Comparative performance of the Platelia Aspergillus Antigen and Aspergillus Galactomannan antigen Virclia Monotest immunoassays in serum and lower respiratory tract specimens: a "real-life" experience.

The Platelia Aspergillus Antigen immunoassay is the "gold standard" for Aspergillus galactomannan (GLM) measurement in sera and bronchoalveolar lavage (BAL) for the diagnosis of invasive pulmonary aspergillosis (IPA). We evaluated the performance of the Aspergillus GLM antigen Virclia Monotest compared to the Platelia assay. A total of 535 specimens [320 sera, 86 bronchial aspirates (BAs), 70 BAL, and 59 tracheal aspirates (TAs)] from 177 adult patients (72 hematological, 32 Intensive Care Unit, and 73 hospitalized in other wards) were processed for GLM testing upon clinical request. One patient had proven IPA, and 11 had probable disease. After excluding indeterminate Virclia results (n = 38), 396 specimens yielded concordant results (56 positive and 340 negative) and 101 discordant results (Virclia positive/Platelia negative, n = 95). The overall agreement between immunoassays was higher for sera (κ 0.56) than for BAL (κ ≤ 0.24) or BAS and TA (κ ≤ 0.22). When considering all specimen types in combination, the overall sensitivity and specificity of the Virclia assay for the diagnosis of proven/probable IPA were 100% and 65%, respectively, and for the Platelia immunoassay, sensitivity and specificity were 91.7% and 89.4%, respectively. The correlation between index values by both immunoassays was strong for serum/BAL (ρ = 0.73; P < 0.001) and moderate for BAS/TA (Rho = 0.52; P = 0.001). The conversion of Virclia index values into the Platelia index could be derived by the formula y = (11.97 * X)/3.62 + X). Data from GLM-positive serum/BAL clinical specimens fitted the regression model optimally (R2 = 0.94), whereas that of BAS and TA data did not (R2 = 0.11). Further studies are needed to determine whether the Virclia assay may be an alternative to the Platelia assay for GLM measurement in sera and lower respiratory tract specimens.IMPORTANCEGalactomannan detection in serum or bronchoalveolar fluid specimens is pivotal for the diagnosis of invasive pulmonary aspergillosis (IPA). The Platelia Aspergillus Antigen immunoassay has become the "gold standard" for Aspergillus GLM measurement. Here, we provide data suggesting that the Virclia Monotest assay, which displays several operational advantages compared with the Platelia assay, may become an alternative to the Platelia assay, although further studies are needed to validate this assumption. We also provide a formula allowing the conversion of Virclia index values into Platelia values. The study may contribute toward positioning the Virclia assay within the diagnostic algorithm of IPA.

Performance of a MALDI-TOF mass spectrometry-based method for rapid detection of third-generation oxymino-cephalosporin-resistant Escherichia coli and Klebsiella spp. from blood cultures.

We optimized and prospectively evaluated a simple MALDI-TOF MS-based method for direct detection of third-generation oxymino-cephalosporin resistance (3rd CephR) in Escherichia coli and Klebsiella spp. from blood cultures (BC). In addition, we assessed the performance of a lateral flow immunochromatographic assay (LFIC) for detecting extended-spectrum ß-lactamases (ESBL) (NG-Test CTX-M MULTI assay) using bacterial pellets from BC. A total of 168 BCs from unique patients were included. A pre-established volume of BC flagged as positive was transferred in brain heart infusion with or without ceftriaxone (2 mg/ml). After 2-h incubation, intact bacterial pellets were used for MALDI-TOF MS testing. Identification of bacterial species (index score > 2) in the presence of CRO was considered marker of 3rd CephR. The LFIC assay was evaluated in 141 BC. Bacteremia episodes were caused by E. coli (n = 115) or Klebsiella spp. (n = 53). A total of 49 strains were 3rd CephR by broth microdilution, of which 41 were ESBL producers, seven expressed ESBL and OXA-48 type D carbapenemase, and one harbored a plasmid-mediated AmpC. The MALDI-TOF MS method yielded four very major errors (false susceptibility) and two major errors (false resistance). The overall sensitivity of the assay was 91.8% and the specificity 98.3%. Concordance between the LFIC assay and the MALDI-TOF MS method for detection of ESBL-mediated 3rd CephR was 100%. Both evaluated methods may prove useful for early adjustment of empirical therapy in patients with E. coli and Klebsiella spp. bloodstream infections. Whether their use has a beneficial impact on patient outcomes is currently under investigation.

Early adjustment of empirical antibiotic therapy of bloodstream infections on the basis of direct identification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Gram staining results.

