Concomitant Vitamin B1 and Vitamin B12 Deficiency Mimicking Thrombotic Thrombocytopenic Purpura.

Vitamin B12 deficiency-induced pseudo-thrombotic thrombocytopenic purpura (pseudo-TTP) is a rare condition. In reported literature, most cases were due to pernicious anemia (confirmed by the presence of anti-parietal cells or anti-intrinsic factor antibodies). Nutritional vitamin B12 deficiency causing pseudo-TTP is a much rarer entity. Differentiating thrombotic thrombocytopenic purpura (TTP) cases from pseudo-TTP (from any cause) should be done as soon as possible since the etiology, treatment, and outcome are different. Hematological findings from pseudo-TTP (when associated with vitamin B12 deficiency) respond to B12 replacement but do not respond to plasmapheresis. Neurological symptoms are one of the criteria for TTP, and altered mentation or psychosis in these cases is presumed secondary to either TTP or vitamin B12 deficiency. However, neurological symptoms are more characteristic of TTP rather than pseudo-TTP. In the rarer subsets of patients concerned with nutritional deficiency and neuropsychiatric symptoms, prompt consideration of concomitant vitamin B1 deficiency and Wernicke encephalopathy is essential. Immediate empiric treatment with high-dose IV thiamine should be started. If unrecognized and left untreated, thiamine deficiency can cause rapid progression to irreversible neurological symptoms, coma, and death, despite hematological improvement with B12 replacement. We report a rare case of concomitant vitamin B12 and vitamin B1 deficiency presenting with confusion, severe hemolytic anemia, acute renal failure, diarrhea, and thrombocytopenia mimicking TTP.

The spectrum of ABO haemolytic disease of the fetus and newborn in neonates born to group O mothers.

BACKGROUND AND OBJECTIVES: ABO haemolytic disease of the fetus and newborn (HDFN) is a lesser recognized entity; however, the severity may vary in neonates. This prospective observational study was performed to determine the severity and risk of ABO-HDFN in neonates born to O group mothers. MATERIALS AND METHODS: A total of 260 neonates born to non-alloimmunized blood group O mothers were recruited. Blood group O neonates were excluded from the study. Neonatal direct antiglobulin test (DAT) was performed using the column agglutination technique. They were monitored for clinical and laboratory parameters and followed up at 6-8 weeks. The maternal anti-A and anti-B titres (IgM and IgG) were also done. RESULTS: A total of 176 neonates with blood group A (77/260; 29.6%) and B (99/260; 38.1%) were finally included in the study, and 15 (8.5%) of them were DAT positive. Overall, 26.7% (47/176) neonates received phototherapy, 172 (97.7%) survived and none required readmission. The median (inter-quartile range [IQR]) maternal IgG anti-B titre (32 [32-64]) was significantly higher (p < 0.001) than the IgG anti-A titre (16 [8-64]). The maximum total serum bilirubin in neonates had a significant positive association with neonatal birth weight (p = 0.045), positive DAT (p = 0.006) and requirement of phototherapy (p < 0.001). The relative risk (95% CI) of a DAT-positive neonate requiring phototherapy was 4.55 (3.12-6.33). CONCLUSION: The frequency of ABO incompatibility in neonates born to group O mothers was 67.69% (176/260). The maternal IgG titre of ≥64 could be a good predictor for identifying the neonates at risk of developing hyperbilirubinaemia requiring phototherapy.

International guidelines regarding the role of IVIG in the management of Rh- and ABO-mediated haemolytic disease of the newborn.

Haemolytic disease of the newborn (HDN) can be associated with significant morbidity. Prompt treatment with intensive phototherapy (PT) and exchange transfusions (ETs) can dramatically improve outcomes. ET is invasive and associated with risks. Intravenous immunoglobulin (IVIG) may be an alternative therapy to prevent use of ET. An international panel of experts was convened to develop evidence-based recommendations regarding the effectiveness and safety of IVIG to reduce the need for ETs, improve neurocognitive outcomes, reduce bilirubin level, reduce the frequency of red blood cell (RBC) transfusions and severity of anaemia, and/or reduce duration of hospitalization for neonates with Rh or ABO-mediated HDN. We used a systematic approach to search and review the literature and then develop recommendations from published data. These recommendations conclude that IVIG should not be routinely used to treat Rh or ABO antibody-mediated HDN. In situations where hyperbilirubinaemia is severe (and ET is imminent), or when ET is not readily available, the role of IVIG is unclear. High-quality studies are urgently needed to assess the optimal use of IVIG in patients with HDN.

