Background

Malaria is still one of the most important life-threatening parasitic diseases in tropical and subtropical areas. There were approximate 219 million malaria cases and 435,000 deaths in the world in 2017 [1]. In Southeast Asia, the Greater Mekong Subregion (GMS) is one of the high malarious areas, with the co-existence of different species and emergence of drug-resistant parasites. In Yunnan Province, the most high malarious endemic region in China, annual incidence has decreased from 196/100,000 in 2006 to 0.7/100,000 in 2016, where indigenous malaria transmission is mostly concentrated in Yingjiang County that is adjacent to the Kachin State of Myanmar [2]. In addition, the malaria cases are also clustered on small spatial scales along the China–Myanmar border, which may be related to climatic, environmental, and ecological factors favoring vector survival [3, 4], as well as to the high malaria endemicity in the adjacent Kachin State of Myanmar.

Genetic diversity of the parasites provides useful information on the parasite populations and control efforts against malaria. Polymorphic genetic markers of P. falciparum include the merozoite surface protein 1 (MSP1) and MSP2 that have been used to evaluate the genetic diversity of malaria parasites [5,6,7,8,9,10]. Based on the sequence analysis of P. falciparum isolates from different endemic areas, the msp1 gene is divided into two allelic types of MAD20 and K1, whereas the highly polymorphic block 2 is represented by three allelic types of K1, RO33 and MAD20 [11]. In contrast, the msp2 gene is grouped into two different allelic types of 3D7 and FC27 [12,13,14]. These two polymorphic markers have been used to study the P. falciparum population in northeastern Myanmar, suggesting a highly diverse parasite population [15]. Due to the dramatic changes of the malaria situation in Yunnan Province, China, in recent years, this study aimed to investigate the genetic diversity of the P. falciparum populations along the China–Myanmar border region using two polymorphic markers MSP1 and MSP2.

Methods

Collection of clinical parasite samples

This study was approved by the Ethical Review Board of Yunnan Institute of Parasitic Diseases, China. A total of 242 P. falciparum clinical samples were collected from malaria patients attending local hospitals along the China–Myanmar border during 2006–2011. These patients came from Laza, Nawei, Mangdong, and Nankajiang in Myanmar, and Tengchong, Yingjiang and Mengla in Yunnan Province, China (Fig. 1). All patients were diagnosed with P. falciparum infection by Giemsa-stained blood smears and microscope examination at the local hospitals, and further confirmed by a nested PCR [16]. Two hundred and fifty microlitres of finger-pricked blood was spotted on the 3 mm Whatman filter paper (GE Healthcare, USA), dried, and stored at 4 °C until further analysis.

Fig. 1
figure 1

Map of the China–Myanmar border region showing the sampling sites that indicated with red star. The map was prepared by using the website of https://map.baidu.com/

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PCR amplification

Genomic DNA of parasite was extracted from the filter paper by using the QIAamp® DNA Mini Kit (Qiagen, Germany). The msp1 and msp2 genes were genotyped by a nested PCR using allele-specific primers as described elsewhere [17]. The PCR products were analysed on 2% agarose gel electrophoresis and stained with GoldView (Shanghai, China), whose size was determined using the standard DNA ladder marker (Toyobo, Japan).

The PCR products were sequenced and deposited in the GenBank with accession numbers MG004320–MG004381 for the K1 type, MG004447–MG004517 for the MAD20 type, and MG004518–MG004527 for the RO33 type of msp1; MG004219–MG004319 for the 3D7 type and MG004382–MG004446 for the FC27 type of msp2.

Multiplicity of infection

Multiplicity of infection (MOI) was defined as the largest number of alleles at each locus, and single infection was that with only one allele per locus at all of the genotyped loci [15, 18].

Statistical analysis

All statistical analyses were conducted with the software Statistical Package for Social Sciences (SPSS) version 23.0. The frequencies of the different combinations of alleles in seven studied areas were assessed by Kruskal–Wallis test, and normally distributed continuous data were evaluated by analysis of variance (ANOVA). The Spearman’s rank correlation coefficient test was calculated to assess relationships between MOI and parasite densities or ages in these patients, respectively. The difference was considered statistically significant when P value was less than 0.05.

Results

General characteristics of included patients

A total of 242 malaria patients were enrolled into this study during 2006–2011. Among these patients, there were 200 patients who were confirmed by a nested PCR to be infected with P. falciparum, 14 patients infected with Plasmodium vivax, and 15 patients co-infected with P. falciparum and P. vivax, and 13 patients excluded from the study as the genomic DNA from these patients were not successfully extracted (Fig. 2). All the P. falciparum clinical isolates (215 patients) were included in this study. Of these, 65 patients were from Yunnan province, China, and 150 from Myanmar. Sixty-eight patients were children under 19 years, 11 patients were more than 49 years old, and other 136 patients were between 19 and 49 years old.

