Rapid identification and detection of β-lactamase-producing Enterobacteriaceae from positive blood cultures by MALDI-TOF/MS.

Abstract Objectives Current evidence suggests that early diagnosis of sepsis and timely detection of antimicrobial resistance are crucial to improve mortality rates among patients. The aim of this study was to evaluate a rapid method for the identification of Gram-negative bacteria from positive blood cultures (BCs), combined with the detection of extended spectrum β-lactamases (ESβL) and carbapenemases production, by means of MALDI-TOF/MS analysis. Methods During the study, all BCs positive for Gram-negative rods were selected. Starting from bacterial pellets obtained directly from BC broths, species identification and hydrolysis assays were achieved through MALDI-TOF/MS (Bruker). In particular, we performed a hydrolysis assays of cefotaxime (CTX) and ertapenem (ERT) for the rapid detection of resistance via ESβL and carbapenemases, respectively. These results were compared with the routine workflow, including BC subcultures and confirmation phenotypic methods. Finally, a comparison of the turnaround-time (TAT) between the two protocols was conducted. Results Overall, 185 BCs positive for Enterobacteriaceae were collected. In terms of species identification, we observed a concordance of 95.9% comparing MALDI-TOF/MS results to the subculture-based method. The sensitivity and specificity for CTX hydrolysis assay were 91.1% and 92%, respectively; ERT hydrolysis assay showed a sensitivity of 96.2% and a specificity of 99.2%. The TAT of the proposed MALDI TOF/MS-based protocol was significantly lower compared with the routine workflow (P  Conclusions The proposed protocol can provide reliable bacterial identification and data concerning β-lactam resistance in only 3 hours, positively improving management of patients in terms of antimicrobial stewardship

Rectal screening of carbapenemase-producing Enterobacteriaceae: a proposed workflow

Abstract Objectives Active screening is a crucial element for the prevention of carbapenemase-producing Enterobacteriaceae (CPE) transmission in healthcare settings. Here, we proposed a culture-based protocol for rectal swab CPE screening that combines the detection of CPE and the identification of carbapenamase type. Methods The workflow integrates an automatic digital analysis of selective chromogenic media (WASPLab, Copan), with subsequent rapid tests for the confirmation of carbapenemase production (i.e. detection of Klebsiella pneumoniae KPC-specific peak by MALDI-TOF mass spectrometry; a multiplex immunochromatographic assay identifying the five commonest carbapenemase types). To in-depth evaluate the performance of this protocol, data about 21 162 rectal swabs submitted for CPE screening at the Microbiology of S. Orsola-Malpighi Hospital in Bologna were analyzed. Results Considering its ability to correctly segregate plates with/without Enterobacteriaceae, WASPLab Image Analysis Software showed globally a sensitivity and a specificity of 100% and 79.4%, respectively. Out of the plates with a bacterial growth (n = 901), 76.9% were found positive for CPE by MALDI-TOF (specific KPC-peak for K. pneumoniae) or by the immunochromatographic assay. Only 2.8% of KPC-positive K. pneumoniae strains were missed by the specific MALDI-TOF MS algorithm, being detected by the immunochromatographic assay. The mean turn-around-time needed from the sample arrival to the final report ranged between 18 to 24 hours, with a significant time saving compared to a manual reading. Conclusions This workflow proved to be fast and reliable, being particularly suitable for KPC-K. pneumoniae endemic areas and for high-throughput laboratories

Different patterns of HIV-1 DNA after therapy discontinuation

Background: By persisting in infected cells for a long period of time, proviral HIV-1 DNA can represent an alternative viral marker to RNA viral load during the follow-up of HIV-1 infected individuals. In the present study sequential blood samples of 10 patients under antiretroviral treatment from 1997 with two NRTIs, who refused to continue any antiviral regimen, were analyzed for 16-24 weeks to study the possible relationship between DNA and RNA viral load. Methods: The amount of proviral DNA was quantified by SYBR green real-time PCR in peripheral blood mononuclear cells from a selected group of ten patients with different levels of plasmatic viremia (RNA viral load). Results: Variable levels of proviral DNA were found without any significant correlation between proviral load and plasma HIV-1 RNA levels. Results obtained showed an increase or a rebound in viral DNA in most patients, suggesting that the absence of therapy reflects an increase and/or a persistence of cells containing viral DNA. Conclusion: Even though plasma HIV RNA levels remain the basic parameter to monitor the intensity of viral replication, the results obtained seem to indicate that DNA levels could represent an adjunct prognostic marker in monitoring HIV-1 infected subjects

