Review Of Antimalarial Drug Resistance Biology Essay

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Biological advantage favoring resistant parasites (Bloland P, 2001). The genetic events that lead to resistance to an antimalarial drug are usually spontaneous mutations or changes in copy number of genes relating to the drug target of the parasite (White, 2004). These events confer reduced sensitivity to a particular drug, or a whole drug class. Over time, resistance becomes stable in the population and it can persist even after drug pressure is removed. Among the species causing human malaria, drug resistance has been reported and characterized the most in P. falciparum, although resistance to antimalarials has been documented for P. malariae and P. vivax, as well. In P. falciparum, resistance has been observed in all currently used antimalarials (including artemisinin derivatives). The geographical distributions and rates of spread have varied considerably (Fig. 1.4). P.

vivax has developed resistance rapidly to SP in many areas, while resistance to CQ is confined largely to Indonesia, Papua New Guinea, Timor-Leste and other parts of

Oceania. There are also reports of CQ resistance from Brazil, Peru, India, and Africa

(Fig. 1.4). However, P. vivax remains sensitive to CQ in most of South-East Asia, the

Indian subcontinent, the Korean peninsula, the Middle East, north-east Africa, and most

of South and Central America (Organisation, 2010).

In response to the increasing burden of malaria caused by P. falciparum resistance to the standard antimalarial medicines, World Health Organization (WHO) recommended the use of combination therapies, ideally those containing artemisinin derivatives in countries where P. falciparum malaria is resistant to the conventional antimalarial medicines chloroquine, SP, and amodiaquine28. Unfortunately, even artemisinin derivatives, the only drugs that had been fully effective against P. falciparum until very recently, seem to be loosing their efficacy along the border between Cambodia and Thailand (Lim et al., 2009; Noedl et al., 2009).

Assessment of drug resistance monitoring

Drug surveillance is necessary to ensure correct management of clinical cases and early detection of changing patterns of resistance to assure that national treatment policies remain effective (W.H.O, 2003). Three approaches have been used to evaluate the efficacy of an antimalarial drug: clinical in vivo studies (also known as therapeutic efficacy testing), in vitro susceptibility testing, and more recently, molecular markers. In discussing these different approaches, it is fundamental to differentiate intrinsic parasite resistance from decreased clinical efficacy. The term resistance means the failure of a drug to prevent parasite growth in culture, at defined drug concentrations, and in the absence of the host immune response. Alterations in efficacy are detected through clinical in vivo studies in which parasite intrinsic susceptibility is one of several factors that determine the outcome (Laufer. M. K, 2009).

In vivo measures of drug resistance.