INTRODUCTION: To assess the potential added value of rapid MALDI-TOF MS-based identification of bacteria in positive blood cultures to the information provided by Gram staining for adequate empirical antibiotic treatment adjustments in patients with bloodstream infections (BSI). METHODS: We conducted a retrospective, single-center, pre-post quasi-experimental study. In the pre-MALDI-TOF MS phase of the study antibiotic adjustments were made on the basis of Gram stain results, whereas in the MALDI-TOF MS phase they were based on information provided by Gram staining and MALDI-TOF MS results. No antimicrobial stewardship program for BSI was in place within the study period. Antibiotic regimens were categorized as correct, improvable or incorrect. RESULTS: Cohorts were matched for demographics, clinical characteristics of patients and bacterial species involved. Enterobacteriales were the most represented in both study periods (67%), followed by Non-fermenting Gram-negative bacilli and Gram-positive cocci. The number of patients receiving correct, improvable and incorrect empirical antibiotic treatments was comparable for both study periods (P = 0.45, P = 0.57, P = 0.87, respectively). The percentage of patients who ended up receiving correct treatment following modified empirical antibiotic regimens was significantly higher (P = 0.008) in the MALDI-TOF MS phase (27 patients/38.6%) than in the pre-MALDI-TOF MS phase of the study (11 patients/15.7%), although overall adequate coverage of the bacteria causing the infection was comparable across the study periods (90%). CONCLUSION: Gram stain results offer valuable information for early adjustment of empirical antibiotic therapies for BSI. Nevertheless, rapid identification of bacteria involved in BSI by MALDI-TOF MS provides added value to achieve this aim.

Vancomycin MICs and risk of complicated bacteremia by glycopeptide-susceptible Staphylococcus aureus.

Vancomycin (VAN) minimum inhibitory concentrations (MICs) at the upper end of the susceptible range for Staphylococcus aureus (S. aureus), as measured by the Etest method, have been associated with poor clinical outcomes of S. aureus bloodstream infections, as has the isolate's genetic background. Here, we assessed the impact of VAN MICs, as determined by a broth microdilution method (BMD) that incorporates incremental VAN concentrations between the conventional log2 dilutions, isolate susceptibility to killing by human phagocytes, acting as a surrogate marker for bacterial cell wall thickness, and S. aureus genetic composition, on the development of complicated S. aureus bacteremia (SAB). We carried out a retrospective, observational single-center cohort study of 148 consecutive patients with SAB caused by methicillin-susceptible (MSSA) isolates (n = 113) or methicillin-resistant (MRSA) isolates (n = 35). S. aureus isolates were genotyped using a commercially available DNA microarray. Overall, VAN MICs of S. aureus isolates taken from complicated and uncomplicated SAB were comparable, irrespective of the testing method (P = 0.19 with BMD, and P = 0.94 with Etest). Likewise, S. aureus isolates in both comparison groups had the same susceptibility to killing by human phagocytes (P = 0.5). Among the genes screened by the S. aureus DNA array, only Sec and Sel were differentially present among S. aureus isolates in both groups (overrepresented in those causing complications) and their presence was associated independently with complicated SAB in multivariate models adjusted for potentially relevant clinical covariates. Separate analysis of MSSA SAB episodes yielded similar results.

Performance of a Highly Sensitive Mycobacterium tuberculosis Complex Real-Time PCR Assay for Diagnosis of Pulmonary Tuberculosis in a Low-Prevalence Setting: a Prospective Intervention Study.

The potential impact of routine real-time PCR testing of respiratory specimens from patients with presumptive tuberculosis in terms of diagnostic accuracy and time to tuberculosis treatment inception in low-prevalence settings remains largely unexplored. We conducted a prospective intervention cohort study. Respiratory specimens from 1,020 patients were examined by acid-fast bacillus smear microscopy, tested by a real-time Mycobacterium tuberculosis complex PCR assay (Abbott RealTime MTB PCR), and cultured in mycobacterial media. Seventeen patients tested positive by PCR (5 were acid-fast bacillus smear positive and 12 acid-fast bacillus smear negative), and Mycobacterium tuberculosis was recovered from cultures for 12 of them. Patients testing positive by PCR and negative by culture (n = 5) were treated and deemed to have responded to antituberculosis therapy. There were no PCR-negative/culture-positive cases, and none of the patients testing positive for nontuberculous mycobacteria (n = 20) yielded a positive PCR result. The data indicated that routine testing of respiratory specimens from patients with presumptive tuberculosis by the RealTime MTB PCR assay improves the tuberculosis diagnostic yield and may reduce the time to antituberculosis treatment initiation. On the basis of our data, we propose a novel mycobacterial laboratory algorithm for tuberculosis diagnosis.

High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes.

