PLAGL2-EGFR-HIF-1/2α Signaling Loop Promotes HCC Progression and Erlotinib Insensitivity.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, hence a major public health threat. Pleomorphic adenoma gene like-2 (PLAGL2) has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. APPROACH AND RESULTS: In this study, we demonstrated that PLAGL2 was up-regulated in HCC compared with that of adjacent nontumorous tissues and also correlated with overall survival times. We further showed that PLAGL2 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo. PLAGL2 expression was positively correlated with epidermal growth factor receptor (EGFR) expression. Mechanistically, this study demonstrated that PLAGL2 functions as a transcriptional regulator of EGFR and promotes HCC cell proliferation, migration, and invasion through the EGFR-AKT pathway. Moreover, hypoxia was found to significantly induce high expression of PLAGL2, which promoted hypoxia inducible factor 1/2 alpha subunit (HIF1/2A) expression through EGFR. Therefore, this study demonstrated that a PLAGL2-EGFR-HIF1/2A signaling loop promotes HCC progression. More importantly, PLAGL2 expression reduced hepatoma cells' response to the anti-EGFR drug erlotinib. PLAGL2 knockdown enhanced the response to erlotinib. CONCLUSIONS: This study reveals the pivotal role of PLAGL2 in HCC cell proliferation, metastasis, and erlotinib insensitivity. This suggests that PLAGL2 can be a potential therapeutic target of HCC.

Reinvigorating exhausted CD8+ cytotoxic T lymphocytes in the tumor microenvironment and current strategies in cancer immunotherapy.

Immunotherapy has revolutionized the treatment of cancer in recent years and achieved overall success and long-term clinical benefit in patients with a wide variety of cancer types. However, there is still a large proportion of patients exhibiting limited or no responses to immunotherapeutic strategy, some of which were even observed with hyperprogressive disease. One major obstacle restricting the efficacy is that tumor-reactive CD8+ T cells, which are central for tumor control, undergo exhaustion, and lose their ability to eliminate cancer cells after infiltrating into the strongly immunosuppressive tumor microenvironment. Thus, as a potential therapeutic rationale in the development of cancer immunotherapy, targeting or reinvigorating exhausted CD8+ T cells has been attracting much interest. Hitherto, both intrinsic and extrinsic mechanisms that govern CD8+ T-cell exhaustion have been explored. Specifically, the transcriptional and epigenetic landscapes have been depicted utilizing single-cell RNA sequencing or mass cytometry (CyTOF). In addition, cellular metabolism dictating the tumor-infiltrating CD8+ T-cell fate is currently under investigation. A series of clinical trials are being carried out to further establish the current strategies targeting CD8+ T-cell exhaustion. Taken together, despite the proven benefit of immunotherapy in cancer patients, additional efforts are still needed to fully circumvent limitations of exhausted T cells in the treatment. In this review, we will focus on the current cellular and molecular understanding of metabolic changes, epigenetic remodeling, and transcriptional regulation in CD8+ T-cell exhaustion and describe hypothetical treatment approaches based on immunotherapy aiming at reinvigorating exhausted CD8+ T cells.

The effectiveness of Hedyotis diffusa Willd extract in a mouse model of experimental autoimmune prostatitis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a frustrating and often debilitating disease. Current studies have shown that Traditional Chinese Medicine (TCM) can improve patients' quality of life and alleviate CP/CPPS symptoms. In this study, the efficacy of Hedyotis diffusa Willd aqueous extraction in experimental autoimmune prostatitis (EAP) mice models was revealed. The C57BL/6 mice were randomly assigned to three groups. Except for the control group, all other groups were subcutaneously injected with 0.2 ml emulsion of T2 peptide, on day 0 and day 14, for inducing EAP models. After the EAP modelling, oral saline was given to the model group, while the H. diffusa group was treated with aqueous extract of H. diffusa Willd. Micturition habits and withdrawal response frequencies were measured. Haematoxylin and eosin staining and immunohistochemistry were used to investigate inflammatory cell infiltration and TNF-α in the prostate tissue respectively. TNF-α levels in the serum were evaluated by ELISA. The H. diffusa Willd aqueous extraction considerably reduced the urine spots number and increased the pain threshold in H. diffusa group. H. diffusa group showed significantly reduced inflammatory lesion and inflammatory cell infiltration than the model group. The levels of TNF-α in H. diffusa group were considerably reduced.

