Incidence of enterovirus in patients with acute gastroenteritis.

Enteroviruses (EV) have been linked to lymphocytic meningitis and exanthems, but they may also be involved in acute gastroenteritis (AGE), a condition whose aetiological agent often remains unidentified. In this work 1214 samples from individuals with AGE were studied with the aim of establishing the incidence of EV. The samples were collected between September and December in three different years and subjected to real-time genomic amplification in order to determine the viral load (VL). Of the 1214 samples studied, infection by a single virus was found in 328 cases (27%) and coinfection in 69 (5.7%). While adenoviruses (AdV) were the most frequent (14.8% of total), EV were present in 126 (10.4%) of the individuals tested. Of the 126 EV-positive samples, this virus was found as a single infection and coinfection in 76 (6.3%) and 50 (4.1%) cases, respectively. VL for EV was 5.58±1.51 log copies/ml (range 3.73-9.69) in the former and 6.27±1.75 (range 3.73-10.5) (p=0.02) in the latter. EV were identified in 97 children under 5 (16.9%) and in 29 (4.5%) patients over 5. Patients less than 5 years showed a higher VL that those more than 5 years age [6.08±1.57 (range 3.82-9.69) vs. 5.07±1.53 (range 3.73-10.58); (p=0.002)]. There was a high incidence of EV in AGE patients, and they were more frequent in those under 5, where they were found to replicate more efficiently. These results therefore indicate that testing for EV should be included in the diagnosis of AGE.

The use of unfixed bone marrow trephines for multicolour flow cytometry.

An adequate bone marrow aspirate is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the simultaneous assessment of multiple antigens on the cell surface as well as intracellular or nuclear ones. In the context of acute leukaemia, it is important to have a diagnosis of the blasts lineage as soon as possible to decide the appropriate treatment. This is sometimes delayed due to difficulties in obtaining a bone marrow aspirate due to a "dry tap". In this study we evaluated retrospectively cell markers results by flow cytometry of unfixed bone marrow trephines of 65 patients with leukaemia at diagnosis and including a few after treatment. Our aims were: 1) To compare cell markers results between bone marrow trephine (BMT) and bone marrow aspirate (BMA) 24 cases and BMT with peripheral blood (PB) 14 cases in paired samples to establish if they were reproducible with results of the unfixed bone marrow trephine biopsies. 2) To ascertain a precise diagnosis in 27 (42%) of the cases in which only a bone marrow trephine was available. We demonstrated that unfixed bone marrow trephine provides an adequate and representative cell suspension for flow cytometry and it is a powerful tool when no other material (bone marrow aspirate or peripheral blood) is available to make a rapid diagnosis. Furthermore when marrow aspirate or peripheral blood paired samples were available, flow cytometry results obtained were identical across all the sample types. Applicability to the clinical laboratory: We described a method to obtain a cell suspension from core biopsies that can easily be implemented routinely in a laboratory that performs diagnostic flow cytometry immunophenotyping. This method is simple, inexpensive and it doesn't require extra equipment.

Morphological characteristics of the optic nerve evaluated by confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population from the city of Barcelona.

OBJECTIVE: To evaluate morphological parameters of optic disc and retinal nerve fiber layer (RNFL) examined with confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population, and analyze correlations of these parameters with demographic variables. PATIENTS AND METHODS: Cross-sectional study in the context of a glaucoma screening campaign in the primary care center of Barcelona. The individuals selected were non-hypertensive Mediterranean Caucasians with risk for glaucoma development (individuals≥60 years old or≥40 years old with family history of glaucoma or intraocular pressure or myopia>3diopter). All subjects underwent a complete ophthalmic examination, confocal laser tomography (HRT3) and scanning laser polarimetry (GDX-VCC), subjects with results within normal limits only being included. Structural parameters were analyzed along with age, refraction, and pachymetry based on the Spearman rank correlation test. RESULTS: A total of 224 subjects included, with a mean age of 63.4±11.1 years. Disc areas, excavation and ring area were 2.14±0.52mm(2), 0.44±0.34mm (2) and 1.69±0.38mm(2), respectively. The mean RNFL (GDX) was 55.9±6.9µm. Age was correlated with lower ring volume, highest rate of cup shape measure, largest mean and maximum cup depth, lower nerve fiber index (NFI) and RNFL (all p-values below .05). CONCLUSION: The mean values and distribution of several parameters of the papilla and the RNFL in normal Mediterranean Caucasians population are presented. A loss of thickness of the RNFL, ring thinning, and enlarged cup was observed with increased age.

