Search

BackgroundMore 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 patients 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. Materials and methods To do so, blood samples were collected from cases diagnosed and revalidated as single infections of P. 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 P. vivax circumsporozoite protein (pvcsp) gene. ResultsSeventy-eight suspected recurrent patients were retrieved from 2484 vivax malaria cases, with a total of 81 recurrent episodes. A total of 159 blood samples from primary infection P. 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 patients. 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 P. 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 P. vivax hypnozoites from the same population as the primary infection. Conclusions The paired analysis of the similarity of Plasmodium high variant genes allowed the identification of recurrent episodes caused by P. vivax homologous hypnozoites, and also demonstrated pvcsp gene as one of the candidate molecular markers. 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.

Background 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. Materials and methods 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. Results 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. Conclusions 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.

BackgroundPrimaquine is one of two medications able to effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for vivax malaria patients. However, the reduced CYP2D6 enzyme activity caused by gene mutations has been considered to result in the failure of primaquine relapse vivax malaria. Based on the analysis of CYP2D6 gene polymorphisms in vivax malaria cases the current study aims to determine the association between human CYP2D6 polymorphism and the relapse of vivax malaria. MethodsBlood samples of vivax malaria cases who received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The suspected relapsed cases were determined by epidemiology and gene sequence alignment. Human genomic DNA was extracted from the blood samples, nine exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and PCR products were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning with the non-mutation reference sequence. The mutation loci, haplotypes (star alleles) and genotypes of CYP2D6 gene were inferred and their association with vivax malaria relapse were analyzed. ResultsA total of 120 blood samples from 45 suspected relapsed cases of vivax malaria and 75 non-relapsed cases were collected and two nested-PCR products (2411bp and 2388bp) containing exon1-4 and exon5-9, were obtained from 119 samples. 119 CDS chains (1491bp length) were formed by splicing and combining sequencing sequences, A total of 12 mutation loci were identified in 238 CDS chains (including 119 chains from direct sequencing and another 119 chains identified from sister chromosome). Among them, the mutation locus at c.886 has the closest relationship with the relapse of vivax malaria (OR=2.167, 95%CI: 1.104~4.252, PP

Background: Primaquine is one of two medications able to effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for the treatment of vivax patients. However, 5-hydroxy-primaquine, the presumed active component, must be generated by CYP2D6 enzyme in human hepatocytes and reduced CYP2D6 enzyme activity caused by gene mutations has been considered cause of primaquine failure. Based on the analysis of CYP2D6 gene polymorphisms in suspected relapsed vivax malaria patients, the current study aims to determine the association between human CYP2D6 genotype and the efficacy of primaquine. Methods: Blood samples from vivax patients in Yunnan Province who received "chloroquine/primaquine eight-day therapy" and experienced suspected relapses were collected from 2014 to 2018. Human genomic DNA was extracted, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and were then sequenced. The mutation types of CDS and their association with CYP2D6 enzyme activity changes were analyzed. Results: A total of 156 blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected and two nested-PCR products, the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp), were obtained from the sample. After splicing and combining these two amplification products, we found that the CDS of CYP2D6 gene in each sample was 1491bp in length. Moreover, we identified 24 haplotypes (Hap_1~Hap_24) in the Clustered CYP2D6 full-CDS of 150 patients, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients (8 haplotypes co-existed in two groups of patients). In addition, we identified 33 diplotypes (A_1~A_33) in 150 patients. The A_10 and A_12 diplotypes showed higher frequency in suspected relapsed group.Conclusion: Among the numerous mutations of CYP2D6 gene， Hap_2 accounted for the largest proportion in 24 haplotypes. And the combined homozygous mutations at c. 408, c. 1457, especially the two loci combined mutations with c. 886, may be related to the poor efficacy of primaquine in the radical cure P. vivax in Yunnan, however, greater sample size is required confirm these findings.