The therapeutic efficacy test remains the "gold standard" method for detecting drug resistance (W.H.O, 2003). These tests reveal the exact biological nature of drug treatment response. This response involves a complex interaction between the drugs, the parasites, and the host response (i.e. the therapeutic response of currently circulating parasites infecting the current population in which the drug will be used), while in vitro tests measure only the interaction between the parasites and the drugs (Talisuna et al., 2004). In vivo tests involve the treatment of symptomatic P. falciparum infected patients with a standard dose of an antimalarial drug and subsequent follow-up of clinical and parasitological outcomes of treatment during a fixed period. The WHO developed a scheme for estimating the degree of antimalarial drug resistance, which involves studying patient parasitemia over 28 days. The in vivo response to drugs was originally defined by WHO in terms of parasite clearance (sensitive [S] and three degrees of resistance [RI, RII, RIII]) 137,155. Blood smears were taken on days 2, 7 and 28 after initiation of antimalarial treatment to grade the resistance as RI-RIII. Sensitivity was classified as reduction of initial parasitemia by ≥75% on day 2 with smears negative for malaria parasites from day 7 to 28 (end of follow up, but could be longer if drugs with longer half lives are used like mefloquine). RI response was classified as initial clearance of parasitemia with negative smear on day 7, but recrudescence on 8th day or more days after treatment started. RII response was classified as an initial clearance or substantial reduction of parasitemia (<25% of the initial count on day 2) but with persistence or recrudescence of parasitemia during days 4-7 after treatment. RIII was classified as no significant reduction of parasitemia at all 28 days after treatment. This scheme of classification still remains valid for areas with low or no malaria transmission, but it is difficult to apply to areas with intensive transmission, because of the chance that new infection can be mistaken for recrudescence (which can also happen after 28 days). Other drawbacks of this method included the fact that RII was too broad of a category, practical difficulties in following a patient for 28 days, and the intermittent nature of parasitemia in the blood of infected patients. Therefore, WHO introduced a modified protocol in 1996 based on clinical outcome targeted at a practical assessment of therapeutic responses in areas with intense transmission, where parasitemia in the absence of clinical signs or symptoms is common (Roll Back Malaria Partnership and World Health Organization, 2001; Wongsrichanalai et al., 2002). The modified classification has established categories of Early treatment failure (ETF) (aggravation/persistence of clinical symptoms in the presence of parasitemia during the first 3 days of follow-up), Late treatment failure (LTF) (reappearance of symptoms in the presence of parasitemia during days 4-14 of follow-up), and Adequate clinical response (ACR) (Absence of parasitemia on day 14 irrespective of fever, or absence of clinical symptoms irrespective of parasitemia, in patients not meeting ETF or LTF criteria). The WHO has continued to update therapeutic efficacy protocols for high transmission areas and validate the therapeutic efficacy protocol for low-to-moderate transmission areas on the basis of feedback from countries and scientific recommendations. Recently, the WHO modified the existing protocol to include applications of the same definitions of treatment responses at all levels of malaria transmission, with slight adjustment of patient inclusion criteria; administration of rescue treatment to patients with parasitological treatment failure at all levels of malaria transmission; requirement for 28 or 42 days of follow-up as a standard, depending on the medicine tested; and requirement for genotyping by PCR to distinguish between recrudescence and re-infection. The 28-day follow-up is recommended as the minimum standard to allow national malaria control programs to capture most failures with most medicines, except mefloquine and piperaquine, for which the minimum follow-up should be 42 days 192. There are now set definitions of treatment response that are used in all areas of malaria transmission. The ETF definition has been modified to the following: danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitemia; parasitemia on day 2 higher than on day 0, irrespective of axillary temperature; parasitemia on day 3 with axillary temperature ≥ 37.5 °C; and parasitemia on day 3 ≥ 25% of count on day 0. Late clinical failure (LCF) is defined as: severe malaria in the presence of parasitemia on any day between day 4 and day 28 (day 42) in patients who did not previously meet any of the criteria of ETF; and presence of parasitemia on any day between day 4 and day 28 (day 42) with axillary temperature ≥ 37.5 °C in patients who did not previously meet any of the criteria of early treatment failure. Late parasitological failure (LPF) is defined as the presence of parasitemia on any day between day 7 and day 28 (day 42) with axillary temperature < 37.5 °C in patients who did not previously meet any of the criteria of early treatment failure or late clinical failure. Adequate clinical and parasitological response (ACPR) is defined as an absence of parasitemia on day 28 (day 42), irrespective of axillary temperature, in patients who did not previously meet any of the criteria of early treatment failure, late clinical failure or late parasitological failure. These tests provide decision-makers with a simple, readily comprehensible indicator of the efficacy of an antimalarial drug with reduced requirement for equipment and supplies (W.H.O, 2003).

In vitro tests

The in vivo method has allowed the thresholds of treatment failure that are crucial for adjusting antimalarial drug policies to be determined but it not sufficient on its own to confirm drug resistance(W.H.O, 2003). To support the evidence of a failing antimalarial, an in vitro test can be used providing a more accurate measure of drug sensitivity under controlled experimental conditions, which removes variables such as patient immune status, re-infection and pharmacokinetics. In vitro tests allow a more objective approach to parasite resistance, since in these studies the parasite will be in direct contact with incremental drug concentrations. Several tests can be carried out with the same sample, and several drugs can be studied at the same time, including drugs that are still at the experimental stage(W.H.O, 2003). Several in vitro tests exist, which differ with respect to the measure effect and the duration of exposure to the test compound. These include microscopic examination of blood films for the WHO mark III test (inhibition of maturation or replication; Giemsa-stained), the radioisotopic test (incorporation of hypoxantine) and the enzyme-linked immunosorbent assay with antibodies directed against Plasmodium lactate dehydrogenase or histidine-rich protein II(Olliaro, 2005). The importance of these tests has become evident with the increasing use of combination therapy, since they can be used to monitor susceptibility to each drug in a combination. It is often impossible to perform in vivo tests for each component, due to ethical problems, non-availability of the drug as monotherapy and the need to study a large number of patients (Vestergaard, Ringwald, 2007). Although this method is useful, its application is limited. In vitro methods require trained personnel with access to a laboratory capable to perform culture of malaria parasites. Even when provided with such facilities, it is often difficult to establish cultures and not all the primary parasites will adapt to in vitro culture conditions (LeRoux et al., 2009). Moreover, in part because these tests remove the host factors, the correlation between results of in vitro and in vivo tests is not always reliable and is not well understood. In vitro drug sensitivity data may provide early evidence of increasing drug tolerance prior to parasitological/clinical resistance. Whereas, this test may give misleading indications if the alterations in sensitivity are so small that they do not result in parasitological/clinical resistance (Hastings et al., 2007). These limitations of in vivo and in vitro methods have led to the search for genetic markers of resistance.

