INTRODUCTION Plasmodium spp which tainting humans. (Hymel and Yang,


Malaria is a protozoan parasitic infection triggered
by Plasmodium spp. which is usually spread by Anopheles spp. Plasmodium
falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium ovale and
Plasmodium malariae are the Plasmodium spp which tainting humans. (Hymel and
Yang, 2008).

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The most common and the lethal
human malaria parasite is P.
falciparum,which is spread by mosquitoes genus Anopheles. . Even
with widespread malaria eradication efforts, 214 million cases of malaria and
438,000 deaths were valued globally in 2015, which largely affected the
sub-Saharan African population and children under 5 years of age. Between 2000
and 2015, malaria frequency and death rates reduced worldwide by 37% and 60%,
respectively.Worldwide the malaria cases are decreases due to extensive usage
of insecticide-treated bed nets (ITN), indoor residual spraying (IRS), larval control,
improved diagnostic testing and treatment by artemisinin- combination therapy
(ACT). (WHO ,2015).  In a country like
Pakistan to which malaria is endemic, continuous molecular surveillance of the
field isolates is required to know the pattern of existing and emerging drug
resistance. This knowledge is the key to effective malaria control program.

Genetic diversity of P.
falciparum plays a crucial role in defining the strength of malaria
transmission. Several P. falciparum genes show wide-ranging genetic
polymorphism however, high polymorphism has been shown in Merozoite surface
proteins 1 and 2 (MSP-1 and MSP-2) and
Glutamate Rich Protein (GLURP) in different geographical locations in malaria
endemic areas. MSP-1, MSP-2 and GLURP genes are broadly used to study the
allelic diversity and frequency of P. falciparum (Mwingira et al.,

cycle of P.falciparum

The complex life cycle of malaria parasite involve an insect
vector (mosquito) and a vertebrate host (human). Humans infect by four species
of plasmodium : P. falciparum, P. vivax, P. malarriae and p.
ovale. All the  four species display
a similar life cycle with only slight dissimilarities (NIH ,2007). Plasmodium vivax & P. ovale have 14 days of incubation period, P.
falciparum 12 days while P. malariae has 30 days of incubation
period (Francis et al., 2010). When
the feeding mosquito infuse sporozoites with saliva the infection is started,
carries to blood circulatory system. Sporozoites are carried by the circulatory
system to the liver and attack the liver cells . The intracellular parasite
undergoes an asexual replication with in the liver cells known as exoerythrocytic schizogony. Merozoites are released into the blood stream
when exoerythrocytic schizogony terminates (vaughan et al., 2017). A part
of the liver-stage parasites from P. vivax and P. ovale go through
a dormant period immediately instead of undergoing asexual replication. These hypnozoites will reactivate after the
primary infection for several weeks to months (or years)  and are responsible for relapses. Merozoites
attack red blood cells enlarge parasite when  undergo through trophic period. The early
trophozoite is frequently referred to as ‘ring form’ due to its morphology. Active metabolism enlarge
trophozoite including the host cytoplasm ingestion and hemoglobin
proteolysis into amino acids. Multiple rounds of nuclear division are shown at
the end of trophic period without cytokinesis resulting is a schizont. Mature schizont of merozoite
bud, also called a segmenter,
and in the rupture infected erythrocytes merozoites are released. Another
round of the blood-stage  reinitiate replicative
cycle resulting invade erythrocytes. The pathology of malaria is related
with the blood stage. The recurrent fever paroxyms is the cause of the
synchronous lysis of the infected erythrocytes. P. malariae shows a 72
hour periodicity, and the other three species show 48 hour cycles. However,
P. falciparum often displays an unceasing fever rather than the periodic
paroxyms. P. falciparum is also responsible for more morbidity and
mortality than the other three species. The higher levels of parasitemia is a part of increase
virulence associated with P. falciparum infections (Arnot et al., 2011).
In addition, more complications are associated with P. falciparum because
of the sequestration of the trophozoite- and schizont-infected
erythrocytes in the deep tissues. As an alternative to the asexual replicative
cycle, the parasite can differentiate into sexual forms known as macro- or microgametocytes. The gametocytes are large parasites which fill
up the erythrocyte, but only contain one nucleus. Ingestion of
gametocytes by the mosquito vector induces gametogenesis (i.e., the
production of gametes) and escape from the host erythrocyte. Factors
which participate in the induction of gametogenesis include: a drop in
temperature, an increase in carbon dioxide, and mosquito metabolites. Microgametes, formed by a process known
as exflagellation, are flagellated forms which will fertilize the macrogamete  leading to a zygote . The zygote develops into a motile ookinete which penetrates the gut
epithelial cells and develops into an oocyst. The oocyst undergoes multiple rounds of asexual
replication resulting in the production of sporozoites. Rupture of the mature oocyst releases the sporozoites
into the hemocoel (i.e., body cavity) of the mosquito. The sporozoites migrate
to and invade the salivary glands, thus completing the life cycle (Talman et al., 2004).

