Background
Malaria is one of the most common tropical infectious diseases worldwide. It is particularly prevalent in the tropics and subtropics. The global burden and economic cost of the disease are still immense: approximately 220 million cases of malaria occur annually worldwide, and in 2018 alone there were nearly half a million malaria-related deaths [
1,
2]. Human malaria is caused by four
Plasmodium species:
Plasmodium falciparum,
Plasmodium vivax,
Plasmodium malariae and
Plasmodium ovale. Of these species,
P. falciparum is considered the most virulent and prevalent, accounting for 99.7%, 62.8% and 69% of the malaria cases reported by the World Health Organization (WHO) Regional Offices for Africa, South-East Asia and the Eastern Mediterranean, respectively [
1].
One of the critical challenges to malaria elimination is the emergence and spread of anti-malarial drug resistance. In response to widespread chloroquine resistance, artemisinin-based combination therapy (ACT) has been adopted by the WHO as the first-line anti-malarial treatment for uncomplicated
P. falciparum malaria [
3]. The artemisinin-based combinations used consist of an artemisinin derivative [artesunate (AS) or artemether] co-administered with a longer-acting partner drug [e.g. sulfadoxine–pyrimethamine (SP) or lumefantrine or mefloquine] [
4,
5]. In some regions, ACT is supplemented with a single, low-dose primaquine for clearance of
P. falciparum gametocytes [
6]. ACT has remained highly efficacious for the last 2 decades, but is now facing a major threat due to the emergence and spread of
P. falciparum strains resistant to both artemisinin and its partner drugs, a phenomenon first observed in the western region of Cambodia in 2009 [
7,
8]. Mutations at the propeller domain of the Kelch 13 protein encoded by the
P. falciparum k13 (
pfkelch13) gene have been associated with delayed parasite clearance due to resistance to artemisinin [
9,
10]. In addition, allelic mutations in
P. falciparum dihydrofolate reductase (
pfdhfr) and dihydropteroate synthetase (
pfdhps) genes have also been associated with resistance to pyrimethamine and sulfadoxine, respectively [
11,
12]. It has also been demonstrated that an accumulation of mutant
pfdhps and
pfdhfr alleles confers clinical resistance to SP. Of these, the quintuple mutant genotype that includes
pfdhfr (N51
I + C59
R + S108
N) and
pfdhps (A437
G + A540
E) has been found to be significantly associated with in-vivo resistance to SP [
13,
14].
In Saudi Arabia, the malaria control programme, which was established in 1956, has achieved tremendous progress in reducing the incidence of malaria cases and interrupting local malaria transmission [
15,
16]. Major progress was seen between 2000 and 2010, when the number of indigenous cases reduced significantly from 511 in 2000 to only 29 in 2010, with the number of imported cases remaining at around 1500 per annum [
17]. Thus, the country has been included in the E-2020 WHO initiative with the aim of achieving a target of zero indigenous cases by 2020 [
1]. However, the numbers have increased, and 5382 cases were reported in 2016, including 272 indigenous cases, mostly in Jazan and Aseer regions [
1]. The first cases of resistance to chloroquine and SP monotherapies were documented in Jazan in 1997 and 2007, respectively [
18,
19]. Thus, ACT was adopted in 2007 for the treatment of uncomplicated
P. falciparum malaria, with AS + SP or artemether–lumefantrine (AL) as first- and second-line treatments, respectively [
20,
21]. Overall, there is a scarcity of information on the molecular markers of anti-malarial drug resistance in Saudi Arabia. While limited studies have investigated selected point mutations for SP resistance [
19,
22], just one recent study has assessed the presence of polymorphism in the
pfkelch13 propeller domain, but only in 13
P. falciparum isolates collected from Taif region, western Saudi Arabia [
23].
Thus, this study aimed to investigate the prevalence and distribution of the mutations present in the pfkelch13 propeller domain, and in the pfdhfr and pfdhps genes of the P. falciparum parasites circulating in Jazan region. Regular assessment of the molecular markers for anti-malarial drug resistance is crucial to the success of malaria control programmes and can help health authorities and policy-makers to predict the effectiveness of and resistance to anti-malarial drugs and thus respond in a timely manner to the emergence of resistance.
