Plasmodium falciparum drug resistance gene status in the Horn of Africa: A systematic review

Antimalarial drug resistance monitoring is the key factor in malaria control policy for early detection and subsequent prevention of drug resistance spread. This review was performed to collate all available data of P. falciparum resistant genes in the Horn of Africa as a baseline for future appraisal of the regional malaria control policy. The search of this review was performed in January 2018 using the scientific databases Pub Med and Google Scholar. The search terms used included: Plasmodium falciparum AND drug resistance genes OR molecular marks AND Somalia OR Ethiopia OR Eritrea OR Djibouti. The majority of studies (9 of 18 studies, 50%) examined pfdhfr, pfcrt and pfmdr 1 genes. Eight (44%), 4 (22%), and 2 (11%) studies analyzed pfdhps, pfk 13 and pfatp 6 genes, respectively. The Pfcytbc1 associated with atovaquone resistance is the only gene with no mutation detected. High frequencies of pfdhfr and pfdhps mutations were reported with an association to treatment failure after the artemisinin-based combination therapy (ACT) artesunate + sulfadoxine/pyrimethamine. The aminoquinoline resistance genes such as pfmdr1, and pfatp 6 were only reported with low frequency. The 76T mutation of pfcrt ranged from 4 to 100%, while pfmdr1 mutations at codon 86 and 184 varied depending on geographical locations. The 402V and 431K mutations of pfatp 6 were found highly prevalent at 93 % and 58 % in Southwestern Ethiopia, respectively. The pfk13 gene mutation at codon 622I was 2.4%, with an association to artemether-lumefantrine efficacy and delay of parasite clearance on day 3.


