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Genetic diversity of Plasmodium falciparum and Plasmodium vivax field isolates from the Nowshera district of Pakistan

Malaria Journal volume 23, Article number: 358 (2024) Cite this article

Abstract

Background

The genetic diversity of malaria parasites contributes to their ability to adapt to environmental changes, develop drug resistance and circumvent the host immune system. This study aimed to analyse the genetic diversity of the Pfmsp1 and Pfmsp2 genes in Plasmodium falciparum and the Pvmsp-3α gene in Plasmodium vivax isolates from District Nowshera in Pakistan.

Methods

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

Results

Of the analyzed P. falciparum, 21 distinct alleles were detected, including 14 alleles for Pfmsp-1 and 7 alleles for Pfmsp-2. The sub-allelic families MAD20 (50%) of Pfmsp-1and FC27 (75%) of Pfmsp-2 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, Pvmsp-3α types A, B, C and D, were detected, while RFLP digestion of amplicons, detected 9 sub-allelic variants (A1-A4, B1, B2, C1, C2 and D1) 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 parasite population from District Nowshera, Pakistan.

Background

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, causing 249 million cases and 608,000 deaths in 2022 [1]. Pakistan is one of the malaria-endemic countries in the world, where 2.65 million cases of malaria were reported by the World Health Organization (WHO) in 2022 [1]. Malaria is moderately endemic in Pakistan with unstable transmission [2, 3]. Approximately 60% of its populace lives in malaria-endemic areas, with 177 million people at risk of contracting malaria [3]. 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 159,912 of malaria cases were reported in 2022 [4].

Among the five human malaria 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 most severe clinical cases and is associated with 95% of malarial deaths, while the latter is the most widely distributed parasite in tropical regions, especially in Asia and America [5, 6]. Plasmodium falciparum remains the major focus by researchers, as the most virulent, genetically complex and contributes to majority of malaria-associated deaths, whereas P. vivax has been comparatively less explored and used to be classified as benign [7]. However, recent studies have revised this classification of P. vivax malaria by reporting severe vivax malaria in most malaria-endemic regions, causing about 300 million episodes of clinical malaria annually [7,8,9,10]. Plasmodium vivax has a wider geographical distribution and is increasingly being recognized as less responsive to malaria control measures than P. falciparum [11]. A number of distinctive features of P. vivax biology are believed to assist in the evasion of control measures, such as infection relapse [12, 13], the early appearance of transmission stages [12, 14] and prompt acquisition of clinical immunity [14, 15]. Plasmodium vivax-based malaria transmission is, therefore, likely to be more stable over time and during control efforts than P. falciparum malaria transmission [15].

Malaria control is confounded by different factors, including drug and insecticide resistance, and lack of vector control measures [1]. Moreso, the unavailability of an effective multivalent vaccine against Plasmodium species is contributory to the high malaria burden [16]. 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 [17]. 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 [18]. For P. falciparum, Pfmsp-1 and Pfmsp-2, and P. vivax, Pvmsp-3α and Pvmsp-3β are among the promising target antigens for candidatevaccine development [19]. The PfMSP-1 (190 kDa protein) has a pivotal role in erythrocyte invasion by merozoites, and its block 2 is considered the most polymorphic region [20]. PfMSP-2 is a glycoprotein consisting of 5 blocks, in which block 3 is a polymorphic region [21]. The Pfmsp-1 gene comprises 3 main allelic variants, K1, MAD20, and RO33, and the Pfmsp-2 gene comprises FC27 and 3D7 allelic variants [22].

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. The Pvmsp-3α is composed of three blocks of alanine-rich domains with heptad repeats [23]. Thus, the Pvmsp-3α and Pvmsp-3β genes are highly polymorphic due to the addition and removal of alanine-rich regions [24]. 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 different regions [25]. Worldwide genetic diversity and genotyping of P. falciparum and P. vivax field isolates have been extensively studied using their MSPs [26,27,28,29,30,31,32,33,34,35]. Although there is very limited information available regarding the genetic diversity of P. falciparum and P. vivax field isolates from Pakistan [26, 32, 36,37,38] 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 generate baseline information on P. falciparum and P. vivax genetic diversity among Pakistani isolates from District Nowshera using their corresponding polymorphic markers, Pfmsp-1, Pfmsp-2 and Pvmsp-3α and the multiplicity of infection (MOI).

Methods

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 situated in the centre 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 km2with 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 [39]. Its location is along the River Kabul, and its rainfall and weather conditions are apparently favourable for malaria transmission.

Fig. 1
figure 1

Map showing the study area of District Nowshera Pakistan

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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, stained with Giemsa. 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), between March and 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). All the samples were transferred to the Molecular Biology Laboratory 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 theThermo Scientific Gene JET Genomic DNA Purification Kit (Cat No. K0721) followed by checking the quality and roughly estimating the quantity of extracted DNA through gel electrophoreses and visualization through an ethidium bromide-based UV gel documentation system.

