Plasmodium Vivax Malaria Research Articles

Plasmodium cynomolgi Infections Not Found in Microscopy-Diagnosed Malaria Cases across Sabah, Malaysia

Zoonotic malaria presents a major public health challenge in Southeast Asia. Plasmodium cynomolgi coinfects the same macaque hosts and mosquito vectors as the most common cause of zoonotic malaria, Plasmodium knowlesi. Plasmodium cynomolgi appears morphologically similar to Plasmodium vivax on microscopy and can amplify P. vivax polymerase chain reaction (PCR) assays, confounding transmission estimates. We screened 2,103 samples for P. cynomolgi across all 26 districts in Sabah, Malaysia, from 2010 to 2021. Samples comprised 1,425 P. knowlesi, 256 P. vivax, 293 P. falciparum, and 31 Plasmodium malariae PCR-confirmed malaria cases and 100 malaria microscopy-positive and species-specific PCR-negative samples. A nested PCR assay targeting P. cynomolgi-specific 18S small subunit ribosomal ribonucleic acid with a detection limit of ∼2 parasites/µL was conducted on whole blood samples. No P. cynomolgi infections were detected. Symptomatic P. cynomolgi co-infections appear rare in Malaysia, although prevalence may be underestimated owing to the absence of routine molecular screening and the sensitivity of available assays.

Association between RANTES/CCL5 levels with Plasmodium infections and malaria severity: a systematic review

BackgroundMalaria continues to be a significant global health concern, and developing effective therapeutic strategies requires an understanding of the immune response to the disease. This systematic review synthesized the current body of research on the role of regulated on activation, normal T cell expressed and secreted (RANTES)—in the pathogenesis and disease severity of malaria.MethodsA systematic review protocol was registered with PROSPERO under the registration number CRD42024535822. The systematic review was conducted following PRISMA guidelines to identify studies examining RANTES levels in individuals infected with Plasmodium species. Searches were performed across multiple databases, including ProQuest, Journals@Ovid, Embase, Scopus, PubMed, and MEDLINE. Further searches were performed in Google Scholar. Quality assessment was done using the Joanna Briggs Institute (JBI) critical appraisal tools. Alterations in RANTES levels in patients with malaria were synthesized narratively.ResultsA comprehensive search of major databases identified 22 studies meeting inclusion criteria, predominantly focusing on Plasmodium falciparum and Plasmodium vivax infections. RANTES levels were found to vary significantly across different severities of malaria, with several studies reporting lower levels in severe cases compared to non-malarial controls. However, inconsistencies were observed in the alterations of RANTES levels between severe and non-severe malaria cases.ConclusionTaken together, the finding of this systematic review underscore the complex regulation of RANTES in malaria pathophysiology. Future research should focus on longitudinal assessments to elucidate the dynamic role of RANTES throughout the course of malaria and recovery, to potentially inform the design of novel therapeutic strategies.

Severe Plasmodium vivax Malaria Complicated with Acute Leukoencephalopathy with Restricted Diffusion.

Severe Plasmodium vivax Malaria Complicated with Acute Leukoencephalopathy with Restricted Diffusion.

Potential Role of the Eastern Mosquitofish (Gambusia holbrooki) in the Spread of the Harmful Fish Parasite, Asian Tapeworm Schyzocotyle (Bothriocephalus) acheilognathi

Biological control of undesirable organisms and pathogens often involves the introduction of alien species into new regions. However, alien species themselves pose a potential threat to local ecosystems and economies. The Eastern mosquitofish Gambusia holbrooki is recognised as a dangerous invasive species, but despite this, it is still used for biological control of mosquito larvae, a potential vector of malaria plasmodium transmission to humans, on the Black Sea coast of the Caucasus. We focused on the species composition of helminths in this fish species. We detected adult nematodes Pseudocapillaria (Pseudocapillaria) tomentosa and cestodes Schyzocotyle acheilognathi (formely Bothriocephalus acheilognathi). The above nematode was observed for the first time in fish of the genus Gambusia. Importantly, the cestode S. acheilognathi, which we found in G. holbrooki, is the most successful invasive alien parasite species in freshwaters of the planet and is extremely hazardous to natural ecosystems and aquaculture as it can cause mass mortality of young fish. Thus, the current practice of transferring mosquitofish between water bodies on the Black Sea coast of the Caucasus to control mosquito larvae may contribute to the undesirable spread of a harmful quarantine fish parasite.

