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Background In recent years, the incidence rate of vivax malaria recurrence still had 3.1% in Yunnan Province population after eradication therapy using primaquine (PQ). In order to understand the specific failure reasons for preventing vivax malaria relapses, a preliminary exploration on the CYP2D6 enzyme activity was carried out in the vivax malaria patients in Yunnan Province population by analysing mutational polymorphism in the coding region of CYP2D6 gene. Methods Blood samples were collected from vivax malaria patients with suspected relapse (SR) and non-relapsed (NR) malaria in Yunnan Province. The DNA fragments containing 9 exons regions of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were aligned by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape. The alleles of CYP2D6 gene were identified according to the criteria prescribed by Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database and the CYP2D6 enzyme activity was predicted based on diploid genotype. Results A total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1,457 (rs1135840) were observed on the 640 CDS chains (including 320 maternal and 320 paternal chains). The high-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group ($${x}^{2}$$ x 2 = 4.468, 5.889, P $${x}^{2}$$ x 2 = 3.911, P $${x}^{2}$$ x 2 = 10.050, P Conclusion In the patients receiving PQ dosage in Yunnan Province population, both rs1135840 single nucleotide polymorphism and *10 allele form was common in the CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse.

Background The Anopheles hyrcanus group, which includes 25 species, is widely distributed in the Oriental and Palaearctic regions. Given the difficulty in identifying cryptic or sibling species based on their morphological characteristics, molecular identification is regarded as an important complementary approach to traditional morphological taxonomy. The aim of this study was to reconstruct the phylogeny of the Hyrcanus group using DNA barcoding markers in order to determine the phylogenetic correlations of closely related taxa and to compare these markers in terms of identification efficiency and genetic divergence among species. Methods Based on data extracted from the GenBank database and data from the present study, we used 399 rDNA–ITS2 sequences of 19 species and 392 mtDNA–COII sequences of 14 species to reconstruct the molecular phylogeny of the Hyrcanus group across its worldwide range. We also compared the performance of rDNA–ITS2 against that of mtDNA–COII to assess the genetic divergence of closely related species within the Hyrcanus group. Results Average interspecific divergence for the rDNA–ITS2 sequence (0.376) was 125-fold higher than the average intraspecies divergence (0.003), and average interspecific divergence for the mtDNA–COII sequence (0.055) was eightfold higher than the average intraspecies divergence (0.007). The barcoding gap ranged from 0.015 to 0.073 for rDNA–ITS2, and from 0.017 to 0.025 for mtDNA–COII. Two sets of closely related species, namely, Anophels lesteri and An. paraliae, and An. sinensis, An. belenrae and An. kleini, were resolved by rDNA–ITS2. In contrast, the relationship of An. sinensis/An. belenrae/An. kleini was poorly defined in the COII tree. The neutrality test and mismatch distribution revealed that An. peditaeniatus, An. hyrcanus, An. sinensis and An. lesteri were likely to undergo hitchhiking or population expansion in accordance with both markers. In addition, the population of an important vivax malaria vector, An. sinensis, has experienced an expansion after a bottleneck in northern and southern Laos. Conclusions The topology of the Hyrcanus group rDNA–ITS2 and mtDNA–COII trees conformed to the morphology-based taxonomy for species classification rather than for that for subgroup division. rDNA–ITS2 is considered to be a more reliable diagnostic tool than mtDNA–COII in terms of investigating the phylogenetic correlation between closely related mosquito species in the Hyrcanus group. Moreover, the population expansion of an important vivax malaria vector, An. sinensis, has underlined a potential risk of malaria transmission in northern and southern Laos. This study contributes to the molecular identification of the Anopheles hyrcanus group in vector surveillance. Graphical abstract

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.

