Vicente Ausina

Prospective Study of Community-Acquired Pneumonia of Bacterial Etiology in Adults

Abstract The aim of this study was to prospectively analyze the bacterial etiology of community-acquired pneumonia in adults in Spain. From May 1994 to February 1996, 392 episodes of CAP diagnosed in the emergency department of a 600-bed university hospital were studied. An etiological diagnosis based on noninvasive microbiological investigations was achieved in 228 cases (58%); 173 of these diagnoses were definitive and 55 probable. Streptococcus pneumoniae, which caused 23.9% of the episodes, was the predominant pathogen observed, followed by Chlamydia pneumoniae (13.5%) and Legionella pneumophila (12.5%). Other less frequent pathogens found were Haemophilus influenzae (2.3%), Pseudomonas aeruginosa (1.5%), Mycoplasma pneumoniae (1.3%), Coxiella burnetii (1%), Moraxella catarrhalis (2 cases), Nocardia spp. (2 cases), and Staphylococcus aureus (2 cases). Streptococcus pneumoniae was significantly more frequent in patients with underlying disease and/or age ≥60 years (28% vs 13%, P=0.002), while Legionella pneumophila was more frequent in patients below 60 years of age and without underlying disease (20% vs 9%, P=0.006). Likewise, Streptococcus pneumoniae and Legionella pneumophila were the most frequent etiologies in patients requiring admission to the intensive care unit, occurring in 29% and 26.3% of the patients, respectively. In addition to Streptococcus pneumoniae, other microorganisms such as Chlamydia pneumoniae and Legionella spp. should be seriously considered in adults with community-acquired pneumonia when initiating empiric treatment or ordering rapid diagnostic tests.

Comparison of Two Commercially Available Gamma Interferon Blood Tests for Immunodiagnosis of Tuberculosis

ABSTRACT We evaluated the T-SPOT.TB and Quantiferon-TB Gold In tube (QFN-G-IT) tests for diagnosing Mycobacterium tuberculosis infection. T-SPOT.TB was more sensitive than QFN-G-IT in diagnosing both active and latent infection. Both gamma interferon tests were unaffected by prior Mycobacterium bovis BCG vaccination. Among children who were not BCG vaccinated but had a positive tuberculin skin test, QFN-G-IT was negative in 53.3% of cases, and T-SPOT.TB was negative in 50% of cases.

Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials

The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.

Usefulness of Urinary Antigen Detection by an Immunochromatographic Test for Diagnosis of Pneumococcal Pneumonia in Children

ABSTRACT We evaluated an immunochromatographic assay detecting pneumococcal antigen in urine samples from children diagnosed with pneumococcal pneumonia. The sensitivity and specificity of the immunochromatographic test with nonconcentrated urine (NCU) were 86.7 and 62.9%, respectively; with concentrated urine (CU), they were 100 and 11.7%, respectively. Pneumococcal antigen was also detected in 42.5% of NCU and 87.1% of CU samples from nasopharyngeal carriers. This is a nonspecific test for the diagnosis of pneumococcal pneumonia in children, particularly the very young.

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.

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).

Comparative Evaluation of the New Version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium Assays for Identification of Mycobacterium Species from MB/BacT Liquid Cultures Artificially Inoculated with Mycobacterial Strains

ABSTRACT The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4 Mycobacterium kansasii strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the Mycobacterium genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.

T-cell responses to the Mycobacterium tuberculosis-specific antigens in active tuberculosis patients at the beginning, during, and after antituberculosis treatment.

The objectives of the study were to assess the performance of the QuantiFERON-TB Gold In-Tube (QFN-G-IT) and the T-SPOT.TB tests in the immunodiagnosis of active tuberculosis (TB) in adult patients, and to study the T-cell interferon gamma (IFN-gamma) responses during treatment and in patients who have recovered after curative treatment and self-healed TB patients. When only analyzing patients included at the beginning of treatment, the sensitivity was 83.3% for T-SPOT.TB and 69.4% for QFN-G-IT. In contrast, when evaluating patients during treatment, the sensitivity of the T-SPOT.TB and QFN-G-IT decreased to 69.8% and 48.8%, respectively. The response to the specific antigens increased after finishing the treatment compared with the values during the treatment. The T-SPOT.TB was more sensitive in diagnosing active TB than the QFN-G-IT. The IFN-gamma tests could be used as a complementary method in the diagnosis of active TB.

