Health Care and Research Institute

1. Measurment of cell-mediated immunity by using ELISA test in extra-pulmonary TB localization: Pediatric experience BAMBINO GESU’ Children Hospital HealthCare and Research Institute Rome - ITALY Poster Board NUMBER H28 russocri@opbg.net Abstract Background and Rationale of the Study Extrapulmonary manifestation of tuberculosis disease is a rare event. In the last three years, at “Bambino Gesù” Children Hospital, Reference Centre for Diagnosis and Healthcare of Mycobacteria Infection in Children, we evaluated the QuantiFERON TB Gold to assess the assay performance in extra-pulmonary TB diagnosis. Four cases referred to TB meningitis, one occurred in a child over 5 years and three in children under 5 years, were investigated with QFT-G in whole blood. During the first evaluation, two cases out of 4 had already QFT-G positive, one generated an Indeterminate result and one a Negative result. These two patients received a second QFT-G test a few days later and both resulted QFT-G Positive Four cases of pleural TB without any pulmonary lesion were collected in this study. Three cases out of four were strongly positive for INF- measured directly on pleural effusion, in one patient an enough amount of pleural effusion for QFT-G was not collected. One case with peritoneal fluid, as only evidence of a peritoneal TB manifestation, was investigated both in blood and in the pleural fluid. QFT-G performed in peripheral blood was negative while QFT-G was strongly positive when tested directly on pleural effusion.All of these cases were confirmed as extra-pulmonary TB by culture isolation of Mycobacterium tuberculosis.In conclusion, in our experience QFT-G has revealed as a powerful tool in a rapid diagnosis of extra-pulmonary TB in children. The clinical presentation of TB in children is extremely variable. It depends by different factors as: the age, the immunocapability of the host response and also the TB spread. In particular Tuberculous meningitis has high rate of morbidity and mortality. Demonstration of tubercle bacilli in cerebrospinal fluid, the only reliable method of diagnosis, is time consuming Surveillance of tuberculosis in Europe Four children received the finaly diagnosis of tuberculous meningitis and has a low yield.Pleural or peritoneal tuberculosis presenting with effusions are not always easy to diagnose because conventional tests for extra-pulmonary TB have several limitations. Also Nucleid Acid Amplification commercial kits have low and varying sensitivities, and therefore should not be used for excluding a diagnosis of tuberculous pleuritis. Moreover, Cutaneous sensitivity to Purified Protein antigen Derivative is not satisfied. In recent years, numerous authors studied possible biochemical markers such as interferon gamma (IFN- ), to improve diagnostic efficiency. All children had foreign origin (three from Est Europe and one from Africa) TST was not reactive (negative) in all children QFT-G was positive during the first evaluation for two children under 2 years of age Computed Tomography images showed hydrocephalus and basilar meningitis but without any evidence of tuberculoma The aims of our study have been: • to apply the new QuantiFeron TB Gold (QFT-G – Cellestis Limited, Carnegie, Victoria, Australia) both in blood and in pleural or peritoneal effusion to asses the performance of QFT-G in samples specimens different from blood as marker of TB; • to test if the new QuantiFeron TB Gold (QFT-G) is able to work in suspected TB meningitis as rapid tool for TB diagnosis. Population and Methods OUR PATIENTS INF- ELISA assay From 2004 to 2007 we investigated children admitted Liquid and In Tube version QuantiFeronTB-Gold • at “Bambino Gesù” Children Hospital (Rome, Italy) presenting with: • Signs or symtoms of meningitis compatible wich a TB suspected • Pleural effusion or • Peritoneal effusion In wich any other etiology were found Step 2 ELISA assay Step 1 samples collection The QFT-G was performed on blood as manufacture instructions and on body fluids both whole and after fluid concentration. Four children received the finaly diagnosis of pleural tuberculosis and one child had a peritoneal TB localization Two children were italian and three had foreign origin (with history of TB immunization) Step 3 Interpretation of INF-γ amount All specimen fluids (cerebrospinal, pleural and peritoneal) were collected and processed by using our Mycobacterial TB laboratory protocol Three children received TST (two were TST positive and one TST negative) QFT-G was always positive when performed on pleural or peritoneal fluid • AFBfluorescence Stain • Solid and liquid media cultures Computed Tomography images showed a large ammount of effusion in pleural cavity but without any parenchymal involvement or typical granulomas • Nucleid Acid Amplification (Cobas Amplicore®- Roche) direct on samples Discussion Conclusions Tuberculosis is a systemic infection caused by Mycobacterium tuberculosis. It is transmitted by coughed aerosol and usually presents with respiratory symptoms; however, it can produce disease in any organ system by haematogenous spread, specially in newborn, children and teen agers. The mean annual number of cases referred to extra-pulmonary TB cases are approximately unchanged during the last five years (about 25-30% of all TB cases) in Europe . Extra-pulmonary TB is often diagnosed on the basis of clinical experience which may lead to diagnostic errors. A correct diagnosis depends on the possibility of obtaining appropriate specimens for cultures and often requiring invasive procedures and more sophisticated laboratory techniques. In conclusion, evaluation of an increase in the IFN- level in the pleural fluid is a good and useful diagnostic marker of pleural tuberculosis and, in our experience, QFT-G has revealed as a powerful tool in a rapid diagnosis approach in case of suspected extra-pulmonary TB in children Our study was carried out prospectively in a clinical routinely situation during the last three years. We assessed whether the new Interferon-γ release assay QuantiFERON-Gold could be used in practice in special setting (as pediatric population) and in extra-pulmonary localization as meningitis, pleural and peritoneal tuberculosis. • Potential limitations of our study • should be: • the low number of cases may affect precision • we did not evaluate children HIV affected Our results reveal that: • in pleural and peritoneal effusion, INF- levels are significantly higher than in whole blood in tuberculous disease; • in case of suspected TB meningitis in children under 2 ys the QFT-G assay gives a positive results Acknowledgements Silvia Gobbi and Eugenia Galeno laboratory technicians – Microbiology Unit – Laboratory Department Stefania Colafati MD - Radiology Department -for supporting in CT images and interpretation C. Russo, L. Coltella, and D. Menichella - "Bambino Gesù" Children Hospital Health Care and Research Institute Results MENINGITIS PLEURAL TB PERITONEAL TB