First Report of Root Rot Caused by Pythium spiculum Affecting Cork Oaks at Doñana Biological Reserve in Spain.

Cork oaks (Quercus suber L.) are key tree species at Doñana Biological Reserve (DBR), Huelva, Spain. Sampling was conducted on a total of 13 trees exhibiting symptoms of decline (foliar wilting and defoliation, branch dieback, and root necrosis). In 2008. Phytophthora cinnamomi was isolated from feeder roots of one tree and Pythium spiculum from two additional oaks. In 2011, both pathogens were isolated from six oaks, only P. cinnamomi from three oaks, and only Py. spiculum from one oak. This expansion was associated with high winter rainfall levels since 2009 that led to long periods of soil flooding. While P. cinnamomi is well known to cause a root disease on Q. suber (2), P. spiculum is a newly described species isolated from Quercus, Vitis, Prunus, Castanea, and Celtis species, but its pathogenicity was demonstrated only on Q. ilex (syn. Q. rotundifolia) (1). Pathogenicity tests were conducted on 4-year-old Q. suber plants. Inocula consisted of two isolates of Py. spiculum from DBR (DO8 and DO36 from Q. suber). For comparison with these, three isolates previously tested on Q. ilex (1) were included: two isolates of Py. spiculum, PA54 (from Q. suber) and PE156 (from Q. ilex); and one isolate of P. cinnamomi, PE90 (from Q. ilex). All these isolates came from the Andalucía region, stored at the oomycete collection of the University of Córdoba, and showed a 99 to 100% homology with their expected ITS sequences in GenBank (DQ196131 for Py. spiculum and AY943301 for P. cinnamomi). Inoculum was prepared by shaking and mixing propagule-bearing mycelium produced in carrot broth petri dishes (20°C, 4 weeks) in sterile water, to produce a concentration of 3 × 104 oospores × ml-1 (Py. spiculum) or 3 × 104 chlamydospores × ml-1 (P. cinnamomi). One hundred milliliters of inoculum was applied to each root (1). There were 10 inoculated plants per isolate and 10 non-inoculated control plants. All plants were waterlogged 2 days per week to favor root infection and maintained in an acclimatised greenhouse (12-28°C). Three months later, the inoculated plants showed symptoms of root necrosis that resulted in foliar wilting followed occasionally by defoliation. Control plants did not develop foliar symptoms nor root necrosis. Root damage severity assessed on a 0 to 4 scale (3) exhibited significant differences (P < 0.05) in relation to the control plants for all the isolates tested, with isolate PE90 (P. cinnamomi) and isolates PA54, DO8, and DO36 (P. spiculum) all averaging a root necrosis value of 2.5. Isolate PE156 of P. spiculum produced values of root necrosis (1.6 in average) significantly lower (P < 0.05) than the rest. This isolate belongs to the low virulence group of P. spiculum described on Q. ilex (1). The inoculated oomycete was always reisolated from necrotic roots and never from roots of control plants. To the best of our knowledge, this is the first report of P. spiculum as the cause of root rot of Q. suber. References: (1) Romero et al. J. Phytopathol. 155:289, 2007. (2) Sánchez et al. For. Pathol. 32:5, 2002. (3) Sánchez et al. For. Pathol. 35:115, 2005.

Use of neuron-specific enolase for assessing the severity and outcome of neurological disorders in patients

Neuron-specific enolase (NSE) is a glycolytic enzyme present almost exclusively in neurons and neuroendocrine cells. NSE levels in cerebrospinal fluid (CSF) are assumed to be useful to estimate neuronal injury and clinical outcome of patients with serious clinical manifestations such as those observed in stroke, head injury, anoxic encephalopathy, encephalitis, brain metastasis, and status epilepticus. We compared levels of NSE in serum (sNSE) and in CSF (cNSE) among four groups: patients with meningitis (N = 11), patients with encephalic injuries associated with impairment of consciousness (ENC, N = 7), patients with neurocysticercosis (N = 25), and normal subjects (N = 8). Albumin was determined in serum and CSF samples, and the albumin quotient was used to estimate blood-brain barrier permeability. The Glasgow Coma Scale score was calculated at the time of lumbar puncture and the Glasgow Outcome Scale (GOS) score was calculated at the time of patient discharge or death. The ENC group had significantly higher cNSE (P = 0.01) and albumin quotient (P = 0.005), but not sNSE (P = 0.14), levels than the other groups (Kruskal-Wallis test). Patients with lower GOS scores had higher cNSE levels (P = 0.035) than patients with favorable outcomes. Our findings indicate that sNSE is not sensitive enough to detect neuronal damage, but cNSE seems to be reliable for assessing patients with considerable neurological insult and cases with adverse outcome. However, one should be cautious about estimating the severity of neurological status as well as outcome based exclusively on cNSE in a single patient.

