Evaluation of Real Time PCR Aspergillus spp. in bronchoalveolar lavage samples.

The present study investigated the improvement in the diagnosis of invasive pulmonary aspergillosis (IPA) adding a molecular test on bronchoalveolar lavage (BAL) to the routine diagnostic approach including microscopy, culture and galactomannan (GM) immunoassay. A total of 133 BAL samples were retrospectively tested for the Aspergillus DNA: 112 samples were from immunocompromised patients at risk of invasive fungal infection and 21 were from patients not at risk and without clinical evidence of IPA. The latter samples were used to identify the cut-off of positivity for the molecular test. Applying the cut-off quantity of 50 copies/reaction, the PCR test had 90% sensitivity and 97% specificity and resulted the most sensitive, specific and accurate among those evaluated. The statistical analysis showed that the probability that a patient is not affected by IPA is 99% when the three tests (PCR, GM and culture) are concordantly negative.

Oxidative status in treatment-naïve essential thrombocythemia: a pilot study in a single center.

Oxidative stress (OS), due to pro-oxidant species [reactive oxygen species (ROS)] excess not counterbalanced by endogenous antioxidant molecules [e.g., reduced glutathione (GSH)], is involved in the pathogenesis of human cancers, but few data are available on essential thrombocythemia (ET). This study aims to investigate OS in ET off-therapy patients. Thirty ET treatment-naïve patients were compared with 26 age-matched and gender-matched controls. Serum ROS, urinary 8-hydroxydeoxyguanosine, full blood GSH levels, and reduced/oxidized GSH ratio (GSH/GSSG) were measured. Data were adjusted for gender, age, JAK2 mutational status, smoking, dyslipidemia, or hypercholesterolemia requiring drug therapy, antiplatelet therapy, treatment with acetylsalicylic acid, high-sensitive C-reactive protein levels, and absolute monocyte count. ROS and GSH levels were increased in both patients and controls. Patients showed increased GSSG (p = 0.05), reduced GSH/GSSG ratio (p = 0.08), and similar 8-hydroxydeoxyguanosine levels when compared with controls. No differences in OS parameters were found between JAK2-positive and JAK2-negative patients. Confounding factors did not modify the results. Our study suggests an OS condition in a cohort of treatment-naïve ET patients, not associated with JAK2 mutational status or with chronic inflammation situation. GSH/GSSG ratio, altered in ET patients because of increased GSSG levels, showed the presence of higher GSH levels in ET than controls as a possible compensatory mechanism of an excess of pro-oxidant production. Copyright © 2015 John Wiley & Sons, Ltd.

Immunosuppressive drugs in whole blood: validation of a commercially available liquid chromatography/tandem mass spectrometry kit and comparison with immunochemical assays.

RATIONALE: In the determination of immunosuppressive drugs cyclosporine A (CSA), tacrolimus (TARO), sirolimus (SIRO), and everolimus (EVE) in whole blood there is an open debate about which is the best assay between immunochemistry and liquid chromatography/tandem mass spectrometry (LC/MS/MS). This work is aimed to explore this topic, focusing on the use of updated assays and the analysis of a large number of samples. METHODS: A certified in vitro diagnostic kit coupled with a medical device LC/MS/MS was validated and applied to the analysis of 1192 blood samples of patients treated with immunosuppressive drugs. The results were compared with those obtained by immunoassays. RESULTS: The LC/MS/MS approach was found to provide linear, stable, precise, and accurate results, with lower limits of quantification of 12.5, 1.1, 1.2, and 1.2 µg/L for CSA, TACRO, SIRO, and EVE, respectively. With this method 80 samples were analysed and reported within a single work shift. A correlation was observed between the LC/MS/MS and immunoassay data, with Spearman correlation coefficients of 0.980 (n = 260) for CSA, 0.836 for TACRO (n = 562), 0.898 for SIRO (n = 113), and 0.904 for EVE (n = 257). Passing-Bablock regression showed the presence of constant and proportional biases for most of the drugs. A Blond-Altman graph showed differences between the assays, with immunoassays generally overestimating the drugs. CONCLUSIONS: The LC/MS/MS certified kit was validated for the detection of immunosuppressant drugs in whole blood and it provided a high-throughput method that is consistent with the requirements of clinical laboratories. The comparison of patient data between LC/MS/MS and up-dated immunoassays shows that a significant discrepancy still exists, especially for CSA and SIRO, confirming the greater specificity associated with use of the LC/MS/MS assay Copyright © 2017 John Wiley & Sons, Ltd.

