Genetic diversity and multiplicity of Plasmodium falciparum merozoite surface protein 2 in field isolates from Sudan

Introduction

Malaria was and still is a major health problem, with over one third of worldwide populations being at risk (World Health Organization, 2015). The burden of this disease falls the heaviest on Sub-Saharan African countries (World Health Organization, 2015). In Sudan, almost 90% of the population live under high malaria transmission levels, as recently reported by the WHO (“WHO | Sudan,” 2016; World Health Organization, 2015). Malaria in Sudan is transmitted mainly by Anopheles arabiensis and the majority of cases are due to P. falciparum (Ageep et al., 2009). The merozoite surface protein-2 (MSP2) is a highly polymorphic plasmodium membrane protein and the most expressed in the surface of the merozoite (Gerold et al., 1996). It has been long studied in vaccine trials for being accessible by the immune system (McCarthy et al., 2011). MSP2 protein is highly polymorphic, which gives an opportunity for molecular epidemiologists to differentiate between recurrent and new infections (Mwingira et al., 2011). Genetic diversity of MSP2 alleles is due to mutations and proliferation of highly polymorphic central repeats (Ferreira & Hartl, 2007). The allelic diversity of MSP2 proteins can be used to calculate multiplicity of infection (MOI), which is the number of different strains of P. falciparum co-infecting the same host and is a valuable for estimating malaria transmission level (Vafa et al., 2008). Sudan experiences a range of climate variations, from arid desert in the north, to tropical areas in the south and a short rainy season (Noor et al., 2012). The current study aimed to determine the level of genetic diversity of the P. falciparum MSP2 gene, using field isolates across different geo-ecological regions in Sudan. The study also aimed to determine the relationship between genetic diversity and the characteristics between different patients. Little has been documented regarding the molecular diversity of P. falciparum in hypo and meso-endemic regions of Sudan, in particular after adoption of ACT as antimalarial treatment.

Methods

Results

P. falciparum rDNA was detected in a total of 241 (88.9%) malaria patients using nested PCR (Figure 1). 158 (65.6%) were male, while 83 (34.4%) were female. The distribution of patients across the four studied states: Khartoum, Gezira, River Nile and the Red Sea was 81 (33.6%), 58 (24.1%), 47 (19.5%) and 55 (22.8%), respectively. The age groups ranged between 3–90 years, with a mean of 31.39 ±14.9 years. The mean parasite density was higher in the River Nile state (15869 parasites/µl) compared to Khartoum, Gezira, and Red Sea states (14563, 11266, and 14603 parasites/µl, respectively), and this difference was statistically significant (p-value = 0.02). The mean hemoglobin level was lowest in Khartoum (5.23 g/dl) compared to the other regions; Gezira, River Nile, and Red Sea (7.94, 9.26, and 7.74 g/dl, respectively), and the difference was statistically significant (p-value = 0.02) (Table 1).

Figure 1. Detection of P. falciparum species.

Amplification of the P. falciparum ribosomal DNA gene using genus and species specific primers by nested PCR, yielding amplicons of 220 bp in size.) Molecular weight marker (MW) is 100 bp (iNTRON, Inc.). Lanes 2, 3, 4, and 6 are positive for samples of P. falciparum; 1 and 5 are negative for samples of P. falciparum; lane 7 is the negative control.

Allelic polymorphism of MSP2, multiplicity of infection, distribution of multiclonal P. falciparum infection, and parasite density across different age groups in Sudan

Allele genotyping revealed the highly polymorphic nature of Sudanese P. falciparum isolates with respect to the MSP2 gene. Both IC1/3D7 and FC27 MSP2 allele types were identified (Figure 2). The total number of different sized alleles detected in this study was 42. Among them IC1/3D7 (160–400 bp) and FC27 (140–400 bp) allele families were noted. Frequencies of different MSP2 alleles and their combinations and multiplicity of infection across the study sites are shown in Table 2. The frequency of samples with only IC1/3D7 and FC27 were 56.4% (136/241), and 17% (41/241), respectively and IC1/3D7/FC27 combinations were found in 26.6% (64/241) of samples. The prevalence of IC1/3D7 and FC27 allelic types was 82.9% (200/241) and 43.5% (105/241), respectively. Multiple clones were detected in all study sites. Multiplicity of infection (MOI) was highest in P. falciparum infected patients from Gezira state (1.67 genotypes per infection) and lowest in those from Red Sea state (1.20 genotypes per infection) and it was statistically significant, (p-value = 0.001) (Table 2). The difference in distribution of allelic polymorphism of MSP2 was only significant for total MSP2 and FC27 (p-value = 0.002 and p-value = 0.042, respectively). However, for IC1/3D7 the result was not statistically significant (p-value = 0.89). The estimated mean MOI of all studied areas was 1.46 genotypes per infection (Table 2). There was a negative correlation between age and parasite density (Spearman rank coefficient = 0.03; p-value = 0.65), though it was not significant. The 18–40 age group had the highest mean parasite density (16354 parasites/µl, Table 3).

