Miguel Ángel Bratos

Evaluation of six serological tests in diagnosis and postoperative control of pulmonary hydatid disease patients

Latex agglutination (LA), passive hemagglutination (PHA), immunoelectrophoresis (IEP) and specific IgE, IgM, IgG enzyme-linked immunosorbent assay (ELISA) tests for diagnosis and postoperative follow-up of 79 patients with surgically confirmed pulmonary hydatidosis were evaluated. Specific IgG ELISA was the most sensitive test (83.5%) and the least sensitive tests were specific IgE ELISA (44.3%) and IEP (50.6%). The specificity obtained for all the serologic test was above 97% in all cases. The greatest number of false positives in all tests (except IEP) occurred in patients with Taenia saginata and Taenia solium cysticerci infestations and in patients with lymphoma and leukemia. Specific IgG ELISA demonstrated the highest negative predictive value (93.8%). No statistically significant differences (p > 0.050) were found in the sensitivity of the tests when patients with only one cyst and patients with various cysts were compared. Considering only the patients without relapse, the percentage of seropositive patients increased in all tests at 1 and 3 months after surgery. After that time the percentage of seropositive patients decreased. At 48 months after surgery all patients without relapse became negative in IEP, specific IgE ELISA, and specific IgM ELISA. The antibody titers in all seropositive patients increased during the 3 months after surgery. From these 3 months onward, antibody levels decreased in all serologic tests studied in the group of patients without relapse. The patients who had relapses during the first year after surgery presented persistently elevated antibody titers in all postoperative sera. The antibody titers of the patients who relapsed between the third and fourth years after surgery decreased progressively the third month after surgery, and increased in the serum obtained at the moment of relapse diagnosis. Our results show that persistence of elevated antibody titers in patients with pulmonary hydatidosis in the year after surgery or titer increase after a progressive decrease are indicative of relapse or reinfection.

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.

Diagnóstico de laboratorio y evolución serológica de pacientes con tularemia

Fundamento La tularemia era una enfermedad practicamente inexistente en Espana hasta finalesde 1997, cuando se declaro un brote epidemico en nuestra comunidad. El objetivo denuestro trabajo ha sido estudiar los datos existentes sobre el diagnostico microbiologico de 55pacientes que sufrieron tularemia. Pacientes Y Metodos Se obtuvieron 32 muestras para cultivo pertenecientes a 19 pacientes y151 sueros correspondientes a 55 pacientes. El diagnostico serologico se realizo mediante seroaglutinacionen tubo y microaglutinacion. En todos los sueros se realizo una seroaglutinacionde Wright (SAW) y un test de Coombs frente a Brucella y seroaglutinaciones frente a Yersiniaenterocolitica O:9, Yersinia enterocolitica O:3 y Proteus OX 19. Resultados Se aislo F. tularensis en dos muestras (6,25%) de las 32 estudiadas. Se obtuvierontitulos mayores o iguales a 1/160 en el 78,2% y en el 74,5% de los sueros iniciales por seroaglutinacionen tubo y microaglutinacion, respectivamente. La correlacion entre las dos pruebasfue de 0,80 (p Conclusiones El cultivo de F. tularensis es poco sensible. La correlacion obtenida entre la seroaglutinacionen tubo y microaglutinacion es buena. Ambas tecnicas son utiles en el diagnosticode la tularemia, con algunas ventajas de la microaglutinacion sobre la aglutinacion. Background Tularemia was practically unknown in Spain until the end of 1997, when an epidemicoutbreak was declared. This paper presents the data on microbiological diagnosis of 55patients who suffered from tularemia. Patients and Methods Thirty-two samples from 19 patients and 151 serum samples from 55 patientswere obtained for culture. Serologic diagnosis was performed by tube seroagglutinationand microagglutination. Three types of tests were performed on all sera: Wright seroagglutination(WSA), Coombs test against Brucella spp. and seroagglutination against Yersinia enterocoliticaO:3, Yersinia enterocolitica O:3, and Proteus OX 19. Results F. tularensis was found in two samples (6.25%) of the 32 received. Titers. 1/160were obtained in 78.2% and 74.5% of the initial sera by tube seroagglutination and microagglutination,respectively. Correlation between the two tests was 0.80 (p Conclusions Culture of F. tularensis has low sensitivity. The correlation obtained between tubeseroagglutination and microagglutination is good. Both techniques are useful in routine diagnosisof tularemia, although microagglutination has some advantages over tube agglutination.

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.

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.

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.

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 manufacturers 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%).