Molecular Markers

Molecular markers for drug resistant malaria provide promising public heath tools of great potential value(Plowe et al., 2007). Studies that can detect genetic markers of drug resistance are relatively fast, quantitative and less expensive compared with clinical studies involving more patient care and follow-up (Ekland, Fidock, 2008). In addition, collection, storage and transport of specimens for subsequent molecular analysis are far easier than for in vitro tests(W.H.O, 2003). Molecular markers for drug resistance malaria are based on genetic changes that confer parasite resistance to drugs. These genetic mechanisms of P. falciparum drug resistance have not been completely elucidated. However, five genes that appear to play a role in regulation of resistance to the principal chemical families of antimalarials in current use have been identified. Multiple mutations in the P. falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine. In particular a substitution at amino acid position 76 (K to T) (Fidock et al., 2000; Sidhu et al., 2005) is crucial to the manifestation of in vitro resistance as well as therapeutic failure. Mutation in the P-glycoprotein homologue (Pgh1) encoded by pfmdr1 (P. falciparum multidrug resistance gene 1) may further modulate the extent of chloroquine resistance53. Polymorphisms and/or amplification of pfmdr1 have also been shown to affect the susceptibility to structurally unrelated antimalarial drugs, including mefloquine, artesunate, lumefantrine and quinine (Price et al., 1996; Sidhu et al., 2005). Resistance to sulphadoxine and pyrimethamine (SP) is conferred by mutations in the dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes, respectively (Brooks et al., 1994; Peterson et al., 1988). One mutation in the plasmodial ATPase6 gene has been proposed as a marker for artemisinin resistance but this association has not yet been confirmed (Jambou et al., 2005).

As it is the case with in vitro tests, the presence of particular molecular markers does not necessarily directly predict treatment outcome. Mutations in the above genes contribute to drug failure but the outcome is not certain: some patients with "resistant" alleles clear the infection, and some patients with "sensitive" alleles failed treatment. Therefore is better to think in terms of mutations encoding rising probabilities of drug failure that ultimately depends on factors such as host immune response, the drug dose taken, and variation in drug absorption and metabolism(Hastings, 2007). Much debate has occurred concerning the relative qualities of one test over another, with the suggestion always being that one type of test should be used preferentially. These methods can be considered as complementary sources of information about resistance, rather than competing, regarding the different types of information each approach gives.

Mechanisms of drug resistance

In general, development of drug resistance occurs in several discrete steps. When

organisms or cells are exposed to suboptimal, and thus sublethal, levels of a drug, they

tend to respond to the stress situation by adaptation involving one or more mechanisms

of drug resistance. In addition to single drug resistance, there is recent evidence for the development of multiple drug resistance mechanisms that allow the organism to survive not only to the exposure to one drug, but also to others with non related structures or mechanisms of action. The exposure to sub-optimal drug levels through self medication in the management of fever in developing countries is probably one of the most important

reasons for the presence of (multiple) drug resistant malaria.

