Cross-sectional study of asymptomatic malaria and seroepidemiological surveillance of seven districts in Gia Lai province, Vietnam.

BACKGROUND: Malaria elimination by 2030 is an aim of many countries in the Greater Mekong Sub-region, including Vietnam. However, to achieve this goal and accelerate towards malaria elimination, countries need to determine the extent and prevalence of asymptomatic malaria as a potential reservoir for malaria transmission and the intensity of malaria transmission. The purpose of this study was to determine the prevalence of asymptomatic malaria and seropositivity rate in several districts of Gia Lai province in the Central Highlands of Vietnam. METHODS: A cross-sectional survey of asymptomatic malaria and serological testing was conducted in 3283 people living at 14 communes across seven districts in Gia Lai province in December 2016 to January 2017. Finger prick capillary blood samples were tested for malaria using rapid diagnostic testing and polymerase chain reaction (PCR), as well as detecting antibodies against 3 Plasmodium falciparum and 4 Plasmodium vivax antigens by indirect enzyme-linked immunosorbent assay (ELISA). Age-seroprevalence curves were fitted using reverse catalytic models with maximum likelihood. RESULTS: The study population was predominantly male (65.9%, 2165/3283), adults (88.7%, 2911/3283) and of a minority ethnicity (72.2%, 2371/3283), with most participants being farmers and outdoor government workers (90.2%, 2960/3283). Using a small volume of blood (≈ 10 µL) the PCR assay revealed that 1.74% (57/3283) of the participants had asymptomatic malaria (P. falciparum 1.07%, P. vivax 0.40%, Plasmodium malariae 0.15% and mixed infections 0.12%). In contrast, the annual malaria prevalence rates for clinical malaria in the communities where the participants lived were 0.12% (108/90,395) in 2016 and 0.22% (201/93,184) in 2017. Seropositivity for at least one P. falciparum or one P. vivax antigen was 38.5% (1257/3262) and 31.1% (1022/3282), respectively. Age-dependent trends in the proportion of seropositive individuals in five of the districts discriminated the three districts with sustained low malaria prevalence from the two districts with higher transmission. CONCLUSIONS: Asymptomatic Plasmodium carriers were found to be substantially more prevalent than clinical cases in seven districts of Gia Lai province, and a third of the population had serological evidence of previous malaria exposure. The findings add knowledge on the extent of asymptomatic malaria and transmission for developing malaria elimination strategies for Vietnam.

Surveillance of pfhrp2 and pfhrp3 gene deletions among symptomatic Plasmodium falciparum malaria patients in Central Vietnam.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) remain the main point-of-care tests for diagnosis of symptomatic Plasmodium falciparum malaria in endemic areas. However, parasites with gene deletions in the most common RDT target, histidine rich protein 2 (pfhrp2/HRP2), can produce false-negative RDT results leading to inadequate case management. The objective of this study was to determine the prevalence of hrp2/3 deletions causing false-negative RDT results in Vietnam (Gia Lai and Dak Lak provinces). METHODS: Individuals presenting with malaria symptoms at health facilities were screened for P. falciparum infection using light microscopy and HRP2-RDT (SD Bioline Malaria Antigen Pf/Pv RDT, Abbott). Microscopically confirmed P. falciparum infections were analysed for parasite species by 18S rRNA qPCR, and pfhrp2 and pfhrp3 exon2 deletions were investigated by nested PCR. pfhrp2 amplicons were sequenced by the Sanger method and HRP2 plasma levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The prevalence of false-negative RDT results among symptomatic cases was 5.6% (15/270). No pfhrp2 and pfhrp3 deletions were identified. False-negative RDT results were associated with lower parasite density (p = 0.005) and lower HRP2 plasma concentrations (p < 0.001), as compared to positive RDT. CONCLUSIONS: The absence of hrp2/3 deletions detected in this survey suggests that HRP2-based malaria RDTs remain effective for the diagnosis of symptomatic P. falciparum malaria in Central Vietnam.

High Proportion of Genome-Wide Homology and Increased Pretreatment pvcrt Levels in Plasmodium vivax Late Recurrences: a Chloroquine Therapeutic Efficacy Study.

