Background
Malaria, one of the most prevalent parasitic diseases in the world, still causes high morbidity and death, mainly in
Plasmodium falciparum-infected, non-treated patients[
1].
Plasmodium vivax causes intense morbidity and contributes to significant political, social and economic instability in developing countries of Latin America and Asia[
2,
3]. CQ is the drug of choice to treat
vivax malaria in endemic areas of Brazil and primaquine (PQ) is used to avoid late malaria relapses[
3]. The recommended dose for adults is 1500 mg of CQ (daily for three days) and 210 mg of PQ (daily for seven days)[
4].
Plasmodium vivax resistance is now widespread and has rendered CQ ineffective in parts of Indonesia and Papua New Guinea[
5‐
7]. Low levels of resistance have also been reported in Myanmar, South Korea, Vietnam, India, Turkey, Ethiopia, and in regions of Southern Africa and South America[
3,
8,
9]. The occurrence of severe
vivax malaria and patient’s deaths has been reported in Brazil[
10‐
12] raising the possibility of an association between malaria severity and drug resistance[
13]. In areas of CQ resistance, treatment of uncomplicated
P. falciparum malaria is carried out with artemisinin-based combination therapy (ACT)[
3]. Drugs that complement ACT include lumefantrine, amodiaquine, AQ, MQ, sulphadoxine-pyrimethamine and antibiotics. In Brazil, the first option for
falciparum malaria treatment is the combination of artemether (480 mg daily for four days) and lumefantrine (2880 mg daily for four days). PQ (45 mg) is administrated on day one to avoid malaria transmission. These doses are recommended for adults with 50 Kg weight or more[
4]. A reduced susceptibility to artemisinin derivatives has been described in
P. falciparum-treated patients[
14,
15].
Increasing evidence of a lower
P. vivax susceptibility to CQ in malaria-endemic areas[
16] includes the state of Amazonas[
17] and is believed to be associated with malaria’s clinical severity[
18].
Molecular markers associated with CQ resistance are non-synonymous mutations in the drug/metabolite transporter gene
pfcrt (C72S, K76T) and in the multidrug resistance protein 1 gene
pfmdr1 (N86Y; Y184F; S1034C; N1042D; D1246Y), described in
P. falciparum[
19‐
21]. One mutation of the
multidrug resistance gene 1 (Y976F) of
P. vivax is also associated with parasite susceptibility to CQ[
8]. A non-synonymous mutation of the
pvdhps gene at codon 382 (S382C) was recently associated with
in vitro susceptibility to CQ[
18]. The present study aimed to examine the phenotypic and genotypic chemoresistance profile of
P. falciparum and
P. vivax to commonly used anti-malarial drugs in a Brazilian malaria-endemic area in the Amazon Region.
Discussion
Due to the strong impact of chemo-resistance among the malaria parasites to most drugs used in the control of the disease, monitoring the development of resistant phenotypes and genotyping are priorities wherever endemic malaria is present. The
in vitro methods used to examine anti-malarial drug sensitivity provide a profile of
Plasmodium sensitivity to a variety of drugs, simultaneously assayed. However, a lower
in vitro sensitivity of a parasite isolate does not imply drug-resistance
in vivo, as other factors can interfere, which are not determined by
in vitro tests. The
ex vivo tests provide an outline of resistant-circulating phenotypes for each tested drug, provided that an adequate number of patients are examined in a given area. This was not the case for
P. falciparum in our study: only nine patients were evaluated, reflecting the reduced transmission of this parasite species in Brazil[
30]. Determining parasite genotype, performed in parallel, provides further information since mutations in the
pfcrt gene alter the CQ flux and/or reduce drug efficacy[
31]; and provides data for policy makers to decide the best drug to be prescribed in that area. Ideally, monitoring anti-malarial chemo-resistance must be continuous since development and spreading of resistance are dynamic events, changing with time and according to human interventions and other factors such as population migration[
32].
