Therapeutic efficacy of pyronaridine-artesunate (Pyramax) in treating Plasmodium vivax malaria in the central highlands of Vietnam.

The emergence and spread of chloroquine-resistant Plasmodium vivax have necessitated the assessment of alternative blood schizonticidal drugs. In Vietnam, chloroquine-resistant P. vivax malaria has been reported. In an open-label, single-arm trial, the safety, tolerability, and efficacy of pyronaridine-artesunate (Pyramax, PA) was evaluated in Dak Nong province, Vietnam. A 3-day course of PA was administered to adults and children (≥20 kg) infected with P. vivax. Patients also received primaquine (0.25 mg/kg daily for 14 days). PA was well tolerated with transient asymptomatic increases in liver transaminases. The per-protocol proportion of patients with day 42 PCR-unadjusted adequate clinical and parasitological response was 96.0% (95% CI, 84.9%-99.0%, n = 48/50). The median parasite clearance time was 12 h (range, 12-36 h), with a median fever clearance time of 24 h (range, 12-60 h). Single nucleotide polymorphisms (SNPs) as potential genetic markers of reduced drug susceptibility were analyzed in three putative drug resistance markers, Pvcrt-o, Pvmdr1, and PvK12. Insertion at position K10 of the Pvcrt-o gene was found in 74.6% (44/59) of isolates. Pvmdr1 SNPs at Y976F and F1076L were present in 61% (36/59) and 78% (46/59), respectively. Amplification of Pvmdr1 gene (two copies) was found in 5.1% (3/59) of parasite samples. Only 5.1% (3/59) of isolates had mutation 552I of the PvK12 gene. Overall, PA rapidly cleared P. vivax blood asexual stages and was highly efficacious in treating vivax malaria, with no evidence of artemisinin resistance found. PA provides an alternative to chloroquine treatment for vivax malaria in Vietnam. CLINICAL TRIALS: This study is registered with the Australian New Zealand Clinical Trials Registry as ACTRN12618001429246.

Multidrug-Resistant Plasmodium falciparum Parasites in the Central Highlands of Vietnam Jeopardize Malaria Control and Elimination Strategies.

Plasmodium falciparum resistance to dihydroartemisinin-piperaquine has spread through the Greater Mekong Subregion to southwestern Vietnam. In 2018 to 2019, we collected 127 P. falciparum isolates from Dak Nong (36), Dak Lak (55), Gia Lai (13), and Kon Tum (23) provinces in Vietnam's Central Highlands and found parasites bearing the Pfkelch13 C580Y mutation and multiple plasmepsin 2/3 genes (mean prevalence, 17.9%; range, 4.3% to 27.8%), conferring resistance to dihydroartemisinin-piperaquine. This information is important for drug policy decisions in Vietnam.

Pyronaridine-Artesunate (Pyramax) for Treatment of Artemisinin- and Piperaquine-Resistant Plasmodium falciparum in the Central Highlands of Vietnam.

The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine in Vietnam justifies the need to evaluate alternative artemisinin-based combination therapies. Between July 2018 and October 2019, a single-arm trial of pyronaridine-artesunate (Pyramax, PA) was conducted in Dak Nong province, Vietnam. PA (3-day course) was administered to adults and children infected with P. falciparum. PA was well tolerated by the participants. The proportion of patients with Day 42 PCR-corrected adequate clinical and parasitological response was 95.2% (95% confidence interval [CI], 82.3 to 98.8, n = 40/42) for treating falciparum malaria. The median parasite clearance half-life was 6.7 h (range, 2.6 to 11.9) and the median parasite clearance time was 72 h (range, 12 to 132) with 44.9% (22/49) of patients having positive blood films at 72 h. The two patients that recrudesced had comparable Day 7 blood pyronaridine concentrations (39.5 and 39.0 ng/ml) to the 40 patients who did not recrudesce (median 43.4 ng/ml, 95% CI, 35.1 to 54.9). Ring-stage and piperaquine survival assays revealed that of the 29 P. falciparum isolates collected from the patients before PA treatment, 22 (75.9%) had reduced susceptibility to artemisinins and 17 (58.6%) were resistant to piperaquine. Genotyping confirmed that 92.0% (46/50) of falciparum patients were infected with parasites bearing the Pfkelch13 C580Y mutation associated with artemisinin resistance. Of these, 56.0% (28/50) of the isolates also had multiple copies of the plasmepsin 2/3 genes responsible for piperaquine resistance. Overall, PA was effective in treating P. falciparum in the Central Highlands of Vietnam. (This study has been registered at AustralianClinicalTrials.gov.au under trial ID ACTRN12618001429246.).

