A metagenomics method for the quantitative detection of bacterial pathogens causing hospital-associated and ventilator-associated pneumonia.

IMPORTANCE: The management of ventilator-associated pneumonia and hospital-acquired pneumonia requires rapid and accurate quantitative detection of the infecting pathogen. To this end, we propose a metagenomic sequencing assay that includes the use of an internal sample processing control for the quantitative detection of 20 relevant bacterial species from bronchoalveolar lavage samples.

Frequently asked questions on seven rare adverse events following immunization.

Routine mass immunization programs have contributed greatly to the control of infectious diseases and to the improvement of the health of populations. Over the last decades, the rise of antivaccination movements has threatened the advances made in this field to the point that vaccination coverage rates have decreased and outbreaks of vaccine-preventable diseases have resurfaced. One of the critical points of the immunization debate revolves around the level of risk attributable to vaccination, namely the possibility of experiencing serious and possibly irreversible adverse events. Unfortunately, the knowledge about adverse events, especially rare ones, is usually incomplete at best and the attribution of a causal relationship with vaccinations is subject to significant uncertainties. The aim of this paper is to provide a narrative review of seven rare or very rare adverse events: hypotonic hyporesponsive episode, multiple sclerosis, apnea in preterm newborns, Guillain-Barré syndrome, vasculitides, arthritis/ arthralgia, immune thrombocytopenic purpura. We have selected these adverse events based on our experience of questions asked by health care workers involved in vaccination services. Information on the chosen adverse events was retrieved from Medline using appropriate search terms. The review is in the form of questions and answers for each adverse event, with a view to providing useful and actionable concepts while not ignoring the uncertainties that remain. We also highlight in the conclusion possible future improvements to adverse event detection and assessment that could help identify individuals at higher risk against the probable future backdrop of ever-greater abandonment of compulsory vaccination policies.

Quadriceps muscles activity during gait: comparison between PFPS subjects and healthy control.

PURPOSE: The purpose of the study was to evaluate if during a common activity as walking, altered quadriceps muscular activity may be present in patellofemoral pain syndrome (PFPS) patients. METHODS: Forty subjects with clinically diagnosed PFPS and forty healthy males matching in age, weight, height and level of sport activity were enrolled in the study. Subjects were asked to walk on an instrumented walking path at their self-selected speed. Force platform and motion tracking system were used for the analysis of the gait. Wireless surface EMG probes were used to evaluate quadriceps muscles activity. Rectus femoris, vastus medialis and lateralis activity percentage, onset and offset time, walking speed, cadence, step length, stride length, knee ROM during gait were measured and reported. Tegner activity questionnaire was reported. RESULTS: Patient group showed a significant increasing in all quadriceps muscles activity when compared to the control (p < 0.05). In particular, for VM and VL muscle onset time was anticipated and offset time was postponed in PFPS group when compared with healthy group (p < 0.05). Knee range of motion during walking was significantly decreased in the patient group. CONCLUSIONS: Young athletes with PFPS showed increased length of quadriceps muscles activity and reduced functional knee Rom while walking, comparing with healthy subjects, in particular muscular onset was anticipated in respect of the loading response event of the gait. Nonetheless, walking parameters were not affected by these alterations.

Nosocomial rotavirus infection: An up to date evaluation of European studies.

Rotavirus (RV) is worldwide considered as the most important viral agent of acute gastroenteritis in children less than 5 y. Since 2006, the availability of anti-RV vaccines has deeply modified the incidence and economic burden of RV infection. In Europe, some countries have introduced an anti-RV vaccination program in the last 10 y. Although community acquired RV (CARV) disease is the most studied condition of RV infection, recently some authors have highlighted the importance of nosocomial RV (nRV) disease as an emerging public health issue. The aim of this review is to summarize the epidemiology of both CARV and nRV, in order to discuss the difficulty of a clear evaluation of the burden of the disease in absence of comparable data. In particular, we focused our attention to European studies regarding nRV in terms of divergences related to definition, report of incidence rate and methodological issues.

Effect of mass media on influenza vaccine coverage in the season 2014/2015: a regional survey in Lazio, Italy.

