DOI: https://doi.org/10.21203/rs.3.rs-1861513/v1
More than 85% of the malaria burden is caused by imported vivax malaria in Yunnan Province and Yunnan is also where the majority of vivax malaria cases are diagnosed across China. Timely removal of the source of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To compensate for the uncertainty of epidemiological surveys in tracing vivax malaria recurrence, this study attempted to use molecular markers for identification.
To do so, blood samples were collected from cases diagnosed and revalidated as single infections of Plasmodium vivax in Yunnan Province from 2013 to 2020. Specifically, samples from suspected relapses with recurrent episodes were subjected to PCR amplification, product sequencing, and analysis of the Plasmodium vivax circumsporozoite protein (pvcsp) gene.
Seventy-eight suspected recurrent cases were retrieved from 2484 vivax malaria cases, with a total of 81 recurrent episodes. A total of 159 blood samples from primary infection Plasmodium vivax and recurrences were subjected to PCR amplification and sequencing to obtain 156 CDS sequences of pvcsp gene, 121 of which can be matched into the paired sequences of 59 cases. There were 475 polymorphic loci and 84 haplotypes (H01-H84) in the 121 sequences. Also, there were 79 and 5 haplotypes with CRR repeat units (PRM) of VK210 and VK247 structure, respectively. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites, meaning the every paired sequences were completely homologous and the paired Plasmodium vivax strains were homologous single clone. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, and except for 2 polymorphic loci (39 and 1027), all single nucleotide polymorphisms were double-equivalent bases differentially transferred between paired sequences, indicating that the paired sequences are "weakly heterologous" with no fragment insertions (or deletions) and only individual site polymorphisms. All 59 vivax malaria recurrences were respectively caused by the activation of Plasmodium vivax hypnozoites from the same population as the primary infection.
The paired analysis of the similarity of Plasmodium high variant genes allowed the identification of recurrent episodes caused by Plasmodium vivax homologous hypnozoites, and also demonstrated pvcsp gene as a good molecular marker. Moreover, the study showed most of the hypnozoites causing vivax malaria recurrence in Yunnan Province belonged to homologous single clone or sibling strains comparison with the original infection strains.
In recent years, as efforts to control malaria have increased, the proportion of Plasmodium vivax infections in many traditionally highly endemic areas, such as Sri Lanka, Thailand and Brazil, has shown a counter-intuitive trend. The cause may be related to the fact that previous prevention and control measures ignored the characteristics of Plasmodium vivax and indirectly contributed to the accumulation of infection sources of Plasmodium vivax [1–2]. It has been observed that mature gametophytes (stage V gametophytes) of Plasmodium vivax appear almost simultaneously with its asexual bodies in the early stages and continue to produce gametophytes throughout the infection period [3–5]. The high efficiency of transmission therefore also means that the most effective measures to remove the infection sources of Plasmodium falciparum, such as early diagnosis and timely treatment, may not be able to contain the danger of an already infectious early episode of Plasmodium vivax. In addition, Plasmodium vivax develops more rapidly inside the Anopheles mosquitoes and can easily circumvent interventions such as insecticide netting and indoor residual spraying. Thus, the maintenance of Plasmodium vivax populations is easier than those applicable to Plasmodium falciparum under the same vector control pressure [6]. Moreover, due to the existence of a parasitic mechanism of hypnozoites, Plasmodium vivax can break through the local and seasonal limits of mosquito vector transmission [7–8]. The covert accumulation of infection sources plays a negative role in increasing the complexity associated with Plasmodium vivxa transmission.
Indigenous cases of malarial infection in China ceased in 2016. However, Yunnan remains the province with the largest number of imported vivax malaria cases, with instances primarily originating in Southeast Asian countries. To wit, in 2018, such cases accounted for 80.8% (172/213) malaria cases caused by Plasmodium vivax infection throughout the province [9], of which there was no shortage of groups with multiple episodes and suspected recurrences of Plasmodium vivax infection [10]. During the period spanning 2013-19, suspected recurrent events constituted approximately 3.5% (83 in total) of the 2364 vivax malaria cases diagnosed in Yunnan Province. This was based on a minimum interval of 45 days between the first recurrence and the original episode [11] (this interval has an average duration of 41 days across those Southeast Asian countries where Plasmodium vivax is widely prevalent [7]), and 7% of vivax malaria recurrence within 6 months in Thailand [11], suggesting the burden of vivax malaria recurrence in Yunnan Province is no less than that of neighboring countries. Of course, other regions of the world are also facing the challenge posed by the Plasmodium vivax recurrence, with the recurrence rate in Papua New Guinea being as high as 80% [12]. 23% of pregnant women in Brazil had a recurrence of due to failure to receive primaquine eradication [13], making its management unavoidable source of difficulty along the process towards the global elimination of malaria [14–15]. One of the three major technical bottlenecks in the control of Plasmodium vivax is the accurate identification of the resurgent infection caused by the activation of Plasmodium vivax hypnozoites [14].