Body mass index of women in Bangladesh: comparing Multiple Linear Regression and Quantile Regression.

This study explored the association between socio-demographic factors and the body mass index (BMI) of women of reproductive age (15-49 years) in Bangladesh. Data from the 2014 Bangladesh Demographic and Health Survey (BDHS-14) were analysed using Multiple Linear Regression (MLR) and Quantile Regression (QR) analyses. The study sample comprised 15,636 non-pregnant women aged 15-49. The mean BMI of the women was 22.35±4.12 kg/m2. Over half (56.75%) had a BMI in the normal range (18

Reduced Cardiac Index Reserve and Hypovolemia in Severe Falciparum Malaria.

BACKGROUND: Impaired microvascular perfusion is central to the development of coma and lactic acidosis in severe falciparum malaria. Refractory hypotension is rare on admission but develops frequently in fatal cases. We assessed cardiac function and volume status in severe falciparum malaria and its prognostic significance. METHODS: Patients with severe (N = 101) or acute uncomplicated falciparum malaria (N = 83) were recruited from 2 hospitals in India and Bangladesh, and healthy participants (N = 44) underwent echocardiography. RESULTS: Patients with severe malaria had 38% shorter left ventricular (LV) filling times and 25% shorter LV ejection times than healthy participants because of tachycardia; however, stroke volume, LV internal diameter in diastole (LVIDd), and LV internal diameter in systole (LVIDs) indices were similar. A low endocardial fraction shortening (eFS) was present in 17% (9 of 52) of severe malaria patients. Adjusting for preload and afterload, eFS was similar in health and severe malaria. Fatal cases had smaller baseline LVIDd and LVIDs indices, more collapsible inferior vena cavae (IVC), and higher heart rates than survivors. The LVIDs and IVC collapsibility were independent predictors for mortality, together with base excess and Glasgow Coma Scale. CONCLUSIONS: Patients with severe malaria have rapid ejection of a normal stroke volume. Fatal cases had features of relative hypovolemia and reduced cardiac index reserve.

Associations Between Restrictive Fluid Management and Renal Function and Tissue Perfusion in Adults With Severe Falciparum Malaria: A Prospective Observational Study.

BACKGROUND: Liberal fluid resuscitation has proved harmful in adults with severe malaria, but the level of restriction has not been defined. METHODS: In a prospective observational study in adults with severe falciparum malaria, restrictive fluid management was provided at the discretion of the treating physician. The relationships between the volume of fluid and changes in renal function or tissue perfusion were evaluated. RESULTS: A total of 154 patients were studied, 41 (26.6%) of whom died. Median total fluid intake during the first 6 and 24 hours from enrollment was 3.3 (interquartile range [IQR], 1.8-5.1) mL/kg per hour and 2.2 (IQR, 1.6-3.2) mL/kg per hour, respectively. Total fluid intake at 6 hours was not correlated with changes in plasma creatinine at 24 hours (n = 116; rs = 0.16; P = .089) or lactate at 6 hours (n = 94; rs = -0.05; P = .660). Development of hypotensive shock or pulmonary edema within 24 hours after enrollment were not related to the volume of fluid administration. CONCLUSIONS: Restrictive fluid management did not worsen kidney function and tissue perfusion in adult patients with severe falciparum malaria. We suggest crystalloid administration of 2-3 mL/kg per hour during the first 24 hours without bolus therapy, unless the patient is hypotensive.

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.

Genetic polymorphisms in DNA repair genes XRCC1 and 3 are associated with increased risk of breast cancer in Bangladeshi population.