Use of neuron-specific enolase for assessing the severity and outcome in patients with neurological disorders.

Neuron-specific enolase (NSE) is a glycolytic enzyme present almost exclusively in neurons and neuroendocrine cells. NSE levels in cerebrospinal fluid (CSF) are assumed to be useful to estimate neuronal injury and clinical outcome of patients with serious clinical manifestations such as those observed in stroke, head injury, anoxic encephalopathy, encephalitis, brain metastasis, and status epilepticus. We compared levels of NSE in serum (sNSE) and in CSF (cNSE) among four groups: patients with meningitis (N=11), patients with encephalic injuries associated with impairment of consciousness (ENC, N=7), patients with neurocysticercosis (N=25), and normal subjects (N=8). Albumin was determined in serum and CSF samples, and the albumin quotient was used to estimate blood-brain barrier permeability. The Glasgow Coma Scale score was calculated at the time of lumbar puncture and the Glasgow Outcome Scale (GOS) score was calculated at the time of patient discharge or death. The ENC group had significantly higher cNSE (P=0.01) and albumin quotient (P=0.005), but not sNSE (P=0.14), levels than the other groups (Kruskal-Wallis test). Patients with lower GOS scores had higher cNSE levels (P=0.035) than patients with favorable outcomes. Our findings indicate that sNSE is not sensitive enough to detect neuronal damage, but cNSE seems to be reliable for assessing patients with considerable neurological insult and cases with adverse outcome. However, one should be cautious about estimating the severity of neurological status as well as outcome based exclusively on cNSE in a single patient.

Effects of intrathecal sufentanil on plasma oxytocin and cortisol concentrations in women during the first stage of labor.

BACKGROUND AND OBJECTIVES: Intrathecal sufentanil provides analgesia comparable to epidural bupivacaine for the first stage of labor. Both epidural local anesthetics and intrathecal opioid reduce some parameters of the neuroendocrine response to labor pain and the reflex release of oxytocin in animals. In humans, epidural local anesthetics only reduce the spurt release of oxytocin. This study compared the effect of intrathecal sufentanil and epidural bupivacaine administration on the plasma concentration of oxytocin and cortisol in women with labor pain during the first stage of labor. METHODS: Thirty healthy parturients requesting analgesia were enrolled in this randomized and open-label study. Each patient was in spontaneous labor at greater than 5 cm cervical dilatation. Using a combined spinal and epidural technique, patients received either intrathecal sufentanil 10 microg (SUF = intrathecal sufentanil group) or epidural plain bupivacaine 0.25%, 12 mL (BUPIV = epidural bupivacaine group). Analgesia was assessed using a visual analog scale, and blood samples for oxytocin and cortisol plasma concentration measurements were collected immediately before analgesia and 15, 30, 60, and 90 minutes after induction of the analgesia. Plasma cortisol and oxytocin concentrations were determined by specific radioimmunoassay. The values were expressed as mean +/- SEM. RESULTS: Intrathecal sufentanil provided faster and more complete analgesia within 15 and 30 minutes of its administration, compared with epidural bupivacaine. Plasma oxytocin concentrations were similar in the 2 groups before analgesia (7.24 +/- 2.1 and 6.6 +/- 3.1 pg/mL SUF and BUPIV, respectively). It decreased significantly in the SUF and increased in the BUPIV after analgesic administration. Cortisol concentrations were elevated in both groups before analgesia (51.6 +/- 5.3 and 54.2 +/- 4.8 microg/dL SUF and BUPIV, respectively). Both analgesic treatments significantly decreased the plasma cortisol levels. CONCLUSIONS: Intrathecal sufentanil analgesia decreases plasma concentrations of oxytocin and cortisol in women with labor pain during the first stage of labor, but epidural bupivacaine only reduced the cortisol concentration.

Labor analgesia in a patient with paroxysmal nocturnal hemoglobinuria with thrombocytopenia.