Copper-induced Haemolytic Anaemia.

Acute copper toxicity is uncommon in Western countries and is often the result of accidental consumption or a suicide attempt. We report the case of a 65-year-old man presenting to the accident and emergency department after a suicide attempt with ingestion of Bordeaux mixture, ibuprofen, acetaminophen and bleach. Primary evaluation showed caustic oesophagitis, toxic hepatitis and acute renal injury, which were treated with supportive care. During admission, he developed a non-immune haemolytic anaemia associated with high levels of copper in urine and blood. Chelation treatment with penicillamine was started and evolution was favourable after 1 month of treatment. Copper poisoning can be lethal. Prompt diagnosis and treatment are key for a favourable prognosis. LEARNING POINTS: Acute copper intoxication is rare and early clinical suspicion and diagnosis are essential to reduce mortality.The diagnosis of copper poisoning should be based on clinical presentation and measurement of urine and blood copper levels in addition to serum ceruloplasmin levels.Treatment includes reduction of absorption, supportive measures, management of complications and chelation therapy.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

A greater awareness of children with glucose-6-phosphate dehydrogenase deficiency is imperative in western countries.

AIM: This national retrospective Danish study described the characteristics of children diagnosed with glucose-6-phosphate dehydrogenase (G6PD) deficiency, an inherited X-linked recessive disorder that often affects children of Middle Eastern descent. METHODS: We studied children born between 1 January 2000 and 31 December 2017 and diagnosed with G6PD deficiency. They were identified from the Danish National Hospital Discharge Register and the Danish Database of Extreme Neonatal Hyperbilirubinaemia. RESULTS: There were 113 children diagnosed with G6PD deficiency, 67% were of Middle Eastern descent and they were frequently diagnosed before the onset of symptoms, based on known heredity. Of the 67 infants born in Denmark, 10% had extreme neonatal hyperbilirubinaemia and one developed kernicterus spectrum disorder, as did one child born in the Middle East. Most (61%) of the 33 children with jaundice received phototherapy, 12% had exchange transfusions and 18% received whole blood transfusions. After the neonatal period, 23% of the cohort had blood transfusions and 4% needed intensive care for acute haemolytic anaemia. The incidence of G6PD deficiency appeared to be severely underestimated. CONCLUSION: Many families from countries where G6PD deficiency is endemic move to Denmark and other Western countries. Greater awareness is essential to avoid chronic and potentially lethal, consequences.

Life-Threatening Microangiopathy or Vitamin Deficiency: A Case Report of the Clinical Manifestations of Pseudo-Thrombotic Microangiopathic Anemia.

Hemolytic anemia with thrombocytopenia and organ damage raises suspicion for thrombotic microangiopathy (TMA), a pathology that results in thrombosis within the small vessels secondary to endothelial injury. While usually attributed to atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP), an increasingly recognized and treatable entity is pseudo-thrombotic microangiopathic anemia (pseudo-TMA) secondary to severe vitamin B-12 deficiency. While TMA often requires expensive diagnostic testing and can lead to invasive treatment options such as plasma exchange, immunosuppression, and/or complement cascade blocking, pseudo-TMA requires only vitamin supplementation. Therefore, the prompt and accurate diagnosis of this entity is important for the clinician to recognize in order to avoid unnecessary health costs and institute appropriate treatment. We present the case of a 51-year-old male without any past medical history, who presented with generalized weakness, dyspnea on exertion, and decreased exercise tolerance for several months and was found to have severe microangiopathic anemia with work-up concerning for TTP. After stabilization, he was found to have severe B-12 deficiency secondary to newly diagnosed pernicious anemia and was treated with subcutaneous B-12 injections with improvement in clinical symptoms and laboratory parameters. This presentation highlights the need for prompt diagnosis and high clinical suspicion for vitamin deficiencies as a source of pseudo-microangiopathy.