The WHO recommends analyzing the homology of single-copy antigenic genes of Plasmodium vivax as a method for the molecular identification of 'new infection' and 'recrudescence' events [16]. Lin et al. [17] identified 37.9% (11/29) of re-emergence vivax malaria episodes caused by the activation of Plasmodium vivax homologous hypnozoites on the basis of paired analysis of genetic similarity between initial infection and the re-emergent strains. Craig et al [18], Imwong et al [19] and Chen et al [20] also assessed the differentiation characteristics of pvama1, pvcsp, pvmsp1, and other genes which revealed the first vivax malaria recurrence was mostly caused by the activation of Plasmodium vivax homologous hypnozoites. The feasibility of molecular identification of recurrent events in Plasmodium vivax has been demonstrated from different perspectives. To establish a practical and reasonable method for evaluating the recurrence resulted from Plasmodium vivax, this study used selective whole genome amplification (SWGA) [21] and deep sequencing of hypervariable genes [15] to investigate the vivax malaria recurrence events in Yunnan Province. The results of the molecular characterization of the pvcsp gene and its marker role in identification of the recurrent strains of Plasmodium vivax are reported below.
The study was conducted on cases drawn from throughout Yunnan Province from January 2013 to December 2020 and confirmed at the Yunnan Province Malaria Diagnosis Referent Laboratory (YPMDRL) using both examination by light microscopy of blood smears and genetic testing (Additional file 1). All cases were officially registered and counted in the "China Disease Surveillance Information Reporting System", from which suspected recurrent instances of Plasmodium vivax with a history of re-emergence were further screened. The following inclusion criteria were applied for the vivax malaria relapse: (1) patients who were clinically cured after an eight-day course with chloroquine/primaquine, had a second episode of appearing Plasmodium vivax in his peripheral blood 28 days later; (2) no further history of exposure to malaria endemic areas was reported after the original episode (Additional file 2). The determining of infection source of Plasmodium vivax is confirmed by epidemiological surveys. The criteria is as follows: those with no history of movement outside Yunnan Province within 30 d prior to the onset of Plasmodium vivax were classified as indigenous Yunnan cases, while those with a history of exposure to Plasmodium vivax in endemic areas such as Myanmar and Africa were classified as imported cases of Myanmar and African infections (Additional file 2).
Venous blood was collected from all vivax malaria cases during the primoinfection and before treatment for recurrent episodes (0 d). The samples were prepared as filter paper dry blood drops for Plasmodium genetic analysis.
Extraction of Plasmodium genomic DNA and PCR amplification of pvcsp gene
Three filter paper dry blood drops with a diameter of 5 mm were used to extract Plasmodium genomic DNA. This was carried out in accordance with the instructions of the QIAgen Mini Kit (QIAamp, Germany), and subsequently stored at -20°C.
The reference sequence (ID: NC_009913.1) was used as the template to design the primers and reaction conditions for the nested PCR amplification of the pvcsp gene. The first round of PCR amplified the region of 1537513–1539033 containing a total of approximately 1521 base pairs (bp), with the upstream and downstream primers as 5'-CCGTTCGAACAAGTTCTGTTC-3' and 5'-GCGCATAATGTGTAAGAGGTGT-
3', respectively. The second round amplified a region of 1341 or so bp from 1537625–1538965, for which the upstream and downstream primers were 5'-GCTTAAG TTAAGCAAGCAAAACAGC-3' and 5'-GCAGGGAACATTCATAAG
AAAGGG-3', respectively. The system for both PCR reactions was the following: 2.6 µl of DNA template; 14.0 µl of PCR mixed with 2× Taq; and 0.7 µl of each of the upstream and downstream primers (10umol/L), then to make up the volume to 25.0 µl with ddH2O. It was conducted under the following sets of PCR reaction conditions: 94°C for 3 min; 94°C for 30 sec, 49°C for 90 sec, 72°C for 2 min, 35 cycles; or 72°C for 7 min. The second round of amplification was carried out using 1.5% agarose gel electrophoresis, and the amplified products were sent to Shanghai Meiji Biomedical Technology Co. Ltd. for Sanger sequencing.
