Acta Tropica 149 (2015) 5963

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Survey of chloroquine-resistant mutations in the Plasmodiumfalciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen

Omar A.A. Bamagaa, Mohammed A.K. Mahdya,b,c,, Yvonne A.L. Lima,

a Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysiab Tropical Disease Research Center, University of Science and Technology, Sanaa, Yemenc 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 2015Received in revised form 14 May 2015Accepted 16 May 2015Available online 19 May 2015

Keywords:MalariaDrug resistanceMolecular markerHadhramout-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 toPlasmodium falciparum . Recently in Yemen, the antimalarial treatment policy was changed from chloro-quine (CQ) to artemisinin combination therapy (ACTs). However, CQ is still available and prescribed inthe Yemeni market. The persistence of CQ resistance will be prolonged if the shift to ACT and the simul-taneous withdrawal of CQ are not rigorously implemented. The aim of the current survey is to detectchloroquine-resistant mutations in P. falciparum chloroquine-resistance transporter (pfcrt) and P. falci-parum multi-drug resistance-1 (pfmdr1) genes. These data will be important for future monitoring andassessment of antimalarial drug policy in Yemen. Blood specimens were collected from 735 individualsfrom different districts of the Hadhramout province, Yemen by house-to-house visit. Mutation-specicnestedpolymerase chain reaction (PCR)and restriction fragment lengthpolymorphism(PCRRFLP)meth-ods 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 were50.4%, 58.7%, 54.3% and 44.9%, respectively. All isolates had wild-type pfcrt 356 allele. The majority ofpfmdr1 86 alleles (83.3%) and all pfmdr1 1246 alleles were wild type. There was no association betweenpfcrt mutations and symptomatology, gender and age groups. In conclusion, point mutations in codons76, 271, 326 and 371 of pfcrt of P. falciparum are high suggesting a sustained high CQ resistance evenafter 4 years of shifting to ACTs. These ndings warrant complete withdrawal of CQ use from the Yemenimarket 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 causesof mortality and morbidity worldwide. In Yemen, malaria is clas-sied as an afro-tropical type and 99% of malaria cases are due toPlasmodium falciparum as the predominant species with Anophe-les arabiensis and Anopheles sergenti as the main mosquito vectors(WHO, 2013). Socotra Island and the eastern governorate of Al-Maharah belong to the oriental type of malaria with Anophelesculicifacies 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: TropicalDiseaseResearchCenter, University of Scienceand 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).

in children (Al-Taiar et al., 2006). Although Hadhramout, a Yemeniprovince in theeast, hasbeen suggested tobe in thepre-eliminationstage, the west provinces of Yemen are still in the control phase. Incontrast, the neighbouring countries, such as Saudi Arabia is in theelimination phase andOman is in the prevention of re-introductionphase (WHO, 2013; Bamaga et al., 2014). The national strategy ofmalaria control involves early diagnosis and proper treatment toprevent mortality and to reduce morbidity, indoor residual spray-ing, distribution of insecticide-treated mosquito nets, and reactiveand proactive case surveillance (WHO, 2013).

For years, CQ had been the rst line treatment. The rst detec-tion of the indigenous chloroquine resistance (CQR) cases in Yemenwas reported in 1989 in Taiz (Mamser, 1989; Alkadi et al., 2006),and then in Hodeidah (Al-Shamahy et al., 2006). In addition, recentstudies have shown high prevelance of CQR marker pfcrt 76T inHodeidah, Dhammar, Rymah and Taiz (Al-Mekhla et al., 2011;Abdul-Ghani et al., 2013; Al-Hamidhi et al., 2013). In November2005, antimalaria drug policy shifted from CQ to a combina-

http://dx.doi.org/10.1016/j.actatropica.2015.05.0130001-706X/ 2015 Elsevier B.V. All rights reserved.