Low-dose steroid-induced bradyarrhythmias and treatment refractory hypokalaemia: a case report.

Corticosteroid therapy has become an important modality of treatment for diseases in which rapid control of immunoinflammatory processes is required. However, one of the serious, but less known adverse effect of this therapy is cardiac arrhythmias. This includes both tachyarrhythmias and bradyarrhythmias. Corticosteroid use may also be associated with electrolyte imbalances like hypokalaemia by its mineralocorticoid activity. Those side effects are mainly seen with high-dose intravenous methyl-prednisolone or oral pulse dose prednisolone therapy. Here we report our experience in a child with warm idiopathic autoimmune haemolytic anaemia who developed sinus bradyarrhythmias and treatment refractory hypokalaemia during low-dose steroid therapy with reduction in heart rate by 60% of baseline.

Leaf and root bark extracts of Ziziphus abyssinica Hochst ex. A. Rich (Rhamnaceae) ameliorate hepatic, renal and splenic injuries induced by phenylhydrazine in rats.

Objectives Ziziphus abyssinica (ZA) is employed in managing several ailments in Traditional African Medicine. Scientific evaluations are necessary to ascertain the medicinal potential of ZA as a source of new drug molecules. This study investigated the possible therapeutic benefit of ZA leaf (ZAL) and root bark (ZARB) extracts in an experimental model of multi-organ injuries induced by phenylhydrazine (PHZ). Methods Hyperbilirubinaemia, hepatotoxicity, nephrotoxicity and splenic injuries were induced by pretreating albino rats with PHZ (40 mg/kg, p.o.) for two alternate days. Afterward, six out of the eight groups of rats (n = 5) used were treated with either ZAL or ZARB (30, 100 and 300 mg/kg/day, p.o.) for seven days. Naïve control rats received saline without PHZ whereas negative control group received saline after PHZ. After one week of treatment, rats were sacrificed and blood collected for assessment of haematological and biochemical parameters. Liver, kidney and spleen sections were processed for histology and examined under light microscope. Results Data indicate that PHZ significantly (p < 0.05) increased total bilirubin, serum alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen (BUN), creatinine and BUN/creatinine ratio whereas red blood cell count was significantly reduced. These anomalies were significantly reversed in rats treated with ZAL or ZARB. The therapeutic effect of the extracts was supported by photomicrographs of the liver, kidney, and spleen of rats which revealed recovery from PHZ-mediated pyknosis, glomerular degeneration and multiple splenic necrosis respectively. Conclusions Overall, data from this study suggest that ZA may be useful in multiple organ injuries associated with PHZ-like xenobiotic toxicity.

A case report of haemolytic disease of the foetus and newborn caused by Alloantibody D and Jka in a Rhesus D negative Nigerian woman: Justification for the implementation of universal access to prophylaxis and evidenced-based best practices.

A case report of a 38 years old ABO group A and Rhesus D negative multiparous, gravidae 8 and para 2, Nigerian woman who had a case of premarital miscarriage and who was not offered anti-D prophylaxis as part of her management. Lady went on to develop alloantibody D and Jka. Lady has had 7 further pregnancies post the miscarriage. The first child who is B Rhesus D positive is the only surviving child. The surviving child was delivered severely jaundiced and needed management post-delivery for haemolytic disease of the foetus and newborn (HDFN). Lady has had a history of a stillbirth. She was given a non-clinically indicated anti-D prophylaxis during the second pregnancy despite having been previously sensitized. The second baby died 3 months after delivery from complications of HDFN. She had had a further history of 5 miscarriages. She has had challenge with conception since 2010. Alloantibody testing confirms the presence alloantibody D and anti-Jka. Finding from this is a clear case of sub-optimal laboratory, obstetric and neonatal care offered particularly to pregnant women who are Rh D negative and those with alloantibodies in Nigeria. The Nigerian government will need to implement evidenced-based best practices; determination of alloantibody status of pregnant women during their first antenatal visit; provision of facilities for alloantibody identification, titration, quantification and feto maternal haemorrhage testing (FMH); implementation of a policy on universal access to anti-D prophylaxis for pregnant Rh D negative women who are not previously sensitized; provision of facilities required for the optimal intrauterine management of HDFN (foetal genotype testing, intrauterine transfusion, doppler ultrasound to diagnose anaemia inutero and provision of donor blood that meet the minimum requirements for intrauterine transfusion); determination of Rh D status of women who require a termination of pregnancy and provision of prophylactic anti-D for those found Rh D negative within 72 hours of procedure and the optimization of the knowledge of Medical Laboratory Scientist, Obstetricians, Neonatologist, Pharmacist and Traditional Birth Attendants in a bid to reduce the residual number of women who become sensitized and the number of preventable deaths of babies with HDFN.