The diagnostic accuracy of biomarkers for diagnosis of primary biliary cholangitis (PBC) in anti-mitochondrial antibody (AMA)-negative PBC patients: a review of literature.

Primary biliary cholangitis (PBC), also known as primary biliary cirrhosis, is an autoimmune disease of the liver characterized by anti-mitochondrial antibodies (AMA) in 90%-95% of patients. The aim of this study was to evaluate the diagnostic value of several serum biomarkers in patients with PBC but negative for AMA. Some antinuclear antibodies (ANA) pattern, detected by indirect immunofluorescence (IIF), such as multiple nuclear dot (MND) and rim-like patterns are well-known to be specific for PBC. The corresponding nuclear antigens are the components of the nuclear pore complex (Gp210 for rim-like pattern) and Sp100, PML proteins (for MND pattern) detectable by immunoblotting and ELISA methods. More recently, new biomarkers have been evaluated in order to improve the diagnostic sensitivity, such as kelch-like 12 (KLHL12) and hexokinase-1. Considering these different serum biomarkers, studies evaluating their diagnostic role in AMA-negative PBC patients compared to AMA-positive ones and controls were included in this review. Pooled sensitivity and specificity were 37% and 85%, respectively. The overall PPV and NPV mean values were 45% and 83%. Even if all biomarkers are very specific for PBC, the overall sensitivity was poor and therefore is necessary to identify a marker with a greater sensitivity for PBC in AMA-negative patients.

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.

Homocysteine metabolism in children and adolescents with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) belongs to a family of rare heterogeneous, genetic disorders characterized by blistering of the skin and mucous membranes in response to minor mechanical trauma. The involvement of the oral mucosa and oesophagus stenosis is suggested to be responsible for severe nutritional deficiencies, but few studies have till now considered this aspect. This observational study aimed to evaluate homocysteine status in children and adolescents with EB by assessing total plasma homocysteine (tHcy) and metabolically related vitamins (B6, B12, folate) concentrations. METHODS: Twenty EB patients (12 M; age range 0.5-19 years) were evaluated for: plasma tHcy, serum B12 and holotranscobalamin (HoloTC, the active fraction of B12), serum and erythrocyte folate (s-F and Ery-F, respectively), plasma B6 and serum high sensitive C-reactive-protein (hsCRP) levels. Clinical severity was also evaluated through the Birmingham Epidermolysis Bullosa Severity (BEBS) score. A sex and age well-matched population was also enrolled. RESULTS: EB patients showed tHcy levels higher (p = 0.04) and B6 levels lower (p = 0.03) than controls. B12, HoloTC, s-F and ery-F concentrations did not differ between patients and controls. Multiple linear regression analysis showed that tHcy levels were independent of the metabolically related vitamins levels. In addition, serum hsCRP levels were higher in EB patients than in controls (p = 0.003) and correlated negatively with B6 concentrations (r = -0.6; p = 0.009). BEBS score correlated negatively with HoloTC (p = 0.022) and B6 (p = 0.005) levels and positively with age (p = 0.031) and hsCRP levels (p < 0.001). CONCLUSIONS: The assessment of tHcy and metabolically related vitamin levels describes an important aspect of EB patients' nutritional status which can result essential for their long term care. Monitoring B6 levels in EB patients could be particularly important in order to prevent several complications associated with B6 deficiency and to avoid a B6 excess which sustains an inflammatory condition.

Interferon-ribavirin therapy induces serum antibodies determining 'rods and rings' pattern in hepatitis C patients.

