1.1MalariaMalaria is a disease transmitted by the female Anopheles mosquito and caused by anintracellular protozoan parasite of the genus Plasmodium.
There are five Plasmodiumspecies that cause malaria in humans: P.falciparum, P. vivax, P. malariae, P.
knowlesi and two ovale subspecies,i.e. P. ovale curtisi and P. ovale wallikeri(1). The first two types are the most dominantspecies causing malaria with P.
falciparum as the most frequent and virulentin sub-Saharan Africa;infection with it can lead to death, whereas the other species cause illnessbut are rarely life-threatening (2,3). The P. falciparum genome is made up of 22.8 megabases (Mb) spreadacross its 14 chromosomes, a nucleotide content of 80% A+T that rises to90% in introns and approximately 5300proteins (4). Comparative genome analysis shows thatthere is synteny between the different Plasmodiumspecies with the exception of genes located in the teleromic regions that areknown to be involved in antigenic variation and immune evasion.1.2The Epidemiology of MalariaIt is difficult to estimate the burden of malaria especially inlow-income countries due to the inconsistencies in data collection and alsobecause not all infections progress to disease manifestation. In 2002, therewere 515 million cases of P.
falciparum malaria reported globally withthe majority of these cases concentrated in sub-Saharan Africa (70%) (2). A recent World malaria report (5) estimates that there were 212 millionmalaria cases and 42,900 deaths in 2015. Infection rates are higher in childrenunder five years, pregnant women, HIV/AIDS patients and migrants or mobilepopulations who have limited access to prevention and diagnostic tests.1.3 LifeCycle of Plasmodium falciparum1.
3.1Exo-erythrocytic CycleThe life cycle of P. falciparum involves two cycles: theasexual and sexual cycles that occur in the human and Anophelesmosquito, respectively as shown in Figure 1-1. Malaria begins when an infectedmosquito bites a human host thereby injecting sporozoites into the dermis (6,7). The highly motile sporozoites travelfrom the bite site to the liver via the bloodstream and start theirintracellular development in the hepatocyte in a process known asexoerythrocyticschizogony. Sporozoites in the hepatocyte begin dividing intoschizonts. Each schizont gives birth to thousands of merozoites which are thenreleased into the bloodstream.
This is a clinically silent stage with theresult being a 10,000 fold amplification of parasite numbers and may takeapproximately 2-10 days, depending on the parasite species (8). P. vivaxand P. ovaleremaindormant in the liver and are reactive weeks to months after the primaryinfection (9,10).1.3.2Erythrocytic CycleMature merozoites from hepatocytes invade the red blood cellsmarking the start of clinical disease. Over a period of 44-72 hours, themerozoites undergo development from ring stages to mature trophozoites andfinally schizonts.
Each schizont gives rise to 10-30 merozoites which are seento circulate through the blood stream when the erythrocyte ruptures. Circulatingmerozoites are able to re-invade other uninfected red blood cells (uRBC) in theend leading to an exponential increase in parasite densities as high as 1010parasites per host. Asexual erythrocytic parasites are primarily responsible forfebrile illness associated with fever and chills but may develop into severedisease affecting organs. After at least two asexual cycles, a small proportionof merozoites (1%) develop into macrogametes (female) and microgametes (male)sexual forms in a process known as gametocytogenesis. These sexual forms of theparasite are known to be infective to mosquitoes and develop as a result ofhost environmental stress or antimalarial drug usage.
1.3.3Mosquito StageThe sexual cycle starts after a female Anopheles mosquito feeds on an infected human host.
Following ablood meal, the mosquito ingests micro- and macrogametes that are taken to themosquito’s midgut. The male gamete fertilizes the female gamete forming a diploidzygote. Following zygote formation, meiosis proceeds and genetic recombination,that is important in generation of multiple drug resistant phenotypes, occurs (11). The zygote further develops into amotile ookinete that perforates the midgut cell and becomes an oocyte. Theoocyte undergoes several mitotic divisions to form sporoblasts after whichbudding takes place 10-14 days later leading to the production of sporozoitesin a process called sporogony.
These midgut sporozoites move to the mosquito’ssalivary gland that can be inoculated into the human host during a blood meal,initiating another life cycle. Figure1-1: The life cycle of the P. falciparum parasite occurring in both the human host and Anopheline mosquito.
