Background
Almost 40 % (approximately three billion) of the world’s population is presently at risk of contracting malaria. The disease causes almost 200 million clinical cases and around 600,000 deaths each year [
1,
2].
Plasmodium vivax is the most geographically widespread of the human malaria parasites, and a serious public health concern in South and Central America, Asia and Southwest Pacific [
3].
In Brazil, endemic regions are restricted to the Legal Amazon (comprising Acre, Amapá, Amazonas, part of Maranhão, Mato Grosso, Pará, Rondonia, Roraima and Tocantis States), a region that presently accounts for 99.6 % of the countrywide malaria burden [
4].
Plasmodium vivax is the predominant species, responsible for 84 % of the reported cases [
5], PNCM, SVS, MS, unpublished data 2015. Extra-Amazonian autochthonous cases account for only 0.04 % of all Brazilian total registered and correspond to the autochthonous malaria existing in the Atlantic Forest, located along the southeastern Atlantic Coast [
6].
Chloroquine resistance (CQR) is the main challenge for national malaria control programmes to control vivax malaria. The first cases of
P. vivax resistant to chloroquine (CQ) were described in Papua New Guinea [
7] and thereafter observed in Indonesia [
8], Oceania, Asian [
9,
10] and South American countries, including Brazil [
11,
12]. In Brazil, CQ treatment failures, presumably related to CQR, have been reported [
13]. The latest 28 day in vivo test conducted to assess the efficacy of standard supervised CQ therapy in 109 volunteers showed a proportion of 10.1 % of treatment failure (n = 11), despite an adequate absorption of CQ in these individuals on day 2 [
14].
Molecular markers can represent a valuable tool for monitoring introduction and spread of drug resistance. Contrarily to
Plasmodium falciparum, mutations at codons in the
pfcrt orthologue (
pvcg10) gene do not seem to mediate CQR in
P. vivax [
15]. On the other hand, the polymorphisms at codons Y976
F and F1076
L in the multidrug-resistant gene 1 (
pvmdr1) has been described as molecular marker associated to CQR [
16]. Indeed, in Thailand, Indonesia [
17] and Myanmar [
18], as well as in Mauritania [
19] and Cambodia [
20], it has been shown that 976
F mutants were associated with clinical resistance to CQ. In Nepal and India, where
P. vivax CQR has not been recorded, prevalence of the 976
F mutation is very low (5 %) [
21] or not detected [
22], while in India the presence of the F1076
L mutation was not associated to CQR. In addition, in Madagascar, despite 5 % of clinical failures more than 90 % of Y976
F mutant parasites were detected [
33].
These polymorphisms also seem to be relatively uncommon in Latin America, where
P. vivax CQR remains relatively infrequent [
23]. In Brazil, different conclusions were drawn: either mutations in
pvmdr1 were reported in CQ-sensitive
P. vivax parasites [
24‐
26] or not detected in resistant
P. vivax isolates [
25,
26], as well as
P. vivax CQR being associated with
pvmdr1 mutants only in patients with severe malaria [
27].
In view of these different epidemiological data, the nucleotide polymorphisms (SNPs) of pvmdr1 gene in successfully cured vivax malaria patients living in endemic (Amazonian) and non-endemic (Extra-Amazonian) Brazilian areas, were investigated in the present study.
Results
The pvmdr1 gene was successfully amplified and DNA sequenced in 49 isolates from the Amazon Region (Acre, Amazonas, Pará, and Rondonia) and the Extra-Amazonian State of Rio de Janeiro.
Globally, 34 (69 %) showed non-synonymous (958
M, 976
F and 1076
L) mutations. 958
M mutant alleles were the more frequent (25/34; 73 %) while 976
F (5/34; 15 %) and 1076
L (10/34; 12 %) were detected at lower frequencies (Table
1). Single mutation was observed in 24 isolates (70.5 %, 24/34), while double mutations were recorded in ten (29.5 %, 10/34)
P. vivax samples. In the isolates presenting single mutant genotype, the
MYF profile was predominant (19/55 %) contrasting with the
FF, which was found in only five isolates (Table
2).
Table 1
Frequency of 958M, 976F and 1076L mutants in pvmdr1 gene among 49 Brazilian P. vivax isolates
No mutation | 15 (31) |
958M
| 25 (51) |
976F
| 5 (10) |
1076L
| 4 (8) |
Table 2
Proportion of the 4 alleles observed among 49 Brazilian P. vivax isolates, according to the sampling location
Wt Sal1 type | 15 (31) | 4 | 5 | 4 | 2 | 0 |
Single FF | 5 (15) | 1 | 4 | 0 | 0 | 0 |
Double FL
| 4 (12) | 2 | 0 | 2 | 0 | 0 |
Single MYF | 19 (55) | 10 | 2 | 5 | 2 | 0 |
Double MYL
| 6 (18) | 0 | 0 | 1 | 1 | 4 |
| | 13 (76 %) | 6 (54 %) | 8 (67 %) | 3 (60 %) | 4 (100 %) |
The
pvmdr1 wild-type allele was prevalent in Pará (54 %) followed by Acre (40 %), Amazonas (33 %), and Rondonia (24 %) states without statistically significant difference in proportion (p > 0.05). Single mutants were observed only in isolates from the Brazilian Amazon: the
MFY allele was prevalent in Rondonia (77 %), followed by Amazonas (62 %) and Acre (67 %) (p > 0.05), although in Pará the
FF single mutant was more frequent (66 %) than the
MYF (p > 0.05). However, when double mutants were investigated, samples presenting
FL (12 %) and
MY
L (20 %) in both Amazonian and Extra Amazonian States, were identified. Irrespective to the Brazilian State
FL double mutant was the less frequent (Table
2) (p > 0.05). Interestingly, all isolates from the Extra Amazon (Rio de Janeiro State) showed double mutant genotype (
MY
L) contrasting with those from the Amazon Basin (6 %) (p = 0.01), where most of the isolates came from.