Discussion
In this study, the prevalence and distribution of polymorphisms associated with
P. falciparum resistance to AS + SP, the first-line ACT in Saudi Arabia, were investigated. The investigation focused on the point mutations within the
pfkelch13,
pfdhfr and
pfdhps genes. Despite intensive efforts to eliminate malaria from Saudi Arabia, active foci for transmission are still reported in Aseer and Jazan regions. Both regions have diverse ecological and natural environments that favour vector breeding sites and local malaria transmission [
16,
31]. Moreover, both regions share borders with Yemen, a country with a high malaria transmission rate [
32,
33]. Between 2015 and 2017, over 32% of all imported malaria cases reported in all regions of Saudi Arabia were of Yemeni origin [
34].
This study demonstrated that all isolates analysed for the potential polymorphism in
pfkelch13 propeller gene were found of wild type showing no mutations throughout the amplified sequences. This finding is consistent with the only previous study on potential mutations in
pfkelch13 in isolates, which was conducted in the Taif region of Saudi Arabia [
23]. Similar findings have also been reported in other countries either in the Arabian Peninsula or in the East Mediterranean Region including Yemen, Sudan and Somalia [
27,
35,
36]. On the other hand, few non-synonymous
pfkelch13 mutations were identified in
P. falciparum isolates from other neighbouring countries including Qatar, Iran and Ethiopia; however, none of those mutations were associated with artemisinin resistance [
36‐
38].
With regards to the partner drug, SP, the current study found a very high prevalence of the
pfdhfr S108N and N51I mutations (84.8% each), with the
pfdhfr double mutant (N51
I + S108
N) reported in 47% of the isolates while 37.8% carried the triple mutant haplotype (N51
I + C59
R + S108
N). In 2012, a previous study on 176 isolates from Jazan region reported 33% and 34% prevalence of
pfdhfr S108
N and N51
I mutations, respectively, with the double mutant (N51
I + S108
N) reported in 33% [
22]. Interestingly, Bin Dajem et al. [
22] found no mutations at other
pfdhfr codons (C59
R and I164
L) and the
pfdhfr triple mutated haplotype was not observed, whereas the current study detected C59
R mutation in 37.7% of the isolates. In Taif region, a recent study among only 13
P. falciparum isolates reported the presence of
pfdhfr double (N51
I + S108
N) and triple (N51
I + C59
R + S108
N) mutations in three (23%) and nine (69%) isolates, respectively [
23]. Moreover, results similar to those obtained by the current study have also been reported among imported
P. falciparum isolates in the neighbouring country of Qatar [
37]. By contrast, previous studies from Yemen have concluded that
P. falciparum with the
pfdhfr triple mutation (N51
I + C59
R + S108
N) is not circulating in the country; however, none of these studies were conducted in areas bordering Jazan, Saudi Arabia [
27,
39,
40].
In respect of
pfdhps, the current study found a 56.3% and 51.7%% prevalence of mutations in the
pfdhps gene at codons A437
G and K540
E, respectively, with 51.7% carrying the
pfdhps double mutant haplotype (S
GEAAI). Until 2016, a prevalence of K540
E exceeding 50% had been reported in 12 African countries including Sudan, Somalia, Ethiopia, Tanzania and Kenya [
11]. While Bin Dajem et al
. [
22] found a single mutation at the
pfdhps codon A437
G in only one isolate from Jazan in 2012, Soliman et al
. [
23] detected mutations at codon A437
G in a single isolate (7.7%) and at codon K540
E in five isolates (38.5%) from Taif region. While not found in the current study, mutations in the
pfdhps gene at codons A581
G and S436
A have been detected in a single (7.7%) and seven (53.8%) isolates from the Taif region [
23]. In Yemen, the results thus far have been mixed. Mutations in the
pfdhps gene have not been detected by some studies [
27,
39], whereas another previous study reported a prevalence of 44.7% of the
pfdhps single mutation at codon A437
G [
40]. Interestingly, a novel mutation at codon I431
V that may favour resistance risk has been found in west and sub-Saharan Africa as well as in imported isolates in the United Kingdom, Qatar and China [
37,
41,
42]. However, this mutation (I431
V) was not detected in the isolates investigated in the current study.
