Search

Immunofluorescenza diretta (DFA) su cellule HEP2 e R-Mix Too per la rilevazione degli Adenovirus

Quantificazione del virus respiratorio sinciziale di tipo A e B mediante saggi real time RT-PCR con standards a cRNA

Introduction: The Cytomegalovirus (CMV) is the major cause of morbidity and mortality in solid organ (SOT) and bone marrow (BMT) transplantation. An early reconstitution of immune response is crucial in limiting CMV replication; on the other hand, a late reconstitution may determine CMV reactivation with possible evolution to symptomatic phase.The Enzyme-Linked Immunosorbent Spot (EliSPOT) assay is a useful tool for monitoring the CMV-specific immune response recovery in transplant patients.This study propose the development and optimization of a interferon-gamma-(IFN-γ)-based EliSPOT assay for the detection of CMV immune response. Methods: CD3 + lymphocytes were separated using Robosep (negative selection, HLA ® EasySep WB Human T Cell Enrichment Kit, Stemcell Technology,Vancouver, Canada).The EliSPOT assay was optimized using the Human IFN gamma ELISPOT Kit and a modified protocol provided by Nanogen Advanced Diagnostics (Buttigliera Alta, Italy). Different parameters were analyzed: number of cells (200,000 - 300,000), antigens (CMV peptide mix and whole CMV antigen), incubation time (18 - 20 - 22 - 24h), number of washes, incubation conditions with secondary antibody and conditions of substrate development. Results: Herein we report the optimal conditions identified. 200,000 CD3+ cells per well were stimulated with CMV peptide mix antigens. Cells were stimulated for 18h at 37 °C in humidified atmosphere; wells were washed and secondary antibody was added and incubated at room temperature for 2h.After incubation a second series of washes were performed. Substrate was added and incubation for 15 minutes was carried out.The reaction was detected by plate reader AID ELISPOT (Strassberg, Germany). Conclusions:We developed an EliSPOT assay for the detection of CMV-immune response and reconstitution.All variables were compared to previous published protocols.The improvement of the EliSPOT assay for the detection of CMV-specific immune response represents the first step for result evaluation in clinical practice.

Introduction. Human Bocavirus (HBov) is a ubiquitous parvovirus predominantly associated with respiratory tract infections in pediatric patients.The results of the few studies conducted in adult patients are conflicting, with data of prevalence around 0.8%, thus making difficult to evaluate the HBOV clinical role. In this study the prevalence and clinical role of HBoV in adult hospitalized patients were evaluated. Methods. The presence of HBoV was evaluated in 514 bronchoalveolar lavages (BAL), obtained over a period of 24 months from 341 adult patients (mean age, 56.5 ± 16.2 years, range 19-85), by real-time TaqMan PCR.The BAL was performed for the presence of symptoms/signs of suspected infection of the lower airways or for surveillance in the transplanted lung patients (month 1 and every 3 months). Results. 12/341 patients (3.5%) were positive for HBoV; in particular: 1/45 (2.2%) lung transplanted, 1/20 (5%) liver transplanted and 2/26 (7.7%) bone marrow transplanted, while no kidney (n=19) or heart (n=13) transplant patients resulted positive. 8/218 (3.6%) not transplantpatient were positive, in particular 1.8% patients with haematological cancers and 1.8% with other disease.The prevalence of HBoV did not differ between transplanted and not transplanted patients, depending on the type of transplant, and between immunocompetent and immunocompromised individuals. Conclusions. HBoV can be detected at low frequency in BAL of adult patients with acute lower respiratory tract, further studies would show whether the virus plays a role as a single pathogen or whether the altered lung background could help the viral infection.

The respiratory syncytial virus (RSV) is the major cause of respiratory infection in children, with bronchopneumonia and bronchiolitis. In the adult the clinical manifestation is lighter than in infant, however, in the case of immunocompromised patients (transplant recipients or patients in immunosuppressive treatment) infection can lead to death. Immunofluorescence is a diagnostic procedures which can detect RSV infection and the presence of viable virus in the biological sample of the patient. In this study, indirect and direct immunofluorescence techniques using monoclonal antibodies directed to RSV have been compared. For the comparison HEP2 shell vials, that are susceptible to RSV, were used.These cells were infected with different virus titres, ranging from 102 to 10-3 TCID50. Subsequently, different primary or Fluorescein isothiocyanate antibody dilutions were tested (1:40, 1:80, 1:160), for indirect and direct immunofluorescence evaluation, respectively, at three incubation periods after infection (48h, 72h, 96h). Indirect immunofluorescence showed a sensitivity of 101 TCID50, with a cells positive detection even at lower dilutions up to 10-1 TCID50 at 72 hours. For direct immunofluorescence, the same sensitivity was observed with 1:40 antibody dilution at 72h, with a detection limit of 10-1 TCID50. Indirect immunofluorescence showed optimal RSV detection at 72h after infection with 1:80 primary antibody dilution, with a sensitivity of 101 TCID50. An equivalent sensitivity has been observed with direct immunofluorescence at 72h, 1:40 antibody dilution, thus representing an advantage in terms of time.