Recurrence patterns and evolution of submicroscopic and asymptomatic Plasmodium vivax infections in malaria-endemic areas of the Peruvian Amazon.

In the Peruvian Amazon, Plasmodium vivax malaria transmission is maintained due to the high frequency of recurrences. By understanding the recurrence rates of submicroscopic and asymptomatic cases, we can develop informed strategies to prevent transmission more efficiently and disrupt the silent transmission cycle. A three-year, population-based cohort study was conducted in two sites, Cahuide and Lupuna, within the Loreto region in Peru from 2013 to 2015. The study included 385 individuals and aimed to examine the temporal dynamics of malaria recurrences and their impact on transmission and control. Individuals from Lupuna presented a higher risk of P. vivax infections compared to Cahuide, where most recurrences were asymptomatic and submicroscopic. It is estimated that a great proportion of these recurrences were due to relapses in both communities. The application of molecular diagnostic method proved to be significantly more effective, detecting 2.3 times more episodes during the follow-up (PCR, 1068; microscopy, 467). PCR identified recurrences significantly earlier, at 151 days after an initial infection, compared to microscopy, which detected them on average after 365 days. Community, occupation and previous malaria infections were factors associated with recurrences. Finally, potential infection evolution scenarios were described where one frequent scenario involved the transition from symptomatic to asymptomatic infections with a mean evolution time of 240 days. This study explores the contrast in malaria recurrence risk among individuals from two endemic settings, a consequence of prolonged exposure to the parasite. Through the analysis of the evolution scenarios of P. vivax recurrences, it is possible to have a more complete vision of how the transmission pattern changes over time and is conditioned by different factors.

Biogenesis of Cytochromes c and c1 in the Electron Transport Chain of Malaria Parasites.

Plasmodium malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and is a key antimalarial drug target. ETC function requires cytochromes c and c1, which are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome c synthase (HCCS) that binds heme and the apo cytochrome to facilitate the biogenesis of the mature cytochrome c or c1. Although humans encode a single bifunctional HCCS that attaches heme to both proteins, Plasmodium parasites are like yeast and encode two separate HCCS homologues thought to be specific for heme attachment to cyt c (HCCS) or cyt c1 (HCC1S). To test the function and specificity of Plasmodium falciparum HCCS and HCC1S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC1S knockdown selectively impaired cyt c1 biogenesis and caused lethal ETC dysfunction that was not reversed by the overexpression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in Escherichia coli of cyt c hemylation by P. falciparum HCCS and HCC1S strongly suggest that both homologues are essential for mitochondrial ETC function and have distinct specificities for the biogenesis of cyt c and c1, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.

Expanding the occurrence of antimalarial metabolites in dorid nudibranch Hypselodoris tryoni

This study examined the antimalarial activity of a furanosesquiterpene, furodysinin, one of the major metabolites of the dorid nudibranch Hypselodoris tryoni. The nudibranchs were collected from Balinese waters and the metabolites were purified by chromatography. Ex vivo rodent malaria Plasmodium berghei assays were conducted to determine the metabolite antimalarial activity. In silico molecular docking was employed to investigate the interaction between furodysinin against wild-type P. berghei and atovaquone-resistant P. berghei (Y268C). This study reported for the first time that the furodysinin displayed a promising antimalarial activity based on the ex vivo tests against wild-type P. berghei and atovaquone-resistant P. berghei. In silico molecular docking study showed that furodysinin inhibits the parasite mitochondrial cytochrome b (cyt b) by binding to the protein Qo pocket (ef-helix) where it interacts with residue 268, the mutation of which is known to confer resistance to atovaquone. Furodysinin binds to the mutated tyrosine at residue 268, which has changed to cysteine, forming an alkyl bond with C268 at a distance of 4.6 Å. Therefore, we predict that furodysinin has a target in Plasmodium mitochondria.