BACKGROUND: Genetic polymorphisms in DNA repair genes, XRCC1 (Arg399Gln) and XRCC3 (Thr241Met), may affect their DNA repair capacity leading to individual variation in breast cancer susceptibility among Bangladeshi females. METHODS: The case-control study comprised 121 breast cancer patients and 133 healthy controls. Genomic DNA isolated from peripheral blood was genotyped for target SNPs using PCR-RFLP method. RESULTS: For XRCC1, heterozygous Arg/Gln and homozygous Gln/Gln genotypes showed 1.78-fold (95% CI 1.0084 to 3.1442, p = 0.0467) and 2.41-fold (95% CI 1.0354 to 5.5914, p = 0.0413) increased risk of breast cancer, respectively, when compared with Arg/Arg genotype. The presence of any XRCC1 Gln showed association with 1.93-fold increased risk. The variant Gln allele was associated with increased risk of breast cancer (95% CI 1.1885 to 2.6805, p = 0.0052). For XRCC3, Thr/Met heterozygous and combined Thr/Met + Met/Met genotypes were associated with 1.85-fold (95% CI 1.0815 to 3.1834, p = 0.0248) and 1.89-fold (95% CI 1.1199 to 3.1908, p = 0.0171) higher risk, respectively, compared to Thr/Thr genotypes. The variant Met allele showed significant association with increased breast cancer susceptibility. Among cases genotype frequencies were significantly different in patients with age 55 or above, and with menopause and diabetes. CONCLUSION: XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) polymorphisms may be associated with increased breast cancer risk in Bangladeshi females.

Genetic polymorphisms in CDH1 and Exo1 genes elevate the prostate cancer risk in Bangladeshi population.

The polymorphisms of invasion suppressor gene CDH1 and DNA mismatch repair gene Exo1 have been reported to play critical role in the development, tumorigenesis, and progression of several kinds of cancers including prostate cancer. This study was designed to analyze the contribution of single-nucleotide polymorphisms of the CDH1 (-160C/A) and Exo1 (K589E) to prostate cancer susceptibility in Bangladeshi population. The study included 100 prostate cancer cases and age-matched 100 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to determine the genetic polymorphisms. A significant association was found between CDH1 -160C/A (rs16260) and Exo1 (rs1047840, K589E) polymorphisms and prostate cancer risk. In case of CDH1 -160C/A polymorphism, the frequencies of the three genotypes C/C,C/A, and A/A were 45%, 48%, and 7% in cases and 63%, 32%, and 5% in controls, respectively. The heterozygote C/A genotype and combined C/A + A/A genotypes showed 2.10-fold (odds ratio = 2.1000, 95% confidence interval = 1.2956-4.0905, p = 0.013) and 2.08-fold (odds ratio = 2.0811, 95% confidence interval = 1.1820-3.6641, p = 0.011) increased risk of prostate cancer, respectively, when compared with homozygous C/C genotypes. The variant A allele also was associated with increased risk of prostate cancer (odds ratio = 1.6901, 95% confidence interval = 1.0740-2.6597, p = 0.0233). In case of Exo1 (K589E) polymorphism, G/A heterozygote, A/A homozygote, and combined G/A + A/A genotypes were found to be associated with 2.30-, 4.85-, and 3.04-fold higher risk of prostate cancer, respectively (odds ratio = 2.3021, 95% confidence interval = 2.956-4.0905, p = 0.0031; odds ratio = 4.8462, 95% confidence interval = 1.0198-23.0284, p = 0.0291; OR = 3.0362, 95% confidence interval = 1.7054-5.4053, p = 0.0001, respectively). The "A" allele showed significant association with increased susceptibility (2.29-fold) to prostate cancer (odds ratio = 2.2955, 95% confidence interval = 1.4529-3.6270, p = 0.0004). Our results suggest that CDH1 -160C/A and Exo1 K589E polymorphisms are associated with increased susceptibility to prostate cancer in Bangladeshi population.

Does reduced oxygen delivery cause lactic acidosis in falciparum malaria? An observational study.

BACKGROUND: Lactic acidosis with an elevated lactate-pyruvate ratio suggesting anoxia is a common feature of severe falciparum malaria. High lactate levels are associated with parasitized erythrocyte sequestration in the microcirculation. To assess if there is an additional contribution to hyperlactataemia from relatively inadequate total oxygen delivery, oxygen consumption and delivery were investigated in patients with malaria. METHODS: Adult Bangladeshi and Indian patients with uncomplicated (N = 50) or severe (N = 46) falciparum malaria or suspected bacterial sepsis (N = 27) and healthy participants as controls (N = 26) were recruited at Chittagong Medical College Hospital, Chittagong, Bangladesh and Ispat General Hospital, Rourkela, India. Oxygen delivery (DO2I) was estimated from pulse oximetry, echocardiographic estimates of cardiac index and haematocrit. Oxygen consumption (VO2I) was estimated by expired gas collection. RESULTS: VO2I was elevated in uncomplicated median (IQR) 185.1 ml/min/m2 (135-215.9) and severe malaria 192 ml/min/m2 (140.7-227.9) relative to healthy persons 107.9 ml/min/m2 (69.9-138.1) (both p < 0.001). Median DO2I was similar in uncomplicated 515 ml/min/m2 (432-612) and severe 487 ml/min/m2 (382-601) malaria and healthy persons 503 ml/min/m2 (447-517) (p = 0.27 and 0.89, respectively). The VO2/DO2 ratio was, therefore, increased by similar amounts in both uncomplicated 0.35 (0.28-0.44) and severe malaria 0.38 (0.29-0.48) relative to healthy participants 0.23 (0.17-0.28) (both p < 0.001). VO2I, DO2I and VO2/DO2 did not correlate with plasma lactate concentrations in severe malaria. CONCLUSIONS: Reduced total oxygen delivery is not a major contributor to lactic acidosis in severe falciparum malaria.

