Malaria is a disease transmitted by the female Anopheles mosquito and caused by an
intracellular protozoan parasite of the genus Plasmodium. There are five Plasmodium
species 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 dominant
species 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 illness
but are rarely life-threatening (2,3). The P. falciparum genome is made up of 22.8 megabases (Mb) spread
across its 14 chromosomes, a nucleotide content of 80% A+T that rises to
90% in introns and approximately 5300
proteins (4). Comparative genome analysis shows that
there is synteny between the different Plasmodium
species with the exception of genes located in the teleromic regions that are
known to be involved in antigenic variation and immune evasion.
The Epidemiology of Malaria
It is difficult to estimate the burden of malaria especially in
low-income countries due to the inconsistencies in data collection and also
because not all infections progress to disease manifestation. In 2002, there
were 515 million cases of P. falciparum malaria reported globally with
the majority of these cases concentrated in sub-Saharan Africa (70%) (2). A recent World malaria report (5) estimates that there were 212 million
malaria cases and 42,900 deaths in 2015. Infection rates are higher in children
under five years, pregnant women, HIV/AIDS patients and migrants or mobile
populations who have limited access to prevention and diagnostic tests.
Cycle of Plasmodium falciparum
The life cycle of P. falciparum involves two cycles: the
asexual and sexual cycles that occur in the human and Anopheles
mosquito, respectively as shown in Figure 1-1. Malaria begins when an infected
mosquito bites a human host thereby injecting sporozoites into the dermis (6,7). The highly motile sporozoites travel
from the bite site to the liver via the bloodstream and start their
intracellular development in the hepatocyte in a process known as
exoerythrocyticschizogony. Sporozoites in the hepatocyte begin dividing into
schizonts. Each schizont gives birth to thousands of merozoites which are then
released into the bloodstream. This is a clinically silent stage with the
result being a 10,000 fold amplification of parasite numbers and may take
approximately 2-10 days, depending on the parasite species (8). P. vivaxand P. ovaleremain
dormant in the liver and are reactive weeks to months after the primary
Mature merozoites from hepatocytes invade the red blood cells
marking the start of clinical disease. Over a period of 44-72 hours, the
merozoites undergo development from ring stages to mature trophozoites and
finally schizonts. Each schizont gives rise to 10-30 merozoites which are seen
to circulate through the blood stream when the erythrocyte ruptures. Circulating
merozoites are able to re-invade other uninfected red blood cells (uRBC) in the
end leading to an exponential increase in parasite densities as high as 1010
parasites per host.
Asexual erythrocytic parasites are primarily responsible for
febrile illness associated with fever and chills but may develop into severe
disease affecting organs. After at least two asexual cycles, a small proportion
of merozoites (1%) develop into macrogametes (female) and microgametes (male)
sexual forms in a process known as gametocytogenesis. These sexual forms of the
parasite are known to be infective to mosquitoes and develop as a result of
host environmental stress or antimalarial drug usage.
The sexual cycle starts after a female Anopheles mosquito feeds on an infected human host. Following a
blood meal, the mosquito ingests micro- and macrogametes that are taken to the
mosquito’s midgut. The male gamete fertilizes the female gamete forming a diploid
zygote. 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 a
motile ookinete that perforates the midgut cell and becomes an oocyte. The
oocyte undergoes several mitotic divisions to form sporoblasts after which
budding takes place 10-14 days later leading to the production of sporozoites
in a process called sporogony. These midgut sporozoites move to the mosquito’s
salivary 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).
Infection with P. falciparum
can either result in uncomplicated malaria, asymptomatic infection or severe
malaria. In malaria-endemic areas, continuous exposure to P. falciparum infection early in life results in an unsterile
development of clinical immunity later in life. In such environments it is
therefore common to find adults with circulating parasites but without any
evident symptoms. These kinds of infections are known as asymptomatic
infections. This is in contrast with malaria infections in low transmission
areas, where there is little acquired immunity and P. falciparum infection leads to symptomatic disease in all age
groups. Young children are however prone to symptomatic infections mainly due
to their underdeveloped humoral response to pathogens. Approximately 99% of
infected individuals have uncomplicated malaria with non-specific symptoms such
as malaise, joint pains and headaches at 48 hour intervals which can be easily
confused with other diseases (13). Due to reasons such as delayed or
inappropriate treatment and low immunity, approximately 0.2-0.5% of P. falciparum infections develop into a
more severe form of disease (14). Common complications of severe malaria
include: cerebral malaria, respiratory failure and severe anaemia or even lead
to a coma (13).
