Phylogenetic characterization of enterovirus 68 strains in patients with respiratory syndromes in Italy.

Enterovirus 68 (EV-D68) was associated with mild to severe respiratory infections. In the last 4 years, circulation of different EV-D68 strains has been documented worldwide. In this study, the phylogenetic characterization of nine EV-D68 strains identified in patients in the 2010-2012 period and 12 additional EV-D68 Italian strains previously identified in 2008 in Italy was described. From January 2010 to December 2012, a total of 889 respiratory specimens from 588 patients stayed or visited at the Fondazione IRCCS Policlinico San Matteo were positive for HRV or HEV. Extracted nucleic acids were amplified by one-step RT-PCR with primer specific for VP1 region of EV-D68 and purified positive PCR products were directly sequenced. Overall, 9/3736 (0.24%) patients were EV-D68 positive. Of these, 7/9 (77.8%) were pediatric and two (22.2%) were adults. Five out of seven (71.4%) pediatric patients had lower respiratory tract infection with oxygen saturation <94%. Four cases were detected from August through October 2010, while five other cases from September through December 2012. The Italian EV-D68 strains in 2008 belonged to clade A (n = 5) and clade C (n = 7). In 2010 all the Italian strains belonged to clade A (n = 4) and in 2012, four Italian strains belonged to clade B and one to clade A. In conclusion, we provide additional evidence supporting a role of EV-D68 in severe respiratory infection in pediatric patients. In addition, all the three EV-D68 clades circulating worldwide were identified in Italy in a 5-year period of time.

Human rhinovirus and human respiratory enterovirus (EV68 and EV104) infections in hospitalized patients in Italy, 2008-2009.

The epidemiology of picornavirus infections along with associated risk factors for lower respiratory tract infections (LRTI) and duration of virus shedding were investigated in 985 hospitalized patients in the period October 2008-September 2009. One-third of patients were human rhinovirus (HRV)-positive. Of 336 HRV-associated episodes, 153 (45.5%) were sustained by HRV-A, 31 (9.2%) by HRV-B, and 93 (27.7%) by HRV-C, while 7 episodes showed multiple HRV types and 52 were sustained by undefined HRV species. Independent risk factors for LRTI included high viral load and age less than 5 years. Twenty (2.1%) patients were enterovirus (EV)-positive (12 had EV-68, 7 EV-104, and 1 E-13 infection). Half of the EV-positive patients had a LRTI and were younger with respect to patients with upper RTI (median 18 months versus 37 years; P < 0.001). HRVs are often the cause of LRTI in children less than 5 years, frequently in association with a high viral load.

Human parechovirus infections in patients admitted to hospital in Northern Italy, 2008-2010.

Human parechoviruses (HPeVs) infection is associated with a wide range of clinical syndromes such as respiratory, gastrointestinal, neurologic diseases, and neonatal sepsis-like illness. The main objective of this study was to investigate the epidemiology of HPeVs infection in hospitalized patients in a period of 2 years. Respiratory samples from 3,525 patients with respiratory syndrome, cerebrospinal fluid (CSF) from 340 patients with neurologic syndrome as well as CSF and plasma samples from five neonatal patients with sepsis-like illness collected from October 2008 to 2010 were tested retrospectively using HPeV-specific real-time RT-PCR. Phylogenetic analysis of VP3/VP1 region was performed on the positive samples. Fourteen out of 3,525 (0.4%) patients with respiratory syndrome and five out of five patients with sepsis-like illness were positive for HPeV. In 3/5 patients with sepsis-like illness multiple samples (e.g., stool, plasma, CSF, or respiratory samples) were available, and HPeV was found in all specimens. In contrast, no positive CSF was detected among the 340 patients with neurologic syndromes. Eleven patients (57.9%) were infected with HPeV1 strain, 7 (36.8%) with HPeV3, and 1 (5.3%) with HPeV6 strains. Ten of the 14 HPeV patients with respiratory syndrome were co-infected with other respiratory viruses (eight with rhinovirus and two with coronavirus OC43). All five patients with sepsis-like illness were less than 1 month of age and were infected with HPeV3. Although not circulating at high frequency and unlikely to cause respiratory syndrome, HPeV was associated with severe clinical syndromes in a minority of newborns.

Impact of child obesity on adipose tissue physiology: assessment of adipocytokines and inflammatory cytokines as biomarkers of obesity.

