Background
Of the five
Plasmodium species infecting humans,
Plasmodium vivax is the most widely distributed species and the cause of 25-40% of malaria cases worldwide [
1], and substantial morbidity associated with vivax malaria has been reported [
2-
4]. Despite the public health importance,
P. vivax malaria has received little attention and limited funds for research and control, since it usually produces less severe symptoms than falciparum malaria [
2,
5,
6]. Current treatment for vivax malaria relies primarily upon two anti-malarial drugs, chloroquine (CQ) and primaquine (PQ), with the latter being the only effective drug against the hypnozoite stage. Indeed, the emergence of drug resistance in
P. vivax particularly to the only class of compounds available for killing the dormant liver stage is alarming and of high priority for research [
7-
10]. It is worth noting that inadequate surveillance tools delayed the detection and containment of CQ-resistant
P. falciparum resulting in increased morbidity and mortality. If a repetition is to be avoided, the threat of emerging CQ-resistant
P. vivax needs to be acknowledged quickly and widely and substantial resources need to be allocated to validate and standardize tools necessary for characterization of drug-resistant
P. vivax [
10]. In Indonesia, East Timor and Papua New Guinea, CQ-resistant vivax malaria has already reached an alarming prevalence [
11]. Furthermore,
P. vivax CQR has occurred in at least three Latin American countries (Guyana, Peru and Brazil) [
12]. The four clinical trials carried out in Asia (Thailand and Pakistan) and Africa (Ethiopia), for instance, showed that CQ alone (25 mg/kg over 3 days) is less effective against
P. vivax asexual blood stages than CQ (25 mg/kg over 3 days) co-administered with PQ (15 mg of PQ base/day for 14 days) over 28 days of follow-up [
9]. To unveil the current knowledge regarding the molecular mechanisms of
P. vivax resistance to CQ and the prospects for developing and standardizing reliable molecular markers of drug resistance, Goncalves
et al. [
13] reviewed the available data by combining published
in vivo and
in vitro studies.
Unlike in
P. falciparum, the molecular mechanism of
P. vivax CQR remains elusive [
7]. This is because, previous studies focusing on genes known to be main determinants of CQR in
P. falciparum have failed to demonstrate a strong correlation between
pvcrt-o and
pvmdr-1 genotypes and the CQR phenotype in
P. vivax. Melo
et al. [
14], on the other hand, showed the association of expression levels of
pvcrt-o and
pvmdr-1 with CQR and severe
P. vivax malaria, because parasites from patients with CQR presented up to 6.1-fold and 2.4-fold increase in
pvcrt-o and
pvmdr-1 expression levels, respectively, compared to the susceptible group in the Brazilian Amazon.
Drug resistance in
P. vivax is becoming more widespread, hindering management of clinical cases and posing a huge threat to the health of millions of people exposed to the risk of vivax malaria. Analysis of the single nucleotide polymorphisms (SNPs) in drug resistant genes has proved to be useful and important in monitoring drug resistance in malaria endemic countries [
15]. Mutations in multidrug resistance 1 (
pvmdr-1) and K10 insertion in the
pvcrt-o genes have been identified as possible molecular markers of CQR in
P. vivax [
16,
17]. Few data are available on the possible relationship between the
pvcrt-o and
pvmdr-1 genes and CQR [
18]. Nevertheless, there are a number of contradicting reports regarding the association between
pvcrt-o and
pvmdr-1 polymorphisms and CQR. Some reports suggest the Y976F mutation in
pvmdr-1 to be associated with an increase in CQ IC
50 value of
P. vivax isolates
in vitro [
19]. Non-synonymous amino acid mutations in codons Y976F and F1076L of the
pvmdr-1 have been reported to have correlation with CQR although much work remains to link these mutations irrefutably with CQR [
16,
18,
20]. The role of Y976F mutation in
pvmdr-1 gene suggested reduced susceptibility to CQ [
20]. Recent experiments have shown that the expression of
pvcrt-o in transgenic lines of
P. falciparum modulates CQ response [
17]. A study by Fernandez-Becerra
et al. [
21] demonstrated up to 21-fold and up to three-fold increases in transcript levels of
pvcrt-o and
pvmdr-1, respectively, in severe vivax malaria cases compared to isolates from non-severe vivax malaria patients. Another study in India showed the predominance of the wild-type
pvmdr-1 and
pvcrt-o alleles [
22] although one isolate had the Y976F mutation in the
pvmdr-1 gene, which could suggest the beginning of a trend towards decreased CQ sensitivity. In Thailand and Indonesia, where CQR is common, the
pvmdr-1 (Y976F and F1076L) polymorphisms were also identified in
P. vivax samples [
16]. In Latin America, where
P. vivax CQR remains relatively uncommon, the Y976F and F1076L polymorphisms are relatively infrequent [
23,
24].
