Search

Abstract Background The efficacy of chloroquine treatment for vivax malaria has been rarely evaluated due to a lack of an appropriate testing method. The objective of this study was to conduct molecular monitoring of chloroquine resistance in Plasmodium vivax strains from vivax malaria patients in Yunnan Province, focusing on the analysis of polymorphism in the P. vivax chloroquine resistance transporter protein orthologous gene (pvcrt-o). Methods In accordance with the principles of a cohort study, blood samples were collected from malaria cases diagnosed with a P. vivax mono-infection in Yunnan Province from 2020 to 2022. Segmental PCR was used to amplify the whole pvcrt-o gene in the blood samples and their products were subsequently sequenced. The sequencing data were arranged to obtain the full coding DNA sequence (CDS) as well as the gene’s promoter region sequences. The CDSs were aligned with the reference sequence (XM_001613407.1) of the P. vivax SalI isolate to identify the mutant loci. Results From a total of 375 blood samples taken from vivax malaria cases, 272 both whole gene CDSs (1272–1275 bp) and promoter DNA sequences (707 bp) of pvcrt-o gene were obtained. Among the whole CDSs, there were 7 single nucleotide polymorphic sites in which c.7 A>G was the minor allele frequency (MAF) site with 4.4% (12/272) detection rate. The mutation detection rate showed a significant decrease from 9.8% (10/102) in 2020 to 1.1% (1/92) in 2021 and 1.3% (1/78) in 2022, indicating statistical significance (χ 2 = 11.256, P

Abstract 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 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. Graphical Abstract

Abstract 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

Abstract 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

Abstract 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

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 (F ST = 0.97824, 0.97343, P

Abstract 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