Comparison of two methods skipping cell lysis and protein extraction for identification of bacteria from blood cultures by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry.

OBJECTIVE: Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) is widely used for fast identification of bacteria from blood cultures (BC). We compared the performance of two procedures, one including a pre-enrichment step in brain heart infusion and the other a direct method using vacutainer separator gel tubes (DI), for identification of bacteria from blood cultures by MALDI-TOF MS. METHODS: We first prepared a training set of 20 simulated bacteremia specimens, including 10 Gram-negative and 10 Gram-positive species. A total of 145 non-consecutive BCs flagged as positive (68 Gram-negative rods, and 77 Gram-positive cocci) were prospectively analyzed (validation set). RESULTS: A total of 82% and 49% of isolates were correctly identified to the species level by the respective methods. CONCLUSIONS: The pre-enrichment method outperformed the DI method for identification of virtually all bacterial species included in the panels.

Human infection by a "fish tapeworm", Diphyllobothrium latum, in a non-endemic country.

We document a case of locally acquired "broad- or fish-tapeworm" infection caused by Diphyllobothrium latum in a 27-year-old Spanish man, confirmed by molecular analysis (COI gene). The patient had naturally expelled a worm of 110 cm in length, but the physical examination did not yield any remarkable findings, and the patient did not suffer from any particular symptoms. Laboratory test results were normal except for a remarkable increase in the red blood cell count, and an evident decrease in the mean corpuscular volume and mean corpuscular hemoglobin. Vitamin B12 and folic acid values were in the normal range without signs of anemia. It was suggested that these anomalies in erythrocyte formation might not be related to the parasite, and analysis of the patient's anamnestic data revealed that the infection could only have been caused by the ingestion of imported fish, although no light could be shed on the specific source of infection. From a public health viewpoint, this human case of fish-borne zoonosis is exemplary, suggesting that not only is control of fish and fish product quality essential, but also increased awareness of the general population with regard to changes in culinary habits.

Grado de conocimientos y aceptación de la vacunación anti-rotavirus por parte de padres con niños ingresados por gastroenteritis / Level of knowledge and acceptance of the anti-rotavirus vaccination by parents of children hospitalised due to gastroenteritis

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Global study of viral diarrhea in hospitalized children in Spain: results of structural surveillance of viral gastroenteritis net work (VIGESS-net) 2006-2008.

BACKGROUND AND OBJECTIVES: Detection and characterization of gastroenteritis cases (viruses) was conducted during eleven years through the surveillance VIGESS-net, which was created in an effort to conduct a structured surveillance of rotavirus genotypes co-circulating in Spain. STUDY DESIGN AND RESULTS: This phase includes the study of 2048 fecal samples from children <5 years old, hospitalized in fifteen different hospitals throughout Spain from March 2006 to March 2008. Of them, 821 (40.1%) samples were rotavirus positive. Rotavirus was identified as the only etiological agent in 694 (33.9%) cases and in 127 (6.2%) was found as coinfection with other enteric viruses, mainly with noroviruses. Predominant G genotypes detected were G1 (49.8%) followed by G9 (32.9%), G3 (2.6%), G8 (1.0%), G4 (0.4%), G6 (0.2%) and G12 (0.2%). The G2 was encountered in 3.2% of cases. Rotavirus mixed G-types infections occurred in 3.9% of cases. The main G/P combinations were G1P[8] (51.9%) and G9P[8] (35.6%), which predominates alternatively in the first and second period of the study. More rare combinations occur in less than 7.4% of cases. CONCLUSION: The diversity of rotavirus circulating strains suggests to maintain a surveillance system through different regions of the country.

Descontaminación digestiva en pacientes con intoxicación medicamentosa aguda. Validación de un algoritmo para la toma de decisiones sobre la indicación y el método prioritario / Method of digestive decontamination in patients with acute medication intoxication. Validation of a decision-making algorithm

Objetivos: Debido a la gran controversia existente sobre las indiciones de la descontaminación digestiva en pacientes con intoxicación medicamentosa aguda (IMA), se decidió diseñar y validar un algoritmo para la elección del método prioritario para prevenir la absorción del tóxico. Métodos: Se diseñó un algoritmo para elegir entre jarabe de ipecacuana, lavado gástrico, carbón activado o nada, en función del tipo de medicamento ingerido, dosis, tiempo transcurrido desde la ingesta y situación clínica del paciente, y se incorporó a la rutina asistencial del Servicio de Urgencias. Para validarlo, se recogieron de forma prospectiva variables epidemiológicas, clínicas y toxicológicas de las IMA, comparando la evolución de los pacientes en los que se siguió el algoritmo con la de aquellos en los que se tomó otra decisión. Resultados: Se incluyeron 117 pacientes. En 84 casos se siguió el algoritmo (Grupo A) y en 33 se tomó otra opción de descontaminación (Grupo B). Ambos grupos fueron comparables a su llegada a Urgencias en su distribución por sexo, edad, tiempo transcurrido desde la ingesta, tipo de medicamento, nivel de consciencia y constantes vitales. Presentaron un deterioro clínico posterior a su ingreso el 12 por ciento de los pacientes del grupo A y el 21 por ciento del grupo B, la necesidad de ingreso en Cuidados Intensivos fue del 2 y 6 por ciento, el tiempo medio transcurrido hasta el alta médica fue de 15 y 52 horas, respectivamente, y se consideró que tuvieron una evolución totalmente satisfactoria el 73 por ciento de los casos del grupo A y el 69 por ciento del grupo B, aunque ninguna de estas diferencias tuvo significación estadística. Conclusiones: El seguimiento de un algoritmo para decidir sobre el método prioritario de descontaminación digestiva en las IMA, además de aportar una unidad de criterio, se ha asociado a una evolución clínica más satisfactoria, aunque sin diferencias estadísticas significativas (AU)

