Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates

Introduction The genomic miscellany of malaria parasites can help inform the intensity of malaria transmission and identify potential deficiencies in malaria control programs. This study was aimed at investigating the genomic miscellany, allele frequencies, and MOI of P. falciparum infection. Methods A total of 85 P. falciparum confirmed isolates out of 100 were included in this study that were collected from P. falciparum patients aged 4 months to 60 years in nine districts of Khyber Pakhtunkhwa Province. Parasite DNA was extracted from 200µL whole blood samples using the Qiagen DNA extraction kit following the manufacturer’s instructions. The polymorphic regions of msp-1, msp-2 and glurp loci were genotyped using nested PCR followed by gel electrophoresis for amplified fragments identification and subsequent data analysis. Results Out of 85 P. falciparum infections detected, 30 were msp-1 and 32 were msp-2 alleles specific. Successful amplification occurred in 88.23% (75/85) isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp gene. In msp-1, the K1 allelic family was predominantly prevalent as 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples with single infection having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75), and 10.67% (8/75), respectively. In msp-2, both the FC27 and 3D7 allelic families revealed almost the same frequencies as 70.14% (47/67) and 67.16% (45/67), respectively. Nine glurp RII region alleles were identified in 60 isolates. The overall mean multiplicity of infection for msp genes was 1.6 with 1.8 for msp-1 and 1.4 for msp-2, while for glurp the MOI was 1.03. There was no significant association between multiplicity of infection and age groups (Spearman’s rank coefficient = 0.050; P = 0.6) while MOI and parasite density correlated for only msp-2 allelic marker. Conclusions The study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates in Khyber Pakhtunkhwa, Pakistan. In the present study the genotype data may provide valuable information essential for monitoring the impact of malaria eradication efforts in this region.

Glurp protein-based antibodies can inhibit the growth of P. falciparum and are suggested to play important role in controlling parasitaemia [20].
To our knowledge, the genetic diversity of P. falciparum has been extensively studied in different parts of the world but there is very limited information on the genetic diversity of msp1, msp2 and glurp genes in our study area. This study is aimed at evaluating the genetic diversity, multiplicity of infection, the level of malaria transmission, and allele frequencies of msp-1, msp-2 and glurp in malarial isolates from 9 districts of Khyber Pakhtunkhwa province of Pakistan.

Study area
This study was carried out in 9 districts of Khyber Pakhtunkhwa province including Mardan, Swat, Hangu, Buner, Swabi, Kohat, Bannu, Timergara, and Peshawar. The latitude and altitude of the study site is 34.9526 N and 72.3311 E. The population of the study area is 35.53 million and the total area is 101,741 km 2 . The region receive an annual rainfall of 384 mm from March to May and August to November. The mean temperature ranges from 20˚C to 40˚C. According to Pakistan Malaria Annual Report 2019 [21], Khyber Pakhtunkhwa Province ranked second in the number of malaria cases (31%) in Pakistan after Sindh province whereas the annual parasite index (API) was 6.3 in the year 2018. Samples for this study were collected during the clinical trial conducted during 2017-2019 in different health facilities of the nine districts (Fig 1).

Study population and blood samples collection
A total of 100 venous blood samples (3mL) were collected from microscopy-confirmed patients having uncomplicated P. falciparum malaria after taking written informed consent from the patients or the parents of patients aged below 18 years. Among 100 samples, 85 were confirmed positive for P. falciparum by species-specific PCR analysis. The patients aged 4 months to 60 years were the residents of the study area who visited the local health centers (hospitals, clinics, laboratories, etc) with body temperature of �37.5˚C. This study was reviewed and approved by the Ethical Committee of the Department of Biochemistry, Abdul Wali Khan University Mardan (AWKUM/Biochem/Dept/Commit/18). The blood was stored at 20˚C before DNA extraction at the Biochemistry research lab, Abdul Wali Khan University Mardan, Pakistan. Genomic DNA was extracted from 200µl whole blood samples using the Qiagen DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.

