Genetic Diversity of Plasmodium falciparum and Plasmodium vivax Field Isolates from the Nowshera District of Pakistan

doi:10.21203/rs.3.rs-5030801/v1

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Genetic Diversity of Plasmodium falciparum and Plasmodium vivax Field Isolates from the Nowshera District of Pakistan

https://doi.org/10.21203/rs.3.rs-5030801/v1

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Background

The genetic diversity of malaria parasites contributes to their ability to adapt to environmental changes, develop drug resistance and escape from the host immune system; hence, it is very important for control measures of malaria. This study aimed to analyse the genetic diversity of the pfmsp1 and pfmsp2 genes in P. falciparum and the Pvmsp-3α gene in P. vivax isolates from District Nowshera in Pakistan.

Methods

Blood samples from 124 consenting patients with uncomplicated malaria presenting to different hospitals of district Nowshera were collected during March-August 2019, representing 28 P. falciparum and 96 P. vivax isolates. DNA from all samples was subjected to nested PCR-based allele-specific marker analysis. Pvmsp-3α amplified fragments were further treated with restriction fragment length polymorphism (RFLP)-based Hha1 restriction enzyme.

Results

In P. falciparum, 21 alleles were detected, including 14 alleles for Pfmsp-1 and 7 alleles for Pfmsp-2. The suballelic families MAD20 (50%) in the Pfmsp-1 family and FC27 (75%) in the Pfmsp-2 family were predominant. The multiplicity of infection (MOI) was calculated as 1.4 and 1.2 for Pfmsp-1 and Pfmsp-2, respectively, with an overall mean MOI of 1.34. In P. vivax, 4 allelic variants, Type A-D, were detected for Pvmsp-3α through nested PCR, while after RFLP digestion of amplicons, 9 suballelic variants (A1-A4, B1, B2, C1, C2 and D1) were observed at the Pvmsp-3α locus.

Conclusion

This first ever report of molecular characterization of P. falciparum and P. vivax genotypes from District Nowshera, Pakistan reveals moderate to high allelic diversity in these parasites from District Nowshera, Pakistan.

Malaria

Pfmsp-1

Pfmsp-2

Pvmsp-3α

genetic diversity

Nowshera

Pakistan

Despite huge investments and efforts to control and eradicate malaria, it is still a major concern for human health and wealth in resource-limited countries of the world. Malaria is one of the most important protozoan infections around the world, inflicting 249 million cases and 608,000 deaths during 2022 alone (World Health Organization, 2023). Pakistan is one of the malaria-endemic countries in the world, where 371,828 cases of malaria were reported by the WHO in 2020 (World Health Organization, 2021). Pakistan is a moderately endemic region for malaria, where approximately 60% of its populace lives in malaria-endemic areas, with 177 million people at risk of contracting malaria (Qureshi et al., 2019). The prevalence of malaria differs in provinces and even in different districts of Pakistan with different climates. Khyber Pakhtunkhwa is one of the most endemic provinces in Pakistan, where approximately 31% of malaria cases were reported in 2018 (World Health Organization, 2019).

Among the five malaria inflicting species, Plasmodium falciparum and Plasmodium vivax are the most prevalent in the world; the former remains the most dominant species in Africa and is responsible for the most severe clinical forms associated with 95% of malarial deaths, while the latter is the most widely distributed parasite in tropical regions, especially in Asia and America (Ouledi, 1995; Mze et al., 2016). P. falciparum remained the major focus of interest by the researchers, as it is accountable for the majority of malaria-inflicted deaths, whereas P. vivax has been comparatively less explored and classified as benign. However, recent studies have revised this classification of P. vivax malaria by reporting severe vivax malaria in most malaria-endemic regions responsible for 300 million episodes of clinical malaria annually (Phyo et al., 2022; Anvikar et al., 2020; Naing et al., 2014; Gething et al., 2012). vivax has a wider geographical distribution and is increasingly being recognized as less responsive to malaria control measures than P. falciparum (Oliveira-Ferreira et al., 2010). A number of distinctive features of P. vivax biology are believed to assist in the evasion of control measures, such as infection relapse (Bousema and Drakeley, 2011; White and Imwong, 2012), the early appearance of transmission stages (Bousema and Drakeley, 2011; Mueller et al., 2013) and prompt acquisition of clinical immunity (Mueller et al., 2013; Feachem et al., 2010). P. vivax-based malaria transmission is therefore likely to be more stable over time and during control efforts than P. falciparum malaria transmission (Feachem et al., 2010).

