Distinct Allelic Diversity of Plasmodium vivax Merozoite Surface Protein 3-Alpha (PvMSP-3α) Gene in Thailand Using PCR-RFLP

Considering the importance of merozoite surface proteins (MSPs) as vaccine candidates, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax merozoite surface protein 3-alpha (PvMSP-3α) in Thailand. To analyze genetic diversity, 118 blood samples containing P. vivax were collected from four malaria-endemic areas in western and southern Thailand. The DNA was extracted and amplified for the PvMSP-3α gene using nested PCR. The PCR products were genotyped by PCR-RFLP with Hha I and Alu I restriction enzymes. The combination patterns of Hha I and Alu I RFLP were used to identify allelic variants. Genetic evaluation and phylogenic analysis were performed on 13 sequences, including 10 sequences from our study and 3 sequences from GenBank. The results revealed three major types of PvMSP-3α, 91.5% allelic type A (∼1.8 kb), 5.1% allelic type B (∼1.5 kb), and 3.4% allelic type C (∼1.2 kb), were detected based on PCR product size with different frequencies. Among all PvMSP-3α, 19 allelic subtypes with Hha I RFLP patterns were distinguished and 6 allelic subtypes with Alu I RFLP patterns were identified. Of these samples, 73 (61%) and 42 (35.6%) samples were defined as monoallelic subtype infection by Hha I and Alu I PCR-RFLP, respectively, whereas 77 (65.3%) samples were determined to be mixed-allelic subtype infection by the combination patterns of Hha I and Alu I RFLP. These results strongly indicate that PvMSP-3α gene is highly polymorphic, particularly in blood samples collected from the Thai-Myanmar border area (the western part of Thailand). The combination patterns of Hha I and Alu I RFLP of the PvMSP-3α gene could be considered for use as molecular epidemiologic markers for genotyping P. vivax isolates in Thailand.


Introduction
Te most recent World Malaria Report reveals that there were 247 million cases of malaria in 84 countries where the disease is prevalent in 2021, as opposed to 245 million cases in 2020. Te estimated number of malaria-related deaths in 2021 was 619,000, compared to 625,000 in 2020 [1]. Tere are fve species of Plasmodium known to cause malaria in humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Among these species, P. vivax is the most geographically widespread species. It is predominant in the region of the Americas and Southeast Asia, comprising 71.5% and 49.9% of estimated malaria cases, respectively [2].
Malaria remains an important public health issue in Tailand, where over 19% of the total population is (13 million people) presently at risk [3]. P. vivax is the most prevalent species of human malaria in Tailand and causes about 93% of malaria infections [4]. Te forested areas bordering especially western and southern Tailand are malaria-endemic areas [3,5].
Plasmodium vivax has been reported to be responsible for relapsing infection at the liver stage called the hypnozoite [6,7] and severe complications, such as cerebral malaria, renal failure, acute respiratory distress, and shock [8,9]. Moreover, chloroquine drug resistance has been reported in vivax malaria, which is mainly endemic in Southeast Asia including the Tai-Myanmar area [10][11][12].
Genetic diversity has been associated with several characteristics of Plasmodium species, including biology, epidemiology, transmissibility by Anopheles mosquitoes, immunological responses, as well as drug resistance [13][14][15]. In endemic areas with high transmission rates, extensively diverse variants are leading to a public health threat because they have likely developed increased virulence and resistance to drugs for their surviving and ftness. Tus, the intensive understanding of parasite genetic diversity plays an important role for designing malaria control programs and developing efective antimalarial drugs and vaccines.
According to treatment failure and multiple drug resistance in malaria disease, the investigation of genetic diversity of Plasmodium spp. in endemic areas is signifcantly considered for antimalarial drugs used in each specifc endemic area, particularly in western and southern Tailand. Terefore, the diferences among allelic subtypes in western and southern Tailand have been focused in this study.
Te purpose of this study was to evaluate the genetic polymorphism of P. vivax feld isolates from malariaendemic areas of western and southern Tailand. Te allelic diversity of PvMSP-3α was analyzed by nested PCR-RFLP using two restriction enzymes (Hha I and Alu I) in order to understand the molecular epidemiology of vivax malaria in Tailand.

