Lurdes Matas

Evaluation of a rapid immunochromatographic assay for the detection of Legionella antigen in urine samples.

Abstract A new immunochromatographic membrane assay for detecting Legionella pneumophila serogroup 1 antigen in urine samples (Binax Now Legionella Urinary Antigen Test; Binax, USA) was evaluated. Its sensitivity, specificity and level of agreement with the Binax enzyme immunoassay were compared using nonconcentrated and concentrated urine samples. The overall agreement between the two tests was 98.1%; the specificity of both was 100%. The sensitivity of the immunochromatographic assay was 55.5% in nonconcentrated urine and 97.2% in concentrated urine in comparison with the enzyme immunoassay, using concentrated urine as the reference test. This immunochromatographic assay screens successfully for Legionella pneumophila serogroup 1 soluble antigen in concentrated urine samples.

Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

ABSTRACT We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5′ noncoding region (5′NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5′NC reverse hybridization (method 2; InnoLiPA HCV II) and 5′NC sequencing (method 3; Trugene HCV 5′NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.

Comparison of Antibody Responses to a Potential Combination of Specific Glycolipids and Proteins for Test Sensitivity Improvement in Tuberculosis Serodiagnosis

ABSTRACT The humoral response to different proteinaceous antigens of Mycobacterium tuberculosis is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to M. tuberculosis glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from M. tuberculosis to develop a serodiagnostic test.

Serodiagnosis of Tuberculosis: Comparison of Immunoglobulin A (IgA) Response to Sulfolipid I with IgG and IgM Responses to 2,3-Diacyltrehalose, 2,3,6-Triacyltrehalose, and Cord Factor Antigens

ABSTRACT Nonpeptidic antigens from the Mycobacterium tuberculosis cell wall are the focus of extensive studies to determine their potential role as protective antigens or serological markers of tuberculous disease. Regarding this latter role and using an enzyme-linked immunosorbent assay, we have made a comparative study of the immunoglobulin G (IgG), IgM, and IgA antibody responses to four trehalose-containing glycolipids purified from M. tuberculosis: diacyltrehaloses, triacyltrehaloses, cord factor, and sulfolipid I (SL-I). Sera from 92 tuberculosis patients (taken before starting antituberculosis treatment) and a wide group of control individuals (84 sera from healthy donors, including purified protein derivative-negative, -positive, healed, and vaccinated individuals, and 52 sera from nontuberculous pneumonia patients), all from Spain, were studied. The results indicated a significantly elevated IgG and IgA antibody response in tuberculosis patients, compared with controls, with all the antigens used. SL-I was the best antigen studied, showing test sensitivities and specificities for IgG of 81 and 77.6%, respectively, and of 66 and 87.5% for IgA. Using this antigen and combining IgA and IgG antibody detection, high test specificity was achieved (93.7%) with a sensitivity of 67.5%. Currently, it is widely accepted that it is not possible to achieve sensitivities above 80% in tuberculosis serodiagnosis when using one antigen alone. Thus, we conclude that SL-I, in combination with other antigenic molecules, could be a useful antigen for tuberculosis serodiagnosis.

Evaluation of Meridian ImmunoCard Mycoplasma test for the detection of Mycoplasma pneumoniae-specific IgM in paediatric patients.

The Meridian ImmunoCard Mycoplasma kit, a 10-min card-based enzyme-linked immunosorbent assay (ELISA) designed to detect immunoglobulin M (IgM) antibodies to Mycoplasma pneumoniae was evaluated. We compared the ImmunoCard with the Fujirebio Serodia Myco II particle agglutination test, as well as with the complement fixation (CF) test to detect M. pneumoniae antibodies in paediatric patients. The ImmunoCard test and Serodia Myco II test agreed in 93.95%, and ImmunoCard test and CF test agreed in 83.51% of the 182 specimens tested. Nine specimens gave negative particle agglutination titres in the acute phase sample, and 28 specimens gave negative CF titres in the acute phase sample, although in the ImmunoCard test they were positive. These results may indicate that the ImmunoCard assay detects lower IgM levels of antibodies than the Serodia Myco II and CF test. The ImmunoCard appears to be a good screening assay test for M. pneumoniae IgM in children in whom M. pneumoniae IgM is found frequently.

Declive de la incidencia de la sepsis perinatal por estreptococo del grupo B (Barcelona 1994-2001). Relación con las políticas profilácticas

Introduccion Analizar la incidencia de la sepsis perinatal por estreptococo del grupo B (EGB) y relacionarla con la aplicacion de las recomendaciones de prevencion consensuadas en 1997 por las sociedades catalanas de Obstetricia, Pediatria y Enfermedades Infecciosas y Microbiologia Clinica. Metodos El estudio se realizo desde 1994 a 2001 y en el participaron 10 hospitales del area de Barcelona, donde se registraron 157.848 nacidos vivos. Resultados Fueron diagnosticados 129 recien nacidos de sepsis perinatal por EGB. La incidencia disminuyo el 86,1%, desde el 1,92/1.000 nacidos vivos en 1994 hasta el 0,26/1.000 en 2001 (p Conclusiones En 8 anos se ha conseguido una importante disminucion de la incidencia de sepsis perinatal por EGB, coincidiendo con la aplicacion de protocolos de prevencion de esta patologia.