Malaria control is confounded by different factors, including drug resistance, insecticide resistance and lack of vector control measures. Moreover, the unavailability of an effective multivalent vaccine against malaria is the main reason behind the high burden of malaria (Kaslow et al., 2015). Investigation of Plasmodium parasite structure and genetic diversity is essential for designing promising vaccines against malaria and for understanding the evolution of parasite virulence and the role of polymorphisms in malaria transmission (Thakur et al., 2008). During the blood stage of the malaria life cycle, merozoite surface proteins are expressed and are the main potential targets for the assessment to develop an effective vaccine (Yang et al., 2006). In P. falciparum, Pfmsp-1 and Pfmsp-2 and in P. vivax, Pvmsp-3α and Pvmsp-3β are among the promising candidate genes for vaccine development (Kumar et al., 2002). PFMSP-1 (190 kDa protein) has a pivotal role in erythrocyte invasion by merozoites, and its block 2 is considered the most polymorphic region (Holder et al., 1992). PfMSP-2 is a glycoprotein consisting of 5 blocks, in which block 3 is a polymorphic region (Kimura et al., 1990). The Pfmsp-1 gene comprises 3 main allelic variants, K1, MAD20, and RO33, and the Pfmsp-2 gene comprises FC27 and 3D7 allelic variants (Snounou et al., 1999). Similarly, merozoite surface proteins expressed on the P. vivax parasite surface are used to study P. vivax genotypes, among which Pvmsp-3α and Pvmsp-3β are highly immunogenic and hence are important vaccine candidate antigens. Pvmsp-3α is composed of three blocks of alanine-rich domains with heptad repeats (Lupas, 1996). Thus, the Pvmsp-3α and Pvmsp-3β genes are highly polymorphic due to the addition and removal of alanine-rich regions (Rayner et al., 2002). The genetic diversity of Plasmodium parasites and multiplicity of infection (MOI) have been reported to vary with the severity and transmission intensity of malaria in a particular region (Ashley et al., 2018). Worldwide genetic diversity and genotyping of P. falciparum and P. vivax field isolates have been extensively studied using their MSPs (Afridi et al., 2018; Anantabotla et al., 2019; Chen et al., 2018; Funwei et al., 2018; Hamid et al., 2016; Kang et al., 2010; Khatoon et al., 2012; Khatoon et al., 2010;Ullah et al., 2022; Verma et al., 2016). Although there is very limited information available regarding the genetic diversity of P. falciparum and P. vivax field isolates from Pakistan (Afridi et al., 2018; Khatoon et al., 2012; Khan et al., 2021; Mohammed et al., 2018; Ullah et al., 2021), no such study delineating the genetic diversity of P. falciparum and P. vivax from district Nowshera of Khyber Pakhtunkhwa Province in Pakistan has been reported thus far. Therefore, the current study seeks to elaborate P. falciparum and P. vivax genetic diversity among Pakistani isolates from District Nowshera using their corresponding polymorphic markers, Pfmsp-1 and Pfmsp-2 in P. falciparum and Pvmsp-3α in P. vivax isolates along with multiplicity of infection (MOI) of P. falciparum and P. vivax isolates.

Ethics statement and consent for participation

The present study was approved by the Ethical Committee of the Department of Biochemistry Abdul Wali Khan University Mardan (AWKUM/Biochem/Dept/Commit/18), and all the participants or their parents/guardians were asked to provide written informed consent before recruiting them for this study.