Sample Collection.
Tis study was approved by the Ethics Committee of Rangsit University, Tailand (RSUERB2018-026). Blood samples were collected during 2013 to 2018 from malaria-infected patients in western Tailand bordering Myanmar (Kanchanaburi, Mae Hong Son, and Tak provinces) and southern Tailand bordering Malaysia (Yala province). Te single infection of P. vivax blood samples confrmed by nested PCR was recruited in this study for genetic diversity analysis of the PvMSP-3α gene. Two hundred microliters of blood samples were spotted onto flter paper (Whatman 3 MM, Whatman International, Maidstone, England), and the dried blood spots were stored in plastic bags at room temperature prior to DNA extraction with ISOLATE II Genomic DNA Kit (Meridian Bioscience, Inc. USA). Te DNA samples were stored at −20°C until analysis. Te blood samples were examined by Giemsa stained blood smear microscopy and nested PCR [28] for diagnosis of P. vivax infection. Te confrmed samples for a single infection of P. vivax were recruited for the genetic diversity analysis of the PvMSP-3α gene.

Nested PCR of PvMSP-3α
Gene. Te allelic types of PvMSP-3α gene were analyzed using the modifed protocol of Bruce et al. [16]. Primary PCR was performed in 20 µl using primers P1 (5′-CAGCAGACACCATTTAAGG-3′) and P2 (5′-CCGTTTGTTGATTAGTTGC-3′); the reaction mixture contained 1 × HSTaq PCR bufer master mix, 0.2 μM each primer, 1 U of Taq HS DNA polymerase (Meridian Bioscience, Inc. USA), and 3 µl of extracted DNA. Te conditions were as follows: initial denaturation at 95°C for 5 min and 24 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 2 min with the fnal extension at 72°C for 5 min. Nested PCR was done in 20 µl with primers N1 (5′-GACCAGTGT GATACCATTAACC-3′) and N2 (5′-ATACTGGTTCTT CGTCTTCAGG-3′), using 1 µl of the primary PCR product with 30 cycles of the conditions and reaction mixture similar to the primary PCR. Under UV transilluminator, the PCR product was visualized on 1.2% agarose gel, stained with NEOgreen DNA staining reagent (GELLGENTEK Co., Ltd., Korea). Te sample that contained a single PCR fragment size indicating a monoallelic type was considered for the allelic subtype analysis using the RFLP pattern. If a sample presented more than one PCR fragment size, indicating mixed allelic types, then it was excluded from the RFLP analysis.

RFLP Analysis of PvMSP-3α PCR Products.
Te allelic subtypes of PvMSP-3α gene were identifed by using the PCR-RFLP technique. Five microliters of each amplifed product were digested with Hha I and Alu I restriction enzymes (NEB, Inc, Beverly, MA, USA) in a total volume of 20 μl as followed by the manufacturer protocol. Te digested products were presented by 2.5% agarose gel electrophoresis. Te restriction patterns of DNA fragments were photographed for the allelic subtype analysis, and each Hha I and Alu I RFLP patterns were classifed as allelic subtype numbers such as A1, A2, and A3. Te allelic subtypes were further diagnosed by the combination patterns of Hha I and Alu I RFLP.
If the sum of digested DNA fragments was less than or equal to those from uncut PCR, the sample was considered infected with a monoallelic subtype (for example, in Figure 1, the monoallelic subtype showed one or more than single fragment after digestion), named as the monoallelic subtype infection. In case of mixed-allelic subtype infection, the sum of digested DNA fragments was more than the uncut PCR product.

Phylogenetic Analysis of PvMSP-3α
Gene. Ten PCR product samples were analyzed for PvMSP-3α nucleotide sequence in both directions by the commercial service at 1st BASE, Malaysia. Tese samples included four collected from Yala province and two each from Kanchanaburi, Mae Hong Son, and Tak provinces. Using basic local alignment search tool (BLAST), phylogenetic relationships among these 10 PvMSP-3α sequences were compared with the three previously published sequences in the GenBank database, EU430585.1, AF491960.1, and AF093584.2 from Myanmar isolate, Salvador I, and Belem strain, respectively. Te phylogenetic tree involving 13 nucleotide sequences was constructed using MEGA7 program with the neighborjoining method. Te bootstrap consensus tree inferred from 1000 replicates.

