blood spots were also collected on Whatman lter
paper 3MM (Whatman International Ltd., Maidstone, England) and

kept separately in clean, dry and well-sealed plastic bag with silica
gel to reduce humidity in the bag and stored at room temperature
until further use for molecular analysis.
2.3. Molecular identication of malaria species
Genomic DNA was extracted from lter paper blood spots.
Briey, by using a sterile-amed puncher, a small disc of the lter paper blood spot (approximately 5 mm diameter) was punched
out and transferred into microcentrifuge tubes using a clean and
methanol-amed forceps. Genomic DNA sample was extracted
using QIAgen DNA Mini Kit for blood and tissue (QIAGEN, Germany)
according to the manufacturers instructions. Extracted DNA was
eluted using 50 L Qiagen AE elution buffer and kept at 20 C until
use. Plasmodium species were identied using nested PCR based
on small subunit ribosomal RNA gene (Singh et al., 1999; Snounou
et al., 1993). PCR master mix and thermal cycling conditions were
carried out as reported previously (Snounou, 1996).
2.4. Molecular detection of pfcrt K76T
A nested mutation-specic PCR was used to detect mutations in
pfcrt K76T (Lopes et al., 2002). Primary PCR was performed using
two sets of primer (CRTP1 and CRTP2) to amplify 537 bp of pfcrt
gene. The secondary PCR was performed using a common inner
primer (CRTP3) together with either CRTP4m or CRTP4w for mutant
and wild types, respectively, resulting in 366 bp of the two alleles.
PCR reaction was carried out in a nal volume of 25 L in a PCR
tube containing 2.5 mM MgCl2 , 200 M of each dNTP, 1.25 units of
Taq polymerase, 1 M of CRTP1 primer, 1 M of CRTP2 primer and
ve microliter of genomic DNA. PCR reagent and primers were from
iNtRON (iNtRON Biotechnology, Inc., Seoul, Republic of Korea). PCR
was performed in the thermal cycler (MyCycler, BioRad Hercules,
USA) with the following cycling conditions: initial denaturation at
95 C for 3 min, 45 cycles of denaturation at 94 C for 30 s, annealing
at 56 C for 30 s, extension at 60 C for 1 min and nal extension at
60 C for 5 min. About 12 L of primary PCR product was used
as template in the secondary PCR reaction which contained the
same PCR reagent concentrations as in the primary PCR except for
using 1.5 mM MgCl2 and amplication cycle of 25 cycles instead
of 45 cycles. The cycling conditions were similar to the primary
PCR except that the annealing and extension temperatures were
47 C and 64 C, respectively. The PCR products were loaded in a
2% agarose gel, stained with SYBER safe DNA gel stain (Invitrogen,
USA) for electrophoresis and visualized by UV transilluminator.
2.5. Molecular detection of mutations in pfcrt gene (positions
Q271E, N326S, I356T, R371I) and pfmdr1 gene (positions N86Y
and D1246Y)
Nested PCR followed by restriction fragment length polymorphism was performed as previously described (Djimd et al.,
2001; Lopes et al., 2002). Briey, the restriction enzymes XMN1,
MSe1, AlwN1 and AII digest pfcrt at codons 271, 326, 356 and
371, respectively. The enzymes AIII and Bg1II digest pfmdr1 at
codons 86 and 1246, respectively. The digestion of PCR product
was achieved by FastDigest restriction enzymes (Thermo Fisher
Scientic Inc., USA) according to the instructions of the manufacturer. Digestion results were analyzed by electrophoresis in a 2.5%
agarose gel containing SYBER safe DNA gel stain (Invitrogen, USA)
and visualized in a UV transilluminator. Genomic DNA from P. falciparum strains HB3, 3D7 and Dd2 supplied by Malaria Research and
Reference Reagents Resources Centre (MR4, ATCC, ManassasVA,
USA) were used as positive controls for mutant and wild types,
whereas nuclease-free water was used as the negative control.

O.A.A. Bamaga et al. / Acta Tropica 149 (2015) 5963

61

Fig. 1. Map of Hadhramout province highlighting the study sites in Hajer and Al-Raydah-Qusyer districts.

2.6. Statistical analysis

Data analysis was performed using the SPSS statistical package version 19. The signicance of the associations between point
mutations and independent variables were assessed using Chisquare test. The signicance level was considered at p < 0.05.

3. Results
3.1. Malaria detection/identication using microscopy and PCR
A total of 735 samples were collected from two districts of
Hadhramout, 423 (42.4%) males and 312 (57.6%) females. The age
of participants ranges from 1 to 75 years with a median of 16 years
and 22 interquartile range. Of the 735 samples, 18.8% (138/735)
were positive for malaria parasite via microscopy. Of the 138 positive samples, 137 (99.3%) had P. falciparum and one case was P.
vivax (0.7%). Nested PCR detected P. falciparum in four samples that
were negative using microscopy and identied three samples with
mixed infections of P. falciparum and P. vivax which were identied
as P. falciparum using microscopy.

