The set of primers amplify the sequence of

The
samples labeled as sample no 1, to 35 were from hari pur; 36, to 48 from
Abbottabad ;49 to  60 from Mansehra  ;And onwaord were from Batagrame
,Sheenkari,Bafa etc  .The Participant
Including were both males and females , with different ages.

DNA
Extraction using GeneJet Genomic DNA purification kit. DNA  was extracted from all Samples by using kit
protocol. After DNA extraction gel electrophoresis was conducted to verify the
extraction of DNA After Gel Electrophoresis, the gel was examined in gel doc
and the pictures were captured.

  

Figure
. An example of extracted DNA samples. Lane 1-8 Genomic DNA

Nested PCR

Nested
PCR give higher and specific amplification of the target sequence in DNA and to
avoid non –specific binding of the primer to sequence other target area. Two
sets of Primers were used in Nested PCR . Like normal PCR, 1st set
of primers amplify sequences Outside the target DNA while the  2nd set of primers amplify the
sequence of the target DNA amplified by the 1st set of Primer.

Gel electrophoresis of
DNA samples after PCR

Total
102 alleles were detected in this study both for pfmsp –l and pfmsp -2 genes of
all the Tested samples during present investigation. In this study the allele sizes
odserved were, K1 100bp to 850 bp , with majority allele size of 850 bp were
present. Allele size of MAD20 was 100bp to 850 bp, Ro 33   150 bpto 200 bp, FC27 600bp – 850 bp and
3D7/ Ic1 allele size ranged from 400bp were detected respectively.

Nested
PCR

PCR
amplification profile of DNA sample 1 and 2 using Pf msp1 and Pf msp2 Primers.

PCR
amplification profile of DNA sample using Pf msp1 and Pf msp2 Primers.

 

 

 

 

 

 

 

 

 

 

The msp1 and msp2 a1 Lelic
frequency was calculated as the proportion of the allele found for the allelic
family out of the alleles detection.

 

The patients
 ages ranged
 from six months  to Above
40  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 of
Plasmodium 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 infections
were
34 (42.5%). Among
Polyclonal infections those that carried two allelic types K1/RO33,
K1/ MAD20, MAD20/RO33 comprised4%, 0% and
 88.8%,
respectively. Trimorphic infections
K1/MAD20/RO33were detected
in 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.   The
frequency   of samples having only FC27
allelic 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  other
hand,  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                                           100-800

MAD20                                   100-800

Ro33                                       100-200

Pfmsp-2                                  

FC27                                       200-500

3D7/IC1                                  100-450

 

 

Discussion

Malaria is a major health problem in Pakistan, and the
circumstances are not helped by the fact that it stakes borders with countries
like Afghanistan, India and southern Iran, where the disease is also highly
endemic. Moreover, there is significant cross-border human migration within
this region, exacerbated by disorders in places like Afghanistan that lead to
refuge invasions, all of which facilitate malaria transmission. The
introduction of new alleles by mutation, migration and recombination generates
genetic diversity in natural populations, while the immune responses of the
human host, as well as chemotherapy play key roles in selection, which affects
the frequency of new alleles in parasite populations.

The
purpose 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 has
been given to investigating the genetic
diversity of P. falciparum than other
countries information
about the genetic diversity of P. falciparum in Hazara division. These finding may be an
important element for implementing malaria control strategies in the country, as removal may
impact genetic diversity and their
heterozygosity. The allele specific P. falciparum msp1 and msp2 has shown that malaria parasite population in
Hazara division is moderate to high allelic
diversity. In their
allelic frequency, out of the 59 allelic types detected in msp1, the RO33 allelic
family with 52%allelic frequency besides its representation in
poly  allelic bands was found predominant. This is in line with previous studies  in Sudan and Malaysia. With regard
to msp2, 8 allelic
types
were found and the
alleles belonging to FC27 family were more frequently detected (14%). This is because high numbers of bands were encountered
with genotypes of these two allelic families. These findings are in agreement
with previous study in Uganda and the
Sudan Hamid et al., 2013;Peyerl et al.,2001 whereRO33and  FC27allelic
families were found predominant.

In
Hazara Division of Pakistan high levels of polymorphism at msp-1 and msp-2 loci for
the P. falciparum isolates. Such high
levels of genetic diversity at these two genes reflect what has been observed
in Bannu district of Pakistan and where all three families of msp-1 (K1, MAD20 and RO33) and the two
of msp-2 families (FC27 and 3D7/IC)
were amplified (Lubna et al., 2010). However, the prevalence of the RO 33 +Mad
20 mixed genotype in Hazara division was 88%, which is substantially higher
than what was observed in Bannu District (5%). Furthermore, the Bannu District
samples showed an over representation of the 200 bps allele of MAD20 (Lubna et
al., 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 in
Bannu District 3D7/IC, has been at high level 65% is reported. However, the
allele sizes in the Hazara Division isolates (100-500bp for 3D7/IC alleles and
150-500 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 results
suggest high spatial heterogenesity of P.
falciparum infections, which may result from geographical isolation,
differences in transmission intensity, and malaria treatment policies. In
particular, geographically isolated parasite populations are expected to be
less diverse than exposed populations, due to lack of a continuous introduction
of new parasite clones from other areas. Similarly, because of high levels of
inbreeding, areas of low transmission intensity tend to have less diverse
parasites than areas of high endemicity, where the rates of cross fertilization
are higher (yang et al., 2006). Similarly by comparing the current study with
Kohat district where15 isolates showed msp1 gene in which  K1 genotype is 20 % (3/15) bps size ranged is
180-250 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 showed
msp2 gene 3D7/IC 20% (2/10)  FC27  30% (3/10) Mixed infaction 50%  (5/10). Which is dominant bps range for
3D7/IC IS 400-580 and FC27 with 300-400bp(Khatoon et al 2012) mixed infaction
is high in this region complexity in this region may be attributed to the free
movement of people through tribal areas b/w Kohat and Afghanista. msp2 gene has
limited diversity in this region as compared to Iran, India and
Karachi.(Khatoon et al., 2012). In the same vein, treatment policies that focus
on using highly potent drugs like artemisinin combination therapies (ACTs) that
kill the asexual blood stage parasites as well as gametocytes are more likely
to decrease parasite transmission and clonal diversity than regimens based on
drugs that act only on the blood stage parasites (Greenwood
et al., 2008 ; Targett et al., 2001) In Pakistan, mono therapies that work on
blood stage parasite are still largely used, with the key first line malaria
drugs being chloroquine for P. vivax and
sulphadoxine pyrimethamine (SP) for P.
falciparum (Asif et al., 2008).

 

 

Conclusion

From
this current study we conclude that Prevalence of P. falciparum is high in Hazara division. The type specific alleles
including K1, Ro33 and MAD20 of pfmsp-1 and 3D7/ICI pfmsp-2 are present in
four samples collected and FC27 allelic family that is present in only three
samples. In pfmsp-1 genotype total 56 alleles of Ro33 are
present with higher frequency of 97% with allele size 150bp-200bp, followed by
MAD20 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 of
3D7/ICI are present having frequency 11% followed by FC27 having 3 alleles with
8% frequency.

Finally
we conclude that most of the cases of malaria in the Hazara Division P. falciparum
and Ro33 allelic family overcome in higher frequency as compared to other
allelic families. This study help in planning new effective treatments against P. falciparum.
Outcome of this study present a significant records on the prevalence of
overall malarial plasmodium and specifically the genetic variation of P. falciparum
that will maintenance epidemiological studies in future.

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