The kinetic profile of clinical and laboratory findings and treatment outcome of patients with toxocariasis.

OBJECTIVES: Human toxocariasis is a widespread zoonosis for which a chemotherapy decision and therapy effectiveness are difficult to determine. We aimed to investigate the kinetic profile of clinical and laboratory findings and treatment outcome of patients with toxocariasis in Vietnam. METHODS: The prospective study was conducted between October 2017 and June 2019. The diagnosis of toxocariasis was established based on clinical, laboratory (eosinophilia, raised IgE concentration) and serological (positive Toxocara IgG ELISA) evaluation as well as the exclusion of another helminthic co-infection. The patients were followed up after seven days, then one, three and six months after chemotherapy by thiabendazole. RESULTS: The study involved 80 patients with a mean age of 41.6 ± 15.2 years of whom 58.8% were female. At three and six months after chemotherapy, most patients demonstrated resolution of clinical signs and symptoms, eosinophil count and IgE concentration but not in the proportion of IgG seropositivity. Skin lesions and eosinophilia resolved earlier than the other symptoms (one month after treatment). About four-fifths of the patients were "cured" after three and six months of follow-up; 33.8% showed side effects to thiabendazole therapy but no severe events were reported. The most common adverse reaction was neurologic symptoms followed by gastrointestinal or skin manifestations which lasted as long as 4 days. CONCLUSIONS: In toxocariasis patients, cutaneous manifestations and eosinophilia resolve more rapidly than other clinical and laboratory findings while IgG titre has a very slow kinetic after therapy. Thiabendazole seems to be a potential alternative for the treatment of human toxocariasis.

Implementing parasite genotyping into national surveillance frameworks: feedback from control programmes and researchers in the Asia-Pacific region.

The Asia-Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia-Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process.

The impact of targeted malaria elimination with mass drug administrations on falciparum malaria in Southeast Asia: A cluster randomised trial.

BACKGROUND: The emergence and spread of multidrug-resistant Plasmodium falciparum in the Greater Mekong Subregion (GMS) threatens global malaria elimination efforts. Mass drug administration (MDA), the presumptive antimalarial treatment of an entire population to clear the subclinical parasite reservoir, is a strategy to accelerate malaria elimination. We report a cluster randomised trial to assess the effectiveness of dihydroartemisinin-piperaquine (DP) MDA in reducing falciparum malaria incidence and prevalence in 16 remote village populations in Myanmar, Vietnam, Cambodia, and the Lao People's Democratic Republic, where artemisinin resistance is prevalent. METHODS AND FINDINGS: After establishing vector control and community-based case management and following intensive community engagement, we used restricted randomisation within village pairs to select 8 villages to receive early DP MDA and 8 villages as controls for 12 months, after which the control villages received deferred DP MDA. The MDA comprised 3 monthly rounds of 3 daily doses of DP and, except in Cambodia, a single low dose of primaquine. We conducted exhaustive cross-sectional surveys of the entire population of each village at quarterly intervals using ultrasensitive quantitative PCR to detect Plasmodium infections. The study was conducted between May 2013 and July 2017. The investigators randomised 16 villages that had a total of 8,445 residents at the start of the study. Of these 8,445 residents, 4,135 (49%) residents living in 8 villages, plus an additional 288 newcomers to the villages, were randomised to receive early MDA; 3,790 out of the 4,423 (86%) participated in at least 1 MDA round, and 2,520 out of the 4,423 (57%) participated in all 3 rounds. The primary outcome, P. falciparum prevalence by month 3 (M3), fell by 92% (from 5.1% [171/3,340] to 0.4% [12/2,828]) in early MDA villages and by 29% (from 7.2% [246/3,405] to 5.1% [155/3,057]) in control villages. Over the following 9 months, the P. falciparum prevalence increased to 3.3% (96/2,881) in early MDA villages and to 6.1% (128/2,101) in control villages (adjusted incidence rate ratio 0.41 [95% CI 0.20 to 0.84]; p = 0.015). Individual protection was proportional to the number of completed MDA rounds. Of 221 participants with subclinical P. falciparum infections who participated in MDA and could be followed up, 207 (94%) cleared their infections, including 9 of 10 with artemisinin- and piperaquine-resistant infections. The DP MDAs were well tolerated; 6 severe adverse events were detected during the follow-up period, but none was attributable to the intervention. CONCLUSIONS: Added to community-based basic malaria control measures, 3 monthly rounds of DP MDA reduced the incidence and prevalence of falciparum malaria over a 1-year period in areas affected by artemisinin resistance. P. falciparum infections returned during the follow-up period as the remaining infections spread and malaria was reintroduced from surrounding areas. Limitations of this study include a relatively small sample of villages, heterogeneity between villages, and mobility of villagers that may have limited the impact of the intervention. These results suggest that, if used as part of a comprehensive, well-organised, and well-resourced elimination programme, DP MDA can be a useful additional tool to accelerate malaria elimination. TRIAL REGISTRATION: ClinicalTrials.gov NCT01872702.