BACKGROUND AND OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH) is a form of acquired hemolytic anemia in which a defect of glycophosphatidylinositol anchor proteins in the cell membrane of bone marrow stem cells leads to activation of the complement system and consequent destruction of defective cells. The characteristics of this disease are an increased frequency of thrombotic events, anemia, and thrombocytopenia. METHODS: We report a case of a pregnant patient with PNH with thrombocytopenia who delivered vaginally after receiving epidural labor analgesia. Prophylaxis of thromboembolism was performed with heparin 1 hour after the removal of the epidural catheter, and repeated at 12-hour intervals. Sensory changes or motor changes and pain were monitored every 10 minutes for 8 hours after delivery. RESULTS: During analgesia, the patient reported complete pain relief. Delivery and the immediate postpartum period were without any untoward events. CONCLUSIONS: Four major factors influenced the anesthetic conduct used for the present patient: (1) the risk of an acute hemolytic crisis, (2) the need to perform prophylaxis for thromboembolism, (3) the need to reduce labor stress, and (4) minimizing the risk of missing an epidural hematoma. We also present a survey of the literature about PNH and discuss the anesthetic conduct in this patient.

Analgesic and adverse effects of a low dose of intrathecally administered hyperbaric neostigmine alone or combined with morphine in patients submitted to spinal anaesthesia: pilot studies.

We report the analgesic and adverse effects of intrathecally administered hyperbaric neostigmine, alone or combined with morphine, in two patients suffering from severe lower limb ischaemic pain (group 1), five patients undergoing Caesarean section (group 2) and 19 patients scheduled for orthopaedic surgery (group 3) under spinal anaesthesia. These patients were enrolled in three pilot studies undertaken before the initiation of the planned controlled studies. Hyperbaric neostigmine (50 micrograms in glucose 8%) produced analgesia lasting more than 6 h in patients of group 1, but the effect was accompanied by episodes of vomiting. A lower dose of hyperbaric neostigmine (25 micrograms), alone (two patients) or combined with morphine (50 micrograms) (one patient) produced no discernible analgesic effect but was followed by severe nausea and vomiting within 15 min of intrathecal injection in patients of group 2. Two patients who received hyperbaric morphine (100 micrograms) had analgesia for more than 24 h and exhibited mild pruritus. In patients of group 3, hyperbaric neostigmine alone (25 micrograms) produced analgesia of shorter duration than neostigmine (25 micrograms) plus morphine (50 micrograms) or morphine (100 micrograms). Neostigmine alone or combined with morphine was associated with adverse events, mainly nausea and vomiting that lasted up to 9-12 in some patients. Other adverse events observed included anxiety, somnolence and involuntary defaecation. Most patients who received the combination of neostigmine and morphine exhibited more severe nausea, vomiting and somnolence. The low clinical efficacy of intrathecally administered neostigmine alone or in combination with morphine impairs the design of a double-blind protocol and might restrict the clinical usefulness of the drug combination.

Improved RPLC determination of acyclovir using hexylamine as silanol masking agent.

The aim of the present work is to improve the sensitivity in the RPLC determination of acyclovir [9-(2-hydroxy ethoxymethyl) guanine] (ACV) and guanine, the major impurity of the drug synthesis and one of the compounds found in the chemical degradation process of ACV. The method was applied to the quantification of drug in liposomal formulations. The most important problem for RPLC analysis of both compounds are their high pKa values, mainly guanine, and the interaction with reactive silanol groups in the stationary phase. In order to avoid these problems there are four basic strategies: (i) ionic pair reagents, (ii) deactivated silica columns, (iii) polymeric based columns and (iv) silanol masking agents. A validation protocol was followed to develop the analytical method, using a Spherisorb ODS (250 x 4.6 mm i.d.) analytical column, with a mobile phase of 95% aqueous phosphate buffer (pH 3.0) and 5% HPLC methanol pumped isocratically at 1.3 ml/min(-1), with ultraviolet detection at 254 nm. The results showed a high reproducibility in retention time value, with R.S.D. of 2.37% for ACV and 0.32% for guanine. The lowest concentration levels assayed, 0.15 microg/ml(-1) for guanine and 1 microg/ml(-1) for ACV, showed good R.S.D. in the quantification parameter (peak area) 11.0% (guanine) and 9.64% (ACV)