Intestinal injury and the gut microbiota in patients with Plasmodium falciparum malaria.

The pathophysiology of severe falciparum malaria involves a complex interaction between the host, parasite, and gut microbes. In this review, we focus on understanding parasite-induced intestinal injury and changes in the human intestinal microbiota composition in patients with Plasmodium falciparum malaria. During the blood stage of P. falciparum infection, infected red blood cells adhere to the vascular endothelium, leading to widespread microcirculatory obstruction in critical tissues, including the splanchnic vasculature. This process may cause intestinal injury and gut leakage. Epidemiological studies indicate higher rates of concurrent bacteraemia in severe malaria cases. Furthermore, severe malaria patients exhibit alterations in the composition and diversity of the intestinal microbiota, although the exact contribution to pathophysiology remains unclear. Mouse studies have demonstrated that the gut microbiota composition can impact susceptibility to Plasmodium infections. In patients with severe malaria, the microbiota shows an enrichment of pathobionts, including pathogens that are known to cause concomitant bloodstream infections. Microbial metabolites have also been detected in the plasma of severe malaria patients, potentially contributing to metabolic acidosis and other clinical complications. However, establishing causal relationships requires intervention studies targeting the gut microbiota.

Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum.

Resistance of the human malaria parasites, Plasmodium falciparum, to artemisinins is now fully established in Southeast Asia and is gradually emerging in Sub-Saharan Africa. Although nonsynonymous SNPs in the pfk13 Kelch-repeat propeller (KREP) domain are clearly associated with artemisinin resistance, their functional relevance requires cooperation with other genetic factors/alterations of the P. falciparum genome, collectively referred to as genetic background. Here we provide experimental evidence that P. falciparum cyclophilin 19B (PfCYP19B) may represent one putative factor in this genetic background, contributing to artemisinin resistance via its increased expression. We show that overexpression of PfCYP19B in vitro drives limited but significant resistance to not only artemisinin but also piperaquine, an important partner drug in artemisinin-based combination therapies. We showed that PfCYP19B acts as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P). Curiously, artemisinin and piperaquine affect eIF2α-P in an inverse direction that in both cases can be modulated by PfCYP19B towards resistance. Here we also provide evidence that the upregulation of PfCYP19B in the drug-resistant parasites appears to be maintained by a short tandem repeat (SRT) sequence polymorphism in the gene's promoter region. These results support a model that artemisinin (and other drugs) resistance mechanisms are complex genetic traits being contributed to by altered expression of multiple genes driven by genetic polymorphism at their promoter regions.

Spatio-temporal spread of artemisinin resistance in Southeast Asia.

Current malaria elimination targets must withstand a colossal challenge-resistance to the current gold standard antimalarial drug, namely artemisinin derivatives. If artemisinin resistance significantly expands to Africa or India, cases and malaria-related deaths are set to increase substantially. Spatial information on the changing levels of artemisinin resistance in Southeast Asia is therefore critical for health organisations to prioritise malaria control measures, but available data on artemisinin resistance are sparse. We use a comprehensive database from the WorldWide Antimalarial Resistance Network on the prevalence of non-synonymous mutations in the Kelch 13 (K13) gene, which are known to be associated with artemisinin resistance, and a Bayesian geostatistical model to produce spatio-temporal predictions of artemisinin resistance. Our maps of estimated prevalence show an expansion of the K13 mutation across the Greater Mekong Subregion from 2000 to 2022. Moreover, the period between 2010 and 2015 demonstrated the most spatial change across the region. Our model and maps provide important insights into the spatial and temporal trends of artemisinin resistance in a way that is not possible using data alone, thereby enabling improved spatial decision support systems on an unprecedented fine-scale spatial resolution. By predicting for the first time spatio-temporal patterns and extents of artemisinin resistance at the subcontinent level, this study provides critical information for supporting malaria elimination goals in Southeast Asia.

Molecular markers of artemisinin resistance during falciparum malaria elimination in Eastern Myanmar.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized. METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance. RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted. CONCLUSION: The malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.