INTRODUCTION
Malaria is an arthropod-borne disease with a major impact on the world's human population health.In 2017, 90% of malaria cases were reported from Africa, 7% from Southeast Asia and 2% from the Eastern Mediterranean *Corresponding author.E-mail: kesaratmu@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Region (WHO, 2017).The Horn of Africa consists of four countries, i.e., Djibouti, Ethiopia, Eritrea, and Somalia (Figure 1).In 2015, about 1.9 million malaria cases were reported from Ethiopia, while 20,963 cases were reported from Somalia and 19,372 from Eritrea (WHO, 2016).Malaria exists as hypo-endemic in Djibouti, varies from hypo-to mesoendemic in Somalia, endemic or epidemic in Eritrea, while in Ethiopia, it seasonally fluctuates (WHO, 2007).The influx of refugees is exacerbating the situation of malaria in the Horn of Africa.Drug resistance is the major problem confronting malaria control in the region (Heuchert et al., 2015).Plasmodium falciparum has developed resistance to multiple drugs including chloroquine (CQ), sulfadoxine-pyrimethamine (SP) and recently, artemisinin (ART) resistance strains have emerged at the Thai-Cambodian border (Golassa et al., 2014;Heuchert et al., 2015).The situation has also jeopardized the use of safe, cheap and affordable drugs in poor countries where control of malaria has been inefficient (Gebru et al., 2005).The spread of antimalarial drug resistance by human migration from one country to another has to be regarded and neighboring countries need to enforce regional instead of national programs to avoid genetic drug resistance spreading among them (Bridges et al., 2009;WHO, 2007).CQ was the first antimalarial drug used in endemic areas and resistance was reported from Thailand in the late 1950's.Since then, it spread out quickly to South Asia, then to Africa in 1974, and finally reaching East Africa in 1980's (Mekonnen et al., 2014).The dispersion of CQ resistance was a paramount factor in the failure of the first malaria control and elimination efforts in the mid of 20 th century (Takala-Harrison and Laufer, 2015).Ethiopia was the first country in the Horn of Africa to document CQ resistance in 1985.At that time, the resistant isolates in Ethiopia were reported from areas bordering Kenya, Somalia, and Sudan, while the central part was apparently free from resistant strains (Alene and Bennett, 1996).In 1998, Ethiopia switched to SP as the first-line treatment for uncomplicated P. falciparum malaria.Unfortunately, within a short period, P. falciparum developed resistance to SP (Lo et al., 2017;Mekonnen et al., 2014;Schunk et al., 2006).Retrospective analysis confirmed that pyrimethamine resistant strains were present in sub-Saharan Africa before implementation of SP (Alifrangis et al., 2014).In 2004, an artemisinin combination therapy (ACT) called artemether-lumefantrine (AL) became Ethiopia's first-line treatment for uncomplicated falciparum malaria (Heuchert et al., 2015;Mekonnen et al., 2014).Eritrea, which was previously using SP, shifted to artesunate-amodiaquine (AS-AQ) in 2007 (Menegon et al., 2016) and Somalia adopted AS plus SP (AS-SP) as first-line therapy in 2007 (Warsame et al., 2015).In 2016, AL became the first line drug in Somalia's national plan for uncomplicated P. falciparum malaria (Warsame et al., 2017).
Drug resistance monitoring is the key factor in malaria control policy for early detection and subsequent prevention of the spread of drug resistance (Takala-Harrison and Laufer, 2015).The three basic approaches used to detect antimalarial drug resistance are in vivo test, in vitro test, and analysis of molecular markers.In vitro test allows investigation of parasite susceptibility to antimalarial drugs in artificial culture media without the influence of host's factors.Numerous tests are available and different drugs can be simultaneously assessed with a single isolate.Nevertheless, the requirement of live parasites and highly skilled personnel are the drawbacks of this approach Bloland et al, 2003;WHO, 2010).In vivo test is the gold standard for guiding malaria control policy since the test accounts for the influence of parasite, host, and drug factors.However, this approach is expensive and difficult to apply in clinical settings in most endemic areas due to limited supplies and equipment, long patient's follow-up for 28 or 48 days, repeated biological samples, and requirement of experienced staff (Bloland et al., 2003).An analysis of validated molecular marker of antimalarial drug resistance is an alternative approach for detecting antimalarial drug resistance associated with previously documented mutations.These markers are useful for monitoring of antimalarial drug resistance and obtaining a picture of the situation at a particular time (Mvumbi et al., 2015).Single or multiple nucleotide mutations in diverse P. falciparum gene alleles are related to a wide range of antimalarial drugs resistance.The prominent genes identified up to now include P. falciparum dihydropteroate synthase gene (pfdhps), P. falciparum dihydrofolate reductase gene (pfdhfr), P. falciparum multidrug resistance 1 gene (pfmdr 1), P. falciparum chloroquine resistance transporter gene (pfcrt), P. falciparum Klech 13 propeller gene (pfk13), P. falciparum adenosine triphosphatase 6 (pfatp6), and P. falciparum cytochrome b1 (pfcytb1).
