Discussion
Malaria is one of the most important infectious diseases in China, and most of the autochthonous malarial cases have been reported in the southern region of China in earlier decades, including Hainan Province and Yunnan Province. Parasites, including
P. falciparum and
P. vivax, are prevalent in those two provinces; however, in the past ten years, malaria has been controlled in China. The autochthonous malaria cases in the corresponding endemic regions have been reduced almost to zero with the support from the Chinese Government and the dedicated efforts of healthcare professionals. Hainan Province, one of the most endemic regions with historically high transmission of
P. falciparum, has not reported any autochthonous
P. falciparum malaria case since 2010 [
16,
17]. Moreover, no
P. vivax autochthonous case has been reported since 2013 [
18]. Information on genetic diversity of malarial parasites is important to understand the dynamics of disease transmission, to develop targeted anti-malarial drugs, and to develop effective methods to trace the origin of infections. Furthermore, knowledge of parasite population genetics would be useful in designing and monitoring strategies for elimination, and provide valuable metrics for monitoring the success of control efforts, if population genetic parameters accurately reflect transmission intensity. However, the information of autochthonous
P. vivax population in Hainan Province is limited.
The
pvmsp-1 locus codes for a major asexual blood-stage antigen had extensive polymorphism in isolates from different geographical regions. Three allele types including Belem, Sal-1, and recombinant variable block 5 have been found in isolates of
P. vivax worldwide. The
pvmsp-1 markers have been used in genetic studies of
P. vivax in many countries, including India, Myanmar, Brazil, Latin America, and Korea [
10,
12,
19,
20]. In 2003, this marker was used for investigating 33 samples collected from various sites of
P. vivax endemic areas in mainland China [
21]. The result of this investigation showed that three allele types were found in China and that Sal-1 and recombinant allelic types were dominant, although the frequency of those types were different in different areas. In Hainan, three allele types were found and some sample had two allele types in 2002 and 2005 [
21,
22]. In the present study, three allele types were detected among the Hainan isolates, with the Sal-1 type being the predominant one. A similar degree of diversity was also found in Hainan Province in 2002 and 2005 [
21,
22]. Since 2003, some new subtypes have been found according to their amino acid sequences. In present study, the sequence of amino acid of
pvmsp-1 was longer than most published sequences and most of subtypes were similar with other stains in phylogeny tree of
pvmsp-1. Only subtype F and subtype I showed 100% identity with AAR30523 and AAR30526 strains isolated from Hainan in 2003, respectively. Comparison with published sequences, some of subtypes had single amino acid inserted or deleted or substituted and became new subtypes. Furthermore, Sal subtype I from Hainan isolates had some similarity with the subtype K and the subtype L, which were collected from Myanmar [
12]. Those isolates were contained the amino acid sequence MKKELLDQYK specific to the Sal 1 type rather than the DKKLLKEYE specific to the Belem type. This kind of isolate was first reported in China.
Currently,
pvcsp acts as an important gene marker and it has been used successfully in epidemiological studies of
P. vivax malaria. This marker has two types: classic type (VK210) and variant type (VK247); the former, is known as classic type with obvious character of repeat motif as GDRAA/DGQPA within the amino acid tandem repeat region whereas, the latter is known as variant type with ANGAGNQPG amino acid repeats within the amino acid tandem repeat region. These two types have a worldwide distribution [
23,
24]. VK210 has been observed as predominant type in many countries, although VK247 was reported to be the predominant type previously [
25-
28]. In 2001, 384 samples were collected from 10 vivax-endemic provinces and the results showed that both types of
csp genes were observed and that VK210 was the dominant type [
29]. Since then, many research devote to investigate the ratio of two types in different regions of China but VK247 and mixed infections also have only been found in south China, including Yunnan Province and Hainan Province [
30,
31]. Moreover, in other endemic areas, including Anhui, Hubei, Guangxi, Guizhou, Guangdong, there was only the VK210 type. In present study, VK210 and VK247 still exist in Hainan but there were no mixed infections. The ratio between VK210 and VK247 decreased from 87:12 in control malaria stage in 2002 to 23:4 in elimination stage of present study [
30]. There are no statistical differences between these data (χ
2 = 0.747,
df = 1, P > 0.05). The changes of ratio of two types of
csp have influenced the decrease of malaria cases in Hainan Province. There is limited information concerning the sequence of the subtypes in the two types of
pvcsp from China. Comparison with published sequences in NCBI and nearby sequences from phylogeny trees of
pvcsp, most of subtypes were new alleles because they had different numbers of repeat motif or had different mutations in motif, except subtype C was showed 100% identity with AAV80840 strains isolated from Mauritania in 2004.
pvmsp-
3α,
pvmsp-3
β and
pvmsp-3γ are members of a multi-allelic diversifying selection to fit the evolution[
31,
32]. This gene family exhibits a high degree of genetic diversity but
pvmsp3 may be analogous with
pfmsp3, not homologous [
31]. The PCR-RFLP method has been applied for analysing the degree of diversity in
pvmsp-
3α and
pvmsp-3
β over many years [
33,
34], but this method has recently been suspected not to be suitable for broad geographic studies or tracking parasite populations [
33]. The larger sample showed that some of identical haplotypes could be produced from analogous bands after PCR-RFLP analysis and revealed incongruence between the observed levels of nucleotide polymorphism and the pattern of PCR-RFLP haplotype [
33].