Expanding the roles of community health workers to sustain programmes during malaria elimination: a meeting report on operational research in Southeast Asia.

In Southeast Asia malaria elimination is targeted by 2030. Cambodia aims to achieve this by 2025, driven in large part by the urgent need to control the spread of artemisinin-resistant falciparum malaria infections. Rapid elimination depends on sustaining early access to diagnosis and effective treatment. In much of Cambodia, rapid elimination will rely on a village malaria worker (VMW) network. Yet as malaria declines and is no longer a common cause of febrile illness, VMWs may become less popular with febrile patients, as VMWs do not diagnose or treat other conditions at present. There is a risk that VMWs become inactive and malaria rebounds before the complete interruption of transmission is achieved.During 2021-23 a large-scale operational research study was conducted in western Cambodia to explore how a VMW network could be sustained by including health activities that cover non-malarial illnesses to encourage febrile patients to continue to attend. 105 VMWs received new rapid diagnostic tests (including dengue antigen-antibody and combined malaria/C-reactive protein tests), were trained in electronic data collection, and attended health education packages on hygiene and sanitation, disease surveillance and first aid, management of mild illness, and vaccination and antenatal care.In August 2023 the National Malaria Control Programme of Cambodia convened a stakeholder meeting in Battambang, Cambodia. Findings from the study were reviewed in the context of current malaria elimination strategies. The discussions informed policy options to sustain the relevance of the VMW network in Cambodia, and the potential for its integration with other health worker networks. This expansion could ensure VMWs remain active and relevant until malaria elimination is accomplished.

Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial.

In early symptomatic COVID-19 treatment, high dose oral favipiravir did not accelerate viral clearance. BACKGROUND: Favipiravir, an anti-influenza drug, has in vitro antiviral activity against SARS-CoV-2. Clinical trial evidence to date is inconclusive. Favipiravir has been recommended for the treatment of COVID-19 in some countries. METHODS: In a multicentre open-label, randomised, controlled, adaptive platform trial, low-risk adult patients with early symptomatic COVID-19 were randomised to one of ten treatment arms including high dose oral favipiravir (3.6g on day 0 followed by 1.6g daily to complete 7 days treatment) or no study drug. The primary outcome was the rate of viral clearance (derived under a linear mixed-effects model from the daily log10 viral densities in standardised duplicate oropharyngeal swab eluates taken daily over 8 days [18 swabs per patient]), assessed in a modified intention-to-treat population (mITT). The safety population included all patients who received at least one dose of the allocated intervention. This ongoing adaptive platform trial was registered at ClinicalTrials.gov (NCT05041907) on 13/09/2021. RESULTS: In the final analysis, the mITT population contained data from 114 patients randomised to favipiravir and 126 patients randomised concurrently to no study drug. Under the linear mixed-effects model fitted to all oropharyngeal viral density estimates in the first 8 days from randomisation (4,318 swabs), there was no difference in the rate of viral clearance between patients given favipiravir and patients receiving no study drug; a -1% (95% credible interval: -14 to 14%) difference. High dose favipiravir was well-tolerated. INTERPRETATION: Favipiravir does not accelerate viral clearance in early symptomatic COVID-19. The viral clearance rate estimated from quantitative measurements of oropharyngeal eluate viral densities assesses the antiviral efficacy of drugs in vivo with comparatively few studied patients.

Clinical Antiviral Efficacy of Remdesivir in Coronavirus Disease 2019: An Open-Label, Randomized Controlled Adaptive Platform Trial (PLATCOV).

