Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

BACKGROUND: The interaction between iron status and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. METHODS: We retrieved data and samples from 55 participants (19 female) enrolled in malaria VIS, and 171 patients (45 female) with malaria and 30 healthy controls (13 female) enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. FINDINGS: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline ferritin was associated with post-treatment increases in liver transaminase levels. In Malaysian patients with malaria, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. By day 28, hepcidin had normalised; however, ferritin and sTfR both remained elevated. INTERPRETATION: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency. FUNDING: National Health and Medical Research Council (Program Grant 1037304, Project Grants 1045156 and 1156809; Investigator Grants 2016792 to BEB, 2016396 to JCM, 2017436 to MJG); US National Institute of Health (R01-AI116472-03); Malaysian Ministry of Health (BP00500420).

Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots.

Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence. Methods: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield™) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls. Results: The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/µL for P. knowlesi and 0.002 parasites/µL for both P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/µL); Divis et al. real-time 18S rRNA (0.0002 parasites/µL); Lubis et al. hemi-nested SICAvar (1.1 parasites/µL) and Lee et al. nested 18S rRNA (11 parasites/µL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/µL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi. Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

Characterisation of Plasmodium vivax lactate dehydrogenase dynamics in P. vivax infections.

Plasmodium vivax lactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway of P. vivax. It is widely used as a diagnostic biomarker and a measure of total-body parasite biomass in vivax malaria. However, the dynamics of PvLDH remains poorly understood. Here, we developed mathematical models that capture parasite and matrix PvLDH dynamics in ex vivo culture and the human host. We estimated key biological parameters characterising in vivo PvLDH dynamics based on longitudinal data of parasitemia and PvLDH concentration collected from P. vivax-infected humans, with the estimates informed by the ex vivo data as prior knowledge in a Bayesian hierarchical framework. We found that the in vivo accumulation rate of intraerythrocytic PvLDH peaks at 10-20 h post-invasion (late ring stage) with a median estimate of intraerythrocytic PvLDH mass at the end of the life cycle to be 9.4 × 10-3ng. We also found that the median estimate of in vivo PvLDH half-life was approximately 21.9 h. Our findings provide a foundation with which to advance our quantitative understanding of P. vivax biology and will facilitate the improvement of PvLDH-based diagnostic tools.

Single cell transcriptomics shows that malaria promotes unique regulatory responses across multiple immune cell subsets.

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

Primaquine-induced Severe Hemolysis in the Absence of Concomitant Malaria: Effects on G6PD Activity and Renal Function.

Primaquine prevents relapses of Plasmodium vivax malaria but can cause severe hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The clinical and laboratory features of this outcome are usually confounded by the clinical and hemolytic effects of concomitant malaria. We describe a case of severe hemolysis occurring after a total dose of 2.04 mg/kg of primaquine used for prophylaxis in a young, G6PD-deficient (Kaiping variant), Australian man without malaria. During acute hemolysis, he had markedly elevated urinary beta-2-microglobulin, suggestive of renal tubular injury (a well-recognized complication of primaquine-induced hemolysis). He also had albuminuria and significantly increased excretion of glycocalyx metabolites, suggestive of glomerular glycocalyx degradation and injury. We show that regularly dosed paracetamol given for its putative renoprotective effect is safe in the context of severe oxidative hemolysis. Acute drug-induced hemolysis transiently increases G6PD activity. Cases such as this improve our understanding of primaquine-induced hemolysis and ultimately will help facilitate widespread safe and effective use of this critically important drug.

Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

Background: The interaction between iron deficiency and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. Methods: We retrieved samples and associated data from 55 participants enrolled in malaria VIS, and 171 malaria patients and 30 healthy controls enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. Results: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline iron status (ferritin) was associated with post-treatment increases in liver transaminase levels. In Malaysian malaria patients, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. Hepcidin normalised by day 28; however, ferritin and sTfR both remained elevated 4 weeks following admission. Conclusion: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency.

Plasmodium vivax malaria serological exposure markers: Assessing the degree and implications of cross-reactivity with P. knowlesi.

Serological markers are a promising tool for surveillance and targeted interventions for Plasmodium vivax malaria. P. vivax is closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivax and P. knowlesi are co-endemic across much of South East Asia, making it important to design serological markers that minimize cross-reactivity in this region. To determine the degree of IgG cross-reactivity against a panel of P. vivax serological markers, we assayed samples from human patients with P. knowlesi malaria. IgG antibody reactivity is high against P. vivax proteins with high sequence identity with their P. knowlesi ortholog. IgG reactivity peaks at 7 days post-P. knowlesi infection and is short-lived, with minimal responses 1 year post-infection. We designed a panel of eight P. vivax proteins with low levels of cross-reactivity with P. knowlesi. This panel can accurately classify recent P. vivax infections while reducing misclassification of recent P. knowlesi infections.