Assessment of Acinetobacter baumannii susceptibility to antiseptics and disinfectants

Disinfection and antisepsis are of primary importance in controlling outbreaks of Acinetobacter baumannii, a nosocomial pathogen that frequently shows multiple antibiotic resistance. In this study we assessed the susceptibility of nine A. baumannii strains isolated during a sustained intensive care unit outbreak, to several antiseptics and disinfectants based on European Standards. While the tested strains showed diverse antibiotic resistance patterns, they were equally sensitive to the biocides assessed in vitro. We observed neither evidence of development of resistance to biocides over time, nor a correlation between resistance to antibiotics and a decreased susceptibility to antiseptics or disinfectants.

ASSESSMENT OF A NEW TEST TO DETECT LEGIONELLA URINARY ANTIGEN FOR THE DIAGNOSIS OF LEGIONNAIRES' DISEASE

Given that the rate of mortality by Legionella pneumonia increases in incorrectly treated patients, rapid diagnosis and early antibiotic treatment are needed. We have assessed the performance of a new enzyme immunoassay (EIA) test (Bartels Inc. Trinity Biotech Company, Wicklow, Ireland) to detect Legionella pneumophila antigen in urine comparing it to Binax EIA (Binax, Portland, Maine). We also evaluated the capability of both EIAs to detect extracted soluble antigens of Legionella strains. Using nonconcentrated urine samples (NCU) the sensitivity of Bartels EIA was 74.1% (66/89) and the sensitivity of Binax EIA was 51.7% (46/89). The sensitivity of both EIA tests were 91.5% (54/59) using concentrated urine samples (CU). Specificity of both EIA tests was 100% in NCU and CU. Bartels EIA was able to detect all serogroup L. pneumophila antigens, achieving a higher sensitivity in the case of L. pneumophila serogroup 1 soluble antigen. The new EIA was found to be a useful test for the rapid diagnosis of Legionella pneumonia, being a better alternative to the Binax EIA if NCU is used.

Comparative evaluation of two commercial assays for direct detection of Mycobacterium tuberculosis in respiratory specimens.

Two commercial systems for the amplification and detection ofMycobacterium tuberculosis directly from respiratory samples were compared. The Roche Cobas Amplicor MTB Test and the Roche manual Amplicor MTB Test (Roche Diagnostic Systems, USA) were applied to 755 decontaminated respiratory specimens collected from 470 patients. Results were compared with those of acid-fast staining and culture. A total of 251 specimens were collected from 156 patients diagnosed with pulmonary tuberculosis, including 28 specimens corresponding to 13 patients that were receiving antituberculous treatment. Given the overall positivity rate of 33.2% (251/755), the sensitivity, specificity, and positive and negative predictive values were 92.4, 100, 100, and 96.5%, respectively, for the Cobas Amplicor MTB Test and 90.8, 100, 100, and 95.8%, respectively, for the Amplicor MTB Test. For 204 (81.3%) smear positive specimens and 47 (19.7%) smear negative specimens, the sensitivity values were 100 and 59.6%, respectively, for the Cobas Amplicor MTB Test and 100 and 51%, respectively, for the Amplicor MTB Test. There were no statistically significant differences in sensitivity or specificity between the two assays and culture (p>0.05). The overall results of both assays were concordant for 99.5% of the samples. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection ofMycobacterium tuberculosis complex in respiratory specimens, the Cobas Amplicor MTB Test appears to be slightly more sensitive than the Amplicor MTB Test when smear negative specimens are investigated.

Utility of a pneumonia severity index in the optimization of the diagnostic and therapeutic effort for community-acquired pneumonia

The objective was to evaluate the utility of the Pneumonia Severity Index (PSI) developed by Fine et al. as a tool to streamline diagnostic and therapeutic effort. Site of care of patients was recommended in accordance with the PSI class: classes I and II underwent treatment at home and classes III, IV, and V were hospitalized. Class I comprised 37 patients; class II had 30, class III had 20, class IV had 31, and class V had 10 patients. 80 patients were admitted into the hospital, 3 of whom required admittance to the intensive care unit, and 48 were managed as outpatients from the emergency room. Overall mortality was 4 patients (3.1%). Of these, 3 belonged to class IV and 1 to class V. The aetiological diagnosis was obtained in 53.9% of the cases (69/128). If classes I to III are analysed together, the percentage of aetiological diagnoses was 47% (41/87), increasing to 68% (28/41) for patients in classes IV and V. In our experience Fines PSI classification, with rationalization and adaptation to the particularities of each centre, is an effective tool for deciding on hospitalization for selecting the most suitable battery of diagnostic tests based on cost-benefit criteria. However, it is inadequate for young patients with hypoxia or pleural effusion. Therefore, although hospitalization of patients with pneumonia should be mainly based on clinical criteria, Fines PSI classification could help physicians in making more rational decisions in this respect.