Gene polymorphism and homology analysis of pvcsp genes in paired blood samples
The sequencing results were collated in DNAStar 11.0 and BioEdit 7.2.5. The obtained DNA sequences were compared with the reference sequences of the pvcsp gene. When the ‘query cover’ and ‘identifies’ were greater than 98%, it indicated that the sequences collated were the intended targets. DnaSP 6.11.01 software was used to confirm the locus mutations and haplotypes of the pvcsp gene, from which the expected heterozygosity (He) and nucleic acid diversity index (π) of the DNA sequences were calculated. The pvcsp gene sequences of paired primary and relapse isolates from the same individual patients with several episodes of Plasmodium vivax were identified [18, 20] separately in MEGA 5.04 software for similarity matching and central repetitive region (CRR) [23–25]. And these regions including 5 'end, coding sequence of KLKQP five amino acids, 3’ end of pvcsp gene were named asα-N、Region I(RI)and Thrombospondin repeat༈TSR༉, respectively [24];When the main peptide repeat motifs (PRMs) of the CRR were "GDRA[D/A]GQPA" and "ANGAGNQPG", they were identified as VK210 and VK247 type sequences, respectively [26, 23, 25], while "APGANQ[E/G]GAA" was confirmed as the PRM of the P. simiovale type sequences [27].
Confirmation of the paired Plasmodium vivax strains and the nature of recurrent episode
When the DNA sequence of the pvcsp gene and the CRR repeat motifs of each group paired samples showed the following characteristics, the Plasmodium vivax strains of both original infection and the recurrent episodes were determined to be from the same time mosquito biting inoculation. Subsequent vivax malaria episodes of the same case were caused by the activation of Plasmodium vivax hypnozoites in this population and were termed as relapse episodes [28]: (1) only one haplotype was shown, and both the expected heterozygosity (He) and quantity of variable sites (V) were equal to 0. This indicated the Plasmodium vivax strains from a paired samples were completely homologous single-clone strains [16, 19–20, 22, 29]; (2) the number of haplotypes was greater than one, and both He > 0 and V > 0. V was confirmed by checking peak plots (Additional file 3), from which it was seen the paired DNA sequences were of the same length and with no fragment deletions (or insertions), suggesting the paired Plasmodium vivax strains were sibling strains which pvcsp gene were weakly heterologous but did not undergo intra-helical recombination events [30, 25].
Seventy-eight reports (3.1%) with recurrent episodes were retrieved from 2484 vivax malaria cases, drawn from the period 2013-19 for patients, all of whom were living in Yunnan Province (97°31′ E to 106°11′ E; 21°8′ N to 29°15′ N). The majority of cases could be traced to origins in Myanmar (98.7%, 77/78), and the male-to-female ratio was 1:5 for all within study’s sample. The majority of cases had one relapse (97.4%, 76/78), while one case had two episodes, and one case had three episodes. General demographic information and original place of infection for the 78 cases are shown in Table 1 (Additional file 2).
| Variable | Total | 2013 | 2014 | 2015 | 2016 | 2017 | 2018 | 2019 | 2020 |
|---|---|---|---|---|---|---|---|---|---|
| Total | 78 | 0 | 14 | 20 | 17 | 9 | 9 | 7 | 2 |
| 1. Gender | |||||||||
| Male | 65 | 0 | 12 | 17 | 13 | 7 | 9 | 5 | 2 |
| Female | 13 | 0 | 2 | 3 | 4 | 2 | 0 | 2 | 0 |
| 2. Age (in years) | |||||||||
| 0–20 | 8 | 0 | 1 | 0 | 4 | 3 | 0 | 0 | 0 |
| 21–60 | 67 | 0 | 12 | 20 | 13 | 5 | 9 | 6 | 2 |
| above 60 | 3 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 0 |
| 3. Malaria recurrence | |||||||||
| 1 episode | 76 | 0 | 13 | 20 | 17 | 9 | 8 | 7 | 2 |
| 2 episodes | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 |
| 3 episodes | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| 4. Infection source | |||||||||
| Myanmar | 77 | 0 | 13 | 20 | 17 | 9 | 9 | 7 | 2 |
| Africa | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Yunnan indigenous | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 5. Interval time of recurrence(days) | |||||||||
| Longest | 1561 | 0 | 939 | 882 | 1561 | 426 | 319 | 367 | 268 |
| Shortest | 28 | 0 | 54 | 46 | 62 | 80 | 43 | 28 | 264 |
| Average | 299 | 0 | 271 | 291 | 292 | 277 | 275 | 285 | 264 |
| NOTE. Data are no. (%) of paired samples, unless otherwise indicated. | |||||||||
A total of 81 relapses occurred across these 78 cases infected with Plasmodium vivax, allowing a total of 159 blood samples which were obtained from all reported original infection and recurrent episodes. From these, 156 PCR amplification products (> 1,000 bp in length) of the pvcsp gene were successfully obtained, with a product acquisition rate of 98.1% (156/159). Of them, paired CDS full strands (807–1179 bp in length) of the pvcsp gene were obtained from blood samples in 75 cases (96.2%, 75/78), but only those from 59 cases could be used for homological analysis of the gene sequences (see Fig. 1).