BACKGROUND: Uncertainty over the therapeutic benefit of parenteral remdesivir in coronavirus disease 2019 (COVID-19) has resulted in varying treatment guidelines. METHODS: In a multicenter open-label, controlled, adaptive, pharmacometric platform trial, low-risk adult patients with early symptomatic COVID-19 were randomized to 1 of 8 treatment arms including intravenous remdesivir (200 mg followed by 100 mg daily for 5 days) or no study drug. The primary outcome was the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance (estimated under a linear model fit to the daily log10 viral densities, days 0-7) in standardized duplicate oropharyngeal swab eluates, in a modified intention-to-treat population. This ongoing adaptive trial is registered at ClinicalTrials.gov (NCT05041907). RESULTS: The 2 study arms enrolled 131 patients (remdesivir n = 67, no study drug n = 64) and estimated viral clearance rates from a median of 18 swab samples per patient (a total of 2356 quantitative polymerase chain reactions). Under the linear model, compared with the contemporaneous control arm (no study drug), remdesivir accelerated mean estimated viral clearance by 42% (95% credible interval, 18%-73%). CONCLUSIONS: Parenteral remdesivir accelerates viral clearance in early symptomatic COVID-19. Pharmacometric assessment of therapeutics using the method described can determine in vivo clinical antiviral efficacy rapidly and efficiently.

Activity of Ivermectin and Its Metabolites against Asexual Blood Stage Plasmodium falciparum and Its Interactions with Antimalarial Drugs.

Ivermectin is an endectocide used widely to treat a variety of internal and external parasites. Field trials of ivermectin mass drug administration for malaria transmission control have demonstrated a reduction of Anopheles mosquito survival and human malaria incidence. Ivermectin will mostly be deployed together with artemisinin-based combination therapies (ACT), the first-line treatment of falciparum malaria. It has not been well established if ivermectin has activity against asexual stage Plasmodium falciparum or if it interacts with the parasiticidal activity of other antimalarial drugs. This study evaluated antimalarial activity of ivermectin and its metabolites in artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and assessed in vitro drug-drug interaction with artemisinins and its partner drugs. The concentration of ivermectin causing half of the maximum inhibitory activity (IC50) on parasite survival was 0.81 µM with no significant difference between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The ivermectin metabolites were 2-fold to 4-fold less active than the ivermectin parent compound (P < 0.001). Potential pharmacodynamic drug-drug interactions of ivermectin with artemisinins, ACT-partner drugs, and atovaquone were studied in vitro using mixture assays providing isobolograms and derived fractional inhibitory concentrations. There were no synergistic or antagonistic pharmacodynamic interactions when combining ivermectin and antimalarial drugs. In conclusion, ivermectin does not have clinically relevant activity against the asexual blood stages of P. falciparum. It also does not affect the in vitro antimalarial activity of artemisinins or ACT-partner drugs against asexual blood stages of P. falciparum.

Severe falciparum malaria in pregnancy in Southeast Asia: a multi-centre retrospective cohort study.

BACKGROUND: Severe malaria in pregnancy causes maternal mortality, morbidity, and adverse foetal outcomes. The factors contributing to adverse maternal and foetal outcomes are not well defined. We aimed to identify the factors predicting higher maternal mortality and to describe the foetal mortality and morbidity associated with severe falciparum malaria in pregnancy. METHODS: A retrospective cohort study was conducted of severe falciparum malaria in pregnancy, as defined by the World Health Organization severe malaria criteria. The patients were managed prospectively by the Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border or were included in hospital-based clinical trials in six Southeast Asian countries. Fixed-effects multivariable penalised logistic regression was used for analysing maternal mortality. RESULTS: We included 213 (123 SMRU and 90 hospital-based) episodes of severe falciparum malaria in pregnancy managed between 1980 and 2020. The mean maternal age was 25.7 (SD 6.8) years, and the mean gestational age was 25.6 (SD 8.9) weeks. The overall maternal mortality was 12.2% (26/213). Coma (adjusted odds ratio [aOR], 7.18, 95% CI 2.01-25.57, p = 0.0002), hypotension (aOR 11.21, 95%CI 1.27-98.92, p = 0.03) and respiratory failure (aOR 4.98, 95%CI 1.13-22.01, p = 0.03) were associated with maternal mortality. Pregnant women with one or more of these three criteria had a mortality of 29.1% (25/86) (95%CI 19.5 to 38.7%) whereas there were no deaths in 88 pregnant women with hyperparasitaemia (> 10% parasitised erythrocytes) only or severe anaemia (haematocrit < 20%) only. In the SMRU prospective cohort, in which the pregnant women were followed up until delivery, the risks of foetal loss (23.3% by Kaplan-Meier estimator, 25/117) and small-for-gestational-age (38.3%, 23/60) after severe malaria were high. Maternal death, foetal loss and preterm birth occurred commonly within a week of diagnosis of severe malaria. CONCLUSIONS: Vital organ dysfunction in pregnant women with severe malaria was associated with a very high maternal and foetal mortality whereas severe anaemia or hyperparasitaemia alone were not associated with poor prognosis, which may explain the variation of reported mortality from severe malaria in pregnancy. Access to antenatal care must be promoted to reduce barriers to early diagnosis and treatment of both malaria and anaemia.