The antiplasmodial activity and the molecular profile of resistance by anti-malarial standards like CQ, ART and MQ, confirms P. falciparum resistance to CQ only, as demonstrated in ex-vivo tests against P. vivax and P. falciparum isolates from patients with naturally acquired malaria in the state of Rondônia in the Brazilian Western Amazon, which is substantiated by sequencing of genes related to resistance to anti-malarial drugs.
There is evidence of
P. vivax resistance to CQ in the state of Amazonas, specifically in the city of Manaus where an increase in the proportion of
P. vivax malaria parallels an increase of unusual clinical complications related to this species[
33]. These authors used an
in vivo test to assess the efficacy of standard supervised CQ therapy. Among 109 volunteers with
P. vivax who completed the
in vivo test, 19 had positive blood smears within the 28-days follow-up (one on day 14, three on day 21, and 15 on day 28). All were then required to undergo an alternative therapy with MQ. In another study performed by the same group, a lower
P. vivax susceptibility to CQ through the attainment of IC
50 by ELISA assay or using traditional methods[
18,
34] was examined; they demonstrated that drug-resistance was related to the presence of non-synonymous mutation at
pvdhps,
pvcrt and
pvmdr1.
All
P. vivax isolates presently studied in the Brazilian Western Amazon were sensitive to CQ in
ex vivo assays. Although the threshold of IC
50 to define a sample as resistant to CQ is not well established for
P. vivax, it has been proposed that the same threshold used for
P. falciparum should be used, with 100 nM threshold of CQ[
35].
The IC
50 for ART and MQ in
P. vivax were also examined and they were higher when compared with the values found in
P. falciparum. It can be due to differences in the stage-specific activities of CQ, MQ and AQ in
P. vivax, demonstrated here and by Marfut
et al.[
28]. It may be interesting to compare the susceptibility of
P. vivax in strains from other regions of the world. This issue remains to be further studied using strains from different places.
Only one point mutation for CQ was studied for
P. vivax in the
pvmdr1 gene codon 976, but other genes were associated with CQ resistance in the Brazilian Amazon, e.g., in the pvdhps gene in codon 382 (S → C)[
18].
Considering that the severity of
P. vivax malaria in the state of Amazonas has been attributed to CQ resistance and to the increased levels of
pvmdr1 and
pvcrt-
o compared to the levels expressed by parasites from patients with mild symptoms[
36], these genes copy number could also be evaluated. In addition, the
mdr1 copy number is strongly associated with recrudescence after artesunate-mefloquine administration, and could be used as a surveillance tool for artesunate-mefloquine resistance, as reported in patients in Cambodia[
37,
38].
In conclusion, in West Amazon, most P. falciparum isolates were CQ resistance, a data confirmed by parasite genotyping. No mutations were found for P. vivax in the region supporting the lower prevalence of this strain in Brazil.
Acknowledgements
We are grateful: to Prof. Luiz Hidelbrando Pereira da Silva, FIOCRUZ-RO, for facilities in the endemic area; Luiz Shozo Ozaki, from Department of Microbiology and Immunology, Virginia Commonwealth- University for suggestions; Karina de Sousa Paula for help with parasitaemia evaluation and Glaecia Nascimento with some in vitro tests ; for financial support from the Conselho Nacional de Pesquisas e Desenvolvimento- CNPq - Edital MCT/CNPq n° 09/2009 – PRONEX Rede Malária (305314/2009-2) ; MCTI/CNPq/MS-SCTIE-Decit N° 40/2012- Doenças Negligenciadas (404455/2012-3 ) and to CNPQ for fellowships to AUK, ACCA, and NSA; for financial support from Fundação de Amparo a Pesquisa de Minas Gerais - FAPEMIG (Universal, CBB, APQ-01692-11).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ACCA and NSA carried out the molecular studies. DBP was the MD who interviewed and treated the patients in the endemic area and ACCA performed the ex vivo and diagnostic exams tests. AUK and LDM conceived the studies, participated in the experimental design and were responsible for the biological tests. AUK was the project leader. All authors read and approved the final manuscript.