Bearing in mind that Bin Dajem et al
. [
22] conducted their study on 176
P. falciparum isolates collected from healthcare facilities in Jazan (i.e. a similar study area, setting and sample size to the current study), the present findings clearly imply an alarmingly increased prevalence (as well as emergence) in
pfdhfr and
pfdhps point mutations in Jazan region, particularly among Saudis, 11 years after the change in the malaria treatment policy. This study found a high prevalence of
pfdhfr double (47%) and triple (37.8%) mutated haplotypes (i.e. 84.8% when combined as double-to-triple mutations) as well as a high prevalence of the
pfdhps double mutant haplotype (51.7%). Interestingly, 23.8% of the isolates harboured a combination of the
pfdhfr triple mutant (N51
I + C59
R + S108
N) and the
pfdhps double mutant (A437
G + K540
E) haplotypes (i.e. quintuple mutant genotype), a combination that confers full resistance to SP, according to a recent classification suggested by Naidoo and Roper [
43].
The present study is the first to report the presence of quadruple and quintuple mutated
pfdhfr–
pfdhps genotypes in Saudi Arabia and the Arabian Peninsula in general. The distribution of different
pfdhfr and
pfdhps point mutations and related haplotypes across the governorates of Jazan region can be considered comparable, despite the absence of quintuple mutant genotypes in a few governorates as this could be due to the variation in the number of isolates collected from each area (Fig.
1 and Additional file
1: Table S2).
Previous studies conducted in different endemic countries have identified a strong association between the
pfdhfr double mutation (N51
I and S108
N) and resistance to pyrimethamine [
44,
45]. In the same vein, other previous studies have demonstrated that the triple mutant
pfdhfr (N51
I + C59
R + S108
N) confers a significant component of in-vitro and in-vivo resistance to SP [
46,
47]. When this
pfdhfr triple mutation is combined with a
pfdhps double mutation, specifically A437
G and K540
E, producing either quadruple or quintuple mutant genotypes, the risk of SP treatment failure can be over 75% [
48‐
50].
This study also found significant associations between the prevalence of mutated haplotypes and age, gender and nationality. Interestingly, the harbouring of
P. falciparum with
pfdhfr–
pfdhps quintuple mutations (AC
IRNI–S
GEAAI) was significantly higher in patients aged ≥ 30 years compared to those aged below 30 years. The number of
pfdhfr and
pfdhps mutations was found to positively correlate with participants’ age, with an age of 20 years or older was identified as a risk factor of harbouring isolates with a high number of mutations [
51]. By contrast, previous studies have concluded that age does not influence the distribution and carriage of resistant
P. falciparum parasites whatever the type of mutation; however, these studies included only children aged ≤ 15 years [
48,
52]. While the explanation for the association with gender is not known, it should be borne in mind that the identified association could be attributed to the low number of female participants involved in this study.
Interestingly, in Jazan region, the C59
R pfdhfr-mutant allele was detected only in two out of 19 isolates in 2007 [
19] and was not detected in any of 176 isolates (80% Saudi) in 2012 [
22]. However, the current study found that the frequency of C59
R point mutation and the quintuple AC
IRNI–S
GEAAI haplotype was significantly associated with participants’ nationality, with the frequency of AC
IRNI–S
GEAAI haplotype was almost double in the Saudi than non-Saudi patients. Despite the intensive efforts to eliminate indigenous malaria in the Kingdom, 52 (34.4%) cases in this study were Saudi. Moreover, as the majority of malaria cases in this study were among foreigners (65 from Yemen and 27 from Southern Asia, mainly Pakistan and India), the possibility of introducing ACT resistance into Saudi Arabia should not be ignored, especially as it has been over 10 years since the policy change to ACT for uncomplicated falciparum malaria treatment.
There are some limitations that should be considered when interpreting the findings of the present study. First, the total number of the collected P. falciparum isolates was small. Second, the small number of blood samples collected from some governorates as well as the small number of females compared to male participants. Third, the molecular findings were not further correlated with treatment outcome among the study participants. However, this study still provides important findings on both the types and prevalence of P. falciparum mutations circulating in southwestern Saudi Arabia and these findings will enable the setting up of a database on molecular markers of anti-malarial drug resistance in Jazan region. Furthermore, these findings “sound the alarm” for the Jazan region, which together with Aseer region, is one of the last remaining foci of malaria transmission in the country and therefore calls for the close monitoring of the efficacy of ACT.
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