Rapid diagnosis of bloodstream infections with PCR followed by mass spectrometry.

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods.

Use of Quantitative and Semiquantitative Procalcitonin Measurements to Identify Children with Sepsis and Meningitis

During infancy and childhood, clinical signs of infection and conventional laboratory markers are not specific in the early phase of disease [1]. The availability of a parameter that more rapidly identifies children suspected to have bacterial sepsis before microbiological results are available would minimize unnecessary treatments and hospitalization. Since its original description, the importance of procalcitonin (PCT) as an indicator of systemic bacterial infection has been demonstrated in many reports [2, 3, 4, 5]. The aim of our study was to evaluate the reliability of PCT measurement by quantitative luminometric immunoassay (LIA) in distinguishing between systemic bacterial infection (sepsis and/or meningitis), localized bacterial infection and aseptic meningitis in children, compared to leukocyte count and C-reactive protein (CRP) levels. We also evaluated the correlation of a rapid immunochromatographic test (ICT) for semiquantitative PCT measurement in comparison with quantitative test results. The study was carried out on selected children aged between 1 month and 12 years who were admitted to the pediatric emergency department of our hospital after presenting with fever of less than 12-h duration. At the time of admission, blood samples were collected for leukocyte count, CRP and PCT measurement. Clinical specimens were collected for microbiological testing in order to correctly establish the etiology. Serial serum samples for PCT and CRP assays were collected daily when possible. Patients were grouped retrospectively according to the type of illness. Group 1 included 25 children diagnosed with bacterial sepsis and/or meningitis by culture of blood and/or cerebrospinal fluid (CSF) samples: Neisseria meningitidis was isolated in 18 cases, Streptococcus pneumoniae in 6 and Haemophilus influenzae in 1. Bacteria were isolated from CSF culture only in 8 patients, blood culture only in 10 and both culture types in 7. Group 2 included 18 children diagnosed with aseptic meningitis by compatible clinical presentation, negative Gram stain, cultures and antigen tests for bacterial infection using blood and CSF samples, compatible CSF analysis (pleocytosis with a predominance of mononuclear cells) and successful recovery without antibiotic therapy. Viral cultures were not performed. Group 3 included 22 children presenting with localized bacterial infection such as purulent conjunctivitis, acute otitis media, streptococcal pharyngitis, cellulitis or sinusitis, confirmed by culture of the site of infection and negative blood cultures. The bacteria isolated were Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes. In order to establish normal PCT levels for comparison, serum samples were also collected from a control group of 25 similarly aged healthy children admitted to the hospital for elective surgery. PCT was measured using LIA (Lumitest PCT; Brahms Diagnostica, Germany) for quantitative detection and ICT (PCT-Q; Brahms Diagnostica) for semiquantitative detection following the manufacturer’s instructions. LIA requires 20 l of serum and 90–120 min to be performed. ICT requires 200 l of serum and results can be obtained in 30 min; samples can be measured individually. CRP was measured using a turbidimetric assay (C-Reactive Protein FlexT reagent cartridge, Dimension; Dade Behring, USA) and leukocyte count was measured with Coulter Counter Maxm (Coulter, USA). Comparison between groups for quantitative parameters was performed using the non-parametric MannWhitney U test for CRP, leukocyte count and PCT. Diagnostic accuracy and optimum cut-off points were determined using a receiver operating characteristic curve. Comparisons between the two methods were made C. Prat ()) · J. Dom nguez · M. Gim nez · S. Blanco · V. Ausina Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, C/Canyet s/n, 08916 Badalona, Spain e-mail: crisprat@ns.hugtip.scs.es Tel.: +34-93-4978894 Fax: +34-93-4978895