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Figure 3 -Plasmodium’s life cycle and its relationship to drug resistance
Legend: Selective forces that drive the emergence and spread of drug resistance differ throughout the life cycle.
Important factors include the parasite numbers and drug pressure at different stages, stage specificity of drug action, the essentiality of the targeted pathways in the mosquito vector and vertebrate host, host immunity, multiplicity of infection, and local factors that affect therapeutics use and compliance .Source: (Blasco et al., 2017)
Treatment failure: is defined as an inability to clear malarial parasitaemia or resolve clinical
symptoms despite administration of an antimalarial medicine. Treatment failure is not, however, always due to drug resistance, and many factors can contribute, mainly by reducing drug concentrations. These factors include incorrect dosage, poor patient compliance in respect of either dose or duration of treatment, poor drug quality and drug interactions. Even after supervised administration of a full regimen of an antimalarial medicine, individual variations in pharmacokinetics might also lead to treatment failure because of poor absorption, rapid elimination (e.g. diarrhoea or vomiting) or poor biotransformation of prodrugs (WHO, 2010a)
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Multidrug resistance: P. falciparum is seen when the parasite is resistant to more than two operational antimalarial compounds of different chemical classes and modes of action.
Generally, the two classes first affected are the 4-aminoquinolines and the antifolates (diaminopyrimidine, sulfonamides). Drug resistance results in a delay in or failure to clear asexual parasites from the blood, which allows production of the gametocytes that are responsible for transmission of the resistant genotype (WHO, 2010a)
2.5.3.1 Genetic basis of antimalarial drug resistance
The genetic events that confer antimalarial drug resistance (while retaining parasite viability) are spontaneous and rare and are thought to be independent of the drug used. They are mutations in or changes in the copy number of genes encoding or relating to the drug’s parasite target or influx/efflux pumps that affect intra parasitic concentrations of the drug (White, 2004). The onset of resistance is thought to occur in two phases. In the first phase, an initial genetic event produces a resistant mutant (de novo mutation) in which a new genetic trait gives the parasite a survival advantage against the drug (White, 2004). As the resistant parasites multiply and are selected, a parasite population which is no longer susceptible to treatment emerges. Depending on the drug, conferment of resistance may involve a single point or multiple point mutation.The acquired mutations allow the survival or reproduction of the resistant parasite whereas drug pressure will eliminate susceptible ones (Bloland, 2001). Figure 4 illustrates factors that drive development and spread of resistance. Mutations may be associated with fitness disadvantages (i.e., in the absence of the drug they are less fit and multiply less well than their drug-sensitive counterparts). Another factor that may explain the discrepancy between in vitro and much lower apparent in vivo rates of spontaneous mutation is host immunity.
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Figure-4 Development and spread of resistance[Source:www.malariasite.com/drug-resistance/]
Drug pressure leads to higher gametocyte release, and this facilitates the propagation of the resistant mutants that have escaped the drugs. Failure to use primaquine as a gametocytocidal agent for P. falciparum further aids such spread of resistance. Therefore, gametocyte production from the recrudescent resistant infection must be prevented by administration of early, appropriate treatment (WHO, 2010a), combined with primaquine. Administration of drugs with long elimination phases facilitates the spread of resistant mutant malaria parasites. The residual antimalarial activity that is present during the post-treatment period serves as a “selective filter”, which prevents infection by sensitive parasites but allows infection by resistant parasites. Drugs such as chloroquine, mefloquine and piperaquine, which persist in the blood for months, provide a selective filter long after their administration has ceased (WHO, 2010a). Inadequate drug exposure either due to improper dosing, poor pharmacokinetic properties, fake drugs, or other infections acquired during the drug elimination phase of a previous infection can expose
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plasmodium to sub-optimal drug concentrations leading to the selection of resistant genotypes (Blasco et al., 2017; Costa et al., 2017)
2.5.3.2 Molecular markers of resistance
Molecular markers that are linked with resistance to different antimalarial drugs have been described and are summarized in Table 2. Mutations in Pfcrt genes at codon72-76 of CVIET haplotype and Pfmdr1 at codon 86Y are known to be associated with resistance in P. falciparum to chloroquine (Babiker et al., 2001; Abdoulaye Djimdé et al., 2001; Fidock et al., 2000). Novel mutations in k13 kelch genes (Ariey et al., 2014), duplication and single nucleotide polymorphism(SNP) of Pfmdr1 genes at codon (N86, 184F, and D1246) have been associated with resistance in Plasmodium falciparum to artemisinin (ART), artemisinin/lumefantrine(AL) (Baliraine and Rosenthal, 2011; Sidhu et al., 2006, 2005) while tolerance to lumefantrine has also been shown in parasites with SNP in Pfcrt at codon K76 (Ashley et al., 2014) . Parasites with mutations in pfdhfr at codon 51, 59, 108 and Pfdhps genes at codon 437 and 540 are sulphadoxine/pyrimethamine resistant (Cowman et al., 1988;Foote et al., 1990; Peterson et al., 1988; Triglia, Menting et al., 1997).
