Current opinion on an emergence of drug resistant strains of Plasmodium falciparum through genetic alterations

The human malarial parasite Plasmodium falciparum is one of the world's most devastating pathogen. Its capability to regulate its genes under various stages of its life cycle as well as under unfavourable environmental conditions has led to the development of vaccine resistant strains. Similarly, under drug pressure it develops mutations in the target genes. These mutations confer mid and high-level resistance to the antimalarial drugs. Increasing a resistance of malaria parasites to conventional antimalarial drugs is an important factor contributing to the persistence of the disease as a major health threat. This article reviews current knowledge of stage specific malarial targets, antimalarial drugs and the mutations that have led to the emergence of resistant strains.


Life cycle in brief
Life cycle of malarial parasite can be dividing into three stages (1) Exoerythrocytic stage; (2) Intraerythrocytic stage and (3) Sporogonic stage. Among these first two stages are completed in humans and next one is in mosquitoes. Infection of Plasmodium is initiated as porozoites (Exoerythrocytic Stage), they are injected into peripheral blood stream through the saliva of a feeding mosquito. These sporozoites are carried to the liver and invade hepatocytes through the circulatory system. After that it forms merozoites and enters into the blood stream and infects the erythrocytes (Intraerythrocytic), which is a pathogenic stage of malaria. Digestion of Hemoglobin is the major catabolic process of Plasmodium sps in merozoitic stage [2], mature merozoites can form tropozoites and these can form gamatocytes or again invade into fresh erythrocytes. Plasmodium completes its life cycle in humans and enters into mosquitoes gut by blood meal for sexual cycle which is sporogonic and enters into salivary gland, ready to infect another human being.

Stage specific protein targets
The protein targets of P. falciparum from sporozoites (the infectious form injected by the mosquito), merozoites (the invasive stage of the erythrocytes), trophozoites (the form multiplying in erythrocytes), and gametocytes (sexual stages) of the human malaria parasite are tabulated Table 1 (see  supplementary material). Genes involved in virulence and antigenic variation (for example, var, vir and rif genes) in Plasmodium falciparum are located in the subtelomeric regions of the chromosomes [3, 4].

Anti malarial drug-target-resistance
A limited number of chemotherapeutic agents for the treatment of malaria are available but the growing problem of drug resistance makes the treatment of malaria increasingly difficult [5]. Its control is hampered by increasing resistance of malaria to the available drugs [6]. Some of the anti malarial drugs available in market are mainly Quinine derivatives, antifolates and arylalcohols. Quinoline antimalarials, i.e. an active ingredient of Cinchona bark, which was used as antimalarial drug since seventeenth century [7]. Quinine requires thrice daily administration over 7 days; this can result in poor compliance. The dependence on raw material for its extraction and the availability of structural elucidation techniques leads to the development of fully synthetic chloroquine and later amodiaquine [8], which are inexpensive and more effective for a long time. Amodiaquine use has been limited since the mid 1980s, as it shows a high degree of efficacy [9]. The other antimalarial drugs structurally similar to chloroquine are Mefloquine and halofantrine, but resistance to these drugs was rapidly developed than chloroquine [10]. Additional limitations to these drugs usage of these drugs are occasionally neuropsychiatric disturbances for mefloquine and halofantrine, it can cause harm to the people with a history of heart disease [9].
Artemisinin the active constituent of the Chinese herb (Artemisia annua), which was used traditionally for treating several types of fevers, have been used increasingly over the past two decades [9, 11]. The derivatives of this include artemether, arteether and artesunate, they are metabolized to dihydroartemisinin the main active agent in the body to treat malaria. These drugs can act against gametocytes, the sexual stages of the parasite that infect mosquitoes. Lumefantrine is an aryl alcohol similar to mefloquine and halofantrine, and neurotoxicity was not reported in animal toxicology studies [12]. Antifolates are not derived from plants and synthesized through knowledge of cell biology and synthetic medicinal chemistry. Fully reduced folate cofactors are essential for nucleotide biosynthesis and amino-acid metabolism [13]. Antifolate used to treat malaria is 2, 4-diaminopyrimidine (Pyrimethamine) and Sulphadoxine, (sulphonamide) an inhibitor of dihydrofolate reductase (DHFR), and dihydroopteroate synthase (DHPS) respectively. These enzymes are the part of folate pathway. Atovaquone/proguanil is another combination similar to that of sulphadoxine/ pyrimethamine [13]. The hydroxynaphthoquinone atovaquone interferes with mitochondrial electron transport. Common antibiotics acting against bacterial protein synthesis such as tetracycline, doxycycline and clindamycin inhibit parasite growth and are being used increasingly in combination with other antimalarial treatments to augment their activity [9].