Global health at local level: characterization of tropical diseases in northern Portugal, 2020-2024

Abstract Background The escalating global prevalence and geographic spread of tropical diseases pose significant global health risks worldwide, with local implications. In Portugal, these diseases require mandatory notification. Concerns were raised following the detection of Aedes albopictus mosquitoes in mainland Portugal in 2017, alongside a Dengue outbreak in Madeira in 2012. Despite Portugal being declared malaria-free, the detection of Anopheles mosquitoes also raises concerns. This study aims at characterizing notified cases of tropical diseases in two cities in Northern Portugal since 2020. Methods A retrospective study of Malaria, Dengue, Zika, and Chikungunya cases in the Maia & Valongo’s Public Health Unit included data from the National Epidemiological Surveillance System covering January 2020 to April 2024. Descriptive analysis was conducted using SPSS IBM for iOS® v29. Results are presented as mean ± standard deviation for continuous variables and proportions for categorical ones. Results Five malaria cases were reported, predominantly affecting males (80%), with an average age of 34±16.96 years. Four were Portuguese and one Angolan. All cases were imported, mostly from Angola (80%). Etiological analysis revealed two cases of Plasmodium malariae and three of Plasmodium falciparum. All were hospitalized, no deaths. Four dengue cases were documented, 50% in males, averaged 33±24.73 years. Three were Portuguese and one Mozambican. One was hospitalized with no deaths. All cases were imported from Brazil, Cuba, Indonesia, and islands of Saint Martin/Saint-Barthélemy. No Zika or Chikungunya confirmed cases were identified during this period. Conclusions The increasing incidence of Dengue and Malaria underscores the critical connection between infectious diseases, vector ecology, and global health security. This study highlights the risk of imported Dengue and Malaria cases, emphasizing the imperative for surveillance and preventive actions to protect public health. Key messages • Dengue and Malaria cases in Portugal underline the importance of international collaboration and robust strategies. • Stepped-up surveillance is key to protecting vulnerable groups from imported infections.

Optimization of loop mediated isothermal amplification assay (LAMP) for detection of chloroquine resistance in P. vivax malaria

Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.

Locally Acquired (Autochthonous) Mosquito-Transmitted Plasmodium vivax Malaria - Saline County, Arkansas, September 2023.

A case of locally acquired (autochthonous) mosquito-transmitted Plasmodium vivax malaria was diagnosed in Arkansas in September 2023. This represents the 10th autochthonous case identified nationally in 2023, after 20 years without recorded local mosquitoborne malaria transmission in the United States. The public health response included case investigation, active case surveillance, mosquito surveillance and control, assessment of medical countermeasures, and clinical and public outreach. Prompt diagnosis and appropriate treatment of malaria can improve clinical outcomes and, in addition to vector control, minimize risk for local transmission. Clinicians should consider malaria among patients who have traveled to countries where malaria is endemic, or with unexplained fever regardless of travel history. Although the risk for autochthonous malaria in the United States remains very low, its reemergence highlights the importance of vectorborne disease preparedness and response. Examples of such efforts include improving awareness among clinicians, access to diagnostics and antimalarial medications, and capacity for mosquito surveillance and control. Collaboration and communication among CDC, health departments, local jurisdictions, clinicians, hospitals, laboratories, and the public can support rapid malaria diagnosis, prevention, and control. Before traveling internationally to areas where malaria is endemic, travelers should consult with their health care provider regarding recommended malaria prevention measures, including chemoprophylaxis and precautions to avoid mosquito bites, to reduce both personal and community risk.

An Uncommon Case of Plasmodium vivax Malaria with Disseminated Intravascular Coagulation: Rare Case Report with Review of Literature.