Malaria parasite density
Thick and thin blood smears were stained with Giemsa stain for 20 minutes. Parasite density was determined by analyzing the number of asexual parasites per 200 white blood cells and calculated per microliter by multiplying the number of parasites with 8000/200 assuming a blood cell count of 8000 cells per microliter. In case of no result using 200 high power ocular fields of the thick film, it was considered negative [22]. The data were graded into 3 categories according to parasitaemia as patients with 50-5000 parasites/µL, 5000-10000 parasites/µL, and patients with more than 10000 parasites /µL [23]. % parasitaemia = Parasitized RBCs /total number of RBCs X 100

Multiplicity of infection
The mean multiplicity of infection (MOI) was calculated by dividing the total number of fragments detected in both msp-1 and msp-2 loci by the number of samples positive for both markers. The isolates with more than one allelic family were considered polyclonal infections and those with single allelic family were classified as monoclonal infections. The number of infecting genotypes present in each sample represents the multiplicity of infection (MOI) for that sample was calculated as the highest number of alleles obtained in the samples [24,25].

Genotyping of P. falciparum msp-1, msp-2 and glurp genes
Nested PCR genotyping of the polymorphic region of msp-1 (block-2), msp-2 (block-3), and glurp (RII repeat regions) was performed using primers and methods as previously described [26,27]. The initial amplification primers pairs corresponding to conserved sequences within polymorphic regions of each gene were used in separate reactions ( and 2 min at 72˚C and final extension of 5 min at 72˚C [28]. The amplified msp-1, msp-2 and glurp fragments were resolved by gel electrophoresis using 2% agarose gel and visualized under UV light using Gel doc system after staining with ethidium bromide. The size of DNA fragments was estimated by visual inspection using a 100 bp DNA ladder marker (New England Biolabs. Inc, UK).

Statistical analysis
All the statistical analyses were performed using SPSS version 22. The msp-1, msp-2 and glurp allelic frequency and their mean MOI were calculated. The spearman's rank correlation coefficient was calculated to evaluate the relationship of MOI with parasite densities and age groups of the patients. A P value of < 0.05 was considered indicative of statistically significant differences.

General characteristics of the study population
Out of 100 jhmicroscopy-based confirmed samples of P. falciparum malaria, 85 PCR-confirmed samples were included in this study.

Multi-clonal infections
The multiplicity of infection (MOI)/number of genotypes per infection was calculated by dividing the total number of fragments detected in one antigenic marker by the number of samples positive for the same marker. Out of 85 samples, 86.1% showed more than one parasite genotype (multi-clonal infections). The overall mean multiplicity of infection for both msp-1 and msp-2 genotypes was detected as 1.6. Multiple clones were detected for all the 3 genes under study with 60% (45/75) multi-clonal infections for msp-1, 37.3% (25/67) for msp-2 and 3.34% (2/60) for glurp genes. The mean MOI for msp1, msp2 and glurp was calculated as 1.8, 1.4 and 1.03, respectively.

Relationship of MOI with age groups and parasite densities
The prevalence of msp-1, msp-2 and glurp allelic families and their sub-allelic types were significantly higher in number among patients' age group 21-40 years as compared to other age Table 3. Frequencies and MOI of Plasmodium falciparum msp-1 and msp-2 isolates. groups. The highest MOI (1.9) for msp-1 was observed in age groups 21-40 and 41-60 years while the lowest MOI (1.0) was in age group >60 years. Likewise for msp-2, the MOI was higher (1.5) in the age group 41-60 years and the lowest (1.0) was detected for the age group >60 years. No statistically significant correlation was found between the multiplicity of infection and age groups of different patients for msp-1 (Spearman rank coefficient = 0.050; P = 0.6), msp-2, (Spearman rank coefficient = 0.094; P = 0.3) and for glurp genes (Spearman rank coefficient = 0.036; P = 0.7) (Table 5). However, an increasing trend of mean MOI of individual msp-1, msp-2 and their overall MOI was observed with the increase in the age of patients below 60 years. The patients with age>60 years were very few and therefore all the genes revealed the lowest MOI values for this age group. The distribution of msp-1 gene and its sub-allelic families according to parasitaemia revealed correlation differences to some extent but not significant (Spearman rank 0.205; P = 0.060). Likewise, for glurp gene and its alleles, this association was non-significant showing the same MOI for all parasitaemia groups (0.026; P = 0.814). On the other hand, MOI and parasite density for msp-2 were significantly correlated (Spearman rank coefficient 0.250; P = 0.021). The MOI and parasite density of all the markers collectively (msp-1, msp-2 and glurp) was not significantly correlated (P = 0.23). For msp-1, the highest MOI (2.1) was observed in the range of 50-5000 parasitaemia and the lowest MOI (1.5) in 5001-10000 parasite density. For msp-2, lower MOI (1.2) was detected for 50-5000 parasitaemia and higher MOI (1.5) for >10000 parasitaemia. The distribution of different alleles of msp-1, msp-2 and glurp according to parasitaemia is shown in Table 6).