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

tion of artesunatesulphadoxine/pyrimethamine as the rst-linetherapy and artemetherlumefantrine as a second line treatmentfor uncomplicated malaria. However, this new policy was onlyimplemented four years later in 2009 after proper training anddistribution of the national guideline for antimalaria drugs werecarried out (WHO, 2012). Despite these efforts, previous studiesconducted in Hadhramout province reported that CQ is still com-monly prescribed (18 out of 42 prescriptions) and some clinicianswere not aware and had poor knowledge about the new nationaldrug policy (Bashrahil et al., 2010; Ghouth, 2013). Continued useof CQ sustains the selection of CQ resistant mutations leading topersistence of mutant parasite. The complete withdrawal of CQ usemay enhance the emergence of CQ sensitive parasite over time andmakeCQpossible to be re-introduced formalaria treatment (Kublinet al., 2003; Laufer et al., 2006). However, the persistence of CQresistancewill beprolonged if the shift toACTand the simultaneouswithdrawal of CQ are not rigorously implemented. The prevalenceof CQ resistant mutations in Hadhramout is not at present known.The aim of the current survey is to obtain such data, since they willbe important for future monitoring and assessment of antimalarialdrug policy in Yemen.

2. Methods

2.1. Survey area/subjects and ethical consideration

This surveywascarriedout inHadhramout province,which rep-resent about 36% of the total area of Yemen and located in thesoutheast corner of the country with a population estimated at1,028,556 (CSO, 2013). Hadhramout has coastal plain region andmountainous area. The coastal plain region (with an altitude of0600m) is characterized by a hot climate throughout the yearwith little rainfall ranging between 50 and 100mm per annum.The coastal region is endemic for malaria (only in valleys) with aseasonal transmission occurring from November to April (NMCP,2011). The sources of income of the population include fundstransferred from natives working in neighbouring Gulf countries,agriculture, shing, livestock or handicraft. A total of 735 partic-ipants of all ages and both genders were enrolled in this survey;ngerprickblood sampleswere collected fromparticipants in threevillages in Hajer and four villages in Al-RaydahQusyer districts(Fig. 1). These villages were selected because they are endemic formalaria and houses were selected by random sampling. The surveywas conductedbetween July 2011 andMay2012. Informed consentwas obtained from each participant, and for children, consent wasobtained from their parents after a clear explanation of the surveyobjectiveswas given. Prior to sample collection, the surveyprotocolwas approved by the Faculty of Medicine, Hadhramout Universityfor Science and Technology, the Ministry of Health and Population,Yemen, and NMCP division in Hadhramout governorate. All posi-tive cases were treated by NMCP following the recommendationsof the national drug policy.

2.2. Samples and microscopy

Blood was collected by nger prick and thin and thick bloodlms were prepared and left to air-dry and thin lm was xedwith methanol. Blood smears were brought back from the eldand stained as soon as possible with 10% diluted Giemsa stain andscreened under amicroscope for the presence ofmalaria in the lab-oratory. A negative result was recorded after screening at least 200elds under the oil immersion lens of the light microscope. Thepositive specimens for P. falciparum were used for molecular stud-ies. Filter paper blood spots were also collected on Whatman lterpaper 3MM (Whatman International Ltd., Maidstone, England) and

kept separately in clean, dry and well-sealed plastic bag with silicagel to reduce humidity in the bag and stored at room temperatureuntil 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 l-ter paper blood spot (approximately 5mm diameter) was punchedout and transferred into microcentrifuge tubes using a clean andmethanol-amed forceps. Genomic DNA sample was extractedusingQIAgenDNAMini Kit for blood and tissue (QIAGEN,Germany)according to the manufacturers instructions. Extracted DNA waselutedusing50LQiagenAEelutionbuffer andkept at20 C untiluse. Plasmodium species were identied using nested PCR basedon small subunit ribosomal RNA gene (Singh et al., 1999; Snounouet al., 1993). PCR master mix and thermal cycling conditions werecarried out as reported previously (Snounou, 1996).