The structural regions of the amino acid chains derived from the 78 CDS strand conversions were completed, including the conserved region near the 5'-end (1st ~ 90th aa) (α-N region), the R I (KLKQP) region, the CRR (96th ~ 275th aa), and the conserved region near the 3'-end (276th ~ 393th aa) (TSR), etc. (see Fig. 2)
Diversity of pvcsp gene and CRR array
The 121 CDS strands of pvcsp gene obtained from paired blood samples of 59 cases showed 475 variable (polymorphic) loci, comprised of 20 singleton variable sites and 455 parsimony informative sites (Additional file 3), with a nucleotide diversity index (π) equal to 0.1384 (± 0.0056). Among them, 32 alleles were double alleles at positions 112, 113, 233, 234, 240, 261, 264, 270, 274, 282, 295, 309, 327, 347, 354, 426, 491, 507, 511, 534, 545, 552, 572, 579, 615, 684, 742, 761, 769, 805, 892, and 999 (bimodal chart). The sequences from the original infection and relapse strains both call only one of the biallelic bases respectively, usually the type with a strong sequencing signal (Additional file 3). The 32 double alleles were distributed in all seats of the pvcsp gene, but were predominantly concentrated in the CRR region (62.5%, 20/32) (Table 2). Furthermore, 56.3% (18/32) of the polymorphic sites
| Regions | Loci | Alleles of call (Major/Minora) | Codings | Amino acid variation | No. of CDS (n = 127) | Frequency |
|---|---|---|---|---|---|---|
| α-N b | c.112 | A/G | AAC/GGC | N38G | 2 | 0.0157 |
| c.113 | A/G | 2 | 0.0157 | |||
| c.233 | A/G | GAG/GGG | E78G | 2 | 0.0157 | |
| c.234 | G/T | GAG/GAT | E78D | 20 | 0.1575 | |
| c.240 | A/G | AAA/AAG | K80K | 18 | 0.1417 | |
| c.261 | A/C | CCA/CCC | P87P | 14 | 0.1102 | |
| c.264 | T/G | CGT/CGG | R88R | 4 | 0.0315 | |
| c.270 | A/T | AAA/AAT | K90N | 6 | 0.0472 | |
| RIc (91th -95th aa) | c.274 | T/C | TTG/CTG | L92L | 16 | 0.1260 |
| c.282 | A/G | CAA/CAG | Q94Q | 2 | 0.0157 | |
| c.295 | C/A | CGA/AGA | R99R | 2 | 0.0157 | |
| c.309 | G/A | CAG/CAA | Q103Q | 2 | 0.0157 | |
| CRR (96th -290th aa) | c.327 | A/C | GGA/GGC | G109G | 2 | 0.0157 |
| c.354 | A/C | GGA/GGC | G118G | 4 | 0.0315 | |
| c.426 | C/A | GGC/GGA | G142G | 2 | 0.0157 | |
| c.491 | G/C | GGT/GCT | G164A | 2 | 0.0157 | |
| c.507 | A/C | GGA/GGC | G169G | 2 | 0.0157 | |
| c.511 | G/A | GGA/AGA | G171R | 2 | 0.0157 | |
| c.518 | C/A | GCT/GAT | A173D | 2 | 0.0157 | |
| c.534 | C/A | GGC/GGA | G178G | 2 | 0.0157 | |
| c.545 | A/C | GAT/GCT | D182A | 2 | 0.0157 | |
| c.552 | T/A | CAT/CAA | Q184R | 2 | 0.0157 | |
| c.572 | G/C | AGG/AGC | R286S | 2 | 0.0157 | |