Adapted from (12).1.4Malaria DiseaseInfection with P. falciparumcan either result in uncomplicated malaria, asymptomatic infection or severemalaria. In malaria-endemic areas, continuous exposure to P.
falciparum infection early in life results in an unsteriledevelopment of clinical immunity later in life. In such environments it istherefore common to find adults with circulating parasites but without anyevident symptoms. These kinds of infections are known as asymptomaticinfections. This is in contrast with malaria infections in low transmissionareas, where there is little acquired immunity and P. falciparum infection leads to symptomatic disease in all agegroups.
Young children are however prone to symptomatic infections mainly dueto their underdeveloped humoral response to pathogens. Approximately 99% ofinfected individuals have uncomplicated malaria with non-specific symptoms suchas malaise, joint pains and headaches at 48 hour intervals which can be easilyconfused with other diseases (13). Due to reasons such as delayed orinappropriate treatment and low immunity, approximately 0.2-0.5% of P. falciparum infections develop into amore severe form of disease (14). Common complications of severe malariainclude: cerebral malaria, respiratory failure and severe anaemia or even leadto a coma (13).1.
5Interventions for Control and Elimination of Malaria1.5.1Prevention of malariaCurrent strategies for the control of malaria mainly depend on theuse of insecticide-treated nets (ITNs), indoor residual spraying (IRS) andartemisinin based combination therapies (ACT). These strategies have resultedin reduced mortality rates by 60% in Africa between the years 2000 and 2015 (15), not excluding the positive impact ofimproved quality of health systems and improved case management due to enhanceddiagnostics.
The most effective malaria prevention strategies recommended forsub-Saharan Africa are ITNs and IRS. ITNs not only provide a physical barrierbetween the mosquito and human, but also repel and kill mosquitoes because theyhave embedded insecticides. There is evidence that a wide scale use of ITNsprovides some extended level of protection to non-ITN users, as it helps reducethe overall malaria transmission (16). IRS involves spraying insecticides onthe ceilings, walls and other indoor resting places of mosquitoes. In mostcases, sleeping and living rooms of a household are targeted for spraying.Countries adopting the use of either ITNs or IRS or in combination, havereported promising declines in malaria related morbidity and mortality (17,18).1.
5.2Chemotherapy for TreatmentFor many years, chloroquine(CQ) has been the major drug for the treatment of uncomplicated malaria. Theappearance of CQ-resistance (CQR) in Southeast Asia and its spread to Africaand Latin America led to the introduction of sulphadoxine-pyrimethamine (SP) towhich parasites developed an even more rapid resistance (19). To reduce the pace of selection of resistance, WHO prescribesthe use of artemisinin (ART) -based combination therapies (ACTs) for treatmentof uncomplicated malaria. ACTs are a co-formulation consisting of a potentartemisinin component that rapidly clears majority of the asexual parasitespaired with a longer acting partner drug that clears the residual parasites (20). WHO recommends the following ACTs:artemether-lumefantrine (AL), artesunate-amodiaquine, artesunate-mefloquine, dihydroartemisinin-piperaquine(DP), artesunate-pyronaridine and artesunate-sulphadoxine-pyrimethamine.
Thechoice of ACT to be used depends on the outcome of therapeutic efficacy studiesagainst the local circulating strains of P.falciparum. In Kenya, the first line treatment of uncomplicated malaria isAL 1.6Artemisinin Based Combination TherapiesArtemisinin,qinghaosu,is derived from the Chinese plant Artemisiaannua. The antimalarial activity of A.
annua, with its active ingredient being the sesquiterpene lactone (21), was discovered throughscreening of an array of medicines that were able to treat monkeys and mice withsimian and rodent malaria, respectively (22,23). Artemisinin has a poorbioavailability that limits its efficacy. Semisynthetic derivatives ofartemisinin (Figure 1-2): artemether, artesunate and dihydroartemisinin havebeen developed with a modified chemical structure and improved pharmacologicalproperties. In humans, these derivatives rapidly achieve optimum plasma levelsand have been shown to have elimination half-lives of approximately 1-3 hours. The 3-day ACTs treatment course reduces thenumber of asexual parasites by approximately 107-fold with an addedbonus of having gametocytocidal activity which is important in reducingtransmissibility of the parasite (24). Figure1?2: The chemical structure of the three artemisininderivatives.