Susceptibility of Plasmodium falciparum to artemisinins and Plasmodium vivax to chloroquine in Phuoc Chien Commune, Ninh Thuan Province, south-central Vietnam.

BACKGROUND: Reduced artemisinin susceptibility and artemisinin-based combination therapy (ACT)-resistance against Plasmodium falciparum and chloroquine (CQ)-resistant P. vivax malaria has been reported in Vietnam. Two therapeutic efficacy studies were conducted in Thuan Bac District (Ninh Thuan Province, Vietnam) in 2015 and 2016 to determine the extent of reduced artemisinin susceptibility and ACT resistant falciparum malaria, and CQ-resistant vivax malaria were present. METHODS: Twenty-seven patients with falciparum malaria were randomized to receive artesunate alone (AS ~ 4 mg/kg/day) for 4 days followed by dihydroartemisinin (DHA) (2.2 mg/kg)-piperaquine (PPQ) (18 mg/kg) daily for 3 days or artemether (AM) (1.7 mg/kg)-lumefantrine (LUM) (12 mg/kg) twice daily for 3 days. Sixteen subjects with vivax malaria received CQ (total 25 mg/kg over 3 days). The therapeutic efficacy study for treating falciparum malaria was complemented with molecular analysis for artemisinin and piperaquine resistance, and in vitro drug susceptibility testing. Patient's drug exposure following both falciparum and vivax treatment studies was determined. RESULTS: Twenty-five of 27 patients treated with the artemisinin regimens completed the 42-day follow-up period. None had parasites present on day 3 after commencing treatment with no incidence of recrudescence (100% curative rate). One patient on AS + DHA-PPQ was lost to follow-up and one patient had Plasmodium falciparum and Plasmodium vivax infection on day 0 by PCR. Of the vivax patients, 15 of 16 completed CQ treatment and two had a recurrence of vivax malaria on day 28, a failure rate of 13.3% (2/15). No mutations in the Pfkelch-13 gene for artemisinin resistance or exo-E415G gene polymorphism and amplification in plasmepsins 2 and 3 for piperaquine resistance were observed. In vitro testing of patient's falciparum parasites indicated susceptibility (low IC50 nM values) to dihydroartemisinin, lumefantrine, piperaquine and pyronaridine. Patient's drug exposure to artesunate and lumefantrine was comparable to published data, however, blood CQ concentrations were lower. CONCLUSIONS: Clinical findings, molecular analysis and in vitro testing revealed that the falciparum parasites at Phuoc Chien Commune were artemisinin susceptible. The clinical failure rate of the 15 vivax patients who completed CQ treatment was 13%. Further studies are required to determine whether CQ-resistant vivax malaria is present at the commune.

Molecular characterization and mapping of glucose-6-phosphate dehydrogenase (G6PD) mutations in the Greater Mekong Subregion.