ANTIMALARIAL DRUGS: MODES OF ACTION AND RESISTANCE

The effect of the bark of the cinchona tree on malaria was first uncovered by the native

Indians of South America, while in China sweet worm-wood has been used for the

treatment of malaria for a very long time (Aydin-Schmidt et al., 2010). Different types

of antimalarial drugs have had different histories, impact and although some with some

similarities, they also differ in the molecular mechanism underling parasite drug

resistance. Table 1 gives a brief overview of P. falciparum proteins with a proven or

likely role in resistance to clinical antimalarial drugs.

Quinolines and related compounds

Introduced during the 17th Century, the use of extracts from the bark of the cinchona

tree was the first effective chemotherapy available in Europe. Its principle active

compound, isolated in the 19th Century, was named quinine (QN). Its structure is built

upon a quinoline ring system. From this basic structure, the group of synthetic

antimalarials collectively named quinolines was created during the 20th Century,

including chloroquine (CQ), amodiaquine (AQ), piperaquine (PQ) and mefloquine

(MQ). Based on more loosely related ring systems are the antimalarials halofantrine

(HF) and lumefantrine (LUM) (Figure 3). These compounds are all thought to share a

common target, with the most widely accepted hypothesis, based on studies with CQ,

proposing the target as being in the parasite heme detoxification systems (Fitch, 2004). CQ, clinically available since 1947, is one of the most successful antimalarial drugs

ever produced, being a safe and cheap compound that is estimated to have saved

countless millions of lives (O'Meara et al., 2010). The resistance of P. falciparum to this drug was identified approximately 10 years after its introduction, with the first pilot reports coming from the Thai-Cambodian borders in 1957, and the first formal complete

records in Northeast South America. CQ resistance further expanded to Africa, where it

first appeared in the late 1970s, further spreading to most sub-Saharan countries by the

end of the 1980s. CQ acts by interfering with the detoxification of the heme group (ferroprotoporphyrin IX) produced when haemoglobin is digested. This process occurs inside the Plasmodium parasite's food vacuole (FV), where CQ typically accumulates due to its di-protonation by the acidic environment of the FV lumen. There, it binds to the toxic heme, preventing the process of biomineralization towards hemozoin. The highly

reactive free heme ultimately becomes lethal to the parasite. Although the long-term accepted view for explaining CQ resistance resides in the accumulation of the drug inside the food vacuole, with higher accumulation of CQ in sensitive parasites compared with the resistant ones (Krogstad et al., 1987), a direct demonstration of the phenomenon at the molecular level occurred only recently (Martin et al., 2009). The discovery of the precise genetic basis of chloroquine resistance was also a long and difficult process, with more then 40 years between its initial clinical recognition and the identification of the chloroquine resistance transporter gene (pfcrt) (Fidock et al., 2000). This gene, located on chromosome 7, is highly polymorphic with at least 20 variable codon positions reported to date (Cooper et al., 2005). A specific polymorphism resulting in a lysine to threonine substitution at amino acid 76 (K76T) was shown to confer in vitro(Fidock et al., 2000; Sidhu et al., 2002) and in vivo (Djimde et al., 2001) CQ resistance to the extent that it became a molecular marker for predictive therapeutic efficacy(WHO, 2002).

Resistance to quinoline drugs, including the afore mentioned CQ, has also been linked to other transporter proteins. This includes the long studied P. falciparum multidrug resistance 1 (PfMDR1), Na+/h+ exchanger (PfNHE) and multidrug resistance associated

protein 1 (PfMRP1). A more detailed description of the importance of pfcrt and the pfmdr1 and pfmrp1 ABC transporter genes in the development of drug resistance will be given in the next chapter.

Antifolates

The antifolate class of drugs consists of compounds that bind enzymes necessary for

parasite folate biosynthesis. The main drugs used against malaria are the combinations

sulphadoxine-pyrimethamine (SP) and chlorproguanil-dapsone (not as accepted due to