Adapted from (25). 1.6.1Mechanism of Action of ACTsArtemisinins target immaturering forms as well as the mature trophozoite stages by effecting up to 10,000fold reductions in parasitemia every 48 hours (26). Artemisinin is activated by Fe2+ heme thatis produced in the process of hemoglobin digestion in the parasite foodvacuole, forming heme-artemisinin compounds invivo (27,28). Activated artemisinin releases carbon centered freeradicals that alkylate parasite biomolecules leading to cell death, as shown infigure 1.2.
In patients treated with artemisinin, reduced parasitemia from theblood stream has been associated with pitting (29,30). Pitting occurs in the spleen, with the removal of theintraerythrocytic parasites from the red blood cell as it crosses the theendothelial wall of the red pulp sinuses in the spleen (31). However, a small population of artemisinin treatedrings enter a state of dormancy rather than being killed, resuming growth onlyafter a period of days to weeks (32). 1.7 Dynamics of ACT resistanceDrug resistance limits the efficacy of manyantimalarial drugs, placing a significant strain on malaria control programs.Parasite resistance is a gradual process by which the parasite is able to withstandor multiply despite administration and absorption of the drug within limits ofhost tolerability.
The key driver of antimalarial drug resistance is thegenetic diversity of the malaria parasite. The development of resistance toantimalarials is a two-step process involving an initial genetic event thatproduces a mutant clone followed by a drug selection process of the arisingmutant. Such resistant-mutant parasites would be further selected uponadministration of antimalarials.
In some cases, changes in drug policy have recordeda resurgence of the sensitive phenotypes, confirming the phenomenon that mutantparasites have reduced biological fitness than sensitive parasites in theabsence of drug pressure (33,34). For this reason, WHOrecommends continued monitoring of the efficacy of ACTs byin vivo tests, in vitrotests and molecular genotyping of known antimalarial resistant markers forearly detection of resistance(35).This study attempts to genotype a number ofantimalarial resistant markers from field isolates obtained during an in vivo study.In vivo studies have been used to assess the effectiveness ofantimalarial drugs. Patients with malaria are recruited into a study andfollowed up at regular intervals. According to the standard WHO protocol, patientenrollment occurs on day 0 and follow-up visits take place on days 1, 2, 3, 7,14, 21, 28 and any time the patient is ill. Other studies have suggested a42-day follow-up to adequately capture the treatment failures after treatmentwith antimalarial drugs with a long plasma half-life(36).1.
8 The role of molecularmarkersEarly detection of antimalarial drug resistanceis greatly enhanced by the identification of molecular markers of resistance.These markers have been utilized to monitor the origin and spread ofantimalarial drug resistance, providing a better understanding to thepopulation dynamics of drug resistant genotypes. The sequencing and annotationof the P. falciparum genome has provided a platform for identifying genecandidates that can be linked to phenotypes such as drug resistance. Thepresence of polymorphisms, which have been selected due to drug pressure, havebeen exploited as markers of drug resistance. Such polymorphisms include:microsatellites, single nucleotide polymorphisms (SNP) and small insertions ordeletions (indels). In this study, we mainly focus on determining the pre- andpost-treatment prevalence of known resistance-mediating mutations in the k13 propeller-domain, Pfcrtand Pfmdr1.
1.8.1 Kelch 13 (k13)The k13gene is found on chromosome 13 with one exon encoding the K13 protein with 726amino acids and a molecular weight of 83.66kDa.
At the C-terminus end of theK13 protein there are six motifs where each motif is made up of 50 amino acidsthat form secondary structured beta-sheets. The six Kelch motifs are seen to foldinto a propeller domain to which multiple protein-protein interaction sites arefound (37). Mutations in the k13-propeller domain have been shown tobe the genetic correlates of in vivoand in vitro resistance toartemisinin in Southeast Asia (38).The first cases of artemisinin resistance weredisplayed as prolonged parasite clearance times of >90 hours, as compared tothe median of 52 hours for patients who were cured, in patients from theThailand-Cambodian border in 2008 after administration of artesunatemonotherapy(39).