More than 85% of the malaria burden in the Yunnan Province is caused by imported vivax malaria, and Yunnan is also where the majority of vivax malaria patients are diagnosed in China. Timely removal of the infection sources of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To that end, blood samples were collected from cases diagnosed and revalidated as single species infection with P. vivax in the Yunnan Province from 2013 to 2020. Specifically, samples from vivax malaria patients with suspected relapses episodes were subjected to PCR amplification, product sequencing, and analysis of the P. vivax circumsporozoite protein (pvcsp) gene. In total, 77 suspected relapse patients were identified out of 2484 cases infected with P. vivax, with a total of 81 recurrent episodes. A total of 156 CDS (coding DNA sequence) chains were obtained through PCR amplification and sequencing of the pvcsp gene from 159 blood samples, 121 of which can be matched to the paired sequences of 59 vivax malaria patients with both primary attack and recurrent experience. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites (VS), meaning every two paired sequence was completely homologous. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, indicating that the paired sequences was “weakly heterologous” with no fragment insertions (or deletions). All 59 vivax malaria patients with recurrences were caused by the activation of P. vivax hypnozoites originated from the same population as the primary infection. The paired analysis of the similarity between high variant genes allowed the identification of relapse episodes caused by P. vivax homologous hypnozoites and also demonstrated pvcsp gene as one of the candidate molecular markers for tracing infection origin.

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.

Background Border malaria is one of the most intractable problems hindering malaria elimination worldwide. Movement of both the human population and anopheline mosquitoes infected with Plasmodium spp. can cause cross-border malaria transmission. The Yunnan border area was still hyperendemic for malaria in the early part of this century. The objective of this case study was to analyze the strategies, interventions and impacts of malaria control and elimination in the Yunnan border area. Main text A total of 10,349 malaria cases and 17.1 per 10,000 person-years of annual parasite incidence (API) were reported in the border area in 2003. Based on natural village-based stratification, integrated interventions, including mass drug administration for radical cures and preventive treatment, clinically presumptive treatment of all febrile patients for malaria and indoor residual spraying or dipping bed nets with insecticides were successfully carried out from 2003 to 2013. The overall API was reduced to 0.6 per 10,000 person-years by 2013, while effective cross-border collaboration interventions dramatically reduced the malaria burden in the neighbouring border areas of Myanmar. From 2014 forward, the comprehensive strategy, including universal coverage of surveillance to detect malaria cases, a rapid response to possible malaria cases and effective border collaboration with neighbouring areas, successfully eliminated malaria and prevented reintroduction of malaria transmission in the Yunnan border area. Conclusions In Yunnan malaria burden has successfully reduced by dynamically accurate stratification and comprehensive interventions; and then the region achieved elimination and prevented reintroduction of malaria transmission through intensive surveillance, rapid response and border collaboration. Other border areas should perform their own intervention trials to develop their own effective strategy. Graphical Abstract

Background Accumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene. Methods Blood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation “reference” sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes (star alleles), genotypes and odds ratios (OR) of all the CDSs were analysed. Results Of the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria (OR = 2.167, 95% CI 1.104–4.252, P Χ2 = 16.177, P Conclusion Mutation of CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D6*2 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.