A cytoplasmic antigen associated to inosine-5'-monophosphatedehydrogenase 2 eliciting specific antibodies (antirods and rings, RR) has been identified in patients with chronic hepatitis C who were exposed to pegylated interferon (PI) and ribavirin (RBV). The significance of anti-RR in these patients merits to be investigated. Sera from 88 chronic hepatitis C virus (HCV)-infected patients undergoing PI-RBV therapy were analysed for the presence of RR pattern by indirect immunofluorescence on HEp-2 substrate (Inova Diagnostics, San Diego, CA, USA). Anti-RR antibodies developed de novo in 32 patients independently of any demographic and virological feature, but with a significant association with cumulative exposure to PI-RBV (P = 0.0089; chi-square test). RR pattern was significantly more frequent in relapsers than in patients achieving sustained virological response (56% vs 30%; P = 0.0282, chi-square test). Anti-RR titre ranged from 1:80 to 1:1280, but significantly declined following treatment cessation. Anti-RR develop de novo in a substantial proportion of patients exposed to PI-RBV in relation to the duration of treatment exposure. Further investigations are necessary to unravel the mechanisms leading to the formation of these autoantibodies.

An encapsulated juice powder concentrate improves markers of pulmonary function and cardiovascular risk factors in heavy smokers.

OBJECTIVE: Cigarette smoking is associated with reduced pulmonary function and increased risk factors for cardiovascular disease. This randomized placebo-controlled double-blind study evaluated the effects of two different combinations of mixed fruit and vegetable juice powder concentrate (Juice Plus+, NSA, Collierville, TN) on heavy smokers. METHODS: At baseline (T 0) and after 3 months' supplementation (T 1), pulmonary function parameters and cardiovascular risk factors-that is, plasma total homocysteine (tHcy) with related B vitamins and cysteine (tCys) concentrations-were assessed in 75 apparently healthy smokers (aged 49.2 ± 10.6 years, >20 cigarettes/d, duration ≥10 years) randomized into 3 groups: placebo (P), fruit/vegetable (FV) and fruit/vegetable/berry (FVB). RESULTS: T 0: most smokers showed abnormalities in tHcy and tCys concentrations. T 1: respiratory function was unchanged in P and slightly, but not significantly, improved in FV, whereas FVB showed a significant improvement in forced expiratory flow at 25% (FEF25; p < 0.0001 vs P and FV) and significant improvement in CO diffusion lung/alveolar volume (DLCO/VA). FV and FVB (50%) showed significant reduction in tHcy and tCys compared to T 0 ( p < 0.0001) and P ( p < 0.0001). CONCLUSIONS: At T 1, both supplemented groups, but to a greater extent the FVB group, showed improvements in some pulmonary parameters, cardiovascular risk factors, and folate status. The beneficial effects of Juice Plus+ supplementation could potentially help smokers, even if smoking cessation is advisable.

Effects of encapsulated fruit and vegetable juice powder concentrates on oxidative status in heavy smokers.

OBJECTIVE: Long-term cigarette smoking has negative effects on oxidative status, promoting low-density lipoprotein (LDL) oxidation and formation of lipid peroxides. We evaluated the effects of 2 different encapsulated formulas, consisting primarily of mixed juice powder concentrate, on oxidative status compared with placebo. METHODS: This randomized, double-blind, placebo-controlled study was performed on 101 apparently healthy heavy smokers (>20 cigarettes/d, duration >10 years; median age 47 years, range 41-57 years; 54 M) before and after 3 months' supplementation. Subjects were randomized into 3 groups, well matched for sex and age: (1) placebo; (2) fruit/vegetable (FV); and (3) fruit/vegetable/berry (FVB). Analysis of oxidative status was performed on 75 (46 M) compliant subjects (>95% of assigned capsules). Changes in lipid panel parameters, oxidative-INDEX (Oxy-I, calculated on the basis of serum hydroperoxides and total antioxidant capacity measured by spectrophotometric methods), oxidized-LDL (ox-LDL; enzyme-linked immunosorbent assay [ELISA] method), and malondialdehyde (MDA; gas chromatography-mass spectrometry method) in free (fMDA), bound (bMDA), and total (tMDA = fMDA + bMDA) forms are reported. Statistical analysis was performed with R statistical software. RESULTS: After supplementation, compared with placebo, both FV and FVB groups showed a significant decrease in total cholesterol (p < 0.05), ox-LDL (p = 0.03), and fMDA levels (p = 0.004) accompanied by a slight increase in bMDA concentrations, possibly as the result of fMDA conjugation. Moreover, a significant decrease in Oxy-I was found in both active groups compared with placebo (p < 0.001). CONCLUSION: Intervention with both nutraceutical formulations resulted in improvement in some oxidative alterations attributed to long-term cigarette smoking.

