Prospective evaluation of epidemiological, clinical, and microbiological features of pandemic influenza A (H1N1) virus infection in Italy.

Since several characteristics of pandemic influenza A (H1N1) virus infection remain to be determined, this study aimed to describe clinical features and complications of patients infected with H1N1. Subjects affected by influenza-like illnesses and a control group of asymptomatic patients were enrolled prospectively at an Emergency Department from October 2009 to April 2010. At enrollment, clinical data and nasopharyngeal swabs for virological analyses were obtained. Ill subjects were followed until recovery and swabs were collected weekly in patients infected with H1N1. Of 318 patients enrolled, 92 (28.9%) were positive to H1N1. Patients infected with H1N1 were mainly young adults and complained classic influenza-like symptoms. Fever was observed for a median time of 5 (IQR 3-7) days. Hospitalization occurred in 27.7% with 2% requiring intensive care unit admission: median length of hospitalization was 6 days (IQR 5-9). Pneumonia was diagnosed in 19.6% of patients. A similar proportion of lower airways involvement and of clinical complications was observed in subjects testing positive or negative for H1N1. However, patients infected with H1N1 were younger and hospitalized for a shorter period as compared to the control group (P = 0.002 and P = 0.045, respectively). Older age, asthma/chronic obstructive pulmonary disease and hypertension were associated with an increased risk of pneumonia. Viral shedding was observed for at least 1 week in 21.3% of patients. Asymptomatic infection was uncommon (1.1%). Respiratory syndromes caused by H1N1 and factors associated with disease severity were investigated and compared to influenza-like illnesses of other origin. Such findings might contribute to improve clinical and epidemiological management of the disease.

Comparison of real-time PCR and pp65 antigen assays for monitoring the development of Cytomegalovirus disease in recipients of solid organ and bone marrow transplants.

Cytomegalovirus (CMV) infection is a frequent complication in transplant recipients. This retrospective study compared real-time PCR (rt-PCR) and a pp65 antigen assay as tools for monitoring CMV infection in solid organ (SOT) and bone marrow (SCT) transplant patients. The study tested 2662 samples by rt-PCR, and 1284 specimens with a pp65 antigen assay. 24.3% of the rt-PCR samples and 4.1% of the pp65 antigen samples were positive. 793 specimens, from 230 patients, were tested with both assays. In 6.7% of samples, both tests were positive; in 72.7% both were negative; in the remaining 20.6% of cases, the results were discordant. CMV disease was diagnosed in 50 patients. Results from the two methods were poorly correlated (r=0.460). The sensitivity of rt-PCR (94%) was higher than that of the pp65 antigen assay (27%). Both assays showed high specificity (92% and 99%, respectively). ROC curve analysis, performed separately for SOT and SCT patients, confirmed that rt-PCR outperformed the pp65 assay in the detection of CMV. These findings provide evidence that rt-PCR is a reliable diagnostic tool, and that it can be more effective than pp65 based assays in monitoring CMV infection progression and in guiding therapy in immunocompromised patients.

Prospective evaluation of epstein-barr virus reactivation after stem cell transplantation: association with monoclonal gammopathy.

Epstein-Barr Virus (EBV) reactivation and EBV-related post-transplant lymphoproliferative disease (PTLD) have emerged as a severe complication after stem cell transplantation (SCT). We prospectively evaluated 104 consecutive patients receiving SCT either autologous or allogeneic. Fifty-two patients (50%) presented EBV DNA-emia and five of them developed PTLD proven or probable. PTLD rate was 9.6% among patients with EBV DNA-emia. One patient developed PTLD without EBV DNA-emia (0.96%). Overall PTLD incidence was 5.7%. No PTLD developed after autologous SCT. EBV DNA-emia was significantly more frequent after allogeneic than autologous SCT (60.7% vs 17.4%, p = 0.0002). At EBV reactivation, serum protein electrophoresis and immunofixation were assessed. Global incidence of γ-peak after allogeneic SCT with EBV reactivation was 65.3% (32/49 patients) and monoclonal gammopathy (MG) was identified in 23/28 evaluable patients (82%). All patients with PTLD developed γ-peak and in five of them MG was identified. MG is consistently associated with EBV DNA-emia and may help identification of progression to PTLD after allogeneic SCT.

Human antibodies from phage display libraries: expression of recombinant full length immunoglobulin G specific to the hepatitis C virus E2 glycoprotein.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 glycoprotein (HCV/E2) represents a major target antigen involved in the containment and resolution of naturally occurring HCV infection. Antibody phage display allows the molecular cloning of cDNA sequences encoding antibody fragments specific to a wide range of diverse antigens. These antibodies may be produced in bacteria as Fab or converted into full length IgG. The latter have a higher serum half life and display Fc encoded function. Using a library prepared from an HCV-infected individual, we selected a panel of Fab fragments for binding to invariant epitopes of the E2 glycoprotein. This work describes a technique used to convert the selected Fab fragments into full length IgG and to express these antibodies in eukaryotic cells. All the recombinant antibodies retained the binding specificity of the parental Fab showing an increase in apparent relative affinity for E2.

Molecular characterization of human adenoviruses isolated in Italy.

There is little information on the epidemiology of Human Adenoviruses (HAdVs) in Italy. In this study, 103 HAdV isolates, collected by the A. Gemelli Hospital (Catholic University Medical School of Rome, Italy) between 1987 and 2005, were genotyped by sequencing and phylogenetic analysis on a partial hexon gene region. Nine different serotypes belonging to all six HAdV species were identified. Serotype 2 was the most frequent (53.4%), followed by serotype 1 (15.53%) and serotype 41 (9.7%). Partial-hexon-based identification was confirmed as an effective tool for studying the molecular epidemiology of HAdVs.

Human monoclonal antibody fragment specific for glycoprotein G in herpes simplex virus type 2 with applications for serotype-specific diagnosis.

A combinatorial library was used to select a human monoclonal antibody fragment (Fab) with high affinity for G glycoprotein in herpes simplex virus type 2 (HSV-2). Tests with 112 clinical specimens demonstrated successful discrimination between HSV-2 and HSV-1, showing the potential of Fab as a low-cost tool for HSV subtyping in clinical diagnosis.

Molecular identification and typing of enteroviruses isolated from clinical specimens.

Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5' noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.