A significantbreakthrough in understanding the genetic architecture of artemisinin resistantparasites came as a result of combining whole genome sequence data ofART-resistant and sensitive in vitroparasites and targeted gene Sanger sequence analysis of both resistant andsensitive parasites (38). Non-synonymousmutations at codons Y493H, R539H, I543T and C580Y were observed in the K13propeller domain and were associated with higher ring-stage parasite survival(RSA0-3h survival assay) rates as compared to the wild type.Additional gene editing studies using CRISPR-Cas9 were then carried out tovalidate the role of the C580Y mutation.
The C580Y mutation has been linked toincreased ring stage parasite survival of ~13.5% (40), almost similar to therate previously reported for the Cambodian resistant parasite isolate (38). Several epidemiological studies conducted inSoutheast Asia have identified multiple occurrences of mutations in the K13propeller domain that result in drug resistance (41–45). Non-synonymous K13mutations have also been identified in Africa but at very low frequencies,known as singletons (41), that have no impact onART efficacy. However, a recent study conducted in China reported a migrantworker with P. falciparum K13-variantinfection from Equatorial Guinea who displayed the delayed parasite clearancephenotype following several rounds of ACT treatment (46). Figure 1-3: TheStructure of the Kelch protein showing the six propeller domains and the fournon-synonymous mutations associated with higher ring-stage parasite survivalrates.
Adapted from (38)1.8.2 P. falciparum multidrug resistance protein 1 (Pfmdr1)ThePfmdr1gene is located on chromosome 5 with one exon encoding the P-glycoproteinhomolog 1 (Pgh-1) protein with 1419 amino acids and a molecular mass of62.25kDa. This gene encodes a digestive vacuole membrane-bound ATP-bindingcassette (ABC) transporter with two domains each consisting of 6 helicaltransmembrane domains.
Pgh-1 is locatedon the parasite food vacuole throughout the asexual cycle, where it has been suggestedto regulate the intracellular drug concentrations (47). Studies using fluorescein derivatives(Fluo-4) provide supporting evidence that PfMDR1 imports solutes, includingantimalarial drugs into the parasite’s food vacuole (48). In humans,P-glycoprotein polymorphisms are linked to resistance to cancer drugs (49). Polymorphisms and copy number variations of Pfmdr1 gene are a major determinant ofparasite resistance or susceptibility to a number of antimalarials (50).
The main mutations inPfMDR1 can be grouped into two: amino-terminal mutations that include N86Y(asparagine changing to a tyrosine), Y184F (tyrosine to a phenylalanine) and acarboxyl-terminal mutation D1246Y (aspartic acid to a tyrosine).The PfMDR1 polymorphisms have been affiliatedwith differential in vivo and in vitro parasite sensitivities to arange of antimalarials includingamodiaquine(51), mefloquine(52), lumefantrine(53) and artemisinin (52). Figure 1-4: The Structure of PfMDR1 showing the 12 transmembrane domains (shadedblue) and the known point mutations indicated as shaded circles. Adapted from (49). 1.8.3 P. falciparum chloroquine resistant transporter (pfcrt)The Pfcrtgene is localized on chromosome 7 encoding 424 amino acids with a molecularmass of 48.
6kDa (54). The PfCRT proteinbelongs to the drug transporter superfamily with 10 putative transmembranedomains spanning the digestive vacuole membrane of the parasite (see Figure 1-3)(55). Mutations in the Pfcrtgene are a key determinant of CQresistant (CQR) both in-vivo and in-vitro(54,56). Analysis of the geneticcross between the CQ-sensitive HB3 isolate and the CQ-resistant Dd2 isolateprovided conclusive evidence that pfcrtis the primary determinant of CQR (57).
A number of studiescomparing the wild and mutant pfcrtallele have shown less CQ accumulation inside the parasite vacuole of the mutant pfcrt(58,59). CQR is linked to 15 different polymorphisms in the pfcrt gene. Mutationson codons 72-76 have been used to distinguish the two different geographicalhaplotypes: CVIET (South-East Asia and Africa) and SVMNT (South America and SoutheastAsia) (60). The causalmutation in pfcrt a change from Lysine (K) to Threonine (T) at codon 76is used as the molecular marker for CQR(60). Figure 1?5: The Structure of PfCRT showing the 10 transmembrane domains (shaded blue) and the known point mutations indicated as shaded circles. Adapted from (49). 1.9 The role of othermolecular markers A number of P.
falciparum genes are known to be highly polymorphic. The occurrence of suchgenes has been exploited in assessing parasite populations in the human host.The probability of a patient, especially in a malaria endemic area, beinginfected with the same P. falciparum genotypeto the former is low(61). The genetic diversityof P. falciparum is performed bygenotyping highly deiverse antigenic markers such as RII repeat of theglutamate rich protein (GLURP) and the merozoite surface proteins (msp1 and msp2) (62). Therefore, by comparingthe genotypes of such loci before treatment and the time of parasiterecurrence, should provide a distinction between recrudescent and newinfections (61).