An artesunate pharmacometric model to explain therapeutic responses in falciparum malaria.

BACKGROUND: The artemisinins are potent and widely used antimalarial drugs that are eliminated rapidly. A simple concentration-effect pharmacometric model does not explain why dosing more frequently than once daily fails to augment parasite clearance and improve therapeutic responses in vivo. Artemisinins can induce a temporary non-replicative or 'dormant' drug refractory state in Plasmodium falciparum malaria parasites which may explain recrudescences observed in clinical trials despite full drug susceptibility, but whether it explains the dosing-response relationship is uncertain. OBJECTIVES: To propose a revised model of antimalarial pharmacodynamics that incorporates reversible asexual parasite injury and temporary drug refractoriness in order to explain the failure of frequent dosing to augment therapeutic efficacy in falciparum malaria. METHODS: The model was fitted using a Bayesian Markov Chain Monte Carlo approach with the parasite clearance data from 39 patients with uncomplicated falciparum malaria treated with artesunate from western Cambodia and 40 patients from northwestern Thailand reported previously. RESULTS: The revised model captured the dynamics of parasite clearance data. Its predictions are consistent with observed therapeutic responses. CONCLUSIONS: A within-host pharmacometric model is proposed in which it is hypothesized that some malaria parasites enter a temporary drug refractory state after exposure to artemisinin antimalarials, which is followed by delayed parasite death or reactivation. The model fitted the observed sequential parasite density data from patients with acute P. falciparum malaria, and it supported reduced ring stage activity in artemisinin-resistant infections.

The impact of anti-malarial markets on artemisinin resistance: perspectives from Burkina Faso.

BACKGROUND: Widespread artemisinin resistance in Africa could be catastrophic when drawing parallels with the failure of chloroquine in the 1970s and 1980s. This article explores the role of anti-malarial market characteristics in the emergence and spread of arteminisin resistance in African countries, drawing on perspectives from Burkina Faso. METHODS: Data were collected through in-depth interviews and focus group discussions. A representative sample of national policy makers, regulators, public and private sector wholesalers, retailers, clinicians, nurses, and community members were purposively sampled. Additional information was also sought via review of policy publications and grey literature on anti-malarial policies and deployment practices in Burkina Faso. RESULTS: Thirty seven in-depth interviews and 6 focus group discussions were conducted. The study reveals that the current operational mode of anti-malarial drug markets in Burkina Faso promotes arteminisin resistance emergence and spread. The factors are mainly related to the artemisinin-based combination therapy (ACT) supply chain, to ACT quality, ACT prescription monitoring and to ACT access and misuse by patients. CONCLUSION: Study findings highlight the urgent requirement to reform current characteristics of the anti-malarial drug market in order to delay the emergence and spread of artemisinin resistance in Burkina Faso. Four recommendations for public policy emerged during data analysis: (1) Address the suboptimal prescription of anti-malarial drugs, (2) Apply laws that prohibit the sale of anti-malarials without prescription, (3) Restrict the availability of street drugs, (4) Sensitize the population on the value of compliance regarding correct acquisition and intake of anti-malarials. Funding systems for anti-malarial drugs in terms of availability and accessibility must also be stabilized.

A framework for stakeholder engagement in the adoption of new anti-malarial treatments in Africa: a case study of Nigeria.