Pre-transplant MRD predicts outcome following reduced-intensity and myeloablative allogeneic hemopoietic SCT in AML.

The presence of minimal residual disease (MRD) by multiparametric flow cytometry (MFC) has been associated with adverse outcomes in AML patients treated with chemotherapy alone, but its impact in the setting of allogeneic hematopoietic SCT (HSCT) is less clear. We studied 88 patients who underwent myeloablative (MA) or reduced-intensity conditioned allogeneic HSCT for AML in first or subsequent remission at our center. MRD status was determined using three-color MFC on pre-HSCT BM aspirates, and patients were stratified by MRD status into MRD-negative, low-level MRD-positive (<1%) or high-level MRD-positive groups (1-4.9%). Two-year survival estimates in these groups were 66.8%, 51% and 30%, respectively (P=0.012), and 2-year estimates of relapse were 7.6, 37 and 70% (P<0.001). Pre-HSCT MRD was related to disease characteristics including secondary AML (P=0.002) and primary induction failure (P=0.005), but, despite these strong correlations, MRD remained independently associated with poorer survival in multivariate analysis (hazard ratio, 1.92; P=0.014). Pre-HSCT MRD is associated with adverse clinical outcomes in AML patients undergoing reduced-intensity or MA HSCT in first or subsequent remission and should be integrated into transplant strategies for patients with AML.

IgVH genes mutation and usage, ZAP-70 and CD38 expression provide new insights on B-cell prolymphocytic leukemia (B-PLL).

B-prolymphocytic leukemia (B-PLL) is a rare disease with poor prognosis. To further characterize the biological features of this disease, we analyzed immunoglobulin heavy chain (IgVH) mutations, ZAP-70 and CD38 in 19 cases with de novo B-PLL. Immunoglobulin heavy chain genes analysis showed an unmutated pattern (>98% homology to germ line) in 9/17 cases (53%), with 100% homology in eight. In the remaining, it ranged from 90 to 97.4%, with three cases slightly mutated (98-95%) and five heavily mutated (<95%). All B-PLL utilized members of VH3 (11/17) and VH4 (6/17) families, with V3-23, V4-59 and V4-34 gene accounting for more than half of them, regardless of mutational status. ZAP-70, assessed by flow cytometry, ranged from 1 to 91% cells, being > or =20% in 57% of cases. CD38 ranged from 1 to 99% (median 21%). There was no correlation between IgVH status and ZAP-70 or CD38 expression, but male gender and del(17p) were more common in the unmutated group. Neither IgVH mutations, CD38 expression nor del(17p) influenced patients' outcome. Unexpectedly, ZAP-70+ B-PLL patients survived longer (40 months) than ZAP-70- B-PLL (8 months). B-PLL appears biologically heterogeneous regarding IgVH mutations, ZAP-70 and CD38 expression, showing a pattern distinct from that of other lymphoproliferative disorders.

CD52 expression in T-cell large granular lymphocyte leukemia--implications for treatment with alemtuzumab.

Few reports on the successful treatment of T-cell large granular lymphocyte (LGL) leukemia with the humanized anti-CD52 monoclonal antibody alemtuzumab are emerging in the literature. The expression of CD52 by LGLs has not been previously investigated. Using semi-quantitative 2- and 3-color flow cytometry, we documented the expression of CD52 in 100% of abnormal cells in T-cell LGL leukemia (n = 11) and natural killer (NK) cell LGL leukemia (n = 2), and showed no significant difference in CD52 expression between T-cell prolymphocytic leukemia (PLL) and T-cell LGL leukemia. Higher CD52 expression has been noted in responders to alemtuzumab in T-cell PLL and in chronic lymphocytic leukemia (CLL), a B-cell disorder. The strong and consistent expression of CD52 shown here highlights the potential role of alemtuzumab in the treatment of refractory T-cell LGL leukemia and possibly aggressive NK cell leukemia.

Prolonged treatment response in aggressive natural killer cell leukemia.