Interventions for Control and Elimination of Malaria
Prevention of malaria
Current strategies for the control of malaria mainly depend on the
use of insecticide-treated nets (ITNs), indoor residual spraying (IRS) and
artemisinin based combination therapies (ACT). These strategies have resulted
in reduced mortality rates by 60% in Africa between the years 2000 and 2015 (15), not excluding the positive impact of
improved quality of health systems and improved case management due to enhanced
The most effective malaria prevention strategies recommended for
sub-Saharan Africa are ITNs and IRS. ITNs not only provide a physical barrier
between the mosquito and human, but also repel and kill mosquitoes because they
have embedded insecticides. There is evidence that a wide scale use of ITNs
provides some extended level of protection to non-ITN users, as it helps reduce
the overall malaria transmission (16). IRS involves spraying insecticides on
the ceilings, walls and other indoor resting places of mosquitoes. In most
cases, sleeping and living rooms of a household are targeted for spraying.
Countries adopting the use of either ITNs or IRS or in combination, have
reported promising declines in malaria related morbidity and mortality (17,18).
Chemotherapy for Treatment
For many years, chloroquine
(CQ) has been the major drug for the treatment of uncomplicated malaria. The
appearance of CQ-resistance (CQR) in Southeast Asia and its spread to Africa
and Latin America led to the introduction of sulphadoxine-pyrimethamine (SP) to
which parasites developed an even more rapid resistance (19). To reduce the pace of selection of resistance, WHO prescribes
the use of artemisinin (ART) -based combination therapies (ACTs) for treatment
of uncomplicated malaria. ACTs are a co-formulation consisting of a potent
artemisinin component that rapidly clears majority of the asexual parasites
paired 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. The
choice of ACT to be used depends on the outcome of therapeutic efficacy studies
against the local circulating strains of P.
falciparum. In Kenya, the first line treatment of uncomplicated malaria is
Artemisinin Based Combination Therapies
is derived from the Chinese plant Artemisia
annua. The antimalarial activity of A.
annua, with its active ingredient being the sesquiterpene lactone (21), was discovered through
screening of an array of medicines that were able to treat monkeys and mice with
simian and rodent malaria, respectively (22,23). Artemisinin has a poor
bioavailability that limits its efficacy. Semisynthetic derivatives of
artemisinin (Figure 1-2): artemether, artesunate and dihydroartemisinin have
been developed with a modified chemical structure and improved pharmacological
properties. In humans, these derivatives rapidly achieve optimum plasma levels
and have been shown to have elimination half-lives of approximately 1-3 hours. The 3-day ACTs treatment course reduces the
number of asexual parasites by approximately 107-fold with an added
bonus of having gametocytocidal activity which is important in reducing
transmissibility of the parasite (24).
Figure1?2: The chemical structure of the three artemisinin
derivatives. Adapted from (25).
Mechanism of Action of ACTs
Artemisinins target immature
ring forms as well as the mature trophozoite stages by effecting up to 10,000
fold reductions in parasitemia every 48 hours (26). Artemisinin is activated by Fe2+ heme that
is produced in the process of hemoglobin digestion in the parasite food
vacuole, forming heme-artemisinin compounds in
vivo (27,28). Activated artemisinin releases carbon centered free
radicals that alkylate parasite biomolecules leading to cell death, as shown in
figure 1.2. In patients treated with artemisinin, reduced parasitemia from the
blood stream has been associated with pitting (29,30). Pitting occurs in the spleen, with the removal of the
intraerythrocytic parasites from the red blood cell as it crosses the the
endothelial wall of the red pulp sinuses in the spleen (31). However, a small population of artemisinin treated
rings enter a state of dormancy rather than being killed, resuming growth only
after a period of days to weeks (32).
1.7 Dynamics of ACT resistance
Drug resistance limits the efficacy of many
antimalarial drugs, placing a significant strain on malaria control programs.
Parasite resistance is a gradual process by which the parasite is able to withstand
or multiply despite administration and absorption of the drug within limits of
host tolerability. The key driver of antimalarial drug resistance is the
genetic diversity of the malaria parasite. The development of resistance to
antimalarials is a two-step process involving an initial genetic event that
produces a mutant clone followed by a drug selection process of the arising
mutant. Such resistant-mutant parasites would be further selected upon
administration of antimalarials. In some cases, changes in drug policy have recorded
a resurgence of the sensitive phenotypes, confirming the phenomenon that mutant
parasites have reduced biological fitness than sensitive parasites in the
absence of drug pressure (33,34). For this reason, WHO
recommends continued monitoring of the efficacy of ACTs byin vivo tests, in vitro
tests and molecular genotyping of known antimalarial resistant markers for
early detection of resistance(35).