Resistance
Chloroquine, which served as a cheap and reliable drug for decades, is becoming ineffective against Plasmodium falciparum in most tropical areas. Thus, there is a need to develop novel antimalarial agents that are effective against drug-resistant malarial parasites [14]. Drug development efforts generally aim for structurally unrelated compounds that work through new, independent mechanisms of action to existing antimalarial agents. Chloroquine and other derivatives such as amodiaquine, mefloquine, quinidine, quinacrine are potent inhibitors of heme polymerization. Target for the Chloroquine is the lysosome or food vacuole of the parasite; it interferes in the detoxification of haemoglobin. Pfmdr1 gene located on chromosome 5 of P. falciparum, encodes a P glycoprotein of 160 kDa which plays a role in drug efflux [15]. Pfmdr1 gene at codons 86, 184, 1034, 1042 and 1246 have been proposed to be associated with chloroquine resistance [16]. N86Y mutation in pfmdr1 seems to be playing some role in the chloroquine resistance but it is unclear. pfcrt is located on chromosome 7 and encodes for a protein named as P. falciparum chloroquine resistance transporter protein (PfCRT), this 36 kb membrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action K76T mutation in pfcrt has been found in all the chloroquine resistant parasites. Besides K76T mutation in pfcrt, mutations at codon 72, 74, 75, 97, 220, 271, 326, 356 and 371 have also been found to be associated with chloroquine resistance [17]. Concomitantly associated with increased sensitivity to mefloquine and halofantrine [18-38] because of the 1042D and 1034C/1042D/1246Y alleles are associated with increased resistance to quinine and increased sensitivity to mefloquine and artemisinin drugs. The increased copy number of the Pfmdr1 gene in plasmodium falciparum leads to treatment failure with mefloquine [19,20].
After the failure of chloroquine, Sulphadoxine-pyrimethamine is used as a second line of drug to treat uncomplicated chloroquine resistant falciparum malaria cases. Use of SP is limited for pregnant women during the early trimester [21,22]. Sulphadoxine and pyrimethamine inhibit the enzymes dihydropteroate synthase and dihydrofolate reductase, respectively, involved in folate pathway. The mutation at codon 108 of P. falciparum DHFR S108N reduces the sensitivity of the pyrimethamine. The parasite isolates which showed pyrimethamine resistance, were found to contain this mutation and other mutations at codons 51 (N51I), 59 (C59R) and 164 (I164L)] were associated with S108N mutation. Majority of Indian isolates were found to contain double DHFR mutations (C59R+ S108N) [24]. Most of the mutation in dihydropteroate synthase are at codons 436 (Ser to Ala/Phe), 437 (Ala to Gly), 540 (Lys to Glu), 581 (Ala to Gly) and 613 (Ala to Ser/ Thr). A437G is the key point mutation in DHPS; it is similar to S108N mutation in DHFR. A single DHFR mutation or double DHFR mutations alone will not cause SP treatment failure but that double DHFR mutations plus a single DHPS mutation or triple DHFR mutations alone can cause higher level of SP resistance [24,25]. Artemisinin was another antimalarial drug adopted by several countries; it inhibits the uptake of host hemoglobin. P. falciparum degrades host hemoglobin in an acidic digestive vacuole in a mid ring stage and reaches a peak in the mid trophozoite stage [17]. Artemisinin distributes like BODIPYthapsigargin to membranous structures in the cytoplasm of parasites, and does not localize to the parasite food vacuole [26]. Artemisinin resistance would be calamitous for global malaria control. Artemisinin resistance is observed because of the polymorphisms in the Plasmodium falciparum merozoite genes encoding the surface proteins of (MSP-1 and MSP-2) and the glutamate-rich protein (GLURP). The point mutations in the PfMDR1 gene at positions N86Y, S1034C, N1042D, and D1246Y, but the mutant codon Y184F was found to be responsible for resistance againt Artemisinin. Sporadic point mutations noted in the sequence of PfSERCA gene revealed that mutations at I89T, N465S, and E847K are also seen in several field isolates. The PfSERCA L263E and S769N polymorphisms, proposed to confer artemisinin resistance, but these two mutations were not detected in fields [27] Table 2 (see supplementary material).
