Progress towards malaria elimination in the Greater Mekong Subregion: perspectives from the World Health Organization.

Malaria remains a global health challenge, disproportionately affecting vulnerable communities. Despite substantial progress, the emergence of anti-malarial drug resistance poses a constant threat. The Greater Mekong Subregion (GMS), which includes Cambodia, China's Yunnan province, Lao People's Democratic Republic, Myanmar, Thailand, and Viet Nam has been the epicentre for the emergence of resistance to successive generations of anti-malarial therapies. From the perspective of the World Health Organization (WHO), this article considers the collaborative efforts in the GMS, to contain Plasmodium falciparum artemisinin partial resistance and multi-drug resistance and to advance malaria elimination. The emergence of artemisinin partial resistance in the GMS necessitated urgent action and regional collaboration resulting in the Strategy for Malaria Elimination in the Greater Mekong Subregion (2015-2030), advocating for accelerated malaria elimination interventions tailored to country needs, co-ordinated and supported by the WHO Mekong malaria elimination programme. The strategy has delivered substantial reductions in malaria across all GMS countries, with a 77% reduction in malaria cases and a 97% reduction in malaria deaths across the GMS between 2012 and 2022. Notably, China was certified malaria-free by WHO in 2021. Countries' ownership and accountability have been pivotal, with each GMS country outlining its priorities in strategic and annual work plans. The development of strong networks for anti-malarial drug resistance surveillance and epidemiological surveillance was essential. Harmonization of policies and guidelines enhanced collaboration, ensuring that activities were driven by evidence. Challenges persist, particularly in Myanmar, where security concerns have limited recent progress, though an intensification and acceleration plan aims to regain momentum. Barriers to implementation can slow progress and continuing innovation is needed. Accessing mobile and migrant populations is key to addressing remaining transmission foci, requiring effective cross-border collaboration. In conclusion, the GMS has made significant progress towards malaria elimination, particularly in the east where several countries are close to P. falciparum elimination. New and persisting challenges require sustained efforts and continued close collaboration. The GMS countries have repeatedly risen to every obstacle presented, and now is the time to re-double efforts and achieve the 2030 goal of malaria elimination for the region.

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 ).

Anopheles diversity, biting behaviour and transmission potential in forest and farm environments of Gia Lai province, Vietnam.

BACKGROUND: Despite recent reductions in Vietnam, malaria transmission persists in some areas in forests and farmlands where a high density of Anopheles mosquitoes relative to other environments occurs. To inform effective malaria control measures, it is important to understand vector bionomics and the malaria transmission role of Anopheles spp. in the highland regions of Vietnam. This study was conducted to quantify the abundance, composition and biting behaviour of the Anopheles mosquito population, and the proportion of Plasmodium spp. infected mosquitoes collected from forest and agricultural farm sites in Gia Lai province, Vietnam. METHODS: Forest and agricultural farm sites in Gia Lai province were selected for mosquito collections (total eight sites). Mosquito collection was performed by Human-baited Double Net Trap (HDNT), animal-baited traps (ABT) using cattle, and CDC light traps. Captured mosquitoes were identified morphologically, and salivary glands of Anopheles mosquitoes were examined for sporozoites using microscopy. Plasmodium infection was determined by Polymerase Chain Reaction (PCR), and identification of blood meal type was determined by PCR and diffuse serum agglutination assay. RESULTS: A total of 1815 Anopheles mosquitoes belonging to 19 species were collected by ABT (n = 1169), HDNT (n = 471) and CDC light trap (n = 175). Anopheles abundance and diversity varied by district and environment. Capture by HDNT of Anopheles of vectorial concern was observed between early evening and early morning. Plasmodium vivax infection was determined by PCR in two Anopheles dirus specimens captured by HDNT in forest sites. Blood from a range of hosts could, including human blood, could be detected in species considered primary and secondary vectors An. dirus, and Anopheles aconitus, and Anopheles maculatus, respectively. CONCLUSIONS: A low number of Anopheles spp. considered primary vectors of concern and very low numbers of Plasmodium spp. infected Anopheles mosquitoes were captured at the end of the rainy season in the Central Highlands of Vietnam. However, capture species of vectorial concern by HDNT throughout the early to late evening demonstrates that use of additional personal protective measures could supplement current preventative measures, such as bed nets to prevent exposure to vectors of concern in this region.

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.

Treatment Failure of Dihydroartemisinin/Piperaquine for Plasmodium falciparum Malaria, Vietnam.