This study attempts to genotype a number of
antimalarial resistant markers from field isolates obtained during an in vivo study.In vivo studies have been used to assess the effectiveness of
antimalarial drugs. Patients with malaria are recruited into a study and
followed up at regular intervals. According to the standard WHO protocol, patient
enrollment 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 a
42-day follow-up to adequately capture the treatment failures after treatment
with antimalarial drugs with a long plasma half-life(36).
1.8 The role of molecular
Early detection of antimalarial drug resistance
is greatly enhanced by the identification of molecular markers of resistance.
These markers have been utilized to monitor the origin and spread of
antimalarial drug resistance, providing a better understanding to the
population dynamics of drug resistant genotypes. The sequencing and annotation
of the P. falciparum genome has provided a platform for identifying gene
candidates that can be linked to phenotypes such as drug resistance. The
presence of polymorphisms, which have been selected due to drug pressure, have
been exploited as markers of drug resistance. Such polymorphisms include:
microsatellites, single nucleotide polymorphisms (SNP) and small insertions or
deletions (indels). In this study, we mainly focus on determining the pre- and
post-treatment prevalence of known resistance-mediating mutations in the k13 propeller-domain, Pfcrtand Pfmdr1.
1.8.1 Kelch 13 (k13)
gene is found on chromosome 13 with one exon encoding the K13 protein with 726
amino acids and a molecular weight of 83.66kDa. At the C-terminus end of the
K13 protein there are six motifs where each motif is made up of 50 amino acids
that form secondary structured beta-sheets. The six Kelch motifs are seen to fold
into a propeller domain to which multiple protein-protein interaction sites are
found (37). Mutations in the k13-propeller domain have been shown to
be the genetic correlates of in vivo
and in vitro resistance to
artemisinin in Southeast Asia (38).
The first cases of artemisinin resistance were
displayed as prolonged parasite clearance times of >90 hours, as compared to
the median of 52 hours for patients who were cured, in patients from the
Thailand-Cambodian border in 2008 after administration of artesunatemonotherapy(39). A significant
breakthrough in understanding the genetic architecture of artemisinin resistant
parasites came as a result of combining whole genome sequence data of
ART-resistant and sensitive in vitro
parasites and targeted gene Sanger sequence analysis of both resistant and
sensitive parasites (38). Non-synonymous
mutations at codons Y493H, R539H, I543T and C580Y were observed in the K13
propeller 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 to
validate the role of the C580Y mutation. The C580Y mutation has been linked to
increased ring stage parasite survival of ~13.5% (40), almost similar to the
rate previously reported for the Cambodian resistant parasite isolate (38).
Several epidemiological studies conducted in
Southeast Asia have identified multiple occurrences of mutations in the K13
propeller domain that result in drug resistance (41–45). Non-synonymous K13
mutations have also been identified in Africa but at very low frequencies,
known as singletons (41), that have no impact on
ART efficacy. However, a recent study conducted in China reported a migrant
worker with P. falciparum K13-variant
infection from Equatorial Guinea who displayed the delayed parasite clearance
phenotype following several rounds of ACT treatment (46).
Figure 1-3: The
Structure of the Kelch protein showing the six propeller domains and the four
non-synonymous mutations associated with higher ring-stage parasite survival
rates. Adapted from (38)
1.8.2 P. falciparum multidrug resistance protein 1 (Pfmdr1)
gene is located on chromosome 5 with one exon encoding the P-glycoprotein
homolog 1 (Pgh-1) protein with 1419 amino acids and a molecular mass of
62.25kDa. This gene encodes a digestive vacuole membrane-bound ATP-binding
cassette (ABC) transporter with two domains each consisting of 6 helical
transmembrane domains. Pgh-1 is located
on the parasite food vacuole throughout the asexual cycle, where it has been suggested
to regulate the intracellular drug concentrations (47). Studies using fluorescein derivatives
(Fluo-4) provide supporting evidence that PfMDR1 imports solutes, including
antimalarial 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 of
parasite resistance or susceptibility to a number of antimalarials (50). The main mutations in
PfMDR1 can be grouped into two: amino-terminal mutations that include N86Y
(asparagine changing to a tyrosine), Y184F (tyrosine to a phenylalanine) and a
carboxyl-terminal mutation D1246Y (aspartic acid to a tyrosine).
The PfMDR1 polymorphisms have been affiliated
with differential in vivo and in vitro parasite sensitivities to a
range of antimalarials including
amodiaquine(51), mefloquine(52), lumefantrine(53) and artemisinin (52).
Figure 1-4: The Structure of PfMDR1 showing the 12 transmembrane domains (shaded
blue) and the known point mutations indicated as shaded circles. Adapted from (49).