Study Area

The current study was conducted in District Nowshera of Khyber Pakhtunkhwa (KP) Province in Pakistan, located between 33° 04′ 00″ to 34° 10′ 00″ N latitude and 71° 44′ 50″ to 72° 15′ 05″ E longitude. District Nowshera is in the center of KP and is bordered by district Swabi from the north and district Charsadda from the northwest, while to its east district Attock of Punjab Province, in the south district Kohat and in the west district Peshawar are located (Fig. 1). Its total area is 1748 km^2, with a total population of 1,518,549 persons in 2017. In Nowshera, summers are hot and humid, and winters are cool and rainy. Its annual rainfall is 131.34 mm, and its temperature typically ranges from 43°F to 111°F (Nasir et al., 2022). Its location is along the River Kabul, and its rainfall and weather conditions are apparently favorable for malaria transmission.

Sample Collection

Blood samples (2 mL) were collected from 124 malaria patients after being confirmed positive for P. vivax (96 samples) and P. falciparum (28 samples) by microscopy using thick and thin blood smears and stained with Giemsa stain. Study samples were collected from patients (aged 6–65 years) visiting three major hospitals of District Nowshera, i.e., DHQ Hospital, Nowshera Medical Complex (NMC) and Combined Military Hospital (CMH), during March-August 2019. Among 124 blood isolates, 114, including 90 P. vivax and 24 P. falciparum isolates, were further confirmed to be positive by RDT (CareStart™: Malaria HRP2/pLDH(Pf/PAN) Combo) and species-specific PCR targeting the 18S ribosomal RNA (rRNA) gene (Snounou et al., 1999). All the samples were shifted to the Molecular Biology Lab in the Department of Biochemistry AWKUM for parasite genomic DNA isolation and genotyping analysis.

DNA Isolation

Collected blood samples of P. vivax and P. falciparum infections were subjected to DNA extraction using a Thermo Scientific Gene JET Genomic DNA Purification Kit (Cat No. K0721) followed by analysing the DNA quality and quantity through gel electrophoreses and visualization through an ethidium bromide-based UV gel documentation system.

PCR amplification of the Pfmsp-1 and Pfmsp-2 genes

Nested PCR genotyping of the Pfmsp-1 (block-2) and Pfmsp-2 (block-3) polymorphic regions of P. falciparum parasites was performed following a previously reported protocol by Snounou et al. (1999) (Table 1). For the negative control, deionized water was used, while for the positive control, DNA from commercially available parasite strains was used. The final volume of the initial PCR mixture for both Pfmsp-1 and Pfmsp-2 was adjusted to 16 µL by adding 7.5 µL master mix (Solis BioDyne), 0.5 µL each forward and reverse primer, 5.5 µL double distilled water and 2 µL genomic DNA. Using the product of the initial amplification as a template for the secondary reaction, 5 reactions were performed, in which specific primers (Table 1) were used to identify allelic variants of Pfmsp-1 (K1, MAD20 and RO33) and Pfmsp-2 (FC27 and 3D7). In secondary reactions 20 µL mixture was prepared for each sample containing 6 µL master mix, 10 µL ddH₂O, 0.5 µL each forward and reverse nested primers and 3 µL PCR product of initial PCR as a template. Nested products were analysed using a 2% agarose gel stained with ethidium bromide under UV light. The suballelic variants of the P. falciparum infections were determined from their banding pattern on agarose gels using a 100 bp DNA ladder.

