Identification of HEV in symptom-free migrants and environmental samples in Italy.

Hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The occurrence of HEV genotypes in samples of faeces from asymptomatic migrants arriving on the coasts of South Italy and environmental samples was investigated. Analyses of sequences were used to compare human and environmental genotypes. A total of 40 stool specimens, 12 samples of untreated urban sewage, 12 samples of treated urban sewage and 12 samples of surface water were analysed. Viruses were concentrated from water samples by the tangential flow ultrafiltration technique. The presence of HEV RNA was detected by nested RT-PCR. Viral isolates were sequenced and phylogenetically characterized. Two (5%) of the 40 faecal samples tested were found to be positive for HEV RNA (G1 and G3 genotypes). The virus was detected in 25% (3/12) of the untreated sewage samples and 25% (3/12) of the surface water samples: all isolates belonged to G3 genotype. None of the treated sewage samples were found to be HEV RNA positive. The virus was detected in the faeces of two asymptomatic subjects, suggesting a potential role for symptom-free HEV carriers as a human reservoir. G3 HEV strains were detected in the untreated sewage, as observed in similar studies conducted in other European countries but differing from another study conducted in Italy recently. Moreover, our results show the first case of HEV isolated from fresh surface waters.

Molecular approaches in the diagnosis of sepsis in neutropenic patients with haematological malignances.

INTRODUCTION: Sepsis is a major cause of significant morbidity and mortality in neutropenic patients. Blood culture remains the gold standard in the microbiological diagnosis of bacterial or fungal bloodstream infections, but it has clear limits of rapidity and sensitivity. The objective of the study was to compare the real-time polymerase chain reaction (RT-PCR) with automated blood cultures (BC) method in detection in whole blood of pathogens in febrile neutropenic patients with hematological malignancies. METHODS: A total of 166 consecutive febrile neutropenic patients were enrolled. Blood samples for cultures and SeptiFast testing were obtained at the onset of fever, before the implementation of empirical antibiotic therapy. RESULTS: Forty (24.1%) samples out of the 166 blood samples tested, were positive by at least one method. Twenty-three (13.9%) samples were positive by blood culture and 38 (22.9%) by multiplex real-time PCR. The analysis of concordance evidenced a low correlation between the two methods (n = 21; 52.5%), mainly due to samples found negative by culture but positive with the Septi-Fast assay. Sensitivity, specificity, and positive and negative predictive values of RT-PCR were 91.3%, 88.1%, 55.3%, and 98.4%, respectively, compared with BC. DISCUSSION: Multiplex real-time PCR assay improved detection of the most bacteria associated with febrile neutropenia episodes. Further studies are needed to assess the real advantages and clinical benefits that molecular biology tests can add in diagnosis of sepsis.

Innate and adaptive immunity in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autosomal recessive syndrome characterised by chronic mucocutaneous candidiasis (CMC) and multiple endocrine failures. While the spectrum and modalities of autoimmunity are the main objects of current research into APECED, unequivocal data on the efficiency of immune responses to infectious agents are still elusive. The in vitro ability of monocytes and polymorphonuclear leucocytes to phagocytise and kill bacteria and fungi, and the degree of activation of lymphocytes cultured with mitogens and Candida albicans were investigated by flow cytometry in 11 APECED patients and healthy subjects. In addition, a comparison of gamma-globulin and immunoglobulin (Ig) concentrations was performed, and a correlation was sought between oral fungal load and the anti-Candida antibody titre. No difference between APECED patients and healthy subjects was observed in the phagocyte function, although the patients had a larger number of monocytes. Similarly, cultured lymphocytes were equally activated in the two groups. The concentration of gamma-globulins was higher among APECED patients, and anti-Candida IgM and IgG correlated with current and past oral candidiasis respectively. APECED patients have efficient innate and adaptive immune responses against exogenous stimuli, and currently, the mechanisms of mucocutaneous anergy leading to the high prevalence of CMC in this syndrome remain to be elucidated.

Epidemiological and virological assessment of influenza activity in Apulia, Italy, during the seasons 2004 - 2005 and 2005 - 2006.