INTRODUCTION: Adherence to vaccination program for Influenza virus is an important issue of Public Health in presence of many no-vaccine tendencies. The media event about some deaths, occurring after MF59 adjuvanted vaccine administration, has characterized the season 2014/15 vaccination program in Italy. Aim of the study is vaccination adherence assessment of the current season with regards to local health units (LHU) coordinators's perceptions in Lazio Region (IT). METHODS: LHU coordinators's perceptions were collected from a questionnaire that was send via email to the all 12 LHU coordinators. The questionnaire was built with 4 questions concerning the impression about the vaccination adherence of elderly people in the current season. Data from questionnaire was compared with the official coverage rate obtained by the Regional Authority. Severe adverse events were collected by 1 LHU. RESULTS: All the 12 LHU coordinators answered to our questionnaire: 7/12 (50%) predicted a coverage rate of at least 50%; 3/12 (25%) referred a coverage rate around 40-45%; 2/12 (17%) predicted a reduction of 5-10% less than the previous season. Indeed, a mean 49.1% vaccination coverage in the elderly has been reported by the Regional Authority highlighting a reduction of 10% less than the 2013/14 season coverage. No severe adverse events were observed. DISCUSSION: In our survey an important effect of media event on anti-flu vaccination program adherence has been evidenced, with a failure in communication and joint management of Public Health Institutions in Italy about efficacy and safety information of flu vaccine.

Does L-arginine supplementation play a role in cerebral small vessels disease? Implication in the treatment of leukoaraiosis.

Although leukoaraiosis can be considered as a part of the normal aging process, it is strongly associated with stroke, cognitive impairment and other disabilities. The pathogenesis of leukoaraiosis is poorly understood, even if chronic ischemia with consequent arteriolosclerosis probably due to endothelial dysfunction has been suggested. To date, treatment focuses only on prevention of lesion formation and progression by aggressive control of risk factors, beginning at an early age and continuing throughout life. L-Arginine, a semi-essential amino acid, is a precursor of NO in the reaction catalyzed by endothelial nitric oxide synthase and, it has been recently found to importantly influence endothelial function. Arginine supplementation has been demonstrated to be safe and effective therapy for many health conditions, particularly vascular diseases such as intermittent claudication, angina pectoris, erectile dysfunction and MELAS. Thus we hypothesize that, since a lack of endothelium-derived NO may be responsible for several features of LA, long-term administration of high oral doses of L-Arg may slow LA progression and the associated functional impairment.

[Early molecular diagnosis of lung cancer: a method to rule out the disease]. / Diagnosi molecolare precoce del tumore polmonare: un metodo per escludere la malattia.

Aim of this study was the determination of new markers for the diagnosis of lung cancer. 61 patients with non-small cell lung cancer (NSCLC) and 42 controls were enrolled. In the NSCLC patients the following markers were increased: H2O2 in exhaled breath condensate, pentane, hexane, nonenal, trans-2-heptanal, trans-2-nonenal in exhaled breath, while pentanal was decreased. Using multivariate statistical models, a sensitivity of 73.8% and a specificity of 76.8% were calculated. This study shows that with this non-invasive test followed by a most powerful test on positives (e.g. PET) it is possible to decrease the number of false positives.

Autoantibodies recognizing citrullinated rat filaggrin in an ELISA using citrullinated and non-citrullinated recombinant proteins as antigens are highly diagnostic for rheumatoid arthritis.