Proteins on the merozoite surface

Early electron microscope discovered that Plasmodium merozoites
were enclosed in a ‘fuzzy’ fibrillar coat of surface proteins; remarkably, this
coat seemed shed during RBC invasion (Aikawa et al., 1978; Ladda et al.,
1969; Langreth et al., 1978 ;  Bannister et al., 1975). Since these early
observations, the function and composition and of merozoite surface proteins
(MSPs) has been of great interest because of extensive role in invasion of red
blood cells and potential as vaccine candidates antigens (Richards et al., 2009)
and, more recently, as drug targets for stop blood-stage replication (Boyle et
al., 2013; Chandramohanadas et al., 2014; Wilson et al.,2015).

Merozoites appears a fibrillar surface
coat to be basically composed of glycosylphosphatidy inositol (GPI) an anchored
proteins, and the peripherally associated surface proteins with integral
membrane proteins representing a small part  of the total surface protein (Gilson et
al., 2006). Up to date many GPI anchored merozoite surface proteins (MSPs)
have been recognized: these include proteins formally known as MSPs (MSP-1, MSP-2,
MSP-4, MSP-5 and MSP-10) and the 6-cysteine domain family proteins, Pf92, Pf38
and Pf12 (Sanders et al., 2005). In addition, other GPI-anchored
proteins, rhoptry associated membrane antigen (RAMA) microneme associated
cysteine-rich protective antigen (CyRPA) (Reddy et al, 2015 ; Topolska et
al., 2004),) and GPI-anchored micronemal antigen (GAMA) (Arumugamet et al.,
2011) migrate to the merozoite surface from organelles during, invasion
of  red blood cells. While many of these
proteins contain binding domains like cysteine-rich EGF and other different  globular domains prophesied function of  mediate receptor binding during the primary
recognition and attachment to RBCs, very little experimental evidence are
insufficient support this function. 
Exceptionally little understanding is available about the functions and  interactions of GPI-anchored surface proteins;
clearly this is an area leads to great advances. Most merozoite surface
GPI-anchored proteins appear to be reported refractory to genetic disruption (MSP-5,Pf-38 and Pf-12
have been successfully disrupted and  play chief role in merozoite invasion. (Arumugam
et al., 2011; Reddy et al., 2015; Sanders et al., 2006 ).

surface protein-1

Merozoite surface protein-1 (MSP-1) is a
major surface protein of P.falciparum,
with an estimated molecular size of 190 kDa which  plays a key role in erythrocyte invasion by
the merozoite (Conway et al., 2000). MSP1
is considered a protein of  high
molecular mass that experiences most  proteolytic processing prior to egress of the
merozoite from the schizont. This processing alter the secondary struction of
MSP1 so that it can fix with spectrin and rupture  RBC (Das et al., 2015). MSP1 is generally observed as dimorphic but it is highly
polymorphic with large polymorphisms across the protein, predominantly in the P.falciparum isolates MSP133 (with two
allelic groups) and MSP1-block 2 regions (with three allelic groups), whereas
the C-terminal MSP119 region is relatively conserved (Barry et al., 2009;
Miller et al. 1993; Holder et al., 2009).The protein is a main target of
human immune responses and is a useful candidate for a blood stage subunit
vaccine (Holder et al., 1999). The
MSP- 1 gene with 7 variable block are separated by conserved and semi-conserved
regions. A region near the N-terminal of the MSP-1 gene Block-2, is the more polymorphic
part of protein and leads strong diversifying selection within natural
populations (Takala et al., 2002). Up
to now, four different types of  alleles
are identified block 2: MAD20, K1, and MR (Happi et al., 2004).