Malaria is a severe health issue in India despite government efforts. Malaria has many complications, some of which can be life-threatening. Disseminated intravascular coagulation (DIC) is a prominent malaria complication, besides renal failure, cerebral malaria, pulmonary edema, and anemia. We report a young man with Plasmodium vivax malaria who developed septic shock due to DIC. A 25-year-old male had a fever, abdominal pain, vomiting, and nausea for 5 days. On clinical examination, the patient was in distress and had a fever of 102.6°F with a blood pressure of 86/46 mmHg. A complete blood count revealed anemia, leukocytopenia, and severe thrombocytopenia. Peripheral blood smear findings showed a P. vivax parasite in red blood cells with the presence of a few schistocytes. Further coagulation tests showed raised prothrombin, partial thromboplastin time, and d-dimer. DIC and septic shock required critical care unit admission. Conservative treatment with artesunate and lumefantrine completely cured him. This unusual case shows that P. vivax causes severe malaria and its morbid consequences. It stresses early diagnosis and treatment to prevent mortality and morbidity.

In vivo efficacy of chloroquine plus primaquine combination therapy against uncomplicated Plasmodium vivax malaria in Limu Kossa District, Jimma Zone, Southwest Ethiopia.

Plasmodium vivaxis the second most common malaria parasite in Ethiopia. It has been treated with chloroquine (CQ) for the past seven decades. However, the emergence of CQ-resistant strains in the nation urged the Federal Ministry of Health of Ethiopia to review its national malaria treatment guideline in 2018.In the revised guideline, thefirst-line treatment for uncomplicatedP. vivaxinfection is a combination of CQ and primaquine (PQ). Thus, the present study was designed to evaluate the in vivo efficacy of CQ and PQ combination therapy against clinicalP. vivaxmono-infection in one of the malaria-endemic areas of Ethiopia. Anopen-label prospective clinical trialwas conducted in the Limmu Kossa District, Jimma zone, Southwest Ethiopia, from September 2023 to March 2024. A total of 108 patients were recruited for the study. All participants received treatment with CQ at a dosage of 25mg/kg over three days, followed by PQ at 0.25mg/kg for 14 consecutive days. Patients were monitored for 42days for any signs of treatment failure and malaria clinical symptoms, as per the World Health Organization (WHO) guidelines for anti-malarial drug evaluation. Additionally, haemoglobin (Hb) levels, body temperature,any adverse events, and signs of haemolysis were assessed.Data was analysed using R-software (version 4.0.0) and a significant level was considered at p < 0.05. The median age of the patients was 23years, ranging from 2.5 to 62years. Of the 108 patients initially recruited, 100 completed the 42-day follow-up period. The combination therapy of CQ and PQ for uncomplicated clinicalP. vivaxmalaria demonstrated excellent therapeutic efficacy, with a 100% cure rate observed at both day 28 and day 42. Additionally, the recommended low dose of PQ (0.25mg/kg) was well-tolerated, with no signs of. Additionally, most common malaria symptoms were disappeared early in the follow-up period. The combination of CQ plus PQ has exhibited excellent efficacy against uncomplicatedP. vivaxmalaria mono-infections. To preserve this efficacy, it is critical to ensure patients adhere to the full course of PQ treatment, despite its extended duration. Therefore, health authorities should put emphasis on the boosting of the public on the importance of finishing the prescribed medication regimen.

Ligand-based pharmacophore modeling, virtual screening, and molecular dynamics simulations of Pfhsp90 fingerprints in Plasmodium malaria treatment

