4.1 and pfmsp-2 genes of all the samples

4.1 Description of Blood Samples
Studied

The
current study was conceded in Malakand division of Khyber Pakhtunkhwa province.
The people are engaged in regular forming and cattle herding. Data of the malaria
affected people were collected from the DHQ hospitals and different laboratories
of the Malakand division including Swat and Dargai with the help of hospital
administration and laboratory technicians.

In
the current study a total of seventy blood sample were analyzed and were
confirmed as positive case of P. falciparum. The effected candidates
included were male, female and children.

4.2 DNA Extraction

Using
Thermo Scientific Gene jet Genomonic DNA Purification Kit method of DNA
extraction, DNA was extracted from total 70 samples. The DNA obtained through
commercial kit was of good quality and quantity as depicted in figure 2 after
gel electrophoresis, visualization and capturing the picture of gel containing
genomic DNA.

 

 

                       

 

 

 

 

 

 

                    Figure 2. An example of DNA
profile extracted from blood samples of malaria     

                     patients. M represents
ladder 1kb.                                                                                          

4.3 Nested PCR

Preventing
of non-specific binding of primer to sequence other than the target region,
Nested PCRs were used which are specific in target sequence of the DNA and have
higher amplification power.  Two sets of primers
were used in Nested PCR. Like initial PCR, 1st primers amplify sequences of the
target DNA outside while 2nd set primers amplify the sequence of the target DNA
amplified by the 1st set of primer.

4.4 Gel Electrophoresis of DNA
Samples after PCR

In
present study total 126 alleles were detected both for pfmsp-1 and pfmsp-2 genes
of all the samples tested during investigation. In this study allele sizes
observed were as K1 100bp to 850, with majority alleles with size of 850 bp
were present. Allele size of Ro33 ranged 150bp to 200bp, MAD20 ranged 100bp to
850bp, FC27 600bp to 850bp and 3D7 allele size from 400bp to 850 bp were
detected respectively (table no 3) Pfmsp-1 and Pfmsp-2 gene were found in the sample of all five families. In pfmsp-1 gene, Ro33 was most prevalent with 68 (54%) alleles followed by
MAD20 carrying 32 (25%) alleles and K1 with 12 (9%) alleles. Correspondingly,
in pfmsp-2, the 3D7/ICI family with 8 (6%) alleles was more prevalent as
compared to FC27 having 6 (4%) alleles.

M   K1  R 
MD  FC   3D 
K1  R  MD FC  
3D   K1  R  
MD  FC  3D 
K1  R   MD 
FC 3D  

 

        Figure 3. PCR amplification of DNA sample
1,2,3 and 4 using pfmsp-1 and pfmsp-2 primers. M= 50 bp DNA Ladder.

M    K1   Ro 
MD  FC  3D  
K1  Ro  MD   
Fc   3D    K1  
Ro   MD   FC  
3D    K1    Ro  
MD  FC  3D   
M

 

 

 

 

 

 

 

 

 

Figure
4. PCR amplification of DNA sample 5, 6, 7 and 8 using pfmsp-1 and pfmsp-2
primers. M= 50 bp DNA Ladder

 

 

 

    M    K1  
Ro    MD   FC  
3D   K1   RO 
MD  FC  3D   
K1    Ro   MD  
FC   3D   K1   
Ro    MD   FC  
3D

Figure
5. PCR amplification of DNA sample 9, 10, 11 and 12 using pfmsp-1 and pfmsp-2
primers. M= 50 bp DNA Ladder.

M  K1  R   
MD  FC  3D  
K1  R   MD 
FC  3D   K1 
R   MD  FC 
3D    K1   R 
MD FC 3D M      

 

Figure
6. PCR amplification of DNA sample 13, 14, 15 and 16 using pfmsp-1 and pfmsp-2
primers. M= 50 bp DNA Ladder.

        M     K1          Ro        MD          FC     3D               K1           Ro        MD        FC     3D       M

 

Figure
7. PCR amplification of DNA sample 17 and 18 using pfmsp-1 and pfmsp-2
primers. M= 50 bp DNA Ladder.

