**4. Resistance facilitates the transmission potential**

mefloquine resistance *in vitro* and hence with *Pfmdr-1* copy number amplification [84–86]. Later findings have shown that the amplification of *Plasmepsin-2* gene (probably *Plasmepsin-3* as well) on chromosome 14 is significantly associated with piperaquine resistance *in vitro* as well as *in vivo* [26, 87]. Worryingly, a recent study in Cambodia has demonstrated the presence of parasite isolates with amplification of both *Pfmdr-1* and *plasmepsin-2* genes [20]. This finding indicates that the parasite has successfully adapted to acquire concomitant mutations

Artemisinins are thought to be inhibitor of *P. falciparum* phosphatidylinositol-3-kinase (*PfPI3K*), which phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate involved in cell survival pathways. Hence, inhibition of *PfPI3K* activity causes a reduction in PI3P level, which subsequently leads to parasite death. After the introduction of artemisinins in the 1990s, the unanimous opinion by the experts was that resistance was unlikely to emerge because of inherent pharmacokinetic-dynamic property of the molecule. However, artemisinins were not everlasting drugs and the artemisinin resistance did emerge

There are two main proposed pathways for artemisinin resistance with the involvement of Kelch (*K-13*) mutations, that is, a cell survival signalling pathway with *PfPI3K* and an unfolded

In Kelch (propeller) mutant alleles, the mutations may alter the topology of the Kelch protein probably by modification of surface charges that disrupt interactions with other enzymes such as *PfPI3K* [89]. This leads to a reduced amount of ubiquitination, as well as degradation of *PfPI3K* associated with increased levels of both the enzyme *PfPI3K* and the substrate *PI3P* [90, 91]. The *PI3P* facilitating the host remodelling is present in the apicoplast and food vacuole and contributes to the cell survival pathways either through redox, transcriptional or DNA repair [90–94]. All of which have been implicated in artemisinin resistance [90, 95–98]. Possible mechanisms proposed by transcriptomic study [99] is through upregulation of genes involved in the UPR pathway (especially two putative chaperonin complexes, *Plasmodium* reactive oxidative stress complex/PROSC and TCP-1 ring complex/TRiC) which enhances the capacity of parasites to quickly repair or degrade proteins or other cellular components. (The UPR pathway is usually damaged by brief artemisinin exposures in patients, but these genes are upregulated in artemisinin resistant parasites) and/or downregulation of genes involved in

DNA replication, which is associated with developmental arrest and dormancy [100].

The role of Kelch non-propeller mutation (before the amino acid position 441) is still unclear. Some SNPs like E252Q emerged earlier along the Thai-Myanmar border and associated with reduced efficacy of ACT [25] but are being taken over by the propeller SNPs particularly C580Y [101]. All these findings indicate that artemisinin resistance is likely to be multi-locus and that other genetic changes, such as P623T polymorphism in *Kelch-10* gene [15] and background mutations (arps10-apicoplast ribosomal protein S10, *Pfmdr-2*, ferredoxin, *Pfcrt* [17], etc.), are providing compensatory fitness for K-13 mutant parasites or perhaps conferring partner drug resistance.

related to resistance to these two different antimalarial partner drugs [20].

**3.9. Artemisinin resistance**

22 Towards Malaria Elimination - A Leap Forward

protein response pathway (*UPR*) [88].

in 2008 [6].

For the newly selected resistant parasites to be propagated, the recrudescent infection is essential [102]. The threshold for successful transmission of malaria is around six viable gametocytes in one blood meal [103]. Post-treatment gametocytaemia is a composite of ongoing gametocytogenesis despite treatment (especially with ineffective drug) and the release of sequestered gametocytes, which is enhanced by drug-induced stress [104]. If the malaria infection is treated with partially effective drugs, post-treatment gametocytaemia is more likely. This was clearly shown for drugs such as CHQ and SP [105] as evidenced in patients with slower parasite clearance after artesunate treatment [106]. Moreover, mutant isolates were also related to pre- and post-treatment gametocytaemia [107–110] and hence possess transmission advantage (**Figure 3**).

**Figure 3.** Postulated flow chart of emergence/spread of drug resistance (copyright permission from Prof Francois Nosten).