Determination of serum holotranscobalamin concentrations with the AxSYM active B(12) assay: cut-off point evaluation in the clinical laboratory.

BACKGROUND: A reliable early marker is required for diagnosis of cobalamin deficiency. We calculated an appropriate holotranscobalamin (HoloTC) cut-off point for identifying cobalamin deficiency using an immunoenzymatic assay. METHODS: Determination of the cut-off threshold and correlation between HoloTC and the other diagnostic parameters routinely used for vitamin B(12) deficiency [total vitamin B(12) (tB(12)), folate, homocysteine] were measured in 250 routine blood specimens from 107 men (mean age 59.0+/-18.8 years) and 143 women (mean age 54.2+/-23.1 years). The inclusion criterion was serum tB(12) concentration

Three-year analysis of repeated laboratory tests for the markers total cholesterol, ferritin, vitamin D, vitamin B12 , and folate, in a large research and teaching hospital in Italy.

RATIONALE, AIMS AND OBJECTIVES: The increasing number of diagnostic tests requests all over the world is a problem that can partially be explained by inappropriate testing. Impact on the total costs of health systems becomes relevant when tests are performed in a large amount. In this paper, retesting of total cholesterol, ferritin, vitamin B12 , vitamin D, and folate is assessed. METHODS: The Quality Unit of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico di Milano (Fondazione) decided to perform a first assessment of the appropriate use of the laboratory tests cholesterol, ferritin, vitamin B12 , vitamin D, and folate focusing on the retesting interval for the same patient in the time period January 1, 2012, to December 31, 2014, in every care setting. The minimum retesting intervals were chosen following the ACB recommendations. The Fondazione is a research and teaching hospital with 3 emergency units (adult, pediatric, and obstetric), kidney, liver, lung, cornea, and bone marrow transplant centers and a medical school. Record linkage of laboratory records selected for the time interval January 1, 2012, to December 31, 2014, was applied using tax code. For each marker, the distribution of retesting intervals was evaluated for every year and the total period. With the same record linkage variable, requests on inpatients were identified from hospital discharge records. A cost analysis of inappropriate retesting was performed for every test. RESULTS: We examined 466 035 requests for 113 019 patients. Proportions of tests judged potentially inappropriate varied between 8.1% for 1,25-dihydroxy vitamin D and 37.1% for total cholesterol. The rates of inappropriate tests from year to year never showed significant decrease, and the maximum increase corresponded to an odds ratio of 1.85 (95% CI, 1.36-2.51) for 1,25-dihydroxy vitamin D from 2012 to 2013. Calculated loss of money was approximately €500 000 in the 3 years. CONCLUSIONS: Inappropriate requests represent a waste of time and money resources. Our analysis highlighted economically unacceptable rates of inappropriate retesting, with no evidence of decreasing trend. Actions to raise awareness in clinicians or automated electronic solutions are necessary to limit unnecessary test repetitions.

Mycoplasma hominis Induces Mediastinitis after a Tonsillar Abscess.

Mycoplasma hominis is commonly involved in genitourinary tract infections. We report a 59-year-old man who developed a M. hominis-associated mediastinitis following acute tonsillar infection.

Oxidative status in different settings and with different methodological approaches compared by Receiver Operating Characteristic curve analysis.

