Marina Luquin

Prospective study on the etiology of community-acquired pneumonia in children and adults in Spain

The cause of primary pneumonia was diagnosed in 157 of 198 children and 165 of 207 adults seen as inpatients or outpatients in a 12-month period. In childrenMycoplasma pneumoniae and pneumococcus were identified in 79 and 29 cases respectively. Twenty-nine of 53 cases of viral infection in children were caused by respiratory syncytial virus, two-thirds of the cases occurring in children under three years of age. No children died of pneumonia. In adults pneumococcus was the most common pathogen, accounting for 81 cases. The overall mortality in adults was 7.7%. A high mortality was found in patients withHaemophilus influenzae and other gram-negative bacilli infections, and in elderly patients with pneumococcal pneumonia. Coagglutination was more sensitive than counterimmuno-electrophoresis for the detection of pneumococcal antigen in respiratory samples (p<0.001). Counterimmunoelectrophoresis was the only useful technique for detection of pneumococcal antigen in urine specimens, concentration, overnight storage at 4 °C and specific staining significantly increasing positivity (p<0.001).

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.

Mycobacterium xenopi Infections in the Acquired Immunodeficiency Syndrome

Abstract To the Editor:Several cases of infection due toMycobacterium xenopihave been reported (1, 2). This organism has been cultured frequently in samples from hot-water generators and storage ta...

Connaught and Russian strains showed the highest direct antitumor effects of different Bacillus Calmette-Guérin substrains.

PURPOSE Evolutionarily early and late bacillus Calmette-Guérin substrains are genetically distinct, showing different antigenic determinants. While it was suggested that this may influence the immunostimulatory effects of bacillus Calmette-Guérin as a vaccine in the context of tuberculosis, to our knowledge the impact of these genetic differences on the antitumor activity of bacillus Calmette-Guérin remains unknown. We compared the direct antitumor capacity and the ability to trigger cytokine production of 8 evolutionarily early and late BCG substrains in urothelial bladder cancer cell lines. MATERIALS AND METHODS The T24, J82 and RT4 bladder tumor cell lines were cultured with different doses of 3 evolutionarily early bacillus Calmette-Guérin substrains (Japan, Moreau and Russian) and 5 evolutionarily late strains (Connaught, Danish, Glaxo, Phipps and Tice). The inhibition of cell proliferation at different time points and the production of interleukin-6 and 8 in cell culture supernatants were measured. RESULTS For T24 and J82 cells Russian and Connaught induced the highest inhibition of cell proliferation and cytokine production, triggering values up to threefold higher than the other bacillus Calmette-Guérin strains. In contrast, Glaxo and Phipps (for T24 cells) and Glaxo and Tice (for J82 cells) were the least efficacious. For RT4 all bacillus Calmette-Guérin strains inhibited cell proliferation to a similar extent and induced low levels of only interleukin-8 except the Danish and Glaxo strains, which were less efficacious. CONCLUSIONS Russian and Connaught, which are evolutionarily early and late substrains, respectively, are the most efficacious bacillus Calmette-Guérin strains for inhibiting cell proliferation and inducing cytokine production. Glaxo is the least efficacious strain.

An ELISA for five glycolipids from the cell wall of Mycobacterium tuberculosis:: Tween 20 interference in the assay

Mycobacterium tuberculosis cell wall contains antigenic glycolipids: phenol-glycolipid (PGL), diacyltrehalose (DAT), triacyltrehalose (TAT), cord-factor (CF), and sulpholipid-I (SL-I). In the last decade, the usefulness of these antigens for the serodiagnosis of tuberculosis has been evaluated mainly using enzyme-linked immunosorbent assays (ELISA). Currently, there are no conclusive results about the utility of these glycolipidic antigens, because the results obtained by different groups are discrepant. In order to explain these discrepancies, we have investigated the methodological variations in the ELISAs used previously. Specifically, we have studied the following: the coating solvent, the optimum amount of glycolipid coated per well, the blocking agent, and the use of detergent (Tween 20) in the washing buffer. The most significant finding was that Tween 20 detaches PGL, DAT, TAT and SL-I from microtitre wells. However, Tween 20 does not remove CF from the wells. In addition, we have found that the best solvent for coating is n-hexane, that the optimum antigen coating concentration is 1000 ng/well, and that BSA and gelatin are equally effective blocking agents. We can therefore conclude that the use of Tween 20 as a detergent, and the lower antigen coating concentrations (100-200 ng/well), may well explain some of the discrepancies in previous studies.

Surface Spreading Motility Shown by a Group of Phylogenetically Related, Rapidly Growing Pigmented Mycobacteria Suggests that Motility Is a Common Property of Mycobacterial Species but Is Restricted to Smooth Colonies

Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.

Demonstration of Cord Formation by Rough Mycobacterium abscessus Variants: Implications for the Clinical Microbiology Laboratory

ABSTRACT In low-income countries some infections caused by nontuberculous mycobacteria are misdiagnosed as multidrug-resistant tuberculosis. In most of these settings the observation of microscopic cords is the only technique used to identify Mycobacterium tuberculosis in the laboratory. In this article we definitively demonstrate that Mycobacterium abscessus, an emerging pulmonary pathogen, also forms microscopic cords.

Production of Antibodies against Glycolipids from the Mycobacterium tuberculosis Cell Wall in Aerosol Murine Models of Tuberculosis

Evolution of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall has been studied for the first time in experimental murine models of tuberculosis induced by aerosol, in which infection, reinfection, reactivation, prophylaxis and treatment with antibiotics have been assayed. Results show a significant humoral response against these antigens, where diacyltrehaloses (DAT) and sulpholipid I (SL‐I) elicited higher antibody levels than protein antigens like antigen 85 protein complex (Ag85), culture filtrate proteins (CFP) and purified protein derivative (PPD). Only immunoglobulin M (IgM) antibodies have been detected against DAT and SL‐I. Their evolution has a positive correlation with bacillary concentration in tissues.

Simple and Rapid Differentiation of Mycobacterium tuberculosis H37Ra from M. tuberculosis Clinical Isolates through Two Cytochemical Tests Using Neutral Red and Nile Blue Stains

ABSTRACT The attenuated Mycobacterium tuberculosis strain H37Ra is one of the most commonly used controls for M. tuberculosis identification in the clinical laboratory and is a source of false-positive results for M. tuberculosis as a consequence of cross-contamination. Therefore, the ability to discriminate between H37Ra and real clinical isolates has important public health implications. To date, differentiation of H37Ra from M. tuberculosis clinical isolates is possible only by IS6110 genotyping and spoligotyping. In the 1950s, some authors reported that the virulent strain H37Rv and M. tuberculosis clinical isolates were able to fix basic dyes in their anionic forms, while H37Ra was not. We have studied the different techniques described for M. tuberculosis cytochemical staining and have chosen the best of these, introducing certain modifications in order to increase their discriminative power and reproducibility. We describe cytochemical staining of M. tuberculosis cells with neutral red and Nile blue, which differentiates H37Ra from virulent strains. This method could be used as an easy laboratory tool for distinguishing between H37Ra and real M. tuberculosis clinical isolates.