2.4. Molecular detection of pfcrt K76T

A nested mutation-specic PCR was used to detect mutations inpfcrt K76T (Lopes et al., 2002). Primary PCR was performed usingtwo sets of primer (CRTP1 and CRTP2) to amplify 537bp of pfcrtgene. The secondary PCR was performed using a common innerprimer (CRTP3) togetherwitheitherCRTP4morCRTP4wformutantand wild types, respectively, resulting in 366bp of the two alleles.PCR reaction was carried out in a nal volume of 25L in a PCRtube containing 2.5mM MgCl2, 200M of each dNTP, 1.25 units ofTaq polymerase, 1M of CRTP1 primer, 1M of CRTP2 primer andvemicroliter of genomicDNA. PCR reagent andprimerswere fromiNtRON (iNtRON Biotechnology, Inc., Seoul, Republic of Korea). PCRwas performed in the thermal cycler (MyCycler, BioRad Hercules,USA) with the following cycling conditions: initial denaturation at95 C for 3min, 45 cycles of denaturation at 94 C for 30 s, annealingat 56 C for 30 s, extension at 60 C for 1min and nal extension at60 C for 5min. About 12L of primary PCR product was usedas template in the secondary PCR reaction which contained thesame PCR reagent concentrations as in the primary PCR except forusing 1.5mM MgCl2 and amplication cycle of 25 cycles insteadof 45 cycles. The cycling conditions were similar to the primaryPCR except that the annealing and extension temperatures were47 C and 64 C, respectively. The PCR products were loaded in a2% 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 (positionsQ271E, N326S, I356T, R371I) and pfmdr1 gene (positions N86Yand D1246Y)

Nested PCR followed by restriction fragment length polymor-phism 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 and371, respectively. The enzymes AIII and Bg1II digest pfmdr1 atcodons 86 and 1246, respectively. The digestion of PCR productwas achieved by FastDigest restriction enzymes (Thermo FisherScientic Inc., USA) according to the instructions of the manufac-turer. 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. falci-parum strains HB3, 3D7 and Dd2 supplied by Malaria Research andReference 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 pack-age version 19. The signicance of the associations between pointmutations and independent variables were assessed using Chi-square test. The signicance level was considered at p

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

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

Districts n (%)

Mutation codons Alleles Hajer Al-RaydahQusyer Total p value

Pfcrt 76 Wild 3 (11.5) 33 (29.5) 36 (26.1) 0.006*

Mutant 11 (42.3) 59 (52.7) 70 (50.7)Mixed 12 (46.2) 20 (17.9) 32 (23.1)

Pfcrt 271 Wild 10 (38.5) 47 (42.0) 57 (41.3) 0.744Mutant 16 (61.5) 65 (58.0) 81 (58.7)

Pfcrt 326 Wild 9 (34.6) 54 (48.2) 63 (45.7) 0.210Mutant 17 (65.4) 58 (51.8) 75 (54.3)

Pfcrt 356 Wild 26 (100.0) 112 (100.0) 138 (100.0) NAMutant 0 0

Pfcrt 371 Wild 8 (30.8) 68 (60.7) 76 (55.1) 0.006*

Mutant 18 (69.2) 44 (39.3) 62 (44.9)

Pfmdr 1-86 Wild 18 (69.2) 97 (86.6) 115 (83.3) 0.032Mutant 8 (30.8) 15 (13.4) 23 (16.7)

Pfmdr1-1246 Wild 26(100.0) 111 (100.0) 137 (100.0) NAMutant 0 0

* Using Pearson Chi-Square.

Laufer et al., 2006). However, the prevalence of pfcrt mutations,in this survey, is still considered high which means that malariaparasite is sustaining the CQ resistance in this region. The possibleexplanation of the sustainability of CQ resistance is the continuedunsupervised use of CQ for the treatment ofmalaria inHadhramout(Bashrahil et al., 2010; Ghouth, 2013). Unfortunately, no previousdata are available about the prevalence of pfcrt 76T gene mutationin Hadhramout which could be used for comparison to show thetrend of CQ resistance in this area over the years. The prevalenceof mutations in pfcrt at loci 271, 326 and 371 in this survey, was58.7%, 54.3% and 44.9%, respectively. These mutations are morecommonly distributed in the Old World CQ resistant strain andaffect the response toCQ in thepresenceof 76Tmutation (Ibraheemet al., 2014). Findings from this survey necessitate the implementa-tionof effective control of CQusage in theYemenimarket especiallyas CQ is still necessary for P. vivax infection. However, this has tobe done based on accurate diagnosis.

