Malaria causes 300–500 million cases worldwide and approximately 0.5–3 million deaths annually, the majority of which are caused by Plasmodium falciparum (Phillips, 2001; Ferreira et al., 2004). Pakistan is a moderate malaria-endemic region, and approximately 60% of its population is living in the endemic areas, whereas 177 million individuals are at risk of malaria (Qureshi et al., 2019). Annually, the estimated number of suspected and confirmed individuals is 3.5 million in Pakistan. The WHO included Pakistan as one of the six Eastern Mediterranean region countries with almost 100% population at risk of malaria (WHO, 2017; 2018). The endemicity of malaria varies in different provinces and even in different cities having variable climates. In 2017, about 30% of total malaria cases were reported from Khyber Pakhtunkhwa only, and the province has the highest reported cases of malaria (WHO, 2017).

The National Malaria Control Programmes (NMCP) has reported a record sixfold increase in P. falciparum during the last decade. The falciparum malaria has acknowledged sparse scientific attention, particularly concerning the molecular characterization of the local parasite population. The rise of P. falciparum in various districts of Pakistan may be attributable to the failed treatment of chloroquine resistance (Nizamani et al., 2006). Besides that, the heavy influx and continued presence of refugees from Afghanistan, where malaria is most prevalent, may contribute not only to the increasing number of cases of malaria but also to its genetic variations in Khyber Pakhtunkhwa province (Murtaza et al., 2004; Sheikh et al., 2005; Howard et al., 2011).

Analyses of the genotypes of Plasmodium spp. by PCR have remarkably improved our understanding of the biology of these parasites. In this regard, genetically distinct P. falciparum has been identified and extensively studied to further unveil its molecular epidemiology, parasite resistance, and potential vaccine candidates (Diggs et al., 1993; Greenhouse et al., 2006). The mainly used genetic markers of P. falciparum are the merozoite surface protein 1 (msp-1), merozoite surface protein 2 (msp-2), and glutamate-rich protein (glurp) (Smythe et al., 1991; Snounou and Beck, 1998). Allelic forms of these polymorphic markers have been reported in various parts of the world (Babiker et al., 1997; Jordan et al., 2001).

The msp-1 and msp-2 are antigenic proteins responsible for immunological responses in humans (Taylor et al., 1995; Aubouy et al., 2003). Block 2 of msp-1 has three polymorphic allelic families identified as MAD20, K1, and RO33 (Contamin et al., 1996). Similarly, the central domain of the msp-2 has two distinct families, i.e., 3D/IC and FC27 (Sallenave-Sales et al., 2000). These markers are unlinked and located on different chromosomes (Färnert et al., 2001). These features make them attractive candidates for studies where identification and enumeration of genetically distinct P. falciparum parasite subpopulations are of interest. As such, they have proven to be useful tools in molecular epidemiology studies in different epidemiological settings as well as to distinguish treatment failures from new infections in antimalarial drug trials (Cattamanchi et al., 2003; Collins et al., 2006).

The genetic diversity of P. falciparum population is an important indicator of the malaria transmission intensity in an area (Babiker et al., 1995; Paul et al., 1998). A high endemic area is generally characterized by extensive parasite diversity, and infected humans often carry multiple genotypes. Conversely, the parasite population in a low transmission area has a limited genetic diversity, and most infections are monoclonal (Babiker et al., 1997; Haddad et al., 1999; Peyerl-Hoffmann et al., 2001; Gómez et al., 2002). The P. falciparum field isolates have been characterized in Afghanistan, Iran, and India and previously from Sindh and Baluchistan in Pakistan, using the above-mentioned molecular markers. Therefore, we investigated the genetic diversity and polymorphic nature of P. falciparum isolates in selective districts of the malaria endemic province Khyber Pakhtunkhwa of Pakistan.

Out of 199 microscopy-positive samples, 192 were further confirmed for P. falciparum using PCR, whereas seven samples (3.2%) were excluded due to negative PCR outcome (Figure 1).

Out of total, 97 amplicons were observed for msp-1 and 95 for msp-2. In msp-1, the highest frequency of K1, MAD20, and RO33 was 44.3% (43/97), 33.0% (32/97), and 23.0% (22/97), respectively (Table 1). In msp-2, the FC27 has the highest frequency of 60.0% (57/95), followed by 3D7/IC with 40.0% (38/95) (Table 2). However, no polyclonal infection was observed in msp-1 and msp-2.

Seventeen alleles were detected in msp-1 comprising eight alleles (180–350 bp) for K1, six alleles (110–250 bp) for MAD20, and three alleles (130–190 bp) for RO33. However, 16 alleles were attributed to msp-2, with nine alleles (400–580 bp) for FC27 and seven alleles (300–430 bp) for 3D7/IC. The district-wise details of allelic frequencies are presented in the supplementary materials (Supplementary Tables S1, S2). The expected heterozygosity was 0.98 and 0.97 for msp-1 and msp-2, respectively.