 

 

 

 M   K1 
Ro  MD  FC  
3D  K1  Ro MD  
FC  3D  K1  
Ro  MD FC  3D 
K1  Ro  MD  
FC  3D

M19K19R19M19F19,3D20K20R20M20F20,3D21K21R21M21F 21,3D22K22R22M 22F 22,3D

Figure
8. PCR amplification of DNA sample 19, 20, 21 and 22 using pfmsp-1 and pfmsp-2
primers. M= 50 bp DNA Ladder.

 

     M      K1      
Ro         MD          FC          3D          K1    
Ro     MD       FC      3D            M

 

            

 

 

 

 

 

           
Figure 4 9. PCR amplification of DNA sample 23and 24 using pfmsp-1 and pfmsp-2       

            
primers.M=50 bp DNA Ladder.

 

  M  K1 Ro MD FC  3D  
K1   Ro  MD 
Fc  3D  K1 
Ro   MD   FC  
3D

 

         Figure 4.9 PCR amplification of DNA
sample 25, 26 and 27 using pfmsp-1
and pfmsp-2                         

         primers. M= 50 bp DNA Ladder.

 

          
Table 3. Alleles size of family pfmsp-1
and pfmsp-2 during present study.

Alleles

Size (bp)

Pfmsp-1

 

K1

100-850

Ro33

150-200

MAD20

100-850

Pfmsp-2

 

FC27

600-850

3D7/ICI

400-850

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4 Gel Wise Distributions of Individual
Primers Bands with Percentages.

S.no of gel

gels

K1 family

Ro33 family

MAD20 family

FC27 family

3D7/ICI family

1

10

0

8

2

0

0

2

10

0

8

2

0

0

3

20

0

8

6

4

2

4

8

2

6

0

0

0

5

12

0

6

6

0

0

6

16

0

6

6

0

4

7

22

8

8

6

0

0

8

10

0

10

0

0

0

9

4

2

2

0

0

0

10

14

0

6

4

2

2

Grand Total

126

12

68

32

6

8

% age

 

9%

54%

25%

4%

6%

 

Discussion

Malaria
poses key threats to the health and economic development of many countries
worldwide. Plasmodium falciparum is
the most dangerous species that causes severe malaria leading to death globally
(Joshi et al., 2007). Malaria till
date is a severe health problem in Pakistan. Approximately 2.6 million cases of
malaria were registered and 5000 deaths occur annually in Pakistan in the year
2008 (Khatoon et al., 2009, Khatoon et al., 2010, Ghanchi et al., 2010). In year 2010 in Eastern
Mediterranean areas over 1 million malaria cases were confirmed by microscopy
and 22% of these were from Pakistan (WHO, 2011). In our study the result of Plasmodium falciparum are closely
comparable with (Jan and Kiani., 2001). The studying malarial parasites in
Kashmiri refugees stabled in Muzaffarabad reported the prevalence of Plasmodium falciparum 0.67% while they
reported the Plasmodium vivax 6.33%
which is little from our results. According to (Yasinzai and Kakarsulemankhel,
2008) reported 88.69% Plasmodium vivax while they reported the Plasmodium falciparum 11.30% in Kharan
region. The seasonal variation was also well-known in Lal Qilla. The maximum
cases of malaria were noted in the month of October where the lowest cases were
arisen in the month of June. In another studies show that the most cases of
malaria was reported in Pakistan in the month of July to November (Jan and
Kiani, 2001; Khadim, 2002; Lathia and Joshi, 2004).

In
human blood prevalence of malarial parasite in Karachi was studied and out of
2457 samples, 311samples were created to be positive. In southern Punjab, 41%
species were infected by Plasmodium
to be found. In Khyber-Pakhtunkhwa, among males (64%) cerebral malaria was more
common and the most vulnerable were pregnant women. Major problem falciparum malaria is among Afghan
refugees in Khyber Pakhtunkhwa. Throughout the present study, no case of P. malariae
and P. ovale was detected. The same was the case in a study done in Multan
(Yar et al., 1998). High rate of P. vivax
(60.5%) was also detected, and the highest P.
vivax (90.4%) was observed in
Kashmiri refugees stabled in Muzaffarabad. Malaria Control Programmed observed
(88.5%) high slide positive rate (SPR) of P.
vivax in Ziarat (Yasinzai et al., 2009). High SPR of P. vivax
was also detected in other portions of Balochistan viz Kohlu and Ziarat. In
southern Punjab, P.vivax was more prevalent to be found
(39.0%) than P. falciparum (36.6%). In Karachi, P.
falciparum was observed to be leading
(90.99%) compared to P. vivax (9.0%) (Faiz et al., 2011).

