Malaria know the pattern of existing and emerging drug

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).

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.

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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.,
2016).

Life
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 ).

Merozoite
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).

Merozoite
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)

Glutamate
Rich Protein

Glutamate Rich Protein
(GLURP) is a 220-kDa exoantigen
expressed
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 determined 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 exhibits N-terminal
non-repeat region (R0), a central repeat region (R1) and an immunodomi­nant
C-terminal repeat region (R2). GLURP is extremely antigenic and there are few
polymorphisms in the encoding genes of GLURP in P. falciparum isolates
from differ­ent geographic regions. Polymorphisms in GLURP is mainly involve
variations in the numbers of repeats of many genomic sequences that therefore
affect the gene size and its protein product. During the blood stages of the
parasite, a single variant of the gene exhibits the presence of more than one
allele and characterizes a multiclonal infection (Theisen et al., 1995;Stricker et al.,
2000).

The
Genome

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

Polymorphism

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

Drug
resistance

Antimalarial multidrug
resistance of the parasite is a chief reason for failure of treatment and reactivation.
Resistance to mefloquine, CQ, quinine, halofantrine, sulpha drugs (e.g.
sulfadoxine), folate antagonists (e.g. pro-/ cycloguanil, pyrimethamine), as
well as launched atovaquone has been reported recently. Although after the application
of artemisinine derivatives repeated parasitemias are being observed, so far no
resistance has been described. Some recent reports of artemether therapeutic
failure have, however, raised that issue (Gogtay et al., 2000). Drug resistance
can be either caused by genetic mutations of of the drug targets (pfdhfr, P.
falciparum dihydrofolate reductase; pfcytb, P. falciparum cytochrome b,  pfdhps, P. falciparum dihydropteroate
synthase, or by drug metabolism startegies of parasite , transport, or the
modification of intracellular conditions (pfmdr1, P. falciparum multidrug
resistance 1; pfcrt, P. falciparum chloroquine resistance transporter; pfgr, P.
falciparum gluthathione reductase) (Thaithong et al., 2001;Inselburg et al.,
1987).

Treatment

Malarial parasites were cultured for the growth of malarial
drugs sexual stages but they gain drug resistance, as culturing of liver
stages, were extra hard to complete, made it possible to figure out and to test
the drugs in opposition to this stage experiemently, this provided important
information about the liver  immune
reaction. Finally, the culture of sporogonic stages, has enabled researchers to
determine that what happens to the parasite in the mosquito vector (NIH ,2007).
Drugs that is required for malarial treatment includes atovaquoneproguanil,sulfadoxine/pyrimethamine,
mefloquine ,quinine or quinidine, chloroquine ,clindamycin, doxycycline, and
primaquine (Kawamoto et al., 1991). The most effective medicines are the
artemisinin that have ever been invented for. With limited success a lot of
work has been done on malarial vaccines but not an effective vaccine prepared
so for because of genetic variation of the parasite and specilly the P.falciparum (WHO ,2005, Tinto et al.,
2006. The circum sporozoite protein (CSP) is a protein of sporozoites that is
present on the outside of surface, is an objective for vaccine development world
wide for effective treatment. World Health Organization recommended an intermittent
preventive treatment (IPT) that is taken time to time regardless of malarial
treatment that immune the body against the 
specific parasite encounters infection  especially for pregnant women. ((Yoshida et
al., 2007;Escalante et al.,1994).

From
different region of the globe, a worldwide study is carry on Plasmodium species,
specially the P.falciparum which
exibits wide range of genetic polymorphism, differentiation of the different
strains and existence of multiple parasite strains in individual host have been
reported. (Basco et al., 2004). However,
in Pakistan limited reports are accessible on the genetic polymorphism prevailing
among P. falciparum population. In this study, polymorphic markers in P.
falciparum isolates are used to examine genetic polymorphism and complexity
of parasite populations in patients with uncomplicated malaria infections in
Southern area of KP Pakistan .

 

 

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