| c.579 | G/A | CAG/CAA | Q193Q | 2 | 0.0157 | |
| c.615 | A/C | GGA/GGC | G205G | 2 | 0.0157 | |
| c.684 | A/C | GGA/GGC | G228G | 6 | 0.0472 | |
| c.742 | C/G | CCA/GCA | P248A | 2 | 0.0157 | |
| c.761 | C/G | AGC/AGG | G254R | 2 | 0.0157 | |
| c.769 | C/G | CCA/GCA | P257A | 2 | 0.0157 | |
| c.805 | A/C | ACC/CCC | P269T | 2 | 0.0157 | |
| TSRd (291th -393th aa) | c.892 | C/T | CTT/TTT | L298F | 2 | 0.0157 |
| c.999 | A/G | AAA/AAG | K333K | 2 | 0.0157 | |
| a: At the double allelic base in the DNA sequencing peak map, the base with higher wave peak is Major allele and the another base with lower wave peak is Minor allele; b: Named the near N-terminal of pvcsp amino acid chain; c: The coding region of KLKQP five amino acids; d: The C-terminal of pvcsp amino acid chain. | ||||||
belonged to the third base of the amino acid codon, and only 27.8% (5/18) of these resulted in amino acid variants. The percentages of the second base and first base were 17.6% (6/34) and 26.5% (9/34) (Table 2), while the highest frequency of the double allele was 0.1575 for 234, the minor allele frequency (MAF) was 0.1417 for 240, and 75.0% (24/32) of the double alleles were present in only one set of paired sequences (Table 2).
Further to the aforementioned, 121 CDS strands were defined as 84 haplotypes (H01 to H84) with a He of 0.9940 (± 0.0040). Of these, only haplotypes H08 and H13 had similar other paired sequences. Haplotypes H05, H50, H51, H63 and H64 had CRR repeat units (PRMs) of the VK247 genotype (Fig. 3B), while the remaining 79 had PRMs characteristic of the VK210 genotype (Fig. 3A).
Among the haplotypes of the VK210 type, there were 39 CRR forms consisting of peptide repeat motifs (PRMs) (Fig. 3A). Of these, there were 15 PRM unit types, with GDRAAGQPA and GDRADGQPA occurring most often, with frequencies of 0.470 (987/2100) and 0.3833 (805/2100), respectively. The remaining 13 PRMs, included the five newly detected PRMs GNRANGQPA (0.0033, 7/2100), GNRANGQAA (0.0001, 1/2100), GDRADGQTA (0.0001, 1/2100), GDRADGHPA (0.0001, 1/2100), and GNGAAGQPA (0.0001, 1/2100) (Fig. 3A). Generally, the CRRs of VK210 type consisted of 14–20 PRMs with 18 being the most common, and 96.8% (38/39) ended with GNGAGGQAA units (Fig. 3A).
Of the five haplotypes of type VK247, three types of CRR consisted of 17–21 PRMs (Fig. 3B) in which there were eight unit types of PRMs. Those with the highest frequency of occurrence were ANGAGNQPG (0.7414, 86/116), ANGAGGQAA (0.0517, 6/116), and ANGDDQPGA (0.0172, 2/116) while the remaining two were newly detected PRMs (Fig. 3B).