PI and OPG/RANKL levels in human osteoblast cells

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Evaluation of HIV-DNA and inflammatory markers in HIV-infected individuals with different viral load patterns

Abstract Background: Persistent residual viremia (RV) and low grade inflammation and immune activation have been associated with non-AIDS defining events. The impact of persistent RV and HIV-DNA load on immune activation/ inflammation remains unclear. The purpose of this study was to gain new insights into the relation between viremia, markers of inflammation and HIV-DNA levels. Methods: Three hundred and twenty-one HIV-infected patients were studied. A retrospective analysis of viremia values, prospectively collected for 48 months, was performed. Patients were separated into three groups: 113 TND (Target Not Detected, patients with sustained undetectable viremia); 113 RV (Residual Viremia, patients who had at least three detectable viral load (VL) values <37 copies/ml); 95 LLV (Low Level Viremia, patients with at least two VL values >37 but <200 copies/ml). HIV-DNA, TNF-α, IL-6 and sCD14 were analyzed. Results: HIV-DNA, sCD14 and TNF-α were significantly lower in the TND group than in the RV and LLV groups. In addition, RV patients showed lower levels of HIV-DNA and sCD14 than LLV individuals. HIV-DNA load was not related to markers of inflammation. The ordinal logistic analysis showed that two independent variables were significantly associated with VL pattern: sCD14, HIV-DNA. In addition NRTIs plus NNRTIs and NRTIs plus PIs were negatively associated to VL pattern compared to INI-containing regimen. Conclusions: Persistent undetectable viremia was associated with lower levels of inflammatory markers and HIVDNA. However, the lack of normalization of these biomarkers in the TND group and the fact that HIV-DNA load was not associated with inflammation strongly suggest that other mechanisms play a major role in maintaining inflammation over time

Comparison of the Aptima HIV-1 Quant Dx Assay with the COBAS\uae AmpliPrep/COBAS\uae TaqMan\uae HIV-1 v2.0 Test for HIV-1 Viral Load Quantification in Plasma Samples from HIV-1-Infected Patients.

Background and aims: HIV\u20101 RNA viral load (VL) in plasma samples of HIV\u20101\u2013positive patients is used to assess the level of viral replication, the risk of disease progression, and the response and efficacy to antiretroviral treatment. Knowing the performance of different tests for HIV\u20101 RNA detection is, therefore, important for clinical care. This study compared the performance of the recently introduced Aptima HIV\u20101 Quant Dx assay (Hologic, Inc) and the standard COBAS AmpliPrep/COBAS TaqMan HIV\u20101 v2.0 Test (CAP/CTM2) (Roche Molecular System, Inc) for HIV\u20101 RNA quantitation. Methods: Assay performance was assessed using 335 clinical samples, a standard HIV\u20101 low VL panel, and 2 diluted samples from well\u2010characterized patients infected with different HIV\u20101 subtypes tested in 5 replicates over 3 days. All samples were tested on both assays to evaluate inter\u2010assay agreement, both qualitatively and quantitively. Altogether, we evaluated assay sensitivity, linearity, accuracy, precision, repeatability, and reproducibility. Results: Assay agreement for qualitative results in 335 clinical samples was fair (80.6%). Correlation of quantitative assay results (n = 164) was excellent (R2 = 0.97), with 96.3% of the results within the 95% limit of assay agreement ( 120.42 to +0.86 log), and 98.8% within 1 log of each other. Aptima\u2010HIV\u20101 yielded results, on average, 0.22 log higher than CAP/CTM2. Both assays accurately quantitated the HIV\u20101 standard at low VL (R2 65 0.94), with all samples within 0.5 log of the target. Conclusion: Aptima\u2010HIV\u20101 assay demonstrated sensitivity, accuracy, reproducibility, and precision for the detection and quantitation of HIV\u20101 RNA across a wide dynamic range of VLs. Its performance, together with full automation and high throughput, suggests that Aptima\u2010HIV\u20101 could be a suitable assay for reliable monitoring of HIV\u20101 VL in patients undergoing treatment

Serum antibody reactivity to human intracisternal A-type particle retrovirus proteins in systemic sclerosis patients.

