The set of primers amplify the sequence of

Thesamples labeled as sample no 1, to 35 were from hari pur; 36, to 48 fromAbbottabad ;49 to  60 from Mansehra  ;And onwaord were from Batagrame,Sheenkari,Bafa etc  .The ParticipantIncluding were both males and females , with different ages.DNAExtraction using GeneJet Genomic DNA purification kit. DNA  was extracted from all Samples by using kitprotocol. After DNA extraction gel electrophoresis was conducted to verify theextraction of DNA After Gel Electrophoresis, the gel was examined in gel docand the pictures were captured.    Figure. An example of extracted DNA samples.

Lane 1-8 Genomic DNANested PCR NestedPCR give higher and specific amplification of the target sequence in DNA and toavoid non –specific binding of the primer to sequence other target area. Twosets of Primers were used in Nested PCR . Like normal PCR, 1st setof primers amplify sequences Outside the target DNA while the  2nd set of primers amplify thesequence of the target DNA amplified by the 1st set of Primer. Gel electrophoresis ofDNA samples after PCRTotal102 alleles were detected in this study both for pfmsp –l and pfmsp -2 genes ofall the Tested samples during present investigation.

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In this study the allele sizesodserved were, K1 100bp to 850 bp , with majority allele size of 850 bp werepresent. Allele size of MAD20 was 100bp to 850 bp, Ro 33   150 bpto 200 bp, FC27 600bp – 850 bp and3D7/ Ic1 allele size ranged from 400bp were detected respectively.NestedPCR PCRamplification profile of DNA sample 1 and 2 using Pf msp1 and Pf msp2 Primers. PCRamplification profile of DNA sample using Pf msp1 and Pf msp2 Primers.                   The msp1 and msp2 a1 Lelicfrequency was calculated as the proportion of the allele found for the allelicfamily out of the alleles detection.  The patients ages ranged from six months  to Above40  y ears.

  The parasite  DNA  P. falciparum isolates was analysed for msp1 and msp2 genes. The esti- mated of frequency of msp-1 and  msp-2  gene  amplifi- cation  reactions with  family specific  primers  was 73% (59/80) and 10% (8/80), nd mixed msp1 and msp2 is 13/80. In msp1, K1, RO33 and  MAD20,FC27,3D7/IC7 allele types were identified.  Frequencies   of  different   msp-1and msp-2   alleles  and their   combinations  and   multiplicity   of  infection   are shown in Tables 2 and 3.The  proportion of K1, MAD20  and RO33,FC27 , 3D7/IC1  types  were 4%, 45% and 52%, 14% 5% respectively. The remaining  nearly           Table 2 Allele typing and  diversity profiles ofPlasmodium falciparum isolates from Hazara divition  based on genetic diversity of msp1  and  msp2 Allelic type                       n (%)               Allelic type                  n (%)MSP-1                                                       MSP-2  K1                                                4(4)                    FC27                              12(80) MAD20                             45(42)                3D7/IC1                       3(20)  RO33                                 52(49)                 FC27 + 3D7/IC1            2(13) RO33 + K1                                 1(3.7) K1+ MAD20                      0 (0) RO33 + MAD20                 24(88)  Mad20 + K1 + Ro33           2(7.

4)Total                                  128                                                        17  27(33.75%) were the  poly-allelic  types  of  msp1   (K1/MAD20, K1/RO33, MAD20/RO33 and K1/MAD20/RO33). The monoclonal infectionswere34 (42.5%). AmongPolyclonal infections those that carried two allelic types K1/RO33,K1/ MAD20, MAD20/RO33 comprised4%, 0% and 88.8%,respectively. Trimorphic infectionsK1/MAD20/RO33were detectedin 7.

4% of cases (Table 2). Among  msp1 isolated,  three for  K1 (200–250 bp),  five for  RO33  (150–225 bp) and three  for MAD20 (100-300  bp)  allele families were ob- served  (Table  3). With  respect to msp2, both FC27and          3D7/IC1  allele types were  detected.

   Thefrequency   of samples having only FC27allelic family (80% (12/15)) was higher than  the sample with  only 3D7/IC1 allelic type(20% (3/15)). Twenty-seven  of the  isolated (69.2%) car- ried  both msp2  allelic  families  (Table  2).

On the  otherhand,  among  cases that were positive for msp2 alleles,86% (13/15)  were monoclonal  infection   while  13.3%(2/15)  were polyclonal  infections. The length  variants of the amplified  products were seven  for FC27 (300–600 bp) and five for 3D7/IC1 (200–500 bp) (Table 3).  Alleles                                  Size (bp)              Pfmsp-1                                     K1                                           100-800MAD20                                   100-800Ro33                                       100-200Pfmsp-2                                   FC27                                       200-500 3D7/IC1                                  100-450  DiscussionMalaria is a major health problem in Pakistan, and thecircumstances are not helped by the fact that it stakes borders with countrieslike Afghanistan, India and southern Iran, where the disease is also highlyendemic. Moreover, there is significant cross-border human migration withinthis region, exacerbated by disorders in places like Afghanistan that lead torefuge invasions, all of which facilitate malaria transmission. Theintroduction of new alleles by mutation, migration and recombination generatesgenetic diversity in natural populations, while the immune responses of thehuman host, as well as chemotherapy play key roles in selection, which affectsthe frequency of new alleles in parasite populations.Thepurpose of this study was to compare the two most polymorphic regions of msp1 and msp2 genes, the genetic diversity of P. falciparum in malaria.