surface protein-2

 MSP2, is another second important GPI anchored
merozoite surface protein with approximately 25 kDa  (Gilson et al., 2006).
MSP2 is another leading antigen subunit of P. falciparum for malaria
vaccine (Happi et al., 2004). It
consists of highly polymorphic central repeats flanked with conserved N- and
C-terminal domains unique variable domains. The MSP-2 has generally two alleles
types, FC27 and 3D7, with different dimorphic structure considerably of the
variable central region, block-3 . The specific region of strains is consists
of repeating units; 3D7 allele  contain
repeating units of Ser, Gly and Ala, while FC27 allelic  forms contain 32-, 12- and 8-mer sequence
repeats. Both allelic forms of MSP2 are basically unstructured, but full length
recombinant proteins under physiological conditions make fibrils (Adda et
al., 2009). Fibril development is mediated through the region of N-terminal
(Low et al., 2007) and this region may also have the properties membrane
interaction(Zhang et al. 2008). It is called whether native fibril like
form of  MSP2 or other complexes;
however, there is some evidence that MSP2 oligomers are placed on merozoites
surface with a number of  MSP-2
interactions molecules being hypothesized (Yang et al., 2010). Recent
studies recommend that the MSP-2, N-terminal region may interact with the lipid
membrane of the merozoite surface (MacRaild et al,. 2012 ; (Adda et
al., 2009)). MSP2 appears to be important for invasion and during invasion retained
on the surface and  soon after degraded
when invasion is complete. However, its exact character is unknown, and no interaction
of receptor ligand or fixing of MSP2 to RBCs have been defined.  MSP-1 and
MSP- 2 genes  because of polymorphic
characters  have been characterized as
polymorphic markers in studies of malaria transmission dynamics in natural
isolatesof P. falciparum (Ferreira
et al., 2007 ; (Boyle et al., 2014)

Rich Protein

Glutamate Rich Protein
(GLURP) is a 220-kDa exoantigen
on the merozoite surface found in the parasitophorous vacuole. The single
full-length GLURP sequence available to date (strain F32) shows two amino acid
repeat regions (R1 and R2) with degenerate repeat motifs found in both.
Diversity in GLURP has been indicated by different sized polymerase chain
reaction (PCR) products from the R2 region of various laboratory-adapted and
field strains.The glutamate- rich protein (GLURP) is expressed in all stages of
the Plasmodium falciparum life cycle in humans (Stricker et al., 2000). GLURP
contains an N-terminal non-repeat region (R0), a central repeat region (R1) and
an immunodomi­nant C-terminal repeat region (R2). GLURP is highly antigenic and
there are few polymorphisms in the gene encoding GLURP in P. falciparum isolates
from differ­ent geographic regions (Theisen et
al., 1995). GLURP polymorphisms mainly involve variations in the numbers of
repeats of certain genomic sequences that therefore affect the size of the gene
and its protein product. Given that a single variant of the gene is found
during the blood stages of the parasite, the presence of more than one allele
represents a multiclonal infection (Stricker et al., 2000).


The genome of P.
falciparum consists of 14 linear chromosomes with a total of 25–30 megabases of
nuclear DNA with approximately 5000 genes, a mitochondrial fragment of a 6 kb
repeat element, and a circular element of 35 kb within the apicoplast. As a
consequence of continuous deletions and crossing-over and rearrangement events
occurring preferably at their telomeric regions, the chromosomes differ
considerably in size (Corcoran et al. 1986). The genome is extremely A/ T rich
(80%), which has led to difficulties in conventional sequencing strategies
because of the instability of genomic fragments in bacterial Escherichia coli
clones. Meanwhile, several yeast artificial clone (YAC) constructs have been
established, allowing for a stable maintenance of  P. falciparum clone fragments. The P. falciparum
genome is now subject of a large DNA-sequencing project, the Malaria Genome
Project, which was established in 1996. Contig arrays and restriction maps have
been produced for mapping of complete chromosomes (Rubio et al. 1995).