Acetaminophen as a Renoprotective Adjunctive Treatment in Patients With Severe and Moderately Severe Falciparum Malaria: A Randomized, Controlled, Open-Label Trial.

Background: Acute kidney injury independently predicts mortality in falciparum malaria. It is unknown whether acetaminophen's capacity to inhibit plasma hemoglobin-mediated oxidation is renoprotective in severe malaria. Methods: This phase 2, open-label, randomized controlled trial conducted at two hospitals in Bangladesh assessed effects on renal function, safety, pharmacokinetic (PK) properties and pharmacodynamic (PD) effects of acetaminophen. Febrile patients (>12 years) with severe falciparum malaria were randomly assigned to receive acetaminophen (1 g 6-hourly for 72 hours) or no acetaminophen, in addition to intravenous artesunate. Primary outcome was the proportional change in creatinine after 72 hours stratified by median plasma hemoglobin. Results: Between 2012 and 2014, 62 patients were randomly assigned to receive acetaminophen (n = 31) or no acetaminophen (n = 31). Median (interquartile range) reduction in creatinine after 72 hours was 23% (37% to 18%) in patients assigned to acetaminophen, versus 14% (29% to 0%) in patients assigned to no acetaminophen (P = .043). This difference in reduction was 37% (48% to 22%) versus 14% (30% to -71%) in patients with hemoglobin ≥45000 ng/mL (P = .010). The proportion with progressing kidney injury was higher among controls (subdistribution hazard ratio, 3.0; 95% confidence interval, 1.1 to 8.5; P = .034). PK-PD analyses showed that higher exposure to acetaminophen increased the probability of creatinine improvement. No patient fulfilled Hy's law for hepatotoxicity. Conclusions: In this proof-of-principle study, acetaminophen showed renoprotection without evidence of safety concerns in patients with severe falciparum malaria, particularly in those with prominent intravascular hemolysis. Clinical Trials Registration: NCT01641289.

Increased salt and drought tolerance by D-pinitol production in transgenic Arabidopsis thaliana.

D-ononitol epimerase (OEP) catalyzes the conversion of D-ononitol to D-pinitol, which is the last step in the biosynthetic pathway, where myo-inositol is converted to pinitol in higher plants. In this study, OEP cDNA was isolated from Glycine max (GmOEP) and was functionally characterized, which confirmed that GmOEP expression was induced by high salinity and drought stress treatments. To understand the biological function of GmOEP, transgenic Arabidopsis plants overexpressing this protein were constructed. The transgenic Arabidopsis plants displayed enhanced tolerance to high salinity and drought stress treatments.

Acidosis and acute kidney injury in severe malaria.

BACKGROUND: In severe falciparum malaria metabolic acidosis and acute kidney injury (AKI) are independent predictors of a fatal outcome in all age groups. The relationship between plasma acids, urine acids and renal function was investigated in adult patients with acute falciparum malaria. METHODS: Plasma and urinary acids which previously showed increased concentrations in proportion to disease severity in patients with severe falciparum malaria were quantified. Patients with uncomplicated malaria, sepsis and healthy volunteers served as comparator groups. Multiple regression and multivariate analysis were used to assess the relationship between organic acid concentrations and clinical syndromes, in particular AKI. RESULTS: Patients with severe malaria (n = 90), uncomplicated malaria (n = 94), non-malaria sepsis (n = 19), and healthy volunteers (n = 61) were included. Univariate analysis showed that both plasma and creatinine-adjusted urine concentrations of p-hydroxyphenyllactic acid (pHPLA) were higher in severe malaria patients with AKI (p < 0.001). Multiple regression analysis, including plasma or creatinine-adjusted urinary acids, and PfHRP2 as parasite biomass marker as independent variables, showed that pHPLA was independently associated with plasma creatinine (ß = 0.827) and urine creatinine (ß = 0.226). Principal component analysis, including four plasma acids and seven urinary acids separated a group of patients with AKI, which was mainly driven by pHPLA concentrations. CONCLUSIONS: Both plasma and urine concentrations of pHPLA closely correlate with AKI in patients with severe falciparum malaria. Further studies will need to assess the potential nephrotoxic properties of pHPLA.