Chloroquine (CQ) is the first-line treatment for Plasmodium vivax malaria in most countries where malaria is endemic. Monitoring P. vivax CQ resistance (CQR) is critical but remains challenged by the difficulty to distinguish real treatment failure from reinfection or liver relapse. The therapeutic efficacy of CQ against uncomplicated P. vivax malaria was evaluated in Gia Lai Province, Vietnam. Sixty-seven patients were enrolled and followed for 42 days using microscopy and quantitative PCR. Adequate clinical and parasitological response (ACPR) was 100% (66/66) on day 28 but 75.4% (49/65) on day 42. Eighteen recurrences (27.7%) were detected, with a median time to recurrence of 42 days (interquartile range [IQR], 35 to 42) and blood CQ concentration of <100 ng/ml. Primary infections leading to recurrence occurred in younger individuals (median age for ACPR = 25 years [IQR, 20 to 28]; recurrences = 18 [16 to 21]; P = 0.002) had a longer parasite clearance time (PCT for ACPR = 47.5 h [IQR, 36.2 to 59.8 h]; recurrences = 54.2 [48.4 to 62.0]; P = 0.035) and higher pvcrt gene expression (median relative expression ratio for ACPR = 0.09 [IQR, 0.05 to 0.22]; recurrences = 0.20 [0.15 to 0.56]; P = 0.002), but showed no differences in ex vivo CQ sensitivity. Parasite genotyping by microsatellites, single nucleotide polymorphism (SNP) barcoding, and whole-genome sequencing (WGS) identified a majority of homologous recurrences, with 80% (8/10) showing >98% identity by descent to paired day 0 samples. This study shows that CQ remained largely efficacious to treat P. vivax in Gia Lai; i.e., recurrences occurred late (>day 28) and in the presence of low blood CQ concentrations. However, the combination of both WGS and gene expression analysis (pvcrt) data with clinical data (PCT) allowed us to identify potential emergence of low-grade CQR, which should be closely monitored. (This study has been registered at ClinicalTrials.gov under identifier NCT02610686.).

Rapid profiling of Plasmodium parasites from genome sequences to assist malaria control.

BACKGROUND: Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the genomic epidemiology of malaria. Genome sequencing is rapidly gaining traction as a diagnostic and surveillance tool for clinical settings, where the profiling of co-infections, identification of imported malaria parasites, and detection of drug resistance are crucial for infection control and disease elimination. To support this informatically, we have developed the Malaria-Profiler tool, which rapidly (within minutes) predicts Plasmodium species, geographical source, and resistance to antimalarial drugs directly from WGS data. RESULTS: The online and command line versions of Malaria-Profiler detect ~ 250 markers from genome sequences covering Plasmodium speciation, likely geographical source, and resistance to chloroquine, sulfadoxine-pyrimethamine (SP), and other anti-malarial drugs for P. falciparum, but also providing mutations for orthologous resistance genes in other species. The predictive performance of the mutation library was assessed using 9321 clinical isolates with WGS and geographical data, with most being single-species infections (P. falciparum 7152/7462, P. vivax 1502/1661, P. knowlesi 143/151, P. malariae 18/18, P. ovale ssp. 5/5), but co-infections were identified (456/9321; 4.8%). The accuracy of the predicted geographical profiles was high to both continental (96.1%) and regional levels (94.6%). For P. falciparum, markers were identified for resistance to chloroquine (49.2%; regional range: 24.5% to 100%), sulfadoxine (83.3%; 35.4- 90.5%), pyrimethamine (85.4%; 80.0-100%) and combined SP (77.4%). Markers associated with the partial resistance of artemisinin were found in WGS from isolates sourced from Southeast Asia (30.6%). CONCLUSIONS: Malaria-Profiler is a user-friendly tool that can rapidly and accurately predict the geographical regional source and anti-malarial drug resistance profiles across large numbers of samples with WGS data. The software is flexible with modifiable bioinformatic pipelines. For example, it is possible to select the sequencing platform, display specific variants, and customise the format of outputs. With the increasing application of next-generation sequencing platforms on Plasmodium DNA, Malaria-Profiler has the potential to be integrated into point-of-care and surveillance settings, thereby assisting malaria control. Malaria-Profiler is available online (bioinformatics.lshtm.ac.uk/malaria-profiler) and as standalone software ( https://github.com/jodyphelan/malaria-profiler ).

Reduction in Plasmodium falciparum Pfk13 and pfg377 allele diversity through time in southern Vietnam.