BACKGROUND: Plasmodium vivax malaria elimination can only be achieved by the deployment of 8-aminoquinolines (primaquine and tafenoquine) in combination with ACT to kill both blood and liver-stage parasites. However, primaquine and the other 8-aminoquinolines cause dose-dependent haemolysis in subjects with G6PD deficiency, an X-linked disorder of red blood cells that is very common in populations living in tropical and subtropical areas. In order to inform safer use of 8-aminoquinolines in the Greater Mekong Subregion, a multi-centre study was carried out to assess the prevalence of G6PD deficiency and to identify the main G6PD variants in samples collected in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. METHODS: Blood samples were collected in the five countries during National Malaria Surveys or during Population Surveys. During Population Surveys samples were characterized for G6PD phenotype using the Fluorescent Spot Test. Samples were then genotyped for a panel of G6PD mutations. RESULTS: G6PD deficiency was found to be common in the region with an overall mean prevalence of deficient or mutated hemizygous males of 14.0%, ranging from a mean 7.3% in Thailand, 8.1% in Lao PDR, 8.9% in Vietnam, 15.8% in Myanmar and 18.8% in Cambodia. Mahidol and Viangchan mutations were the most common and widespread variants found among the nine investigated. CONCLUSIONS: Owing to the high prevalence of G6PD deficiency in the Greater Mekong Subregion, strategies for vivax malaria elimination should include point-of-care G6PD testing (both qualitative and quantitative) to allow safe and wide treatment with 8-aminoquinolines.

Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam.

The aim of this study is to delineate 'admixed hybrid' and 'introgressive' Fasciola genotypes present in the Fasciola population in Vietnam. Adult liver flukes collected from ruminants in 18 Provinces were morphologically sorted out by naked eyes for small (S), medium (M) and large (L) body shapes; and human samples (n=14) from patients. Nuclear ribosomal (rDNA) ITS1 and ITS2, and mitochondrial (mtDNA) nad1 markers were used for determination of their genetic status. Total 4,725 worm samples of ruminants were tentatively classified by their size: 6% (n=284) small (S)-, 13% (n=614) medium (M)-, and 81% (n=3,827) large (L)-forms. All the representative (n=120, as 40 each group) and 14 human specimens, possessed maternal mtDNA of only F. gigantica and none of F. hepatica. Paternally, all (100%) of the L-(n=40) and 77.5% (n=31) of the M-flukes had single F. gigantica rDNA indicating 'pure' F. gigantica. A majority (90%, n=36) of the S- and 15% (n=6) of the M-worms had single F. hepatica rDNA, indicating their introgressive; the rest (10%, n=4) of the S- and 7.5% (n=3) of the M-flukes had mixture of both F. gigantica and F. hepatica rDNAs, confirming their admixed hybrid genetic status. Fourteen human samples revealed 9 (64%) of pure F. gigantica, 3 (22%) of introgressive and 2 (14%) of admixed hybrid Fasciola spp. By the present study, it was confirmed that the small worms, which are morphologically identical with F. hepatica, are admixed and/or introgressive hybrids of Fasciola spp., and able to be the pathogens of human fascioliasis.

K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016.

The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites.

Rapid decline in the susceptibility of Plasmodium falciparum to dihydroartemisinin-piperaquine in the south of Vietnam.

BACKGROUND: Artemisinin resistant Plasmodium falciparum has emerged in the countries of the Greater Mekong sub-region posing a serious threat to global malaria elimination efforts. The relationship of artemisinin resistance to treatment failure has been unclear. METHODS: In annual studies conducted in three malaria endemic provinces in the south of Vietnam (Binh Phuoc, Ninh Thuan and Gia Lai) between 2011 and 2015, 489 patients with uncomplicated P. falciparum malaria were enrolled in detailed clinical, parasitological and molecular therapeutic response assessments with 42 days follow up. Patients received the national recommended first-line treatment dihydroartemisinin-piperaquine for three days. RESULTS: Over the 5 years the proportion of patients with detectable parasitaemia on day 3 rose steadily from 38 to 57% (P < 0.001). In Binh Phuoc province, the parasite clearance half-life increased from 3.75 h in 2011 to 6.60 h in 2015 (P < 0.001), while treatment failures rose from 0% in 2012 and 2013, to 7% in 2014 and 26% in 2015 (P < 0.001). Recrudescence was associated with in vitro evidence of artemisinin and piperaquine resistance. In the treatment failures cases of 2015, all 14 parasite isolates carried the C580Y Pfkelch 13 gene, marker of artemisinin resistance and 93% (13/14) of them carried exoE415G mutations, markers of piperaquine resistance. CONCLUSIONS: In the south of Vietnam recent emergence of piperaquine resistant P. falciparum strains has accelerated the reduced response to artemisinin and has led to treatment failure rates of up to 26% to dihydroartemisinin-piperaquine, Vietnam's current first-line ACT. Alternative treatments are urgently needed.