Obesity could be interpreted as a low grade inflammatory state. The role of cytokines for innate and acquired immune response and adipocytokines in pathogenesis of obesity is not completely understood. The aim of the study was to evaluate anthropometric parameters, adipocytokines and inflammatory cytokine levels as biomarkers of childhood obesity. This investigation was designed as a longitudinal observational study. Forty-seven obese children (19 males and 28 females) were enrolled by Pediatric Clinic of the Foundation IRCCS Policlinico San Matteo, Pavia, Italy. For each patients a blood sample, used for other biochemical evaluations, was collected. Cytokines and adipocytokines plasmatic levels were determined using an ELISA method. Plasma leptin levels are in correlation with age (r=0.5; P<0.001) and BMI-z score (r=0.36; P<0.001), particularly in girls; plasma resistin levels are in inverse correlation with age, particularly in boys (r=-0.67; P<0.001) and in correlation with BMI-z score (r=0.52; P=0.002). Plasma leptin and resistin levels show a good correlation with antrophometric parameters of child obesity (sex and BMI z score). This study suggests that leptin and resistin can be considered as biomarker of childhood obesity and its comorbility. We observed a statistically significant correlation between plasma leptin and resistin levels and antrophometric parameters of child obesity (sex and BMI z score). This study suggests that adipocytokines, such as leptin and resistin, can be considered as biomarkers of childhood obesity.

Surveillance of influenza virus B circulation in Northern Italy: summer-2008 isolation of three strains and phylogenetic analysis.

Influenza virus type B strains were unexpectedly detected and isolated in Italy during summer-fall 2008 from three patients travelling to Italy from Lebanon, Senegal and Uzbekistan. The three summer-fall strains matched to a high degree the hemagglutinin (HA1) of influenza virus type B strains circulating in Italy in the second part (January through April) of the 2007/2008 season, and HA1 of the type B strains included in the 2008/2009 vaccine (B/Yamagata/16/88 lineage). Surveillance of influenza virus circulation in Western countries also during the summer-fall season may help to trace and anticipate the appearance of new influenza virus variants.

Rapid typing, subtyping and RNA quantification of influenza virus type A strains in respiratory secretions.

During the winter-spring season 2006-2007, 38 influenza virus strains were identified in patients admitted to hospital with an acute respiratory tract infection. Infections were diagnosed in parallel by direct fluorescent antibody (DFA) staining using type-specific monoclonal antibodies and real-time reverse transcription (RT)-PCR targeting the gene M (nt 25-124). In addition, virus strains were isolated in MDCK cells. Overall, 37 influenza virus strains were type A, and one type B. Of these, 35 (80.4%) were detected and typed by real-time RT-PCR, 34 (80.1%) by DFA, and 27 (71.0%) by virus isolation. Subtyping of 37 influenza virus A strains by RT-PCR and DFA gave the following results: 4/6 H1 strains were correctly subtyped by both methods, while of the 29 H3 strains subtyped by RT-PCR 7 were missed by DFA. Thus, the overall concordance of the two subtyping methods was 28/37 (75.7%). Viral RNA quantification by real-time PCR showed that when respiratory secretion collection was done within 5 days after the onset of symptoms, viral load was greater than 1 x 10(6) RNA copies/ml. In conclusion, typing and subtyping of influenza virus type A strains may benefit from both MAbs and RT-PCR, while viral RNA quantification may provide an indication of symptom onset.

The human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification.

The major objective of this study was to investigate the pathogenic role of human bocavirus (hBoV) in patients hospitalized with acute respiratory tract infection (ARTI). Overall, 685 respiratory samples from 426 patients were examined by PCR for human bocavirus, as well as for other known human respiratory viruses. HBoV was quantified by PCR. Forty/283 (14.1%) pediatric patients, and 2/143 (1.4%) adult patients were found to harbor hBoV for a total of 45 episodes (16 detected as single infection, and 29 as coinfection) of hBoV-associated respiratory infection. HBoV DNA quantification revealed the presence of an NPA viral load > 1.0 x 10(5) DNA copies/ml in respiratory secretions from 17/40 (42.5%) children and 0/2 adults. Below this cut-off, hBoV appeared to be an innocent bystander or a persistent virus. Although hBoV may be frequently detected in children with upper or lower ARTI, in less than 50% young patients it appears to be potentially pathogenic.