3.2. Frequency and distribution of pfcrt and pfmdr1 gene alleles

in Hadhramout
The results for pfcrt and pfmdr1 gene alleles are shown in Table 1.
Of 138 falciparum positive samples, pfcrt K76T mutant, wild and
mixed type alleles were detected in 50.7%, 26.1% and 23.1% of positive specimens, respectively. The prevalence of the mutant alleles
of pfcrt 271, pfcrt 326 and pfcrt 371 was 58.7%, 54.3% and 44.9%,
respectively. However, all alleles of pfcrt 356 were wild type. The
majority of pfmdr1 Y86 alleles (83.3%) and all pfmdr1 1246 alleles
were wild type.

The survey showed a signicant difference in the distribution of

pfcrt 76 alleles between Hajer and Al-RaydahQusyer districts with
pfcrt 76 mutant allele being higher in Al-RaydahQusyer (52.7%)
(p = 0.006). In contrast, pfcrt 371 mutant allele had higher frequency
in Hajer (69.2%) compared to Al-RaydahQusyer (39.3%). The distribution of pfcrt 371 alleles between the two districts was statistically
signicant (p = 0.006). Pfmdr 186 mutant allele was more prevalent in Hajer than Al-RaydahQusyer while the wild type allele was
higher in Al-RaydahQusyer district (p = 0.03).
This survey indicated that there is no signicant association
between the distribution of pfcrt and pfmdr1 gene alleles with age
and gender. Table 2 shows no signicant difference in the frequency
of pfcrt and pfmdr1 gene alleles between symptomatic and asymptomatic malaria cases.

4. Discussion
This survey is a community based study conducted to monitor CQ resistance based on the detection of mutations in the pfcrt
and pfmdr1 genes. In the present survey, the prevalence of pfcrt
76T mutation, the highly sensitive marker for CQ resistance in
Hadhramout was 50.7%. This result is lower than the prevalence
of pfcrt 76T mutation reported from west of Yemen (8182%) (AlMekhla et al., 2011; Abdul-Ghani et al., 2013) and Lahj in the south
of Yemen (98%) (Mubjer et al., 2011). In contrast, recently published
study from Taiz province (south of Yemen) revealed similar ndings (50.9%) (Al-Hamidhi et al., 2013). These differences could be
explained by the time period between the implementation of the
new drug policy and screening for CQ resistance markers. This survey was conducted four years after the launch of the new drug
policy while previous reports were carried out either before or
12 years after the initiation of the new antimalaria drug policy.
The decline in prevalence of CQ resistant parasite after the withdrawal of CQ use has been well documented (Kublin et al., 2003;

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O.A.A. Bamaga et al. / Acta Tropica 149 (2015) 5963

Table 1
Frequency and distribution of pfcrt and pfmdr1 alleles in Hadhramout (n = 138).
Districts n (%)
Mutation codons

Alleles

Hajer

Pfcrt 76

Wild
Mutant
Mixed

3 (11.5)
11 (42.3)
12 (46.2)

33 (29.5)
59 (52.7)
20 (17.9)

36 (26.1)
70 (50.7)
32 (23.1)

0.006*

Pfcrt 271

Wild
Mutant

10 (38.5)
16 (61.5)

47 (42.0)
65 (58.0)

57 (41.3)
81 (58.7)

0.744

Pfcrt 326

Wild
Mutant

9 (34.6)
17 (65.4)

54 (48.2)
58 (51.8)

63 (45.7)
75 (54.3)

Pfcrt 356

Wild
Mutant

26 (100.0)
0

112 (100.0)
0

138 (100.0)

Pfcrt 371

Wild
Mutant

8 (30.8)
18 (69.2)

68 (60.7)
44 (39.3)

76 (55.1)
62 (44.9)

Pfmdr 1-86

Wild
Mutant

18 (69.2)
8 (30.8)

97 (86.6)
15 (13.4)

115 (83.3)
23 (16.7)

0.032

Pfmdr1-1246

Wild
Mutant

26(100.0)
0

111 (100.0)
0

137 (100.0)

NA

Al-RaydahQusyer

Total

p value

0.210
NA
0.006*

Using Pearson Chi-Square.