It
is not easy to guess exact rate of Plasmodium
infection due to parasites spreading and diverse prevalence in many areas has
not been completely reported. Malaria case management disk guide has reported
that KP stands third in Pakistan that contributes to malaria that was reported
in 2007 (Atroosh et al., 2011). The
current study confirmed the old results of P.
falciparum are found in KP (WHO, 20
11; Khatoon et al., 2009).

This
current study was carried out to give information about the molecular profile
of msp1 and msp2 allelic polymorphism in Plasmodium falciparum species of
malaria parasites in the Malakand Division of KP. During 2015 to 2016, 70
samples were collected. Five malaria causative alleles including K1, Ro33 and
MAD20 of msp-1, FC27 and 3D7/ICI of msp-2 were studied in the current work.
Study of these alleles by Nested PCR confirmed high range of polymorphism for
the P. falciparummsp-1 and msp-2
isolates in Malakand Division. All the families of pfmsp-1(K1, Ro33 and
MAD20) and pfmsp-2 (FC27 and 3D7/ICI) gave amplification and Ro33 alleles were
leading. Outcome was in contradiction with the previous study of (Mamillapalli et al., 2007) in which MAD20 allelic
family was predominant. A possible reason for this conflict may be the
difference in areas of studies. Ro33 family was found in all the samples
amplified by nested PCR. A total 126 alleles were found after amplification by
Nested PCR. All these samples analyzed had all three pfmsp-1 alleles. The Ro33
had highest frequency (54%) followed by MAD20 (25%) and K1 (9%). However, the
allele size of Ro33 (150bp to 200bp) is shorter than the Ro33 size (360 bps to
460 bps) recognized before.

In
Pfmp-2, the 3D7ICI allelic family was leading in the Malakand division,
same as described in the country of Iran (Heidari et al., 2007). The allele size isolated (400-850bp for 3D7ICI
alleles and 600-850bp for FC27 alleles) in study was greater as compared to
allele detected in Iran whose size was 400-600bp for 3D7ICI alleles and
200-400bp for FC27 alleles. Both of family Pfmsp-1 and Pfmsp-2 alleles are
polymorphic detected in study showing an amazing contrast with P.falciparum
isolated from South America showed monomorphic at msp-1 (Legrand et al., 2005) and also in Colombia,msp-1
showed monomorphic as msp-2 had 3 alleles only (Montoya et al., 2003). This result proposes that
P.falciparum
is highly hetero-genetic infection that might be referred from environmental
isolation, spread concentration alterations or handling procedures. In
Pakistan, sulphadoxine  pyrimethamine
(SP) is used for the treatment of P.falciparum (Asif, 2008). Moreover,
present molecular studies approve that parasite of this locality is getting
resistant of the prearranged drugs (Khatoon et
al., 2009). Worrying a lot, the gametocytes release, being the infective
period of mosquito vector, is stimulated by antimalarial medicines such as SP
(Targett et al., 2001)

 

 

 

 

 

 

Conclusion

From
the present study it was concluded that Prevalence of P. falciparum is high in
Malakand Division. Out of 70 samples having 126 alleles, in pfmsp-1 genotypes total 112 alleles were present, Ro33 had 68 alleles showing
higher frequency of 54% with allele size 150bp-200bp, followed by MAD20 with 32
alleles having frequency of 25% and K1 exhibited 12 alleles with 9% frequency. In
family pfmsp-2 eight alleles of 3D7/ICI were present having frequency of 6%
followed by FC27 having six alleles with 4% frequency.

It
was concluded that most of the cases of malaria in the Malakand Division with P. falciparum
exhibited higher frequency of Ro33 allelic family as compared to other allelic
families. This study will helps 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 be helpful in
continuation of molecular research with the aim of designing potential vaccines
and improved diagnosis and treatment of malaria studies in future.

 

 

 

 

 

 

 

 

 

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