Background Vector control is still a pivotal method for preventing malaria, and its potency is weakened by the increasing resistance of vectors to chemical insecticides. As the most abundant and vital malaria vector in Southeast Asia, the chemical insecticide resistance status in Anopheles sinensis remains elusive in Laos, which makes it imperative to evaluate the true nature of chemical insecticide resistance-associated genetic mutations in An. sinensis in Laos. Methods Adult An. sinensis were collected from three border regions in Laos. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of a fragment containing codon 1014 of the voltage-gated sodium channel (vgsc) gene were completed to study the kdr allele frequency distribution, kdr intron polymorphism, population genetic diversity, and the evolutionary status of the kdr codon. The mitochondrial cytochrome c oxidase subunit II gene (COII) was amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion, and gene flow patterns. Results Nine wild kdr haplotypes of the vgsc gene were detected in this study, and eight of them, namely 1014L1, 1014L2, 1014L4, 1014L7, 1014L9, 1014L10, 1014L11, and 1014L21, were discovered in the China–Laos border (northern Laos), while 1014L3 was only detected in the Thailand–Laos border (northwestern Laos) and Cambodia–Laos border (southern Laos). The newly identified haplotype, 1014L21, was uniquely distributed in the China–Laos border and was not identified in other countries. Based on sequence analysis of the mitochondrial COII genes, significant genetic differentiation and limited gene flow were detected between the China–Laos and Cambodia–Laos An. sinensis populations, which suggested that those two regions were genetically isolated. The distinct distribution of the kdr haplotype frequencies is probably the result of geographical isolation in mosquito populations. Conclusions Lack of kdr mutations in the vgsc gene was probably due to genetic isolation and the absence of intense selection pressure in the three border regions of Laos. This study reveals that pyrethroid-based chemical insecticides are still appropriate for battling An. sinensis in parts of Laos, and routine monitoring of chemical insecticide resistance should be continuously implemented and focused on more restricted areas as part of chemical insecticide resistance management. Graphical Abstract

Objectives The China–Laos border has been identified as an important origin of imported malaria outside China. The aim of this study was to describe the asymptomatic malaria infections and epidemic trend of malaria in the China–Laos border region. Methods A prevalence survey and surveillance of mosquito vectors was conducted in Muang Khua District of Phongsaly Province, China–Laos border, to determine the parasite carriage rate using nested PCR and microscopy. The species composition of malaria vectors was determined by overnight trapping. Blood samples were collected from 354 local residents aged 1–72 years in Sankang village in 2016. A total of 2430 adult mosquitoes were collected from four other villages in Muang Khua District from June to August 2016. Results The parasite carriage rate was 7.63% (27/354) by microscopy or 7.91% (28/354) by nested PCR. The results of surveillance of the mosquito vectors revealed that the predominant genera of adult mosquitoes were Culex (69.92%, 1699/2430) and Anopheles (21.48%, 522/2430). Anopheles sinensis (82.95%, 433/522) was identified as the predominant species among the seven members of Anopheles found in this border region. Conclusions A high prevalence of asymptomatic malaria was present and the most important malaria vector was Anopheles sinensis, suggesting that the malaria epidemic situation on the China–Laos border is serious.

Background To develop an effective malaria vector intervention method in forested international border regions within the Greater Mekong Subregion (GMS), more in-depth studies should be conducted on local Anopheles species composition and bionomic features. There is a paucity of comprehensive surveys of biodiversity integrating morphological and molecular species identification conducted within the border of Laos and Cambodia. Methods A total of 2394 adult mosquitoes were trapped in the Cambodia–Laos border region. We first performed morphological identification of Anopheles mosquitoes and subsequently performed molecular identification using 412 recombinant DNA–internal transcribed spacer 2 (rDNA-ITS2) and 391 mitochondrial DNA–cytochrome c oxidase subunit 2 (mtDNA-COII) sequences. The molecular and morphological identification results were compared, and phylogenetic analysis of rDNA-ITS2 and mtDNA-COII was conducted for the sequence divergence among species. Results Thirteen distinct species of Anopheles were molecularly identified in a 26,415 km2 border region in Siem Pang (Cambodia) and Pathoomphone (Laos). According to the comparisons of morphological and molecular identity, the interpretation of local species composition for dominant species in the Cambodia–Laos border (An. dirus, An. maculatus, An. philippinensis, An. kochi and An. sinensis) achieved the highest accuracy of morphological identification, from 98.37 to 100%. In contrast, the other species which were molecularly identified were less frequently identified correctly (0–58.3%) by morphological methods. The average rDNA-ITS2 and mtDNA-COII interspecific divergence was respectively 318 times and 15 times higher than their average intraspecific divergence. The barcoding gap ranged from 0.042 to 0.193 for rDNA-ITS2, and from 0.033 to 0.047 for mtDNA-COII. Conclusions The Cambodia–Laos border hosts a high diversity of Anopheles species. The morphological identification of Anopheles species provides higher accuracy for dominant species than for other species. Molecular methods combined with morphological analysis to determine species composition, population dynamics and bionomic characteristics can facilitate a better understanding of the factors driving malaria transmission and the effects of interventions, and can aid in achieving the goal of eliminating malaria. Graphical Abstract