Comparison of paired blood samples of the pvcsp gene and confirmation of relapse episode
Results from the comparison of the pvcsp gene CDS chains of the 59 paired blood samples showed the paired CDS chains of 31 groups (52.5%, 31/59) had only one haplotype and no variant sites, and the He and V values were both 0. This indicated each of the 31 pairs was homologous and the source of the paired Plasmodium vivax was a single clone with complete genetic homology, belonging to the same mosquito-bite inoculated population (Table 3). Subsequent episodes of Plasmodium vivax were caused by the activation of hypnozoites from the same population as primary infection strains. The paired blood samples of CDS chains of the other 28 groups (47.5%) had varying numbers of polymorphic sites (1⁓6 loci) between the paired sequences. However, there were two exceptions, at 39 (0.0082, 1/121) and 1027 (0.0082, 1/121), which were true base substitutions (Table 3, Additional file 3), while the remaining sites were all double allelic bases (Table 2). These 28 sequences showed no evidence of DNA fragment insertion (or deletion), suggesting they were weakly heterologous for each other, but did not experience intra-helical recombination events. Also, their source pairs were "weakly heterologous" parallel sibling strains
| No. of paired samples | Length of aa chains | Nucleotide diversity (Pi) | Ins or Del in CRR (bp) | Combined variable (polymorphic) sites | Paired strains | Single infection | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. | α-N (1th -90th aa) | RI (91th -95th aa) | CRR (96th -290th aa) | TSP (291th -393th aa) | |||||||||||||
| Ⅰ. Paired (groups = 59) | |||||||||||||||||
| 31 | 364–393 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | com-Homo | Yes | |||||||
| 8 | 340–376 | 0.0009 | 0 | 1a | 234, 240, 261, 264 | 274 | 295 | 0 | non-re-Sibl | Yes | |||||||
| 9 | 340–376 | 0.0018 | 0 | 2b | 234, 240, 261, 270 | 274 | 327, 354, 507, 552, 572, 615, 742 | 999, 1027* | non-re-Sibl | Yes | |||||||
| 4 | 333–386 | 0.0030 | 0 | 3c | 234, 240, 261, 264 | 274, 282 | 491, 511, 545 | 0 | non-re-Sibl | Yes | |||||||
| 3 | 372–376 | 0.0036 | 0 | 4d | 234, 240, 261 | 274 | 426, 518, 534, 742, 761, 769 | 0 | non-re-Sibl | Yes | |||||||
| 3 | 336–375 | 0.0049 | 0 | 5e | 112, 113, 233, 234, 240, 261, 270 | 274 | 309, 579, 684, 805 | 892 | non-re-Sibl | Yes | |||||||
| 1 | 358–368 | 0.0054 | 0 | 6f | 39*, 234, 240, 261, 270 | 274 | 0 | 0 | non-re-Sibl | Yes | |||||||
| Ⅱ. non-Paired | |||||||||||||||||
| 69–109 | 372 and 368 | 0.0263 | + 12g | 29f | 240, 247*, 259*, 264 | 0 | 291*,294*,318*,321*,345*, 348*,363*,444*,453*,552, 561*,572,579,588*,599*, 606*,615,642*,783*,798*, 800*,803*,805 | 987*, 1015* | dif-Popu | No | |||||||
| 156 − 110 | 393 and 368 | 0.0245 | + 54g | 27f | 261 | 0 | 291**,294*,318*,321*,345, 348*,363*,364*,372*,373*, 376*,390*,444*,453*,552, 561*,572,579,588*,599*,669*,687*,696*,707*,714*,796* | 0 | dif-Popu | No | |||||||
| 49–125 | 275 and 249 | 0.0204 | +(51 + 27)h | 22f | 240, 261, 264 | 274 | 404*,435*,567*,570*,591*, 594*,597*,598*,621*,645*, 648*,650*,651*,652*,702*, 705*,729*,780* | 0 | dif-Popu | No | |||||||
| a-e: Existing one locus polymorphic and any two, three, four, five loci polymorphic; f: Existing all loci polymorphic at the same time; g: “+” Including one inserted peptide composed of many amino acids; h: Including two inserted peptides composed of many amino acids; *༚No showing the double peaks at the base site in sequencing peak map. | |||||||||||||||||
which were not genetically homologous and belonged to the latter generations bred from the common ancestor inoculated by a time mosquito-biting population (Table 3). Subsequent episodes of Plasmodium vivax are still caused by the activation of hypnozoites in the same population as primary infection strains.
However, similarity matching of two randomly selected sequences from the 121 CDS strands of the unpaired samples showed there were significantly more base substitutions between the two sequences. Furthermore, the whole strand showed length polymorphism due to the presence of oligonucleotide strand insertions (or deletions) of different lengths (Table 3). This indicated the two sequences of the unpaired samples were more heterologous and their corresponding genomic donors, Plasmodium vivax, were more likely to belong to different populations (Table 3).
The single-copy pvcsp gene commences at the tip of Plasmodium vivax chromosome 8 [30, 25, 31], extends for 807–1179 bp, and does not possess introns, but has a coding DNA sequence (CDS) which encodes polypeptides between 269 and 393 aa long. The structural diversity of the pvcsp gene is concentrated in the mid-segment CRR repeat region encoding the 90th to 275th aa and its flanks (Fig. 1) [23–25, 32]. It is generally characterized by insertions and deletions of repetitive units, mostly due to sexual recombination during meiosis or intra-helical strand-slippage events during mitotic DNA replication [33–35], as well as frequent base substitutions within the locus. Using the repeat units of the CRR as markers, Plasmodium vivax can be defined as three genotypes: VK210; VK247; and the P. semiovale which is Plasmodium. vivax-like [27, 36–37]. Therefore, pvcsp gene is commonly used as a molecular marker for the evolutionary description of populations of Plasmodium vivax [30, 38–40].