Red-blood-cell alloimmunization and prophylactic antigen matching for transfusion in patients with warm autoantibodies.

BACKGROUND: Warm autoantibodies (WAA) are antibodies that react with an antigen on a patient's own red-blood-cells and can complicate compatibility testing whether or not they cause clinical haemolysis. The goal of this study was to understand the overall prevalence of WAA, the risk of RBC alloimmunization and determine whether RBC selection practices have an impact on alloimmunization. MATERIALS AND METHODS: Records of patients (>1 year of age) with an indirect antibody detection test (IAT) and serologic evidence of WAA over a 10-year-period were included. Eight centres from 5 countries collectively reviewed 1 122 245 patients who had an IAT. RESULTS: Of patients having IAT, 1214 had WAA (0·17%). Transfusion information for 1002 of the patients was available; 631 were transfused after identification of the WAA (63%); of the transfused patients, 390 received prophylactic antigen-matched (PAM) RBCs and 241 did not. Of the 372 patients with WAA who were transfused and had serologic testing 30+ days following transfusion (30-2765 days), 56 developed new RBC alloimmunization (15·1%). Patients who were transfused using a PAM strategy were not protected from new RBC alloimmunization [14·6% (31 of 212 patients) having PAM transfusion approach compared with those not receiving PAM approach (15·6%, 25 of 160 patients, P = 0·8837)]. CONCLUSIONS: The prevalence of WAA in patients having an IAT is low (<1%). A significant portion of patients with WAA form new RBC alloimmunization (15·1%); however, the use of PAM approach for RBC selection was not found to be protective against new alloimmunization.

Delayed cord clamping in Rh-alloimmunised infants: a randomised controlled trial.

Despite advancement in medical care, Rh alloimmunisation remains a major cause of neonatal hyperbilirubinaemia, neuro-morbidity, and late-onset anaemia. Delayed cord clamping (DCC), a standard care now-a-days, is yet not performed in Rh-alloimmunised infants due to paucity of evidence. Hence, we randomised these infants of 28- to 41-week gestation to delayed cord clamping (N = 36) or early cord clamping (N = 34) groups. The primary outcome variable was venous packed cell volume (PCV) at 2 h of birth. The secondary outcomes were incidence of double volume exchange transfusion (DVET) and partial exchange transfusion (PET), duration of phototherapy (PT), functional echocardiography (parameters measured: superior vena cava flow, M-mode fractional shortening, left ventricular output, myocardial perfusion index, and inferior vena cava collapsibility) during hospital stay, and blood transfusion (BT) until 14 weeks of life. Neonates were managed as per unit protocol. The baseline characteristics of enrolled infants were comparable between the groups. The median (IQR) gestation and mean (SD) birth weight of enrolled infants were 35 (33-37) weeks and 2440 (542) g, respectively. The DCC group had a higher mean PCV at 2 h of life (48.4 ± 9.2 vs. 43.5 ± 8.7, mean difference 4.9% (95% CI 0.6-9.1), p = 0.03). However, incidence of DVET and PET, duration of PT, echocardiography parameters, and BT until 14 weeks of postnatal age were similar between the groups.Conclusion: DCC in Rh-alloimmunised infants improved PCV at 2 h of age without significant adverse effects.Trial registration: Clinical Trial Registry of India (CTRI), Ref/2016/11/012572 http://ctri.nic.in/Clinicaltrials, date of trial registration 19.12.2016, date of first patient enrolment 1 January 2017.What is Known:•Delayed cord clamping improves haematocrit, results in better haemodynamic stability, and decreases the need of transfusion in early infancy.•However, due to lack of evidence, potential risk of hyperbilirubinaemia, and exacerbation of anaemia (following delayed cord clamping), early cord clamping is the usual norm in Rh-alloimmunised infantsinfants.What is New:•Delayed cord clamping in Rh-alloimmunised infants improves haematocrit at 2 h of life without any increase in incidence of serious adverse effects.