Vancomycin minimum inhibitory concentrations (MICs) at the upper end of the susceptible range for Staphylococcus aureus have been associated with poor clinical outcomes of bloodstream infections. We tested the hypothesis that high vancomycin MICs in S. aureus bacteraemia isolates are associated with increased cell wall thickness and suboptimal bacterial internalisation or lysis by human phagocytes. In total, 95 isolates were evaluated. Original vancomycin MICs were determined by Etest. The susceptibility of S. aureus isolates to killing by phagocytes was assessed in a human whole blood assay. Internalisation of bacterial cells by phagocytes was investigated by flow cytometry. Cell wall thickness was evaluated by transmission electron microscopy. Genotypic analysis of S. aureus isolates was performed using a DNA microarray system. Vancomycin MICs were significantly higher (P=0.006) in isolates that were killed suboptimally (killing index <60%) compared with those killed efficiently (killing index >70%) and tended to correlate inversely (P=0.08) with the killing indices. Isolates in both killing groups were internalised by human neutrophils and monocytes with comparable efficiency. The cell wall was significantly thicker (P=0.03) in isolates in the low killing group. No genotypic differences were found between the isolates in both killing groups. In summary, high vancomycin MICs in S. aureus bacteraemia isolates were associated with increased cell wall thickness and reduced intracellular killing by phagocytes.

Clostridium difficile heterogeneously impacts intestinal community architecture but drives stable metabolome responses.

Clostridium difficile-associated diarrhoea (CDAD) is caused by C. difficile toxins A and B and represents a serious emerging health problem. Yet, its progression and functional consequences are unclear. We hypothesised that C. difficile can drive major measurable metabolic changes in the gut microbiota and that a relationship with the production or absence of toxins may be established. We tested this hypothesis by performing metabolic profiling on the gut microbiota of patients with C. difficile that produced (n=6) or did not produce (n=4) toxins and on non-colonised control patients (n=6), all of whom were experiencing diarrhoea. We report a statistically significant separation (P-value <0.05) among the three groups, regardless of patient characteristics, duration of the disease, antibiotic therapy and medical history. This classification is associated with differences in the production of distinct molecules with presumptive global importance in the gut environment, disease progression and inflammation. Moreover, although severe impaired metabolite production and biological deficits were associated with the carriage of C. difficile that did not produce toxins, only previously unrecognised selective features, namely, choline- and acetylputrescine-deficient gut environments, characterised the carriage of toxin-producing C. difficile. Additional results showed that the changes induced by C. difficile become marked at the highest level of the functional hierarchy, namely the metabolic activity exemplified by the gut microbial metabolome regardless of heterogeneities that commonly appear below the functional level (gut bacterial composition). We discuss possible explanations for this effect and suggest that the changes imposed by CDAD are much more defined and predictable than previously thought.

Antihypertensive activity of angiotensin II AT1 receptor antagonists: a systematic review of studies with 24 h ambulatory blood pressure monitoring.

OBJECTIVE: To perform a systematic review of the antihypertensive activity of the angiotensin II AT1 receptor antagonists (ARB). METHODS: Studies in which blood pressure (BP) was measured using ambulatory BP monitoring for at least 24 h were collected from MEDLINE. Data for each treatment group, ARB, placebo or the drug used for its comparison were obtained from the selected studies. Only studies with a minimum of quality criteria were selected. The final study group contained 36 publications, with a total of 47 patient cohorts receiving ARB in monotherapy, 10 with placebo, 10 with amlodipine, and five with enalapril. The reduction in clinical and ambulatory BP during 24 h, day, night and the last 4-h period for each of the drugs analysed were calculated and adjusted by age, sex, number of participants and by the initial BP level. RESULTS: The global antihypertensive activity of ARB differs from that observed with amlodipine in the sense that the magnitude of the reduction in the BP values does not essentially depend on the initial BP values nor on the dose used. When only ARB were considered, the drug used was a determinant for systolic BP reduction, whereas for diastolic BP the influence was on the BP reduction and the duration of the antihypertensive activity. The dose used had a particular influence on the duration of the antihypertensive activity for both systolic and diastolic BP. CONCLUSION: Among the ARB, the influence is on duration more than on the magnitude of BP reduction. Dose, therefore, is an important factor in the duration of antihypertensive activity.

[Factors related to the risk of thrombosis in patients with lupus and antiphospholipid antibodies]. / Factores relacionados con el riesgo de trombosis en pacientes con lupus y positividad para anticuerpos antifosfolipídicos.