Background Eighteen imported ovale malaria cases imported from Myanmar and various African countries have been reported in Yunnan Province, China from 2013 to 2018. All of them have been confirmed by morphological examination and 18S small subunit ribosomal RNA gene (18S rRNA) based PCR in YNRL. Nevertheless, the subtypes of Plasmodium ovale could not be identified based on 18S rRNA gene test, thus posing challenges on its accurate diagnosis. To help establish a more sensitive and specific method for the detection of P. ovale genes, this study performs sequence analysis on k13-propeller polymorphisms in P. ovale. Methods Dried blood spots (DBS) from ovale malaria cases were collected from January 2013 to December 2018, and the infection sources were confirmed according to epidemiological investigation. DNA was extracted, and the coding region (from 206th aa to 725th aa) in k13 gene propeller domain was amplified using nested PCR. Subsequently, the amplified products were sequenced and compared with reference sequence to obtain CDS. The haplotypes and mutation loci of the CDS were analysed, and the spatial structure of the amino acid peptide chain of k13 gene propeller domain was predicted by SWISS-MODEL. Results The coding region from 224th aa to 725th aa of k13 gene from P. ovale in 83.3% of collected samples (15/18) were amplified. Three haplotypes were observed in 15 samples, and the values of Ka/Ks, nucleic acid diversity index (π) and expected heterozygosity (He) were 3.784, 0.0095, and 0.4250. Curtisi haplotype, Wallikeri haplotype, and mutant type accounted for 73.3% (11/15), 20.0% (3/15), and 6.7% (1/15). The predominant haplotypes of P. ovale curtisi were determined in all five Myanmar isolates. Of the ten African isolates, six were identified as P. o. curtisi, three were P. o. wallikeri and one was mutant type. Base substitutions between the sequences of P. o. curtisi and P. o. wallikeri were determined at 38 loci, such as c.711. Moreover, the A > T base substitution at c.1428 was a nonsynonymous mutation, resulting in amino acid variation of T476S in the 476th position. Compared with sequence of P. o. wallikeri, the double nonsynonymous mutations of G > A and A > T at the sites of c.1186 and c.1428 leads to the variations of D396N and T476S for the 396th and 476th amino acids positions. For P. o. curtisi and P. o. wallikeri, the peptide chains in the coding region from 224th aa to 725th aa of k13 gene merely formed a monomeric spatial model, whereas the double-variant peptide chains of D396N and T476S formed homodimeric spatial model. Conclusion The propeller domain of k13 gene in the P. ovale isolates imported into Yunnan Province from Myanmar and Africa showed high differentiation. The sequences of Myanmar-imported isolates belong to P. o. curtisi, while the sequences of African isolates showed the sympatric distribution from P. o. curtisi, P. o. wallikeri and mutant isolates. The CDS with a double base substitution formed a dimeric spatial model to encode the peptide chain, which is completely different from the monomeric spatial structure to encode the peptide chain from P. o. curtisi and P. o. wallikeri.