OBJECTIVES: To test the performance of different analytical approaches in highlighting the occurrence of deregulated redox status in various physio-pathological situations. DESIGN AND METHODS: 35 light and 61 heavy smokers, 19 chronic renal failure, 59 kidney transplanted patients, and 87 healthy controls were retrospectively considered for the study. Serum oxidative stress and antioxidant status, assessed by spectrophotometric Reactive Oxygen Metabolites (d-ROMs) and Total Antioxidant Capacity (TAC) tests, respectively, were compared with plasma free (F-MDA) and total (T-MDA) malondialdehyde, both quantified by isotope-dilution-gas chromatography-mass spectrometry (ID-GC-MS). Sensitivity, specificity and cut-off points of T-MDA, F-MDA, d-ROMs and TAC were evaluated by both Receiver Operating Characteristic (ROC) analyses and area under the ROC curve (AUC). RESULTS: Only T-MDA assay showed a clear absence of oxidative stress in controls and significant increase in all patients (AUC 1.00, sensitivity and specificity 100%). Accuracy was good for d-ROMs (AUC 0.87, sensitivity 72.8%, specificity 100%) and F-MDA (AUC 0.82, sensitivity 74.7%, specificity 83.9%), but not high enough for TAC to show in patients impaired antioxidant defense (AUC 0.66, sensitivity 52.0%, specificity 92.9%). CONCLUSIONS: This study reveals T-MDA as the best marker to detect oxidative stress, shows the ability of d-ROMs to identify modified oxidative status particularly in the presence of high damages, and evidences the poor TAC performance. d-ROMs and TAC assays could be useful for routine purposes; however, for an accurate clinical data evaluation, their comparison versus a "gold standard method" is required.

Diagnostic role of copeptin in patients presenting with chest pain in the emergency room.

BACKGROUND: Chest pain is a frequent symptom leading patients to the Emergency Room. Copeptin, the C-terminal fragment of arginin-vasopressin, is a marker of stressful situations. Recent studies showed that normal levels of copeptin combined with normal troponin accurately rule out the diagnosis of acute coronary syndrome (ACS). In this observational, prospective, multicenter study we evaluated if negative levels of copeptin combined with negative troponin (Tn-T) can correctly rule out the diagnosis of ACS and also of other life-threatening causes of chest pain. RESULTS: Of 472 enrolled patients (64.6% males, mean age 60.1yrs), 28 (5.9%) were diagnosed with ST-elevation myocardial infarction (STEMI), 28 (5.9%) with non ST-elevation myocardial infarction (NSTEMI), 43 (9.1%) with unstable angina (UA), 13 (2.8%) with potentially life-threatening non-ACS pathologies (aortic dissection, pulmonary embolism, pulmonary edema, sepsis), 360 (76.2%) with benign causes of chest pain. Copeptin levels were significantly higher in ACS patients with STEMI and NSTEMI than in those with other diagnoses, but not in those with UA. The combination of copeptin and troponin-T attained a negative predictive value of 86.6% for ACS, of 97.9% for other potentially life-threatening non-ACS diseases and of 85% for all potentially lethal diseases (ACS plus others). CONCLUSIONS: The combined use of troponin and copeptin significantly improved the diagnostic accuracy of troponin alone both in ACS (STEMI and NSTEMI) and in other life-threatening diseases. Measurement of this marker might be therefore considered not only for a rule-out strategy but also as a warning sign of a life-threatening disease.

A biomarker of oxidative stress as a nontraditional risk factor in obese subjects.

BACKGROUND: Oxidative stress has been postulated as an additive factor linking obesity to cardiovascular disease. MATERIALS & METHODS: Derivatives of reactive oxygen species metabolites (d-ROMs) were measured in 136 obese (42 males, 94 females; mean age: 47 ± 12 years; BMI: 36 ± 5 kg/m(2)) and in 306 over- and normal-weight subjects (112 males, 194 females; age: 47 ± 12 years; BMI: 24 ± 3 kg/m(2)). RESULTS: d-ROMs levels were higher in obese than in over- and normal-weight subjects (395 ± 104 vs 362 ± 102 and 351 ± 84 arbitrary units (AU); p < 0.001), in women than males (390 ± 104 vs 327 ± 68 AU; p < 0.001), in subjects with than those without hypertension (390 ± 103 vs 360 ± 95 AU; p < 0.01) and in smokers than former and nonsmokers (380 ± 97 vs 358 ± 97 AU; p < 0.05). A positive correlation was found between d-ROMs and BMI (r = 0.25; p < 0.001) and age (r = 0.13; p < 0.01). Levels of d-ROM (>75th percentile: 420 AU) remained as an independent obesity predictor (odds ratio: 2.5; p < 0.001) in women. Continuous variables are reported as mean ± standard deviation. CONCLUSION: d-ROMs are a powerful obesity predictor, and could represent a reliable tool in obesity and cardiovascular risk evaluation, especially in women.