Fig. 2
figure 2

The procedure of samples from fieldwork to laboratory detection

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Allelic polymorphism of msp1 and msp2

Among 215 P. falciparum clinical isolates, 201 (93.49%) and 204 (94.88%) samples were genotyped at the msp1 and msp2, respectively. For msp1 gene, the MAD20 family was dominant (53.49%) and included 7 different band sizes (120–280 bp), followed by the K1 family (44.65%) that included 9 different band sizes (150–320 bp), and the RO33 family (12.56%) that contained 3 different band sizes (150–200 bp) (Table 1, Fig. 3). The alleles with a high frequency for MAD20, K1 and RO33 were 180 bp (30.38%), 180 bp (30%) and 150 bp (64.10%), respectively.

Table 1 Prevalence of msp1 and msp2 allelic types in the China–Myanmar border region
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Fig. 3
figure 3

Prevalence of msp1 and msp2 alleles classified by fragment length (in base pairs) from allele-specific nested-PCR in different regions of the China–Myanmar border region

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For msp2 gene, the FC27 family (80.93%) showed the higher number of alleles, with 8 different band sizes (250–700 bp) (Table 1, Fig. 3), followed by the 3D7 family (75.81%) that included 9 different band sizes (400–760 bp). The most frequent alleles of FC27 and 3D7 were 700 bp (19.56%) and 550 bp (28.22%), respectively.

The total rate of MOI for msp1 and msp2 was 64.19% and 72.09%, respectively. The alleles of P. falciparum clinical isolates were K1 (30.23%), MAD20 (39.07%), RO33 (8.84%), K1 + MAD20 (11.63%), KI + RO33 (0.93%), MAD20 + RO33 (0.93%), K1 + MAD20 + RO33 (1.86%) for msp1 and FC27 (19.07%), 3D7 (13.95%), FC27 + 3D7 (61.86%) for msp2, respectively, while none of above-mentioned combination alleles of msp1 (such as K1 + MAD20, KI + RO33, MAD20 + RO33, and K1 + MAD20 + RO33) were be found in the isolates from Tengchong of Yunnan and Nankajiang of Myanmar (Table 1 and Fig. 4). There were a statistical difference in prevalences of the K1, MAD20, and RO33 families and their different MOI of msp1 (X2 = 14.478; P = 0.025) or FC27, 3D7, and FC27/3D7 alleles of msp2 (X2 = 30.617; P = 0.000) between the seven studied areas.

Fig. 4
figure 4

Multiplicity of infection of msp1 and msp2 alleles in different regions of the China–Myanmar border region

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The MOI distribution of allelic families across different parasite density and age groups

Almost all patients were detected to have multiclonal infections, with a mean MOI of 1.76 ± 0.85 for msp1 and 2.21 ± 1.29 for msp2. The MOI values of msp1 and msp2 for each parasite density and age groups are summarized in Tables 2 and 3. There was a significant positive correlation between the MOI and parasite density for msp1 (Spearman’s rank coefficient = 0.208; P = 0.002) (Additional file 1: Table S1), but no positive correlation for msp2 (Spearman’s rank coefficient = − 0.040; P = 0.564) was found. Additionally, no significant correlation between age and MOI (Spearman’s rank coefficient = − 0.12; P = 0.08 for msp1 and Spearman’s rank coefficient = 0.007; P = 0.917 for msp2) was found. Furthermore, there was a significant difference of the MOI for the msp1 gene among the groups with different parasite densities (F = 2.588; P = 0.028), while no significant difference for msp2 (F = 0.245; P = 0.942) or for the MOI of msp1 (F = 0.443; P = 0.818) and msp2 (F = 0.433; P = 0.825) among groups with different ages were found.

Table 2 Distribution of msp1 and msp2 allelic types of P. falciparum among different age groups in the China–Myanmar border region
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Table 3 Distribution of msp1 and msp2 allelic types of P. falciparum among different parasite densities in the China–Myanmar border region
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Sequence analysis of MSP1 and MSP2

A total of 38 different alleles of msp1 were used for sequence analysis, including 14 for K1 family, 23 for MAD20, and 1 for RO33 (Additional file 2: Figure S1, Additional file 3: Figure S2). All K1-type alleles were found to have a 24-amino acid sequence: SPSSRSNTLPRSNTSSGASPPADA at the 5′-end and 10-amino acid sequence: NEEEITTKGA at the 3′-end. The central variable region always started with SAQ and terminated with SGT containing the difference number of tripeptide repetition, such as SAQ, SGP, and SGT. The diversity of MAD20 family was also caused by differences in repetitions of SGG, SVA and SVT, but the central variable region always started with SKG or SGG and ended with SVA. Only one amino acid sequence type of amino acids LKDGANTQVVAKPAGAVSTQSAKNPPGATVPSGTASTKGAIRSPGAANPSD was identified in the RO33 family. A total of 37 alleles of 3D7 and 15 alleles of FC27 in the msp2 gene were detected by sequence analysis of the block 3 region (Additional file 4: Figure S3, Additional file 5: Figure S4). The central variable region of 3D7 family included diverse amino acid repeat motifs that contained the different combinations of three amino acids: S, A, and G. The 3D7 family consisted of 12 different amino acids repeats and the 17 single amino acids.