Table 1

The primer sequences, base pairs and PCR conditions for *P. falciparum* and *P. vivax* genes in District Nowshera, Pakistan.
Primers	Sequences	Base pairs	PCR conditions	References
K1 family (N2)	F: 5'-AAATGAAGAAGAAATTACTACAAAAGGTGC-3'	30	95°C 4 min/[95°C 30 sec, 55°C 45 sec, 72°C 3 min] × 30 cycles 72°C 4 min.	(Snounou et al., 1999)
K1 family (N2)	R: 5'-GCTTGCATCAGCTGGAGGGCTTGCACCAGA-3'	30		(Snounou et al., 1999)
MAD20 family (N2)	F: 5 '-AAATGAAGGAACAAGTGGAACAGCTGTTAC-3'	30	95°C 4 min/[95°C 30 sec, 55°C 45 sec, 72°C 3 min] × 30 cycles 72°C 4 min.	(Snounou et al., 1999)
MAD20 family (N2)	R: 5 '-ATCTGAAGGATTTGTACGTCTTGAATTACC-3'	30		(Snounou et al., 1999)
RO33 family (N2)	F: 5 '-TAAAGGATGGAGCAAATACTCAAGTTGTTG-3	30	95°C 4 min/[95°C 30 sec, 55°C 45 sec, 72°C 3 min] × 30 cycles 72°C 4 min.	(Snounou et al., 1999)
RO33 family (N2)	R: 5 '-CATCTGAAGGATTTGCAGCACCTGGAGATC-3	30		(Snounou et al., 1999)
Pfmsp-2 (N1)	F: 5 '-ATGAAGGTAATTAAAACATTGTCTATTATA-3'	30	95°C 5 min/[94°C 1:45 sec, 54°C 30 sec, 72°C 3 min] × 35 cycles 72°C 5 min.	(Snounou et al., 1999)
Pfmsp-2 (N1)	R: 5'-CTTTGTTACCATCGGTACATTCTT-3'	24		(Snounou et al., 1999)
FC27 family (N2)	F: 5'-AATACTAAGAGTGTAGGTGCARATGCTCCA-3'	30	95°C 4 min/[95°C 30 sec, 55°C 45 sec, 72°C 3 min] × 30 cycles 72°C 4 min.	(Snounou et al., 1999)
FC27 family (N2)	R: 5 '-TTTTATTTGGTGCATTGCCAGAACTTGAAC-3'	30		(Snounou et al., 1999)
3D7 family (N2)	F: 5 '-AGAAGTATGGCAGAAAGTAAKCCTYCTACT-3'	30	95°C 4 min/[95°C 30 sec, 55°C 45 sec, 72°C3 min] × 30 cycles 72°C 4 min.	(Snounou et al., 1999)
3D7 family (N2)	R: 5 '-GATTGTAATTCGGGGGATTCAGTTTGTTCG-3'	30		(Snounou et al., 1999)
Pvmsp-3a (N1)	F: 5 '-CAGCAGACACCATTTAAGG-3'	19	95°C 3 min/[94°C 40 sec, 54°C 30 sec, 68°C 30 sec] × 35 cycles 68°C 5 min.	(Snounou et al., 1999) (Bruce et al., 1999)
Pvmsp-3a (N1)	R: 5'-CCGTTTGTTGATTAGTTGC-3'	19		(Snounou et al., 1999) (Bruce et al., 1999)
Pvmsp-3a (N2)	F: 5'-GACCAGTGTGATACCATTAACC-3'	22	95°C 5 min/[94°C 30 sec, 55°C 30 sec, 68°C 30 sec] × 30 cycles 68°C 5 min.	(Bruce et al., 1999)
	R: 5'-ATACTGGTTCTTCGTCTTCAGG-3'	22
	R: 5'-GCTGCTTCTTTTGCAAAGG-3'	19

Multiplicity of infection and heterozygosity (H _E ) of P. falciparum genotypes

The mean multiplicity of infection (MOI) was estimated by dividing the total number of distinct Pfmsp-1 or Pfmsp-2 genotypes detected by the number of positive samples for the same marker (Kobbe et al., 2006). Samples with more than one allelic family were considered polyclonal infections, and those with a single allelic family were considered monoclonal infections. The expected heterozygosity (H_E) was calculated using the formula

H_E = [n/(n − 1)] [1 − ∑P_i²], where n is the sample size and Pi represents the allele frequency of the ith allele.

PCR-RFLP analysis of the Pvmsp-3α gene in P. vivax isolates

The Pvmsp-3α gene of P. vivax was amplified by nested PCR followed by digestion of the PCR amplicons with RFLP restriction enzymes according to previously described protocols by Bruce et al. (1999) and Yang et al. (2006). Briefly, the Pvmsp-3α gene was amplified using nested PCR conditions and primers (Table 1) in a total reaction volume of 20 µL comprising 1 µL genomic DNA, 7 µL ddH₂O, 10 µL master mix and 1 µL each of the forward and reverse oligonucleotide primers. In second amplification reaction, 2 µL product of primary reaction was used along with 8 µL master mix, 6 µL ddH₂O and 1 µL each forward and reverse primers making a total volume of 18 µL reaction mixture. All PCR amplicons were confirmed by electrophoresis using a 2% agarose gel and visualized under UV light. The size of the PCR products was estimated using a 1 kb DNA ladder.