This study evaluated the impact of influenza and vaccination coverage in Apulia, Italy, during the 2004 - 2005 and 2005 - 2006 seasons, using epidemiological and virological surveillance data collected through the Italian Net of Surveillance of Influenza (InfluNet) organized by the Superior Institute of Health (ISS) and the Inter-University Centre of Research on Influenza (CIRI). Vaccination coverage was calculated from the number of doses administered to individuals aged >or= 65 years. Sentinel physicians reported weekly influenza-like illness (ILI) and acute respiratory illness (ARI) occurrences among patients. Influenza viruses were isolated and identified by cell culture on Madin-Darby Canine Kidney cells and polymerase chain reaction techniques. Vaccination coverage reached 72.7% and 77.0% during the 2004 - 2005 and 2005 - 2006 seasons, respectively. Incidence of ILI was higher during the 2004 - 2005 season compared with the 2005 - 2006 season, whereas the incidence ARI appeared to show a more constant trend. Incidence rates for ILI and ARI were higher in the 0 - 14-year age group. The increase in vaccination coverage and implementation of a network of epidemiological and virological surveillance are fundamental for the control and prevention of influenza.

Organ-specific and non-organ-specific autoantibodies in children and young adults with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

OBJECTIVE: The aim of the study was to assess the complex of autoantibodies which can be detected in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare autosomal recessive disease in which the extent of autoimmunity is still unknown. DESIGN: Antibodies (A) to parathyroid glands, adrenal cortex (AC-A), ovary and testis (steroid cell antibodies, SC-A), pancreatic islet cells (IC-A), gastric parietal cells, and non-organ-specific antigens were investigated in 11 APECED patients living in the Salento region of southern Italy. Further measurements included antibodies to cytochrome P450 (CYP) enzymes: cholesterol side-chain cleavage enzyme (CYP11A), 21-hydroxylase (CYP21) and 17alpha-hydroxylase (CYP17); and to glutamic acid decarboxylase 65-kDa isoform (GAD65), tyrosine phosphatase-like protein IA2, thyroglobulin (TG), thyroperoxidase (TPO), thyrotropin receptor, liver CYP enzymes and intrinsic factor. METHODS: Antibodies to organs and subcellular fractions were detected by immunofluorescence. Radiobinding, immunoradiometric, and immunoblotting assays were used for the other measurements. RESULTS: AC-A and SC-A were positive in all sera; among antibodies to adrenal CYP enzymes, only CYP21-A were present in all the patients with Addison's disease of short-medium duration (<15 years). Of three patients with Addison's disease of long duration (>15 years), two tested positive for antibodies to all three CYP enzymes, and the other for only CYP11A-A. In all sera CYP11A-A and/or CYP17-A were found. Two patients tested positive for both IC-A and GAD65-A, one for both IC-A and IA2-A, and one for GAD65-A; the fasting C-peptide assay showed no statistical difference between these four subjects and the others. All four hypothyroid patients were positive for TPO-A, while two of them were positive and two were negative for TG-A; two euthyroid subjects had positivity for TG-A. Liver-kidney microsomal antibodies reacting against the CYP2A6 were detected in two patients with autoimmune hepatitis. All but one sera contained anti-nuclear antibodies at a titre ranging between 1:20 and 1:80; however, only two patients had a connective tissue disease (Sjögren's syndrome). CONCLUSIONS: Several autoantibodies may be detected in any APECED patient. Our data confirm that CYP21-A and TPO-A are major autoantibodies involved in APECED-associated Addison's disease and hypothyroidism respectively, while CYP11A-A and CYP17-A correlate with positivity for SC-A. Markers of islet cell autoimmunity are frequent, but prevalence and modalities of progression to overt beta-cell failure have to be clarified. Low-titre non-organ-specific autoantibodies are a feature of autoimmunity in APECED, but their role has yet to be fully explained.

Prevalence and clinical features of cryoglobulinaemia in multitransfused beta-thalassaemia patients.

OBJECTIVE: The aim of the study was to determine the prevalence of cryoglobulinaemia and its clinical features among beta-thalassaemia patients. METHODS: Eighty eight multitransfused beta-thalassaemia patients were studied. They were physically examined and asked about the presence of cryoglobulinaemia related symptoms. Hepatitis C virus (HCV) serology, HCV-RNA, HCV subtypes, viraemia, serum ferritin, liver and kidney function tests, rheumatoid factor (RF), circulating immune complexes (CIC), complement levels and autoantibodies were all evaluated. The patients were divided into four groups: HCV-RNA positive patients with and without cryoglobulinaemia (groups A and B), HCV-Ab positive/HCV-RNA negative patients (group C), HCV-Ab negative patients (group D). RESULTS: Cryoglobulinaemia was present in 35 of 53 (66.0%) patients with chronic HCV infection. They had higher viraemia than non-cryoglobulinaemic viral carriers, but no statistical difference relating to sex or HCV subtypes was found. In comparison with the other groups, group A patients were older, had undergone transfusion therapy for a longer period, had received a higher number of transfusions, and had increased levels of RF and CIC, as well as consumption of C4; in addition, they had a higher prevalence of cirrhosis. Cutaneous lesions (purpura, Raynaud's phenomenon, nodules and leg rash), peripheral neuropathy and sicca syndrome symptoms were present only in group A. Musculoskeletal symptoms (bone pain, arthralgia and myalgia), weakness, splenomegaly, lymphadenopathy, skin ulcers and proteinuria were also commoner in group A, but the difference did not reach statistical significance, possibly because of partial overlap between cryoglobulinaemia and beta-thalassaemia syndromes. CONCLUSION: Because of its high prevalence in multitransfused beta-thalassaemia patients, cryoglobulinaemia needs to be systematically studied and considered in the differential diagnosis of various beta-thalassaemia manifestations.