The full sequences of 3D7 family were compared to the reference 3D7 (GenBank accession number X53832) (Additional file 4: Figure S3), showing deletion of 10 or 11 amino acids PKG(K/N)G(E/G/K/Q)VQ(E/K/P)(N/P/S) or PKG(K/N)G(E/G/K/Q)VQ(E/K/P)(N/P/S)N in the reference 3D7, which was common in field isolates from China–Myanmar border region. The dominant genotype of FC27 family was found to have one 32-amino acid ADTIASGSQSSTNSASTSTTNNGESQTTTPTA, followed by a conserved sequence ADTPTAT(E/K), and a two type of tandemly repeated units, SNSPSPPITTTE or SNSRSPPITTTE, repeated from two up to five times. All FC27 family had a 10-amino acid SSSSGNAPNK at the 5′-end and 3-amino acid AP(N/K) or 6-amino acid APNAP(K/N) at the 3′-end (Additional file 5: Figure S4).

Discussion

The genetic diversity of P. falciparum may affect the model of transmission and control strategies for the parasite. The pathogenicity, antigen specificity and anti-malarial drug sensitivity of P. falciparum can be associated with their malarial genetic structure [19, 20]. The genetic polymorphism analysis of P. falciparum field isolates is necessary and might shed light on the development of control strategies and effective vaccines against P. falciparum.

In the present study, a high genetic diversity of msp1 and msp2, including 19 different PCR products for msp1 (7 MAD20, 9 K1 and 3 RO33) and 17 for msp2 (8 FC27 and 9 3D7) was found in the P. falciparum population in the Myanmar-China border regions. Two PCR products of msp1 are similar to that in Thailand and western Cambodia [17, 21]. Allele typing for msp1 showed that MAD20 (115/215, 53.49%) was the predominant allelic type in studied areas, which is consistent with the situations in Thailand, Myanmar, Vietnam, Colombia, Equatorial Guinea, and Yunnan Province, China [15, 22,23,24,25,26,27]. On the contrary, the K1 family is the most frequent genotype in Laos, Peru, India, Pakistan, Tanzania, Malaysia, and Senegal [28,29,30,31,32,33,34,35]. Allele typing for msp2 also showed the highest prevalence of FC27 (174/215, 80.93%) and 3D7 (163/215, 75.81%), which is consistent with these situations in Benin [36]. However, 3D7 is the most frequent in Thailand, Myanmar, Colombia, Malaysia, Senegal, India, Equatorial Guinea and Pakistan [16, 21,22,23, 26, 30, 32, 34, 35, 37, 38]. The overall multiplicity of infection (MOI) of msp1 and msp2 was 1.76 and 2.21, respectively. They were similar to those of Thailand and Laos [17, 28], higher than Malaysia, India and Senegal [31, 35, 38], but lower than Ethiopia [39]. The difference may result from the different geographical areas, intensity of malaria transmission and studied populations.

The present study identified 38 alleles of msp1, including 14 for the K1 family, 23 for the MAD20 family, and 1 for the RO33 family. This genetic diversity of msp1 in P. falciparum isolates resulted from the different numbers of tripeptide repeat that includes the SAQ, SGP and SGT for K1 and SGG, SVA and SVT for MAD20, which is consistent with the previous studies [15, 22, 28, 30, 40]. Similarly, msp2 also showed a high genetic diversity, with 37 alleles for the 3D7 family and 15 alleles for the FC27 family. This study showed a highly complicated amino acid repeat motifs in the central variable region of 3D7 alleles that contained the different combinations of S, A and G. There were 4 different continual amino acids repeats, including GASGSA (repeat numbers 2 to 4) [41], GGSGSA (repeat numbers 3 to 9) [41,42,43], GAVASAGS (repeat numbers 2 to 3) [44] and GAGAVAGS (repeat numbers 3), as well as 2 single amino acid that including GGSA and GAGASAGN, which have also been reported in other studies [14, 41,42,43,44,45]. Several new continual amino acids repeats and single amino acid were also found in this study, including the continual amino acids repeat GAGASGSA (repeat numbers 2), GAGAGAVAGS (repeat numbers 2 to 3), GASGSASGSA (repeat numbers 4 to 5), GAVASAGSRD (repeat numbers 5 to 7), GAGAGAGAVAGS (repeat numbers 2 to 3), PAT (repeat numbers 2 to 6) at the central variable region followed by poly-threonine stretch (repeat numbers 4 to 13) and the intermittent amino acids repeat GAGAGASGSA, GAGASGSAGSGD, GAGAGAGASGSA, as well as the above-mentioned single amino acid except for GGSA at the 3′-end. As reported in previous studies on parasites from Papua New Guinea, Cameroon, Myanmar and China [14, 22, 41, 44], the sequences of the FC27 family of P. falciparum isolates in the China–Myanmar border region are conserved at the 3′ and 5′-end, but varied in the number of repeats on SNSPSPPITTTE or SNSRSPPITTTE in the central region.

Conclusion

The findings of this study have demonstrated that P. falciparum clinical isolates in the China–Myanmar border region had a high genetic polymorphism in the msp1 and msp2 genes as well as a high multiplicity of infection, suggesting the highly complex population structure of the parasite.