Screening of antifungal activity of 12 essential oils against eight pathogenic fungi of vegetables and mushroom.

The antifungal properties of 12 Eos, that is, Syzygium aromaticum, Pelargonium graveolens, Lavandula angustifolia, Cupresus sempervirens, Mentha piperita, Santolina chamaecyparissus, Citrus sinensis, Pogostemon patchouli, Thymus mastichina, Thymus vulgaris, Eucalyptus globulus and Rosmarinus officinalis, were screened. The influence of five doses of each EOs was tested against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Phytophthora parasitica, Pythium aphanidermatum, Alternaria brassicae, Cladobotryum mycophilum and Trichoderma aggressivum f.sp. europaeum using disc-diffusion method. The mycelial growth inhibition and ED50 were calculated. The chemical analysis of the EOs was analysed using gas chromatography-mass spectroscopy. A total of 58 compounds were identified in the 12 EOs. All essential oils (EOs) analysed showed antifungal activity against the test pathogens in the range of 5·32-100%. The inhibitory effect of oils showed dose-dependent activity on the tested fungus. Based on the ED50 values, clove, rose geranium, peppermint and patchouli were the most effective. This study warrants further research into the practical use of EOs for the control of important myco- and phytopathogens in intensive horticulture. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the serious damage caused by fungal pathogens of vegetables and mushrooms, it is necessary to search for integrated strategies of disease control. This study provides relevant information about the effects of 12 essential oils (EOs) against eight pathogens of agricultural interest, included mycopathogens with emphasis on the possible future application of the EOs as alternative antifungal agents.

Effectiveness and safety of sofosbuvir-based regimens plus an NS5A inhibitor for patients with HCV genotype 3 infection and cirrhosis. Results of a multicenter real-life cohort.

Patients with HCV genotype 3 (GT3) infection and cirrhosis are currently the most difficult to cure. We report our experience with sofosbuvir+daclatasvir (SOF+DCV) or sofosbuvir/ledipasvir (SOF/LDV), with or without ribavirin (RBV) in clinical practice in this population. This was a multicenter observational study including cirrhotic patients infected by HCV GT3, treated with sofosbuvir plus an NS5A inhibitor (May 2014-October 2015). In total, 208 patients were included: 98 (47%) treatment-experienced, 42 (20%) decompensated and 55 (27%) MELD score >10. In 131 (63%), treatment was SOF+DCV and in 77 (37%), SOF/LDV. Overall, 86% received RBV. RBV addition and extension to 24 weeks was higher in the SOF/LDV group (95% vs 80%, P=.002 and 83% vs 72%, P=.044, respectively). A higher percentage of decompensated patients were treated with DCV than LDV (25% vs 12%, P=.013). Overall, SVR12 was 93.8% (195/208): 94% with SOF+DCV and 93.5% with SOF/LDV. SVR12 was achieved in 90.5% of decompensated patients. Eleven treatment failures: 10 relapses and one breakthrough. RBV addition did not improve SVR (RR: 1.08; P=.919). The single factor associated with failure to achieve SVR was platelet count <75×10E9/mL (RR: 3.50, P=.019). In patients with MELD <10, type of NS5A inhibitor did not impact on SVR12 (94% vs 97%; adjusted RR: 0.49). Thirteen patients (6.3%) had serious adverse events, including three deaths (1.4%) and one therapy discontinuation (0.5%), higher in decompensated patients (16.7% vs 3.6%, P<.006). In patients with GT3 infection and cirrhosis, SVR12 rates were high with both SOF+DCV and SOF/LDV, with few serious adverse events.

Undetectable HCV-RNA at treatment-week 8 results in high-sustained virological response in HCV G1 treatment-experienced patients with advanced liver disease: the International Italian/Spanish Boceprevir/Peginterferon/Ribavirin Name Patients Program.