A number of drug trialshave used this strategy to correct the outcomes of drug efficacy trials (63–65). However, it isimportant to note that the discriminatory power of these loci is dependent onthe polymorphisms, the reported frequency of each allele within the populationand the genotyping protocol used (66,67).1.9.1 msp2The merozoite surface protein-2 is a 45kDaglycoprotein anchored in the merozoite surface by a glycophosphatidylinositol(GPI) anchor and is one of the most abundant proteins on the surface of themerozoite gene (68).
Sequence variation hasbeen detected in the msp2 gene forboth laboratory maintained P. falciparum(69) and field isolates (70–72). The comparison of the msp2 sequences from the two isolate classesreveals highly conserved 5′ and 3′ sequences that flank a central variableregion, block 3 that includes both repetitive and non-repetitive sequences.The non-repetitive allelic families areclassified into two forms that define two allelic families, FC27 and 3D7/IC (69). Each allelic family has its unique pattern ofrepetitive sequences. The FC27 allele-family shares a 12-mer and 32-mer repeatregion, whereas the 3D7 allele-family share a 4-mer repeat sequence. Bothallele families have an N- and C-terminal conserved region with a GPI-anchorfor attachment to the merozoite surface at the C-terminal end (see Figure 1-5).
Figure 1-6: The Structure of the FC27 and3D7 allelic families found on block 3 of the msp2 gene. Adapted from (73).1.9.2 glurpThe glutamate-rich protein (glurp) encodes a 220kDa protein that is expressed at thepre-erythrocytic and erythrocytic stages of the parasite life cycle (74). GLURP contains an N-terminal non-repeat region (R0), a central repeat region (R1) and theC-terminal immunodominant region (R2), as shown in Figure 1-6. The R0 region has beenshown to be highly conserved and elicits stable antibody responses over time (75,76), while the R2 region isgenetically heterogenous(77).
The glurpgene is known to be less genetically diverse, as reported by studies indifferent geographic regions, but highly immunogenic. Polymorphisms in the glurp gene are mainly due to variationsin the numbers of nucleotide repeats, which in turn affects the gene size.Given that a single gene-variant is found in the blood stage of the parasite,the presence of more than one allele confirms a multiclonal infection. Figure1-7: The Protein structure of theglutamate rich protein. Adapted from (78) 1.10 JustificationDespite a recorded 60% decrease in mortalityrates between 2000 and 2015, the spread of drug-resistant malaria parasites hasproven to be one of the greatest challenges to malaria control. This is partlyowing to the use of low quality drugs or counterfeit drugs and poorpatient-treatment compliance.
Following the recommendation of ACT use in 2006by WHO, the increasing deployment of ACTs has been one of the main factorsbehind the reduction in malaria. Unfortunately, over the last decade evidencehas grown that artemisinin resistance has emerged and is spreading withinSoutheast Asia. Southeast Asia has long been considered the epicenter ofantimalarial drug resistance; resistance to chloroquine, proguanil,sulpohadoxine-pyrimethamine, mefloquine and piperaquine has emerged there andhas spread globally. Even though artemisinin resistance has not yet beendocumented in Africa, monitoring parasite resistance to artemisinins and to ACTpartner drugs is crucial for malaria control programs. WHO (2015) recommendscontinued monitoring of resistant markers after every 2 years for earlydetection of artemisinin resistance, to ensure the first-line treatments arestill effective and for prompt changes in national treatment policies. Whenchoosing the ideal ACT for a specific region, validated antimalarial resistancemarkers provide useful data that complement the results of clinical trials and in vitro studies.
The use of molecularmarkers such as Pfk13, Pfmdr1 and Pfcrt is essential for malaria surveillance programs.