Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000-2016).

Emergence and spread of Plasmodium falciparum resistance to artemisinin-based combination therapies (ACT) is a major challenge for Greater Mekong Subregion countries in their goal to eliminate malaria by 2030. Tools to efficiently monitor drug resistance beyond resource-demanding therapeutic efficacy studies are necessary. A custom multiplex amplicon sequencing assay based on Illumina technology was designed to target the marker of partial resistance to artemisinin (K13), five candidate modulators of artemisinin resistance, the marker of resistance to chloroquine (crt), and four neutral microsatellite loci. The assay was used to genotype 635 P. falciparum-positive blood samples collected across seven provinces of Vietnam and one of Cambodia between 2000 and 2016. Markers of resistance to artemisinin partner-drugs piperaquine (copy number of plasmepsin-2) and mefloquine (copy number of multidrug-resistance 1) were determined by qPCR. Parasite population structure was further assessed using a 101-SNP barcode. Validated mutations of artemisinin partial resistance in K13 were found in 48.1% of samples, first detection was in 2000, and by 2015 prevalence overcame > 50% in Central Highlands and Binh Phuoc province. K13-C580Y variant became predominant country-wide, quickly replacing an outbreak of K13-I543T in Central Highlands. Mutations in candidate artemisinin resistance modulator genes paralleled the trends of K13 mutants, whereas resistance to piperaquine and mefloquine remained low (≈ 10%) by 2015-2016. Genomic tools applied to malaria surveillance generate comprehensive information on dynamics of drug resistance and population structure and reflect drug efficacy profiles from in vivo studies.

Assessing the Operational Feasibility of Integrating Point-of-Care G6PD Testing into Plasmodium vivax Malaria Management in Vietnam.

Plasmodium vivax cases represent more than 50% of a diminishing malaria case load in Vietnam. Safe and effective radical cure strategies could support malaria elimination by 2030. This study investigated the operational feasibility of introducing point-of-care quantitative glucose-6-phosphate dehydrogenase (G6PD) testing into malaria case management practices. A prospective interventional study was conducted at nine district hospitals and commune health stations in Binh Phuoc and Gia Lai provinces in Vietnam over the period of October 2020 to October 2021. The STANDARD™ G6PD Test (SD Biosensor, Seoul, Republic of Korea) was incorporated to inform P. vivax case management. Case management data and patient and health care provider (HCP) perspectives, as well as detailed cost data were collected. The G6PD test results were interpreted correctly by HCP and the treatment algorithm was adhered to for the majority of patients. One HCP consistently ran the test incorrectly, which was identified during the monitoring and resulted in provision of refresher training and updating of training materials and patient retesting. There was wide acceptability of the intervention among patients and HCP albeit with opportunities to improve the counseling materials. Increasing the number of facilities to which the test was deployed and decreases in the malaria cases resulted in higher per patient cost for incorporating G6PD testing into the system. Commodity costs can be reduced by using the 10-unit kits compared to the 25 unit kits, particularly when the case loads are low. These results demonstrate intervention feasibility while also highlighting specific challenges for a country approaching malaria elimination.

A randomized, double-blind, phase 2b study to investigate the efficacy, safety, tolerability and pharmacokinetics of a single-dose regimen of ferroquine with artefenomel in adults and children with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: For uncomplicated Plasmodium falciparum malaria, highly efficacious single-dose treatments are expected to increase compliance and improve treatment outcomes, and thereby may slow the development of resistance. The efficacy and safety of a single-dose combination of artefenomel (800 mg) plus ferroquine (400/600/900/1200 mg doses) for the treatment of uncomplicated P. falciparum malaria were evaluated in Africa (focusing on children ≤ 5 years) and Asia. METHODS: The study was a randomized, double-blind, single-dose, multi-arm clinical trial in patients aged > 6 months to < 70 years, from six African countries and Vietnam. Patients were followed up for 63 days to assess treatment efficacy, safety and pharmacokinetics. The primary efficacy endpoint was the polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR) at Day 28 in the Per-Protocol [PP] Set comprising only African patients ≤ 5 years. The exposure-response relationship for PCR-adjusted ACPR at Day 28 and prevalence of kelch-13 mutations were explored. RESULTS: A total of 373 patients were treated: 289 African patients ≤ 5 years (77.5%), 64 African patients > 5 years and 20 Asian patients. None of the treatment arms met the target efficacy criterion for PCR-adjusted ACPR at Day 28 (lower limit of 95% confidence interval [CI] > 90%). PCR-adjusted ACPR at Day 28 [95% CI] in the PP Set ranged from 78.4% [64.7; 88.7%] to 91.7% [81.6; 97.2%] for the 400 mg to 1200 mg ferroquine dose. Efficacy rates were low in Vietnamese patients, ranging from 20 to 40%. A clear relationship was found between drug exposure (artefenomel and ferroquine concentrations at Day 7) and efficacy (primary endpoint), with higher concentrations of both drugs resulting in higher efficacy. Six distinct kelch-13 mutations were detected in parasite isolates from 10/272 African patients (with 2 mutations known to be associated with artemisinin resistance) and 18/20 Asian patients (all C580Y mutation). Vomiting within 6 h of initial artefenomel administration was common (24.6%) and associated with lower drug exposures. CONCLUSION: The efficacy of artefenomel/ferroquine combination was suboptimal in African children aged ≤ 5 years, the population of interest, and vomiting most likely had a negative impact on efficacy. Trial registration ClinicalTrials.gov, NCT02497612. Registered 14 Jul 2015, https://clinicaltrials.gov/ct2/show/NCT02497612?term=NCT02497612&draw=2&rank=1.