We describe a case of natural killer (NK) cell leukemia with acute presentation, systemic symptoms and hepatosplenomegaly. The uniform and aberrant phenotype of NK cells with infiltration of bone marrow and spleen was in keeping with a malignant diagnosis. Aggressive presentation was demonstrated by marked constitutional symptoms and significant tumor burden (liver, spleen, blood, bone marrow). The subsequent clinical course has been indolent, but this may have been influenced by treatment. Treatment consisted sequentially of splenectomy, intravenous pentostatin and the combination of cyclosporine A and recombinant human erythropoietin and has resulted in survival of over 48 months. We discuss the difficulties in the diagnosis of this condition, explore possible causes of cytopenia(s), and highlight the role of immunosuppression in controlling disease manifestations in large granular lymphocyte proliferative disorders.

Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry.

The clinical significance of detecting minimal residual disease (MRD) in B-lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B-cell precursor ALL were followed during and after completion of treatment (median follow-up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5-15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease-free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups (P<0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B-lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with 'true' remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT-negative B-cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B-lineage ALL and should be tested in prospective clinical trials.

Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b).

A scoring system, based on the immunophenotypic analysis of a panel of five membrane markers (CD5, CD22, CD23, FMC7, SmIg) was shown to be useful in the distinction between chronic lymphocytic leukemia (CLL) and other B-cell lymphoproliferative diseases (non-CLL). We investigated whether the monoclonal antibody SN8 (CD79b) could improve our previous scoring system. Peripheral blood samples of 298 patients with CLL and 166 patients with non-CLL were analyzed by flow cytometry. Using the five standard markers, the accuracy of the scoring system was 91.8%, using a cutoff of 4 points or higher, to distinguish CLL from non-CLL. This was increased to 96.6% if SN8 was added and a cutoff of 4 points or higher was also used. A similar accuracy, 96.8%, was observed if CD22 was excluded and a cutoff of 3 points or higher was used. Thus, the replacement of CD22 by SN8 in the original scoring system significantly increases its potential to discriminate between CLL and other B-cell lymphoproliferative diseases.

Effect of hybrid peptides of cecropin A and melittin in an experimental model of bacterial keratitis.

Synthetic peptides, ranging from 12 to 18 residues, containing partial sequences from natural cecropin A and melittin were tested for activity in an experimental pseudomonas keratitis model in rabbits. In separate experiments, two Pseudomonas aeruginosa strains: (a) a clinical isolated strain, and (b) an American Type Culture Collection (ATCC) strain, were inoculated into the stroma of one cornea of each rabbit. Peptides were topically applied at 0.1% in phosphate-buffered saline (PBS) and compared with PBS alone and 0.3% gentamicin eye drops. Clinical evaluation, based on the McDonald-Shadduck scale, was performed during a > 48-h period after the bacterial inoculation. The peptide-treated animals showed significantly lower (p < 0.05) inflammatory signs and lower anterior-segment bacterial damage compared with PBS-treated animals, after the first 6 h. The antiinflammatory/antimicrobial activity was non significantly differnt (p > 0.05) from that in animals treated with gentamicin. We conclude that peptides keeping the sequence KWKLFKK from cecropin A and at least the sequence VLKVL from melittin show promise as novel agents in topical ocular therapy of bacterial keratitis.

Estudio microscópico de fenómenos biológicos asociados a infecciones por cateteres venosos centrales / Microscopic study of biological phenomenae associated to infections related to central venous catheters

Las complicaciones infecciosas son la principal limitación del uso de cateteres venosos centrales. Se presenta aquí un estudio descriptivo, mediante microscopía de barrido, de la formación de una matriz biológica en el interior y exterior del catéter como condicionante de la adherencia y crecimiento de bacterias, comprobándose que es un fenómeno que se presenta con alta frecuencia al permanecer estos dispositivos de poliuretano en el medio intravascular y subcutáneo. En este estudio preliminar se observa una adecuada correlación ente la observación de colonización del catéter por microscopía y el resultado de cultivo semicuantitativo de la punta de éste. La colonización del catéter, si bien representa uno de los eventos iniciales asociados a infección, no siempre se acompaña de signos clínicos o de bacteremia

Fasciola hepatica: characterization of somatic and excretory-secretory antigens of adult flukes recognized by infected sheep.