In many countries, first-generation protease inhibitors (PIs)/peginterferon/ribavirin (P/R) still represent the only treatment option for HCV-infected patients. Subjects with advanced disease and previous failure to P/R urgently need therapy, but they are under-represented in clinical trials. All treatment-experienced F3/4 Metavir patients who received boceprevir (BOC)+P/R in the Italian-Spanish Name Patient Program have been included in this study. Multivariate logistic regression analysis (MLR) was used to identify baseline and on-treatment predictors of SVR and adverse events (AEs). Four hundred and sixteen patients, mean age 57.7 (range 25-78 years), 70% males, 69.5% (289/416) F4, 14% (41/289) Child-Pugh class A6, 24% (70/289) with varices and 42% (173/416) prior null responders to P/R, were analysed. Overall, SVR rate (all 381 patients who received one dose of BOC) was 49%, (58% in F3, 45% in F4, 61% in relapsers, 51% in partial, 38% in null responders, and 72% in subjects with undetectable HCV-RNA at treatment-week (TW)8. Among patients with TW8 HCV-RNA ≥ 1000 IU/L, SVR was 8% (negative predictive value = 92%). Death occurred in 3 (0.8%) patients, while decompensation and infections were observed in 2.9% and 11%, respectively. At MLR, SVR predictors were TW4 HCV-RNA ≥ 1log10 -decline from baseline, undetectable TW8 HCV-RNA, prior relapse, albumin levels ≥3.5 g/dL and platelet counts ≥100 000/µL. Metavir F4, Child-Pugh A6, albumin, platelets, age and female gender were associated with serious and haematological AEs. Among treatment-experienced patients with advanced liver disease eligible for IFN-based therapy, TW8 HCV-RNA characterised the subset with either high or poor likelihood of achieving SVR. Using TW8 HCV-RNA as a futility rule, BOC/P/R appears to have a favourable benefit-risk profile.

[Runners with back pain: to run or not to run?]. / Coureurs souffrant de maux de dos: courir ou ne pas courir?

We evaluated the perceived impact of physical activity on back pain in runners. Information from 777 runners participating in a half marathon was obtained with a questionnaire about basic data, features of the weekly training and the relationship between running activity and back pain. Half the runners (54.1%) reported a history of back pain. Among them, almost twice as many reported an improvement (49%) than a worsening (27%) of pain with running. No significant associations were found between perceived impact of running on back pain and other factors. In our study favorable effects were much more frequent than unfavorable ones. Further studies are needed to better understand these effects.

First Report of Cladobotryum mycophilum Causing Cobweb on Cultivated King Oyster Mushroom in Spain.