Community perceptions of targeted anti-malarial mass drug administrations in two provinces in Vietnam: a quantitative survey.

BACKGROUND: As part of a targeted malaria elimination project, mass drug administrations (MDAs) were conducted in Vietnam. The impact of MDAs on malaria transmission depends largely on the efficacy of the anti-malarial drug regimen, the malaria epidemiology in the site and the population coverage. To explore why some people participate in MDAs and others do not, a quantitative survey of the villagers' perceptions was undertaken in Vietnam. METHODS: In 2013/2014 MDAs were conducted in a village in Binh Phuoc province and a village in Ninh Thuan province. Within three months of the drug administration, 59 respondents in a village in Binh Phuoc and 79 respondents in a village in Ninh Thuan were randomly selected and interviewed. RESULTS: Comprehension of the purpose of the intervention was of paramount importance for participation in the intervention. Respondents aware that the intervention aims to protect against malaria were significantly more likely to participate than respondents who were unaware of the MDA's purpose. Secondly, how and by whom villagers were informed was critical for participation. There was a strong association between sensitization by an informant such as a member of the local health team with participation in the intervention. CONCLUSIONS: The study suggests several approaches to increase participation in mass drug administration campaigns. Training trustworthy informants to sensitize the study population is critical to maximize village participation in this setting. To achieve high coverage the entire community must understand and agree with the intervention.

Measuring effects of ivermectin-treated cattle on potential malaria vectors in Vietnam: A cluster-randomized trial.

BACKGROUND: Malaria elimination using current tools has stalled in many areas. Ivermectin (IVM) is a broad-antiparasitic drug and mosquitocide and has been proposed as a tool for accelerating progress towards malaria elimination. Under laboratory conditions, IVM has been shown to reduce the survival of adult Anopheles populations that have fed on IVM-treated mammals. Treating cattle with IVM has been proposed as an important contribution to malaria vector management, however, the impacts of IVM in this One Health use case have been untested in field trials in Southeast Asia. METHODS: Through a randomized village-based trial, this study quantified the effect of IVM-treated cattle on anopheline populations in treated vs. untreated villages in Central Vietnam. Local zebu cattle in six rural villages were included in this study. In three villages, cattle were treated with IVM at established veterinary dosages, and in three additional villages cattle were left as untreated controls. For the main study outcome, the mosquito populations in all villages were sampled using cattle-baited traps for six nights before, and six nights after a 2-day IVM-administration (intervention) period. Anopheline species were characterized using taxonomic keys. The impact of the intervention was analyzed using a difference-in-differences (DID) approach with generalized estimating equations (with negative binomial distribution and robust errors). This intervention was powered to detect a 50% reduction in total nightly Anopheles spp. vector catches from cattle-baited traps. Given the unusual diversity in anopheline populations, exploratory analyses examined taxon-level differences in the ecological population diversity. RESULTS: Across the treated villages, 1,112 of 1,523 censused cows (73% overall; range 67% to 83%) were treated with IVM. In both control and treated villages, there was a 30% to 40% decrease in total anophelines captured in the post-intervention period as compared to the pre-intervention period. In the control villages, there were 1,873 captured pre-intervention and 1,079 captured during the post-intervention period. In the treated villages, there were 1,594 captured pre-intervention, and 1,101 captured during the post-intervention period. The difference in differences model analysis comparing total captures between arms was not statistically significant (p = 0.61). Secondary outcomes of vector population diversity found that in three villages (one control and two treatment) Brillouin's index increased, and in three villages (two control and one treatment) Brillouin's index decreased. When examining biodiversity by trapping-night, there were no clear trends in treated or untreated vector populations. Additionally, there were no clear trends when examining the components of biodiversity: richness and evenness. CONCLUSIONS: The ability of this study to quantify the impacts of IVM treatment was limited due to unexpectedly large spatiotemporal variability in trapping rates; an area-wide decrease in trapping counts across all six villages post-intervention; and potential spillover effects. However, this study provides important data to directly inform future studies in the GMS and beyond for IVM-based vector control.

