Malaria a crucial role in defining the strength

Malaria is a protozoan parasitic infection triggeredby Plasmodium spp.

which is usually spread by Anopheles spp. Plasmodiumfalciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium ovale andPlasmodium malariae are the Plasmodium spp which tainting humans. (Hymel andYang, 2008).The most common and the lethalhuman malaria parasite is P.

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falciparum,which is spread by mosquitoes genus Anopheles. . Evenwith widespread malaria eradication efforts, 214 million cases of malaria and438,000 deaths were valued globally in 2015, which largely affected thesub-Saharan African population and children under 5 years of age. Between 2000and 2015, malaria frequency and death rates reduced worldwide by 37% and 60%,respectively.

Worldwide the malaria cases are decreases due to extensive usageof 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 likePakistan to which malaria is endemic, continuous molecular surveillance of thefield isolates is required to know the pattern of existing and emerging drugresistance. This knowledge is the key to effective malaria control program.Genetic diversity of P.falciparum plays a crucial role in defining the strength of malariatransmission. Several P.

falciparum genes show wide-ranging geneticpolymorphism however, high polymorphism has been shown in Merozoite surfaceproteins 1 and 2 (MSP-1 and MSP-2) andGlutamate Rich Protein (GLURP) in different geographical locations in malariaendemic areas. MSP-1, MSP-2 and GLURP genes are broadly used to study theallelic diversity and frequency of P. falciparum (Mwingira et al.,2016).

Lifecycle of P.falciparumThe complex life cycle of malaria parasite involve an insectvector (mosquito) and a vertebrate host (human). Humans infect by four speciesof plasmodium : P. falciparum, P. vivax, P. malarriae and p.ovale.

All the  four species displaya 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 incubationperiod (Francis et al., 2010). Whenthe feeding mosquito infuse sporozoites with saliva the infection is started,carries to blood circulatory system. Sporozoites are carried by the circulatorysystem to the liver and attack the liver cells . The intracellular parasiteundergoes an asexual replication with in the liver cells known as exoerythrocytic schizogony. Merozoites are released into the blood streamwhen exoerythrocytic schizogony terminates (vaughan et al.

, 2017). A partof the liver-stage parasites from P. vivax and P. ovale go througha dormant period immediately instead of undergoing asexual replication. These hypnozoites will reactivate after theprimary infection for several weeks to months (or years)  and are responsible for relapses. Merozoitesattack red blood cells enlarge parasite when  undergo through trophic period.

The earlytrophozoite is frequently referred to as ‘ring form’ due to its morphology. Active metabolism enlargetrophozoite including the host cytoplasm ingestion and hemoglobinproteolysis into amino acids. Multiple rounds of nuclear division are shown atthe end of trophic period without cytokinesis resulting is a schizont. Mature schizont of merozoitebud, also called a segmenter,and in the rupture infected erythrocytes merozoites are released. Anotherround of the blood-stage  reinitiate replicativecycle resulting invade erythrocytes. The pathology of malaria is relatedwith the blood stage. The recurrent fever paroxyms is the cause of thesynchronous lysis of the infected erythrocytes. P.

malariae shows a 72hour periodicity, and the other three species show 48 hour cycles. However,P. falciparum often displays an unceasing fever rather than the periodicparoxyms. P. falciparum is also responsible for more morbidity andmortality than the other three species. The higher levels of parasitemia is apart 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- andschizont-infected erythrocytes in the deep tissues. As an alternative tothe asexual replicative cycle, the parasite can differentiate into sexual formsknown as macro- or microgametocytes.

The gametocytes arelarge parasites which fill up the erythrocyte, but only contain onenucleus. Ingestion of gametocytes by the mosquito vector inducesgametogenesis (i.e., the production of gametes) and escape from the hosterythrocyte.

Factors which participate in the induction of gametogenesisinclude: a drop in temperature, an increase in carbon dioxide, andmosquito metabolites. Microgametes,formed by a process known as exflagellation, are flagellated forms whichwill fertilize the macrogamete  leading to a zygote . The zygote develops into a motile ookinete which penetrates the gutepithelial cells and develops into an oocyst. The oocyst undergoes multiple rounds of asexualreplication resulting in the production of sporozoites. Rupture of the mature oocyst releases the sporozoitesinto the hemocoel (i.e., body cavity) of the mosquito.

The sporozoites migrateto 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 merozoiteswere enclosed in a ‘fuzzy’ fibrillar coat of surface proteins; remarkably, thiscoat 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 functionand composition and of merozoite surface proteins (MSPs) has been of greatinterest because of extensive role in invasion of red blood cells and potentialas vaccine candidates antigens (Richards et al., 2009) and, more recently, asdrug targets for stop blood-stage replication (Boyle et al., 2013;Chandramohanadas et al., 2014; Wilson et al.,2015).