For RFLP analysis, PCR products (6µL) of Pvmsp-3α gene were digested with 2 units (1µL) of Hha I restriction enzyme in 16µL reaction volume including 7µL PCR water and 2µLbuffer supplied with the enzyme, at 37°C for 4 hours. Alleles were classified based on undigested PCR product size and RFLP banding patterns after running on 2% agarose gel and visualizing the fragments under UV light. The amplicon size was estimated using a 100 bp and 1 kb DNA ladder (New England Biolab). The mean MOI and H_E index for Pvmsp-3α genotypes were calculated.

Size polymorphism in P. falciparum and multiplicity of infection

For genotyping of P. falciparum parasites in district Nowshera, a total of 24 isolates were successfully amplified for Pfmsp-1 alleles (K1, RO33, MAD20) and Pfmsp-2 alleles (FC27 and 3D7). Pfmsp-1 and Pfmsp-2 allelic families were classified on the basis of PCR-amplified fragment size. Both Pfmsp-1 and Pfmsp-2 with respect to their corresponding genotypes were highly diverse (Table 2). A total of 21 alleles were detected, including 14 for the Pfmsp-1 gene and 7 alleles for the Pfmsp-2 gene. At the Pfmsp-1 locus, four K1 alleles (180–350 bp), four RO33 alleles (150–300 bp) and six MAD20 alleles (100–300 bp) were detected, while at the Pfmsp-2 locus, six FC27 alleles (150–700 bp) and one 3D7 allele (300 bp) were observed. Among the Pfmsp-1allelic families, MAD20 was predominant, comprising nearly half of the isolates, while in the Pfmsp-2 family, FC27 was the most dominant (75%). The mean MOIs for Pfmsp-1 and Pfmsp-2 were calculated as 1.4 and 1.2, respectively, while the overall mean MOI was 1.34 for both genes in P. falciparum isolates. The expected H_E index was higher for Pfmsp-1 genotypes (0.68) but lower for Pfmsp-2 genotypes (0.26) (Table 2).

Table 2

Multiplicity of infection and expected heterozygosity (H_E) of the *Pfmsp-1* and *Pfmsp-2* genes in *P. falciparum* isolates
Genes	Alleles	No of alleles observed (%)	Alleles size	Mean MOI	H_E
Pfmsp-1	K1	4 (28.5)	180–350 bp	1.40	0.68
	MAD20	4 (28.5)	100–300 bp
	RO33	6 (42)	150–300 bp
Pfmsp-2 Pfmsp-1 + Pfmsp-2	FC27	6 (85.7)	150–700 bp	1.20	0.26
Pfmsp-2 Pfmsp-1 + Pfmsp-2	3D7	1 (14.3) 21	300 bp 100–750 bp	1.34

Genetic Diversity of Pvmsp-3α in P. vivax isolates

A total of 90 P. vivax field isolates were successfully analysed using the PCR-RFLP technique to investigate the genetic variation in their highly polymorphic marker Pvmsp-3α gene from the study area. Briefly, after nested PCR amplification of Pvmsp-3α, 4 major allelic variants were detected, labelled Type A (2.5 kb) in 36% of isolates, Type B (1.7 kb) in 32% of isolates, Type C (1.5 kb) in 30% of isolates and Type D (~ 0.65 kb) in 2% of isolates (Table 3). Among these variants, Type A was predominant, followed by types B and C at the Pvmsp-3α locus. Amplicons of the Pvmsp-3α gene acquired from the nested PCR were further digested with the restriction enzyme Hha 1 to obtain better resolution at the diversity level. Following amplicon digestion with Hha 1, 9 allelic variants designated A1-A4, B1, B2, C1, C2 and D1 were detected, where A4 [17% (16/90)] and B2 [15% (14/90)] were dominant among Pvmsp-3α suballelic variants (Table 3). Mixed infections with more than 1 suballelic variant of the Pvmsp-3α gene were also detected with 11% frequency in the study isolates (Fig. 2).

