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

INTRODUCTIONMalaria 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.

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(Hymel andYang, 2008).The most common and the lethalhuman malaria parasite is P.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 a part of increasevirulence associated with P. falciparum infections (Arnot et al., 2011).In addition, more complications are associated with P. falciparum becauseof the sequestration of the trophozoite- and schizont-infectederythrocytes in the deep tissues. As an alternative to the asexual replicativecycle, the parasite can differentiate into sexual forms known as macro- or microgametocytes.

The gametocytes are large parasites which fillup the erythrocyte, but only contain one nucleus. Ingestion ofgametocytes by the mosquito vector induces gametogenesis (i.e., theproduction of gametes) and escape from the host erythrocyte.

Factorswhich participate in the induction of gametogenesis include: a drop intemperature, an increase in carbon dioxide, and mosquito metabolites. Microgametes, formed by a process knownas exflagellation, are flagellated forms which will 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 earlyobservations, the function and composition and of merozoite surface proteins(MSPs) has been of great interest because of extensive role in invasion of redblood cells and potential as vaccine candidates antigens (Richards et al., 2009)and, more recently, as drug targets for stop blood-stage replication (Boyle etal., 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-12have 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 processing prior to egress of themerozoite from the schizont. This processing alter the secondary struction ofMSP1 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.Diversity in GLURP has been indicated by different sized polymerase chainreaction (PCR) products from the R2 region of various laboratory-adapted andfield strains.The glutamate- rich protein (GLURP) is expressed in all stages ofthe Plasmodium falciparum life cycle in humans (Stricker et al., 2000). GLURPcontains an N-terminal non-repeat region (R0), a central repeat region (R1) andan immunodomi­nant C-terminal repeat region (R2).

GLURP is highly antigenic andthere are few polymorphisms in the gene encoding GLURP in P. falciparum isolatesfrom differ­ent geographic regions (Theisen etal., 1995). GLURP polymorphisms mainly involve variations in the numbers ofrepeats of certain genomic sequences that therefore affect the size of the geneand its protein product. Given that a single variant of the gene is foundduring the blood stages of the parasite, the presence of more than one allelerepresents a multiclonal infection (Stricker et al.

, 2000). TheGenomeThe genome of P.falciparum consists of 14 linear chromosomes with a total of 25–30 megabases ofnuclear DNA with approximately 5000 genes, a mitochondrial fragment of a 6 kbrepeat element, and a circular element of 35 kb within the apicoplast. As aconsequence of continuous deletions and crossing-over and rearrangement eventsoccurring preferably at their telomeric regions, the chromosomes differconsiderably in size (Corcoran et al. 1986). The genome is extremely A/ T rich(80%), which has led to difficulties in conventional sequencing strategiesbecause of the instability of genomic fragments in bacterial Escherichia coliclones. Meanwhile, several yeast artificial clone (YAC) constructs have beenestablished, allowing for a stable maintenance of  P. falciparum clone fragments.

The P. falciparumgenome is now subject of a large DNA-sequencing project, the Malaria GenomeProject, which was established in 1996. Contig arrays and restriction maps havebeen produced for mapping of complete chromosomes (Rubio et al. 1995).PolymorphismThe inherent variabilityof P. falciparum provides multiple effective immune evasion and drug resistancemechanisms for the parasite. Many of the studies on the parasite’s polymorphismhave focused on variants exhibiting mutations that lead to amino acidsubstitutions (non-synonymous mutations) that are likely subjected toselection, such as immunogenic proteins and resistance phenotypes.

Other studieshave examined sequences which are rather unlikely to be subjected to adaptivepressure and therefore allow considerations on the phylogeny and the age of theparasite. Polymorphism of the P. falciparum genome has mainly evolved throughDNA 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) contributelargely to the variability (Rich & Ayala 2000). Genotyping ofpolymorphisms is not only a tool for the description of a distinct clonalparasite strain, but also for defining multiplicity of infections with clonallyvariable P.

falciparum strains. Genotyping in field studies mostly allows onlyan approximation to the estimation of different strains in individual infections.The precise estimation of the number of parasite clones is complicated by thehigh proportion of low-parasitemic P. falciparum infections (e.g. Roper et al.

,1996; May et al., 2000). Genes where polymorphism has arisen through intragenicrecombination in repetitive segments are characterized by repeat motifs withlength variability differing between strains. Among these genes are thoseencoding the P. falciparum circumsporozoite protein (Arnot et al.

, 1993), aglutamate-rich protein (GLURP) (Borre et al., 1991), two merozoite surfaceproteins (MSP-1 and MSP-2) (Kimura et al. 1990; Fenton et al. 1991) and theapical membrane antigen AMA-1 (Marshall et al. 1996).Drugresistance Parasite antimalarialmultidrug 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), aswell as to the recently launched atovaquonehas been reported. Although recurrent parasitemiasare being observed after the application of artemisinine derivatives, resistancehas not been described so far. Some recent reports of artemether therapeuticfailure have, however, raised that issue (Gogtay et al., 2000).

Drug resistancecan 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 drugmetabolism, transport, or the modification of intracellular conditions (pfmdr1,P. falciparum multidrug resistance 1; pfcrt, P. falciparum chloroquine resistancetransporter; pfgr, P. falciparum gluthathione reductase) (Inselburg et al.1987; Thaithong et al.

, 2001).TreatmentFor the development of malarial drugs sexual stages malarialparasites were cultured but they gain resistance to drug, as culturing of liverstages, were extra hard to accomplish, made it probable to build up andexperimentally test the drugs in opposition to this stage, this providedsignificant information about the immune reaction in the liver. Finally, theculture of sporogonic stages has enabled researchers to discover that in themosquito vector what happens to the parasite (NIH ,2007). Drugs that isrequired for malarial treatment includes sulfadoxine/pyrimethamine, mefloquine,atovaquoneproguanil, quinine or quinidine, clindamycin, doxycycline,chloroquine, and primaquine (Kawamoto et al.

, 1991. The artemisinin are themost effective medicines that have ever been invented for (WHO ,2005). A lot ofwork 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 theoutside of sporozoites, has been used broadly as an objective for thedevelopment of vaccines (Yoshida et al., 2007). Intermittent preventivetreatment (IPT) that is taken time to time regardless of malarial infection isrecommended by the World Health Organization especially for pregnant women. IPTimmune the body for the specific parasite encounters (Escalante et al.,1994).Studies ongenetic diversity, differentiation of the different strains within a Plasmodiumspecies and presence of multiple parasite strains in individual host havebeen reported from different regions of the globe (Basco et al.

, 2004). However, limited reports are available on thegenetic diversity existing among P. falciparum population of Pakistan.In this study, polymorphic markers in P.

falciparum isolates are used toexamine genetic diversity and complexity of parasite populations in patientswith uncomplicated malaria infections in Southern area of KP  Pakistan .