Diagnostic performance of a 5-plex malaria immunoassay in regions co-endemic for Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae and P. ovale.

Commercial point-of-care tests remain insufficient for accurately detecting and differentiating low-level malaria infections in regions co-endemic with multiple non-falciparum species, including zoonotic Plasmodium knowlesi (Pk). A 5-plex chemiluminescent assay simultaneously measures pan-Plasmodium lactate dehydrogenase (pLDH), P. falciparum (Pf)-LDH, P. vivax (Pv)-LDH, Pf-histidine-rich protein-2 (HRP2), and C-reactive protein. We assessed its diagnostic performance on whole blood (WB) samples from 102 healthy controls and 306 PCR-confirmed clinical cases of Pf, Pv, Pk, P. malariae (Pm) and P. ovale (Po) mono-infections from Southeast-Asia. We confirm its excellent HRP2-based detection of Pf. Cross-reactivity of Pf-LDH with all non-falciparum species tested was observed (specificity 57.3%). Pv-LDH performance was suboptimal for Pv (93.9% sensitivity and 73.9% specificity). Poor specificity was driven by strong Pk cross-reactivity, with Pv-LDH detecting 93.9% of Pk infections. The pan-LDH-to-Pf-LDH ratio was capable of discerning Pv from Pk, and robustly differentiated Pf from Pm or Po infection, useful in regions with hrp2/3 deletions. We tested the platform's performance in plasma for the first time, with WB outperforming plasma for all analytes except Pv-LDH for Pk. The platform is a promising tool for WB malaria diagnosis, although further development is warranted to improve its utility in regions co-endemic for multiple non-falciparum species.

The Effect of Regularly Dosed Acetaminophen vs No Acetaminophen on Renal Function in Plasmodium knowlesi Malaria (PACKNOW): A Randomized, Controlled Trial.

BACKGROUND: Acetaminophen inhibits cell-free hemoglobin-induced lipid peroxidation and improves renal function in severe falciparum malaria but has not been evaluated in other infections with prominent hemolysis, including Plasmodium knowlesi malaria. 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.

Longitudinal ex vivo and molecular trends of chloroquine and piperaquine activity against Plasmodium falciparum and P. vivax before and after introduction of artemisinin-based combination therapy in Papua, Indonesia.

Drug resistant Plasmodium parasites are a major threat to malaria control and elimination. After reports of high levels of multidrug resistant P. falciparum and P. vivax in Indonesia, in 2005, the national first-line treatment policy for uncomplicated malaria was changed in March 2006, to dihydroartemisinin-piperaquine against all species. This study assessed the temporal trends in ex vivo drug susceptibility to chloroquine (CQ) and piperaquine (PIP) for both P. falciparum and P. vivax clinical isolates collected between 2004 and 2018, by using schizont maturation assays, and genotyped a subset of isolates for known and putative molecular markers of CQ and PIP resistance by using Sanger and next generation whole genome sequencing. The median CQ IC50 values varied significantly between years in both Plasmodium species, but there was no significant trend over time. In contrast, there was a significant trend for increasing PIP IC50s in both Plasmodium species from 2010 onwards. Whereas the South American CQ resistant 7G8 pfcrt SVMNT isoform has been fixed since 2005 in the study area, the pfmdr1 86Y allele frequencies decreased and became fixed at the wild-type allele in 2015. In P. vivax isolates, putative markers of CQ resistance (no pvcrt-o AAG (K10) insertion and pvmdr1 Y967F and F1076L) were fixed at the mutant alleles since 2005. None of the putative PIP resistance markers were detected in P. falciparum. The ex vivo drug susceptibility and molecular analysis of CQ and PIP efficacy for P. falciparum and P. vivax after 12 years of intense drug pressure with DHP suggests that whilst the degree of CQ resistance appears to have been sustained, there has been a slight decline in PIP susceptibility, although this does not appear to have reached clinically significant levels. The observed decreasing trend in ex vivo PIP susceptibility highlights the importance of ongoing surveillance.

Endothelial glycocalyx degradation and disease severity in Plasmodium vivax and Plasmodium knowlesi malaria.