Merozoites appears a fibrillar surfacecoat to be basically composed of glycosylphosphatidy inositol (GPI) an anchoredproteins, and the peripherally associated surface proteins with integralmembrane proteins representing a small part  of the total surface protein (Gilson etal., 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, Pf38and Pf12 (Sanders et al., 2005). In addition, other GPI-anchoredproteins, rhoptry associated membrane antigen (RAMA) microneme associatedcysteine-rich protective antigen (CyRPA) (Reddy et al, 2015 ; Topolska etal., 2004),) and GPI-anchored micronemal antigen (GAMA) (Arumugamet et al.,2011) migrate to the merozoite surface from organelles during, invasionof  red blood cells.

While many of theseproteins contain binding domains like cysteine-rich EGF and other different  globular domains prophesied function of  mediate receptor binding during the primaryrecognition and attachment to RBCs, very little experimental evidence areinsufficient 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 surfaceGPI-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.

(Arumugamet al., 2011; Reddy et al., 2015; Sanders et al., 2006 ).Merozoitesurface protein-1Merozoite surface protein-1 (MSP-1) is amajor surface protein of P.

falciparum,with an estimated molecular size of 190 kDa which  plays a key role in erythrocyte invasion bythe merozoite (Conway et al., 2000). MSP1is considered a protein of  highmolecular mass that experiences most  proteolytic processingprior to egress of the merozoite from the schizont.

This processing alter thesecondary 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 highlypolymorphic with large polymorphisms across the protein, predominantly in the P.

falciparum isolates MSP133 (with twoallelic groups) and MSP1-block 2 regions (with three allelic groups), whereasthe 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 ofhuman immune responses and is a useful candidate for a blood stage subunitvaccine (Holder et al., 1999). TheMSP- 1 gene with 7 variable block are separated by conserved and semi-conservedregions.

A region near the N-terminal of the MSP-1 gene Block-2, is the more polymorphicpart of protein and leads strong diversifying selection within naturalpopulations (Takala et al., 2002). Upto now, four different types of  allelesare identified block 2: MAD20, K1, and MR (Happi et al.

, 2004).Merozoitesurface protein-2 MSP2, is another second important GPI anchoredmerozoite surface protein with approximately 25 kDa  (Gilson et al., 2006).MSP2 is another leading antigen subunit of P. falciparum for malariavaccine (Happi et al., 2004). Itconsists of highly polymorphic central repeats flanked with conserved N- andC-terminal domains unique variable domains. The MSP-2 has generally two allelestypes, FC27 and 3D7, with different dimorphic structure considerably of thevariable central region, block-3 .

The specific region of strains is consistsof repeating units; 3D7 allele  containrepeating units of Ser, Gly and Ala, while FC27 allelic  forms contain 32-, 12- and 8-mer sequencerepeats. Both allelic forms of MSP2 are basically unstructured, but full lengthrecombinant proteins under physiological conditions make fibrils (Adda etal., 2009). Fibril development is mediated through the region of N-terminal(Low et al.

, 2007) and this region may also have the properties membraneinteraction(Zhang et al. 2008). It is called whether native fibril likeform of  MSP2 or other complexes;however, there is some evidence that MSP2 oligomers are placed on merozoitessurface with a number of  MSP-2interactions molecules being hypothesized (Yang et al., 2010). Recentstudies recommend that the MSP-2, N-terminal region may interact with the lipidmembrane of the merozoite surface (MacRaild et al,.2012 ; (Adda etal.

, 2009)). MSP2 appears to be important for invasion and during invasion retainedon the surface and  soon after degradedwhen invasion is complete. However, its exact character is unknown, and no interactionof receptor ligand or fixing of MSP2 to RBCs have been defined.

 MSP-1 andMSP- 2 genes  because of polymorphiccharacters  have been characterized aspolymorphic markers in studies of malaria transmission dynamics in naturalisolatesof P. falciparum (Ferreiraet al., 2007 ; (Boyle et al., 2014)GlutamateRich Protein Glutamate Rich Protein(GLURP) is a 220-kDa exoantigenexpressedon the merozoite surface found in the parasitophorous vacuole. The singlefull-length GLURP sequence available to date (strain F32) shows two amino acidrepeat regions (R1 and R2) with degenerate repeat motifs found in both. Diversityin GLURP has been determined by different sized polymerase chain reaction (PCR)products from the R2 region of various laboratory-adapted and field strains.Theglutamate- rich protein (GLURP) is expressed in all stages of the Plasmodiumfalciparum life cycle in humans (Stricker et al.

, 2000). GLURP exhibits N-terminalnon-repeat region (R0), a central repeat region (R1) and an immunodomi­nantC-terminal repeat region (R2). GLURP is extremely antigenic and there are fewpolymorphisms in the encoding genes of GLURP in P. falciparum isolatesfrom differ­ent geographic regions. Polymorphisms in GLURP is mainly involvevariations in the numbers of repeats of many genomic sequences that thereforeaffect the gene size and its protein product. During the blood stages of theparasite, a single variant of the gene exhibits the presence of more than oneallele and characterizes a multiclonal infection (Theisen et al.