BACKGROUND AND OBJECTIVE: Antiphospholipid antibodies (aPL) are frequently associated with eritematosus systemic lupus (SLE) and increases the risk of thrombosis. The aim of the study was to analize risk factors of thrombosis and its temporal profile in subjects with SLE. PATIENTS AND METHOD: One hundred and two SLE patients -mean age: 37.5 years (range: 8-85); 90 women; mean of follow-up: 72 months (range: 9-324); 41 (40.2%) with aPL positive- were included in the study. Actuarial Kaplan-Meier curves were used to assess the thrombosis risk and Cox proportional hazard model was used to evaluate factors associated with the risk. RESULTS: 13 thrombotic events occurred in the group with aPL positive (mean of follow up: 83.5 months) and 5 events in aPL negative group (mean of follow up: 72 months). The event-rates were 3.93 and 0.96/100 patients/year for each group, respectively. Survival curves showed a significantly higher risk of thrombotic events in the patients with positive aPL as compared to the aPL negative group, and the risk still present throughout the observational time. Activated partial thromboplastine time up to 37 s was significantly associated with thrombosis risk (p = 0.003). Furthermore, positivity of lupus anticoagulant and proteinuria > 2.5 g/day tended to increase thrombotic risk, although they did not achieve statistical significance. CONCLUSIONS: In patients with SLE and aPL, risk of first thrombosis remains over the years, and a large activated partial thromboplastine time was the most important risk factor.

Factores relacionados con el riesgo de trombosis en pacientes con lupus y positividad para anticuerpos antifosfolipídicos / Factors related to the risk of thrombosis in patients with lupus and antiphospholipid antibodies

Fundamento y objetivo: Los anticuerpos antifosfolipídicos (AAF) aparecen con frecuencia asociados al lupus eritematoso sistémico (LES), con lo que aumenta el riesgo de trombosis. El objetivo del presente trabajo ha sido analizar el perfil temporal y los factores de riesgo de accidentes trombóticos en pacientes con LES. Pacientes y método: Se ha realizado un estudio retrospectivo en 102 pacientes con LES controlados en nuestra unidad ­edad media de 37,5 años (extremos, 8-85); 90 mujeres­, con una mediana de seguimiento de 72 meses (extremos, 9-324). Del total de la muestra, 41 (40,2%) presentaban AAF positivos. La probabilidad de presentar un accidente trombótico durante el seguimiento se calculó utilizando el método de Kaplan-Meier, y los factores asociados a este riesgo se determinaron según el método de riesgo proporcional de Cox. Resultados: Se registraron 13 accidentes trombóticos en el grupo de pacientes con AAF positivos (mediana de seguimiento, 83,5 meses) y 5 en el grupo con AAF negativos (mediana de seguimiento, 72 meses). La tasa de aparición de episodios fue de 3,93 y 0,96/100 pacientes/año, respectivamente. Las curvas de riesgo demostraron que los pacientes con LES y AAF tenían un elevado riesgo de trombosis desde el inicio y éste se mantenía a lo largo de los años. La presencia de un tiempo de tromboplastina parcial activada superior a 37 s fue el factor asociado significativamente al riesgo de accidentes trombóticos (test de rangos logarítmicos = 0,003), en estrecha relación con la positividad del anticoagulante lúpico. Los pacientes con proteinuria superior a 2,5 g/día y aquellos con anticoagulante lúpico también mostraron mayor tendencia a presentar trombosis. Conclusiones: En pacientes con LES y AAF positivos el riesgo de un primer episodio trombótico se mantiene sin cambios a lo largo de los años, y la determinación del tiempo de tromboplastina parcial activada fue el factor más determinante para el desarrollo de accidentes trombóticos

Background and objective: Antiphospholipid antibodies (aPL) are frequently associated with eritematosus systemic lupus (SLE) and increases the risk of thrombosis. The aim of the study was to analize risk factors of thrombosis and its temporal profile in subjects with SLE. Patients and method: One hundred and two SLE patients ­mean age: 37.5 years (range: 8-85); 90 women; mean of follow-up: 72 months (range: 9-324); 41 (40.2%) with aPL positive­ were included in the study. Actuarial Kaplan-Meier curves were used to assess the thrombosis risk and Cox proportional hazard model was used to evaluate factors associated with the risk. Results: 13 thrombotic events occurred in the group with aPL positive (mean of follow up: 83.5 months) and 5 events in aPL negative group (mean of follow up: 72 months). The event-rates were 3.93 and 0.96/100 patients/year for each group, respectively. Survival curves showed a significantly higher risk of thrombotic events in the patients with positive aPL as compared to the aPL negative group, and the risk still present throughout the observational time. Activated partial thromboplastine time up to 37 s was significantly associated with thrombosis risk (p = 0.003). Furthermore, positivity of lupus anticoagulant and proteinuria > 2,5 g/day tended to increase thrombotic risk, although they did not achieve statistical significance. Conclusions: In patients with SLE and aPL, risk of first thrombosis remains over the years, and a large activated partial thromboplastine time was the most important risk factor