The immune response of sheep to somatic components and excretory/secretory products of adult Fasciola hepatica, was studied during an experimental infection. Antibodies against adult fluke somatic and excretory/secretory antigens were detected by thin layer immunoassay from the second week post infection. Similarly, the results of Western blot analysis showed specific recognition of several components as early as two weeks after infection. However, an increase in the number and intensity of bands with time of infection was observed in the patterns of antigenic recognition. Most notorious was the strong reactivity of all infected sheep sera towards components of 20-23 kDa in the somatic preparation and components of 23-27 kDa in the excretory/secretory products of adult F. hepatica, specially noticeable after the sixth week post-infection. Since these polypeptides were recognized by all infected animals, they could play an important role in the diagnosis of sheep fascioliasis.

Direct cytotoxicity mediated by allogeneic and isogeneic pig lymphocytes modified with fluorescein isothiocyanate.

Cell mediated cytotoxicity (CMC) is restricted by the major histocompatibility complex (MHC) in some animal models but not in others. In order to determine if there was MHC restriction in a CMC experimental model, mononuclear cells from blood of two lines of pigs, (1/1) and (6/6) which are homogeneous to the MHC products (SLA-ABCD), were used. Cytotoxic (1/1) cells were induced in vitro and in vivo against (1/1) mononuclear cells modified with fluorescein isothiocyanate (FITC) and tested against (1/1)-FITC isogeneic and (6/6)-FITC allogeneic cells, without showing MHC restriction of the cytotoxicity. The cytolysis was not due to antibody and K cells because fluoresceinated ovalbumin did not inhibit the test. It was not due to the "altered self" reaction against its own MHC products that sometimes may be induced by allogeneic responses, because the (1/1) anti (6/6) cytotoxic cells did not kill haptenated cells (1/1)-FITC. According to the results there was no evidence of MHC restriction of the CMC response of haptenated cells from pigs of (1/1) haplotype.

[Levels of oligo-elements and trace elements in patients at the time of admission in intensive care units]. / Valores de oligoelementos y elementos traza al ingreso de pacientes en UCI.

Routinely, the macronutrient contribution is thoroughly studied when a patient is included in a total parenteral nutrition (TPN) program. However, it must be kept in mind that along with macronutrients, micronutrients, oligoelements and vitamins are also administered, and these must be taken into account. At present, for instance, the importance of oligoelements has become evident, and clinical conditions of oligoelement deficiency have been described in many cases even when the minimum daily requirements had been met. This study centers on the importance of evaluating oligoelement and trace element levels when patients are admitted into the ICU and were included into a total parenteral nutrition (TPN) program. An assessment of the serum calcium (Ca), phosphorus (P), magnesium (Mg), zinc (Zn), copper (Cu), iron (Fe) and transferrin (Tfe) levels was done on 55 septic patients who underwent abdominal surgery was done on admission to the ICU. Transferrin was measured of serve as an indicator of metabolic damage. A description of the methods used in analytical assessment, a presentation of normal values, the statistical management of each of the elements under study and the interpretation of the results obtained has been done. The discussion is based on the changes detected in the patients' serum levels on admission to the unit, showing low iron, transferrin, zinc and calcium values and normal magnesium, phosphorus and copper figures. The conclusion arrived at suggests that the daily parenteral supplements of these elements should be higher than those recommended by the American Medical Association (AMA) and by other authors.

Complejos inmunes circulantes en ovinos infestados con Fasciola hepatica / Circulating immune complex in sheep infected with Fasciola hepatica

Se determinó la presencia de complejos inmunes circulantes (CIC) en 38 sueros de ovinos infestados con Fasciola hepatica y 15 sueros de ovinos libres del parásito, por precipitación con polietilenglicol 6000 (PEG) al 4, 6, 8 y 10%. La cantidad de proteína precipitada fue mucho mayor en los ovinos parasitados que en los testigos en todas las concentraciones de PEG utilizadas, observándose la mayor diferencia con PEG al 10%. Para corroborar la presencia de CIC se precipitaron los sueros con PEG al 2.5% y se determinó el consumo de complemento (CC) de estos precipitados obteniéndose valores promedio de 57% en los ovinos con fasciolasis y de 29% en los ovinos testigos. Se determinaron los títulos de anticuerpos contra F. hepatica por hemoaglutinación pasiva (HP), difusión doble en agar (DD) e inmunoensayo en capa delgada (ICD). No hubo relación entre la presencia de CIC y los títulos determinados en las pruebas serológicas, excepto en ICD dondo los sueros en títulos más altos fueron los sueros que dieron mayor preciptación con PEG y mayor CC