**3.5 Artemisinin-based combination therapy (ACTs)**

Artemisinin is a sesquiterpene lactone, containing the peroxide group, extracted and isolated from the leaves of *Artemisia annua.* by Chinese scientists in 1972 [75]. The drug and its derivatives play a role in killing *Plasmodium falciparum* by inhibiting the activity of phosphatidylinositol-3-kinase (PfPI3K) [76]. Initially, it was used as monotherapy to treat uncomplicated malaria but due to high recrudescence rate, a combination therapy was advised. Artemisinin-based Combination Therapy (ACTs), particularly artesunate-mefloquine, was introduced in Thailand during the early 1990s [77]. Since 2001, artemisinin (ART) combination therapy (ACT) has been recommended as the first-line treatment in the national treatment guidelines of most malaria endemic countries and have played an important role in reducing global malaria-associated mortality and morbidity [78].

Resistance to artemisinin was first detected in the Greater Mekong Subregion (GMS) region in 2008 [79]. Since then, ART resistance has spread and/or emerged in other areas of the GMS [80–83]. Exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, and the availability of substandard artemisinin, have probably been the main driving force in the selection of the resistant phenotype in the region. ART resistance is defined as the parasite clearance half-life of >5 h or presence of parasites in patients 3 days after treatment but has been more challenging to define, mostly because artemisinin act potently and rapidly clear parasites from the bloodstream by a unique mechanism involving the spleen [84, 85].


Currently, several drugs have been recommended (**Table 1**) for the treatment of severe and uncomplicated vivax malaria [34, 63, 86–90] and WHO is considering

### **Table 1.**

*Antimalarial drugs for the treatment of* Plasmodium vivax *malaria.*

the use of artesunate-pyronaridine, in areas where other ACTs are failing. In the absence of resistance, all six partner drugs would be highly efficacious as monotherapies at the dose used in the ACTs. Two injectable treatments, artesunate and artemether, are recommended for the treatment of severe malaria and should be followed by an ACTs once the patient can tolerate oral therapy [34].

Studies to monitor the efficacy of the ACTs on both *P. falciparum* and *P. vivax* have been conducted since the introduction of this drug in 2001. Evidence to date revealed that resistance of *P. falciparum* to artemisinin so far is not only confined to the Greater Mekong Subregion (GMS). Recent evidence indicated that *P. falciparum* isolates carrying the kelch13 C580Y mutation has been found in Papua New Guinea [91]. The finding is quite worrying as both PNG and Indonesia shared terrestrial border and the mutations may have spread to Indonesia. Therapeutic efficacy studies (TES) conducted during the period of 2009–2018 in various sites in Indonesia, including the Indonesia-PNG border documented no cases of either *P. falciparum and P. vivax* resistance nor treatment failure associated with artemisinin in Indonesia [92–96]. Nevertheless, recurrent parasite at late observation day was reported and this recurrence certainly nothing to do with artemisinin but rather with partner drug.

## **4. Molecular basis of** *P. vivax* **resistance to antimalarial drugs**

The advent of molecular and cellular parasitology within the last 4 decades have brought along a lot of substantial innovations in the antimalarial drug testing platforms, molecular assays to phenotype as well as genotype the malarial parasite, although it mainly attributed to *P. falciparum*. In *P. vivax*, attempts to develop a repeatable *in vitro* drug resistance test continue to elude us, although certain progress has been achieved [23]. As a consequence, progress on the studies to elucidate the molecular basis of the *P. vivax* resistance to antimalarial drugs, particularly chloroquine and artemisinin is lagged far behind *P. falciparum*. While studies on molecular basis of resistance to chloroquine and artemisinin successfully identified candidate gene (s) through a clear phenotypic and genotypic assay, similar progress in *P. vivax* could not be achieved. The molecular basis of *Plasmodium* resistance to antifolates and sulpha drugs had been well described [97–99]. This evidence also applies to *P. vivax*, and the underlying genetic polymorphisms in dhfr and dhps genes, conferring resistance to antifolates and sulpha drugs, respectively. Likewise, resistance to atovaquone, a partner drug of proguanil has been associated genetic polymorphisms in the c*ytb* gene of the malarial parasite [73, 74].