The decline in SP sensitivity is a blend of singlenucleotide polymorphisms (SNPs) in two different genes encoding enzymes involved in the synthesis of the folate cofactor which is essential for parasite growth and survival.Mutation of the pfdhps gene is known to be related to sulfadoxine resistance, while that of the pfdhfr gene is associated with pyrimethamine resistance (Hailemeskel et al., 2013;Tessema et al., 2015).Both are often investigated in combination because of their correlation to SP resistance.The resistance level depends on the number of point mutations in these genes.Thus, multiple changes in the genes are accountable for SP treatment failure in P. falciparum malaria (Gebru et al., 2005).Mutations related to SP treatment failure have been identified at codons 613, 540, 437, and 436 in the pfdhps gene and at codons 164, 108, 59, 51, and 16 in the pfdhfr gene (Gebru et al., 2005;Warsame et al., 2015).The presence of the pfdhps double mutant A437G and K540E along with the pfdhfr triple mutant N51I, C59R, and S108N, known as a quintuple mutant, strongly correlates with SP resistance (Warsame et al., 2017).
The mutation at K76T of the pfcrt gene is strongly related to CQ resistance (Golassa et al., 2014) and is also proposed to affect the susceptibility of P. falciparum to ART, quinine, and amodiaquine (Heuchert et al., 2015).Pfmdr 1 mutation has been associated with resistance of P. falciparum to several antimalarial drugs including CQ, mefloquine, halofantrine, quinine, and ART (Eshetu et al., 2010;Heuchert et al., 2015).Mutations in both pfcrt and pfmdr1 alleles are associated with CQ resistance, although mutation of the pfcrt gene is a stronger indicator for CQ resistance (Golassa et al., 2014;Mekonnen et al., 2014).The pfcrt K76T together with other pfcrt mutations(C72S, M74I, N75E, A220S, I356K, and R371I) is often used as markers of CQ resistance but the clinical association has not been fully validated (Wurtz et al., 2012).The pfcrt CVMNK wild-type can mutate either into the CVIET or CVMNT haplotype, both of which are related to the regional evolution of CQ resistance (Heuchert et al., 2015;Menegon et al., 2016).The pfmdr1 mutant allele 86Y has been associated with a decline in CQ sensitivity in areas where the prevalence of the parasite is low to moderate (Wurtz et al., 2012).The return of CQ sensitivity was reported after long time abandonment of CQ use in several endemic countries (Mvumbi et al., 2015) and this proposes the possibility of re-introduction of CQ for treatment of P. falciparum malaria in the future (Golassa et al., 2014;Golassa et al., 2015).Nevertheless, the decision will require close regional monitoring because studies of CQ resistance after discontinuation of drug pressure are considerably inconsistent among parasitic populations (Golassa et al., 2015).In Ethiopia for instance, the prevalence of P. falciparum clinical isolates carrying CQ resistant pfcrt K76T varied from 16% (Mekonnen et al., 2014) to 100% (Golassa et al., 2015).
The recent emergence of P. falciparum ART resistance strains in Greater Mekong Sub-region (GMS) represents a challenge to the efficacy of ART and also will postpone the goal of malaria elimination by 2030 in the region (WHO, 2015).The dissemination of these strains to Africa, where the majority of deaths due to malaria occurs, will have catastrophic results (Bayih et al., 2016).Mutation of pfatp 6 (encodes P. falciparum SERCA-type ATPase 6) has initially been linked to resistance of P. falciparum to artemether (Heuchert et al., 2015).Currently, the pfk13 gene mutation, firstly reported at the Thai-Cambodian border (Heuchert et al., 2015), is suggested as the accurate marker of ART resistance.The pfcyt b1 mutation at codon 268 was associated with delay recrudescence of parasites after atovaquoneproguanil therapy (Musset et al., 2006;Sutherland et al., 2008;Wichmann et al., 2004).
In the present review, a qualitative analysis was systematically conducted to obtain relevant data available on P. falciparum molecular markers of antimalarial resistance (pfdhps, pfdhfr, pfcrt, pfmdr 1, pfk13, pfatp6, and pfcyt b1) in the Horn of Africa as a baseline for future assessments.
Knowing how malaria parasites disseminate along with monitoring the prevalence of drug-resistant markers in the high-risk endemic areas is imperative for antimalarial policymaking.