Prospective study of human metapneumovirus infection: diagnosis, typing and virus quantification in nasopharyngeal secretions from pediatric patients.

STUDY DESIGN: During the winter-spring seasons 2004-2005 and 2005-2006, 965 nasopharyngeal aspirates from 871 patients were examined for human metapneumovirus (hMPV) by both monoclonal antibodies (MAbs) and reverse transcription (RT)-PCR. RESULTS: Overall, 46 samples (4.8%) from 37 patients were positive for hMPV. Of these, 39 were positive by RT-PCR, and 35 by MAbs. Thus, using RT-PCR as a reference assay, the sensitivity, specificity, positive and negative predictive values of MAbs were 71.8%, 99.2%, 80.0% and 98.8%, respectively. Typing showed that concordant results were obtained in 32/46 (69.6%) strains (five untyped), whereas three strains were typed by MAbs only, and 11 by RT-PCR only. Finally, quantification of hMPV RNA allowed to correlate high viral load in nasopharyngeal secretions with acute respiratory symptoms in a group of 11 infants with acute lower respiratory tract infection examined upon admission and discharge from hospital, and a group of nine infants examined upon admission only. Conversely, hMPV etiology was questioned in a group of 14 infants with low viral load. CONCLUSIONS: MAbs may represent an alternative to or a complement to RT-PCR for detection and typing of hMPV strains, while hMPV RNA quantification may help in associating viral load with clinical symptoms.

Poly-epiphyseal overgrowth: description of a previously unreported skeletal dysplasia.

A skeletal dysplasia with previously unreported features is presented. Its evolution was characterized by growth abnormalities of bones without involvement of other organs. Advanced bone age, increased stature and irregular epiphyseal ossification with stippling of the main long bones were documented. Physeal overgrowth was massive in the left proximal humerus and femur. Furthermore, the hip joint appeared fused with an abundant mass of pathological calcific tissue extending from the femur to the ilium. Pathological epiphyses were characterized by anarchic cartilaginous proliferation with multiple ossification centres, while lamellar bone apposition and remodelling were normal. The observed bone changes were different from those in any previously reported syndrome, metabolic defect or bone dysplasia. However, they clearly indicated a defect of endochondral ossification with some resemblance to phenotypes observed in dysplasia epiphysealis hemimelica.

Human respiratory syncytial virus (hRSV) RNA quantification in nasopharyngeal secretions identifies the hRSV etiologic role in acute respiratory tract infections of hospitalized infants.

BACKGROUND: Human respiratory syncytial virus (hRSV) detection in nasopharyngeal aspirates (NPAs) from infants with acute respiratory tract infection (ARTI) does not prove the hRSV etiology of the current ARTI episode. HRSV RNA quantification may help in affording this issue. OBJECTIVES: hRSV was detected by quantitative reverse transcription-PCR in NPAs taken upon admission to hospital and, whenever possible, at discharge and subsequent medical visits. STUDY DESIGN: Prospective study, including 63 infants affected by either hRSV upper or lower ARTI. RESULTS: Based on the kinetics of viral load, hRSV etiology was identified in 25 infants in whom hRSV load dropped from 2.5 x 10(6) upon admission (presence of respiratory symptoms) to 7.5 x 10(2)RNAcopies/ml NPA upon discharge (absence of symptoms) after a median time of 5 days, and in 19 infants, in whom hRSV load was determined at admission only, in association with clinical symptoms (2.4 x 10(6)copies/ml). Furthermore, low levels of hRSV RNA (<1 x 10(5)copies/ml NPA) identified 14 patients with non-hRSV ARTI. Finally, in 14 infants with hRSV coinfections or sequential infections, hRSV quantification defined the hRSV role in the current ARTI episode. CONCLUSIONS: hRSV RNA quantification is critical in defining the hRSV role in respiratory infections.

Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients.

BACKGROUND: Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. OBJECTIVE: To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. STUDY DESIGN: Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. RESULTS: Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January-May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. CONCLUSION: HKU1 hCoV strains circulated in northern Italy during the winter-spring season 2005-2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR.

Insights into central and peripheral factors affecting the "oxidative performance" of skeletal muscle in aging.