Although pfmdr1 plays an important role in modulating levelsof antimalarial drug resistance, CQ resistance has been correlatedwith the pfmdr1 86Y (Djimd et al., 2001), while the other point

mutations at codons 184, 1034, 1042 and 1246 do not conferresistance to CQ and are correlated to meoquine, artesunate,amodiaquine, halofantrine and quinine resistance (Duraisingh andCowman, 2005). However, the mechanism of pfmdr1 gene muta-tions in drug resistance is controversial. In the present survey,prevalence (16.7%) of pfmdr1 86Ymutant type among P. falciparumisolates was observed, whereas there is no mutation of the pfmdr1at codon 1246. This ndings are in agreement with recent studiesin Yemen and neighboring countries such as Kingdomof Saudi Ara-bia and Iran (Zakeri et al., 2008; Bin-Dajem et al., 2012; Al-Hamidhiet al., 2013). Although the decrease prevalence of pfmdr186Ymuta-tion has been attributed to almost complete withdrawal of CQ inthe community, this is not the case in our survey since CQ is stillprescribed in Hadhramout (Bashrahil et al., 2010; Ghouth, 2013).

In the present survey, the association of pfcrt and pfmdr1mutations with gender and age of participants was not signi-cant. Similar ndings have been reported from Malaysia and Iran(Rastaghi et al., 2008; Atroosh et al., 2012). No signicant differencein theprevalenceofpfcrtandpfmdr1mutationsbetween subclinicaland clinical infection of malaria parasites was noted in this survey.

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

Symptoms n (%)

Mutation Codons Alleles Symptomatic* Asymptomatic p value

Pfcrt 76 Wild 17 (25.8) 19 (26.4) 0.596Mutant 36 (55.5) 34 (47.2)Mixed 13 (19.7) 19 (26.4)

Pfcrt 271 Wild 31 (47.0) 26 (36.1) 0.196Mutant 35 (53.0) 46 (63.9)

Pfcrt 326 Wild 26 (39.4) 37 (51.4) 0.158Mutant 40 (60.6) 35 (48.6)

Pfcrt 356 Wild 66 (100.0) 72 (100.0) NAMutant 0 0

Pfcrt 371 Wild 37 (56.1) 39 (54.2) 0.823Mutant 29 (43.9) 33 (45.8)

Pfmdr 1-86 Wild 54 ( 81.8) 61 ( 84.7) 0.647Mutant 12 ( 18.2 ) 11 ( 15.3 )

Pfmdr1-1246# Wild 65 (100.0) 72 (100.0) NAMutant 0 0

* 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 63

This nding support previous studies conducted in Kenya (Zhonget al., 2008) and the border regions of Burma/Myanmar (Brownet al., 2012).

In conclusion, this survey detected high frequency of mutationsin pfcrt gene at codons 76, 271, 326 and 371. This suggests the sus-tainability of CQ resistance after 4 years of implementing ACTs as anewdrugpolicy inYemen. Surveyndingswarrant for effective andstricter control of CQ usage and its open availability in the Yemenimarket.

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 ofthe 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 ques-tionnaire 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 inter-

pretation.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 andlaboratory expert group for their assistance in the laboratory work,theMalaria National Control Program inHadhramout governorate-Yemen especially Prof. Abdulla Salim Bin Ghouth, Ministry ofHealth and Population in Hadhramout for their cooperation duringthis study.

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Survey of chloroquine-resistant mutations in the Plasmodium falciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen1 Introduction2 Methods2.1 Survey area/subjects and ethical consideration2.2 Samples and microscopy2.3 Molecular identification of malaria species2.4 Molecular detection of pfcrt K76T2.5 Molecular detection of mutations in pfcrt gene (positions Q271E, N326S, I356T, R371I) and pfmdr1 gene (positions N86Y and D1246Y)2.6 Statistical analysis

3 Results3.1 Malaria detection/identification using microscopy and PCR3.2 Frequency and distribution of pfcrt and pfmdr1 gene alleles in Hadhramout

4 DiscussionCompeting interestsFinancial supportAuthors' contributionsAcknowledgmentsReferences