METHODS: Pkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein-ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates.
RESULTS: Five nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein-ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr-pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates.
CONCLUSIONS: A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.
METHODS: We measured PS immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in Malaysian patients with vivax, falciparum, knowlesi, and malariae malaria, and in healthy controls, and correlated antibody titres with hemoglobin. PS antibodies were also measured in volunteers experimentally infected with Plasmodium vivax and Plasmodium falciparum.
RESULTS: PS IgM and IgG antibodies were elevated in patients with vivax, falciparum, knowlesi, and malariae malaria (P < .0001 for all comparisons with controls) and were highest in vivax malaria. In vivax and falciparum malaria, PS IgM and IgG on admission correlated inversely with admission and nadir hemoglobin, controlling for parasitemia and fever duration. PS IgM and IgG were also increased in volunteers infected with blood-stage P. vivax and P. falciparum, and were higher in P. vivax infection.
CONCLUSIONS: PS antibodies are higher in vivax than falciparum malaria, correlate inversely with hemoglobin, and may contribute to the early loss of uninfected red blood cells found in malarial anemia from both species.
METHODS: Under Thailand's integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed.
RESULTS: The results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance.
CONCLUSIONS: This report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.
METHODS: Plasmodium falciparum DNA was collected from the Thailand-Myanmar, Thailand-Malaysia and Thailand-Cambodia borders during 2008-2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated.
RESULTS: Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr-pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr-pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand-Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr-pfdhps haplotypes. In particular, the prevalence of pfdhfr-pfdhps, septuple mutation was observed in the Thailand-Myanmar (50%, 73/146) and Thailand-Cambodia (65%, 26/40) border. In Thailand-Malaysia border, majority of the pfdhfr-pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008-2016. Within the pfdhfr-pfdhps haplotypes, during 2008-2013 the pfdhps A/S436F mutation was observed only in Thailand-Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification.
CONCLUSIONS: Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.
METHODS: PACKNOW is a two-arm, open-label randomised controlled trial of adjunctive paracetamol versus no paracetamol in patients aged ≥ 5 years with knowlesi malaria, conducted over a 2-year period at four hospital sites in Sabah, Malaysia. The primary endpoint of change in creatinine from enrolment to 72 h will be evaluated by analysis of covariance (ANCOVA) using enrolment creatinine as a covariate. Secondary endpoints include longitudinal changes in markers of oxidative stress (plasma F2-isoprostanes and isofurans) and markers of endothelial activation/Weibel-Palade body release (angiopoietin-2, von Willebrand Factor, P-selectin, osteoprotegerin) over 72 h, as well as blood and urine biomarkers of AKI. This study will be powered to detect a difference between the two treatment arms in a clinically relevant population including adults and children with knowlesi malaria of any severity.
DISCUSSION: Paracetamol is widely available and has an excellent safety profile; if a renoprotective effect is demonstrated, this trial will support the administration of regularly dosed paracetamol to all patients with knowlesi malaria. The secondary outcomes in this study will provide further insights into the pathophysiology of haemolysis-induced oxidative damage and acute kidney injury in knowlesi malaria and other haemolytic diseases.
TRIAL REGISTRATION: Clinicaltrials.gov, NCT03056391 . Registered on 12 October 2016.
METHODS: PACKNOW was an open-label, randomized, controlled trial of acetaminophen (500 mg or 1000 mg every 6 hours for 72 hours) vs no acetaminophen in Malaysian patients aged ≥5 years with knowlesi malaria of any severity. The primary end point was change in creatinine at 72 hours. Secondary end points included longitudinal changes in creatinine in patients with severe malaria or acute kidney injury (AKI), stratified by hemolysis.
RESULTS: During 2016-2018, 396 patients (aged 12-96 years) were randomized to acetaminophen (n = 199) or no acetaminophen (n = 197). Overall, creatinine fell by a mean (standard deviation) 14.9% (18.1) in the acetaminophen arm vs 14.6% (16.0) in the control arm (P = .81). In severe disease, creatinine fell by 31.0% (26.5) in the acetaminophen arm vs 20.4% (21.5) in the control arm (P = .12), and in those with hemolysis by 35.8% (26.7) and 19% (16.6), respectively (P = .07). No difference was seen overall in patients with AKI; however, in those with AKI and hemolysis, creatinine fell by 34.5% (20.7) in the acetaminophen arm vs 25.9% (15.8) in the control arm (P = .041). Mixed-effects modeling demonstrated a benefit of acetaminophen at 72 hours (P = .041) and 1 week (P = .002) in patients with severe malaria and with AKI and hemolysis (P = .027 and P = .002, respectively).
CONCLUSIONS: Acetaminophen did not improve creatinine among the entire cohort but may improve renal function in patients with severe knowlesi malaria and in those with AKI and hemolysis.
CLINICAL TRIALS REGISTRATION: NCT03056391.
