Utilización de MALDI-TOF en el diagnóstico rápido de la sepsis / Use of MALDI-TOF in the rapid diagnosis of sepsis

La introducción de la espectrometría de masas mediante MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) en el diagnóstico de bacteriemias y fungemias supone una revolución por la rapidez y fiabilidad de los resultados que pueden ofrecer los servicios y laboratorios de microbiología mediante el análisis del espectro proteico bacteriano directamente a partir del frasco de hemocultivo positivo. Estos datos son más útiles si se integran con otras técnicas capaces de ofrecer el patrón de resistencia antibiótica del microorganismo. Debe llevarse a cabo un proceso de estandarización de los protocolos de procesamiento de las muestras y perfeccionar la identificación de los agentes causales de bacteriemias, especialmente de algunas especies de cocos grampositivos y en los procesos polimicrobianos. La introducción de esta metodología proporciona una información precoz muy importante para el manejo clínico de las bacteriemias. Si el hospital dispone de un grupo de trabajo multidisciplinar que aplica de forma rápida y correcta toda esta información, mejorará la calidad de la atención sanitaria del paciente, disminuirá el gasto en antibióticos y la estancia hospitalaria, y contribuirá a controlar el grave problema de la resistencia a los antibióticos

The introduction of mass spectrometry through MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) in the diagnosis of bacteraemia and fungaemia has represented a revolution due to the rapidity and reliability of the results that it can offer to microbiology services and laboratories through analysis of the mass spectrum of the bacterial protein directly from positive blood culture bottles. These data are more useful if they are used in conjunction with other techniques able to identify the antibiotic resistance pattern of the microorganism. There is a need for a process of standardising sample processing protocols and for perfecting the identification of the agents causing bacteraemia, especially in some species of Gram-positive cocci and in polymicrobial processes. The introduction of this methodology provides rapid information that is highly important for the clinical management of bacteraemia. The availability of a multidisciplinary working group that applies all this information quickly and correctly in hospitals will improve the quality of care, reduce antibiotic expenditure and hospital stay and help to control the serious problem of antibiotic resistance

Sugerencias sobre el análisis molecular microbiológico post mortem / Suggestions on post mortem molecular microbiology analysis

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Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a Spanish hospital over a 4-year period: clonal replacement, decreased antimicrobial resistance, and identification of community-acquired and livestock-associated clones.

This study was carried out on 189 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates collected in a third-level hospital in Valladolid, Spain, between 2005 and 2008 in order to investigate the changes in molecular epidemiology and genetic backgrounds associated with the changes in resistance phenotypes produced over time. The MRSA isolates were classified as belonging to 10 different clones, including the identification of a novel MRSA clone, ST2422-MRSA-IV, belonging to CC121; 1 CA-MRSA strain from a USA300 clone; another from ST97-MRSA-IV, associated with clones adapted to livestock (LA-MRSA); and 2 strains belonging to a new spa type (t10258) related to the ST8-MRSA-IV clone. Sixty-two percent of the strains belonging to Spanish-prevalent MRSA sequence type ST125 harboured composite or multiple SCCmec elements including SCCmec type IV plus ccrA/B4 (ST125-SCCmec IV/VI). In the years studied, it was observed that ST125-SCCmec IV/VI replaced the multiresistant ST228-SCCmec I previously prevalent, and, as a consequence, decreased gentamicin and clindamycin resistance was further observed.

Clonal nature and diversity of resistance, toxins and adhesins genes of meticillin-resistant Staphylococcus aureus collected in a Spanish hospital.

In this study we determined the prevalence of genes coding for antimicrobial resistance, toxins, enzymes, immunoevasion and adhesins factors among 189 meticillin-resistant Staphylococcus aureus (MRSA) strains isolated from a third level hospital in Valladolid (Spain) between 2005 and 2008 in order to examine the relationship between these pathogenicity determinants, both individually and in combination, and the genetic background of main MRSA strains that are presents in Spanish hospitals. MRSA isolates were first characterised epidemiologically by a combination of molecular typing strategies like spa, SCCmec and multilocus sequence typing, and then, a cluster analysis based on pathogenicity factors genes was performed according to the hybridisation pattern of 65 virulence, 36 resistance, 15 adhesins, and 11 set/ssl genes on a Diagnostic DNA microarray (Alere StaphyType DNA microarray Jena, Germany). CC5-agr type II [ST125-SCCmecIV/VI (32.2%) or ST125-IV (19.1%), ST228-I (19.1%), ST146-IV (13.7%) and ST5- IV (0.5%)] isolates was widely distributed. CC8-agr type I [ST8-IV (11.5%), USA300 clone (0.5%), and ST239-III (1.1%)]; CC45-agr type II [ST45- IV (1.6%)], and the CC97-agr type I [ST97-IV] were also detected. We identified 42 different resistance genes profiles, 22 set/ssl genes profiles, and 91 different virulence profiles. However although the high genetic diversity of MRSA strains, mainly with respect to virulence factors genes, the results of the simultaneous assessment of resistance and virulence genes and the genetic background illustrated a correspondence relationship (p<0.001) between the different clones and same resistance and virulence genes or clusters of them. During the study period we observed changes in molecular epidemiology of MRSA isolates and as a consequence we report the changes of the resistance and virulence potential of MRSA strains produced over time in our institution.