First detection of group C rotavirus in children with acute diarrhea in Spain.

Group C rotavirus causes sporadic cases and outbreaks of acute diarrhea in children and adults in many countries, but has never been detected among children in Spain. In a recently conducted surveillance study to screen fecal specimens for bacteria and viruses from a cohort of 822 young children who were treated for acute diarrhea in Madrid, no pathogens were detected in fecal specimens from 238 (29%) children. In this study, we examined 147 of those specimens for group C rotavirus by EIA and PCR and found 22 (15%) were positive. Our findings demonstrate that group C rotavirus is an important cause of childhood diarrhea in Spain.

New immunochromatographic method for rapid detection of rotaviruses in stool samples compared with standard enzyme immunoassay and latex agglutination techniques.

Three different commercial immunologic tests for rapid detection of group A rotavirus (an immunochromatographic method, latex agglutination, and enzyme immunoassay) were used to evaluate 228 faecal specimens obtained from Spanish children with acute gastroenteritis. After resolution of 30 (13.2%) discordant results by reverse transcription-polymerase chain reaction for rotavirus, the statistical values of the enzyme immunoassay, latex agglutination, and immunochromatographic method were respectively 96%, 68%, and 99% for sensitivity; 99%, 99%, and 96% for specificity; 98%, 96%, and 92% for positive predictive value; and 98%, 88%, and 99% for negative predictive value. The immunochromatographic technique showed high sensitivity and specificity and was rapid and easy to perform in the routine clinical laboratory.

Determinación de tiempos de respuestas en un Laboratorio de Urgencias / Determination of turnaround time in Urgency Laboratory

Los programas de garantía de calidad consideran el tiempo de respuesta (TR) como un indicador válido para evaluar los laboratorios de urgencia. En el presente estudio, hemos analizado el TR intralaboratorio y sus variaciones a lo largo del día mediante distintos parámetros estadísticos. El número de muestras estudiado fue3781: el 71 por ciento procedían de Urgencias y el 29 por ciento de las Unidades de Cuidados Intensivos y de Reanimación. La distribución de las muestras en función del tipo de análisis fue: 46 por ciento bioquímica sérica, 37 por ciento hematología, 7 por ciento orinas, 6 por ciento coagulación y 4 por ciento gasometrías. El TR fue inferior a 45 min en el 74 por ciento de las muestras. La mediana de los TR osciló entre 8 min para las gasometrias y 30 min para la bioquímica sérica. Las variaciones diarias mostraron en la mayoría de casos un TR entre 15-30 y 30-45 min, excepto en la franja horaria de 9-12 h donde fue superior a 45 min. Consideramos aconsejable evitar cualquier TR superior a 60 min, siendo nuestro objetivo conseguir valores cercanos a los 30 min (AU)

Perfiles enzimáticos detectados en enteropatógenos bacterianos mediante el sistema API ZYM / Enzymatic profiles detected in bacterial enteropathogens by APIZYM system

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Rotavirus-specific cytotoxic T lymphocytes recognize overlapping epitopes in the amino-terminal region of the VP7 glycoprotein.

Rotavirus-specific cytotoxic T lymphocytes (CTL) play an important role in the resolution of rotavirus infection. The outer capsid glycoprotein, VP7, elicits a class I MHC-restricted CTL response. Vaccinia virus recombinants expressing the VP7 genes from simian rotavirus SA11 (serotype G3) and from the RF strain of bovine rotavirus (serotype G6) were used to analyze the CTL activity to this antigen in BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice neonatally infected with homologous and heterologous rotaviruses. A vaccinia virus recombinant expressing the first amino-terminal 88 amino acids of VP7 was constructed and used to search for cross-reactive CTL against this region of the protein. By using synthetic Kb, Db, and Kd motif-fitting peptides two overlapping CTL epitopes have been identified located in the first hydrophobic domain (H1) of VP7. Splenocytes obtained from rotavirus SA11-infected C57BL/6 mice induced the strongest CTL response against target cells sensitized with a peptide containing a Kb-restricted CTL epitope (amino acids 8-16). A second Kd-restricted epitope (residues 5-13) was recognized by splenocytes derived from rotavirus-infected BALB/c mice. These findings reveal the existence of CTL epitopes in the H1 signal sequence of the VP7 glycoprotein that coexist with a CTL epitope (residues 31-40) previously described within the H2 region.

