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.

Molecular identification of two genotypes of mumps virus causing two regional outbreaks in Asturias, Spain.

BACKGROUND: In spite of universal vaccination, several sporadic cases of mumps infection, which could produce outbreaks, are detected every year in different countries. OBJECTIVE: Mumps virus strains causing two regional outbreaks in Asturias (Spain) were phylogenetically characterized. STUDY DESIGN: Mumps virus strains, which were detected in samples from patients belonging to two regional outbreaks in Asturias, were characterized by sequencing of the SH gene and further alignment to homologous sequences of representative strains of the different mumps genotypes. RESULTS: Two different strains (Ast/SP02 and Ast/SP07) were isolated. Sequence analysis revealed that while Ast/SP02 belonged to genotype H, Ast/SP07 was phylogenetically close to UK02-19, a reference strain for a new genotype. Both strains belonged to different genotypes from those used in the vaccination (Jeryl-Lynn strain is genotype A). CONCLUSION: Mumps virus strains different from those used in vaccination program can cause mumps outbreaks even in vaccinated patients.

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

Detection of circulating Trichinella spiralis muscle larva antigens in serum samples of experimentally and naturally infected swine.

A follow up study was carried out to determine the kinetics of appearance of surface/stichosomal (S/S) components, recently included in the TSL-1 group of Trichinella spiralis muscle larva (ML), in serum samples from 13 experimentally infected pigs. Detection of circulating antigens in these animals was done by a sandwich enzyme-linked immunosorbent assay (ELISA) using T. spiralis specific rabbit polyclonal immunoglobulins to capture free antigens and monoclonal antibody NIM-M1 to recognize S/S antigens. The assay developed was able to detect as little as 35 ng ml-1 of S/S components added to normal pig serum. Antigenemia was observed in 54% of the experimentally infected swine with two peaks of appearance, one early at 1-4 weeks post-infection (pi) and one late at 10-14 weeks pi. Specific antibodies against S/S components were demonstrated in serum samples from all experimentally infected pigs starting at 3-4 weeks pi. Free antigen was also detected in serum samples from naturally infected backyard pigs with a sensitivity of 56% compared with 94% when antibody production was determined using purified S/S components in an ELISA.

Interlaboratory assessment of the bovine corneal opacity and permeability (BCOP) assay.

A multinational interlaboratory study to investigate the bovine corneal opacity and permeability (BCOP) assay is presented. The aim of this work was to determine the capability and possible limitations of this method to predict ocular irritancy of a large set of chemicals. The assays were carried out in 12 European laboratories with different types of activity. In each of these laboratories 52 substances, with a wide range of structure, physical form and irritant properties, were tested and in vitro scores were compared with those obtained from concurrent rabbit eye (Draize) tests. The technique was easily learned by workers in the participating laboratories, as shown by the fact that there were consistent responses between treated corneas within an individual laboratory. Interlaboratory variability was also very good. It was found that a given laboratory had a 96% chance of classifying irritants or non-irritants similarly to the other laboratories. In addition, it was observed that corneas preserved overnight responded similarly to freshly prepared tissues, thus allowing flexibility for those laboratories where the availability of corneas is limited. Comparisons between in vivo and in vitro data showed that the BCOP data correctly predicted whether a compound would be irritating or non-irritating for 44 of the 52 compounds (84.6%). Specificity and sensitivity were also greater than 84%, and the same number of substances were overestimated as were underestimated (four out of 52). All of the false negatives were solids whereas most of false positives were liquids, indicating that some adjustment in the protocol may be required depending on the physical state of the substance to be tested. All of the substances selected could be evaluated, with no limitation such as colour, insolubility, low or high pH. Given the number of products evaluated and the reproducibility within and among the laboratories involved, the overall results are quite satisfactory and therefore confirm the usefulness of the assay for screening chemicals for ocular irritation.

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.

Chronic morphine administration impairs cell-mediated immune responses in swine.

Swine were used as an experimental animal model to evaluate immunomodulating effects of the opiate drug, morphine, on antigen-specific humoral and cell-mediated immune responses. Morphine free base in peanut oil was administered at 4-day intervals for up to 42 days to maintain drug levels at or above 100 ng/ml. The effect of morphine administration on humoral immune responses was evaluated by immunization on day 7 of morphine treatment with a battery of antigens, including swine herpes virus (also known as pseudorabies virus, PRV), Brucella abortus and the Escherichia coli pilus antigens K88, K99, 987P and F41. Fourteen days later, swine were reimmunized with B. abortus and E. coli pilus antigens. Antibody titers to these antigens were evaluated on a weekly basis. Cell-mediated immunity was evaluated by measuring skin immune responses to the antigen 2,4-dintroflurobenozene (DNFB). In one experiment, swine were sensitized with DNFB on day 7 of morphine treatment at the same time as immunization with the other antigens. In a second series of experiments, swine were sensitized either 7 days before or 7 days after initiation of morphine treatment. Pigs were challenged with DNFB administered 27 days after the initiation of morphine treatment and skin responses were evaluated 24 h later. The ability of swine to resist PRV infection was evaluated by exposure to virulent virus on day 28 of morphine treatment. Chronic morphine administration did not impair the induction of the humoral immune responses to bacterial or viral antigens. In addition, morphine treatment did not alter the resistance of immunized swine to PRV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

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.