Discussion
CQ and PQ remain the drugs of choice to treat vivax malaria, but recent studies have reported
P. vivax cases of resistance to CQ in different regions of the world [
9,
31], including Brazil [
11‐
13]. Therefore, monitoring the efficacy of CQ in the treatment of vivax malaria is essential for early warning systems to promote drug policies.
To circumvent the limitations of in vivo and in vitro studies and to assess chemo-resistance, identification of mutations in target genes has been proposed, such as those in the
pvmdr1 gene at codons 976 and 1076, as well as the increased expression of
pvcrto transcripts [
12,
32]. Similar to previous studies performed with samples from western Brazilian Amazon [
24,
25], in this work the T958
M mutant was found to be the more frequent in the Brazilian Amazon and even in isolates from the Extra Amazonian regions. Additionally, in Madagascar [
33], Nepal [
21] and Thailand [
34], most of the samples presented mutations at 958 position, although all isolates have been obtained from individuals with successful response to CQ therapy. No later than November 2015, Schousbe and colleagues [
35] reported a high prevalence of 958
M (97.6 %) among
P. vivax samples from six different geographical sites, suggesting that this allelic variant is most likely not associated with CQR and could be an allele characteristic of Asia and Africa isolates. The present data reinforce the lack of association of 958
M with CQR, but are not in agreement with Asian and African geographical characteristic of this allele, since this allele was present in 51 % of the Brazilian (South American) samples.
Previous studies seemed to indicate that both SNP and amplification of
pvmdr1 are associated with variation in in vitro CQ susceptibility of
P. vivax [
17,
32]. It has been shown that the geometric mean of the CQ inhibitory concentration 50 % (IC50) was significantly higher in isolates carrying the Y976
F mutation when compared to wild-type isolates in samples from Indonesia and Thailand [
17]. However, the clear association between the clinical outcome following a three-day CQ treatment and non-synonymous mutations in this gene has never been demonstrated elsewhere. In fact, the single 976
F mutant was not very common worldwide (7.4 %) [
22], and the
FF double mutant genotype was detected only in endemic regions of three countries: Brazil [
36,
37], Honduras [
38] and Papua New Guinea [
39]. In the samples herein analysed, no significant difference was observed between the presence of double 976
F/1076
L mutant (12 %) and single 976
F mutant (15 %) and no single 1076
L mutant was noted. These findings suggest that polymorphisms at codons 976 and 1076 may not be strong indicators of CQ resistance since all
P. vivax isolates were obtained from patients with good response to CQ therapy. In addition, 976
F and 1076
L mutants were also detected in
P. vivax isolates in several countries in Africa and in South America from patients with no history of CQ recrudescence [
23]. Probably, these mutations might have been introduced in these countries from Asia where these mutations are prevalent [
23]. Interestingly, the 976
F mutation in
P. vivax isolates from Extra-Amazonian were not detected in areas where autochthonous malaria cases from Brazilian Atlantic Forest can occur. Thus, it seems that 976
F mutations are more associated to geographical characteristics than to CQR.
Concerning codon 958, only samples from the Amazon Basin showed the
MYF single mutant genotype, and double
MY
L mutants were observed in Amazonas and Acre State isolates. On the other hand, all isolates from the Extra Amazon State of Rio de Janeiro had the double mutant 958+1076 (
MY
L) genotype and these samples were wild type for codon 976. Once again, the heterogeneity in these
P. vivax populations could reflect the genetic diversity rather than an association with CQR in endemic areas with different endemic profiles [
6].
Considering that
pvmdr1 mutations should not be expected in CQ-sensitive parasites and that PQ efficacy is highly dependent on concurrent administration of a blood schizontocidal agent [
40] and thus PQ could not circumvent CQR, the present findings seem to indicate that the
pvmdr1 gene is not a reliable marker of CQR.
Authors’ contributions
MFFC carried out the study and the manuscript. LRG performed and analysis DNA sequencing and statistical analysis and drafted the manuscript. DM and CTDR participated in the discussions and reviewed the final manuscript. NKAO, SRFL and AL performed DNA extraction and PCRs. APC and PB recruited the patients. All authors read and approved the final manuscript.