The World Health Organization (WHO) documents malaria as one of the leading causes of high morbidity and mortality worldwide. The disease affects millions and kills thousands of people annually. Efforts to reduce the global burden of malaria have prompted the WHO to recommend prevention strategies such as using antimalarial drugs. However, these strategies have been ineffective because of antimalarial drug resistance. The only efficacious malaria treatment is Artemisinin-based Combination Therapy (ACT). However, the extended ACT clearance times, linked to the emergence of artemisinin monotherapy resistance recorded most recently in Africa and the Great Mekong region, pose a danger to its efficacy. Therefore, better efficacious antimalarial drugs are required. Since P. falciparum heat shock protein 90 (PfHsp90) is a well-characterized malaria drug target, this study uses it to discover more efficacious antimalarial drugs. An in silico approach was used to discover PfHsp90 inhibitors with pharmacological properties against Plasmodium malaria using a molecular dynamics simulation (MDS) and hierarchical virtual screening. Geldanamycin (GDM), a well-known anti-PfHsp90 compound, was used to identify PfHsp90 inhibitors with pharmacological properties against Plasmodium malaria by screening it against the ZINC20 database via the ZINCPHARMER web server. This virtual screening process resulted in 17 hits. These ZINCPHARMER hits were subjected to drug-likeness and pharmacokinetics properties analysis in the SwissADME web server, and nine of them satisfied the requirements. The nine ZINC20 compounds were docked with PfHsp90 using the PyRx software version 0.8 to understand their interactions. From the molecular docking results, ZINC09060002 (−8.2 kcal/mol), ZINC72133064 (−7.8 kcal/mol), ZINC72163401 (−7.7 kcal/mol), ZINC72358537 (−8.1 kcal/mol), and ZINC72358557 (−7.6 kcal/mol) had better binding affinities to PfHsp90 than GDM (−7.5 kcal/mol). The stability of these molecularly docked protein–inhibitor complexes was assessed through MDS using GROMACS 2022. ZINC72163401, ZINC72358537, and ZINC72358557 formed stable complexes with PfHsp90. The lead compounds were subjected to in vitro validation for their inhibitory capability. They showed promising inhibition of parasite growth with IC50 values ranging between 200 and 400 ng/mL. In this regard, the three PfHsp90 inhibitors can be further developed as potential antimalarial drugs. However, further structural optimization studies and clinical (in vivo) tests are necessary to ascertain the antimalarial activity of these compounds in humans.

Development and longevity of naturally acquired antibody and memory B cell responses against Plasmodium vivax infection.

Plasmodium vivax malaria causes significant public health problems in endemic regions. Considering the rapid spread of drug-resistant parasite strains and the development of hypnozoites in the liver with potential for relapse, development of a safe and effective vaccine for preventing, controlling, and eliminating the infection is critical. Immunity to malaria is mediated by antibodies that inhibit sporozoite or merozoite invasion into host cells and protect against clinical disease. Epidemiologic data from malaria endemic regions show the presence of naturally acquired antibodies to P. vivax antigens during and following infection. But data on the persistence of these antibodies, development of P. vivax-specific memory B cells (MBCs), and their relation to reduction of malaria severity and risk is limited. This review provides an overview of the acquisition and persistence of naturally acquired humoral immunity to P. vivax infection. Also, we summarize and discuss current progress in assessment of immune responses to candidate vaccine antigens in P. vivax patients from different transmission settings. Longitudinal studies of MBC and antibody responses to these antigens will open new avenues for developing vaccines against malaria infection and its transmission.

Relationship between red blood cell polymorphisms and effectiveness of seasonal malaria chemoprevention in 2020 in Dangassa, Mali.

Treatment failure with amodiaquine was reported in Dangassa, where red blood cell (RBC) polymorphisms are found and seasonal malaria chemoprevention (SMC) is underway. Here, we aimed at assessing the influence of RBC polymorphisms on SMC effectiveness. This was a secondary analysis of data from a study conducted in Dangassa. Children aged 5 to 14years enrolled in an open randomized study were assigned either to receive SMC (intervention arm) or not (control arm). SMC was implemented from July to November. For all children, hemoglobin type and blood group were determined at enrolment in July, and parasitemia and hemoglobin level were monthly monitored by finger-prick. Overall, 166 children were enrolled among which 82 (49.40%) in the control arm and 84 (50.60%) in the SMC arm. The prevalence of HbAS was 10.24% (17/166) with 12.20% in the control and 8.33% in the SMC arm. O group was the most common overall (45%) and in the SMC arm (54%), but the control arm had more B (39.02%) than O (36.59%). In the SMC arm, no case of Plasmodium infection and malaria disease was observed in the 7 HbAS children while in Non-HbAS children, peaks of infection and disease prevalence were respectively observed in October (24.66%) and November (7.14%). For the SMC arm, in group O and Non-group O, Plasmodium infection cases were observed from August to December. Plasmodium infection and malaria disease were more frequently observed in HbAS children in the control arm than in the SMC arm. Further studies are needed to assess factors associated with the asymptomatic carriage of parasites during SMC in Dangassa. NCT04149106.