The inherent variability
of P. falciparum provides multiple effective immune evasion and drug resistance
mechanisms for the parasite. Many of the studies on the parasite’s polymorphism
have focused on variants exhibiting mutations that lead to amino acid
substitutions (non-synonymous mutations) that are likely subjected to
selection, such as immunogenic proteins and resistance phenotypes. Other studies
have examined sequences which are rather unlikely to be subjected to adaptive
pressure and therefore allow considerations on the phylogeny and the age of the
parasite. Polymorphism of the P. falciparum genome has mainly evolved through
DNA rearrangements such as gene duplication events, gene conversions,
translocations, deletions and insertions (Wellems et al., 1990; Kemp 1992;
Deitsch et al., 1997). Single nucleotide polymorphisms (SNPs) contribute
largely to the variability (Rich & Ayala 2000). Genotyping of
polymorphisms is not only a tool for the description of a distinct clonal
parasite strain, but also for defining multiplicity of infections with clonally
variable P. falciparum strains. Genotyping in field studies mostly allows only
an approximation to the estimation of different strains in individual infections.
The precise estimation of the number of parasite clones is complicated by the
high proportion of low-parasitemic P. falciparum infections (e.g. Roper et al.,
1996; May et al., 2000). Genes where polymorphism has arisen through intragenic
recombination in repetitive segments are characterized by repeat motifs with
length variability differing between strains. Among these genes are those
encoding the P. falciparum circumsporozoite protein (Arnot et al., 1993), a
glutamate-rich protein (GLURP) (Borre et al., 1991), two merozoite surface
proteins (MSP-1 and MSP-2) (Kimura et al. 1990; Fenton et al. 1991) and the
apical membrane antigen AMA-1 (Marshall et al. 1996).


Parasite antimalarial
multidrug resistance is a main reason for treatment failure and recrudescence.
Resistance to CQ, mefloquine, halofantrine, quinine, sulpha drugs (e.g.
sulfadoxine), folate antagonists (e.g. pyrimethamine, pro-/ cycloguanil), as
well as to the recently launched atovaquone

has been reported. Although recurrent parasitemias
are being observed after the application of artemisinine derivatives, resistance
has not been described so far. Some recent reports of artemether therapeutic
failure have, however, raised that issue (Gogtay et al., 2000). Drug resistance
can be either caused by mutations of genes of the drug targets (pfdhps, P.
falciparum dihydropteroate synthase; pfdhfr, P. falciparum dihydrofolate reductase;
pfcytb, P. falciparum cytochrome b), or by parasite strategies of drug
metabolism, transport, or the modification of intracellular conditions (pfmdr1,
P. falciparum multidrug resistance 1; pfcrt, P. falciparum chloroquine resistance
transporter; pfgr, P. falciparum gluthathione reductase) (Inselburg et al.
1987; Thaithong et al., 2001).


For the development of malarial drugs sexual stages malarial
parasites were cultured but they gain resistance to drug, as culturing of liver
stages, were extra hard to accomplish, made it probable to build up and
experimentally test the drugs in opposition to this stage, this provided
significant information about the immune reaction in the liver. Finally, the
culture of sporogonic stages has enabled researchers to discover that in the
mosquito vector what happens to the parasite (NIH ,2007). Drugs that is
required for malarial treatment includes sulfadoxine/pyrimethamine, mefloquine,
atovaquoneproguanil, quinine or quinidine, clindamycin, doxycycline,
chloroquine, and primaquine (Kawamoto et al., 1991. The artemisinin are the
most effective medicines that have ever been invented for (WHO ,2005). A lot of
work has been done on malarial vaccines with limited success (Tinto et al.,
2006. The circum sporozoite protein (CSP) is an antigen that is present on the
outside of sporozoites, has been used broadly as an objective for the
development of vaccines (Yoshida et al., 2007). Intermittent preventive
treatment (IPT) that is taken time to time regardless of malarial infection is
recommended by the World Health Organization especially for pregnant women. IPT
immune the body for the specific parasite encounters (Escalante et al.,1994).

Studies on
genetic diversity, differentiation of the different strains within a Plasmodium
species and presence of multiple parasite strains in individual host have
been reported from different regions of the globe (Basco et al., 2004). However, limited reports are available on the
genetic diversity existing among P. falciparum population of Pakistan.
In this study, polymorphic markers in P. falciparum isolates are used to
examine genetic diversity and complexity of parasite populations in patients
with uncomplicated malaria infections in Southern area of KP  Pakistan .