Search strategy
The search of relevant research articles was performed in January 2018 through the two scientific databases, namely, PubMed and Google Scholar.The following medical subject heading (MeSH) terms were used: Plasmodium falciparum AND drug resistance genes OR molecular marks AND Somalia OR Ethiopia OR Eritrea OR Djibouti.The references of retrieved articles were searched to obtain additional relevant articles.The inclusion criteria were (a) studies related to P. falciparum malaria in the Horn of Africa, (b) studies that analyzed P. falciparum resistance molecular markers, and (c) studies presented as original research articles.The exclusion criteria were: (a) studies that only assessed the clinical efficacy of antimalarial drugs, (b) non-molecular studies, (c) studies conducted outside the Horn of Africa (d) studies without full-text articles available online, (e) review articles or case reports, or (f) articles in language other than English.Title and abstract of each article were initially screened, followed by a full-text assessment.

Data extraction
The following information was extracted from the eligible articles: first author, publication year, P. falciparum molecular markers (type and frequency), techniques used to detect these markers, sample size, year of sample collection, and geographic location of the study.The frequencies of mutant alleles for pfdhps, pfdhfr, pfcrt, pfmdr1, pfk13, pfatp 6, and pfcyt b1 genes were then extracted.Screening, selection, and extraction of data were performed by two independent researchers.When disagreement arose at any stage of the process, a higher professional person was consulted for a final decision.

Study selection
A total of 81 articles were obtained from Google Scholar and PubMed databases.Six duplicate articles were removed using EndNote X7; 39 articles were excluded after screening their titles and abstracts against the inclusion criteria; and 19 articles were excluded from the review after full-text assessment due to following reasons: in vivo studies, studies conducted outside the Horn of Africa, studies focusing on treatment adherence, studies with repeated sample analysis, and studies with unsuitable findings.Eighteen eligible articles were therefore selected for this review (Figure 2).Seven P. falciparum genes were investigated in these articles: pfdhps, pfdhfr, pfcrt, pfmdr1, pfk13, pfatp6, and pfcyt b1.The majority of studies (9 of 18 studies, 50%) examined pfdhfr, pfcrt, and pfmdr1genes.Eight (44%), 4 (22%), and 2 (11%) studies analyzed pfdhps, pfk13 and pfatp 6 gene, respectively (Table 1).

Pfdhps and pfdhfr
Of the 16 studies, 8 studies analyzed pfdhfr and pfdhps together, while 1 study analyzed only pfdhfr along with other genes.Mutation at codons 108N, 59R and 51I of the pfdhfr and at codons 540E and 437G of the pfdhps were reported.Eight studies reported the frequency of either pfdhps double mutants (K540E andA437G) or the pfdhfr triple mutant (S108N, C59R, and N51I) or both (Table 2).

Pfcrt
Of the 18 studies, 9 studies investigated K76T mutation, of which 3 studies, also investigated C72S mutation with 3.6% prevalence reported in 1 study.The SVMNK and CVIET at positions72-76 of the pfcrt gene were reported in 4 studies, while the SVMNT haplotype was reported in 2 studies.A high-frequency rate of CVIET haplotype was reported in 3 studies (Table 3).

Pfcyt b1
Two (both from Ethiopia) out of 18 studies analyzed pfcytbc1 gene mutation and none of them showed gene variation from the reference strain.

Association of molecular markers and clinical efficacy
Association of molecular markers and clinical efficacy of antimalarials was evaluated in 3 studies (2 from Somalia and 1 from Ethiopia).The clinical efficacy of AS-SP and AL were investigated with 28 days follow-up period.The PCR-corrected treatment failure was observed in 33 and 3 patients following treatment with AS-SP and AL, respectively.The cumulative efficacy of AS-SP and AL were 78 and 98%, respectively (Table 7).
The association between AS-SP clinical efficacy and the molecular markers pfdhps and pfdhfr were reported in 2015 and 2017 in Somalia (Warsame et al., 2017;Warsame et al., 2015), with failure rates of approximately 22.2 and 12.1%, respectively.For the 2015 study, the quadruple 51I/108N+437G/540E and quintuple 51I/59R/108N+437G/540E mutations of pfdhfr+pfdhps were shown to be associated with high risk of treatment failure with odd ratios of 5.5 and 3.5 respectively.The double 437G/540E mutation of pfdhps was associated with the highest risk of treatment failure (OR =22.4).On the other hand, two years later, all parasites isolated from patients with treatment failure after AS-SP carried the quintuple mutation 51I/108N + 437G/540E/581G.
The efficacy of AL was investigated in two studies in Ethiopia and Somalia (Bayih et al., 2016;Warsame et al., 2017).In the first study (Bayih et al., 2016), the association between AL clinical efficacy and the pfk13 mutation was found.The 622I mutation was found in 2.4% of all samples with association with the delay in parasite clearance on day 3 (all mutant parasites isolated from patients who parasitemic at day 3).In the second study (Warsame et al., 2017), treatment failure rate of AL was <6%; however, no molecular marker was investigated.