Background Few studies have been conducted to investigate the distribution of mosquito vectors and the population structure of secondary vectors in the border region of Cambodia-Laos. The aim of this work was to study the mosquito diversity and molecular phylogeny of secondary vectors, i.e., Anopheles nivipes in this area. Methods 1440 adult mosquitoes were trapped in the Cambodia-Laos border. mtDNA-COII were amplified and sequenced from 53 An. nivipes DNA samples. Together with COII sequences deposited in GenBank, a total of 86 COII sequences were used for examining population variations, genetic differentiation, spatial population structure, population expansion, and gene flow patterns. Results The adult mosquitoes were classified into 5 genera and 27 species in this border region. The predominant genera were Culex (60.07%, 865/1440) and Anopheles (31.25%, 450/1440), and the major Anopheles species were An. nivipes (73.56%, 331/450) and Anopheles maculatus (14.22%, 64/450). Based on sequences analysis of COII, a high level of genetic differentiation was reported in two Northwest India (Cheema and Bathinda, Punjab) and Cambodia-Laos (Siem Pang, Stung treng) populations (FST = 0.97824, 0.97343, P An. nivipes populations. Phylogenetic analysis and SAMOVA revealed a gene barrier restricting gene flow among three An. nivipes populations. Mantel test suggested a significant correlation between geography and gene distance in all An. nivipes populations (Z = 44,983.1865, r = 0.5575, P = 0.0070). Neutrality test and Mismatch distribution revealed a recent population expansion of An. nivipes in the Cambodia-Laos population. Conclusions Anopheles nivipes was one of the major Anopheles species in the Cambodia-Laos border. Based on sequences analysis of COII, a genetic barrier between Cambodia-Laos and two Indian populations was found, and a recent population expanding or selecting of An. nivipes occurred in the Cambodia-Laos population, suggesting that COII might be an effective marker for describing the molecular phylogeny of An. nivipes. Further investigation and continuous surveillance of An. nivipes are warranted in this region.

Additional file 3. Epidemiological information and alignment results of the genes of suspected recurrent cases of vivax malaria.

Additional file 5. Analysis the CYP2D6 genotypes in SR group and NR group.

Additional file 4. Electrophoretic map of PCR products in the coding region of CYP2D6 gene.

Additional file 4. Electrophoretic map of PCR products in the coding region of CYP2D6 gene.

Additional file 3. Epidemiological information and alignment results of the genes of suspected recurrent cases of vivax malaria.

Additional file 2. Sequencing Peak Map of CYP2D6 Gene Polymorphic loci.

Additional file 5. Analysis the CYP2D6 genotypes in SR group and NR group.

Additional file 1. The details of nested PCR testing for differentiating between various Plasmodium species.

Additional file 2. Sequencing Peak Map of CYP2D6 Gene Polymorphic loci.

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.

Additional file 1. Malaria case confirmation by Yunnan Provincial Reference Laboratory.

Additional file 4. Analysis of the inconsistency between initial diagnosis and re-testing in Yunnan Province from 2013 to 2018.

Additional file 3. Accuracy of initial malaria diagnoses in Yunnan Province prefectures (2013 to 2018).

Additional file 1. Re-testing of negative samples by YPMDRL in Yunnan Province (2016 to 2018).

Additional file 2. The details of nested PCR testing for differentiating between various Plasmodium species.