Results
A total of 143 single P. vivax infected samples were confrmed by both microscopy and PCR. Out of these, 118 samples (82.5%) were successfully amplifed for the PvMSP-3α gene by nested PCR (   and multiple infections. If the sum of the digested DNA fragments was less than or equal to that in the uncut product, the sample was designated as a single infection with a monoallelic subtype. Multiple infections with mixed allelic subtypes were defned when the sum of the digested DNA fragments was more than that in the uncut product. Among the single infection samples in this study, 16 allelic subtypes A (A1-A16, shown in Figure 1(a)) and 3 allelic subtypes B (B1-B3 shown in Figure 1(b)) of PvMSP-3α gene were identifed by Hha I restriction enzyme, while 5 allelic subtypes A (A1-A5) and 1 allelic subtype C (C1) were classifed by Alu I restriction enzyme (Figure 1(c)). In Table 2, the subtype A allele with Hha I RFLP pattern was found to have the highest frequency (61%; 73/118), and the subtype C allele with Alu I RFLP pattern was found to have the lowest frequency (0.8%; 1/118). Besides these, samples with multiple infections that showed multiple bands (named as mixed type) were detected at the frequency of 33.9% (40/ 118) Hha I RFLP pattern, 63.6% (75/118) Alu I RFLP pattern, and 65.3% (77/118) combination patterns of Hha I and Alu I RFLP (Table 2).
For western Tailand, allelic subtypes A and B with Hha I RFLP patterns were found, in addition to allelic subtypes A and C with Alu I RFLP patterns (Figures 2(a) and 2(b)). In southern Tailand, only allelic subtypes A with Hha I RFLP pattern and Alu I RFLP pattern were found. Among all allelic subtypes analyzed based on combination patterns of Hha I and Alu I RFLP (HA1-HA21), 2 allelic subtypes (HA1 and HA3) were found in both endemic areas (Figure 2(c)).

Phylogenetic Analysis of PvMSP-3α
Gene. Among the 10 selected isolates, phylogenetic results showed the identity ranging from 90% to 100% with PvMSP-3α gene in the GenBank database. Te P. vivax isolates examined in this study obtained greater than 95% similarity with the isolates from Myanmar, Korea, Tailand, and Mauritania and with GenBank accession numbers EU430583.1, AY266090.1, AY833021.1, and KC935445.1, respectively. Te phylogenetic relationships are shown in Figure 3. Te selected sequences in this study had a nonspecifc relationship to the areas included. From Yala province, isolates were found to be closer to themselves and Salvador I (AF491960.1), while the samples from Kanchanaburi, Mae Hong Son, and Tak provinces were found to have some likeness between themselves and Myanmar (EU430583.1).