Influence of brucellosis history on serological diagnosis and evolution of patients with acute brucellosis.

Serological diagnosis of human brucellosis is problematic in endemic brucellosis regions and with patients having a history of brucellosis. The aim of this study is to ascertain the serologic and evolutionary behavior of the tests of serum agglutination, Coombs anti-Brucella, immunocapture-agglutination, enzyme-linked immunosorbent assay (ELISA) IgG, IgA, IgM and ELISA-IgG avidity against Brucella lipopolysaccharide (S-LPS), in patients with acute brucellosis based on whether or not a history of brucellosis exists. Titers and seropositivity in all the tests assayed were higher in the patients having brucellosis history (from 90.9% in ELISA-IgM to 100% in ELISA-IgG) than in the patients lacking such history (from 79.3% in ELISA-IgM to 86.2% in Coombs, immunocapture-agglutination, and ELISA-IgG). IgG S-LPS avidity results in patients with brucellosis history were significantly higher (always over 84%) than in patients without brucellosis history (from 48.0% in the initial sera to 81% ten months later) (p<0.001). The titers of antibodies against Brucella in the initial sera and ELISA-IgG avidity against S-LPS may allow distinguishing patients with brucellosis caused by primary infection in the initial stages of the disease from patients seropositive due to prior infections from Brucella.

[Seroprevalence of viral hepatitis in a representative general population of an urban public health area in Castilla y León (Spain)]. / Seroprevalencia de las hepatitis virales en población general representativa de una zona básica de salud urbana en Castilla y León.

INTRODUCTION: Viral hepatitis is a major social, health and economic problem worldwide, requiring strict epidemiological control. METHODS: This study presents the viral hepatitis seroprevalence in a representative sample from an urban health care area in Valladolid (Spain). RESULTS: Antibody prevalence was as follows: anti-HAV 52%; anti-HBc, 8.2%; anti-HCV, 1.1%; anti-HEV, 0.8%; and anti-HGV 5.8%. Prevalence of anti-HAV, anti-HBc and anti-HGV increased significantly with age (P < 0.005 in all cases). In individuals younger than 20, prevalence of anti-HAV was 3.8%, anti-HBc < 0.28% and anti-HGV 1.3%. In the 20-39 year-old group, seroprevalence against HAV was associated with low educational levels (P = 0.009) and with birth in other provinces (P = 0.016). Anti-HBc seroprevalence was mainly associated with three factors: prior hospitalization before 1990 (P = 0.002; OR 3.32 [1.48-7.42]); compulsory military service before 1990 (P < 0.0001; OR 37.33 [3.68-378.03]); and acupuncture treatments (P = 0.018; OR 57.75 [26.17-127.42]). Seroprevalence against HGV was associated with hospitalizations before 1990 (P = 0.019; OR = 2.969 [1.154-7.639]). Seropositive status to HCV revealed a transfusion history (2 cases), hospitalization (1 case) or drug addiction (1 case). Only one case among those seropositive to HEV had a history of a prior trip to a HEV-endemic area. CONCLUSIONS: Our study shows that the seroprevalences of viral hepatitis in a representative sample of urban population of Castille and Leon are similar to the seroprevalences observed in the rest of Spain and other developed countries, lower than the ones observed in the studies performed in Spain in the last 20 years due to the measures of prophylaxis that were taken.