The New Spectrum of Plasmodium Vivax Malaria Severity: A Single-Center Experience

Plasmodium vivax malaria, once considered a benign and self-limiting disease, has emerged as a significant public health concern, with increasing reports of severe and even fatal cases. Objective: To evaluate the clinical severity and complications associated with Plasmodium vivax malaria in patients in Peshawar. Methods: A descriptive cross-sectional study was conducted at Lady Reading Hospital, Peshawar, for about two months. A total of 160 patients diagnosed with Plasmodium vivax malaria were enrolled. Participants were divided into two groups based on platelet count. Laboratory tests, including complete blood count, liver function tests, and Renal Function Tests were performed. Data were analyzed using SPSS version 20.0 , An Independent sample t-test was used to assess the significant difference between the two groups with statistical significance set at p&lt;0.05. Results: A total of 165 patients were included in the study. In the severe thrombocytopenia group (&lt;100,000/µL), there were 23 cases of anemia, jaundice (n=25), acute renal failure (n=7), cerebral malaria (n=2), seizures (n=1) and hypoglycemia (n=1). In the less severe thrombocytopenia group (&gt;100,000/µL), there were 14 cases of anemia, jaundice (n=17), and acute renal failure (n=2). The blood glucose level (p=0.37), systolic blood pressure (p=0.18) and pulse rate (p=0.21) revealed no significant differences between the two groups. Conclusions: It was concluded that severe thrombocytopenia in P. vivax malaria was associated with more severe clinical manifestations, with a few cases requiring transfusions. Patients with less severe thrombocytopenia had fewer complications

Methaemoglobin as a surrogate marker of primaquine antihypnozoite activity in Plasmodium vivax malaria: A systematic review and individual patient data meta-analysis.

The 8-aminoquinolines, primaquine and tafenoquine, are the only available drugs for the radical cure of Plasmodium vivax hypnozoites. Previous evidence suggests that there is dose-dependent 8-aminoquinoline induced methaemoglobinaemia and that higher methaemoglobin concentrations are associated with a lower risk of P. vivax recurrence. We undertook a systematic review and individual patient data meta-analysis to examine the utility of methaemoglobin as a population-level surrogate endpoint for 8-aminoquinoline antihypnozoite activity to prevent P. vivax recurrence. We conducted a systematic search of Medline, Embase, Web of Science, and the Cochrane Library, from 1 January 2000 to 29 September 2022, inclusive, of prospective clinical efficacy studies of acute, uncomplicated P. vivax malaria mono-infections treated with radical curative doses of primaquine. The day 7 methaemoglobin concentration was the primary surrogate outcome of interest. The primary clinical outcome was the time to first P. vivax recurrence between day 7 and day 120 after enrolment. We used multivariable Cox proportional-hazards regression with site random-effects to characterise the time to first recurrence as a function of the day 7 methaemoglobin percentage (log base 2 transformed), adjusted for the partner schizonticidal drug, the primaquine regimen duration as a proxy for the total primaquine dose (mg base/kg), the daily primaquine dose (mg/kg), and other factors. The systematic review protocol was registered with PROSPERO (CRD42023345956). We identified 219 P. vivax efficacy studies, of which 8 provided relevant individual-level data from patients treated with primaquine; all were randomised, parallel arm clinical trials assessed as having low or moderate risk of bias. In the primary analysis data set, there were 1,747 patients with normal glucose-6-phosphate dehydrogenase (G6PD) activity enrolled from 24 study sites across 8 different countries (Indonesia, Brazil, Vietnam, Thailand, Peru, Colombia, Ethiopia, and India). We observed an increasing dose-response relationship between the daily weight-adjusted primaquine dose and day 7 methaemoglobin level. For a given primaquine dose regimen, an observed doubling in day 7 methaemoglobin percentage was associated with an estimated 30% reduction in the risk of P. vivax recurrence (adjusted hazard ratio = 0.70; 95% confidence interval [CI] [0.57, 0.86]; p = 0.0005). These pooled estimates were largely consistent across the study sites. Using day 7 methaemoglobin as a surrogate endpoint for recurrence would reduce required sample sizes by approximately 40%. Study limitations include the inability to distinguish between recrudescence, reinfection, and relapse in P. vivax recurrences. For a given primaquine regimen, higher methaemoglobin on day 7 was associated with a reduced risk of P. vivax recurrence. Under our proposed causal model, this justifies the use of methaemoglobin as a population-level surrogate endpoint for primaquine antihypnozoite activity in patients with P. vivax malaria who have normal G6PD activity.