Amodiaquine, artemisinin-based combination therapy (ACT), introduced in 2001has the World Health Organization recommended ACTs as the first-line treatment for uncomplicated malaria. The mode of action of the combination therapy appears to be similar, and known to be concentrated in the parasite's lysosome like Chloroquine(CQ) [28]. Most countries have now adapted to either Artemether Lumefantrine (AL) or ArteSunate (AS) and AmodiaQuine (AQ) as their firstline ACT, AQ is a 4-aminoquinoline related to chloroquine (CQ), and Although CQ resistance is now widespread across the world, the efficacy of AQ is variable in treatment trials [29]. A mutation from 76K-T of the P. falciparum chloroquine resistance transporter (Pfcrt) gene is associated with resistance to both CQ and AQ [30]. Amplification of Pfmdr1gene modulate susceptibility to the artemether-lumefantrine ACT, leading to poor treatment response Drugs that are potent inhibitors of heme polymerization, such as amodiaquine, mefloquine, quinidine, quinacrine, and chloroquine, competed well, although quinine competed to a lesser extent, Mefloquine is a lipophilic quinoline alcohol which is in wide clinical use. Like chloroquine and quinidine, it also binds to heme polymer in a heme-dependent fashion and diminishes binding of quinolines associates with hemozoin. Mefloquine and a protease inhibitor blocks heme release in the food vacuole [31]. Chloroquine accumulates in food vacuole and is bound to free heme and results in the inhibition of formation of hemozoin. Over expression of pfmdr1 related to the mafloquine resistance because it is also sharing same mechanism as other quinine derivatives [32]. Polymorphisms in the P. falciparum multidrug-resistant 1 (Pfmdr1) gene have concluded that its protein product, Pgh1, plays a role in modulating levels of resistance to several structurally unrelated drugs [18,38]. All resistant parasites had the K76T mutation in the PfCRT gene. Some antimalarials, including artemisinin, are reported to inhibit hemoglobin uptake [34], but it is not clear if this is a direct effect, or a consequence of parasite killing. Artemisinin is highly potent against hemoglobin-digesting stages of malaria parasites [34, 37] than other hemoglobindegrading drugs, but it is much less potent against other pathogens [35]. Falcipain-2 is the only cysteine protease hemoglobinase expressed early in erythrocytic parasite development, and knockout parasites have markedly diminished hemoglobinase activity in the early trophozoite stage [36]. These parasites exhibit a swollen vacuole phenotype, consistent with defective haemoglobin digestion, but the deletion is not lethal, apparently due to rescue by the expression of falcipain-2 and falcipain-3 beginning at the mid trophozoite stage [36].

Conclusion:
Antimalarial drugs play very important role in controlling of malaria at individual as well as at epidemiological level. Careful treatment can slow down the development of resistance against the available drugs, mostly due to regular usage leads to development of mutations in the target gene. It is advisable to check the efficacy of the drug at regular intervals so as to take a policy decision in advance on its continued usage in the field. Molecular surveillance can give an advanced indication that a particular drug is going to lose its efficacy in near future. However, molecular markers for several antimalarial drugs are yet to be identified and much improvement is required on the currently used method.