Serum antibodies against human intracisternal A-typeparticle (HIAP) endogenous retrovirus have been foundto be associated with various autoimmune pathologies.To evaluate the presence of serum antibody reactivityto HIAP proteins in systemic sclerosis, a Western blotanalysis was performed on sera from 42 patients withsystemic sclerosis, in comparison with 18 sera frompatients with primary biliary cirrhosis and 52 healthysubjects. A positive Western blot was found in 55.5% ofserum samples from patients with primary biliarycirrhosis and in 66.0% of patients with systemic sclerosis.None of the 52 healthy subjects showed positive results.Although this difference may be attributable either to anautoimmune response to antigenically related cellularproteins or to a specific antibody response to HIAPproteins expressed as an incidental consequence ofattendant pathological processes, the high prevalence ofantibodies against HIAP proteins demonstrated inpatients with systemic sclerosis may be considered ahallmark of this disease.(Accepted November 10, 2003.)Acta Derm Venereol 2004; 84: 177–180.Dr Michelangelo La Placa, Department of Clinicaland Experimental Medicine, Section of Dermatology,Via Massarenti, 1, 40138 Bologna, Italy. E-mail:[email protected] sclerosis (SSc) is a connective tissue diseasecharacterized by excessive deposition of collagen inthe skin and various internal organs, and by vascularabnormalities (1). SSc is considered to be an auto-immune disease. Although both cellular microchimer-ism (2) and molecular mimicry of some commoninfectious agents, such as cytomegalovirus and parvo-virus B19 (3), have been implicated in its pathogenesis,the aetiology of SSc remains unknown.Several publications have described the presence ofretroviral sequences associated with virions, producedby cells of patients with autoimmune diseases. In recentreviews (4, 5), these viruses, identified as human endo-genous retroviruses (HERVs), have been associatedwith Sjo¨grens's syndrome, type 1 or insulin-dependentdiabetes mellitus, multiple sclerosis, rheumatoid arthri-tis, congenital heart block, systemic lupus erythemato-sus and SSc. Serum antibodies specific for humanintracisternal A-type particles (HIAP), a HERV recog-nized by monoclonal antibody against HIV-1 p24capsid protein (6), have been found in primary biliarycirrhosis (PBC) (7), familial erosive arthritis (8) andsome patients with SSc, systemic lupus erythematosus,Still's disease and idiopathic T-lymphocytopenia (9,10).To further investigate serum antibody reactivity toHIAP proteins in SSc, we performed a Western blotanalysis of a substantial number of sera from SScpatients, in comparison with sera from PBC patientsand healthy subjects.MATERIALS AND METHOD

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients

BACKGROUND: The routine determination of drug resistance in newly HIV-1 infected individuals documents a potential increase in the transmission of drug-resistant variants. Plasma samples from twenty seven therapy naive HIV-1 infected Italian patients were analyzed by the line probe assay (LIPA) and the TruGene HIV-1 assay for the detection of mutations conferring resistance to HIV-1. RESULTS: Both tests disclosed amino-acid substitutions associated with resistance in a variable number of patients. In particular, two mutations (K70R and V118I), detectable by LIPA and by sequencing analysis respectively, revealed resistance to NRTIs in two plasma samples. At least three mutations conferring resistance to NNRTIs, not detectable by commercial LIPA, able to reveal mutations associated only with nucleoside reverse transcriptase analogues, were disclosed by viral sequence analysis. Moreover, most samples showed mutations correlated with resistance to protease inhibitors. Remarkably, a key mutation, like V82A (found as a mixture), and some "indeterminate" results (9 samples), due the absence of signal on the lines corresponding to a specific probe, was revealed only by LIPA, while a variable number of secondary mutations was detectable only by TruGene HIV-1 assay. CONCLUSION: Even if further studies are necessary to establish the impact of different tests on the evaluation of drug-resistant strains transmission, LIPA might be useful in a wide population analysis, where bulk results are needed in a short time, while sequencing analysis, able to detect mutations conferring resistance to both NRTIs and NNRTIs, might be considered a more complete assay, albeit more expensive and more technically complex

Universal and Specific Services for University Students with Specific Learning Disabilities: The Relation to Study Approach, Academic Achievement, and Satisfaction

In recent years, an increasing number of students with specific learning disabilities (SLDs) have enrolled in universities. The present exploratory study examined the frequency of use and appreciation of universal (open to every student) and specific services (offered to students with SLDs) and their relation to age, academic achievement, satisfaction, self-efficacy, and use of self-regulated learning (SRL) strategies. Participants were 147 Italian university students with SLD diagnoses (42 males; mean age: 22.49, SD = 3.29). Results showed that, overall, the frequency of use and appreciation of specific services were positively related to academic satisfaction, self-efficacy, and SRL strategies. Furthermore, frequency of use of compensatory tools and dispensatory measures was positively associated with academic achievement. These findings suggest that universities play an important role in supporting students with SLDs during their academic years by providing them with useful services and accommodations