During exercises with relatively small muscle masses, limitations to exercise performance by the cardiovascular system should be significantly reduced, allowing one to fully-test the "oxidative potential" of the investigated muscles. Ten elderly males (E, 77.8 +/- 2.9 years [x +/- SD]) and eight young controls (Y, 26.6 +/- 3.0) underwent incremental exercises to voluntary exhaustion on a dynamic leg-extension (dominant limb) machine (knee-extension, KE) and on a cycloergometer (CYCLO). During KE the load was increased every 3 min to loads corresponding to 20, 40 and 60% of the force of one-repetition maximum (1RM). The following variables were determined (vastus lateralis muscle): concentration changes of deoxygenated haemoglobin and myoglobin (Delta[deoxy(Hb + Mb)]) by near-infrared spectroscopy (NIRS), expressed as percentage of the maximal value obtained during transient limb ischemia, and taken as an index of O2 extraction; root mean square (RMS) and median power frequency (MDF) by electromyography. The total lifted load during KE and peak workload during CYCLO were lower in E versus Y (620.4 +/- 321.9 kg vs. 1347.4 +/- 458.7; 113.5 +/- 23.9 W vs. 224.3 +/- 41.0, respectively). During CYCLO Delta[deoxy(Hb + Mb)] peak (i.e. the value determined at exhaustion) was lower in E (44.5 +/- 17.7%) versus Y (67.1 +/- 22.9), whereas during KE Delta[deoxy(Hb + Mb)] peak was higher in E (56.8 +/- 20.9%) versus Y (38.6 +/- 15.8). "Thresholds", that is abrupt increases in RMS slopes, were detected in Y but not in E, suggesting less recruitment or a preferential atrophy of type 2 fibers in the elderly. These findings, associated with the preserved capacity of O2 extraction, suggest a shift towards oxidative metabolism in skeletal muscles of 78 year-old subjects, which could preserve, at least in part, their capacity to carry out exercise.

Genetic variability of human coronavirus OC43-, 229E-, and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients.

In the winter-spring seasons 2003-2004 and 2004-2005, 47 (5.7%) patients with acute respiratory infection associated with human coronavirus (hCoV) 229E-, NL63-, and OC43-like strains were identified among 823 (597 immunocompetent and 226 immunocompromised) patients admitted to hospital with acute respiratory syndromes. Viral infections were diagnosed by either immunological (monoclonal antibodies) or molecular (RT-PCR) methods. Each of two sets of primer pairs developed for detection of all CoVs (panCoV) failed to detect 15 of the 53 (28.3%) hCoV strains identified. On the other hand, all hCoV strains could be detected by using type-specific primers targeting genes 1ab and N. The HuH-7 cell line was found to be susceptible to isolation and identification of OC43- and 229E-like strains. Overall, hCoV infection was caused by OC43-like, 229E-like, and NL63-like strains in 25 (53.2%), 10 (21.3%), and 9 (19.1%) patients, respectively. In addition, three patients (6.4%) were infected by untypeable hCoV strains. NL63-like strains were not found to circulate in 2003-2004, and 229E-like strains did not circulate in 2004-2005, while OC43-like strains were detected in both seasons. The monthly distribution reached a peak during January through March. Lower predominated over upper respiratory tract infections in each age group. In addition, hCoV infections interested only immunocompetent infants and young children during the first year of life, while all adults were immunocompromised patients. Coinfections of hCoVs and other respiratory viruses (mostly interesting the first year of life) were observed in 14 of the 47 (29.8%) patients and were associated with severe respiratory syndromes more frequently than hCoV single infections (P = 0.002). In conclusion, the use of multiple primer sets targeting different genes is recommended for diagnosis of all types of hCoV infection. In addition, the detection of still untypeable hCoV strains suggests that the number of hCoVs involved in human pathology might further increase. Finally, hCoVs should be screened routinely for in both infants and immunocompromised patients with acute respiratory infection.

Detection and pathogenicity of human metapneumovirus respiratory infection in pediatric Italian patients during a winter--spring season.