Seroprevalencia de las hepatitis virales en población general representativa de una zona básica de salud urbana en Castilla y León / Seroprevalence of viral hepatitis in a representative general population of an urban public health area in Castilla y León (Spain)

Introducción. Las hepatitis virales constituyen uno de los principales problemas sociosanitarios y económicos a nivel mundial por lo que precisan un estrecho control epidemiológico. Métodos. En el presente trabajo estudiamos la seroprevalencia de las hepatitis virales una muestra representativa de la población de una zona básica de salud urbana en Valladolid (España). Resultados. La prevalencia de anticuerpos anti-VHA (AcVHA) fue del 52%, de HBcAc del 8,2%, de AcVHC del 1,1%, de AcVHE 0,8% y AcVHG 5,8%. La prevalencia de AcVHA, HBcAc y AcVHG aumenta significativamente con la edad (p < 0,005 en todos los casos). En menores de 20 años la prevalencia de AcVHA es del 3,8%, HBcAc < 0,28% y AcVHG 1,3%. En el grupo de edad de 20-39 años, la seroprevalencia frente al VHA se asocia con niveles educativos bajos (p 5 0,009) y con el nacimiento en otras provincias (p 5 0,016). La seroprevalencia de HBcAc se asocia principalmente con hospitalizaciones anteriores a 1990 (p 5 0,002; OR: 3,32 [1,48-7,42]), realización del servicio militar obligatorio anterior a 1990 (p < 0,0001; OR: 37,33 [3,68-378,03]) y prácticas de acupuntura (p 5 0,018; OR: 57,75 [26,17-127,42]). La seroprevalencia frente a VHG se asocia con hospitalizaciones antes de 1990 (p 5 0,019; OR: 2,969 [1,154-7,639]). Los seropositivos frente a VHC tenían antecedentes de transfusiones (2 casos) hospitalización (1 caso) o drogadicción (1 caso). De los seropositivos frente a VHE sólo un caso tenía antecedentes de viaje a zona endémica para VHE. Conclusiones. Nuestro estudio muestra que las seroprevalencias de las hepatitis virales en una muestra representativa de población urbana de Castilla y León son similares a las seroprevalencias obtenidas en el resto de España y de los países desarrollados, inferior a la observada en los estudios realizados en España en los últimos 20 años consecuencia de las medidas profilácticas adoptadas (AU)

Introduction. Viral hepatitis is a major social, health and economic problem worldwide, requiring strict epidemiological control. Methods. This study presents the viral hepatitis seroprevalence in a representative sample from an urban health care area in Valladolid (Spain). Results. Antibody prevalence was as follows: anti-HAV 52%; anti-HBc, 8.2%; anti-HCV, 1.1%; anti-HEV, 0.8%; and anti-HGV 5.8%. Prevalence of anti-HAV, anti-HBc and anti-HGV increased significantly with age (P < 0.005 in all cases). In individuals younger than 20, prevalence of anti-HAV was 3.8%, anti-HBc < 0.28% and anti-HGV 1.3%. In the 20-39 year-old group, seroprevalence against HAV was associated with low educational levels (P 5 0.009) and with birth in other provinces (P 5 0.016). Anti-HBc seroprevalence was mainly associated with three factors: prior hospitalization before 1990 (P 5 0.002; OR 3.32 [1.48-7.42]); compulsory military service before 1990 (P < 0.0001; OR 37.33 [3.68-378.03]); and acupuncture treatments (P 5 0.018; OR 57.75 [26.17-127.42]). Seroprevalence against HGV was associated with hospitalizations before 1990 (P 5 0.019; OR 5 2.969 [1.154-7.639]). Seropositive status to HCV revealed a transfusion history (2 cases), hospitalization (1 case) or drug addiction (1 case). Only one case among those seropositive to HEV had a history of a prior trip to a HEV-endemic area. Conclusions. Our study shows that the seroprevalences of viral hepatitis in a representative sample of urban population of Castille and Leon are similar to the seroprevalences observed in the rest of Spain and other developed countries, lower than the ones observed in the studies performed in Spain in the last 20 years due to the measures of prophylaxis that werw taken (AU)

Utility of an immunocapture-agglutination test and an enzyme-linked immunosorbent assay test against cytosolic proteins from Brucella melitensis B115 in the diagnosis and follow-up of human acute brucellosis.

The utility of an immunocapture-agglutination (Brucellacapt, Vircell SL, Granada, Spain) test and an enzyme-linked immunosorbent assay IgG, IgA, and IgM (ELISA-IgG, ELISA-IgA, ELISA-IgM) against cytosolic proteins from Brucella melitensis B115 (R) was compared with ELISA-IgG, ELISA-IgA, and ELISA-IgM against smooth lipopolysaccharide (S-LPS) from B. melitensis 16M (S), serum agglutination test (SAT), and Coombs test in the diagnosis and follow-up for 10 months of 51 patients with acute brucellosis. The sensitivities of ELISA tests against cytosolic proteins varied from 49.0 % for ELISA-IgG to 64.7% for ELISA-IgM and were lower than the sensitivities showed by ELISA S-LPS (from 88.2% to 92.2%), SAT (88.2%), Coombs (96.1%), and Brucellacapt (98.0%) tests. Specificity was over 93% in all cases. The evolutionary behavior of the SAT, Coombs, and Brucellacapt tests was similar. There was a decrease of between 20% and 40% in antibody titer in the 10th month of evolution after treatment. The evolutional curves of IgG, IgA, and IgM against cytosolic protein increased slightly till the eighth month. The specific IgM and IgA antibodies against protein fractions began to show a drop from the eighth month on, showing levels slightly lower than the initial sera values by the end of the 10th month. In this month, titers of specific IgG against proteins fractions remained higher than the titers showed by the initial sera.

