Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

Background: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. Methods: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. Results: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. Conclusion: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis

Studies on the Tannin Decomposing Enzyme of Molds (Part 9) : Acidimetric method for the determination of the enzyme activity

Lymphoproliferative responses (LPRs) to recall antigens (Ags) and human immunodeficiency virus type 1 (HIV-1) Gag and frequencies of circulating HIV-1-specific cytotoxic T lymphocyte precursors (CTLps) were measured in 12 patients undergoing highly active antiretroviral therapy (HAART) after long-standing HIV-1 infection. LPRs to at least 1 recall Ag became detectable or increased in all patients during HAART. No significant LPRs to Gag-p24 were observed, whereas 4 of 8 patients tested presented with Gag-p17-specific LPRs. HIV-1-specific CTLp frequencies became measurable or increased early during therapy in 6 of 10 patients tested and were maintained or decreased thereafter. Increasing HIV-1-specific CTLp frequencies were seen only in association with partial HAART failure in 1 patient. In conclusion, restoration of CD4+ T lymphocyte responsiveness to recall Ags is achieved during HAART. The data provide evidence for limited HIV-1-specific CD4+ memory T cells during advanced HIV-1 infection and suggest that both CD4+ and CD8+ HIV-1-specific T cells are poorly stimulated when viral load is suppresse

Short-term beneficial effects of methylene blue on kidney damage in septic shock patients

Contains fulltext : 71022.pdf (publisher's version ) (Closed access)OBJECTIVE: We previously demonstrated that upregulation of renal inducible nitric oxide synthase (iNOS) is associated with proximal tubule injury during systemic inflammation in humans. In this study we investigated the short-term effect of methylene blue (MB), an inhibitor of the NO pathway, on kidney damage and function in septic shock patients. DESIGN AND SETTING: A prospective clinical study conducted in an intensive care unit. PATIENTS: Nine patients (four men, five women, mean age 71 +/- 3 years) with confirmed or suspected bacterial infection and with refractory septic shock defined as a mean arterial pressure or = 0.2 microg/kg per minute. INTERVENTIONS: A 4 h continuous intravenous infusion of 1 mg/kg MB per hour. MEASUREMENTS AND RESULTS: The urinary excretion of NO metabolites decreased with median 90% (range 75-95%) from baseline to 6 h after MB administration. The first 24 h creatinine clearance improved by 51% (18-173%) after MB treatment but was still strongly impaired. During the first 6 h after the start of MB treatment both the urinary excretion of cytosolic glutathione S-transferase A1-1 and P1-1, markers for proximal and distal tubule damage, respectively, decreased by 45% (10-70%) and 70% (40-85) vs. baseline. After termination of the MB infusion the NO metabolites and markers of tubular injury returned to pretreatment levels. CONCLUSIONS: In septic patients with refractory shock short-term infusion of MB is associated with a decrease in NO production and an attenuation of the urinary excretion of renal tubular injury markers

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 β-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem

While Extended-Spectrum β-Lactamases (ESBL) and AmpC β-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC β-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC β-lactamase, and the plasmid-mediated CMY-2 β-lactamase. Bla CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like β-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli

Myoclonus in comatose patients with electrographic status epilepticus after cardiac arrest: corresponding EEG patterns, effects of treatment and outcomes

Objective: To clarify the significance of any form of myoclonus in comatose patients after cardiac arrest with rhythmic and periodic EEG patterns (RPPs) by analyzing associations between myoclonus and EEG pattern, response to anti-seizure medication and neurological outcome.Design: Post hoc analysis of the prospective randomized Treatment of ELectroencephalographic STatus Epilepticus After Cardiopulmonary Resus-citation (TELSTAR) trial.Setting: Eleven ICUs in the Netherlands and Belgium.Patients: One hundred and fifty-seven adult comatose post-cardiac arrest patients with RPPs on continuous EEG monitoring. Interventions: Anti-seizure medication vs no anti-seizure medication in addition to standard care.Measurements and Main Results: Of 157 patients, 98 (63%) had myoclonus at inclusion. Myoclonus was not associated with one specific RPP type. However, myoclonus was associated with a smaller probability of a continuous EEG background pattern (48% in patients with vs 75% without myoclonus, odds ratio (OR) 0.31; 95% confidence interval (CI) 0.16-0.64) and earlier onset of RPPs (24% vs 9% within 24 hours after cardiac arrest, OR 3.86;95% CI 1.64-9.11). Myoclonus was associated with poor outcome at three months, but not invariably so (poor neurological outcome in 96% vs 82%, p = 0.004). Anti-seizure medication did not improve outcome, regardless of myoclonus presence (6% good outcome in the intervention group vs 2% in the control group, OR 0.33; 95% CI 0.03-3.32).Conclusions: Myoclonus in comatose patients after cardiac arrest with RPPs is associated with poor outcome and discontinuous or suppressed EEG. However, presence of myoclonus does not interact with the effects of anti-seizure medication and cannot predict a poor outcome without false positives.Neurological Motor Disorder

Insights into pathogenic events of HIV-associated Kaposi sarcoma and immune reconstitution syndrome related Kaposi sarcoma

A decrease in the incidence of human immune deficiency virus-associated Kaposi sarcoma (HIV-KS) and regression of some established HIV-KS lesions is evident after the introduction of highly active anti-retroviral treatment (HAART), and is attributed to generalized immune restoration, to the reconstitution of human herpesvirus (HHV)-8 specific cellular immune responses, and to the decrease in HIV Tat protein and HHV-8 loads following HAART. However, a small subset of HIV-seropositive subjects with a low CD4+ T cell count at the time of introduction of HAART, may develop HIV-KS as immune reconstitution inflammatory syndrome (IRIS) within 8 weeks thereafter

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Iatrogenic topiramate poisoning in an ICU patient: Focus on topiramate peak time prolongation

A 35-year-old man with generalized insults was admitted to the intensive care unit because of third-line treatment of persistent epileptic insults with antiepileptic drug therapy. Topiramate was added on top of his outpatient regimen in combination with intravenous antiepileptic drugs. Miscommunication and inappropriate topiramate dosing (2,500 mg twice) resulted in an acute topiramate intoxication. Toxicokinetic assessment showed toxic serum topiramate concentration of 55 mg/L and a dose-dependent shift of peak time t(max). According to our modulations, t(max) follows Y = 0.0009X + 2.65, where X is the topiramate dose. Our results have important implications for effectiveness of gut decontamination modalities