We conducted a study in Binh Phuoc, Vietnam, in 2015 on the therapeutic efficacy of dihydroartemisinin/piperaquine for Plasmodium falciparum malaria. A high number of treatment failures (14/40) was found, and piperaquine resistance in Vietnam was confirmed. A change in the malaria treatment policy for Vietnam is in process.

Risk factors for malaria infection among ethnic minorities in Binh Phuoc, Vietnam.

A cross-sectional study was undertaken to identify the prevalence of malaria infection, behavioral patterns of inhabitants and risk factors for malaria infection in a minority village in an area endemic for malaria in Vietnam. The target population were all the inhabitants of a village in Binh Phuoc Province, Vietnam. Interviews using a structured questionnaire and blood examination for malaria infection were conducted in house-to-house visits. Relationships between malaria infection and variables were examined using uni- and multivariate adjusted analysis. A total of 682 individuals from 159 households participated in both the interview and blood examinations. All households earned income through farming without forest activities at night, and the socio-economic status was generally not very low. The total prevalence of malaria infection was 6.2%, with a peak among 3- to 5-year-old children. Univariate analysis identified 3 - 5 year olds, a family size of > or =5 people, sleeping with >3 people in a bed, and living in a wooden/bamboo house as factors associated with malaria infection. Multivariate adjusted analysis after variable selection identified age 3 - 5 years old, a family size of > or =5 people and living in a wooden/bamboo house were significantly related to malaria infection. Malaria in this area can be controlled by basic activities, such as early diagnosis and treatment and prevention using bednets, since risk factors for malaria infection did not include forest activities, but were young age, living in a wooden/bamboo house and belonging to a large family. Continuous and intensive expansion of existing malaria control activities are required.

Efficacy of bentonite clay in ameliorating aflatoxicosis in piglets fed aflatoxin contaminated diets.

The efficacy of locally produced bentonite was evaluated with respect to ameliorating the adverse effects of aflatoxins (AF) in piglets fed AF contaminated diets. Forty eight piglets were randomly assigned to one of four treatments: 1) 0 g of bentonite and 0 microg AF/kg feed (control); 2) 4 g of bentonite plus 200 microg AF/kg feed (AF + Bento 4); 3) 5 g of bentonite plus 200 microg AF/kg feed (AF + Bento 5) and 4) 0 g of bentonite plus 200 microg AF/kg feed (AFA). Piglets in the AFA treatment had lower overall average daily weight gain (ADG), feed conversion efficiency, albumin (ALB) and total protein (TP) compared to the control diet, while mean serum leukocyte and enzyme activities (glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gamma glutamyltransferase (GGT), alkaline phosphate (ALP) and lactic dehydrogenase (LDH)) were significant increased. The inclusion of bentonite at 0.4% or 0.5% in the AF contaminated diet restored the lower performance, feed efficiency and abnormal blood profiles of the piglets given AF and no differences between 0.4 and 0.5% inclusion of bentonite. The findings in the present study provide critically needed confirmation that bentonite has the ability to reduce the adverse effects of AF.

Screening of Aflatoxins and Zearalenone in feedstuffs and complete feeds for pigs in Southern Vietnam.

A survey ofaflatoxins and zearalenone (ZEA) in 120 samples, including cassava chip (12), maize (12), rice bran (24), broken rice (24), and complete feeds for growing pigs (24) and sows (24) was carried out in southern Vietnam. Aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) were determined by an HPLC method using an immunoaffinity column for clean-up. Zearalenone was analysed by Enzyme-Linked Immunosorbent Assay (ELISA) AgraQuant. High incidences of aflatoxins and ZEA in feedstuffs and pig feeds (83.3%-100%) were detected. Aflatoxins and ZEA were detected in all commodities analyzed. Aflatoxin B1 was found in 100, 92, 92, 83, 100 and 96%, respectively, of samples of cassava chip, maize, rice bran, broken rice, and complete feeds for growing pigs and sows, with mean concentrations of 0.86, 77.5, 1.3, 1.6, 4.7 and 7.5 microg/kg, respectively. AFB2 and AFG1 were found in low concentrations and co-occurred with AFB1 in all samples where they were found. ZEA was detected in 8%, 33%, 36%, 21%, 67% and 67%, respectively, of the samples of cassava chip, maize, rice bran, broken rice, and complete feeds for growing pigs and sows, with average concentrations of 10.0, 163.5, 83.1, 31.4, 86.0 and 101.4 microg/kg, respectively. The high incidence of mycotoxins found in feedstuffs highlights the need for periodic monitoring and prevention ofmycotoxins, not only in feeds, but also in food for human consumption.