A study implementing real-time PCR to identify Strongyloides species of third-stage larvae in human stool samples from Southern Vietnam.

Background: Strongyloidiasis, a neglected disease caused by intestinal nematodes of the genus, is endemic to tropical and subtropical areas such as Vietnam. Morphological methods only identify the genus, while DNA-molecular techniques are susceptible in Strongyloides spp. detection. The study aims to determine the prevalence of dominant Strongyloides species among the population in Duc Hoa district, Long An, Vietnam. Methods: A cross-sectional study used 1190 stool specimens collected from July 2017 to November 2018. All samples were transported within 2 h, stored at 2-8°C, and processed within 48 h for microscopy smear and culture at the Laboratory of Medical Parasitology, Pham Ngoc Thach University of Medicine (PNT). Then all positive samples with the above 2 methods were verified by real-time PCR technique. Real-time PCR amplification was conducted at the Laboratory of Molecular Biology, PNT. Results: Direct microscopy and modified Harada-Mori culture detected Strongyloides spp. larvae in 79/1190 samples (6.6%). About 94.2% of the DNA samples were Strongyloides stercoralis, 2.9% were co-infections with Strongyloides ratti and S. stercoralis, and 2.9% were patients with S. ratti. The identity of 12/14 sequences was confirmed as S. stercoralis with a high level of similarity (91.3%-100%) and over 98% for S. ratti. Conclusion: DNA-molecular techniques and sequence analysis are highly suitable for identifying Strongyloides species isolated from stool samples. It is remarkable evidence of the presence of zoonosis S. ratti disease in human, not just the known S. stercoralis. It is likely to result in a certain proportion of people being infected by this animal-borne infectious pathogen.

Genetic polymorphism and natural selection of the erythrocyte binding antigen 175 region II in Plasmodium falciparum populations from Myanmar and Vietnam.

Plasmodium falciparum erythrocyte binding antigen 175 (PfEBA-175) plays essential role in erythrocyte invasion by the parasite and is a leading vaccine candidate. However, its genetic diversity in global isolates is a concern in developing an universal vaccine incorporating this protein. This study aimed to investigate genetic polymorphisms and natural selection of pfeba-175 region II (RII) in Myanmar and Vietnam P. falciparum isolates. Vietnam pfeba-175 RII displayed a low genetic polymorphism, while Myanmar pfeba-175 RII showed high levels of genetic diversity across the region. Point mutations, deletion, and recombinations were main factors contributing to genetic diversities in P. falciparum populations. Global pfeba-175 RII revealed similar, but not identical, genetic polymorphisms and natural selection profiles. Despite profiles of amino acid substitutions differed among populations, five major amino acid changes (K279E, E403K, K481I, Q584K, and R664) were commonly detected in global pfeba-175 RII populations. Haplotype network and genetic differentiation analyses of global pfeba-175 RII populations demonstrated no geographical relationships. Non-neglectable level of genetic diversity was observed in global pfeba-175 RII populations, emphasizing the need to consider this when designing an effective vaccine based on this protein. This study underscores the importance of the continuous monitoring of genetic diversity of pfeba-175 RII in the global P. falciparum populations.

The efficacy and tolerability of artemisinin-piperaquine (Artequick®) versus artesunate-amodiaquine (Coarsucam™) for the treatment of uncomplicated Plasmodium falciparum malaria in south-central Vietnam.