Presently, Ethiopia maintains a species-specific treatment policy: CQ without PQ is the first-line treatment for
P. vivax and artemether-lumefantrine (AL) for
P. falciparum. Unlike in many malaria endemic countries in Africa, both
P. falciparum and
P. vivax substantially contribute to malaria morbidity in Ethiopia in relative proportions of 60 and 40%, respectively [
25,
26]. In 1996, Ethiopia published its first report of CQR, with 2% (5/255) of study patients on CQ with persistent parasitaemia on day 7 [
27] although 13% of treatment failures and subsequent reports CQR have been documented [
28-
30]. Indeed, data on the presence and prevalence of mutations in
pvmdr-1 and
pvcrt-o genes are limited in Ethiopia. The study was, therefore, initiated to determine the SNPs in the
pvmdr-1 and
pvcrt-o genes.
Discussion
CQ continues to be used for the treatment of
P. vivax infection in Ethiopia despite reports of CQR from various studies in the country [
28-
30,
32]. It is, therefore, important to investigate the prevalence of drug-resistance associated markers in
P. vivax clinical isolates in this country. In
P. falciparum, mutations in the
pfcrt and
pfmdr-1 genes have been linked to CQR but in
P. vivax the picture is still unclear regarding the possible relationship between the
pvcrt-o and
pvmdr-1 genes and CQR. However, the Y976F substitution in the
pvmdr-1 gene is thought to be involved in CQR in
P. vivax [
19] because the geometric mean 50% inhibitory concentration of CQ was shown to be significantly higher in
P. vivax isolates carrying the Y976F mutation than in isolates with the wild-type allele. On the other hand, the ubiquitous presence of Y976F in all patients presenting to a clinic in Papua, where CQ resistance
P. vivax is both at high and prevalent, precluded correlation with
ex vivo drug susceptibility to CQ [
10]. In the Thai isolates, the Y976F substitution was associated with a 1.7-fold higher IC50 to CQ [
10]. Unlike the Y976F mutation, Suwanarusk
et al. [
20] found the
pvmdr-1 F1076L mutation in all the isolates (wild type and mutants).
In the present study, 75% of the
P. vivax isolates had the Y976F mutation in
pvmdr-1. Sequencing results of the
pvmdr-1 fragment showed the presence of two non-synonymous mutations at positions 976 and 1076. The Y → F change at codon 976 (TAC → TTC) was observed in 26 (75%) of 28 isolates. The second F → L change (at codon 1076), which was due to a single mutation (TTT → CTT), was observed in 100% of isolates. Whether isolates carrying the
pvmdr-1 Y976F mutation responded to CQ treatments differently from those isolates with the wild-type sequence necessitates further
in vivo therapeutic efficacy study in Ethiopia, but reports from Indonesia and Thailand suggest this to be the case [
19]. The difference in the prevalence of
pvmdr-1 Y976F in areas where CQR
P. vivax prevails
versus CQ remain efficacious may indicate the correlation between CQR and sequence polymorphisms in
pvmdr-1. In Papua Indonesia, where CQR
P. vivax is present at high prevalence (>65%) and high level [
19], the
pvmdr-1 Y976F mutation was present in all patients presenting to a clinic. In contrast, the sequence polymorphism in
pvmdr-1 conferring Y976F was identified in only 25% of Thai isolates from an area where CQ remains efficacious. Ninety-six percent of Indonesian isolates (where clinical resistance to CQ prevails) had Y976F mutation, compared to 25% of Thai isolates where CQ sensitivity was almost uniform [
19]. The fact that that all parasites with the Y976F substitution in Ethiopia also carried the F1076L mutation, as originally described by Brega
et al. [
22] the F1076L mutation could be a background mutation that precedes the Y976F substitution and could potentially provide an early warning on emerging CQR.
In Ethiopia, CQR
P. vivax has been reported from various studies. Given the high prevalence of the Y976F mutations in
pvmdr-1 in Southeast Asia where CQR prevails [
19], the high prevalence of
pvmdr-1 Y976F mutations identified in this study may be associated with the CQ treatment failure reported in Ethiopia. But the exact role of this mutation needs to be determined by a combination of
in vitro and clinical observation studies in this country. The fact that all isolates carried the
pvmdr-1 F1076L mutation, substitution in this codon may be less involved in the modulation of
P. vivax susceptibility to CQ than the
pvmdr-1 Y976F mutation given that CQ is still effective and widely used in Ethiopia. Indeed, the presence of
pvmdr-1 F1076L mutation in all susceptible and mutant isolates challenged the role of
pvmdr-1 polymorphisms in modulating CQ responses in
P. vivax [
33]. On the other hand,
pvmdr-1 polymorphisms have been recently suggested to be associated with CQR in Southeast Asia [
19] unlike polymorphisms in
pvcrt-o that have not been associated with CQR in
P. vivax. The limitation of this study was that it did not determine drug resistance phenotype (either
in vivo or
in vitro) for the isolates undergoing molecular characterization at the
pvmdr-1 and
pvcrt-o genes. Indeed, withdrawal of a given drug is recommended when 10% of infections are not responding to treatment, although in practice, governments of poor countries leave it longer [
34]. The fact that CQ treatment failure reported earlier in Ethiopia did not exceed the level to withdrawal CQ, periodic assessment of the current status of CQR
P. vivax has great public health significance
.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LG collected the samples. LG, BE, AA, FNB and GS conceived the idea. LG and GS did molecular analysis and drafted the manuscript. BE, AA, FBN and GS critically reviewed the manuscript. All authors read and approved the final manuscript.