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Background: Anopheles maculatus, Anopheles minimus and Anopheles dirus are the major vectors of malaria transmission in the Greater Mekong Subregion (GMS). The malaria burden in this region has decreased significantly in recent years as all GMS countries progress towards malaria elimination. It is necessary to investigate the Anopheles diversity and abundance status and assess the Plasmodium infection rates to understand the malaria transmission potential of these vector species in GMS countries to guide the development of up-to-date vector control strategies and interventions. Methods: A survey of mosquitoes was conducted in Stung Treng, Sainyabuli and Phongsaly Provinces on the Cambodia-Laos, Thailand-Laos and China-Laos borders, respectively. Mosquito collection was done by overnight trapping at sentinel sites in each province. After morphological identification, the 18S rRNA-based nested-PCR was performed to detect malaria parasites in the captured Anopheles mosquitoes. Results: A total of 18 965 mosquitoes comprising of 35 species of 2 subgenera (Subgenus Anopheles and Subgenus Cellia) and 4 tribes (Tribes Culicini, Aedini, Armigerini and Mansoniini) were captured. Tribe Culicini accounted for 85.66% of captures, followed by Subgenus Anopheles (8.15%). Anopheles sinensis dominated the Subgenus Anopheles by 99.81%. Plasmodium-infection was found in 25 out of the 1 683 individual or pooled samples of Anopheles. Among the 25 positive samples, 19, 5 and 1 were collected from Loum, Pangkhom and Siem Pang village, respectively. Eight Anopheles species were found infected with Plasmodium, i.e., An. sinensis, Anopheles kochi, Anopheles vagus, An. minimus, Anopheles annularis, Anopheles philippinensis, Anopheles tessellatus and An. dirus. The infection rates of Plasmodium falciparum, Plasmodium vivax and mixture of Plasmodium parasite species were 0.12% (2/1 683), 1.31% (22/1 683) and 0.06% (1/1 683), respectively. Conclusions: Overall, this survey re-confirmed that multiple Anopheles species carry malaria parasites in the international border areas of the GMS countries. Anopheles sinensis dominated the Anopheles collections and as carriers of malaria parasites, therefore may play a significant role in malaria transmission. More extensive investigations of malaria vectors are required to reveal the detailed vector biology, ecology, behaviour, and genetics in GMS regions in order to assist with the planning and implementation of improved malaria control strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 < 0.05), as well as lower gene flow (Nm = 0.01112, 0.01365) in the 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• A high proportion of asymptomatic parasite carriers of Plasmodium vivax was found in the China–Laos border region. • A questionnaire survey showed that the awareness rate of malaria prevention and control was extremely low in these remote areas. • The most important malaria vector is Anopheles sinensis , which might be widely distributed in this border region. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Background: Chloroquine (CQ) has been the preferred clinical treatment for vivax malaria in Yunnan Province since 1958, with over 300,000 patients. This study aimed to help make trend predictions regarding variations the in anti-malarial drug susceptibility of Plasmodium vivax distributed in Yunnan Province and effectively implement monitoring measures on the efficacy of anti-malarial drugs for vivax malaria. Methods: Blood samples collected from patients with mono-P. vivax infections were employed in this study based on the principle of cluster sampling. The whole gene of P. vivax multidrug resistance 1 protein gene (pvmdr1) was amplified by nested-PCR techniques and the PCR amplification produce were sequenced by Sanger bidirectional sequencing. The mutant loci and haplotypes of coding DNA sequence (CDS) were identified by comparison with the reference sequence (NC_009915.1) of the P. vivax Sal I isolate. Parameters such as Ka/Ks ratio were calculated using MEGA 5.04 software. Results: A total of 753 blood samples from patients infected with mono-P. vivax were collected, of which 624 blood samples yielded the full gene sequence (4392 bp) of the pvmdr1 gene, with 283, 140, 119, and 82 sequences from 2014, 2020, 2021 and 2022, respectively. A total of 52 single nucleotide polymorphic (SNP) loci were detected for the 624 CDSs, of which 92.3% (48/52), 34.6% (18/52), 42.3% (22/52), and 36.5% (19/52) SNPs were detected in 2014, 2020, 2021 and 2022, respectively. All of 624 CDSs were defined for a total of 105 mutant haplotypes, with CDSs of 2014, 2020, 2021, and 2022 containing 88, 15, 21, and 13 haplotypes, respectively. Of the 105 haplotypes, the threefold mutant haplotype (Hap_87) was the starting point for stepwise evolution, and the most drastic tenfold mutations were Hap_14 and Hap_78, and the fivefold, sixfold, sevenfold, and eightfold mutations. Conclusions: In the majority of vivax malaria cases in Yunnan Province, most of them were infected with strains carrying demonstrating highly mutated in pvmdr1 genes. However, the dominant mutation strains types varied from year to year, which warrants further exploration in order to confirm the correlation between with phenotypic changes in P. vivax strains and their susceptibility to anti-malarial drugs such as chloroquine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 = 4.468, 5.889, P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency (36.9%, 236/640), and Hap_9 had the most complex sequence structure, containing 7 loci mutations. Allele *10 was the most frequent among these genotypes (0.423). Among the allele *10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group ( x 2 = 3.911, P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group ( x 2 = 10.050, P < 0.05), and genotype *4/*4was detected only in the SR group. 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. [ABSTRACT FROM AUTHOR]

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 < 0.05). Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, *10 and *39, were confirmed by comparison, and CYP2D6*10 allele accounted for the largest percentage (45.4%, 108/238). The frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (Χ2 = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]