Table 2. MSP2 block 3 allelic type frequencies and multiplicity of infection of P. falciparum isolates from different study sites in Sudan.

allelic type	Khartoum	Gezira	River Nile	Red Sea	Total	Fragment size*	No. of alleles	p-value
	n=81	n=58	n=47	n=55	n=241
	n(%)	n(%)	n(%)	n(%)	n(%)
IC1/3D7	42(51.9)	21(36.2)	28(59.6)	45(81.8)	136(56.4)	450–750	11
FC27	14(17.2)	14(24.1)	10(21.3)	3(5.5)	41(17.0)	250–500	10
IC1/3D7/FC27	25(30.9)	23(39.7)	9(19.1)	7(12.7)	64(26.6)
	81(100)	58(100)	47(100)	55(100)	241(100)		21	0.002
Total IC1/3D7	67(82.7)	44(75.9)	37(78.2)	52(94.5)	200(82.9)			0.893
Total FC27	39(48.1)	37(63.8)	19(40.4)	10(18.2)	105(43.5)			0.042
Multiclonal isolates	25(30.9)	23(39.7)	9(19.1)	7(12.7)	64(26.6)
MOI	1.58	1.67	1.32	1.20	1.46			0.001

Key:

*: Fragment size by base pair

Table 3. Distribution of MSP2 block 3 allelic types among age groups of P. falciparum infected patients from Sudan.

	age groups
allelic type	<18	18–40	>40	Total
	n=34	n=156	n=51	n=241
	n(%)	n(%)	n(%)	n(%)
IC1/3D7	20(58.8)	86(55.1)	30(58.8)	136(56.4)
FC27	3(8.8)	31(19.9)	7(13.7)	41(17.0)
IC1/3D7/FC27	11(32.4)	39(25.0)	14(27.5)	64(26.6)
Total IC1/3D7	31(91.1)	125(80.1)	44(86.3)	200(82.9)
Total FC27	14(41.1)	70(44.9)	21(41.1)	105(43.5)
Multiclonal isolates	11(32.4)	39(25.0)	14(27.5)	64(26.6)
MOI	1.50	1.44	1.52	1.46

Figure 2. PCR genotyping of MSP2 polymorphic alleles.

PCR typing of MSP2 multiple clones (FC27) band size range (250–400 bp) and (IC1/3D7) band size range (500–750 bp). MW: 100 bp ladder (iNTRON, Biotechology). Lanes 1, 3 and 7 are positive for IC1/3D7, lane 9 is negative for the IC1/3D7 allelic family. Lanes 2, 4, and 6 are positive for the FC27 allelic family, 8 is negative for FC27 and lane 11 is a negative control. Lane 5 is positive for IC1/3D7, showing multiple clones (500 and 600 bp).

Discussion

Increasing our knowledge of the genetic diversity of P. falciparum induced malaria will certainly help us understand its pathogenesis, acquired immunity and drug resistance. 14, 15, 13 and 12 different alleles of MSP2 were identified in Khartoum, Gezira, River Nile and Red Sea states, respectively. This data is consistent with previous studies in low and unstable malaria transmission regions in eastern Sudan (A-Elbasit et al., 2007), central Sudan (Hamid et al., 2013), and other areas of seasonal unstable malaria transmission (Elmahdi et al., 2012). 3D7 alleles were more prevalent in the four states (Khartoum, Gezira, River Nile and Red Sea) compared to FC27 alleles. This finding differs from those of previous studies in central and eastern Sudan, where FC27 was the most predominant allelic family (A-Elbasit et al., 2007; Babiker et al.,1997; Hamid et al., 2013). In our study, 25% of studied samples were found to have mixed infections with multiple parasite clones; a result similarly reported in eastern Sudan (Babiker, 1998).

In our study, multiplicity of infection was highest in the >40 age group (with an average MOI of 2 and 1.68 in Khartoum and Gezira states, respectively), whereas the MOI was highest in the <18 age group (with an average of 1.37 and 1.33 in River Nile and Red Sea states, respectively), although the difference was not statistically significant. Recent studies on the variation of MOI with age have suggested that the influence of age on MOI is highly affected by the endemicity of malaria (Pinkevych et al., 2015). this is consistent with studies that have shown an age-dependent MOI in villages with intense perennial malaria transmission (Smith et al., 1999) and some areas with hypo-meso-endemic malaria transmission like Ghana (Agyeman-Budu et al., 2013),

The relatively higher number of alleles detected at high parasite densities might also mean that more diverse parasite populations are present in such infections. This suggestion is partly supported by the finding that all the different allelic types were more often concurrently detected in the high-density samples (Färnert et al., 2001). The present study reported that the increasing level of multiplicity was seen with increased parasite density of the samples. These findings are compatible with a previous study that has shown a clear trend of increasing parasite density with increased multiplicity for genetic markers on both genes, MSP1 and MSP2 (Peyerl-Hoffmann et al., 2001). The results of our study will likely influence current and future malaria control strategies since MOI can predict antimalarial treatment response (Kyabayinze et al., 2008).

Conclusions

Allele genotyping revealed the highly polymorphic nature of Sudanese P. falciparum isolates with respect to the MSP2 gene. The results of our study are expected to have an influence on the current and future malaria control strategies, since MOI predicts development of clinical malaria and subsequent efficacy of antimalarial treatment.

Abbreviations

MSP2: merozoite surface protein 2, PCR: polymerase chain reaction, MOI: multiplicity of infection, ANOVA: analysis of variance, WHO: World Health Organization, rDNA: ribosomal DNA, ACT: artemisinin based combination therapy.

Ethical statement

The study received approval from the scientific and research ethics committee of the Institute of Endemic Diseases; University of Khartoum, Sudan. Informed consent was obtained from all participants (or from the parents/legal guardians when the participant was a minor), prior to their enrolment. All malaria patients approached in the study were treated using the standard treatment of WHO protocol for malaria (Olumese, 2017).

Data availability

Dataset 1: Raw data supporting the findings presented in this study. The dataset is available both in XLSX and SPPS format. DOI, 10.5256/f1000research.12585.d179181 (Mustafa et al., 2017).