Degradation of the endothelial glycocalyx is associated with mortality in adult falciparum malaria. However, its role in the pathogenesis of non-falciparum malaria is unknown. In Malaysian patients with knowlesi (n = 200) and vivax (n = 61) malaria, and in healthy controls (n = 50), we measured glycocalyx breakdown products plasma syndecan-1 and urinary glycosaminoglycans, and evaluated correlations with biomarkers of disease severity. Urinary glycosaminoglycans were increased in patients with knowlesi and vivax malaria compared to healthy controls, and in knowlesi malaria were highest in those with severe disease. In knowlesi malaria, plasma syndecan-1 was also highest in those with severe disease, and correlated with markers of endothelial activation (angiopoietin-2, osteoprotegerin, ICAM-1), asymmetric dimethylarginine (ADMA) and impaired microvascular reactivity. Syndecan-1 also correlated with endothelial activation (ICAM-1, angiopoietin-2) and ADMA in vivax malaria. In knowlesi malaria increased syndecan-1 was associated with acute kidney injury, after controlling for age and parasitemia. In knowlesi malaria, the difference in median syndecan-1 between severe and non-severe disease was more marked in females than males. Endothelial glycocalyx degradation is increased in knowlesi and vivax malaria, and associated with disease severity and acute kidney injury in knowlesi malaria. Agents that inhibit glycocalyx breakdown may represent adjunctive therapeutics for severe non-falciparum malaria.

Reduced circulating dendritic cells in acute Plasmodium knowlesi and Plasmodium falciparum malaria despite elevated plasma Flt3 ligand levels.

BACKGROUND: Plasmodium falciparum malaria increases plasma levels of the cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic factor associated with dendritic cell (DC) expansion. It is unknown if the zoonotic parasite Plasmodium knowlesi impacts Flt3L or DC in human malaria. This study investigated circulating DC and Flt3L associations in adult malaria and in submicroscopic experimental infection. METHODS: Plasma Flt3L concentration and blood CD141+ DC, CD1c+ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria. RESULTS: Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141+ DCs, CD1c+ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi. CONCLUSIONS: In adults, submicroscopic Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141+ DCs, CD1c+ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.

A population of CD4hiCD38hi T cells correlates with disease severity in patients with acute malaria.

OBJECTIVE: CD4+ T cells are critical mediators of immunity to Plasmodium spp. infection, but their characteristics during malarial episodes and immunopathology in naturally infected adults are poorly defined. Flow cytometric analysis of PBMCs from patients with either P. falciparum or P. knowlesi malaria revealed a pronounced population of CD4+ T cells co-expressing very high levels of CD4 and CD38 we have termed CD4hiCD38hi T cells. We set out to gain insight into the function of these novel cells. METHODS: CD4+ T cells from 18 patients with P. falciparum or P. knowlesi malaria were assessed by flow cytometry and sorted into populations of CD4hiCD38hi or CD4norm T cells. Gene expression in the sorted populations was assessed by qPCR and NanoString. RESULTS: CD4hiCD38hi T cells expressed high levels of CD4 mRNA and canonical type 1 regulatory T-cell (TR1) genes including IL10, IFNG, LAG3 and HAVCR2 (TIM3), and other genes with relevance to cell migration and immunomodulation. These cells increased in proportion to malaria disease severity and were absent after parasite clearance with antimalarials. CONCLUSION: In naturally infected adults with acute malaria, a prominent population of type 1 regulatory T cells arises that can be defined by high co-expression of CD4 and CD38 (CD4hiCD38hi) and that correlates with disease severity in patients with falciparum malaria. This study provides fundamental insights into T-cell biology, including the first evidence that CD4 expression is modulated at the mRNA level. These findings have important implications for understanding the balance between immunity and immunopathology during malaria.

Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia.

BACKGROUND: The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. METHODS: Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 Plasmodium vivax, 21 Plasmodium falciparum, and 10 Plasmodium malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. RESULTS: The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95% CI 89.7-100) and 100% (95% CI 93.2-100), respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95% CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi. The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤ 0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95% CI 65.3-88.9) and specificity of 80.4% (95% CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi. CONCLUSION: The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

Transcriptional profiling and immunophenotyping show sustained activation of blood monocytes in subpatent Plasmodium falciparum infection.

OBJECTIVES: Malaria, caused by Plasmodium infection, remains a major global health problem. Monocytes are integral to the immune response, yet their transcriptional and functional responses in primary Plasmodium falciparum infection and in clinical malaria are poorly understood. METHODS: The transcriptional and functional profiles of monocytes were examined in controlled human malaria infection with P. falciparum blood stages and in children and adults with acute malaria. Monocyte gene expression and functional phenotypes were examined by RNA sequencing and flow cytometry at peak infection and compared to pre-infection or at convalescence in acute malaria. RESULTS: In subpatent primary infection, the monocyte transcriptional profile was dominated by an interferon (IFN) molecular signature. Pathways enriched included type I IFN signalling, innate immune response and cytokine-mediated signalling. Monocytes increased TNF and IL-12 production upon in vitro toll-like receptor stimulation and increased IL-10 production upon in vitro parasite restimulation. Longitudinal phenotypic analyses revealed sustained significant changes in the composition of monocytes following infection, with increased CD14+CD16- and decreased CD14-CD16+ subsets. In acute malaria, monocyte CD64/FcγRI expression was significantly increased in children and adults, while HLA-DR remained stable. Although children and adults showed a similar pattern of differentially expressed genes, the number and magnitude of gene expression change were greater in children. CONCLUSIONS: Monocyte activation during subpatent malaria is driven by an IFN molecular signature with robust activation of genes enriched in pathogen detection, phagocytosis, antimicrobial activity and antigen presentation. The greater magnitude of transcriptional changes in children with acute malaria suggests monocyte phenotypes may change with age or exposure.