In 2010, symptoms of cobweb were observed on cultivated king oyster mushroom (Pleurotus eryngii) in Castilla-La Mancha (Spain) affecting 16% of the blocks of substrate cultivated. Cobweb appeared at the end of the crop cycle, first as small, white patches on the casing soil, subsequently spreading to the nearest king oyster mushroom by means of a fine gray-white mycelium, and eventually sporulating to produce masses of dry spores. The mycelium can quickly cover pinheads, stalks, pileus, and gills, eventually resulting in decomposition of the entire fruit body. Infected tissues of P. eryngii were plated onto potato dextrose agar (PDA) and the parasitic fungus was isolated. Fungal colonies consisted of abundant and cottony aerial mycelium spreading rapidly on PDA and red pigment spreading in the agar. Conidiogenous cells were 24 to 35 µm long, 3.5 to 5 µm wide basally, and tapered slightly to the tip. Conidia were cylindrical to narrowly ellipsoidal, 17 to 25 (-28) × 8 to 10 µm, and zero to three septate. Total DNA was extracted and the internal transcribed spacer (ITS) region of rDNA was amplified for one isolate using ITS1F/ITS4 primers (1,3). The amplicon was sequenced (GenBank Accession No. JF505112). BLAST analysis showed 100% similarity of the obtained ITS sequence with two sequences of Cladobotryum mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. Y17096 and Y17095) (2). Pathogenicity tests were performed using 24 blocks containing sterilized, spawned, and incubated P. eryngii substrate (3.6 kg, 352 cm2 in area). The blocks were placed in a mushroom-growing room and cased with a 40-mm layer of a casing soil (0.7 liter block-1) made with mineral soil + Sphagnum peat 4:1 (vol/vol). Five days after casing, a conidial suspension (7 × 103 conidia ml-1) of one isolate of C. mycophilum was sprayed (5 ml per block) onto the surface of the casing layer at a rate of 106 conidia m-2. Twenty-two blocks were sprayed with sterile distilled water as a control. A temperature of 17 to 18°C and 85 to 90% relative humidity were maintained throughout cropping. The first cobweb symptoms developed 23 days after inoculation and C. mycophilum was consistently reisolated from nine (37.5%) of the inoculated blocks. Noninoculated blocks remained healthy. In a second test, conidial suspensions (3.4 × 105 conidia ml-1) of one isolate of C. mycophilum were inoculated onto 20 P. eryngii fruit bodies. Ten fruit bodies were inoculated externally while the other 10 fruit bodies were cut in half and inoculated internally with 50 µl of conidial suspension per fruit body. Sterilized distilled water was used as a control. All fruit bodies were then incubated at 22°C in a moist chamber. Assays were conducted twice and the results were recorded after 7 days. C. mycophilum grew on 85% of the internally inoculated fruit bodies and on 40% of those inoculated superficially, while the control mushrooms remained symptomless. To our knowledge, this is the first report of C. mycophilum causing cobweb in king oyster mushroom in Spain. This finding will have a potentially significant impact on button mushroom farms where cobweb is one of the most common diseases. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) G. J. McKay et al. Appl. Environ. Microbiol. 65:606, 1999. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Brown Blotch Caused by Pseudomonas tolaasii on Cultivated Pleurotus eryngii in Spain.

In 2006, symptoms of brown blotch were observed on cultivated Pleurotus eryngii (king oyster mushroom) in Castilla-La Mancha (Spain). Subsequently, between January and May of 2008, brown blotch affected 39.75% of the blocks of substrate cultivated, resulting in a considerable loss of production. Symptoms observed were principally characterized by a yellowish brown-to-orangish color, first of the cap and then of the stalk. Some samples also showed a slightly concave cap. From samples collected from four different king oyster mushroom farms, a fluorescent gram-negative bacterium was recovered on King's B medium and identified as Pseudomonas tolaasii by the LOPAT scheme and other tests (2). The bacterium isolated had the following characteristics: oxidative, positive for oxidase and arginine dihydrolase, negative for levan production, pectinolitic activity on potato slices, and tobacco hypersensitivity. Results from other tests were as follows: negative for esculin hydrolysis and positive for gelatine, casein, and Tween 80 hydrolysis; mannitol, erythritol, sorbitol, m-inositol, and adonitol were used as a sole carbon source, but not sucrose, d-tartrate, or trigonelline. The white line test was performed (4) using P. reactans LPPA 540 and the presumptive isolates of P. tolaasii were positive. The gene encoding the 16S rRNA from two isolates (LPPA532 and LPPA533) was sequenced after PCR amplification (2) and their nucleotide sequences (1,400 bp; EMBL Accession No. FM864215 for LPPA 532) proved to be identical. The amplified sequences were compared with DNA sequences available in databases (GenBank, EMBL, DDBJ, and PDB) by using BLAST. An identity of 99% was obtained with 16S rDNA of three P. tolaasii strains (GenBank Accession Nos. AF320990, AF094750, and AF255336). Four isolates were selected for pathogenicity tests. Bacterial suspensions were grown for 16 h in yeast peptone glucose broth (approximately 108 CFU/ml) and were inoculated by puncture into 10 mushroom caps using sterilized wooden toothpicks (4). Sterilized distilled water was used as a control. These were then incubated at room temperature in glass dishes. Assays were conducted twice and the results were recorded after 10 days. The symptoms that developed after infection were similar to those observed in the crop, while the control mushrooms remained symptomless. Bacteria sharing the characteristics of the inoculated isolates were recovered from symptomatic caps. P. tolaasii has been described as causing brown blotch on Pleurotus eryngii (1,3), but to our knowledge, this is the first report of P. tolaasii causing brown blotch on Pleurotus eryngii in Spain. References: (1) J. F. Bradbury. No. 891 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1987. (2) A. J. González et al. Appl. Environ. Microbiol. 69:2936, 2003. (3) A. Russo et al. Microbiol. Res. 158:265, 2003. (4) J. M. Wells et al. Phytopathology 86:1098, 1996.