Genotypic and phenotypic characterization of G6PD deficiency in Bengali adults with severe and uncomplicated malaria.

BACKGROUND: Control of malaria increasingly involves administration of 8-aminoquinolines, with accompanying risk of haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Few data on the prevalence and genotypic basis of G6PD deficiency are available from Bangladesh, where malaria remains a major problem in the South (Chittagong Division). The aim of this study was to determine the prevalence of G6PD deficiency, and associated G6PD genotypes, in adults with falciparum malaria in southern Bangladesh. METHODS: G6PD status was assessed via a combination of fluorescent spot testing (FST) and genotyping in 141 Bengali patients admitted with falciparum malaria to two centres in Chittagong Division from 2012 to 2014. In addition, an analysis of genomic data from 1000 Genomes Project was carried out among five healthy Indian subcontinent populations. RESULTS: One male patient with uncomplicated malaria was found to have G6PD deficiency on FST and a genotype associated with deficiency (hemizygous Orissa variant). In addition, there were two female patients heterozygous for deficiency variants (Orissa and Kerala-Kalyan). These three patients had a relatively long duration of symptoms prior to admission compared to G6PD normal cases, possibly suggesting an interaction with parasite multiplication rate. In addition, one of 27 healthy local controls was deficient on FST and hemizygous for the Mahidol variant of G6PD deficiency. Examination of 1000 Genomes Project sequencing data across the Indian subcontinent showed that 19/723 chromosomes (2.63%) carried a variant associated with deficiency. In the Bengali from Bangladesh 1000 Genomes population, three of 130 chromosomes (2.31%) carried deficient alleles; this included single chromosomes carrying the Kerala-Kalyan and Orissa variants. CONCLUSIONS: In line with other recent work, G6PD deficiency is uncommon in Bengalis in Bangladesh. Further studies of particular ethnic groups are needed to evaluate the potential risk of wide deployment of primaquine in malaria control efforts in Bangladesh.

Cell-free hemoglobin mediated oxidative stress is associated with acute kidney injury and renal replacement therapy in severe falciparum malaria: an observational study.

BACKGROUND: Intravascular hemolysis is an intrinsic feature of severe malaria pathophysiology but the pathogenic role of cell-free hemoglobin-mediated oxidative stress in severe malaria associated acute kidney injury (AKI) is unknown. METHODS: As part of a prospective observational study, enrolment plasma cell-free hemoglobin (CFH), lipid peroxidation markers (F2-isoprostanes (F2-IsoPs) and isofurans (IsoFs)), red cell deformability, and serum creatinine were quantified in Bangladeshi patients with severe falciparum malaria (n = 107), uncomplicated malaria (n = 80) and sepsis (n = 28). The relationships between these indices and kidney function and clinical outcomes were examined. RESULTS: AKI was diagnosed at enrolment in 58% (62/107) of consecutive patients with severe malaria, defined by an increase in creatinine ≥1.5 times expected baseline. Severe malaria patients with AKI had significantly higher plasma cell-free hemoglobin (geometric mean CFH: 8.8 µM; 95% CI, 6.2-12.3 µM), F2-isoprostane (56.7 pg/ml; 95% CI, 45.3-71.0 pg/ml) and isofuran (109.2 pg/ml; 95% CI, 85.1-140.1 pg/ml) concentrations on enrolment compared to those without AKI (CFH: 5.1 µM; 95% CI, 4.0-6.6 µM; P = 0.018; F2-IsoPs: 27.8 pg/ml; 95% CI, 23.7-32.7 pg/ml; P < 0.001; IsoFs: 41.7 pg/ml; 95% CI, 30.2-57.6 pg/ml; P < 0.001). Cell-free hemoglobin correlated with markers of hemolysis, parasite burden (P. falciparum histidine rich protein 2 (PfHRP2)), and F2-IsoPs. Plasma F2-IsoPs and IsoFs inversely correlated with pH, positively correlated with creatinine, PfHRP2 and fractional excretion of sodium, and were higher in patients later requiring hemodialysis. Plasma F2-IsoP concentrations also inversely correlated with red cell deformability and were higher in fatal cases. Mixed effects modeling including an interaction term for CFH and time showed that F2-IsoPs, IsoFs, PfHRP2, CFH, and red cell rigidity were independently associated with increasing creatinine over 72 h. Multivariable logistic regression showed that admission F2-IsoPs, IsoFs and red cell deformability were associated with the need for subsequent hemodialysis. CONCLUSIONS: Cell-free hemoglobin and lipid peroxidation are associated with acute kidney injury and disease severity in falciparum malaria, suggesting a pathophysiological role in renal tubular injury. Evaluation of adjunctive therapies targeting cell-free hemoglobin-mediated oxidative stress is warranted.