Molecular studies provide an insight into the transmission intensity and genetic variation of parasite population within a region. The genetic diversity may be linked with the cross-border movement of populations living in the Frontier Regions of Pakistan (Ghanchi et al., 2010; Zakeri et al., 2010). Usually, areas with high malaria transmission are observed to have an extensive genetic diversity (Paul et al., 1998; Peyerl-Hoffmann et al., 2001). This study provides the basis to explore the genetic diversity of P. falciparum in the endemic regions of Khyber Pakhtunkhwa.

In the present study, the number of successfully genotyped samples for msp-1 was higher as compared with that for msp-2. This result is consistent with studies reported from Cote d’Ivoire and Gabon (Yavo et al., 2016) and Burkina Faso (Soulama et al., 2009; Somé et al., 2018). However, in contrast, Mohammad et al. (2019) from Ethiopia, Soe et al. (2017) from Myanmar, and A-Elbasit et al. (2007) from Sudan reported a relatively high frequency of msp-2 genotyped samples. Furthermore, of 192 positive samples for P. falciparum, less than half showed an amplicon for msp-1 (n = 97) or msp-2 (n = 95). In previous studies, relatively higher numbers of amplicons were observed for msp-1 or msp-2 (Somé et al., 2018; Mohammed et al., 2019; Eltayeb et al., 2020; Papa Mze et al., 2020). The nonspecific binding during P. falciparum identification by PCR may justify the smaller number of amplicons for msp-1 and msp-2 in the current study.

It was observed that 17 allelic variants of msp-1 and 16 of msp-2 were present in the studied areas. This result is in comparison with a similar study from the south of Pakistan (Ghanchi et al., 2010), Iran, South Africa, Myanmar, Sudan, Senegal, and Thailand (Heidari et al., 2007; Soe et al., 2017; Somé et al., 2018; Ndiaye et al., 2019; Eltayeb et al., 2020). However, in two southern districts Bannu and Kohat of Khyber Pakhtunkhwa, less allelic variants of P. falciparum were reported (Khatoon et al., 2010; Khatoon et al., 2012). Similarly, a study from the hypo-endemic area of Colombia reported only one allele of msp-1 and three alleles of msp-2 (Montoya et al., 2003). This difference might be due to low malaria endemicity since higher allele frequencies have been reported with high malaria transmission (Konaté et al., 1999; Soulama et al., 2009), suggesting that malaria endemicity affects the circulating strain number. However, high genetic diversity was observed in the Kingdom of Eswatini, which is regarded as a low transmission area for P. falciparum (Roh et al., 2019). Therefore, the genetic diversity may also be attributed to the effects of several factors such as indiscriminate use of long-lasting insecticide-treated nets (LLINs), indoor insecticide spraying, and antimalarial pressure (Soulama et al., 2009).

The K1 and FC27 alleles of msp-1 and msp-2 were predominant, respectively. The K1 and FC27 allelic families were previously reported from Kohat district (Khatoon et al., 2012). Nevertheless, the present findings also showed slight discrepancy with the previous studies (Zakeri et al., 2005; Ghanchi et al., 2010). The current study and previous findings suggest that K1 and FC27 allelic families might have prominent roles in clinical malaria at least in southern Khyber Pakhtunkhwa. However, further in-depth investigation with larger dataset should be carried out to unveil the genetic diversity and prevailing genotypes.

Interestingly, no polyclonal infection was detected in the present study. Malaria treatment policies, geographic isolation, and transmission intensities may result in spatial heterogeneity of P. falciparum (Khatoon et al., 2010). Most importantly, the use of highly potent antiplasmodial drugs that kill the asexual blood stage parasites and gametocytes are more likely to decrease parasite transmission and clonal diversity (Targett et al., 2001; Greenwood et al., 2008). However, in the adjacent districts (Bannu and Kohat) of Khyber Pakhtunkhwa, polyclonal infections were reported previously (Khatoon et al., 2010; Khatoon et al., 2012). It is worth mentioning that these two districts (Bannu and Kohat) accommodated one million internally displaced persons (IDPs) from tribal areas of North Waziristan Agency (NWA) sharing its border with Afghanistan, which may have introduced new genetically distinct variants of P. falciparum. It shows that the huge influx and migration of people between the study area and the neighboring countries like Iran and especially Afghanistan may introduce different alleles of P. falciparum into Khyber Pakhtunkhwa province of Pakistan.

Malaria transmission in Pakistan is markedly seasonal and prone to outbreaks, in particular geographical areas, especially Khyber Pakhtunkhwa, Baluchistan, and Sindh province (Yasinzai and Kakarsulemankhel, 2009). Pakistan is considered to be endemic for malaria, but the precise data on the genetic diversity of malaria in Pakistan are still lacking (Khan et al., 2005). As limitations, a small number of samples were amplified for msp-1 and msp-2, and the use of nested PCR instead of DNA sequencing could possibly underestimate the genetic diversity. Therefore, studies with larger datasets and more robust techniques should be used to explore the genetic diversity in the future. Furthermore, only microscopy-positive samples for P. falciparum were further subjected to nested PCR, which is another limitation of the present study.

It was concluded that extensively diverse and polymorphic P. falciparum populations of merozoite surface protein 1 (msp-1) and 2 (msp-2) are present in Khyber Pakhtunkhwa, Pakistan.