BACKGROUND: Recent reports of artemisinin partial resistance from Rwanda and Uganda are worrisome and suggest a future policy change to adopt new anti-malarials. This is a case study on the evolution, adoption, and implementation of new anti-malarial treatment policies in Nigeria. The main objective is to provide perspectives to enhance the future uptake of new anti-malarials, with an emphasis on stakeholder engagement strategies. METHODS: This case study is based on an analysis of policy documents and stakeholders' perspectives drawn from an empirical study conducted in Nigeria, 2019-2020. A mixed methods approach was adopted, including historical accounts, review of programme and policy documents, and 33 qualitative in-depth interviews and 6 focus group discussions. RESULTS: Based on policy documents reviewed, the adoption of artemisinin-based combination therapy (ACT) in Nigeria was swift due to political will, funding and support from global developmental partners. However, the implementation of ACT was met with resistance from suppliers, distributors, prescribers, and end-users, attributed to market dynamics, costs and inadequate stakeholder engagement. Deployment of ACT in Nigeria witnessed increased developmental partner support, robust data generation, ACT case-management strengthening and evidence on anti-malarial use in severe malaria and antenatal care management. A framework for effective stakeholder engagement for the future adoption of new anti-malarial treatment strategies was proposed. The framework covers the pathway from generating evidence on drug efficacy, safety and uptake; to making treatment accessible and affordable to end-users. It addresses which stakeholders to engage with and the content of engagement strategies with key stakeholders at different levels of the transition process. CONCLUSION: Early and staged engagement of stakeholders from global bodies to community level end-users is critical to the successful adoption and uptake of new anti-malarial treatment policies. A framework for these engagements was proposed as a contribution to enhancing the uptake of future anti-malarial strategies.

In vitro activity of rhinacanthin analogues against drug resistant Plasmodium falciparum isolates from Northeast Thailand.

BACKGROUND: New anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand. METHODS: Using a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates. RESULTS: The 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9-791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6-63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83-0.97, P < 0.001). There were no P. falciparum cytochrome b mutations observed in the field isolates, indicating that P. falciparum isolates from this area remained ATQ-sensitive. Pfkelch13 mutations and the ring-stage survival assay confirmed that most isolates were resistant to ART. CONCLUSIONS: Two rhinacanthin analogues showed parasiticidal activity against multi-drug resistant P. falciparum isolates, although less potent than ATQ. Rhinacanthin analogue 2 was more potent than analogue 1, and can be a lead compound for further optimization as an anti-malarial in areas with multidrug resistance.

Exploring the feasibility of introducing triple artemisinin-based combination therapy in the malaria treatment policy in Vietnam.

BACKGROUND: This study investigates the processes regarding changing malaria treatment policies in Vietnam. Moreover, it explores the feasibility of introducing triple artemisinin-based combination therapy (TACT) in Vietnam to support the national malaria control and elimination plan. METHODS: Data were collected via 12 in-depth interviews with key stakeholders, combined with a review of policy documents. RESULTS: TACT is considered as a useful backup strategy in case future treatment failures with current artemisinin-based combination therapy (ACT) would occur. Moreover, TACT is also considered as a promising strategy to prevent the re-establishment of malaria. However, regulatory procedures and implementation timelines for TACT were expected to be lengthy. Therefore, strategies to engage national decision-makers, regulators, and suppliers should be initiated soon, stipulating the benefits of TACT deployment. In Vietnam, a procedure to apply for an import permit without registration that has previously been applied to the introduction of artesunate-pyronaridine was proposed to accelerate the introduction of TACT. Global-level support through the World Health Organization recommendations and prequalification were considered critical for supporting the introduction of TACT in Vietnam. CONCLUSIONS: Appropriate approach strategies and early stakeholder engagement will be needed to accelerate the introduction of TACT in Vietnam.

Strategies for deploying triple artemisinin-based combination therapy in the Greater Mekong Subregion.