T-cell activation, expression of adhesion molecules and response to ethanol in alcoholic cirrhosis.

Abnormal immune function is a well-recognized feature in patients with alcoholic cirrhosis. It may contribute to the pathogenesis of the disease and to the clinical consequences. Nevertheless, a potential role of ethanol to elicit immune disturbances in patients is still unclear. To further examine the immune mechanisms which potentially are involved in alcoholic cirrhosis and the relationship to ethanol, we have determined the expression of surface antigens CD4, CD8, and of adhesion molecules CD25, LFA-1, ICAM-1 and LFA-3 in patients and in response to stimulation with OKT-3, IL-2 and with ethanol in vitro. In addition, we quantified the production of IL-2, TNF-alpha and IFN-gamma by lymphocytes of alcoholic cirrhosis patients compared to controls. Lymphocytes from patients showed increased basal and stimulated expression of CD4, CD25, LFA-1, ICAM-1 and LFA-3 molecules and increased TNF-alpha production in comparison to controls. When lymphocytes from patients were co-cultured with ethanol, the overexpression of activation markers and TNF-alpha production was similar to that obtained with mitogens. In contrast, a predominant suppressive effect of ethanol was observed in lymphocytes from controls. Our study underlines the importance of a chronic state of immune activation in alcoholic cirrhosis. The data further suggest a role of ethanol to stimulate immune response and to be directly involved in the development of disease.

[Hepatic fascioliasis. Resistance to praziquantel treatment and the radiological findings]. / Fasciolasis hepática. Resistencia al tratamiento con praziquantel y hallazgos radiológicos.

We report a case of hepatic fascioliasis. Fever and epigastric pain was the clinical presentation. We describe the features of the ultrasound and computed tomography examination. Symptoms were persistent after praziquantel treatment. Bithionol was effective and cured the patient.

[Portal hypertension secondary to an arterioportal fistula. Treatment by embolization]. / Hipertensión portal secundaria a fístula arterioportal. Tratamiento mediante embolización.

We report a case of arterioportal fistula which was probably the result of percutaneous liver biopsy. Portal hypertension with hemorrhage by esophageal varices was the clinical presentation. We describe the features of the ultrasound, selective arteriography and Doppler ultrasound examination. The fistula was catheterized superselectively and successfully embolized with steel coils.

[Severe digestive hemorrhage of an unusual etiology. Visible isolated and bleeding esophageal vessels]. / Hemorragia digestiva grave de etiología inusual. Vasos visibles esofágicos aislados y sangrantes.

We describe two patients with massive upper gastrointestinal bleeding. The upper gastrointestinal endoscopy revealed jet haemorrhage in the distal portion of the esophagus, from a visible vessel, without esophageal mucosal damage. In both cases arteriography was performed, and did not show vascular malformations or fistulous points. The patients were successfully treated, one with endoscopic sclerosis and balloon tamponade, and the other with endoscopic sclerosis and transcatheter embolization through left gastric artery. For the time being we do not know the true importance of these findings, but we hope that other reports help us to understand the clinical and pathologic features of this vascular lesion, and the best therapeutic approach.