BACKGROUND: In Vietnam, the artemisinin-based combination therapy (ACT) of dihydroartemisinin-piperaquine is currently used for first-line treatment of uncomplicated Plasmodium falciparum malaria. However, limited efficacy and tolerability data are available on alternative forms of ACT in Vietnam in case there is a reduction in the susceptibility of dihydroartemisinin-piperaquine. A study was conducted to compare the efficacy and tolerability of two fixed-dose formulations of ACT, artemisinin-piperaquine (Artequick®, ARPQ) and artesunate-amodiaquine (Coarsucam™, ASAQ) for the treatment of P. falciparum malaria in south-central Vietnam. METHODS: A randomized, open-label trial was conducted comparing the efficacy of a two-day regimen of ARPQ (~2.8 mg/kg artemisinin plus ~17.1 mg/kg of piperaquine per day) and a three-day regimen of ASAQ (~4.7 mg/kg of artesunate plus ~12.6 mg/kg of amodiaquine per day) for the treatment of children and adults with uncomplicated falciparum malaria. Primary efficacy endpoint was day 42, PCR-corrected, parasitological cure rate. Secondary endpoints were parasite and fever clearance times and tolerability. RESULTS: Of 128 patients enrolled, 63 were administered ARPQ and 65 ASAQ. Of the patients who completed the 42 days follow-up period or had a recurrence of malaria, 55 were on ARPQ (30 children, 25 adults) and 59 were on ASAQ (31 children, 28 adults). Recrudescent parasitaemia was PCR-confirmed for one patient in each treatment group, with cure rates at day 42 of 98% (95% CI: 88-100) for both forms of ACT. The median parasite clearance time was significantly slower in the ARPQ group compared with the ASAQ group (48 h vs. 36 h, P<0.001) and fever clearance times were shorter in the ASAQ group (12 h vs. 24 h, P=0.07). The two forms of ACT were well tolerated with no serious adverse events. CONCLUSION: Both forms of ACT were highly efficacious in the treatment of uncomplicated P. falciparum malaria. Although the two-day course of ARPQ was equally as effective as the three-day course of ASAQ, parasite and fever clearance times were shorter with ASAQ. Further studies are warranted in different regions of Vietnam to determine the nationwide efficacy of ASAQ. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry Number, ACTRN12609000816257.

Genetic Diversity of Circumsporozoite Surface Protein of Plasmodium vivax from the Central Highlands, Vietnam.

The circumsporozoite surface protein of Plasmodium vivax (PvCSP) plays a critical role in parasite biology. It has been extensively studied as a leading vivax-malaria-vaccine candidate. In this study, the genetic polymorphism and natural selection of pvcsp in P. vivax isolates collected from the Central Highlands, Vietnam were analyzed to understand the genetic structure of the parasite circulating in the endemic area and to provide baseline information for effective vaccine development based on the protein. Only two major alleles, VK210 and VK247, were detected in Vietnamese pvcsp, with VK247 being the predominant one. The N-terminal and C-terminal regions of Vietnamese VK210 and VK247 variants showed a low genetic diversity. Amino acid substitutions, insertions of a single amino acid or octapeptide (ANKKAEDA in VK210 and ANKKAGDA in VK247), and tetrapeptide repeat motifs (GGNA) were the main factors generating genetic diversity in the two regions of the Vietnamese VK210 and VK247 variants. Interestingly, these two regions of Vietnamese pvcsp displayed a unique natural selection pressure distinct from global pvcsp, particularly with the neighboring Southeast Asian pvcsp population. Meanwhile, the central repeat region (CRR) in both the VK210 and VK247 variants showed a high degree of polymorphic characters, caused by varying numbers, types, and combinations of peptide repeat motifs (PRMs) in Vietnamese pvcsp. Highly complicated polymorphic patterns of the CRR were also detected in global pvcsp. These results expand our understanding of the genetic structure of Vietnamese pvcsp and the population dynamics of P. vivax in the Central Highlands, Vietnam.

Cross-sectional survey of asymptomatic malaria in Dak Nong province in the Central Highlands of Vietnam for the malaria elimination roadmap.