DISCUSSION
A total of 18 P. falciparum eligible molecular studies conducted in the Horn of Africa were included in the analysis.These studies were conducted 16 years after the completion of malaria genome sequencing in 2002 (Carlton et al., 2004).Three studies particularly addressed the correlation between results of the in vivo assessment and molecular markers of antimalarial drug resistance (Bayih et Warsame et al., 2015).The development and spread of antimalarial drug resistance is attributed to several factors including inappropriate dosing, poor treatment practice, substandard or counterfeit drugs, the use of ART monotherapy, and the ability of the parasite to modify its genome at any time (Bloland et al., 2003).Even though the usual treatment for malaria has followed the World Health Organization (WHO)'s recommendation Bloland et al., 2003;WHO, 2010), antimalarial drug resistance has continuously been emerging.In this systematic review, the frequencies of P. falciparum gene polymorphisms linked with different types of antimalarial drug resistance were analyzed.Pfcyt bc1 associated with atovaquone resistance is the only gene with no mutation detected since the drug has never been used in this part of Africa.
The high level of pfdhps/pfdhfr mutations shown in the majority of the extracted data is likely caused by the wide use of SP prior to AL introduction in the region, or the concurrent use of SP in some countries in the region for intermittent prophylactic treatment for pregnancy (IPTp) and children as recommended by WHO WHO, 2010).In a study conducted in the northern zone of Somalia, the prevalence of the pfdhfr mutant alleles 59R, 51I, and 108N were 100, 22 and 100%, respectively.The pfdhfr triple mutations (51I,59R, and 108N) associated with pyrimethamine resistance was 22% of the isolates.Likewise, the prevalence of pfdhps double mutation (437G and 540E) was 24%.The PCR-corrected AS-SP treatment failure rate reported in the study was 12% (Warsame et al., 2017).The findings of this study support previous work conducted in Jamame in the Southern zone of Somalia for the relatively high failure rate (22%) after AS-SP treatment (Warsame et al., 2015).This most likely occurred due to SP resistance since both studies showed high prevalence rates of mutation rate in the pfdhps/pfdhfr genes (Warsame et al., 2017;Warsame et al., 2015).In the Jamame study, the odds ratio (OR) of treatment failure within 28-days follow-up period was increased among patients harboring the pfdhps/pfdhfr quintuple mutant (OR 3.5,95% CI=1.4-8.8) and those carrying pfdhps double mutant (OR 22,95% CI:5.1-98.1)(Warsame et al., 2015).A study from Ethiopia reported mutations at codons R59, I51, and N108 in the pfdhfr gene at the frequencies of 100, 91 and 98%, respectively.The frequency of triple mutation (51I, 59R, and 108N) of this gene was 22%, while that of the pfdhps mutations at codons 437G, 540E, and both (437G and 540E) was 68, 92 and 91%, respectively (Mula et al., 2011).The observation of high prevalence rate corroborates with findings from Congo and Ghana.This indicates that SP resistance had not yet been declined (Marks et al., 2005;Ndounga et al., 2007).In the above-cited studies from the northern zone of Somalia and Ethiopia, the quintuple (double pfdhps plus triple pfdhfr) mutation which is used as a marker of SP resistance was 11 and 83% respectively.The SP failure due to pfdhfr triple mutation alone has been suggested in other reports from Kenya (Nzila et al., 2000) and Cameroon (Basco et al., 2000).It is worth noting that two studies from northwest Ethiopia investigated pfdhfr/pfdhps genes at two different time points (2005/2008) recording a marked reduction in pfdhfr triple mutation and pfdhps/pfdhfr quintuple mutation in field isolates.Nevertheless, this is not sufficient to conclude the return of SP sensitivity since a high rate of mutation was found (Hailemeskel et al., 2013;Tessema et al., 2015).Eight out of the nine studies detected a high mutation at the core codon 76T of the pfcrt gene, the CQ resistance molecular marker (Mekonnen et al., 2014).The WHO recommends withdrawal of a drug if the prevalence of resistance exceeds 10% Bloland et al., 2003).The 76T mutant often occurred concurrently with other mutants, though their role has not been fully defined (Golassa et al., 2015).In Ethiopia for instance, the frequency of 76T mutation was 100% in a study conducted in South-central Oromia in 2012 (Golassa et al., 2014), while a year later, the frequency of 4.4% was reported in a similar study conducted in Southwest Ethiopia (Heuchert et al., 2015).The recovery of CQ susceptibility is a controversial issue in this country since CQ is still used for management of P. vivax malaria in areas with CQ resistant P. falciparum, resulting in a persistent selective pressure of pfcrt 76T (Golassa et al., 2015).Another reason is the availability of CQ from the drug stores and self-medication without the prescription