the high risk of severe anaemia after treatment in patients with G6PD deficiency (Fanello et al., 2008). Pyrimethamine and chlorcycloguanil (the active metabolite of chlorproguanil), target the dihydrofolate reductase (DHFR) activity of the parasite's bifunctional DHFR-thymidylate synthase protein, whereas the sulfa drugs, sulphadoxine and dapsone, affect dihydropteroate synthase (DHPS). Their inhibition leads to decreased production of tetra-hydrofolate, a cofactor necessary for the production of a number of folate precursors which ultimately disturbs the biosynthesis of nucleotides and subsequent DNA synthesis (Ferone, 1977; Sridaran et al., 2010). Resistance emerged rapidly when these drugs were introduced alone as a monotherapy, but synergistic combinations like SP (Fansidarâ„¢), first introduced in the late 1960s, have proved to be of long-term utility, especially as an inexpensive alternative to combat CQ-resistant parasites. Treatment with Fansidarâ„¢ is taken in a single dose which results in high compliance. However, this has not stopped the spread of resistance, as recently witnessed in retrospective studies conducted in Mozambique (Raman et al., 2010). Nowadays, artemisinin-based combination therapy (ACT) has taken over the first-line regimen of choice in many areas of the world. Nevertheless, antifolates still play a key role in intermittent preventive treatment (IPT) in areas of high transmission of high risk groups (pregnant woman and infants) regardless of their infection status (Warsame. M, 2010).

Mutations in the dhfr and the dhps genes of P. falciparum parasites have been associated with decreased parasite sensitivity to the antifolate drugs. A change from wild type Ser108 to Asn108 (S108N) in pfdhfr is sufficient to cause low level pyrimethamine resistance in vivo and by 100-fold relative to wild type in vitro (Cowman et al., 1988; Peterson et al., 1988). The triple pfdhfr mutant genotype consisting of N51I, C59R and S108N shows in vitro resistance to pyrimethamine with significantly higher inhibitory concentration values than with the single mutation at position 108 (Nzila-Mounda et al., 1998) and have demonstrated strong association with in vivo SP treatment failure (Basco et al., 1998; Happi et al., 2005; Kublin et al., 2002). Data from various malaria endemic areas suggest asymmetric selection of resistant genotypes starting with mutations in pfdhfr and followed by mutations in the pfdhps gene by which A437G and K540E SNPs have been associated with in vivo clinical failure (Happi et al., 2005). The quintuple mutant genotype consisting of the double pfdhps mutant mentioned above in combination with the pfdhfr triple mutant genotype (N51I, C59R, S108N) is a better predictor of clinical failure than either the multiple mutant genotype alone (Happi et al., 2005; Kublin et al., 2002; Mugittu et al., 2006). The amino acid alterations mentioned above are to date the key factors associated with parasite antifolate resistance and other factors may play a role in the levels of clinical failure after SP treatment. Higher serum folate concentrations, as a result of a dietary folate supplementation in children and pregnant women have been reported to be associated with SP treatment failure (Dzinjalamala et al., 2005; van Eijk et al., 2008). The raised endogenous folate pools in parasites will compete with antifolate drugs at enzyme binding sites. Folates are the endogenous substrate, that is transported by MRPs (multidrug resistance-associated protein) (Deeley, Cole, 2006).

Artemisinins

For more than 2000 years, artemisinins have been used in traditional Chinese medicine

for the treatment of febrile illness. But it was only in 1971 that Chinese scientists

discovered its specific antimalarial properties extracted from the ubiquitous annual

wormwood Artemisia annua (White, 2008). Also known as qinghaosu, artemisinin (ART) and its derivatives (mainly utilized in ACT: artemether, artesunate and dihydroartemisinin (DHA)) are a group of sesquiterpene lactone endoperoxides that possess the most rapid action of all current drugs used against P. falciparum, being able to reduce the parasite biomass up to 10000 fold per asexual cycle. They also have a very short elimination half-life in the human body (around 1 hour), reducing the opportunity for the parasite to develop resistance. Artemisinins are active in nearly all of the asexual stages of parasite development in the blood, and also affect the sexual stages of P. falciparum (gametocytes) which are essential for transmission. The specific mechanisms of action of the ART derivatives are still unsolved. Most studies agree that their activity involves the break of the intramolecular endoperoxide bridge, and a recent study suggests that the digestive vacuole is an important initial site of endoperoxide antimalarial activity (del Pilar Crespo et al., 2008). The active endoperoxide compound is assumed to interact with reduced heme (ferroprotoporphyrin IX) or iron to form free-radical by-products of both the drug and the heme (Jefford, 2001; Paitayatat et al., 1997). The radicals are thought to react with susceptible groups within parasite enzymes and lipids; however, the exact sites of action are still unresolved. The first protein to be suggested to be a target for ART was the Plasmodium translationally controlled tumor protein (TCTP) homologue (Bhisutthibhan et al., 1998), a protein that binds heme. One report has shown that there is less incorporation of radiolabeled DHA in a resistant murine plasmodial strain in vivo and a 2.5 fold overexpression of TCTP in a rodent malaria model (Walker et al., 2000), but no genetic alterations have been described regarding ART susceptibility.