Efficacy of dihydroartemisinin/piperaquine and artesunate monotherapy for the treatment of uncomplicated Plasmodium falciparum malaria in Central Vietnam.

BACKGROUND: Artemisinin-based combination therapies (ACTs) have significantly contributed to reduce Plasmodium falciparum malaria burden in Vietnam, but their efficacy is challenged by treatment failure of dihydroartemisinin/piperaquine ACT in Southern provinces. OBJECTIVES: To assess the efficacy of dihydroartemisinin/piperaquine for uncomplicated P. falciparum malaria in Gia Lai, Central Vietnam, and determine parasite resistance to artemisinin (ClinicalTrials.gov identifier NCT02604966). METHODS: Sixty patients received either dihydroartemisinin/piperaquine (4 mg/kg/day, 3 days; n = 33) or artesunate monotherapy (4 mg/kg/day, 3 days; n = 27) followed by dihydroartemisinin/piperaquine (AS + DHA/PPQ). Clinical phenotypes were determined during a 42 day follow-up and analysed together with ex vivo susceptibility to antimalarials and molecular markers of drug resistance. RESULTS: Day 3 positivity rate was significantly higher in the AS + DHA/PPQ arm compared with dihydroartemisinin/piperaquine (70.4% versus 39.4%, P = 0.016). Parasite clearance time was 95.2 h (AS + DHA/PPQ) versus 71.9 h (dihydroartemisinin/piperaquine, P = 0.063) and parasite clearance half-life was 7.4 h (AS + DHA/PPQ) versus 7.0 h (dihydroartemisinin/piperaquine, P = 0.140). Adequate clinical and parasitological response at Day 42 was 100% in both arms. By RT-qPCR, 36% (19/53) patients remained positive until Day 7. No recurrences were detected. kelch13 artemisinin resistance mutations were found in 87% (39/45) of isolates and 50% (20/40) were KEL1/C580Y. The piperaquine resistance marker plasmepsin-2 was duplicated in 10.4% (5/48). Isolates from Day 3-positive patients (n = 18) had higher ex vivo survival rates to artemisinin compounds (P < 0.048) and prevalence of kelch13 mutations (P = 0.005) than Day 3-negative patients (n = 5). The WHO definition of artemisinin resistance was fulfilled in 60% (24/40) of cases. CONCLUSIONS: Although dihydroartemisinin/piperaquine remained effective to treat P. falciparum, the high Day 3 positivity rate and prevalence of KEL1 strains calls for continuous monitoring of dihydroartemisinin/piperaquine efficacy in Central Vietnam.

African isolates show a high proportion of multiple copies of the Plasmodium falciparum plasmepsin-2 gene, a piperaquine resistance marker.

BACKGROUND: Today, the development of new and well-tolerated anti-malarial drugs is strongly justified by the emergence of Plasmodium falciparum resistance. In 2014-2015, a phase 2b clinical study was conducted to evaluate the efficacy of a single oral dose of Artefenomel (OZ439)-piperaquine (PPQ) in Asian and African patients presenting with uncomplicated falciparum malaria. METHODS: Blood samples collected before treatment offered the opportunity to investigate the proportion of multidrug resistant parasite genotypes, including P. falciparum kelch13 mutations and copy number variation of both P. falciparum plasmepsin 2 (Pfpm2) and P. falciparum multidrug resistance 1 (Pfmdr1) genes. RESULTS: Validated kelch13 resistance mutations including C580Y, I543T, P553L and V568G were only detected in parasites from Vietnamese patients. In Africa, isolates with multiple copies of the Pfmdr1 gene were shown to be more frequent than previously reported (21.1%, range from 12.4% in Burkina Faso to 27.4% in Uganda). More strikingly, high proportions of isolates with multiple copies of the Pfpm2 gene, associated with piperaquine (PPQ) resistance, were frequently observed in the African sites, especially in Burkina Faso and Uganda (> 30%). CONCLUSIONS: These findings were considered to sharply contrast with the recent description of increased sensitivity to PPQ of Ugandan parasite isolates. This emphasizes the necessity to investigate in vitro susceptibility profiles to PPQ of African isolates with multiple copies of the Pfpm2 gene and estimate the risk of development of PPQ resistance in Africa. Trial registration Clinicaltrials.gov reference: NCT02083380. Study title: Phase II efficacy study of artefenomel and piperaquine in adults and children with P. falciparum malaria. https://clinicaltrials.gov/ct2/results?cond=&term=NCT02083380&cntry=&state=&city=&dist= . FSFV: 23-Jul-2014; LSLV: 09-Oct-2015.