Laufer et al., 2006). However, the prevalence of pfcrt mutations,

in this survey, is still considered high which means that malaria
parasite is sustaining the CQ resistance in this region. The possible
explanation of the sustainability of CQ resistance is the continued
unsupervised use of CQ for the treatment of malaria in Hadhramout
(Bashrahil et al., 2010; Ghouth, 2013). Unfortunately, no previous
data are available about the prevalence of pfcrt 76T gene mutation
in Hadhramout which could be used for comparison to show the
trend of CQ resistance in this area over the years. The prevalence
of mutations in pfcrt at loci 271, 326 and 371 in this survey, was
58.7%, 54.3% and 44.9%, respectively. These mutations are more
commonly distributed in the Old World CQ resistant strain and
affect the response to CQ in the presence of 76T mutation (Ibraheem
et al., 2014). Findings from this survey necessitate the implementation of effective control of CQ usage in the Yemeni market especially
as CQ is still necessary for P. vivax infection. However, this has to
be done based on accurate diagnosis.
Although pfmdr1 plays an important role in modulating levels
of antimalarial drug resistance, CQ resistance has been correlated
with the pfmdr1 86Y (Djimd et al., 2001), while the other point

mutations at codons 184, 1034, 1042 and 1246 do not confer

resistance to CQ and are correlated to meoquine, artesunate,
amodiaquine, halofantrine and quinine resistance (Duraisingh and
Cowman, 2005). However, the mechanism of pfmdr1 gene mutations in drug resistance is controversial. In the present survey,
prevalence (16.7%) of pfmdr1 86Y mutant type among P. falciparum
isolates was observed, whereas there is no mutation of the pfmdr1
at codon 1246. This ndings are in agreement with recent studies
in Yemen and neighboring countries such as Kingdom of Saudi Arabia and Iran (Zakeri et al., 2008; Bin-Dajem et al., 2012; Al-Hamidhi
et al., 2013). Although the decrease prevalence of pfmdr1 86Y mutation has been attributed to almost complete withdrawal of CQ in
the community, this is not the case in our survey since CQ is still
prescribed in Hadhramout (Bashrahil et al., 2010; Ghouth, 2013).
In the present survey, the association of pfcrt and pfmdr1
mutations with gender and age of participants was not signicant. Similar ndings have been reported from Malaysia and Iran
(Rastaghi et al., 2008; Atroosh et al., 2012). No signicant difference
in the prevalence of pfcrt and pfmdr1 mutations between subclinical
and clinical infection of malaria parasites was noted in this survey.

Table 2
Frequency and distribution of pfcrt and pfmdr1 alleles according to symptomatology (n = 138).
Symptoms n (%)
Mutation Codons

Alleles

Symptomatic*

Asymptomatic

p value

Pfcrt 76

Wild
Mutant
Mixed

17 (25.8)
36 (55.5)
13 (19.7)

19 (26.4)
34 (47.2)
19 (26.4)

0.596

Pfcrt 271

Wild
Mutant

31 (47.0)
35 (53.0)

26 (36.1)
46 (63.9)

0.196

Pfcrt 326

Wild
Mutant

26 (39.4)
40 (60.6)

37 (51.4)
35 (48.6)

0.158

Pfcrt 356

Wild
Mutant

66 (100.0)
0

72 (100.0)
0

NA

Pfcrt 371

Wild
Mutant

37 (56.1)
29 (43.9)

39 (54.2)
33 (45.8)

0.823

Pfmdr 1-86

Wild
Mutant

54 ( 81.8)
12 ( 18.2 )

61 ( 84.7)
11 ( 15.3 )

0.647

Pfmdr1-1246#

Wild
Mutant

65 (100.0)
0

72 (100.0)
0

NA

*
#

Symptomatic was dened by the presence of fever (>37.5 C) with or without shivering and headache.
One sample was missing due to PCR failure for this marker.

O.A.A. Bamaga et al. / Acta Tropica 149 (2015) 5963

This nding support previous studies conducted in Kenya (Zhong

et al., 2008) and the border regions of Burma/Myanmar (Brown
et al., 2012).
In conclusion, this survey detected high frequency of mutations
in pfcrt gene at codons 76, 271, 326 and 371. This suggests the sustainability of CQ resistance after 4 years of implementing ACTs as a
new drug policy in Yemen. Survey ndings warrant for effective and
stricter control of CQ usage and its open availability in the Yemeni
market.
Competing interests
The authors have declared that no competing interests exist.
Financial support
The study was funded by University of Malaya Research Grant
(UMRG - RG503-13HTM). The funder had no role in study design,
data collection and analysis, decision to publish or preparation of
the manuscript.
Authors contributions
MAKM and YALL planned and designed the protocols.
OAAB conducted the eld study and the study programme,
including the collections of blood samples and data from the questionnaire interviews, as well as the management of collected data.
MAKM and YALL supervised all the laboratory work.
OAAB, MAKM and YALL carried out the data analysis and interpretation.
OAAB, MAKM and YALL prepared the rst draft of the
manuscript and all authors revised the manuscript critically.
All authors read and approved the nal version of the
manuscript.
Acknowledgments
The authors thank all the technical staff in the eld of study and
laboratory expert group for their assistance in the laboratory work,
the Malaria National Control Program in Hadhramout governorateYemen especially Prof. Abdulla Salim Bin Ghouth, Ministry of
Health and Population in Hadhramout for their cooperation during
this study.
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