[The molecular epidemiology of the rotavirus in Spanish children. The Rotavirus Study Group (GER)]. / Epidemiología molecular de rotavirus en niños españoles. Grupo de Estudio de Rotavirus (GER).

BACKGROUND: Rotavirus are the most common etiologic agent of acute gastroenteritis in childhood. The knowledge of the circulating antigenic types is important in development of future vacunes. METHODS: Faeces from children (age < 4 years) with acute gastroenteritis admitted in the two hospitals (Hospital Severo Ochoa-Madrid and Hospital General Vic-Barcelona) have been studied prospectively during one year (October-1996 to October-1997). The detection of rotavirus was performed by ELISA (IDEIA, Dako). All samples were G-serotyping by EIA-Mabs (Silenius Laboratories) and the indeterminate or non-serotypable samples were G-genotyping by RT-PCR. P genotypes were identified by RT-PCR. RESULTS: 322 (45%) patients with acute diarrhoea causing for rotavirus were confirmed, 242 coming from the Madrid metropolitan area and other 80 from the Barcelona area. The EIA-Mabs technique made it possible to identify the G serotypes in 287 cases (89%), corresponding 207 to G1 serotype, 70 to G4 serotype and 6 to G3 serotype. In 4 patients both G1 and G4 serotypes were detected. The EIA-Mabs could not determined the serotype in 35 (11%) patients, all of whom were confirmed by RT-PCR (12 belonged to serotype G1 and 23 to serotype G4). Analysis of P genotypes was carried out in 25 patients obtained from Madrid and 17 from Barcelona; all cases were classified in the P[8] genotype. CONCLUSIONS: The most frequent serotype in both hospitals was G1. The EIA-Mabs technique were showed a high sensitivity, however, the RT-PCR technique used were even more efficient, making it possible for us to identify all the non-serotypable EIA-Mabs cases. The temporal study of circulating serotypes/genotypes of rotavirus is necessary to evaluate the efficiency of vaccines.

Viral proteins VP2, VP6, and NSP2 are strongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection.

Rotavirus-specific IgA has been correlated with immune protection against rotavirus reinfection and symptomatic disease. Systemic and mucosal antibody responses were determined by an enzyme-linked immunosorbent assay in 11 infants with severe rotavirus gastroenteritis. Geometric mean titers of antirotavirus serum IgG and IgA antibodies were significantly higher during the convalescence of the disease (P < 0.001 vs. acute-phase titers). Rotavirus-specific fecal sIgA antibodies increased 4 times during the convalescence in 9 (81.8%) children (P < 0.001). The serum IgG and IgA antibody and fecal sIgA antibody responses to individual rotavirus polypeptides were characterized by radioimmunoprecipitation assay (RIPA) using Staphylococcus aureus protein A and the lectin jacalin to precipitate IgG- and IgA-immune complexes, respectively. The main IgG response was directed toward the structural viral proteins VP2, VP4, and VP6 and toward the nonstructural protein NSP2. Serum IgA reactivity was detected by RIPA in all serum samples, with major responses to VP2, VP6, and NSP2. Interestingly, fecal sIgA in convalescent samples reacted strongly toward NSP2 and VP6. These data reinforce the antigenic importance of rotaviral proteins other than VP4 and VP7, such as VP2, VP6, and NSP2, as main targets in the immune response to rotavirus.

[Virus-specific serum and fecal antibodies response in children with acute rotavirus gastroenteritis]. / Respuesta de anticuerpos séricos y coproanticuerpos específicos en niños con gastroenteritis aguda por rotavirus.

BACKGROUND: The analysis of the immune response to rotavirus infection and the characterization of the viral antigens recognized by specific antibodies are of great concern in evaluating the protection against rotavirus. MATERIAL AND METHODS: The levels of rotavirus-specific fecal (sIgA) and serum antibodies (IgM, IgG and IgA) were evaluated by ELISA in 25 children with acute gastroenteritis for rotavirus, in 11 of them during the acute and convalescent phases. The specificity of serum antibodies to viral polypeptides was characterized by immunoblotting. RESULTS: Serum IgM antibodies with a geometric mean titer (GMT) of 1/3,973 were the predominant antibodies detected during the acute phase. In comparison, IgG and IgA serum antibodies and sIgA coproantibodies levels were higher in the convalescent phase (GMT = 1/5,799, 1/257 and 1/137 respectively). Significant differences were observed for all the isotypes of immunoglobulins evaluated during the infection and in the convalescence (p < 0.01). Rotavirus-specific serum antibodies recognized mainly the structural VP6, VP7 and VP3/VP4 proteins. Other polypeptides also detected were VP1, VP5 and the non-structural NS34 protein. CONCLUSIONS: Rotavirus infection produce an intense humoral immune response both in serum and in the gut. Specific antibodies react against structural proteins of the internal (VP6) and external (VP7) capsids of rotavirus.