BACKGROUND: This is a qualitative study to identify implementation challenges for deploying triple artemisinin-based combination therapy (TACT) in the Greater Mekong Subregion (GMS) of Southeast Asia and to explore strategies to overcome these challenges. METHODS: In-depth interviews were conducted in three countries that have repeatedly been confronted with ACT failures: Cambodia, Vietnam, and Lao PDR. Thirty-nine key stakeholders in the healthcare systems in these countries were interviewed. One participatory workshop was conducted in Cambodia, where scenarios for potential TACT deployment were discussed. RESULTS: The results section is organized around four strategic themes that emerged from the data: policy support, data and evidence, logistics and operation, and downstream engagement. The study revealed that countries in the GMS currently rely on ACT to eliminate Plasmodium falciparum malaria by 2025. TACT is, however, considered to be a useful backup strategy in case of future treatment failures and to prevent the re-establishment of malaria. The study showed that a major challenge ahead is to engage decision makers and healthcare providers into deploying TACT, given the low case incidence of falciparum malaria in the GMS. Interview respondents were also skeptical whether healthcare providers would be willing to engage in new therapies for a disease they hardly encounter anymore. Hence, elaborate information dissemination strategies were considered appropriate and these strategies should especially target village malaria workers. Respondents proposed several regulatory and programmatic strategies to anticipate the formation of TACT markets in the GMS. These strategies include early dossier submission to streamline regulatory procedures, early stakeholder engagement strategies to shorten implementation timelines, and inclusion of TACT as second-line therapy to accelerate their introduction in case they are urgently needed. CONCLUSIONS: This paper presents a qualitative study to identify implementation challenges for deploying TACT in the GMS and to explore strategies to overcome these challenges. The findings could benefit researchers and decision makers in strategizing towards potential future deployment of TACT in the GMS to combat artemisinin and partner drug resistance.

Evolution of genetic markers for drug resistance after the introduction of dihydroartemisinin-piperaquine as first-line anti-malarial treatment for uncomplicated falciparum malaria in Indonesia.

BACKGROUND: Dihydroartemisinin-piperaquine has been Indonesia's first-line anti-malarial treatment since 2008. Annual therapeutic efficacy studies (TES) done in the last 12 years showed continued high treatment efficacy in uncomplicated Plasmodium falciparum malaria. Although these studies did not show evidence for artemisinin resistance, a slight increase in Late Treatment Failure was observed over time. It is highlight to explore the evolution of genetic markers for ACT partner drug resistance since adopting DHA-PPQ. METHODS: Dry blood spots were identified from a mass blood survey of uncomplicated falciparum malaria patients (N = 50) in Sumba from 2010 to 2018. Analysis of genotypic profile (N = 51) and a Therapeutic Efficacy Study (TES) from Papua (N = 142) from 2020 to 2021, 42-day follow-up. PCR correction using msp1, msp2, and glurp was used to distinguish recrudescence and reinfection. Parasite DNA from DBSs was used for genotyping molecular markers for antimalaria drug resistance, including in Pfk13, pfcrt, and pfmdr1, as well as gene copy number variation in pfpm2/3 and pfmdr1. RESULTS: The study revealed the absence of SNPs associated with ART resistance and several novel SNPs such as L396F, I526V, M579I and N537S (4.25%). In Sumba, the mutant haplotype SDD of pfmdr1 was found in one-third of the isolates, while only 8.9% in Papua. None of the pfcrt mutations linked to piperaquine resistance were observed, but 71% of isolates had pfcrt I356L. Amplification of the pfpm2/3 genes was in Sumba (17.02%) and Papua (13.7%), while pfmdr1 copy number prevalence was low (3.8%) in both areas. For the TES study, ten recurrences of infection were observed on days 28, 35, and 42. Late parasitological failure (LPF) was observed in 10/117 (8.5%) subjects by microscopy. PCR correction revealed that all nine cases were re-infections and one was confirmed as recrudescence. CONCLUSION: This study revealed that DHA-PPQ is still highly effective against P. falciparum. The genetic architecture of the parasite P. falciparum isolates during 2010-2021 revealed single copy of Pfpm2 and pfmdr1 were highly prevalent. The slight increase in DHA-PPQ LTF alerts researchers to start testing other ACTs as alternatives to DHA-PPQ for baseline data in order to get a chance of achieving malaria elimination wants by 2030.