Resistance to chloroquine, has long been subject for research in many laboratories around the globe. A yet unclear mechanism of action of this compound making it more attractive for elucidation using molecular tool. Initially the role of *Plasmodium falciparum* multidrug resistance 1 (pfmdr1), homologous to the mammalian multiple drug resistance (MDR) gene were incriminated [100–102]. The product of thePfmdr1gene, P-glycoprotein homolog 1 *(Pgh1)* has been localized to the membrane of the digestive vacuole of mature blood stage parasites. This model predicted that thePfmdr1gene would be amplified and/or over expressed in CQ-resistant isolates. Further study, however identified different mechanism for chloroquine resistance but support for the role of this pfmdr1 in other antimalarials such as mefloquine, halofantrine and quinine [103–106]. Chloroquine-resistant parasites pump chloroquine out at 40 times the rate of chloroquine-sensitive parasites; the pump is coded by the *P. falciparum* chloroquine resistance transporter (*PfCRT*) gene [107, 108]. The natural function of the chloroquine pump is to transport peptides: mutations to the pump that allow it to pump chloroquine out impairs its function as a peptide pump and comes at a cost to the parasite, making it less fit.

#### Plasmodium vivax *and Drug Resistance DOI: http://dx.doi.org/10.5772/intechopen.97320*

Several genetic polymorphisms at the PfCRT gene have been associated with resistance to chloroquine in a wide geographic regions of malaria endemic areas [108]. Nevertheless, attempts to prove this finding in CRPV still fail, primarily because the technical difficulties in proving the resistant phenotype in *P. vivax*. Molecular analysis of the *P. vivax* isolates that have been phenotypically determined to be resistant in a rigorous *in vivo* and limited in vitro tests did not reveal any polymorphisms in the PvCRT gene as that of PfCRT. Instead, amplification of Pvmdr1 and several SNPs in the pvmdr1 was found to associate with CRPV [62]. Recent evidence found out that increases in PvCRT copy number associated with the *P. vivax* resistance to chloroquine [109–113].

The molecular basis for artemisinin resistance in the malarial parasite have also been described recently. Since mammalian kelch proteins can detect oxidants and other stressors, mutations in K13-propeller were reasonably implicated in mediating resistance to artemisinin and have been proposed as molecular marker [114–117]. Subsequent studies provided a more detail biochemical impact of the *PfKelch13* mutations on the decreased abundance of PfKelch13 protein, decreased haemoglobin digestion, and enhanced glutathione production [118]. However, the finding on the interaction of dihydroartemisinin with phosphatidylinositol-3-phosphate kinase, and that elevated phosphatidyl-inositol-3 phosphate can be associated with resistance in the absence of PfK13 mutations suggested for other mechanism [119]. In line with this evidence, Tyagi *et al* [120] raised a clear-cut artemisinin resistant isolates of *P. falciparum* following artesunate drug pressure in humanised mouse and the molecular analyses of the ART-resistant isolates revealed no mutations in PfK13 gene. Instead, an obvious selective pressure on RAD5 gene. Interestingly, the ART-resistant isolates also exhibited concomitant resistance to quinine, a second line drug used for treating severe malaria cases. The association between mutations in RAD5 gene and the resistance to artemisinin require further confirmation through either reverse genetics or genetic gross in mosquito.

Resistance of *P. vivax* to artemisinin so far has never been reported in areas where ACTs have long been used as first line drug for *P. vivax* malaria in South and Southeast Asia and the Pacific islands to replace chloroquine. This evidence, however, has to be carefully considered as *P. vivax* perhaps has long experienced with artemisinin pressure as that of *P. falciparum*, particularly in the GMS region where both species are distributed in sympatry and undetectable mixed species infection are common [121]. In support of this assumption, molecular analysis of *P. vivax* isolate from the GMS region revealed a high diversity and *ex vivo* analysis indicate reduced sensitivity to chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin [122, 123]. In this context, regular monitoring of the antimalarial treatment as well as genomic surveillance of the PvK12 gene, orthologues of the PfK13, in *P. vivax* and other relevant gene (s) should be conducted to monitor the emergence of artemisinin-resistant *P. vivax* and to contain the spread of the resistance to other regions [124, 125].