A prospective study of the importance of enteric fever as a cause of non-malarial febrile illness in patients admitted to Chittagong Medical College Hospital, Bangladesh.

BACKGROUND: Fever is a common cause of hospital admission in Bangladesh but causative agents, other than malaria, are not routinely investigated. Enteric fever is thought to be common. METHODS: Adults and children admitted to Chittagong Medical College Hospital with a temperature of ≥38.0 °C were investigated using a blood smear for malaria, a blood culture, real-time PCR to detect Salmonella Typhi, S. Paratyphi A and other pathogens in blood and CSF and an NS1 antigen dengue ELISA. RESULTS: We enrolled 300 febrile patients with a negative malaria smear between January and June 2012: 156 children (aged ≤15 years) and 144 adults with a median (interquartile range) age of 13 (5-31) years and median (IQR) illness duration before admission of five (2-8) days. Clinical enteric fever was diagnosed in 52 patients (17.3 %), lower respiratory tract infection in 48 (16.0 %), non-specific febrile illness in 48 (16.0 %), a CNS infection in 37 patients (12.3 %), urinary sepsis in 23 patients (7.7 %), an upper respiratory tract infection in 21 patients (7.0 %), and diarrhea or dysentery in 21 patients (7.0 %). Malaria was still suspected in seven patients despite a negative microscopy test. S. Typhi was detected in blood by culture or PCR in 34 (11.3 %) of patients. Of note Rickettsia typhi and Orientia tsutsugamushi were detected by PCR in two and one patient respectively. Twenty-nine (9 %) patients died during their hospital admission (15/160 (9.4 %) of children and 14/144 (9.7 %) adults). Two of 52 (3.8 %) patients with enteric fever, 5/48 (10.4 %) patients with lower respiratory tract infections, and 12/37 (32.4 %) patients with CNS infection died. CONCLUSION: Enteric fever was confirmed in 11.3 % of patients admitted to this hospital in Bangladesh with non-malaria fever. Lower respiratory tract and CNS infections were also common. CNS infections in this location merit more detailed study due to the high mortality.

Microvascular obstruction and endothelial activation are independently associated with the clinical manifestations of severe falciparum malaria in adults: an observational study.

BACKGROUND: Microvascular obstruction and endothelial dysfunction have both been linked to tissue hypoperfusion in falciparum malaria, but their relative contributions to the disease's pathogenesis and outcome are unknown. METHODS: Microvascular blood flow was quantified in adults with severe falciparum malaria on their admission to hospital; plasma biomarkers of endothelial function were measured simultaneously. The relationship between these indices and the patients' clinical findings and in-hospital course was examined. RESULTS: Microvascular obstruction was observed in 119/142 (84 %) patients; a median (interquartile range (IQR)) of 14.9 % (6.6-34.9 %) of capillaries were obstructed in patients that died versus 8.3 % (1.7-26.6 %) in survivors (P = 0.039). The proportion of obstructed capillaries correlated with the estimated parasite biomass (rs = 0.25, P = 0.004) and with plasma lactate (rs = 0.38, P <0.0001), the strongest predictor of death in the series. Plasma angiopoietin-2 (Ang-2) concentrations were markedly elevated suggesting widespread endothelial activation; the median (IQR) Ang-2 concentration was 21.9 ng/mL (13.4-29.4 ng/mL) in patients that died versus 14.9 ng/mL (9.8-29.3 ng/mL) in survivors (P = 0.035). Ang-2 concentrations correlated with estimated parasite biomass (rs = 0.35, P <0.001) and plasma lactate (rs = 0.37, P <0.0001). Microvascular obstruction and Ang-2 concentrations were not significantly correlated with each other (rs = 0.17, P = 0.06), but were independently associated with plasma lactate (P <0.001 and P = 0.002, respectively). CONCLUSIONS: Microvascular obstruction and systemic endothelial activation are independently associated with plasma lactate, the strongest predictor of death in adults with falciparum malaria. This supports the hypothesis that the two processes make an independent contribution to the pathogenesis and clinical manifestations of the disease.