Community engagement among forest goers in a malaria prophylaxis trial: implementation challenges and implications.

BACKGROUND: Malaria transmission in Southeast Asia is increasingly confined to forests, where marginalized groups are exposed primarily through their work. Anti-malarial chemoprophylaxis may help to protect these people. This article examines the effectiveness and practical challenges of engaging forest-goers to participate in a randomized controlled clinical trial of anti-malarial chemoprophylaxis with artemether-lumefantrine (AL) versus a control (multivitamin, MV) for malaria in northeast Cambodia. METHODS: The impact of engagement in terms of uptake was assessed as the proportion of people who participated during each stage of the trial: enrolment, compliance with trial procedures, and drug intake. During the trial, staff recorded the details of engagement meetings, including the views and opinions of participants and community representatives, the decision-making processes, and the challenges addressed during implementation. RESULTS: In total, 1613 participants were assessed for eligibility and 1480 (92%) joined the trial, 1242 (84%) completed the trial and received prophylaxis (AL: 82% vs MV: 86%, p = 0.08); 157 (11%) were lost to follow-up (AL: 11% vs MV: 11%, p = 0.79); and 73 (5%) discontinued the drug (AL-7% vs MV-3%, p = 0.005). The AL arm was associated with discontinuation of the study drug (AL: 48/738, 7% vs 25/742, 3%; p = 0.01). Females (31/345, 9%) were more likely (42/1135, 4%) to discontinue taking drugs at some point in the trial (p = 0.005). Those (45/644, 7%) who had no previous history of malaria infection were more likely to discontinue the study drug than those (28/836, 3%) who had a history of malaria (p = 0.02). Engagement with the trial population was demanding because many types of forest work are illegal; and the involvement of an engagement team consisting of representatives from the local administration, health authorities, community leaders and community health workers played a significant role in building trust. Responsiveness to the needs and concerns of the community promoted acceptability and increased confidence in taking prophylaxis among participants. Recruitment of forest-goer volunteers to peer-supervise drug administration resulted in high compliance with drug intake. The development of locally-appropriate tools and messaging for the different linguistic and low-literacy groups was useful to ensure participants understood and adhered to the trial procedures. It was important to consider forest-goers` habits and social characteristics when planning the various trial activities. CONCLUSIONS: The comprehensive, participatory engagement strategy mobilized a wide range of stakeholders including study participants, helped build trust, and overcame potential ethical and practical challenges. This locally-adapted approach was highly effective as evidenced by high levels of trial enrolment, compliance with trial procedures and drug intake.

The heterogeneity of symptom reporting across study sites: a secondary analysis of a randomised placebo-controlled multicentre antimalarial trial.

INTRODUCTION: Symptoms reported following the administration of investigational drugs play an important role in decisions for registration and treatment guidelines. However, symptoms are subjective, and interview methods to quantify them are difficult to standardise. We explored differences in symptom reporting across study sites of a multicentre antimalarial trial, with the aim of informing trial design and the interpretation of safety and tolerability data. METHODS: Data were derived from the IMPROV trial, a randomised, placebo-controlled double blinded trial of high dose primaquine to prevent Plasmodium vivax recurrence conducted in eight study sites in Afghanistan, Ethiopia, Indonesia and Vietnam. At each follow up visit a 13-point symptom questionnaire was completed. The number and percentage of patients with clinically relevant symptoms following the administration of primaquine or placebo, were reported by study site including vomiting, diarrhoea, anorexia, nausea, abdominal pain and dizziness. Multivariable logistic regression was used to estimate the confounder-adjusted site-specific proportion of each symptom. RESULTS: A total of 2,336 patients were included. The greatest variation between sites in the proportion of patients reporting symptoms was for anorexia between day 0 and day 13: 97.3% (361/371) of patients in Arba Minch, Ethiopia, reported the symptom compared with 4.7% (5/106) of patients in Krong Pa, Vietnam. Differences attenuated slightly after adjusting for treatment arm, age, sex, day 0 parasite density and fever; with the adjusted proportion for anorexia ranging from 4.8% to 97.0%. Differences between sites were greater for symptoms graded as mild or moderate compared to those rated as severe. Differences in symptom reporting were greater between study sites than between treatment arms within the same study site. CONCLUSION: Despite standardised training, there was large variation in symptom reporting across trial sites. The reporting of severe symptoms was less skewed compared to mild and moderate symptoms, which are likely to be more subjective. Trialists should clearly distinguish between safety and tolerability outcomes. Differences between trial arms were much less variable across sites, suggesting that the relative difference in reported symptoms between intervention and control group is more relevant than absolute numbers and should be reported when possible. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01814683; March 20th, 2013.