, 1995;Stricker et al.,2000). TheGenomeThe P. falciparum genomeis consists of 14 linear chromosomes and a total of 25–30 megabases of nuclearDNA with approximately 5000 genes, a circular element of 35 kb and amitochondrial fragment of a 6 kb repeat element within the apicoplast. As aconsequence of crossing-over and continuous deletions and preferablyrearrangement events taking place at their telomeric regions, the chromosomesconsiderably differ in size.

The genome of P.falciparumis extremely (80%)  A/ T rich, which hasled to complications in conventional sequencing approaches because of genomicfragments 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, severalyeast artificial clone (YAC) constructs have been established, allowing for astable maintenance of P. falciparum clone fragments.

 Restriction maps and Contig arrays have beenproduced for mapping of complete chromosomes (Rubio et al., 1995).PolymorphismP. falciparum inherentvariability provides drug resistance mechanisms for the parasite and multiple effectiveimmune evasion. Many of the studies are focused on the parasite’s polymorphismwhich cause different exhibiting mutations and lead to amino acid substitutions(non-synonymous mutations) which are likely subjected to selection, such asimmunogenic proteins and resistance phenotypes. The genome of P. falciparumshow more polymorphism has mainly evolved through rearrangements of DNA, suchas gene duplication events, gene translocations, gene conversions , insertionand deletions (Deitsch et al.

, 1997 ;Wellems et al., 1990; Kemp et al., 1992).Other studies have observed sequences which are rather unlikely to be exposedto adaptive pressure and therefore allow considerations on the phylogeny andthe age of the parasite. Genotyping of polymorphisms is not only a descriptiontool of different strains of  clonalparasite, but also for defining multiplicity of infections (MOI) with clonallyvariable P. falciparum strains.

Single nucleotide polymorphisms (SNPs) largely contributeto the variability (Rich et al., 2000).Genotypingin field studies allows approximation only to the estimation of differentstrains in individual infections. Genes where intragenic recombinition mayarise polymorphism in repetitive segments are categorized by repeat motifs withlength variability opposing between strain. The precise estimation of thenumber of parasite clones is complicated by the high proportion oflow-parasitemic P.

falciparum infections (e.g. May et al., 2000;Roper et al.,1996;). Among these encoding proteins are P. falciparum circumsporozoiteprotein, which are two merozoite surface proteins (MSP-1 and MSP-2) and a glutamate-richprotein (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).Drugresistance Antimalarial multidrugresistance 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), aswell as launched atovaquone has been reported recently.

Although after the applicationof artemisinine derivatives repeated parasitemias are being observed, so far noresistance has been described. Some recent reports of artemether therapeuticfailure have, however, raised that issue (Gogtay et al., 2000).

Drug resistancecan be either caused by genetic mutations of of the drug targets (pfdhfr, P.falciparum dihydrofolate reductase; pfcytb, P. falciparum cytochrome b,  pfdhps, P. falciparum dihydropteroatesynthase, or by drug metabolism startegies of parasite , transport, or themodification of intracellular conditions (pfmdr1, P. falciparum multidrugresistance 1; pfcrt, P.

falciparum chloroquine resistance transporter; pfgr, P.falciparum gluthathione reductase) (Thaithong et al., 2001;Inselburg et al.,1987).TreatmentMalarial parasites were cultured for the growth of malarialdrugs sexual stages but they gain drug resistance, as culturing of liverstages, were extra hard to complete, made it possible to figure out and to testthe drugs in opposition to this stage experiemently, this provided importantinformation about the liver  immunereaction.

Finally, the culture of sporogonic stages, has enabled researchers todetermine 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, andprimaquine (Kawamoto et al., 1991). The most effective medicines are theartemisinin that have ever been invented for. With limited success a lot ofwork has been done on malarial vaccines but not an effective vaccine preparedso 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 ispresent on the outside of surface, is an objective for vaccine development worldwide for effective treatment. World Health Organization recommended an intermittentpreventive treatment (IPT) that is taken time to time regardless of malarialtreatment that immune the body against the specific parasite encounters infection  especially for pregnant women.

((Yoshida etal., 2007;Escalante et al.,1994).Fromdifferent region of the globe, a worldwide study is carry on Plasmodium species,specially the P.falciparum whichexibits wide range of genetic polymorphism, differentiation of the differentstrains and existence of multiple parasite strains in individual host have beenreported.

(Basco et al., 2004). However,in Pakistan limited reports are accessible on the genetic polymorphism prevailingamong P. falciparum population. In this study, polymorphic markers in P.falciparum isolates are used to examine genetic polymorphism and complexityof parasite populations in patients with uncomplicated malaria infections inSouthern area of KP Pakistan .

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