A randomised, double-blind clinical phase II trial of the efficacy, safety, tolerability and pharmacokinetics of a single dose combination treatment with artefenomel and piperaquine in adults and children with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: The clinical development of a single encounter treatment for uncomplicated malaria has the potential to significantly improve the effectiveness of antimalarials. Exploratory data suggested that the combination of artefenomel and piperaquine phosphate (PQP) has the potential to achieve satisfactory cure rates as a single dose therapy. The primary objective of the study was to determine whether a single dose of artefenomel (800 mg) plus PQP in ascending doses is an efficacious treatment for uncomplicated Plasmodium falciparum malaria in the 'target' population of children ≤ 5 years of age in Africa as well as Asian patients of all ages. METHODS: Patients in six African countries and in Vietnam were randomised to treatment with follow-up for 42-63 days. Efficacy, tolerability, safety and pharmacokinetics were assessed. Additional key objectives were to characterise the exposure-response relationship for polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response at day 28 post-dose (ACPR28) and to further investigate Kelch13 mutations. Patients in Africa (n = 355) and Vietnam (n = 82) were included, with 85% of the total population being children < 5 years of age. RESULTS: ACPR28 in the per protocol population (95% confidence interval) was 70.8% (61.13-79.19), 68.4% (59.13-76.66) and 78.6% (70.09-85.67) for doses of 800 mg artefenomel with 640 mg, 960 mg and 1440 mg of PQP respectively. ACPR28 was lower in Vietnamese than in African patients (66.2%; 54.55-76.62 and 74.5%; 68.81-79.68) respectively. Within the African population, efficacy was lowest in the youngest age group of ≥ 0.5 to ≤ 2 years, 52.7% (38.80-66.35). Initial parasite clearance was twice as long in Vietnam than in Africa. Within Vietnam, the frequency of the Kelch13 mutation was 70.1% and was clearly associated with parasite clearance half-life (PCt1/2). The most significant tolerability finding was vomiting (28.8%). CONCLUSIONS: In this first clinical trial evaluating a single encounter antimalarial therapy, none of the treatment arms reached the target efficacy of > 95% PCR-adjusted ACPR at day 28. Achieving very high efficacy following single dose treatment is challenging, since > 95% of the population must have sufficient concentrations to achieve cure across a range of parasite sensitivities and baseline parasitaemia levels. While challenging, the development of tools suitable for deployment as single encounter curative treatments for adults and children in Africa and to support elimination strategies remains a key development goal. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02083380 . Registered on 7 March 2014.

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.

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.

Delayed parasite clearance after treatment with dihydroartemisinin-piperaquine in Plasmodium falciparum malaria patients in central Vietnam.

Reduced susceptibility of Plasmodium falciparum toward artemisinin derivatives has been reported from the Thai-Cambodian and Thai-Myanmar borders. Following increasing reports from central Vietnam of delayed parasite clearance after treatment with dihydroartemisinin-piperaquine (DHA-PPQ), the current first-line treatment, we carried out a study on the efficacy of this treatment. Between September 2012 and February 2013, we conducted a 42-day in vivo and in vitro efficacy study in Quang Nam Province. Treatment was directly observed, and blood samples were collected twice daily until parasite clearance. In addition, genotyping, quantitative PCR (qPCR), and in vitro sensitivity testing of isolates was performed. The primary endpoints were parasite clearance rate and time. The secondary endpoints included PCR-corrected and uncorrected cure rates, qPCR clearance profiles, in vitro sensitivity results (for chloroquine, dihydroartemisinin, and piperaquine), and genotyping for mutations in the Kelch 13 propeller domain. Out of 672 screened patients, 95 were recruited and 89 available for primary endpoint analyses. The median parasite clearance time (PCT) was 61.7 h (interquartile range [IQR], 47.6 to 83.2 h), and the median parasite clearance rate had a slope half-life of 6.2 h (IQR, 4.4 to 7.5 h). The PCR-corrected efficacy rates were estimated at 100% at day 28 and 97.7% (95% confidence interval, 91.2% to 99.4%) at day 42. At day 3, the P. falciparum prevalence by qPCR was 2.5 times higher than that by microscopy. The 50% inhibitory concentrations (IC50s) of isolates with delayed clearance times (≥ 72 h) were significantly higher than those with normal clearance times for all three drugs. Delayed parasite clearance (PCT, ≥ 72 h) was significantly higher among day 0 samples carrying the 543 mutant allele (47.8%) than those carrying the wild-type allele (1.8%; P = 0.048). In central Vietnam, the efficacy of DHA-PPQ is still satisfactory, but the parasite clearance time and rate are indicative of emerging artemisinin resistance. (This study has been registered at ClinicalTrials.gov under registration no. NCT01775592.).

Severe malaria not responsive to artemisinin derivatives in man returning from Angola to Vietnam.

Resistance to artemisinin derivatives, the most potent antimalarial drugs currently used, has emerged in Southeast Asia and threatens to spread to Africa. We report a case of malaria in a man who returned to Vietnam after 3 years in Angola that did not respond to intravenous artesunate and clindamycin or an oral artemisinin-based combination.