Antibacterial activities, proposed mode of action and cytotoxicity of leaf extracts from Triumfetta welwitschii against Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa has become a main cause of Gram-negative infection, particularly in patients with compromised immunity. High rates of resistance to antibiotics are associated with nosocomial infections caused by P. aeruginosa strains. The search for novel antimicrobials has been necessitated by the emergence of antimicrobial resistance in some bacteria Plant-based antimicrobials has great potential to combat microbial infections using a variety of mechanisms. Triumfetta welwitschii plant roots are traditionally used to treat symptoms of diarrhoea and fever, suggesting that it possess antimicrobial and immunomodulatory effects. Since research investigating antimicrobial properties of the roots of Triumfetta welwitschii has been explored, there is need to investigate the antimicrobial activity of its leaf extracts in order to probe their prospective use as new antimicrobial agents that can be used to combat nosocomial infections. The objective of this study was to evaluate the antibacterial activities, the mode of action and cytotoxicity of T. welwitschii leaf extracts. METHOD: Extracts of T. welwitschii leaves were obtained using eight different solvents, the serial exhaustive extraction method and the cold maceration technique. In vitro antibacterial activity evaluation of the extracts was done on eight bacterial isolates using the broth microdilution method. The mode of action for the most potent extracts was investigated using the rhodamine 6G efflux assay and the propidium iodide-based membrane damage assay. Toxicity of the extracts was evaluated using the haemolytic and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. RESULTS: The results showed that acetone, ethanol and dichlorometane: methanol extracts had the most potent antibacterial activities against Pseudomonas aeruginosa (ATCC 27853). All three extracts caused membrane disruption of P. aeruginosa as shown by nucleic acid leakage. All three extracts were unable to inhibit efflux pumps. CONCLUSION: The presence of antibacterial activities and low toxicity shown by the extracts indicates that the plant may be a source of effective antibacterial against some bacterial infections caused by P. aeruginosa. The disruption of membrane integrity is one possible mode of action of antibacterial activity of the potent extracts.

Biogenic approach to synthesize rod shaped Gd2 O3 nanoparticles and its optimization using response surface methodology-Box-Behnken design model.

The rare earth metal oxide nanoparticles such as gadolinium oxide nanoparticles (Gd2 O3 NPs) have been synthesized by green synthesis process using methanolic extract of Moringa oleifera (M oleifera) peel. In this process, the Gd2 O3 NPs formation was observed at 280-300 nm in UV-Vis spectroscopy. The XRD pattern of the synthesized Gd2 O3 NPs was exactly matched with JCPDS No 3-065-3181which confirms the crystalline nature of Gd2 O3 NPs. In addition, Energy-dispersive X-ray spectroscopy (EDX) analysis was stated that Gd and O elements were present as 70.31 and 29.69%, respectively in Gd2 O3 NPs. The SEM and TEM analysis were said Gd2 O3 NPs are in rod shape and 26 ± 2 nm in size. Further the synthesized Gd2 O3 NPs were confirmed by X-ray photoemission spectroscopy (XPS). The synthesized Gd2 O3 NPs were further examined for anti-fungal activity against Alternaria saloni (A saloni) and Sclerrotium rolfsii (S rolfsii) and it showed moderate activity. Also, Gd2 O3 NPs evaluated as good antibacterial agent against different Gram +ve and Gram -ve bacteria. Moreover, the toxicity of the Gd2 O3 NPs on red blood cells (RBCs) of the human blood was determined using hemolytic assay, the obtained results were stated the synthesized Gd2 O3 NPs are nontoxic to the human erythrocytes. The photocatalytic activity against malachite green (MG) dye was tested and confirmed as 92% of dye was degraded within 2 hr by Gd2 O3 NPs. The results were stated the green synthesized Gd2 O3 NPs are good anti-fungal agents, nontoxic and we can use as a photocatalyst. Copyright © 2019 John Wiley & Sons, Ltd.