Early Endothelial Activation Precedes Glycocalyx Degradation and Microvascular Dysfunction in Experimentally Induced Plasmodium falciparum and Plasmodium vivax Infection.

Endothelial activation and microvascular dysfunction are key pathogenic processes in severe malaria. We evaluated the early role of these processes in experimentally induced Plasmodium falciparum and P. vivax infection. Participants were enrolled in induced blood-stage malaria clinical trials. Plasma osteoprotegerin, angiopoietin-2, and von Willebrand Factor (vWF) levels were measured as biomarkers of endothelial activation. Microvascular function was assessed using peripheral arterial tonometry and near-infrared spectroscopy, and the endothelial glycocalyx was assessed by sublingual videomicroscopy and measurement of biomarkers of degradation. Forty-five healthy, malaria-naive participants were recruited from 5 studies. Osteoprotegerin and vWF levels increased in participants following inoculation with P. vivax (n = 16) or P. falciparum (n = 15), with the angiopoietin-2 level also increasing in participants following inoculation with P. falciparum For both species, the most pronounced increase was seen in osteoprotegerin. This was particularly marked in participants inoculated with P. vivax, where the osteoprotegerin level correlated with the levels of parasitemia and the malaria clinical score. There were no changes in measures of endothelial glycocalyx or microvascular function. Plasma biomarkers of endothelial activation increased in early P. falciparum and P. vivax infection and preceded changes in the endothelial glycocalyx or microvascular function. The more pronounced increase in osteoprotegerin suggests that this biomarker may play a role in disease pathogenesis.

Cell-Free Hemoglobin Is Associated With Increased Vascular Resistance and Reduced Peripheral Perfusion in Severe Malaria.

BACKGROUND: In severe falciparum malaria, unlike sepsis, hypotension on admission is uncommon. We hypothesized that low nitric oxide bioavailability due to the presence of cell-free hemoglobin (CFH) increases vascular tone in severe malaria. METHODS: Patients with severe malaria (n = 119), uncomplicated malaria (n = 91), or suspected bacterial sepsis (n = 56), as well as healthy participants (n = 50), were recruited. The systemic vascular resistance index (SVRI) was estimated from the echocardiographic cardiac index and the mean arterial pressure. RESULTS: SVRI and hematocrit levels were lower and plasma CFH and asymmetric dimethylarginine levels were higher in patients with malaria, compared with healthy participants. In multivariate linear regression models for mean arterial pressure or SVRI in patients with severe malaria, hematocrit and CFH but not asymmetric dimethylarginine were significant predictors. The SVRI was lower in patients with suspected bacterial sepsis than in those with severe malaria, after adjustment for hematocrit and age. Plasma CFH levels correlated positively with the core-peripheral temperature gradient and plasma lactate levels and inversely with the perfusion index. Impaired peripheral perfusion, as reflected by a low perfusion index or a high core-peripheral temperature gradient, predicted mortality in patients with severe malaria. CONCLUSIONS: CFH is associated with mean arterial pressure, SVRI, and peripheral perfusion in patients with severe malaria. This may be mediated through the nitric oxide scavenging potency of CFH, increasing basal vascular tone and impairing tissue perfusion.

Novel RNA viruses associated with Plasmodium vivax in human malaria and Leucocytozoon parasites in avian disease.

Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle.

Loss of complement regulatory proteins on red blood cells in mild malarial anaemia and in Plasmodium falciparum induced blood-stage infection.

BACKGROUND: Anaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection. METHODS: The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated. RESULTS: CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM. CONCLUSIONS: Removal of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia.

Whole-Genome Sequencing to Differentiate Relapse From Reinfection in Community-Onset Bacteremic Acinetobacter baumannii Pneumonia.

Community-onset bacteremic Acinetobacter baumannii pneumonia recurred in 3 of 30 (10%) patients followed prospectively, all with ongoing hazardous alcohol intake, 3-56 months after initial pneumonia. Paired isolates underwent whole-genome sequencing. Phylogenetic analysis showed that recurrence strains were all distinct from preceding strains, indicating reinfection in susceptible individuals rather than relapse.