Discussion
The present study provides a detailed analysis and assessment of genetic diversity and polymorphism of P. falciparum isolates in Khyber Pakhtunkhwa, Pakistan. The purpose of this study was to determine the genetic diversity and molecular characterization of P. falciparum isolates using their different vaccine candidates genes viz. msp-1, msp-2, and glurp from nine districts of Khyber Pakhtunkhwa province. Genetic diversity and polymorphism play an important role in the acquisition of anti-malaria parasite immunity [29,30], to identify the genotypes circulating in different geographical settings and to effectively monitor malaria control measures.
The allele specific genotyping of msp-1, msp-2 and glurp in P. falciparum isolates revealed high allelic diversity in the nine districts of Khyber Pakhtunkhwa. This trend of high allelic diversity in the study area may be attributed to high-level transmission of malaria and subsequent exposure of inhabitants to mosquito bites. This suggests that the occurrence of mixed infections in a population is affected by the intensity of transmission. Nevertheless, there is a possibility of underestimating the exact number of alleles due to limitations in the analytical techniques used in genotyping as it is difficult to distinguish the fragments with length differences of less than 20bp as distinct alleles [17]. This low to moderate rate of MOI in the present study may be due to intensified malaria control interventions by WHO and low migration inflows across the border from Afghanistan during the last decade due to stability and peace in Afghanistan.  In our study, the genetic diversity of msp-1 and msp-2 alleles were higher than glurp alleles. This result is in the line with the previous studies [24,31]. Among the allelic families of msp-1 gene, a total of 30 allelic types were detected in which K1 was the predominant allelic type. This was in close agreement with those reported for Southwest Ethiopia [17], Cote d' Ivoire and Gabon [32], Central Africa, Gabon, Benin and Ghana [26,28,33] where K1 was the highly prevalent allelic family. In contrast, the isolates from Indonesia For msp-2, a total 32 allelic types were detected, among which the alleles belonging to 3D7 family and FC27 were equal in number (16 each where FC27 was reported as the more prevalent allelic family of msp-2 gene. For glurp gene, the RII region of the glurp also showed a high degree of polymorphism in the parasite population of P. falciparum across Khyber Pakhtunkhwa with 9 different allelic fragments detected. The high number of glurp alleles indicates a high malaria endemicity which is also in agreement with the previous studies [39,42]. For msp-1, msp-2 and glurp, the high level of genetic diversity is compatible with the high level of malaria transmission. A total of 30, 32 and 9 distinct alleles for msp-1, msp-2 and glurp, respectively, were obtained in isolates from 9 districts of Khyber Pakhtunkhwa. In this study, the genetic diversity found was higher in contrast to those reported from countries like Nigeria (where only 5 and 15 alleles were identified for msp-1 and msp-2 genes, respectively) [8], . For glurp gene the same result was reported for Northwest Ethiopia (9 glurp alleles) [46] and from India where 8 glurp alleles were recorded [47]. Conversely, studies from several regions have reported higher polymorphism frequency in the RII region of glurp gene than our reported one, for instance, 11 glurp genotypes have been reported from Southwestern Nigeria [48] and 14 genotypes from Sub-Sahara Africa [39]. Most of the infections were multi-clonal (60%) in msp-1 family while msp-2 isolates were observed with fewer multi-clonal infections (37%) than monoclonal infections (63%). The overall mean multiplicity of infection was 1.6 for both msp genes, while the individual multiplicity of infection was detected as 1.8 for msp-1, 1.4 for msp-2 and 1.03 for glurp gene. MOI reported in this study was higher than reported from countries like Malaysia where the MOI of P. falciparum infection for msp-1 was 1.37 and for msp-2 was 1.20 [49]. However, the MOI for msp-2 was lower than in Cote d' Ivoire (MOI = 2.8) [50]. The overall MOI in our study was 1.6, which is in close agreement with the studies previously conducted in Southern Ghana (MOI = 1.48) [51], Republic of Congo (MOI = 1.7) [45], Southwest Ethiopia (1.8) [17], while it  -1 (blocks-2), msp-2 (block-3) and glurp RII allelic types among parasitaemia groups in the study area.  [53]. This inconsistency in the pattern of allele frequencies in these very important vaccine candidate genes (msp-1, msp-2 and glurp) along with drug resistance genes of malarial parasites may be due to geographical variations, their transmission pattern, use of different malarial drugs, and also samples population determination. When there is a higher malaria transmission level, there will be more chances of getting a higher MOI and mean number of alleles per locus. It was also found that 81% of samples harbored multi-clonal infections (having more than one allele types) which is in line with the previous studies from Mauritania with 82.3% multiple infections [53], Brazzaville Republic of Congo (83%) [38] and Iran (87%) [30]. Conversely, our reported multiple infections cases were higher than those reported from Nigeria (70%) [47], Southwest Ethiopia (59%) [17] and Sudan (62%) [9] showing a lower frequency of samples with multi-clonal infections. Also observed in this research was a high-level prevalence of multi-clonal infections of msp-1 and msp-2 genotypes. This high level of genetic diversity is an indication that the parasite population size remained high enough to allow better mixing of different genotypes and also human migration brought a change in genetic diversity by introducing an additional number of parasites alleles [54]. Age is a key factor affecting MOI in P. falciparum infection and is also involved in the acquisition of immunity against P. falciparum species [55]. In young patients, a low level of immunity may be the major factor contributing to their vulnerability to control the infection [41,56]. The high level of malarial transmission in the region may be expected to lead to a higher risk of severe malaria in younger patients where immunity is lower [57]. In our study, MOI was not correlated with patients' age and the same association has been reported by studies conducted in other countries like Sudan [11], Ethiopia [33], Benin [58] and Senegal [59] which suggests that the MOI is not directly related to the period of acquisition of immunity in asymptomatic patients but reflects the exposure of subjects to malaria in the endemic area. Conversely, our findings disagree with those results reported from Central Sudan [9], Burkina Faso [60] and Bioko Island, Equatorial Guinea [52] where MOI has been reported to be linked with the age of malarial patients. However, partial positive correlations between MOI and parasite density for one of these genes have been reported by studies from Congo Brazzaville [38,59] and West Uganda [61].
Among the categorized three groups of parasite density i.e. 50-5000, 5001-10000 and >10000 parasite/µL, we found that there was no significant association between MOI and parasitaemia (P = 0.23). This trend is in congruity with previous studies reported from Benin [58] and Nigeria [40]. In contrast to our findings, different studies have shown a direct relationship between the MOI and parasite density like studies conducted in Congo, Brazzaville [38] and West Uganda [61] where the MOI showed an increase accordingly to the increase in parasite densities. These results are consistent with many reports demonstrating that high parasite densities increase the probability of detecting concurrent clones in individuals [61]. This finding reveals that high malaria transmission and also parasite density may have a strong association with the genetic diversity of P. falciparum in the Khyber Pakhtunkhwa province of Pakistan.

Conclusions
Field isolates of P. falciparum in nine districts of Khyber Pakhtunkhwa are highly diverse based on allelic variation in msp-1, msp-2 and glurp genes. This study provides basic information on the genetic diversity and multiple infections of P. falciparum msp-1, msp-2 and glurp isolates important for future studies on dynamics of parasite transmission and to evaluate the malaria control interventions in our study area.  (3)