Asymptomatic parasite carriers represent a "silent" infective reservoir for malaria transmission and contributes to malaria persistence. However, limited data are available on asymptomatic malaria in Vietnam. Between November 2018 and March 2019, we conducted a malaria epidemiological survey of asymptomatic people (children ≥ 10 years old and adults ≥18 years old, n = 2,809) residing in three communes in Tuy Duc district, Dak Nong province in the Central Highlands of Vietnam. Based on the national stratification of malaria risk, Dak Buk So, Dak Ngo and Quang Truc communes were classified by the National Malaria Control Programme as low, moderate and high malaria endemic areas, respectively. Using participants' finger prick blood samples, malaria parasites were detected by one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The median age (Interquartile Range) for adults and children were 35 years (26-50) and 12 years (11-14), respectively. The prevalence of asymptomatic malaria was 1.7% (22/1,328), 3.5% (31/890) and 12.2% (72/591) for participants from Dak Buk So, Dak Ngo and Quang Truc, respectively. The prevalence of asymptomatic malaria was lower in children compared to adults: 2.6% (9/352) versus 4.7% (116/2,457) (Odds Ratio 0.53, 95% Confidence Interval 0.28 to1.02). Ownership of long-lasting insecticide-treated bed nets and hammocks was 97.1%, 99.0% and 94.7% for participants in Dak Buk So, Dak Ngo and Quang Truc, respectively, however, only 66.0%, 57.3% and 42.8% of the participants reported using bed nets every night. Of the several risk factors examined, going to the forest two weeks prior to enrolment into the study and sleeping in the forest had a significant association with participants being infected with asymptomatic malaria in Quang Truc, but not in the other two communes. Knowledge of the prevalence and distribution of asymptomatic malaria will help design and evaluate future intervention strategies for malaria elimination in Vietnam.

Ivermectin Treatment for Cattle Reduced the Survival of Two Malaria Vectors, Anopheles dirus and Anopheles epiroticus, Under Laboratory Conditions in Central Vietnam.

Ivermectin is a low-cost and nontoxic mosquitocide that may have a role in malaria elimination. However, the extent to which this drug impacts the mortality of Anopheles dirus and Anopheles epiroticus, two important malaria vectors in Southeast Asia, is unknown. This study compared and quantified anopheline mortality after feeding on ivermectin-treated cattle and control cattle in Vietnam. Local anopheline colonies fed on cattle 1 to 3, 6 to 8, 13 to 15, 20 to 22, and 28 to 30 days after injection (DAI) with ivermectin (intervention) or saline (control). An. dirus that fed on ivermectin-treated cattle had higher mortality rates than controls for up to 20 DAI (P < 0.05); An. epiroticus that fed on ivermectin-treated cattle had consistently higher mortality rates than controls for up to 8 DAI (P < 0.05). Feeding on ivermectin-treated cattle increased the mortality rate of these vector species for biologically relevant time periods. Therefore, ivermectin has the potential to become an important tool for integrated vector management.

Genetic Polymorphism and Natural Selection of Apical Membrane Antigen-1 in Plasmodium falciparum Isolates from Vietnam.

Apical membrane antigen-1 of Plasmodium falciparum (PfAMA-1) is a leading malaria vaccine candidate antigen. However, the genetic diversity of pfama-1 and associated antigenic variation in global P. falciparum field isolates are major hurdles to the design of an efficacious vaccine formulated with this antigen. Here, we analyzed the genetic structure and the natural selection of pfama-1 in the P. falciparum population of Vietnam. A total of 37 distinct haplotypes were found in 131 P. falciparum Vietnamese isolates. Most amino acid changes detected in Vietnamese pfama-1 were localized in the ectodomain, domains I, II, and III. Overall patterns of major amino acid changes in Vietnamese pfama-1 were similar to those of global pfama-1, but the frequencies of the amino acid changes slightly differed by country. Novel amino acid changes were also identified in Vietnamese pfama-1. Vietnamese pfama-1 revealed relatively lower genetic diversity than currently analyzed pfama-1 in other geographical regions, and suggested a distinct genetic differentiation pattern. Evidence for natural selection was detected in Vietnamese pfama-1, but it showed purifying selection unlike the global pfama-1 analyzed so far. Recombination events were also found in Vietnamese pfama-1. Major amino acid changes that were commonly identified in global pfama-1 were mainly localized to predicted B-cell epitopes, RBC-binding sites, and IUR regions. These results provide important information for understanding the genetic nature of the Vietnamese pfama-1 population, and have significant implications for the design of a vaccine based on PfAMA-1.