Retrospective analysis of HDFN due to ABO incompatibility in a single institution over 6 years.

OBJECTIVES: To study the rate of ABO haemolytic anaemia of fetus and newborn (HDFN) in one institution over 6 years. BACKGROUND: ABO major incompatibility between mothers and their newborns occurs in about 10% of births. So, mothers with an O blood group may form IgG-class antibodies against A and B antigens, which could pass across the placenta and lead to a variable degree of HDFN in the newborn. METHODS: At our institution, we have reviewed data regarding ABO-based HDFN in the last 6 years. RESULTS: We found that, in 28 089 deliveries, an ABO major incompatibility between mothers and newborns occurs in 11% of cases, with 72% of O/A and 28% of O/B incompatibility. In turn, 23% of these newborns had an eluate-confirmed positive direct antiglobulin test [DAT; 74% (511) were due to anti-A and 26% (179) to anti-B], with 1·0% requiring invasive treatments (exchange transfusion or intravenous immunoglobulin). Overall, 2·5% of the total newborns had a positive DAT for an anti-A or anti-B antibody, and 0·11% required invasive treatment in addition to phototherapy for their HDFN. CONCLUSIONS: Serological ABO HDFN is a relatively frequent event when an O-A/O-B incompatibility between mothers and their newborn occurs and, in most cases, translates into a self-limiting disease, with a small number of newborns requiring invasive treatments. The DAT test, although not predictive of disease severity, appears to be a useful tool to monitor babies born from O-A/O-B-incompatible pregnancies and to identify those who may require treatment.

Production of bioactive cyclotides in somatic embryos of Viola odorata.

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.

Identification of risk for neonatal haemolysis.

AIM: To identify neonates at risk of haemolytic hyperbilirubinaemia through near-concurrent measurements of total serum/plasma bilirubin (TB) or transcutaneous bilirubin (TcB) and end-tidal breath carbon monoxide (CO), corrected for ambient CO (ETCOc), an index of bilirubin production and haemolysis. METHODS: Paired TB/TcB (mg/dL) and ETCOc (ppm) measurements were obtained in newborns (n = 283) at 20 to <60 hours of age in five nurseries. TB/TcB values were assigned TB/TcB percentile risk values using the Bhutani hour-specific nomogram. In infants having two serial TB/TcB measurements (n = 76), TB rate of rise (ROR, mg/dL/h) was calculated. RESULTS: For the entire cohort (n = 283), 67.1% and 32.9% had TB/TcB<75th and ≥75th percentile, respectively. TB/TcB (5.79 ± 1.84 vs 9.14 ± 2.25 mg/dL) and ETCOc (1.61 ± 0.45 vs 2.02 ± 1.35 ppm, p = 0.0002) were different between the groups. About 36.6% of infants with TB/TcB ≥75th percentile had ETCOc ≥ 2.0 ppm. In the subcohort of infants with serial TB/TcB measurements (n = 76), 44.7% and 55.3% had TB/TcB<75th and ≥75th percentile, respectively. TB/TcB (5.28 ± 1.97 vs 9.53 ± 2.78 mg/dL), ETCOc (1.72 ± 0.48 vs 2.38 ± 1.89 ppm, p = 0.05) and TB ROR (0.011 ± 0.440 vs 0.172 ± 0.471 mg/dL/h) were different between the groups. CONCLUSION: The combined use of TB/TcB percentile risk assessments and ETCOc measurements can identify infants with haemolytic hyperbilirubinaemia. The addition of TB ROR can identify those infants with elimination disorders.

