Antonio Orduña

COMPARATIVE SENSITIVITY OF SIX SEROLOGICAL TESTS AND DIAGNOSTIC VALUE OF ELISA USING PURIFIED ANTIGEN IN HYDATIDOSIS

Most serodiagnostic techniques have been evaluated for diagnosis of cystic hydatid disease caused by Echinococcus granulosus. Each, to varying degrees, has been shown to give false results, with considerable variation between laboratories. The comparative study was made concerning the sensitivity of the immunodiagnostic methods based on 58 sera from hydatid disease with different cyst locations. Latex agglutination, immunoelectrophoresis (IEP), and specific IgE, IgG enzyme‐linked immunosorbent assay (ELISA) tests were studied. Specific IgG ELISA AgB (antigen B‐rich fraction) was the most sensitive test (96.5%) and the least sensitive tests were specific IgE ELISA (24.1%) and IEP (25.8%). The low sensitivity of these two tests was due partly to the low reactivity detected in the sera of patients with lung hydatidosis. Initial laboratory studies showed purified antigens to be preferable to crude cyst fluid, regardless of the type of test used. For this reason, we evaluated the sensitivity and specificity of ELISA by using the purified antigen‐B‐rich fraction. In all, 117 sera were examined: 78 sera from patients with hydatidosis surgically confirmed, 15 sera from healthy control subjects, and 24 sera from patients with diseases other than hydatidosis. The method gave good results: 93.5% sensitivity, 89.7% specificity, and 92.3% diagnostic efficacy. J. Clin. Lab. Anal. 15:14–18, 2001.

Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells.

The role of the outer membrane proteins of the Omp25/Omp31 family in invasiveness and intracellular survival of virulent B. ovis in phagocytes was analyzed. The absence of Omp25d or Omp22 in B. ovis abolished its invasive capacity in HeLa cells and reduced it in J774.A1 cells. Additionally, in J774.A1 cells, the Deltaomp25d mutant was unable to multiply, whereas the Deltaomp22 mutant was cleared at 24h post-infection. These findings demonstrate that Omp25d and Omp22 are essential for the invasion and survival of B. ovis inside host cells, and justify the strong attenuation in virulence of the Deltaomp25d and Deltaomp22 mutants.

Serologic evidence of human infection by Francisella tularensis in the population of Castilla y León (Spain) prior to 1997

Prior to an outbreak in Castilla y León in December 1997, tularaemia was practically non-existent in Spain. In this paper we studied the prevalence of antibodies against Francisella tularensis in a representative sample of the population (4825 people) from Castilla y León (Spain) in samples collected before this outbreak. Antibodies against F. tularensis were detected in nine (0.19%) of the 4825 sera, with antibody titres ranging from 1/20 to 1/160. Of these nine sera, one was positive in seroagglutination against Brucella. Seroagglutination against other bacteria (Yersinia enterocolitica O:9 and O:3 and Proteus OX19) was negative in all sera. Seroprevalence of antibodies in females was 0.20% and 0.17% in males; no statistically significant differences were found in prevalence in terms of sex, age or province.

Seroprevalencia de infección por Echinococcus granulosus en la población de Castilla y León

Introduccion La hidatidosis es una de las zoonosis mas importantes en la Comunidad Autonoma de Castilla y Leon. Este estudio pretende conocer la seroprevalencia de infeccion por Echinococcus granulosus en dicha comunidad autonoma. Metodos Se han estudiado 4.824 muestras de suero pertenecientes a 4.824 personas seleccionadas de forma aleatoria y que constituian una muestra representativa de la poblacion de las provincias de Castilla y Leon. En cada suero se estudio la presencia de anticuerpos de clase IgG frente a Echinococcus granulosus mediante una prueba de enzimoinmunoanalisis indirecto de fabricacion propia. Resultados Se detectaron anticuerpos de clase IgG frente a Echinococcus granulosus en el 3,40% de los sueros estudiados (164 positivos de 4.824), oscilando entre el 1,26 y el 7,10% segun la provincia de origen. La seroprevalencia de anticuerpos aumentaba significativamente con la edad. No se observaron diferencias estadisticamente significativas entre las seroprevalencias halladas en mujeres y en varones (3,14% frente a 3,66%). Conclusion La seroprevalencia de infeccion por E. granulosus en la Comunidad Autonoma de Castilla y Leon es todavia alta. Estos datos de seroprevalencia contribuyen a la vigilancia de la hidatidosis dentro de un programa control de esta enfermedad.

Fas-Activated Serine/Threonine Phosphoprotein Promotes Immune-Mediated Pulmonary Inflammation

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST−/−) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST−/− mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST−/− mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-α and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST−/− neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

A new approach to determine the susceptibility of bacteria to antibiotics directly from positive blood culture bottles in two hours.

The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test was performed in the same way that was carried out in artificially prepared blood culture bottles. Our results indicate that antibiotic susceptibility test can be determined as early as 120min since a blood culture bottle is flagged as positive. The essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identification and antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3h.

Influence of syphilis in hepatitis B transmission in a cohort of female prostitutes.

BACKGROUND AND OBJECTIVES. Prostitutes are a greater risk for hepatitis B virus (HBV) infection than the general population. We studied the influence of age and time as prostitute on HBV infection. We also examined the relationship between syphilis and HBV infection in a cohort of female prostitutes. STUDY DESIGN. The presence of hepatitis B surface antigen (HBsAg), antibodies to hepatitis B core antigen (anti-HBc), antibodies to hepatitis D virus (anti-HD) and treponemal antibodies (FTA-ABS) were determined in 368 prostitutes, of whom 147 were submitted to medical and serological follow-up every six months to evaluate the influence of syphilis in the transmission of hepatitis B. RESULTS AND CONCLUSION. The prevalence of HBsAg was 4.6%, of anti-HBc 31.2%, anti-HD 0.5% and FTA-ABS 35.0%. There was a statistical association between the presence of treponemal antibodies and anti-HBc (P = 0.022). The cohort study performed shows that the accumulated incidence of HBV infection in the FTA-ABS positive prostitutes (24.6%) was significantly higher than that of the FTA-ABS negative group (9.7%) (RR = 2.544; P = 0.034). Our results indicate that syphilis could facilitate the heterosexual transmission of HBV infection.

Clinical evaluation of a commercial ligase-based gene amplification method for detection of Mycobacterium tuberculosis

The purpose of this study was to evaluate the clinical usefulness of a commercial ligase-based gene amplification method (LCxMycobacterium tuberculosis test; Abbott Laboratories, USA) for detection ofMycobacterium tuberculosis. The tuberculosis infection rate among clinical samples was 10.6%. The sensitivity, specificity, and positive and negative predictive values were 23.5%, 100%, 100%, and 91.7%, respectively, with the fluorochrome auramine stain; 32.4%, 100%, 100%, and 92.6%, respectively, with culture; and 76.5%, 95.8%, 68.4% and 97.2%, respectively, with the gene amplification method. When only samples from patients without current or previous treatment were studied, the sensitivity was 36.4% with the auramine stain, 63.6% with culture, and 100% with the gene amplification assay. The mean treatment time for culture-negative and assay-negative samples was greater than that of culture-negative and assay-positive samples. The LCxMycobacterium tuberculosis test is a sensitive method for detection and identification ofMycobacterium tuberculosis. It produces few false-positive results. However, as it can remain positive after the culture becomes negative, it is not recommended for evaluation of treatment efficiency.

The translational repressor T-cell intracellular antigen-1 (TIA-1) is a key modulator of Th2 and Th17 responses driving pulmonary inflammation induced by exposure to house dust mite

T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.

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.