due to poor management of drugs.However, a study from Kenya reported partial re-emergence of CQ sensitive strains after removal of selective pressure for this drug; thus this appears to be a country-specific drug change policy (Mwai et al., 2009).Studies conducted in Ethiopia and Eritrea identified the CVIET haplotype at the frequencies of 50 and 85%, respectively (Heuchert et al.,2015;Menegon et al., 2016), while the frequency reported in another study in Ethiopia was 0% (Mekonnen et al., 2014).Two Ethiopian studies aiming at an investigation of the SVMNT haplotype reported low mutant frequency (4 and 0%) (Heuchert et al., 2015;Mekonnen et al., 2014).The CVIET and SVMNT haplotypes are related to geographic origin of CQ tolerance (Mekonnen et al., 2014), the first haplotype predominates in Africa and Southeast Asia, whereas the other is more prevalent in some South American countries (Mehlotra et al., 2001).Concerning pfmdr1 polymorphisms, disparate codons (86Y, 184F, 1242D, and 1246Y) with different mutation frequencies were noted in isolates from Eritrea and Ethiopia.The majority of studies showed mutation at codons 86Y and 184F.The 86Y is associated with a decline in the CQ sensitivity.Two studies in Southwest Ethiopia reported the decline in the prevalence of pfmdr1 86Yfrom 85% in 2006 to 1.2% in 2013 (Eshetu et al., 2010;Heuchert et al., 2015), while another study in Gondar Ethiopia reported the prevalence of 46% in 2014 (Tajebe et al., 2015).The frequency variation in these studies is likely reflecting the discrepancy of drug pressure in different geographical areas.The prevalence of the pfcrt 76T point mutation (55%) and high copy number variability (CNV) at codon pfmdr1 86Y from Gondar study, could provoke resistance for the new drug AL and delay the re-introduction of CQ in this location.In this study, high frequency of P. falciparum multidrug resistance copy number variants (54.2%) with 25.6% of isolates carried  multi-copies (1.5-2.5 copies) and 28.6% (> 2.5 copies) were found.This is likely to be a risk factor of resistance development for the artemether (A) partner (lumefantrine), since AL is Ethiopia's first-line treatment for falciparum malaria but the prevalence rate of multidrug resistance CNVs was not statistically significant (P>0.05)(Tajebe et al., 2015).In vitro study at the Thai-Cambodian border showed that parasites with higher copy number were remarkably decreased susceptibility to lumefantrine, mefloquine, and artesunate (Lim et al., 2009).
Artemisinins are potent and rapid-acting compounds against multidrug-resistant P. falciparum strains.Eckstein-Ludwig et al suggested that artemisinins selectively inhibit Pfatpase6, the only SERCA-type Ca 2+ -ATPase in the P. falciparum genome (Ariey et al., 2014).Two studies have targeted this gene; the sequencing of field isolates reported by Jimma et al (Eshetu et al., 2010) showed a variety of previously identified (L402V, E431K, A623E, N683, N683K, and S769N) and novel (E237A, L263L, R682R, K766K, K767E, and K767R) mutations in the pfatp6 gene.Most of these mutations are globally dispersed as reports from South Africa and Asia indicate, but they were not correlated with ART resistance except for the S769N mutant being associated with in vitro resistance.An extremely low prevalence rate (3.7%) of S769N mutation was reported (Eshetu et al., 2010).The prevalence of the pfatp 6 E431K mutation was declined from 58.3% in 2006 (Eshetu et al., 2010) to 18.8% in 2013 (Heuchert et al., 2015).This did not occur under ART selective pressure because the drug was still in use and in addition, E431K was not detected along with A623E mutations which have been associated with a reduction of ART sensitivity.The recent emergence of ART-resistant strains in the Southeast Asian region has led many African countries to adopt ACT as the first-line treatment for uncomplicated P. falciparum malaria to become more vigilant since GMS was the previous spreading origin of CQ and SP resistance parasites.However, four studies conducted in Ethiopia showed no mutation at codons580Y, 543T, and 493H in the pfk13 propeller domain which was previously correlated with artemisinin resistance in Southeast Asia (Ménard et al., 2016).These studies are in accordance with reports from Sub-Saharan Africa (Taylor et al., 2015).Only 1(4%) isolate was found harboring a new mutation at codon N531I in one study (Heuchert et al., 2015), while in another study, 3 (2.5%)isolates were identified as having a novel mutation at R622T.Only one out of the three patients was found malaria positive on day-3 by microscopy with no information on in vitro parasite sensitivity.Interestingly, the day-3 positive patients cleared the parasite before day-28 (Bayih et al., 2016).This is unlikely to be resistance strains due to the lack of strong relationship between K13-propeller domain and delayed parasitic clearance after ART treatment as detected in Southeast Asia (Ariey et al., 2014).