BACKGROUND: Some diagnostic, epidemiological and clinical features of the recently discovered human metapneumovirus remain to be investigated. OBJECTIVES: To study the best approach for the diagnosis of human metapneumovirus infections by both conventional and molecular methods, along with the human metapneumovirus circulation rate in northern Italy and the severity of human metapneumovirus respiratory infections in a pediatric patient population. STUDY DESIGN: Nasopharyngeal aspirates (NPA) were taken from 306 pediatric patients during the winter-spring season 2003-2004, and examined for conventional respiratory viruses by direct fluorescent staining and cell culture, while human coronavirus and human metapneumovirus were sought by RT-PCR. RESULTS: RT-PCR detected human metapneumovirus in 40/306 (13.1%) children positive for respiratory viruses, with an incidence intermediate between that of respiratory syncytial virus (58 patients, 18.9%) and that of influenzavirus infections (29 patients, 9.5%). Phylogenetic analysis showed cocirculation of both human metapneumovirus types (A and B) as well as their relevant subtypes (A1-A2 and B1-B2). Clinically, human metapneumovirus was found to be second to human respiratory syncytial virus alone, as a cause of respiratory tract infections, while duration of virus excretion appeared to correlate with severity of infection, and virus load in NPA with the stage of respiratory infection. CONCLUSION: (i) Human metapneumovirus is a major viral pathogen in the Italian pediatric patient population; (ii) the severity of lower respiratory tract infections approaches that of human respiratory syncytial virus; (iii) there are preliminary indications that the duration of virus excretion may reach 2-3 weeks and that the level of viral load in NPA correlates with the clinical stage of human metapneumovirus infection.

Web versus press with a high circulation: an Italian case study.

THE PROBLEM: the quality of health-related information available for general public on the web has been widely debated, often expressing concern, in the last few years, but also other mass media supply medical contents for health consumer. THE OBJECTIVE: To evaluate the quality of information available by the Italian people through Internet in comparison with the one published on the press with a high circulation. A general interest topic such as over-weight and obesity has been identified as a convenient field for the comparison. METHODS: A sample of web sites and articles have been collected--excluding the promotional ones- in order to compare their completeness and correctness. National guidelines were the golden standard. For the web sites, the availability of indications to let the user know the reliability of the site--we will refer to these as "transparency"--has been evaluated through 9 parameters. RESULTS: 45 informative sites and 35 articles have been found: the Italian population has a good chance of getting information. Web sites are much more complete than articles. Correctness of both is quite good. Transparency of web sites leaves much to desire, independently from the quality of the content. LESSON LEARNT: There is probably an excessive confidence about the transparency and caution of articles in dealing with health-related topics. CONCLUSION: The web publishers must necessarily be educated to apply the existing good practice rules, but in the meantime also the press with a high circulation, that is read by a high percentage of people, has to improve its quality in order to co-operate in supplying adequate information. In general, from the traditional exceptionalist approach for medical information on the internet, a definition of media quality standard could be derived.

Analysis of secondary V(D)J rearrangements in mature, peripheral T cells of ataxia-telangiectasia heterozygotes.

Ataxia-telangiectasia (AT) is a rare recessive disease with pleiotropic involvement of the nervous and lymphoid systems. AT heterozygotes have a population frequency of about 1%, and although not manifesting any overt clinical symptoms, they have an increased mortality, mainly because of cancer and ischemic heart disease. We and others have described a mature T lymphocyte population with an altered T cell receptor surface expression ("TCR variant") that reactivates the recombination activating genes (RAG) and is expanded in the blood of patients with AT. In view of the known role of V(D)J recombination in the onset of tumorigenic translocations, we proposed that the increased RAG activity was responsible for the predisposition of AT homozygotes to develop mature-type T leukemia/lymphoma. In the present report, we used cytofluorimetry to quantify the TCR variant population and the memory/naïve T-cell compartments in the blood of AT heterozygotes compared with AT patients and controls. We assessed the expression of different recombinase genes through RT-PCR/oligotyping and cytofluorometric analysis and searched for rearrangement intermediates by ligase-mediated PCR in T-cell lines from four heterozygous carriers. We found the TCR variant population was increased on average 2x in AT heterozygotes (vs 10x in homozygotes) compared with controls, and naïve CD4(+) T lymphocytes were reduced on average 0.5x (vs 0.1x in homozygotes). We were able to demonstrate recombinase gene expression in all four heterozygous T-cell lines, and rearrangement intermediates, indicative of ongoing V(D)J recombination, in two. These rearrangements were compatible with V-gene replacement, a mechanism of receptor editing described for Ig and TCRalpha genes, to our knowledge not previously documented for TCRbeta. In conclusion, we found that RAG reactivation and secondary V(D)J rearrangements, potential risk factors of mature-type leukemia in AT homozygotes, also take place in AT heterozygous carriers and might place this large population fraction at an increased risk of leukemia/lymphoma.