[Seroprevalence of infection due to Echinococcus granulosus in the population of Castilla and León (Spain)]. / Seroprevalencia de infección por Echinococcus granulosus en la población de Castilla y León.

INTRODUCTION: Human hydatidosis is a prevalent zoonotic disease in the Castilla y León region of Spain. The aim of this study is to investigate the seroprevalence of Echinococcus granulosus infection in this region. METHODS: We studied 4824 serum samples from a random, representative population of healthy individuals from each province of Castilla y León, obtained over one year. An indirect enzyme-immunoassay developed in our laboratory was used to determine the presence of IgG antibodies against Echinococcus granulosus in these samples. RESULTS: IgG antibodies against Echinococcus granulosus were detected in 3.4% (164/4824) of samples studied, with a range of 1.26% to 7.10%, depending on the province. Antibody seroprevalence increased significantly with age, but there was no significant sex-related difference (3.66% men vs. 3.14% women). CONCLUSION: The seroprevalence of Echinococcus granulosus infection in Castilla y León is still high. These data contribute to hydatidosis surveillance within the control program for this disease.

Seroprevalencia de infección por Echinococcus granulosus enla población de Castilla y León / Seroprevalence of infection due to Echinococcus granulosus in the population of Castilla and León (Spain)

INTRODUCCIÓN. La hidatidosis es una de las zoonosis más importantes en la Comunidad Autónoma de Castilla y León. Este estudio pretende conocer la seroprevalencia de infección por Echinococcus granulosus en dicha comunidad autónoma. MÉTODOS. Se han estudiado 4.824 muestras de suero pertenecientes a 4.824 personas seleccionadas de forma aleatoria y que constituían una muestra representativa de la población de las provincias de Castilla y León. En cada suero se estudió la presencia de anticuerpos de clase IgG frente a Echinococcus granulosus mediante una prueba de enzimoinmunoanálisis indirecto de fabricación propia. RESULTADOS. Se detectaron anticuerpos de clase IgG frente a Echinococcus granulosus en el 3,40 por ciento de los sueros estudiados (164 positivos de 4.824), oscilando entre el 1,26 y el 7,10 por ciento según la provincia de origen. La seroprevalencia de anticuerpos aumentaba significativamente con la edad. No se observaron diferencias estadísticamente significativas entre las seroprevalencias halladas en mujeres y en varones (3,14 por ciento frente a 3,66 por ciento). CONCLUSIÓN. La seroprevalencia de infección por E. granulosus en la Comunidad Autónoma de Castilla y León es todavía alta. Estos datos de seroprevalencia contribuyen a la vigilancia de la hidatidosis dentro de un programa control de esta enfermedad (AU)

Utility of a commercial immunoblot kit (BAG-Borrelia blot) in the diagnosis of the preliminary stages of Lyme disease.

The aim of this study was to evaluate the usefulness of a commercial immunoblot (IgG and IgM BAG-Borrelia blot) in the serologic diagnosis of the early stages of Lyme disease. A total of 42 sera from patients with Lyme disease (24 patients with localized early stage (LES) and 18 patients with disseminated early stage (DES)) and 129 sera from patients with non-Lyme diseases (specificity control sera) were studied. IgG anti-p41 from Borrelia burgdorferi s.l. was present in 95.2% of patients followed by anti-p41/I PBi (16.7%), anti-p100 (9.5%) and anti-OspA (9.5%). IgM anti-p41 was present in 66.7% of patients, p41/iPBi (54.8%) and OspC (33.3%). IgM against p100, OspA and OspC were more frequent in DES patients (16.7%, 27.8% and 44.4%) than in LES patients (0.0%, 4.2% and 25.0%). In 4.8% of the cases no IgG bands were present and in 26.2% no IgM bands were present. With the exception of isolated p41 bands (59.5%), no band pattern exceeded 17%. Using manufacturer's instructions, test sensitivity in diagnosis of the early stage of Lyme disease is 61.9%, specificity 98.4% and positive and negative predictive values 92.8% and 88.8% respectively. Applying the EUCALB 5, 6 or 7 rules sensitivity increased to 73.8% although specificity decreased to 89.9%. Of the 129 specific control sera, 41.8% presented IgG anti-p41 and 10.8% IgM anti-p41. Patients with non-Lyme diseases that presented more IgG and IgM bands were those patients with syphilis (88.2%), patients with anti-HIV antibodies (57.8%) and patients with anti-nuclear antibodies (ANA) (52.3%).