The objective of the study was to determine the diagnostic value for rheumatoid arthritis (RA) of anti-filaggrin autoantibodies (autoAb) recognizing citrullinated recombinant rat filaggrin (ACRF) in community cases of very early arthritis. To evaluate the diagnostic value of ACRF, were studied sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n= 422) and 314 community cases of very early arthritis (group 2) that were classified as RA (n = 176), non-RA (n = 63) and undifferentiated (n = 75) arthritides after 1 years of follow-up. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on noncitrullinated rat filaggrin (differential ACRF; dACRF). For both groups, rheumatoid factors (RF), anti-keratin autoAb (AKA) and anti-perinuclear factor (APF) were tested; for group 2, anti-CCP autoAb were also tested. Different reactivity patterns against citrullinated and noncitrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not noncitrullinated filaggrin were from RA patients. Among RA and non-RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF sensitivity was 58.4% and 30.7%, and specificity for RA was 99.5% and 98.4%, respectively. In group 2, dACRF specificity for RA was better than that of RF (92.1%), APF (95.2%), AKA (96.8%) and anti-CCP (95.2%). dACRF positive predictive value was high (98.2) and close to that given by the concomitant positivity of RF and anti-CCP autoAb. Despite a high positive correlation between AKA, APF, anti-CCP and dACRF test results, they were complementary since some sera were positive for only one test. Thus, in a community setting, anti-citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serum's reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than other autoAb.

Surveillance for endometrial cancer with transvaginal ultrasonography of breast cancer patients under tamoxifen treatment.

The association of endometrial thickness with the risk of developing endometrial cancer (EC) within 2 years was investigated in a consecutive cohort of 1205 breast cancer patients under tamoxifen treatment, undergoing transvaginal ultrasonography (TVUS) for follow-up purpose (asymptomatic, 1068) or for abnormal uterine bleeding (AUB, 137). Linkage with tumour registry allowed for the follow-up of 3184.3 person-years. According to underlying incidence, 1.85 EC cases were expected in the study cohort while 12 were observed (observed/expected ratio=6.49, 95% CI 3.35-11.33; asymptomatic=4.09, 95% CI 1.65-8.43, symptomatic=35.71, 95% CI 11.59-83.34). No EC was observed with thickness (half layer) <3 mm. Raising this threshold increased specificity with a substantial loss of sensitivity (>or=3, >or=4, >or=6, >or=9 mm; spec.=25.8, 44.5, 76.1, 91.5%, sens.=100, 91.6, 75.0, 66.6%). The presence of AUB was rather specific (88.94%) but poorly sensitive (41.67%). A combination of AUB presence/absence and thickness allowed the best accuracy (AUB + thickness >or=3, >or=4 or >or=5; sens.=100, 81.6 or 91.6%; spec.=22.8, 40.4, or 56.7%). Breast cancer patients under tamoxifen might be selected for further invasive assessment on the basis of AUB and endometrial thickness assessed at TVUS.

Analysis of PSA velocity in 1666 healthy subjects undergoing total PSA determination at two consecutive screening rounds.

The study purpose was to assess PSA velocity (PSAV) in healthy subjects in order to establish a reliable cutoff for the differential diagnosis of prostate cancer in a screening setting. We studied a series of 1666 healthy men aged 55 to 74 years undergoing two total PSA determinations at a four-year interval within a population-based randomized screening trial at the Centro per lo Studio e la Prevenzione Oncologica of Florence. First and second screening round PSA assays (PSA1 and PSA2) were carried out with the same method and by the same laboratory. PSAV (PSA1-PSA2/year) was determined in non-cancer subjects in the overall series or in specific age and PSA subgroups, and in subjects with cancer detected at the second screening round. Average PSAV in 1648 non-cancer subjects was 0.07 ng/mL/year (range -2.18+5.99, 95% CI 0.05-0.09); at least one third of subjects showed a decrease in PSA (negative PSAV), mostly of limited magnitude and in the low PSA range. Average PSAV in the 18 cancer patients was 1.16 ng/mL/year (range 0.10-5.6, 95% CI 0.56-1.77), which is significantly higher (p<0.01) than in non-cancer subjects. None of the cancer patients showed a PSA decrease over time. Whatever cutoff was taken for PSAV, its power to discriminate cancer was limited: in particular the previously used PSAV cutoff of 0.75 ng/mL/year would have included only 42 of the 1648 non-cancer subjects (specificity 97.5%) but excluded eight of the 18 cancer patients (sensitivity 55.5%). At best, with the adopted screening protocol PSAV (cutoff 0.10 ng/mL/year) could have spared 27.9% of non-cancer subjects with PSA > or =2.5 ng/mL further diagnostic assessment and 22.7% of non-cancer subjects with PSA > or =4 ng/mL random sextant biopsy, while missing no cancers. This study provides a reliable estimate of PSAV based on a large unbiased population sample. PSAV is widely variable over time, particularly at low PSA values. PSAV might be of value as an indicator for diagnostic assessment and random sextant biopsy in a screening setting.