Molecular survey of pfmdr-1, pfcrt, and pfk13 gene mutations among patients returning from Plasmodium falciparum endemic areas to Turkey

BackgroundIn recent years, there has been an increasing trend in the number of imported Plasmodium falciparum cases in Turkey. To improve treatment success and to better understand malaria epidemiology among imported cases, it is necessary to determine anti-malarial drug resistance. This study aimed to survey polymorphisms of resistance genes in imported P. falciparum patients using archived thin smear preparations and EDTA blood samples.MethodsA total of 100 imported P. falciparum patients admitted to Bakırköy Dr. Sadi Konuk Research and Training Hospital between 2017 and 2022 were included in this study. DNA extraction was performed using an archived slide and EDTA blood samples that were microscopically diagnosed. After confirming the samples by real-time PCR, the pfmdr1, pfcrt, and pfk13 genes were amplified and sequenced. Single nucleotide polymorphisms (SNPs) were screened using Geneious R9 software, with the reference P. falciparum clone 3D7 isolate.ResultsAll studied samples were confirmed to be P. falciparum using real-time PCR. Nested PCR was conducted and the pfcrt (92 samples), pfmdr1 (91 samples), and pfk13 (93 samples) genes were successfully amplified. Sequence analysis revealed the highest mutation rate in the pfmdr1 (74.5%) gene, with the identification of five haplotypes: NYSND (wild-type, 23%), NFSND (56%), NYSDD (2.2%), NFSDD (15.4%), and YFSND (3.4%)]. The pfcrt mutation was identified in 11 samples (12.2%), whereas the pfk13 mutation was found in only two samples.ConclusionThis study is the first molecular survey of anti-malarial drug resistance genes in Turkey. With the increasing number of imported Plasmodium malaria cases and recent reports of sporadic indigenous P. falciparum cases, malaria is becoming a growing concern in Turkey. Although molecular screening for resistance markers in P. falciparum malaria is not routinely conducted, the data from this study will enhance treatment success rates and contribute to global malaria elimination.

Active diagnostic ingredients (ADIs) for PCR: A mini-bioreactor producing dNTPs with silica immobilized R5-kinases.

Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2'-deoxynucleoside 5'-triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self-immobilizing R5-silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5-kinase mini-bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in Plasmodium malariae NAATs, a high linear correlation was shown between the Ct value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini-bioreactor investigated as a proof-of-principle for work-flow integration with NAAT in low-resource research and diagnostics labs.

Population Genomics of Plasmodium malariae from Four African Countries.

Plasmodium malariae is geographically widespread but neglected and may become more prevalent as P. falciparum declines. We completed the largest genomic study of African P. malariae to-date by performing hybrid capture and sequencing of 77 isolates from Cameroon (n=7), the Democratic Republic of the Congo (n=16), Nigeria (n=4), and Tanzania (n=50) collected between 2015 and 2021. There is no evidence of geographic population structure. Nucleotide diversity was significantly lower than in co-localized P. falciparum isolates, while linkage disequilibrium was significantly higher. Genome-wide selection scans identified no erythrocyte invasion ligands or antimalarial resistance orthologs as top hits; however, targeted analyses of these loci revealed evidence of selective sweeps around four erythrocyte invasion ligands and six antimalarial resistance orthologs. Demographic inference modeling suggests that African P. malariae is recovering from a bottleneck. Altogether, these results suggest that P. malariae is genomically atypical among human Plasmodium spp. and panmictic in Africa.