PCR amplification of Pfmsp-1 and Pfmsp-2 genes

A nested PCR protocol with species-specific primers targeting the 18S ribosomal RNA (rRNA) of P. falciparum and P. vivax was carried out as previously described [22]. PCR-confirmed isolates were further genotyped for the Pfmsp-1 (block-2) and Pfmsp-2 (block-3) polymorphic regions of P. falciparum was performed following a previously reported protocol by Snounou et al. [22] (Table 1). For the negative control, deionized water was used, while for the positive control, DNA from commercially available parasite reference strain 3D7 (ATCC, cat# PRA-405D) was used. The final volume of the initial PCR mixture for both Pfmsp-1 and Pfmsp-2 was adjusted to 16µL by adding 7.5µL master mix (Solis BioDyne), 0.5µL each forward and reverse primer, 5.5µL double distilled water and 2µL genomic DNA. Using the product of the initial amplification as a template for the secondary reaction, 5 separate 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 the secondary reaction, 20µL mixture was prepared for each sample containing 6µL master mix, 10µL ddH2O, 0.5µL each forward and reverse nested primers and 3µL PCR product of initial PCR as template. Nested products were analysed using2% agarose gel stained with ethidium bromide under UV light. The sub-allelic 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
Full size table

Multiplicity of infection and heterozygosity (HE) 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 [40]. 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 (HE) was calculated using the formula.\

$${\text{H}}_{{\text{E}}} \, = \,\left[ {{\text{n}}/\left( {{\text{n}}\, - \,{1}} \right)\left] \, \right[{1}\, - \,\sum {\text{P}}_{{\text{i}}}^{{2}} } \right]$$

where n is the sample size and Pi represents the allele frequency of the -ith allele.

PCR–RFLP analysis of the Pvmsp-3α gene

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. [41] and Yang et al. [18]. Briefly, the Pvmsp-3α gene was amplified using nested PCR conditions and primers (Table 1) in a total reaction volume of 20µL comprising 1 µL genomic DNA, 7µL ddH2O, 10µL master mix and 1µL each of the forward and reverse oligonucleotide primers. In the nested reaction, 2 µL product of the primary reaction was used along with 8 µL master mix, 6 µL ddH2O and 1µL 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 (expected to be 0.5 to 2.5 kb) was estimated using a1 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 2μL buffer supplied with the enzyme, at 37 °C for 4 h. 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 digested and undigested amplicon sizes ranging from 120 bases to 2 kb, were estimated using 100 bp and 1 kb DNA ladders (New England Biolab), respectively. The mean MOI and HE index for Pvmsp-3α genotypes were calculated.

Results

Allelic frequency distribution of Pfmsp-1 , Pfmsp-2 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). The Pfmsp-1 and Pfmsp-2 allelic families were classified on the basis of PCR-amplified fragments 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 from Pfmsp-1 and 7 alleles from Pfmsp-2, respectively. At the Pfmsp-1 locus, 4 K1 alleles (180–350 bp), 4 RO33 alleles (150–300 bp) and 6 MAD20 alleles (100–300 bp) were detected, while at the Pfmsp-2 locus, 6 FC27 alleles (150–700 bp) and 1 3D7 allele (300 bp) were observed. Among the Pfmsp-1 allelic families, MAD20 was predominant, comprising nearly half of the isolates, while FC27 was the most dominant (75%) of Pfmsp-2. 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. The expected HE index was higher for Pfmsp-1 genotypes (0.68) compared to Pfmsp-2 genotypes (0.26) (Table 2).

Table 2 Multiplicity of infection and expected heterozygosity (HE) of the Pfmsp-1 and Pfmsp-2 genes in P. falciparum isolates
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Genetic diversity of Pvmsp-3α

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 Hha1 to obtain better resolution at the diversity level. Following amplicon digestion with Hha1, 9 allelic variants designated as 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 sub-allelic variants (Table 3). Mixed infections with more than 1 sub-allelic 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
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Fig. 2
figure 2

Frequency distribution of Pvmsp-3α suballelic variants in the Nowshera district

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Discussion

The genetic diversity of malarial parasites plays a major role in malaria transmission intensity, drug resistance, and the effectiveness of control measures. Hence, understanding the genetic population structure of these parasites in malaria-endemic regions is essential [28].

Globally, several vaccine candidate genes in P. falciparum and P. vivax have been extensively studied to understand parasite genetic diversity, population dynamics, drug resistance mechanism, and to inform malaria elimination strategies, with particular focus on polymorphic antigenic markers like merozoite surface proteins (MSPs) as promising vaccine targets [34, 42,43,44,45]. 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 [34], Pvmsp-3α, Pvmsp-3β [29], Pvcsp and Pvmsp-1 [46].