Table 3

PCR-based allelic polymorphism of the *Pvmsp-3α* gene in *P. vivax* isolates
Allelic variants	Size in kb (Frequency)	Mean MOI	H_E
Type A	2.5 kb (36%)	1.11	0.685
Type B	1.7 kb (32%)
Type C	1.5 kb (30%)
Type D	0.65 kb (2%)
H_E = Expected heterozygosity
MOI = multiplicity of infection

The genetic divergence of P. falciparum and P. vivax based on their potential vaccine candidate markers largely affects malaria transmission, drug resistance and malaria control measures. Consequently, resolving the genetic population structure of these parasites in malaria-endemic regions is necessary (Chen et al., 2018).

A variety of vaccine candidate genes in malarial parasites, especially in P. falciparum and P. vivax, have been studied worldwide to infer parasite genetic diversity and population dynamics, drug resistance mechanisms and malaria elimination strategies. In these parasites, different polymorphic markers, especially those that contain surface antigens with potential role in ineffective vaccine design, have been studied (Véron et al., 2009; Ullah et al., 2022; Orjuela-Sánchez et al., 2009; Kim et al., 2006; Leclerc et al., 2006). PCR-based genotyping has been successfully applied for genetic diversity analysis of P. falciparum and P. vivax parasites based on their corresponding antigenic markers, such as Pfmsp-1, Pfmsp-2, Pfglurp, Pvmsp-3α, Pvmsp-3β, Pvcsp and Pvmsp-1.

This study aimed to decipher the genetic diversity of P. falciparum and P. vivax isolates collected from Nowshera district, Khyber Pakhtunkhwa, Pakistan. P. falciparum and P. vivax isolates from Nowshera showed moderate to high levels of genetic diversity. In our study, a higher number of successfully genotyped samples was detected for Pfmsp-1 compared to that for Pfmsp-2 infections. This result is congruent with those reported previously from Burkina Faso (Soulama et al., 2009; Somé et al., 2018), Cote d’Ivoire and Gabon (Yavo et al., 2016) and Pakistan (Khan et al., 2021). Conversely, some studies from Ethiopia (Mohammad et al., 2019), Myanmar (Soe et al., 2017) and Sudan (A-Elbasit et al., 2007) have reported a somewhat higher frequency of Pfmsp-2 genotypes than Pfmsp-1 genotypes.

In P. falciparum, all allelic families of Pfmsp-1 (K1, RO33, MAD20) and Pfmsp-2 (FC27, 3D7) were successfully amplified as previously reported from other districts of Pakistan (Khan et al., 2021; Khatoon et al., 2010), Iran (Heidari et al., 2007), Honduras (Lopez et al., 2012), India (Joshi et al., 2007), Burkina Faso (Soulama et al., 2009) and southeast Gabon (Aubouy et al., 2003). Conversely, this study was in contradiction with some studies, such as that reported by the Khyber Agency in Pakistan (Khatoon et al., 2013), where only K1 and MAD20 alleles were reported for Pfmsp-1 isolates, while for Pfmsp-2, only 3D7 was reported, showing a lower level of genetic diversity in P. falciparum isolates than in our present study. The current study reports MAD20 as a highly prevalent variant of Pfmsp-1 infections, showing close agreement with several studies reported from Bannu, Pakistan (Khatoon et al., 2010), Northwest Ethiopia (Mohammed et al., 2018), India (Mamillapalli et al., 2007), and Myanmar (Kang et al., 2010). However, a remarkably incongruent result was reported from the Republic of Congo (Mayengue et al., 2011) and some southern regions of Khyber Pakhtunkhwa in Pakistan (Khan et al., 2021). This contradiction in allelic variation might be due to differences in the geographic and environmental conditions of these regions. In our Pfmsp-2 isolates, FC27 was dominant, as previously observed in Northwest Ethiopia (Mohammed et al., 2018), Nigeria (Ojurongbe et al., 2011) and Gabon (Aubouy et al., 2003). However, some studies conducted 11 years ago by Khatoon et al. (2010) from the Bannu district of Pakistan and Mayengue et al. (2011) from the Republic of Congo have reported a higher prevalence of the 3D7 allele than FC27. MSP-based MOI is considered the most potent tool for identifying the number of distinctive P. falciparum populations during an infection. The mean MOI observed in our study was 1.40 for Pfmsp-1 and 1.20 for Pfmsp-2, with an overall mean MOI of 1.34, reflecting low malaria transmission intensity in the study area. These results are in close agreement with a number of previously reported studies (Huang et al., 2018; Mze et al., 2020; Aroosh et al., 2011). The MOI for Pfmsp-1 and Pfmsp-2 infections in our study was very lower than that reported from Bioko Island, Equatorial Guinea (5.51) by Chen et al. (2018), Nigeria (2.6–2.8) by Funwei et al. (2018) and Gabon (4.0) by Ndong Ngomo et al. (2018). This huge difference in MOI confirms the high intensity and transmission of P. falciparum infection in these African countries compared to the low prevalence of P. falciparum malaria in Pakistan.