Transperineal sonography guided biopsy of the prostate: critical review of 1107 cases.

PURPOSE: To assess the predictive value for a positive biopsy of different indicators (rectal examination, transrectal ultrasonography, total PSA, PSA density). MATERIAL AND METHODS: Positive predictive value was assessed on 1107 consecutive US-guided transperineal biopsies performed from 1991 to 2001 (cancer=344, dysplasia (PIN)=64, atypical hyperplasia=4, benign hyperplasia=686, inadequate=9) with univariate and multivariate analysis. RESULTS: Increasing age (chi square for trend 52.2, p <0.001), positive rectal examination (chi square 233, df=1, p<0.001) or ultrasonography (chi square 191, df=1, p<0.001), total PSA (chi square for trend 68.9, p<0.001) and PSA density (cutoff 0.15, chi square 104, df=1, p<0.001; cutoff 0.20, chi square 104, df=1, p<0,001) were all significantly associated to the likelihood of a positive biopsy outcome. Multivariate analysis stresses the independent role of Psa density over total PSA. If the parameters studied had determined the biopsy, spared benign biopsies [positive rectal examination=505 (66%), positive ultrasonography=467 (61%), PSA>4=124 (16%), PSA>10=159 (74%), PSA density >0,15=426 (62%), PSA density >0.20=517 (75%)] would not have justified the amount of delayed cancer biopsies [positive rectal examination=103 (29%), positive ultrasonography=55 (15%), PSA>4=42 (12%), PSA>10=569 (46%), PSA density >0,15=73 (25%), PSA density >0,20=107 (37%)]. CONCLUSIONS: The parameters currently available prior to biopsy, if used alone, allow no reliable prediction of biopsy outcome. Positive predictive value, particularly for PSA density, allows a better evaluation of biopsy indication, particularly for random sextant biopsies in the 4-10 ng/ml PSA range, which are frequently negative.

Enantiopure 3-(arylmethylidene)aminoxy-2-methylpropionic acids: synthesis and antiinflammatory properties.

Some optically active 3-(arylmethylidene)aminoxy- (3a-g, 4a-g) and fluorenylideneaminoxy-2-methylpropionic acids (5, 6), were prepared as analogues of the antiinflammatory arylpropionic acids of type B, in which the aromatic group is substituted by an MAOM moiety. Some of the new compounds, tested in vivo for their antiinflammatory properties by means of the carrageenan-induced paw edema method in rats, exhibited activity indices similar to that shown in the same test by ibuprofen. Compounds 3a,b and 4a,b, for which at least one of the two enantiomers had shown an inhibition value higher than 40% in the in vivo test, were assayed for their in vitro enzymatic inhibitory activity, showing percentage inhibition values between 40 and 50% at a concentration of 10 microM against COX-2; at the same concentration, they appeared to be devoid of any activity towards COX-1. Compounds 3a,b and 4a,b also proved to possess a similar toxicity. The lack of enantioselectivity shown by compounds 3-6 was tentatively explained in terms of a conformational freedom of the enantiomers which allows their quasi-superimposition.

Predicting prostate biopsy outcome by findings at digital rectal examination, transrectal ultrasonography, PSA, PSA density and free-to-total PSA ratio in a population-based screening setting.