[Esophageal perforation and postoperative fistulae of the upper digestive tract treated endoscopically with the application of Tissucol]. / Perforación esofágica y fístulas posquirúrgicas del tracto digestivo superior, tratadas endoscópicamente con la aplicación de Tissucol.

We present three patients with serious gastro-esophageal complications which were treated with Tissucol. The first patient developed a rare postoperative oesophago-pleural fistula. The second suffered a traumatic esophageal perforation (possibly iatrogenic) that was discovered at the end of the removal procedure of an alimentary bolus impacted in the distal esophagus. Attempts to close the high output oesophago-pleural fistula with standard treatment were unsuccessful. It was closed with Tissucol at the third attempt, in conjunction with oesophago-jejunal stenosis by means of endoscopic dilatation. In the second patient, early Tissucol application after detection of pneumomediastinum was an effective complementary treatment to the conservative approach and rapidly closed the perforation. The third patient developed a low debit postoperative gastro-cutaneous fistula that did not resolve with conservative treatment. It was closed with only one session of Tissucol sealing. We consider that the endoscopic application of fibrin glue should become the first step in the conservative treatment of small esophageal perforations or postoperative esophageal pleural fistulae, especially in cases of high output fistulae. The success of this technique depends on the localization and selective catheterization of the fistula and on brushing the fistular opening. Total resolution of any distal stenosis is necessary to prevent reopening of the fistula.

[Treatment of inoperable malignant gastric stenosis with self-expanding prosthesis]. / Tratamiento de las estenosis gástricas malignas irresecables mediante la colocación de prótesis autoexpandibles.

We report two patients with inoperable malignant stenosis of gastric antrum who were treated with endoscopic placement of a 22 mm Wallstent metallic prosthesis. In one patient, the endoscope was introduced simultaneously with the prosthesis. Different types of pincers were introduced through the endoscope's canal, which aided the movement and placement of the prosthesis. In the other patient, to broaden the stenosis a pediatric endoscope was introduced with a guide inside the canal, which was held straight externally and which facilitated the positioning of the prosthesis. There were no complications and the patients were discharged after 48 hours able to follow a normal oral diet. Both patients are still living, six and four months respectively after the procedure.

[Lymphocyte subpopulations in rheumatoid arthritis patients, their relatives and spouses]. / Subpoblaciones linfocitarias en pacientes con artritis reumatoide, sus familiares y cónyuges.

A study of lymphocytic sub-populations of patients with rheumatoid arthritis, their families and partners, is presented. The determination was carried out by means of monoclonal antibodies, analysing: T cells sub-population (CD3, CD4, CD8 and quotient CD4/CD8); B cells sub-population (B1); activated cells (OK1a); monocytes and NK cells (OKM1 and BMA 070). The results showed an increase of CD3 sub-population (T total lymphocytes) in patients with RA and their families. The meaning of these results is difficult to evaluate as lymphocyte sub-population is affected by several variables. The lack of modification of suppressor population, CD4/CD8 quotient and IKM1 cells in RA patients is probably due to the moderate activity of the disease.

[HLA antigens in patients with rheumatoid arthritis, their relatives, and spouses]. / Estudio de los antígenos HLA en pacientes con artritis reumatoide, sus familiares y cónyuges.

The results of the comparison between phenotypic frequency of HLA antigen classes I and II in patients afflicted with rheumatoid arthritis (RA) and their relatives (study carried out in the east of Andalusian), showed a significant increase of HLA A24, B51, B44, CW1 ans CW5 class I antigen in the group of relatives. We found a statistically significant DR4 antigen class II increase in both, patients and relative groups. The marriage parther of patients with RA did not have variations in class I or II HLA antigens compared to the control group. We concluded that RA is probably related to the following antigens: A24, B51, B44, CW1, CW5 and DR4.