METHODS AND ANALYSIS: PRoVENT-iMIC (study of PRactice of VENTilation in Middle-Income Countries) is an international multicentre observational study to be undertaken in approximately 60 ICUs in 11 Asian countries. Consecutive patients aged 18 years or older who are receiving invasive ventilation in participating ICUs during a predefined 28-day period are to be enrolled, with a daily follow-up of 7 days. The primary outcome is ventilatory management (including tidal volume expressed as mL/kg predicted body weight and positive end-expiratory pressure expressed as cm H2O) during the first 3 days of mechanical ventilation-compared between patients at no risk for acute respiratory distress syndrome (ARDS), patients at risk for ARDS and in patients with ARDS (in case the diagnosis of ARDS can be made on admission). Secondary outcomes include occurrence of pulmonary complications and all-cause ICU mortality.
ETHICS AND DISSEMINATION: PRoVENT-iMIC will be the first international study that prospectively assesses ventilation practices, outcomes and epidemiology of invasively ventilated patients in ICUs in Asia. The results of this large study, to be disseminated through conference presentations and publications in international peer-reviewed journals, are of ultimate importance when designing trials of invasive ventilation in resource-limited ICUs. Access to source data will be made available through national or international anonymised datasets on request and after agreement of the PRoVENT-iMIC steering committee.
TRIAL REGISTRATION NUMBER: NCT03188770; Pre-results.
METHODS AND FINDINGS: Clinical efficacy studies of uncomplicated P. vivax treated with DP or AL and published between January 1, 2000, and January 31, 2018, were identified by conducting a systematic review registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42016053310. Investigators of eligible studies were invited to contribute individual patient data that were pooled using standardised methodology. The effect of mg/kg dose of piperaquine/lumefantrine, ACT administered, and PQ on the rate of P. vivax recurrence between days 7 and 42 after starting treatment were investigated by Cox regression analyses according to an a priori analysis plan. Secondary outcomes were the risk of recurrence assessed on days 28 and 63. Nineteen studies enrolling 2,017 patients were included in the analysis. The risk of recurrent P. vivax at day 42 was significantly higher in the 384 patients treated with AL alone (44.0%, 95% confidence interval [CI] 38.7-49.8) compared with the 812 patients treated with DP alone (9.3%, 95% CI 7.1-12.2): adjusted hazard ratio (AHR) 12.63 (95% CI 6.40-24.92), p < 0.001. The rates of recurrence assessed at days 42 and 63 were associated inversely with the dose of piperaquine: AHRs (95% CI) for every 5-mg/kg increase 0.63 (0.48-0.84), p = 0.0013 and 0.83 (0.73-0.94), p = 0.0033, respectively. The dose of lumefantrine was not significantly associated with the rate of recurrence (1.07 for every 5-mg/kg increase, 95% CI 0.99-1.16, p = 0.0869). In a post hoc analysis, in patients with symptomatic recurrence after AL, the mean haemoglobin increased 0.13 g/dL (95% CI 0.01-0.26) for every 5 days that recurrence was delayed, p = 0.0407. Coadministration of PQ reduced substantially the rate of recurrence assessed at day 42 after AL (AHR = 0.20, 95% CI 0.10-0.41, p < 0.001) and at day 63 after DP (AHR = 0.08, 95% CI 0.01-0.70, p = 0.0233). Results were limited by follow-up of patients to 63 days or less and nonrandomised treatment groups.
CONCLUSIONS: In this study, we observed the risk of P. vivax recurrence at day 42 to be significantly lower following treatment with DP compared with AL, reflecting the longer period of post-treatment prophylaxis; this risk was reduced substantially by coadministration with PQ. We found that delaying P. vivax recurrence was associated with a small but significant improvement in haemoglobin. These results highlight the benefits of PQ radical cure and also the provision of blood-stage antimalarial agents with prolonged post-treatment prophylaxis.
METHODS: Using rapid evaluation methods, we will use four data collection methods: 1) registry embedded indicators to assess quality of care processes and their associated outcomes; 2) process mapping to provide a preliminary framework to understand gaps between current and desired care practices; 3) structured observations of processes of interest identified from the process mapping and; 4) focus group discussions with stakeholders to identify barriers and enablers influencing the gap between current and desired care practices. We will also collect self-assessments of readiness for quality improvement. Data collection and analysis will be led by local stakeholders, performed in parallel and through an iterative process across eight countries: Kenya, India, Malaysia, Nepal, Pakistan, South Africa, Uganda and Vietnam.
CONCLUSIONS: The results of our study will provide essential information on where and how care processes can be improved to facilitate better quality of care to critically ill patients in LMICs; thus, reduce preventable mortality and morbidity in ICUs. Furthermore, understanding the rapid evaluation methods that will be used for this study will allow other researchers and healthcare professionals to carry out similar research in ICUs and other health services.