Based on the length of PCR products, allelic type A and allelic type B of
pvmsp-
3α have been detected among the 27 tested isolates. The observation was different from a previous report from several locations in China, in which type A, B, C, and mixed infection were detected in
P. vivax isolates from Sanya city of Hainan Province [
7,
35]. The lack of type C allele types in the present study could be due to a disappearance of this allele before implementation of the NMEP. Taking consideration of frequency of genotype, type A had a high frequency (96%, 26/27) and was a predominant type at elimination stage of malaria in Hainan Province. The present study results are consistent with previously published reports indicating type A as the most prevalent type around the world with a frequency 70 to 100% (average ~80%) in many regions of China and countries of Asia and South America [
7,
11,
13,
34].
In the analysis of
pvmsp-
3α, PCR-RFLP allele types and frequencies, 11 patterns were detected in the
pvmsp-
3α gene after digestion of the PCR products with
Hha I, and the most frequent allele variant were H1 and H3 subtypes. The
Hha I allelic types, including H1, H3, H4, H5 H6, H7, H8, H10, and H11 were found. Those
Hha I allelic types had been found in other regions of China and in parts of the world. Similarly, variants H1, H3, H5, H7, and H10 were found in Myanmar [
7]. The variants, including H4, H6, and H11 isolated in the present study, have high similarity with variants A3, A6 and A4 isolated in Thailand [
36], respectively. Variant H8 has extensive similarity with variant A6 isolated in Brazil. In China, variants H1, H3, H6, H7, and H10 were reported earlier in Hainan Province [
7] and allelic variants H4 and H11 were also identified in Anhui Province [
35]. Others variants, including A9 and A11, were found only in Anhui Province. Moreover, variants A2, A3, A5, and A10 were not found in present study. However, new allelic variants H2 and H9 have been reported in the present study, which were not described in previous reports.
Nine patterns were detected in the Pv
msp-
3α gene after digestion of PCR products with
Alu I, with PA1 pattern being the predominant in Hainan Province at the elimination stage of malaria. Some allele types of
Alu I digestion are comparable to those reported in other parts of the world, such as allele types A2, A3, A4, and A5 isolated in Anhui Province of China [
35], types A1, A2, A3, A4, A5, and A8 in Pakistan [
11], types A1, A2, A3, A4, and A5 in Iran [
37], and types A1, A2, and A5 in Colombia [
38], which suggests that these allele types of
P. vivax may have a global distribution. The allele variant B1 was a new allele identified in the present study. The analysis of Pv
msp-
3α gene marker suggested that
P. vivax populations in Hainan Province showed diversity at elimination stage of malaria and shared the majority of allelic variants with other parts of China and the world.
pvmsp-3
β gene encodes merozoite surface protein dominated by alanine-rich central domains, which is strongly predicted to form a coil-like tertiary structure. The structure of central domain is radically divergent with the majority bearing large insertion/deletion mutations and it has been employed as a molecular marker to evaluate genetic diversity of
P. vivax. Only type A and type B alleles were detected among four types of alleles in
pvmsp-3
β marker at the elimination stage of malaria, with type A being more abundant (62.9%) than type B. In comparison with previous study on
P. vivax isolates from Hainan Province in 2006 [
7], the multiple genotypes of
pvmsp-3
β marker have been decreased from four to two types. In addition, in the present study, RFLP analysis revealed different allelic compositions and a total of nine alleles were identified. Interestingly, despite the differences in geographic regions, some allele types of
Pst I digestion were comparable to those reported in other parts of the world, such as allele types PA1, PA3, PB1, and PB2 isolated in China and Myanmar [
7], type PA5 in Pakistan [
15], types PA2, PA3, PB1, and PB2 in China and Thailand [
35], suggesting that these allele types of
P. vivax may have a global distribution. In the present study, new allele variants PA1, PA4, PA5, PB3, and PM were identified. The levels of mixed-genotype infections were correlated with the levels of endemicity and the power of sensitivity on selected gene marker. The discrimination of
pvmsp-3
β gene marker for mixed-genotype infections in several endemic regions has more sensitivity than other markers, especially in endemicity; for example, genotyping the Thai samples for
pvmsp-3
β detected 20.5%, whereas combination with genotyping
pvmsp-
3α increased the mixed infection level to 29.5% [
33]. On the other hand, genetic diversity of the malarial parasites is associated with the levels of endemicity and transmission intensity [
15]. In hyperendemic areas, such Myanmar [
12], and Thailand [
39],
P. vivax is highly diverse with multiplicity of infections. However, in Korea and Iran [
23,
40],
P. vivax was resurgent, and mixed infections were detected; however, the infection proportion was very low. In the present study, mixed infection was still detected by
pvmsp-3
β at the elimination stage of malaria. This information could suggest that the genetic diversity of the malaria parasites is not only associated with the levels of endemicity and transmission intensity, but could also be associated with epidemic times in history.
pvmsp-3α and
pvmsp-3
β are promising markers for epidemiological applications. Compared with the simple PCR-RFLP method, sequencing of these markers may offer significantly higher power for determining parasite genetic diversity. Analysis of diversity in
pvmsp-3α and
pvmsp-3
β by sequencing opens new perspectives for diversity analysis and this method had been used at the Thai-Myanmar border area and revealed that the extent of allelic diversity in
P. vivax populations in Thailand [
41].