Discussion
Malaria remains a major health risk in Tailand, and P. vivax is the most prevalent causative species. Information on the genetic diversity of P. vivax is important to understand the dynamics of its populations, support the design of efective malaria control programs, and play an important role in drug and vaccine development. Terefore, this study was carried out to identify P. vivax variants and to determine PvMSP-3α polymorphism of P. vivax isolates collected in malaria-endemic areas of Tailand. Plasmodium vivax isolates were collected from western Tailand, bordering Myanmar (Kanchanaburi, Mae Hong Son, and Tak provinces), and southern Tailand, bordering Malaysia (Yala province), during 2013-2018. Nested PCR-RFLP with Hha I and Alu I restriction enzymes was used to assess the number of distinguishable fragment allelic variants.
Out of 143 samples in the current study, 118 (82.5%) (118/ 143) were successful for PvMSP-3α amplifcation. Te differences in the percentage of successful amplifcation (57.1% to 96.3%) were probably due to the time and sites of sample collection. Samples from Yala Province had a high successful amplifcation rate of 96.3% and were mostly obtained during 2016-2018, whereas those from Kanchanaburi, Mae Hong Son, and Tak had lower success rates (57.1%-65.4%) and were mostly collected between 2013 and 2015.
Based on nested PCR, our results show three allelic types of PvMSP-3α gene, type A (∼1.9 kb), type B (∼1.5 kb), and type C (∼1.2 kb), in agreement with various worldwide reports. Among these isolates, type A allele had the highest frequency (91.5%), which was consistent with reports from China, India, Pakistan, and Tailand [17,19,[23][24][25]. Tere was no allelic type D and mixed allelic type in this study in contrast to previous reports [26,29]. Moreover, we found diferent frequencies of allelic types in each province. Yala and Kanchanaburi revealed only type A, Mae Hong Son contained types A and B, and Tak presented with types B and C. On the contrary, previous reports from Tailand presented all three allelic types in study areas that included western and southern Tailand [23,24,30,31]. Tese differences may be due to variations in sample collection time and sites of sample collection. In addition, the allelic distribution at the Tai-Malaysia border revealed only type A alleles, but all three allelic types distributed in the Tai-Myanmar border that imply to the origin of type A alleles and genetic diversity in each endemic area.
Our fndings with PCR-RFLP demonstrated distinct allelic subtypes of PvMSP-3α gene with a high genetic diversity. Te results on PvMSP-3α polymorphisms from PCR-RFLP with Hha I restriction enzyme revealed 19 allelic subtype patterns in 78 P. vivax monoallelic subtype samples. Te number of allelic subtype variants was more than in previous reports conducted in several diferent geographical areas including Nepal [32], China [17], Pakistan [29], and Tailand [23,31,33].
Te lower numbers of allelic subtypes (six allelic subtypes) and faint, indistinct bands of DNA fragments in the gel detected by Alu I RFLP patterns accorded with a report by Rice [27] in which in silico digestion with Alu I resulted in complicated patterns. Terefore, using the Hha I restriction enzyme was suggested for identifcation of P. vivax strain. A high frequency of migration occurs among people along the Tai-Myanmar border, and high polymorphism of PvMSP-3α gene was observed in this area [23,24,27,31,34,35]. As shown in Figure 2(a), the common subtypes (A1 and A13) in both endemic areas are most likely refective to the genetic movement of malarial parasites between western and southern Tailand.
Te PvMSP-3α RFLP with Hha I and Alu I results showed a high prevalence of mixed subtypes infection (33.9% and 63.6%) among P. vivax isolates in Tailand that   Journal of Tropical Medicine was higher than in other reports from India, Pakistan, China, and Tailand [17,18,25,26,29]. Tese diferent frequencies might have occurred because of diferences in the period of sample collection, geographic area, and detection method. Due to the absence of parasitemia data, this study's limitation lies in the inability to analyze the correlation between parasite density and genotypes of P. vivax infection.
In addition, the observation of P. vivax variants in diferent endemic areas most likely indicates diferent virulence and severity of complications, which has previously been described as an association between multiple genotype infections of P. vivax and disease severity [36]. Although several reports have demonstrated the use of PCR-RFLP with various restriction enzymes for detecting malaria multiplicity [16-18, 37, 38], the current study used the combination patterns of Hha I and Alu I RFLP and detected 21 allelic subtypes, which were more allelic subtypes than classifed individually by Hha I RFLP or Alu I pattern.

Conclusion
Our study demonstrates a high level of polymorphic of PvMSP-3α genes in P. vivax isolates that were remarkably distributed in the Tai-Myanmar border area (western Tailand), in the opposite way of Yala (the Tai-Malaysian border area, southern Tailand). Te fndings of higher numbers of allelic subtypes suggest that the combination patterns of Hha I and Alu I RFLP could be considered to be used in the molecular epidemiological study of P. vivax. An understanding of the allelic diversity  among P. vivax populations plays an important role in drug and vaccine development for the treatment and control of malaria.

Data Availability
All data supporting the fndings of this study are included within the paper; however, details of the full data may be obtained from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
KK designed the study, performed the genotyping, and drafted the manuscript. KC was involved in experiments, data analysis, and bioinformatics analysis. WN supervised data analysis and manuscript writing. RP substantially consulted the results interpretation. SK prepared DNA extraction and data collection. All authors read and approved the fnal manuscript.