Molecular markers that are associated with resistance to the older ineffective drugs have also been linked to resistance to the newly introduced effective regimen especially to the artemisinin partner drugs (Sidhu et al., 2006). Presence of such markers in parasite population could predict treatment outcome to the artemisinin combination therapy. For example, it has been established that presence of Pfmdr1 gene N86 and increased copy number of Pfmdr1 were independent significant risk factor for recrudescence in patients following treatment with artemether-lumefantrine (Happi et al., 2009; Venkatesan et al., 2014).
34 2.5.3.3 Epidemiology of drug resistance
Chloroquine and sulphadoxine/pyrimethamine (SP), which are very affordable was once effective against malaria but are no longer first line drugs due to resistance, prompting recommendation of artemisinin combination therapy (ACT) as the first line therapy by the WHO (WHO, 2001). The adoption of ACT as the first line treatment by many endemic countries led to significant reduction in malaria cases and mortality. This achievement is however being threatened by reports of resistance to the main line drug artemisinin in South-East Asia which manifests itself as delayed clearance of parasitaemia following treatment with artemisinin derivatives (Dondorp et al., 2009; Kyaw et al., 2013; Noedl et al., 2008; Phyo et al., 2012). P.
falciparum resistance to artemisinin has been detected in five countries in South-East Asia and a high failure rate reported after treatment for four artemisinin combination therapies (ACTs) in Cambodia.
P. falciparum parasites from South-East Asia have been shown to have an increased propensity to develop drug resistance (Rathod et al., 1997). Molecular determinant associated with resistance are mutations within a kelch protein located on P. falciparum chromosome 13 (k13 propeller) (Ariey et al., 2014; Straimer et al., 2015). It has been characterized and found in parasites from many countries around the region. It has been reported that secondary loci may also be involved (Takala-Harrison et al., 2015). Resistance due to k13 propeller mutations have been shown to have spread between countries as well as to have emerged independently in different countries (Ashley et al., 2014; Takala-Harrison et al., 2015).The possibility of this resistance parasite being exported to Africa or other regions of the world exists as was the case when resistance to chloroquine and SP spread from South-East Asia to Africa and other parts of the world.
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Recent studies have evaluated the emergence of resistance to piperaquine, an artemisinin partner drug (Amaratunga et al., 2016; Veiga et al., 2016), with one study confirming that 14 out of 40 clinical isolates exhibited piperaquine resistance, leading to the recommendation for a change of treatment in Vietnam (Phuc et al., 2017). To date, no molecular marker for piperaquine resistance has been identified (WHO, 2014a), although a recent study has linked a surrogate marker to piperaquine resistance (Witkowski et al., 2017), highlighting the need for the use of molecular studies (involving whole genome sequencing) as a realistic approach to detect early emergence of resistant populations of parasites
In some studies conducted in African populations, low levels of k13 mutations was detected in parasite isolates obtained from different parts of Africa (Ashley et al., 2014; Kamau et al., 2014;
Amato et al., 2015 ; Talundzic et al., 2017).These findings have serious implication for malaria control efforts especially in Africa where malaria transmission is high and highlight the importance of surveillance studies as a mechanism to detect early the emergence of resistant population of parasites in areas where ACTs are currently used.
2.5.3.4 Current scenario of drug resistance
Resistance to the aminoquinoline, chloroquine which was once widely used for many decades because of its safety, efficacy and affordability is now widespread in malaria endemic countries.
At present, chloroquine remains effective only in some parts of Central America, where clinical studies have confirmed its efficacy (Londono et al., 2009) However, subsequent data on the prevalence of chloroquine-resistant genotypes in these areas present an alarming situation for health officials (ElBadry et al., 2013). Amodiaquine has been observed to be more effective than chloroquine mainly in areas of persistent chloroquine resistance. Thus, amodiaquine in combination with artesunate was adopted as the first-line treatment by several countries. Parasite
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strains that are highly resistant to amodiaquine have however been reported in Tanzania, which may additionally compromise the use of artesunate-amodiaquine in Africa (Sá et al., 2009) Another antimalarial, sulphadoxine-pyrimethamine, has been widely used by several countries to treat chloroquine-resistant malaria. Nonetheless, the treatment failure rate of this combination has been found to be low in several countries of South America and Central and Middle East Asia but high in Eastern Africa (52.8%) (WHO, 2010b) Presently, resistance to mefloquine continues to be a concern in the Greater Mekong sub-region, particularly in Thailand and Cambodia, where artesunate-mefloquine is still used as first-line treatment (Satimai et al., 2012) To maximize the effectiveness of artemisinin and its derivatives and to protect them from the development of resistance, WHO has recommended combination of artemisinin with other drugs that have different mechanisms of action and longer half-lives. The five currently recommended combinations are: artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate-sulphadoxine-pyrimethamine and dihydro-artemisinin piperaquine (WHO, 2010b).
However, remarkable failure rates to some of these combinations have been observed in several African countries where resistance to one drug has been previously encountered. Artemether-lumefantrine remains highly effective in most parts of the world, except for Cambodia. This combination mostly shows failure rates less than 10% (WHO, 2010b) However, resistance to most of these combinations will lead to resurgence in global epidemic of malaria.
In the area of vaccine development, RTS, S/AS01, the most advanced candidate, which has 30-50% protective efficacy in children(Aide et al., 2010) and is believed to represent the first-generation malaria vaccine have been developed by GlaxoSmithKline (GSK) along with the PATH Malaria Vaccine Initiative (MVI).(WHO recommendation expected by 2015).
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