Severe falciparum malaria treated with artesunate complicated by delayed onset haemolysis and acute kidney injury.

BACKGROUND: Severe falciparum malaria may be complicated by haemolysis after parasite clearance, however the mechanisms remain unclear. Recent reports describe a pattern of delayed onset haemolysis among non-immune travellers with hyperparasitaemia treated with intravenous artesunate, termed post-artesunate delayed haemolysis (PADH). The occurrence and clinical impact of PADH following severe malaria infections in areas of unstable transmission are unknown. CASE: A 45-year-old Bangladeshi male was initially admitted to a local hospital with severe falciparum malaria complicated by hyperparasitaemia and treated with intravenous artesunate. Twenty days from his first presentation he was readmitted with delayed onset haemolytic anaemia and acute kidney injury. Multiple blood transfusions and haemodialysis were required. Renal biopsy revealed acute tubular injury and haem pigment nephropathy. His haemoglobin and renal function recovered to baseline after 62 days from his second admission. DISCUSSION: This case highlights the differential diagnosis of post-malaria delayed onset haemolysis, including the recently described syndrome of post-artemisinin delayed haemolysis. The pathophysiology contributing to acute kidney injury in this patient and the limited treatment options are discussed. CONCLUSIONS: This report describes PADH complicated by acute kidney injury in an adult patient living in a malaria hypoendemic region who subsequently required blood transfusions and haemodialysis. This case emphasizes the importance of routine follow up of haemoglobin and renal function in artesunate-treated patients who have recovered from severe malaria.

The role of previously unmeasured organic acids in the pathogenesis of severe malaria.

INTRODUCTION: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. METHODS: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. RESULTS: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean ± SD) was elevated in severe malaria (8.2 mEq/L ± 4.5) and severe sepsis (8.6 mEq/L ± 7.7) compared with uncomplicated malaria (6.0 mEq/L ± 5.1) and encephalopathy (6.6 mEq/L ± 4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and ß-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6-7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5-7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). CONCLUSIONS: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.

Randomized controlled trial of levamisole hydrochloride as adjunctive therapy in severe falciparum malaria with high parasitemia.

BACKGROUND: Cytoadherence and sequestration of erythrocytes containing mature stages of Plasmodium falciparum are central to the pathogenesis of severe malaria. The oral anthelminthic drug levamisole inhibits cytoadherence in vitro and reduces sequestration of late-stage parasites in uncomplicated falciparum malaria treated with quinine. METHODS: Fifty-six adult patients with severe malaria and high parasitemia admitted to a referral hospital in Bangladesh were randomized to receive a single dose of levamisole hydrochloride (150 mg) or no adjuvant to antimalarial treatment with intravenous artesunate. RESULTS: Circulating late-stage parasites measured as the median area under the parasite clearance curves were 2150 (interquartile range [IQR], 0-28 025) parasites/µL × hour in patients treated with levamisole and 5489 (IQR, 192-25 848) parasites/µL × hour in controls (P = .25). The "sequestration ratios" at 6 and 12 hours for all parasite stages and changes in microvascular blood flow did not differ between treatment groups (all P > .40). The median time to normalization of plasma lactate (<2 mmol/L) was 24 (IQR, 12-30) hours with levamisole vs 28 (IQR, 12-36) hours without levamisole (P = .15). CONCLUSIONS: There was no benefit of a single-dose of levamisole hydrochloride as adjuvant to intravenous artesunate in the treatment of adults with severe falciparum malaria. Rapid parasite killing by intravenous artesunate might obscure the effects of levamisole.