In our study, PCR/RFLP analyses of the Pvmsp-3α gene in P. vivax isolates were performed, and 4 distinct fragments of PCR products (A, B, C and D) were observed for Pvmsp-3α, in which the type A (2.5 kb) allele was the most prevalent, followed by type B (1.7 kb). This study agrees with previous studies that reported A allelic variant as the most frequent variant from Khyber Pakhtunkhwa, Sindh, Baluchistan and Punjab provinces of Pakistan (Khan et al., 2014) and from district Bannu (Khatoon et al., 2010) in Pakistan, suggesting the uniform distribution of Pvmsp-3α allelic families in different districts of Khyber Pakhtunkhwa province in Pakistan. However, in terms of the number of distinct allelic variants for Pvmsp-3α, the current study is different from those reported from Iran (Zakeri et al., 2010), Thailand (Cui et al., 2003) and Afghanistan (Zakeri et al., 2009), where only 3 allelic variants (A, B, C) for Pvmsp-3α were observed, which indicates the pattern of parasite diversity across the different regions of the world. Restriction digestion of the Pvmsp-3α amplified product displayed the presence of 9 unique allelic families among the 91 resolved amplicons, with allelic variant types A4 and B2 being the most frequent, while previous studies from Pakistan (Khan et al., 2014; Khatoon et al., 2010) reported 12 allelic variant types for the Pvmsp-3α gene, with the A3 allele being the most frequent. PCR-RFLP data from Pvmsp-3α loci exhibited 11% mixed-strain infections, revealing a comparatively higher frequency than that reported from China (5.6%) and much lower than that found in Thailand (20.5%) and FATA Pakistan (30%) (Yang et al., 2006; Zakeri et al., 2010). The higher rate of mixed infections from FATA Pakistan is reflected by the fact that FATA shares a border with Afghanistan, owing to which human migration was at its peak at that time. Unlike our study, no mixed infections were reported from Iran (Zakeri et al., 2010) and Hongshuihe (China) (Yang et al., 2006).

The variations observed in parasite species are likely attributed to elements such as sampling bias, host immune selective pressure on certain types and spatiotemporal changes in diverse circulating mosquito species that can transmit particular parasite types in a particular area during different seasons, with regions that have the same mosquito types tending to have similar parasite types. Limitations in our study were the small sample size of P. falciparum isolates collected during the study period and the use of nested PCR-based genotyping techniques instead of sequencing the genes under study, which might underestimate the actual level of genetic diversity in the study area.

Overall, medium to higher genetic diversity was found in P. falciparum and P. vivax field isolates from the Nowshera district of Khyber Pakhtunkhwa, Pakistan, with a low value of MOI for P. falciparum and P. vivax infections, inferring low infection intensity and transmission of malaria in the region.

Ethics approval and consent to participate

The study protocol was approved by the Ethical Review Committee of Abdul Wali Khan University Mardan under the letter of AWKUM/ERC/578. Consent was obtained from all participants before conducting the study.

Consent for publication

All authors have given their consent for publication of this manuscript.

Competing interests

The authors declare no competing interests.

Data Availability

All the data is available within the manuscript

Consent for publication

All authors have given their consent for publication of this manuscript.

Acknowledgement

We appreciate study participants for their voluntary participation. We extend our special gratitude to the laboratory technician and field data collectors who took part in the research.

Funding

Not applicable

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Genetic Diversity of Plasmodium falciparum and Plasmodium vivax Field Isolates from the Nowshera District of Pakistan

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