A cautionary note on the use of unsupervised machine learning algorithms to characterise malaria parasite population structure from genetic distance matrices.

Genetic surveillance of malaria parasites supports malaria control programmes, treatment guidelines and elimination strategies. Surveillance studies often pose questions about malaria parasite ancestry (e.g. how antimalarial resistance has spread) and employ statistical methods that characterise parasite population structure. Many of the methods used to characterise structure are unsupervised machine learning algorithms which depend on a genetic distance matrix, notably principal coordinates analysis (PCoA) and hierarchical agglomerative clustering (HAC). PCoA and HAC are sensitive to both the definition of genetic distance and algorithmic specification. Importantly, neither algorithm infers malaria parasite ancestry. As such, PCoA and HAC can inform (e.g. via exploratory data visualisation and hypothesis generation), but not answer comprehensively, key questions about malaria parasite ancestry. We illustrate the sensitivity of PCoA and HAC using 393 Plasmodium falciparum whole genome sequences collected from Cambodia and neighbouring regions (where antimalarial resistance has emerged and spread recently) and we provide tentative guidance for the use and interpretation of PCoA and HAC in malaria parasite genetic epidemiology. This guidance includes a call for fully transparent and reproducible analysis pipelines that feature (i) a clearly outlined scientific question; (ii) a clear justification of analytical methods used to answer the scientific question along with discussion of any inferential limitations; (iii) publicly available genetic distance matrices when downstream analyses depend on them; and (iv) sensitivity analyses. To bridge the inferential disconnect between the output of non-inferential unsupervised learning algorithms and the scientific questions of interest, tailor-made statistical models are needed to infer malaria parasite ancestry. In the absence of such models speculative reasoning should feature only as discussion but not as results.

Determinants of Implementation of a Critical Care Registry in Asia: Lessons From a Qualitative Study.

BACKGROUND: The Collaboration for Research, Implementation, and Training in Critical Care in Asia (CCA) is implementing a critical care registry to capture real-time data to facilitate service evaluation, quality improvement, and clinical studies. OBJECTIVE: The purpose of this study is to examine stakeholder perspectives on the determinants of implementation of the registry by examining the processes of diffusion, dissemination, and sustainability. METHODS: This study is a qualitative phenomenological inquiry using semistructured interviews with stakeholders involved in registry design, implementation, and use in 4 South Asian countries. The conceptual model of diffusion, dissemination, and sustainability of innovations in health service delivery guided interviews and analysis. Interviews were coded using the Rapid Identification of Themes from Audio recordings procedure and were analyzed based on the constant comparison approach. RESULTS: A total of 32 stakeholders were interviewed. Analysis of stakeholder accounts identified 3 key themes: innovation-system fit; influence of champions; and access to resources and expertise. Determinants of implementation included data sharing, research experience, system resilience, communication and networks, and relative advantage and adaptability. CONCLUSIONS: The implementation of the registry has been possible due to efforts to increase the innovation-system fit, influence of motivated champions, and the support offered by access to resources and expertise. The reliance on individuals and the priorities of other health care actors pose a risk to sustainability.