Molecular profiling and bioactive potential of an endophytic fungus Aspergillus sulphureus isolated from Sida acuta: a medicinal plant.

CONTEXT: Sida acuta Burm.f. (Malvaceae) extracts are reported to have applications against malaria, diuretic, antipyretic, nervous and urinary diseases. No fungal endophytes of S. acuta are reported. OBJECTIVE: Isolation, identification and evaluation of antibacterial, antioxidant, anticancer and haemolytic potential of fungal endophytes from the ethnomedcinal plant S. acuta. MATERIALS AND METHODS: Sida acuta stem segments were placed on PDA medium to isolate endophytic fungi. The fungus was identified by genomic DNA analysis and phylogenetic tree was constructed using ITS sequences (GenBank) to confirm species. The antibacterial efficacy of Aspergillus sulphureus MME12 ethyl acetate extract was tested against Gram-positive and Gram-negative pathogenic bacteria. DPPH free radical scavenging activity, anticancer and DNA fragmentation against EAC cells, and direct haemolytic activity (100-500 µg/mL) using human erythrocytes were determined. RESULTS AND DISCUSSION: The ethyl acetate extract of A. sulphureus (Fresen.) Wehmer (Trichocomaceae) demonstrated significant antibacterial potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi compared to streptomycin. MIC against test pathogens was in the range of 15.6-62.5 µg/mL. The antioxidant results revealed significant RSA from 12.43% to 62.02% (IC50 = 350.4 µg/mL, p ≤ 0.05). MME12 offered considerable inhibition of EAC proliferation (23% to 84%, IC50 = 216.7 µg/mL, p ≤ 0.05) supported by DNA fragmentation studies. The extract also offered insignificant haemolysis (5.6%) compared to Triton X-100. CONCLUSIONS: A single endophytic fungus, A. sulphureus MME12 was isolated and identified using molecular profiling. The above-mentioned findings support the pharmacological application of A. sulphureus MME12 extract and demand for purification of the active principle(s).

Quality assessment and antiplasmodial activity of West African Cochlospermum species.

The present study focuses on development of phytochemical methods for quality assessment of two West-African Cochlospermum species (Cochlospermum planchonii and Cochlospermum tinctorium) traditionally used for malaria treatment in Burkina Faso. Antimalarial activity of preparations from dried rhizomes (decoction) was tested against the chloroquine-sensitive Plasmodium strain 3D7 using the histidine-rich protein II (HRP2) drug susceptibility assay and compared with extract preparations using organic solvents of different polarity. Two main apocarotenoids were isolated from rhizomes of C. planchonii and unambiguously identified as dihydrocochloxanthine and cochloxanthine by spectroscopic methods. Comparative HPLC analyses of thirty-nine (39) samples from markets and from collections in natural habitats of both species showed a high variability in the accumulation of cochloxanthines and related carotenoids which were proven to be characteristic for rhizomes of both species and generally absent in leaves. Furthermore, content of total phenolics and antioxidant activities (DPPH and FRAP) as well as haemolytic activity of various extracts was tested. The HPLC method presented here was validated and provides a good separation of both compounds including 10 minor carotenoids. Extracts from both species and pure cochloxanthine offered pronounced antioxidant activities and weak haemolytic activity while, in contrast, dihydrocochloxanthine had a strong haemolytic effect at the highest concentration analysed. However, cochloxanthine as well as dihydrocochloxanthine showed erythroprotective effects against the haemolytic activity of the reference saponin. Moderate antiplasmodial activity between 16 and 63 µg/ml were observed with all tested extracts, and lower IC50 values were obtained with pure dihydrocochloxanthine (IC50=6.9 µg/ml), cochloxanthine (IC50=6.8 µg/ml), the DCM fraction (IC50=2.4 µg/ml) and the ethyl acetate fraction (IC50=11.5µg/ml) derived from a methanolic extract of C. planchonii. This study shows a major variability of carotenoid content and antiplasmodial activity of both C. planchonii and C. tinctorium. The high haemolytic activity of dihydrocochloxanthine (at 100 µg/ml) should be considered as a selection criterion for choosing species phenotypes for treatment.