Less attention hasbeen given to investigating the geneticdiversity of P. falciparum than othercountries informationabout the genetic diversity of P. falciparum in Hazara division. These finding may be animportant element for implementing malaria control strategies in the country, as removal mayimpact genetic diversity and theirheterozygosity. The allele specific P. falciparum msp1 and msp2 has shown that malaria parasite population inHazara division is moderate to high allelicdiversity.

In theirallelic frequency, out of the 59 allelic types detected in msp1, the RO33 allelicfamily with 52%allelic frequency besides its representation inpoly  allelic bands was found predominant. This is in line with previous studies  in Sudan and Malaysia. With regardto msp2, 8 allelictypeswere found and thealleles belonging to FC27 family were more frequently detected (14%). This is because high numbers of bands were encounteredwith genotypes of these two allelic families. These findings are in agreementwith previous study in Uganda and theSudan Hamid et al.

, 2013;Peyerl et al.,2001 whereRO33and  FC27allelicfamilies were found predominant.InHazara Division of Pakistan high levels of polymorphism at msp-1 and msp-2 loci forthe P.

falciparum isolates. Such highlevels of genetic diversity at these two genes reflect what has been observedin Bannu district of Pakistan and where all three families of msp-1 (K1, MAD20 and RO33) and the twoof msp-2 families (FC27 and 3D7/IC)were amplified (Lubna et al., 2010). However, the prevalence of the RO 33 +Mad20 mixed genotype in Hazara division was 88%, which is substantially higherthan what was observed in Bannu District (5%). Furthermore, the Bannu Districtsamples showed an over representation of the 200 bps allele of MAD20 (Lubna etal., 2010).  Whereas shorter (150 bps)MAD20 allele was predominant in the Hazara Division  samples.

For msp2, the FC27 alleles were predominant in Hazara Division While inBannu District 3D7/IC, has been at high level 65% is reported. However, theallele sizes in the Hazara Division isolates (100-500bp for 3D7/IC alleles and150-500 bps for FC27 alleles) were smaller than those observed in Bannu(250-500bp for 3D7/ IC alleles and 400-800 bp for FC27 alleles). These resultssuggest high spatial heterogenesity of P.

falciparum infections, which may result from geographical isolation,differences in transmission intensity, and malaria treatment policies. Inparticular, geographically isolated parasite populations are expected to beless diverse than exposed populations, due to lack of a continuous introductionof new parasite clones from other areas. Similarly, because of high levels ofinbreeding, areas of low transmission intensity tend to have less diverseparasites than areas of high endemicity, where the rates of cross fertilizationare higher (yang et al., 2006). Similarly by comparing the current study withKohat district where15 isolates showed msp1 gene in which  K1 genotype is 20 % (3/15) bps size ranged is180-250 were detected .26%(4/15) Mad20 is dominant having bps range 120-150 .mixed genotype K1+Mad20 +RO33 were observed 7% 1/15  while K1+MAD20+RO33   40% (7/15)IS dominant .

10 isolate showedmsp2 gene 3D7/IC 20% (2/10)  FC27  30% (3/10) Mixed infaction 50%  (5/10). Which is dominant bps range for3D7/IC IS 400-580 and FC27 with 300-400bp(Khatoon et al 2012) mixed infactionis high in this region complexity in this region may be attributed to the freemovement of people through tribal areas b/w Kohat and Afghanista. msp2 gene haslimited diversity in this region as compared to Iran, India andKarachi.

(Khatoon et al., 2012). In the same vein, treatment policies that focuson using highly potent drugs like artemisinin combination therapies (ACTs) thatkill the asexual blood stage parasites as well as gametocytes are more likelyto decrease parasite transmission and clonal diversity than regimens based ondrugs that act only on the blood stage parasites (Greenwoodet al., 2008 ; Targett et al., 2001) In Pakistan, mono therapies that work onblood stage parasite are still largely used, with the key first line malariadrugs being chloroquine for P. vivax andsulphadoxine pyrimethamine (SP) for P.falciparum (Asif et al.

, 2008).  ConclusionFromthis current study we conclude that Prevalence of P. falciparum is high in Hazara division. The type specific allelesincluding K1, Ro33 and MAD20 of pfmsp-1 and 3D7/ICI pfmsp-2 are present infour samples collected and FC27 allelic family that is present in only threesamples. In pfmsp-1 genotype total 56 alleles of Ro33 arepresent with higher frequency of 97% with allele size 150bp-200bp, followed byMAD20 16 alleles having frequency as 45% and K1 6 alleles with 17% frequency.The Ro33 is predominant in the Hazara Division.

In family pfmsp-2 four alleles of3D7/ICI are present having frequency 11% followed by FC27 having 3 alleles with8% frequency.Finallywe conclude that most of the cases of malaria in the Hazara Division P. falciparumand Ro33 allelic family overcome in higher frequency as compared to otherallelic families. This study help in planning new effective treatments against P. falciparum.Outcome of this study present a significant records on the prevalence ofoverall malarial plasmodium and specifically the genetic variation of P. falciparumthat will maintenance epidemiological studies in future.