Computerised counting of tumour infiltrating lymphocytes in 90 breast cancer specimens.

Tumour infiltrating lymphocytes (TILs) implicated in immunologic cytotoxicity were evaluated by immunohistochemistry and digitally counted in serial sections from 90 breast cancers in order to assess their number, the relationships between them and to tumour histology. CD3+, CD4+, CD8+, CD20+, CD25+ and CD56+ lymphocytes were found in 58 (64.4%), 52 (57.7%), 50 (55.5%), 22 (24.4%), 11 (12.2%) and 21 (23.3%) tumours, respectively. There was no difference in the number of TILs between pure infiltrating ductal (NOS) and non-ductal carcinomas, and no relationship between TILs and histological grades was found. CD3+ TILs directly correlated to age, while lymph node negative patients had tumours infiltrated by fewer CD4+ TILs with respect to lymph node positive patients. In 25/90 patients, randomly chosen, the status of peripheral blood lymphocytes was evaluated but no differences with respect to the status found in healthy blood donors was obtained; nonetheless while in some patients CD8+ TILs outnumbered CD4+ TILs in situ, the CD4/CD8 ratio was normal in their peripheral blood. The results show a considerable diversity of TILs among breast tumours, their lack of relationship with the status of the peripheral blood cells, and their potential important relationship with age (CD3+) and lymph node status (CD4+).

Cryoglobulins and pyroglobulins: an overview.

Cryoglobulins are serum proteins with heterogeneous etiopathogenetic and immunochemical properties. What they have in common is temperature-dependent insolubility, in that at temperatures below 37 degrees C (often around 4 degrees C) they precipitate, and then redissolve at 37 degrees C. When the etiopathogenesis of the cryoglobulinemia is unknown, which is true for many patients, the condition is called idiopathic or essential cryoglobulinemia, whereas it is termed secondary whenever it appears to be associated with one of several diseases. Cryoglobulinemia has indeed been found in patients with lymphoproliferative and autoimmune disorders, liver diseases, infectious (viral, bacterial, fungal and parasitic) diseases, and so on. Cryoglobulins are usually classified according to their immunochemical properties as single-type monoclonal, mixtures of a monoclonal Ig with non-immunoglobulin material (DNA, lipoprotein, complement), mixed with one monoclonal Ig or mixed polyclonal, in which constitutive Ig fractions are polyclonal. As compared with normal Ig, cryoimmunoglobulins have sometimes been found to exhibit a peculiar amino acid structure of their heavy chains, less often of their light chains as well, and to have a lower carbohydrate content. Such structural abnormalities may contribute to their loss of solubility at low temperatures, possibly associated to the steric changes induced by the low temperature, causing the precipitate to form. The most common clinical features of cryoglobulins are correlated with vasculitis in the various organs and sometimes with increased viscosity of the plasma. Signs and symptoms include purpura, ulcers of the extremities, arthralgia, proteinuria, hepatic damage, abdominal pain, congestive heart failure, mental confusion, oligo-anuria, hemorrhagic diathesis, and coma. Pyroglobulins are also serum proteins with temperature-dependent insolubility. However, although they precipitate out of serum heated at 56 degrees C for half an hour, they do not resolubilize when the serum is returned to 37 degrees C. Pyroglobulins have been mainly found in patients with lymphoproliferative diseases (especially Waldenström's macroglobulinemia, with or without cryoglobulinemia), systemic lupus erythematosus, and neoplasia. So far, only single monoclonal IgG, IgM or IgA pyroglobulins have been described. Since they precipitate only at 56 degrees C, pyroglobulins do not cause clinical symptoms and they are usually discovered by chance.