Background: Primaquine is the traditional medicine could effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for the treatment of vivax malaria patient. However, 5-hydroxy-primaquine, the active component in primaquine to kill P. vivax hypnozoite has to be catalyzed by Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocytes. Nevertheless, suppressed CYP2D6 enzyme activity caused by CYP2D6 gene mutation has been considered as a risk factor for the efficacy of primaquine. Based on the analysis of CYP2D6 gene polymorphism in relapsed vivax malaria patients, the current study aims to preliminarily reveal the genetic association between human CYP2D6 genotype and the curative effect of primaquine in vivax malaria. Methods: Blood samples of vivax malaria cases received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The relapsed cases were determined by identifying the homogeneity of P. vivax circumsporozoite protein (pvcsp) gene sequences between the Plasmodiums isolated from patients’ infected blood samples. Human genomic DNA was extracted from the blood samples, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning with the wild-type reference sequence. The mutation types of CDS and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by the allelic form of CYP2D6 gene. Results: A total of 156 blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected for nested-PCR amplification. Two nested-PCR products, the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp), were obtained from the sample. After splicing and combining these two amplification products, we found that the CDS of CYP2D6 gene in each sample was 1491bp in length. Moreover, we identified 24 haplotypes (Hap_1~Hap_24) in the Clustered CYP2D6 full-CDS of 150 patients, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients (8 haplotypes co-existed in two groups of patients). Hap_6 showed G>C, C>T and G>C base substitutions at c.408, c.886 and c.1457, resulting in V136V invariance, and variations of R296C and S486T at 136th, 296th and 486th amino acid sites, respectively. The risk of relapsing vivax malaria in Hap_6 was about 5.615-fold higher than in other haplotypes. In the relapsed cases, c.408 locus mutation of CYP2D6 gene was 100% homozygous allele (C/C, 10/10), whereas c.886 locus mutation was 80% homozygous allele (T/T, 8/10) and 20% heterozygous allele (C/T, 2/2). However, the non-relapsed cases only showed mutation heterozygote allele (C/T, 2/2) at c.886 locus. Moreover, most of mutant allele types in relapsed patients were CYP2D6 *2, and the CYP2D6 enzyme activity was predicted as normal metabolizer (NM) according to the scoring of diploid allele of Hap_6 sequence.Conclusion: Among the numerous mutations of CYP2D6 gene, triple mutations at three loci (c.408, c.886, c.1457) are most closely related to impaired CYP2D6 enzyme activity, and greater sample size should be applied to ascertain whether c.886 locus mutation plays a critical role.

Background: So far, it has been confirmed that primaquine is the only medicine could effectively kill the hypnozoites of P. vivax. Therefore, World Health Organization (WHO) recommends primaquine as the only anti-relapsing drug for the treatment of vivax malaria patient. However, due to the active component of primaquine for killing P. vivax hypnozoite is the 5-hydroxy-primaquine, which has to be catalyzed by the Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocytes, the decrease of CYP2D6 enzyme activity caused by CYP2D6 gene mutation has been considered as an risk factor for the use of primaquine. Based on the analysis of CYP2D6 gene polymorphisms in relapsed vivax malaria patients, this study preliminarily revealed the genetic association between human CYP2D6 genotype and the curative effect of primaquine in vivax malaria. Methods: Blood samples of vivax malaria cases treated with "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. Then, the relapsed cases were determined by identifying the homogeneity of P. vivax circumsporozoite protein（pvcsp）gene sequences between the Plasmodiums isolated from patients’ first and second infected blood samples. Human genomic DNA was extracted from blood samples, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing after alignment with the wild-type reference sequence. The mutation types of CDS and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by the allelic form of CYP2D6 gene. Results: One hundred and fifty-six blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected for nested-PCR amplification. Two kinds of nested-PCR products，the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp)，were obtained from every sample. After splicing and combining these two amplification products, the complete CDS of CYP2D6 (1491 bp) of each sample could be gotten. Clustered CYP2D6 full-CDS of 150 patients, 24 haplotypes (Hap_1~Hap_24) were defined, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients. The Hap_6 showed G>C, C>T and G>C base substitutions at c.408, c.886 and c.1457, resulting in V136V invariance and the variations of R296C and S486T at 136th, 296th and 486th amino acid sites. The odds ratio of Hap_6 sequence type was 5.615 (P˂0.05) as compared with the relapse of vivax malaria. For the relapsed cases, the mutation at c.408 of CYP2D6 gene is 100% homozygous allele (C/C, 10/10), while at c.886 is 80% homozygous allele (T/T, 8/10) and 20% heterozygous allele (C/T, 2/2). But the non-relapsed cases only showed mutation heterozygote allele (C/T, 2/2) at c.886 locus. Moreover, most of mutant allele types in relapsed patients were CYP2D6 *2, and the CYP2D6 enzyme activity of them was predicted as normal metabolizer (NM) by scoring the diploid allele of Hap_6 sequence. Conclusion: Among the numerous mutations of CYP2D6 gene, the triple mutations at three loci (c.408, c.886, c.1457) are most closely related to the decreased CYP2D6 enzyme activity, while whether the c.886 locus mutation plays a critical role needs to be further verified by expanded sample size.