1.8.3 P. falciparum chloroquine resistant transporter (pfcrt)
gene is localized on chromosome 7 encoding 424 amino acids with a molecular
mass of 48.6kDa (54). The PfCRT protein
belongs to the drug transporter superfamily with 10 putative transmembrane
domains spanning the digestive vacuole membrane of the parasite (see Figure 1-3)
(55). Mutations in the Pfcrtgene are a key determinant of CQ
resistant (CQR) both in-vivo and in-vitro(54,56). Analysis of the genetic
cross between the CQ-sensitive HB3 isolate and the CQ-resistant Dd2 isolate
provided conclusive evidence that pfcrt
is the primary determinant of CQR (57). A number of studies
comparing the wild and mutant pfcrt
allele 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. Mutations
on codons 72-76 have been used to distinguish the two different geographical
haplotypes: CVIET (South-East Asia and Africa) and SVMNT (South America and Southeast
Asia) (60). The causal
mutation in pfcrt a change from Lysine (K) to Threonine (T) at codon 76
is 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 other
A number of P.
falciparum genes are known to be highly polymorphic. The occurrence of such
genes has been exploited in assessing parasite populations in the human host.
The probability of a patient, especially in a malaria endemic area, being
infected with the same P. falciparum genotype
to the former is low(61). The genetic diversity
of P. falciparum is performed by
genotyping highly deiverse antigenic markers such as RII repeat of the
glutamate rich protein (GLURP) and the merozoite surface proteins (msp1 and msp2) (62). Therefore, by comparing
the genotypes of such loci before treatment and the time of parasite
recurrence, should provide a distinction between recrudescent and new
infections (61). A number of drug trials
have used this strategy to correct the outcomes of drug efficacy trials (63–65). However, it is
important to note that the discriminatory power of these loci is dependent on
the polymorphisms, the reported frequency of each allele within the population
and the genotyping protocol used (66,67).
The merozoite surface protein-2 is a 45kDa
glycoprotein anchored in the merozoite surface by a glycophosphatidylinositol
(GPI) anchor and is one of the most abundant proteins on the surface of the
merozoite gene (68). Sequence variation has
been detected in the msp2 gene for
both laboratory maintained P. falciparum(69) and field isolates (70–72). The comparison of the msp2 sequences from the two isolate classes
reveals highly conserved 5′ and 3′ sequences that flank a central variable
region, block 3 that includes both repetitive and non-repetitive sequences.
The non-repetitive allelic families are
classified into two forms that define two allelic families, FC27 and 3D7/IC (69). Each allelic family has its unique pattern of
repetitive sequences. The FC27 allele-family shares a 12-mer and 32-mer repeat
region, whereas the 3D7 allele-family share a 4-mer repeat sequence. Both
allele families have an N- and C-terminal conserved region with a GPI-anchor
for attachment to the merozoite surface at the C-terminal end (see Figure 1-5).
Figure 1-6: The Structure of the FC27 and
3D7 allelic families found on block 3 of the msp2 gene. Adapted from (73).
The glutamate-rich protein (glurp) encodes a 220kDa protein that is expressed at the
pre-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 the
C-terminal immunodominant region (R2), as shown in Figure 1-6. The R0 region has been
shown to be highly conserved and elicits stable antibody responses over time (75,76), while the R2 region is
gene is known to be less genetically diverse, as reported by studies in
different geographic regions, but highly immunogenic. Polymorphisms in the glurp gene are mainly due to variations
in 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.
1-7: The Protein structure of the
glutamate rich protein. Adapted from (78)
Despite a recorded 60% decrease in mortality
rates between 2000 and 2015, the spread of drug-resistant malaria parasites has
proven to be one of the greatest challenges to malaria control. This is partly
owing to the use of low quality drugs or counterfeit drugs and poor
patient-treatment compliance. Following the recommendation of ACT use in 2006
by WHO, the increasing deployment of ACTs has been one of the main factors
behind the reduction in malaria. Unfortunately, over the last decade evidence
has grown that artemisinin resistance has emerged and is spreading within
Southeast Asia. Southeast Asia has long been considered the epicenter of
antimalarial drug resistance; resistance to chloroquine, proguanil,
sulpohadoxine-pyrimethamine, mefloquine and piperaquine has emerged there and
has spread globally. Even though artemisinin resistance has not yet been
documented in Africa, monitoring parasite resistance to artemisinins and to ACT
partner drugs is crucial for malaria control programs. WHO (2015) recommends
continued monitoring of resistant markers after every 2 years for early
detection of artemisinin resistance, to ensure the first-line treatments are
still effective and for prompt changes in national treatment policies. When
choosing the ideal ACT for a specific region, validated antimalarial resistance
markers provide useful data that complement the results of clinical trials and in vitro studies. The use of molecular
markers such as Pfk13, Pfmdr1 and Pfcrt is essential for malaria surveillance programs.