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 genetic diversity. In current study, a higher number of alleles was detected for Pfmsp-1 compared to Pfmsp-2. This result is in agreement with previous reports from Burkina Faso [47, 48], Côte d’Ivoire and Gabon [49], and Pakistan [38]. Conversely, some studies from Ethiopia [36], Myanmar [50] and Sudan [51] reported higher frequency of Pfmsp-2 than Pfmsp-1 genotypes.

The allelic families of Pfmsp-1 (K1, RO33, MAD20) and Pfmsp-2 (FC27, 3D7) were successfully amplified as previously reported from other districts of Pakistan [33, 36], Iran [52], Honduras [53], India [54], Burkina Faso [47] and southeast Gabon [55]. Conversely, results from this study differ from earlier findings reported from Khyber Agency in Pakistan [56], where only K1 and MAD20 alleles were reported for Pfmsp-1, and 3D7 for Pfmsp-2, showing low genetic diversity than the 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 [33], Northwest Ethiopia [39], India [57], and Myanmar [31]. However, a remarkably incongruent result was reported from the Republic of Congo [58] and some southern regions of Khyber Pakhtunkhwa in Pakistan [36]. The allelic variation might be due to differences in the geographic and environmental conditions. The FC27 allele of Pfmsp-2 was dominant in the present study, as previously observed in Northwest Ethiopia [39], Nigeria [59] and Gabon [55]. However, some studies conducted 13 years ago from the Bannu district of Pakistan [33] and from the Republic of Congo [58] have reported a higher prevalence of the 3D7 allele than FC27. The MOI is a potent tool to identify the number of distinct parasite clones and transmission intensity in different geographical regions. The mean MOI observed was 1.40 for Pfmsp-1 and 1.20 for Pfmsp-2, with an overall mean MOI of 1.34, reflecting low to moderate malaria transmission intensity in the study area. These results are in close agreement with a number of previously reported studies [60, 61]. The MOI for Pfmsp-1 and Pfmsp-2 infections in present study was lower than reported from Bioko Island, Equatorial Guinea (5.51) [28], Nigeria (2.6–2.8) [29] and Gabon (4.0) [62]. This huge difference in MOI confirms the high malaria transmission intensity in sub-Saharan Africa compared to Pakistan.

The Pvmsp-3α gene in P. vivax shows 4 distinct variants (A, B, C and D).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 the type A allelic variant as the most frequent from Khyber Pakhtunkhwa, Sindh, Baluchistan and Punjab provinces [63] and from district Bannu [33], suggesting the uniform distribution of Pvmsp-3α allelic families in most of the provinces 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 [46], Thailand [64] and Afghanistan [65], where only 3 allelic variants (A, B, C) for Pvmsp-3α were observed, which indicates the pattern of parasite diversity across the different geographical regions.

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 [33, 63] 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%) [18, 46]. 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 present study, no mixed infections were reported from Iran [46] and Hongshuihe (China) [18].

The limitations of this study include small sample size of P. falciparum isolates and the use of only one RFLP marker for Pvmsp-3α genotyping, which may underestimate genetic diversity. Future studies should involve larger sample size and cover broader region, utilizing multiple restriction enzymes and sequencing to provide a more comprehensive assessment of genetic diversity.

Conclusion

Overall, moderate to high genetic diversity was observed 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 to moderate malaria transmission intensity in the region.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biochemistry Abdul Wali, Khan University Mardan, Mardan, Pakistan

    Chandni Hayat, Asifullah Khan & Sahib Gul Afridi

  2. Department of Biotechnology Abdul Wali, Khan University Mardan, Mardan, Pakistan

    Atif Kamil

  3. School of Public Health, University of Alabama at Birmingham, Birmingham, USA

    Aniqa Sayed

  4. Department of Biochemistry, Abbottabad International Medical College, Abbottabad, Pakistan

    Kehkashan Akbar

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  1. Chandni Hayat
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  3. Asifullah Khan
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Contributions

Conceptualization: S. G. A. Data curation: C. H., A. K., A. K. Formal analysis: C. H., K. A., S. G. A. Investigation: C. H., S. G. A., A. K. Methodology: C. H., S. G. A., A. K.. Resources: A. K., A. S. Supervision: S. G. A., A. K. Writing – original draft: C. H., K. A., A. S. Writing – review & editing: A. K., S. G. A.

Corresponding author

Correspondence to Sahib Gul Afridi.

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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.

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The authors declare no competing interests.

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Hayat, C., Kamil, A., Khan, A. et al. Genetic diversity of Plasmodium falciparum and Plasmodium vivax field isolates from the Nowshera district of Pakistan. Malar J 23, 358 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12936-024-05190-9

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12936-024-05190-9

Keywords

Malaria Journal

ISSN: 1475-2875

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