Structural similarities of ART to thapsigargin, also a sesquiterpene lactone, led to the

identification of another potential target related to the endoplasmic reticulum, PfATP6

(Eckstein-Ludwig et al., 2003). This represents the only sarco/endoplasmatic reticulum

calcium-dependent ATPase (SERCA) ortholog in P. falciparum, a central player in the

crucial Ca2+ homeostasis of the parasite. A mutation in the pfatp6 gene, noted exclusively in isolates from French Guiana, was reported to be associated with significantly increased IC50 values for artemether in ex vivo tests (Jambou et al., 2005). More recently, in vivo artemisinin resistance has been proposed (Noedl, 2005) and identified by the presence of significantly decreased parasite reduction rates, manifested clinically by markedly longer parasite clearance times from the body (Dondorp et al., 2009; Noedl, 2005; Noedl et al., 2008; Noedl et al., 2010). With only a few individual cases matching as resistant: "adequate plasma drug absorption, prolonged parasite clearance times, increased ARTs IC50s, and reemergence of parasites within 28 days"(Noedl et al., 2008; Noedl et al., 2010), the molecular basis for this phenomenon is uncertain, based on the fact that most of these observations are not clearly associated with altered artemisinin IC50 in vitro. So far only minor determinants with likely roles in resistance, such as pfmdr1 amplifications, correlate (Table 1) (Imwong et al., 2010; Price et al., 2004).

ACT- artemisinin based combination therapy

Combination therapy is well established for the treatment of other diseases such as tuberculosis and infection by the human immunodeficiency virus (HIV). The rationale for combination therapy is based on the diminutive probability of resistance arising after simultaneous use of two or more antimalarials with different modes of action which do not share the same resistance mechanisms (White, 1999b). Artemisinins are currently the most important class of antimalarial drugs, presenting many advantages over other compounds. These advantages include several possible routes of administration, suitability for treating severe malaria replacing quinine and avoiding its side effects, rapid activity and activity against trophozoites, blood schizonts, and also against ring forms and gametocytes (Krishna et al., 2004). This latter property may help diminish transmission rates34. Altogether, artemisinin derivatives reduce the incidence of malaria, reduce drug use, and thus contribute to slowing the evolution of drug resistance (Price et al., 1996; White, 1999a). Artemisinin-based combination therapy (ACT), the combination of an artemisinin derivative with another structurally unrelated antimalarial, is now being proposed as the best therapeutic alternative for treating drug-resistant malaria and delaying the development of resistance (WHO, 2006). Artemisinins are highly effective, short-acting drugs derived from an herb, qinghao (Artemisia annua), a plant found in China. This class of compounds can reduce the number of parasites faster than any other class of antimalarial drug and this is also the reason for their ability to provide clinical relief fast (Hien, White, 1993; White, 1997). Because of the short elimination half-lives of the artemisinin compounds, the administration of artemisinins with longer-acting agents is required (Nyunt, Plowe, 2007). In an ACT, the artemisinin derivative kills rapidly and drastically most of the parasites and those that remain are then eliminated by a high concentration of the longer-lasting partner drug, after the short-lived artemisinin has dropped below therapeutic levels. In this way the artemisinin derivative should protect its partner drug. The time interval when the drug concentrations are at sub-therapeutic levels but still significantly present has been referred as the "selective window". This is the period of time when the drug level is adequate to suppress the growth of susceptible parasites, but too low to prevent replication of less sensitive sub-populations, leading to their selection (Hastings, Ward, 2005; Stepniewska, White, 2008). If the treatment is successful, the partner drug also protects the artemisinin derivative by removing all the remaining parasites that were initially exposed to the artemisinin. Consequently the probability that mutant parasites survive and emerge from these two drugs is low (Nosten, White, 2007; White, 1997). However, this question is under debate. Accordingly to Hastings and Watkins, combination therapies containing drugs with mismatched half-lives could jeopardize the efficacy of the artemisinins, in particular combination with partner drugs against which resistance is already spread (Hastings, Watkins, 2006). Discrepancy in the half-lives of the partner drugs leaves windows of monotherapy during which parasites can acquire resistance, or those with pre-existing resistance to the partner drug, re-emerge and cause drug failure. Ideally, drugs with short half-lives should be preferred, in order to reduce the exposure of reinvading parasites to suboptimal drug levels which may induce the selection for tolerance and eventually the development of resistance. So far, the introduction of ACT as first line treatment has been very successful. The commonly used ACTs are: artemether + lumefantrine (AL); artesunate + amodiaquine (AS-AQ); artesunate + mefloquine (AS-MQ); artesunate + sulphadoxine + pyrimethamine (AS-SP) and dihydroartemisinin + piperaquine (DHA-PQ). Countries where ACT and other malaria controls activities have been introduced have witnessed significant reductions in endemicity (Dondorp et al., 2010).