This study provides preliminary evidence on the pathogenic nature of recurrent episodes of Plasmodium vivax which occurred in Yunnan Province based on the analysis of pvcsp gene differentiation in the sample group. Although the Plasmodium vivax population included in this study was not truly a natural population, 97.4% of the strains in the sample were nonetheless harvested from cases infected in Myanmar. Consequently, the pvcsp genes of the entire sample group showed similar findings to those reported by Thanapongpichat et al. [41] and Võ et al. [32], which related to the Myanmarese population. These included, the CRR region, the same significant polymorphism in length and composition of PRMs, the same predominance of VK210 types (95.0%, 115/121), and the absence of Plasmodium vivax-like variants. Furthermore, there was the same extremely low DNA sequence homology, with up to 84 haplotypes in 121 pvcsp gene sequences, though the expected heterozygosity (He, 0.9940 ± 0.0040) was greater than that observed by Võ et al. (He, 0.096 ± 0.034) [32]. However, at the same time, the RI of the pvcsp gene in this set of samples had only one arrangement of KLKOP, far fewer than the seven described by Võ et al. [32]. In addition to this, there was a reduction in variety of PRMs units constituting the CRR region (VK210: 15, VK247: 8), and less complex arrangements and length polymorphisms than those reported in the aforementioned research. These may be justified by this study having involved a relatively homogeneous population of primarily Myanmarese strains of Plasmodium vivax, unlike the wider range and a more complex population composition of samples taken by Võ et al. However, the displayed high variability of the pvcsp gene was largely consistent with the results obtained in current study. Furthermore, it is agreed the CRR is suitable as a molecular marker for separating genetic differences in Plasmodium vivax populations due to its poor conservation, whereas the α-N and TSR regions on both flanks are suitable as candidate antigen genes for polyclonal antibody serum preparation because of their highly conserved sequences [24–25].
In addition to confirming the high degree of polymorphism in the pvcsp gene structure at the population level, the similarity comparison performed with paired blood samples (n = 59) from the vivax malaria relapse cases showed it was not an accident that there were the Plasmodium vivax strains with highly convergent gene sequences. In 52.5% (31/59) of the cases, the paired pvcsp gene sequences of Plasmodium vivax strains from the original infection and relapse were not only highly consistent in length but also completely similar in sequence structure, with both He and Variable sites equal to 0, indicating complete homology of the paired sequences. Even among the 28 sets of paired sequences with double allelic expression (Table 2, Table 3), the pvcsp gene sequences were not only highly concordant in length. There were notable oligonucleotide fragment insertions (or deletions) in any two of the pvcsp gene sequences from non-paired blood samples (Table 3). There was also a significant increase in the number of base substitutions, suggesting length polymorphism in the pvcsp gene may be an important point of differentiation between populations of Plasmodium vivax, before the degree of single nucleotide polymorphism,
Besides the possible causes of reinfection and relapse [42–43], validation of the activation of the intrahepatic Plasmodium vivax hypnozoites is imperative due to the frequent recurrent episodes after treatment for vivax malaria [28, 44–45]. In the 78 vivax malaria cases with recurrent episodes included in this study, history of exposure in malaria endemic areas outside the country was before the original infection episode only. Therefore, the possibility of recurrent caused by reinfection is basically excluded. The molecular identification of relapse in only 59 (75.6%, 59/78) cases were conducted by alignment of paired pvcsp gene sequences. In 31 of these cases, including those with two or three times relapses, the paired pvcsp gene sequences of Plasmodium vivax strains from the original infection and the relapse episode were identical as the same length and had no variant sites within the sequences. This observation suggested the single clone of a genetically identical strain existed in every paired blood samples, and the vivax malaria relapse could be attributed to the activation of a intrahepatic hypnozoites which gene structure was identical to the original infection strain [17–18, 20, 28, 46]. In the other 28 cases, there was still a large degree of reproducibility in gene structure between the paired pvcsp gene sequences, which were identical in length and the single nucleotide polymorphic sites mostly were also double alleles (Tables 2 and 3). Such an appeared any locus polymorphism may have been due to differential calling of double allelic bases in paired sequences by generation sequencing [45], suggesting the sibling strains [25, 30, 47] with identical genetic structure existed in every group of paired blood samples, and the two batches of Plasmodium vivax populations from primary infection and relapse actually reflects parallel homologs of polyclonal strains. Therefore, the authors suggested that the relapse of these 28 cases with Plasmodium vivax still belonged to the activation event of intrahepatic hypnozoites coming from common ancestor strains inoculated by the same time mosquito-biting [17, 47–49]. It is worth examining whether the polymorphism form resulting from the differential mobilization of the double alleles is similar to the previously considered "weakly heterologous" [50–53].