Background According to China’s Malaria Eradication Action Plan, malaria cases diagnosed and reported by health authorities at the county level must be further re-confirmed by provincial laboratories. The Yunnan Province Malaria Diagnostic Reference Laboratory (YPMDRL) began the synchronous implementation of microscopic examinations and nested polymerase chain reaction (nested-PCR) testing to re-test the malaria cases initially diagnosed by county-level laboratories and to evaluate the consistency of Plasmodium species identified between by YPMDRL and by the county-level laboratories from 2013 to 2018 in Yunnan Province. Methods Data on malaria initial diagnosis completed by county-level laboratories in Yunnan Province were collected weekly from the “China Disease Prevention and Control Information System” from 2013 to 2018. The YPMDRL performed Plasmodium microscopic examination and 18S rRNA gene nested-PCR testing on every malaria case managed by the China Disease Prevention and Control Information System. The re-testing detection results were fed back to the initial diagnosis and reporting unit for revision of malaria case types. Results A total of 2,869 malaria cases were diagnosed and reported by county-level laboratories in Yunnan Province from 2013 to 2018. The re-testing rate was 95.6% (2,742/2,869), and the re-testing rate increased from 2013 to 2018. Among the re-tested 2,742 cases, 96.7% (2651/2742), 2.2% (59/2742), and 1.1% (32/2742) were doubly examined by microscopy and by nested-PCR, only by microscopy, and only by nested-PCR, respectively. The total Plasmodium species accuracy rate at county-level laboratories was 92.6% (2,543/2,742) reference to the diagnosis by YPMDRL. Among the inconsistent 199 cases, they were identified as including 103 negative cases, 45 falciparum malaria cases, 30 vivax malaria cases, 11 ovale malaria cases, and 10 malariae malaria cases by YPMDRL. From 2013 to 2018, the revised and registered malaria cases by the China Disease Prevention and Control Information System in Yunnan Province was 2,747 cases, including 2,305 vivax malaria cases, 421 falciparum malaria cases, 11 ovale malaria cases, and 10 malariae malaria cases. Conclusions The double re-testing strategy by microscopy and by gene testing increases the accuracy of diagnoses malaria in Yunnan Province, and gene testing can reliably differentiate Plasmodium species. The re-testing results provided by YPMDRL are the authoritative basis for revising malaria kind in Yunnan Province.

Background This paper seeks to assess the function of malaria control consultation and service posts (MCCSPs) that are located on the border areas of Yunnan province, P.R. China, as a strategy for eliminating malaria among the mobile and migrant population in these areas. Methods A retrospective descriptive analytical study was conducted. Blood smear examinations conducted at all MCCSPs in Yunnan from 2008 to 2014 were analysed. A cross-sectional survey was conducted in 2014 to understand how the MCCSPs function and to elucidate the quality of the blood smear examinations that they conduct. Results Out of the surveyed MCCSPs, 66 % (39/59), 22 % (13/59), and 12 % (7/59) were attached to local township hospitals, village health clinics, and the county centre for disease control and prevention or private clinics, respectively. More than 64 % (38/59) of the posts’ staff were part-time workers from township hospitals and village health facilities. Less than 31 % (18/59) of the posts’ staff were full-time workers. A total of 35 positive malaria cases were reported from seven MCCSPs in 2014. Four MCCSPs were unable to perform their functions due to under staffing in 2014. There was a small fluctuation in blood smear examinations from January 2008 to June 2009, with two peaks during the period from July 2009 to October 2010. The number of blood smear examinations has been increasing since 2011. The yearly mean number of blood smear examinations in each post increased from 44 per month in 2011 to 109 per month in 2014, and the number of positive malaria cases detected by blood smear examinations has declined (χ2 = 90.67, P = 0.000). The percentage of people from Yingjiang county getting blood smear examinations increased between 2008 and 2014, while percentages of the mobile population including Myanmar people, people from other provinces, and people from other Yunnan counties getting blood smear examinations decreased. Conclusion MCCSPs face challenges in the phase of malaria elimination in Yunnan, China. New case detection strategies should be designed for MCCSPs taking into account the current trends of migration. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0185-y) contains supplementary material, which is available to authorized users.