BACKGROUND: Plasmodium falciparum has acquired resistance to artemisinin in Southeast Asia, with mutations in the P. falciparum Kelch-13 (Pfk13) gene associated with the resistance phenotype. The widespread use of Artemisinin-based combination therapy (ACT)s in Southeast Asia has led to the selection and spread of parasites carrying mutations in Pfk13. We characterised the allele diversity of Pfk13 and pfg377, an artemisinin-resistance neutral polymorphic gene, in parasite DNA extracted human blood from in southern Vietnam in 2003, 2012, 2015 and 2018. METHOD: This study was conducted in Bu Gia Map commune, Binh Phuoc province, Vietnam, from May 2018 to January 2019. Twenty-four samples from 2018 to 2019, 30 from 2003, 24 from 2012 and 32 from 2015 were analysed. Malaria-infected human blood was collected by finger-prick and used for molecular analysis. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification, followed by amplification and nucleotide sequencing of Pfk13 and region 3 of pfg377. Archived blood samples collected in the same region in 2012 and 2015 were also analysed as above for comparison. RESULTS: The genetic diversity of Pfk13 and pfg377 was lower in 2018-2019 compared to 2012 and 2015. The number of distinct Pfk13 mutants decreased from three in 2012 and 2015, P553L, V568G and C580Y, to one, C580Y in 2018-2019. In 2018-2019, the frequency of C580Y mutant strains was 71% (17/24 isolates). All samples were wild type in 2003. In 2012 and 2015, there were single-strain infections as well as co-infections with two mutant strains or with mutant and wild strains, whereas there were no co-infections in 2018. pfg377 allele diversity decreased from five alleles in 2012 to two alleles in 2018-2019. CONCLUSION: The genetic diversity of P. falciparum was reduced at the two genetic loci surveyed in this study, Pfk13 and pfg377. In the case of the former gene, we observed an increase in the prevalence of parasites carrying the C580Y gene, known to confer reduced susceptibility to ACTs. The reduction in the diversity of pfg377 may be linked to the clonal expansion of parasite strains carrying the C580Y mutation, leading to an overall reduction in parasite genetic diversity across the population.

Transcriptome Sequencing and Analysis of Changes Associated with Insecticide Resistance in the Dengue Mosquito (Aedes aegypti) in Vietnam.

The mosquito Aedes aegypti is a transmission vector for dangerous epidemic diseases in humans. Insecticides have been used as the most general vector control method in the world. However, Ae. aegypti have developed many resistant mechanisms such as reduced neuronal sensitivity to insecticides (target-site resistance), enhanced insecticide metabolism (metabolic resistance), altered transport, sequestration, and other mechanisms. It has become a major problem for vector control programs. Transcriptome sequencing and bioinformatic analysis were used to compare transcription levels between a susceptible strain (Bora7) and a resistant strain (KhanhHoa7) collected from the field. A total of 161 million Illumina reads, including 66,076,678 reads from the Bora7 strain and 69,606,654 reads from the KhanhHoa7 strain, were generated and assembled into 11,174 genes. A comparison of the KhanhHoa7 transcriptome to that of Bora7 showed 672 upregulated genes and 488 downregulated genes. We identified the highly upregulated genes: cytochrome P450 4C1, 4C3, 4C21, 4D1, 4D1 isoform X2, 4D2, 4D2 isoform X2, 4G15, 6A2, 6A8, 6D3, and 9E2; Glutathione S transferase (GST1), UGT1-3, 1-7, 2B15, and 2B37; binding cassette transporter (ABC) transporter F family member 4 and ABC transporter G family member 20. Interestingly, there was a significant increase in the expression of the genes such as CYP9E2 (8.3-fold), CYP6A8 (5.9-fold), CYP6D3 (5.4-fold), CYP4C21 (5.4-fold), CYP4G15 (5.2-fold), GST1 (3.5-fold), and ABC transporter 4 (2.1-fold). Our results suggested a potential relationship between the expression of the genes in metabolic processes and insecticide resistance in the studied strain. These results may contribute to the understanding of the mechanisms of insecticide resistance in Ae. aegypti.

Population Genetics of Plasmodium vivax in Four Rural Communities in Central Vietnam.

BACKGROUND: The burden of malaria in Vietnam has drastically reduced, prompting the National Malaria Control Program to officially engage in elimination efforts. Plasmodium vivax is becoming increasingly prevalent, remaining a major problem in the country's central and southern provinces. A better understanding of P. vivax genetic diversity and structure of local parasite populations will provide baseline data for the evaluation and improvement of current efforts for control and elimination. The aim of this study was to examine the population genetics and structure of P. vivax isolates from four communities in Tra Leng commune, Nam Tra My district in Quang Nam, Central Vietnam. METHODOLOGY/PRINCIPAL FINDINGS: P. vivax mono infections collected from 234 individuals between April 2009 and December 2010 were successfully analyzed using a panel of 14 microsatellite markers. Isolates displayed moderate genetic diversity (He = 0.68), with no significant differences between study communities. Polyclonal infections were frequent (71.4%) with a mean multiplicity of infection of 1.91 isolates/person. Low but significant genetic differentiation (FST value from -0.05 to 0.18) was observed between the community across the river and the other communities. Strong linkage disequilibrium ([Formula: see text] = 0.113, p < 0.001) was detected across all communities, suggesting gene flow within and among them. Using multiple approaches, 101 haplotypes were grouped into two genetic clusters, while 60.4% of haplotypes were admixed. CONCLUSIONS/SIGNIFICANCE: In this area of Central Vietnam, where malaria transmission has decreased significantly over the past decade, there was moderate genetic diversity and high occurrence of polyclonal infections. Local human populations have frequent social and economic interactions that facilitate gene flow and inbreeding among parasite populations, while decreasing population structure. Findings provide important information on parasites populations circulating in the study area and are relevant to current malaria elimination efforts.