In summary, this paper analyze the possibility of relapse episodes caused after one time Plasmodium vivax infection using the principle of identifying the strong similarity of highly variable genes in closely related Plasmodium vivax. Thus, methodological implications are provided for the identification of relapse episodes in malaria-free transmission areas, although there are shortcomings. First, the study may still be incomplete in excluding recrudescence events caused by chloroquine-resistant strains or continued proliferation of myeloplasmosis [54–55] due to the lack of feasible experimental verification means, although the chloroquine resistance associated molecular marker have been detected in some Plasmodium vivax strains from individual paired samples (Additional 4). Secondly, the relapse event was confirmed by the similarity of Plasmodium vivax genes between the paired samples from the original infection and the reactivation. It included two results of 100% homology and "weakly heterologous" with only a few base substitutions between paired gene sequences and it remains to be confirmed whether or not the latter was part of the activation of heterologous hypnozoites populations that scholars previously studied. Thirdly, no definitive confirmation could be provided concerning the nature of the paired Plasmodium vivax strains in 24.4% (19/78). Although the authors are continuing to mitigate the limitations of single genetic markers to identify homologous strains, as well as for other aetiological investigations of these relapse episodes, the length of this article does not allow for further elaboration, which may somewhat reduce its apparent completeness of this paper.
In conclusion, this study identified the vivax malaria relapse episodes caused by homologous hypnozoites by deep sequencing and analyzing the similarities between pvcsp genes of paired Plasmodium vivax strains from primary infection and relapse. The pvcsp gene was found to be simple in structure, while the traces of recombined gene are easy to observe, and it is suitable as a molecular marker to identify the Plasmodium vivax homologous strains. This study was also demonstrated that, because most of the blood-stage Plasmodium vivax resulting in relapse among the cases reported in Yunnan Province were single clone or sibling strains with homologous to the original infection, the using gene similarity rules is suitable for identifying homologous hypnozoites activation.
Plasmodium vivax circumsporozoite protein
Selective whole genome amplification
Yunnan Province Malaria Diagnosis Referent Laboratory
Central repetitive region
Peptide repeat motifs
Region I
Thrombospondin repeat
Minor allele frequency
Coding DNA sequence
Completely Homologous
non-recombinated sibling
Different population
Plasmodium vivax chloroquine resistance proteinortholog
Acknowledgements
Our sincere gratitude got to the provided initial diagnosis information on malaria cases and their blood samples for the Centers for Disease Control and Prevention in prefectures and counties: Dehong, Baoshan, Kunming, Pu’er, Lincang, Dali, Nujiang, Lijiang, Xishuangbanna, Yuxi, Chuxiong, Honghe, Zhaotong, Diqing, Qujing, and Whenshan.
Authors’ contributions
YX was responsible for the study design, carried out genetic testing and wrote the contents of microscopy examination in the manuscript; YD wrote the manuscript, and was responsible for the study design, coordinated the project, and statistical analysis; Yan Deng, HH, MC, YL, JW, CZ and ZW performed the collection of blood samples and microscopy examination. All authors reviewed the manuscript.
Funding
The current study was funded by National Science Foundation, China (Nos. 81660559, 82160637).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The design and protocol of the study was approved by the Yunnan Institute of Parasitic Diseases and Ethical Committee (Ethical Approval No.: 2019 Yunnan Ethics Auditing No. 5). The purpose of the study was explained to the subjects or their relatives, genetic testing was performed on stored blood samples obtained as part of the routine diagnostic work-up of patients with fever who were suspected to have malaria. The patients were fully informed on the aims of the study and signed an informed consent agreement after confirmation Plasmodium infection.
Consent for publication
All authors provided their consent for the publication of this report.
Competing interests
The authors declare no financial and/or non-financial conflicted interest regarding the publication of the present manuscript.
Author details
1 Yunnan Institute of Parasitic Diseases Control, Yunnan Provincial Key Laboratory of Vector-Borne Diseases Control and Research, Yunnan Centre of Malaria Research, Pu’er 665000, China. 2 Department of Basic Medical Sciences, Clinical College of Anhui Medical University, Hefei, 230031, China. 3 Center for Disease Control and Prevention, Baoshan, 678000, China.