The study offers a retrospective analysis of the positive predictive value (PPV) of several variables, i.e. digital rectal examination (DRE), transrectal ultrasonography (TRUS), PSA value, PSA density (PSAD), and free/total PSA ratio (F/T), for the histologic outcome of 179 prostate biopsies performed within a population-based screening trial. The ratio of spared benign biopsies to missed cancers (SBB/MC) if biopsy results had been decided on the basis of single variables was also evaluated. PPV was 82.9% for DRE, 56.3% for TRUS, 26.6% for PSA (cutoff > or =4 ng/mL), 47.4% for PSA (cutoff > or =10 ng/mL), 42.0% for PSAD (cutoff 0.15), 59.2% for PSAD (cutoff 0.20), 34.9% for F/T (cutoff 0.20) and 40.0% for F/T (cutoff 0.15). SBB/MC was 121/23 for DRE, 96/12 for TRUS, 11/10 for PSA (cutoff > or =4 ng/mL), 107/34 for PSA (cutoff > or =10 ng/mL), 87/23 for PSAD (cutoff 0.15), 109/26 for PSAD (cutoff 0.20), 45/8 for F/T (cutoff 0.20) and 70/14 for F/T (cutoff 0.15). Multivariate analysis of the association with biopsy outcome showed the highest odds ratio for TRUS (13.24, 95% CI=4.4-30.7), and considerably lower values for DRE (4.17, 95% CI=2.0-8-9), PSAD (cutoff 0.20: 3.24, 95% CI=-1.8-5.7) and F/T (cutoff <0.15: 3.16, 95% CI=1.7-1.8). None of the possible variable combinations was clinically useful: the highest PPV (83.3%) was obtained with a combination of suspicious DRE/TRUS, PSAD >0.20 and F/T <0.15, which nevertheless missed 20 of 52 cancers.

Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation.

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site. The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography. Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, which suggested that the O-glycosylations had no major effect on the protein conformation. However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.

Prostate cancer screening: the problem of overdiagnosis and lessons to be learned from breast cancer screening.

Screening for prostate cancer is a relatively new procedure, still under experimental evaluation within prospective randomised trials. The design of prospective studies has been mainly based on the experience of other cancer screenings, particularly breast cancer, for which data of several controlled studies are available. Unfortunately, breast cancer is very different from prostate cancer, particularly for aspects such as early diagnosis and, thus the screening process, originally modelled on the basis of the lesson taught by breast cancer screening, needs continuous re-evaluation and adjustment, based on data which are now being produced from ongoing screening experiences. In this paper, we will consider the most controversial aspects of prostate cancer screening and compare prostate screening with breast cancer screening in order to promote a better understanding of the current problems and lessons to be learned.

Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride.

SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.

Effects of oral administration of heparan sulphate in the rat remnant kidney model.

Heparins are useful for the protection of residual renal function in several nephropathies, but the anticoagulant action and the need of parenteral administration are two main drawbacks limiting their use in chronic renal failure patients. Heparan sulphate (HS) is a heparin-like mucopolysaccharide devoid of anticoagulant action and active orally. In this study, the effects of HS oral administration have been evaluated in 18 subtotally nephrectomized rats;18 untreated remnant kidney rats served as control. No mortality was observed in the HS-treated rats, whereas in the control rats the survival rate was 72.2% at 18 weeks. At the end of the study, HS-treated rats showed lower urinary protein excretion (44 +/- 22 vs. 80 +/- 54 mg/24 h, p < 0.01), lower urea plasma levels (75 +/- 34 vs. 134 +/- 105 mg/dl, p < 0.01) and higher creatinine clearance (66 +/- 15 vs. 47 +/- 21 ml/min. 10(2), p < 0.05) than control rats. Remnant kidney weight (2.3 +/- 1.1 vs. 1.3 +/- 0.2 g, p < 0.01) and heart weight (1.3 +/- 0.2 vs. 1.1 +/- 0.1 g, p < 0.05) were greater in the control than in the HS-treated rats, as well as the systemic blood pressure values (167 +/- 19 vs. 115 +/- 32 mm Hg, respectively, p < 0.001). The remnant kidney histological examination in the HS-treated rats showed a lower prevalence of glomerular sclerosis, mesangial proliferation, and a much less evident tubulointerstitial damage than in controls. The antiproliferative and anti-inflammatory actions of HS together with its protective action on the endothelium are the putative mechanisms that could account for our findings. In conclusion, the present study supports evidence of an antiproteinuric and a renoprotective effect of orally administered HS in subtotally nephrectomized rats. This is in keeping with the well-known effects exerted also by other heparins, but the effectiveness of an orally available heparin-like product in this animal model could suggest the possibility of a clinical use also in progressing chronic renal failure patients.