Genetic basis of Drug resistance

The development of resistance is a two steps process; de-novo emergence and subsequent spread. Resistance arise through point mutations or gene duplications, mainly during asexual reproduction, a single genetic event may be all that is required, or multiple events (epistasis). The rare de-novo emergence event is thought to be independent of drug selection pressure59. These mutants are then selected for survival, multiplication, and subsequent spread as a result of the drug pressure which provides a selective advantage to resistant parasites2,59,60. Resistant parasites are then transmitted to other individuals by mosquitos61. In addition, sometimes mosquitoes bite two gametocyte-carrying humans offering the possibility of recombination with the formation or breakdown of multigenic resistance2. The parasites carrying the mutant alleles are selected if antimalarial drug concentrations are sufficient to inhibit the development of susceptible parasites but are insufficient to inhibit the mutants, a phenomenon known as "drug selection"62.

Advances in the understanding of the mechanisms of drug action during the last two decades led to the identification of the putative molecular targets and the genetic basis responsible for parasite resistance to antimalarial drugs. The genetic events that confer antimalarial drug resistance include single point mutations in or changes of copy numbers of genes encoding drug targets, such as important enzymes or pumps that affect intraparasitic drug concentrations.

In the following sections the genes implicated in the resistance to the drugs investigated in this thesis, the 4-aminoquinoline (Chloroquine), is described in detail.

Genes Influencing 4-aminoquinolines Susceptibility

Resistance to 4-aminoquilolines is multigenic and relates to changes in drug accumulation in the parasite's food vacuole. In 2000, the P. falciparum chloroquine resistance transporter (pfcrt) gene was identified as a primary candidate gene for chloroquine resistance by extensive mapping of genetic cross between chloroquine sensitive and resistant clones. A single nucleotide polymorphism (SNP) at position 76 (replacement of lysine by threonine), has been reported to be completely associated with chloroquine resistance in 40 laboratory-adapted P. falciparum clones from wide geographic areas51. Other pfcrt mutations occur, but their patterns tend to vary in different geographical areas and their role remains undetermined63. The causal relationship between pfcrt mutations and chloroquine resistance was further confirmed by genetic transfection experiments64. Several in vivo studies showed total selection of the pfcrt K76T mutant allele in clinical failure65. However, the presence of parasites carrying this allele was not always predictive of the clinical outcome or predictive of in vitro resistance66-68. This mutation appears to be a prerequisite for resistance but may not be sufficient on its own69,70. In addition to mutations in pfcrt, polymorphisms in the P. falciparum multidrug resistance gene 1 (pfmdr1) have been suggested to modulate the degree of resistance in parasites harbouring the pfcrt T76 mutation71. Transfection and allelic exchange tests have shown strong associations between pfmdr1 polymorphisms and enhancement of the degree of in vitro chloroquine resistance72. However, many clinical studies have failed to find associations between pfcrt and pfmdr1 mutations and chloroquine resistance67,73,74 and isolates carrying the same pfcrt and pfmdr1 genotypes often reveal different IC50 values, indicating that several other genes may also be responsible for the increase level of chloroquine resistance75. This suggests involvement of additional genetic loci in modulating chloroquine resistance, more studies are necessary to elucidate the mechanisms of resistance to this class of drugs.

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