Actividad antiplasmódica y hemolítica de extractos etanólicos y fracciones obtenidas de Cecropia membranacea Trécul. y Cecropia metensis Cuatrec. (sin. Cecropia peltata var. candida Velásquez) / Antiplasmodial and haemolytic activity of ethanolic extracts and fractions obtained from Cecropia membranacea Trécul. and Cecropia metensis Cuatrec. (syn. Cecropia peltata var. candida Velásquez)

Introducción: el estudio de las plantas medicinales ha permitido el desarrollo de productos fitoterapéuticos y fármacos para el tratamiento de diferentes enfermedades, entre estas la malaria. En Brasil las plantas usadas en la medicina tradicional por sus antecedentes como febrífugos o antimaláricos incluyen Cecropia hololeuca, Cecropia sp., Cecropia pachystachya y Cecropia glaziovii. Aún no ha sido comprobada la actividad antimalárica de Cecropia membranacea y Cecropia metensis, especies distribuidas en América Central y América del Sur, incluida Colombia. Objetivo: evaluar la actividad antiplasmódica frente a Plasmodium falciparum de extractos y fracciones de Cecropia membranacea y Cecropia metensis. Métodos: a partir de las hojas con peciolo de las dos especies se prepararon extractos etanólicos utilizando el método de percolación, se llevó a cabo el fraccionamiento del extracto etanólico, de cada especie, utilizando un sistema de partición con solventes de diferente polaridad (éter de petróleo, acetato de etilo, n-butanol y agua). Con los extractos y fracciones obtenidas se realizó un estudio fitoquímico preliminar. La actividad de extractos y fracciones se evaluó in vitro frente a Plasmodium falciparum FCB-2 y se estudió la actividad hemolítica. Resultados: las fracciones acetato de etilo de Cecropia membranacea (CI50 10,12 µg/mL) y Cecropia metensis (CI50 12,52 µg/mL) presentaron actividad antiplasmódica sin generar daño a la membrana de la célula hospedera (CH50> 1 000 µg/mL). La evaluación fitoquímica evidenció en estas fracciones una mezcla de compuestos tipo esteroide, terpénico y flavonoide. Conclusiones: las fracciones acetato de etilo de Cecropia membranacea y Cecropia metensis presentan actividad antiplasmódica promisoria, no asociada con propiedades líticas sobre las células eritrocitarias hospederas. Las dos especies son de interés para profundizar su estudio, en cuanto a la actividad antimalárica y composición fitoquímica(AU)

Introduction: the study of medicinal plants has led to the development of phytotherapeutic products and drugs for the treatment of various diseases, including malaria. Among the plants used in Brazilian traditional medicine for their febrifuge and antimalarial effects are Cecropia hololeuca, Cecropia sp., Cecropia pachystachya and Cecropia glaziovii. The antimalarial activity of Cecropia membranacea and Cecropia metensis has not been demonstrated. These two species may be found in Central and South America, including Colombia. Objective: evaluate the antiplasmodial activity of extracts and fractions of Cecropia membranacea and Cecropia metensis against Plasmodium falciparum. Methods: ethanolic extracts were obtained from petiolate leaves of the two species using the percolation method. The ethanolic extract of each species was then fractionated, using a partition system based on solvents of varying polarity (petroleum ether, ethyl acetate, n-butanol and water). The extracts and fractions obtained underwent preliminary phytochemical examination. Extracts and fractions were evaluated for their in vitro antiplasmodial activity against Plasmodium falciparum FCB-2, as well as for their haemolytic activity. Results: ethyl acetate fractions of Cecropia membranacea (IC50 10.12 µg/mL) and Cecropia metensis (IC50 12.52 µg/mL) showed antiplasmodial activity without damaging the host cell membrane (HC50 >1000 µg/mL). Phytochemical evaluation of these fractions revealed a mixture of steroid, terpene and flavonoid compounds. Conclusions: ethyl acetate fractions of Cecropia membranacea and Cecropia metensis showed promising antiplasmodial activity, not associated to lytic properties, over erythrocyte host cells. The two species are good ground for further study of their antimalarial activity and phytochemical composition(AU)