Conclusions
This systematic review presents a picture of the geographical distributions of P. falciparum drug resistance genes during 1998-2014.The distribution pattern could be changed over time and it will be crucial Jalei et al. 371 to maintaining tracking through systematic sampling around lineage distribution boundaries.In this regard, a complete picture of P. falciparum resistance allele distribution can be generated.This record will enhance our knowledge of the real challenges on spreading of P. falciparum molecular markers at the extremes of the geographical distributions outlined here.Limitation of the current review includes the lack of standardized reporting layout of the data extracted from the published articles.
Information on the prevalence of some point mutations was not available.Some articles included in the analysis still require a certain level of interpretation due to variations in methodological and reporting format.Furthermore, molecular studies with high prevalence mutant alleles were more likely to be published than those with wild-type alleles.

Figure 1 .
Figure 1.Map of Horn of Africa (white area).

Figure 2 .
Figure 2. Flow chart showing article selection process.

Table 1 .
Characteristics of the 18 studies included in the current systematic review.
*Considering falciparum malaria samples only and samples collecting from the Horn of Africa.ND: no data available, PCR: Polymerase chain reaction, RFLP: Restrict fragment length polymorphism.

Table 2 .
The pfdhps/pfdhfr mutant allele frequencies reported in the articles included in the analysis.

Table 3 .
The pfcrt mutant allele and haplotype frequencies reported in the articles included in the analysis.
ND: no data available

Table 4 .
The pfmdr1 mutant allele and haplotype frequencies reported in the articles included in the analysis.

Table 5 .
The pfk13 mutant allele and haplotype frequencies reported in the articles included in the analysis.

Table 6 .
The pfatp 6 mutant allele and haplotype frequencies reported in the articles included in the analysis.

Table 7 .
Clinical efficacy of antimalarial drugs used in the Horn of Africa reported in the articles included in the analysis.