[Prevalence of antibodies against Francisella tularensis in Castilla y León (Spain) before 1997]. / Prevalencia de anticuerpos frente a Francisella tularensis en la población de Castilla y León con anterioridad a 1997.

BACKGROUND AND OBJECTIVES: The aim of this paper was determine the prevalence of antibodies against Francisella tularensis in the representative sample of people from Castilla-León (Spain) before epidemic outbreak of end 1997. SUBJECTS AND METHOD: We obtain 4,825 sera (between april-1996 and april-1997) of people from Castilla-León. All sera were tested by a microagglutination technique to detect antibodies against Francisella tularensis. The positive sera were tested to determine cross-reaction with Brucella, Yersinia enterocolitica and Proteus in the tube agglutination tests. RESULTS: We detected antibodies against Francisella tularensis in 9 (0,19%) of the 4,825 sera. Only one serum from the 9 seropositive was positive in the tube agglutination against Brucella. None of the 9 sera were positive against the remaining bacterial antigen tested. CONCLUSIONS: In the people of Castilla-León before 1997 the prevalence of antibodies against Francisella tularensis was 0,19%.

Prevalencia de anticuerpos frente a Francisella tularensis en la población de Castilla y León con anterioridad a 1997 / Prevalence of antibodies against Francisella tularensis in Castilla y León (Spain) before 1997

FUNDAMENTO Y OBJETIVOS: Este trabajo pretende conocer la prevalencia de infección por Francisella tularensis en la población de Castilla y León previa al brote de tularemia humana de finales de 1997. SUJETOS Y MÉTODO: Se obtuvieron 4.825 sueros (entre abril de 1996 y abril de 1997) de residentes en Castilla y León. Se realizó una prueba de microaglutinación en placa para detectar anticuerpos anti-F. tularensis. En los sueros positivos se llevaron a cabo seroaglutinaciones en tubo frente a Brucella, Yersinia enterocolitica y Proteus. RESULTADOS: Se detectaron anticuerpos anti-F.tularensis en 9 (0,19 per cent) de los 4.825 sueros. De esos 9 sueros, uno fue positivo en la seroaglutinación frente a Brucella, siendo todos negativos frente a las otras bacterias. CONCLUSIONES: Antes de 1997 la seroprevalencia de anticuerpos anti-F. tularensis en la población de Castilla y León era baja (0,19 per cent) (AU)

Diagnóstico de laboratorio y evolución serológica de pacientes con tularemia / Laboratory diagnosis and serologic evolution of patients with tularemia

Fundamento: La tularemia era una enfermedad prácticamente inexistente en España hasta finales de 1997, cuando se declaró un brote epidémico en nuestra comunidad. El objetivo de nuestro trabajo ha sido estudiar los datos existentes sobre el diagnóstico microbiológico de 55 pacientes que sufrieron tularemia. Pacientes y métodos: Se obtuvieron 32 muestras para cultivo pertenecientes a 19 pacientes y 151 sueros correspondientes a 55 pacientes. El diagnóstico serológico se realizó mediante seroaglutinación en tubo y microaglutinación. En todos los sueros se realizó una seroaglutinación de Wright (SAW) y un test de Coombs frente a Brucella y seroaglutinaciones frente a Yersinia enterocolitica O:9, Yersinia enterocolitica O:3 y Proteus OX 19. Resultados: Se aisló F. tularensis en dos muestras (6,25 por ciento) de las 32 estudiadas. Se obtuvieron títulos mayores o iguales a 1/160 en el 78,2 por ciento y en el 74,5 por ciento de los sueros iniciales por sero-aglutinación en tubo y microaglutinación, respectivamente. La correlación entre las dos pruebas fue de 0,80 (p < 0,001). Se observó fenómeno de prozona en el 59,9 por ciento de los sueros, y reactividad cruzada frente a Brucella y Proteus OX19 en el 9,3 y el 22,8 por ciento, respectivamente. No se observó reactividad cruzada con Y. enterocolitica O:3 y O:9. Conclusiones: El cultivo de F. tularensis es poco sensible. La correlación obtenida entre la seroaglutinación en tubo y microaglutinación es buena. Ambas técnicas son útiles en el diagnóstico de la tularemia, con algunas ventajas de la microaglutinación sobre la aglutinación. (AU)