Development and validation of a multiplex polymerase chain reaction assay for detection of the five families of plasmid-encoded colistin resistance

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.     

Abnormal activation of autophagy-induced crinophagy in Paneth cells from patients with Crohn's disease

Autophagy-related 16 like-1 (ATG16L-1), immunity-related GTPase-M (IRGM), and nucleotide-binding oligomerization domain-containing 2 (NOD2) regulate autophagy, and variants in these genes have been associated with predisposition to Crohn's disease (CD). However, little is known about the role of autophagy in CD. Intestinal biopsies from untreated pediatric patients with CD, celiac disease, or ulcerative colitis were analyzed by immunohistochemistry and electron microscopy. We observed that autophagy was specifically activated in Paneth cells from patients with CD, independently of mucosal inflammation or disease-associated variants of ATG16L1 or IRGM. In these cells, activation of autophagy was associated with a significant decrease in number of secretory granules and features of crinophagy. These observations might account for the disorganization of secretory granules previously reported in Paneth cells from patients with CD.     

In vitro investigation of fluorescence of carious dentin observed with a Soprolife® camera

Objectives

Our aim was to determine the origin of the red fluorescence of carious dentine observed with the Soprolife® camera.

Methods

We conducted in vitro studies to evaluate the origin of the red fluorescence using acids and matrix metalloproteinase (MMP) to mimic caries and methylglycoxal (MGO) to evaluate the effect of glycation reactions on the red fluorescence. In every step of these models, we detected the changes of dentin photonic response with Soprolife® in daylight mode and in treatment mode. A Raman spectroscopy analysis was performed to determine the variations of the dentin organic during the in vitro caries processes. Raman microscopy was performed to identify change in the collagen matrix of dentine.

Results

The red fluorescence observed in carious dentine using a Soprolife® camera corresponds to the brownish color observed using daylight. Demineralization using nitric acid induces a loss of the green fluorescence of dentine. The red fluorescence of carious dentine is resistant to acid treatment. Immersion of demineralized dentine in MGO induces a change of color from white to orange-red. This indicates that the Maillard reaction contributes to lesion coloration. Immersion of demineralized dentine in an MMP-1 solution followed by MGO treatment results in a similar red fluorescence. Raman microspectroscopy analysis reveals accumulation of AGE's product in red-colored dentine.

Conclusions

Our results provide important information on the origin of the fluorescence variation of dentine observed with the Soprolife® camera. We demonstrate that the red fluorescence of carious dentine is linked to the accumulation of Advanced Glycation End products (AGE).

Clinical relevance

The study provides a new biological basis for the red fluorescence of carious dentine and reinforces the importance of the Soprolife® camera in caries diagnostics.     

Prediction of evolution toward brain death upon admission to ICU in comatose patients with spontaneous intracerebral hemorrhage using simple signs

The aim of the study was to identify the predictors of brain death (BD) upon admission to the intensive care unit (ICU) of comatose patients with spontaneous intracerebral hemorrhage (ICH). Patients admitted in our ICU from 2002 to 2010 for spontaneous ICH and placed under mechanical ventilation were retrospectively analyzed. Of the 72 patients, 49% evolved to BD, 39% died after withdrawal of life support, and 12% were discharged alive. The most discriminating characteristics to predict BD were included in two models; Model 1 contained ≥3 abolished brainstem responses [adjusted odds ratios (OR) = 8.4 (2.4, 29.1)] and the swirl sign on the baseline CT‐scan [adjusted OR = 5.0 (1.6, 15.9)] and Model 2 addressed the abolition of corneal reflexes [unilateral/bilateral: adjusted OR = 4.2 (0.9, 20.1)/8.8 (2.4, 32.3)] and the swirl sign on the baseline CT‐scan [adjusted OR = 6.2 (1.9, 20.0)]. Two scores predicting BD were created (sensitivity: 0.89 and 0.88, specificity: 0.68 and 0.65). Risk of evolution toward BD was classified as low (corneal reflexes present and no swirl sign), high (≥1 corneal reflexes abolished and swirl sign), and intermediate. Simple signs at ICU admission can predict BD in comatose patients with ICH and could increase the potential for organ donation.     

Alterations in the motor neuron–renshaw cell circuit in the Sod1G93A mouse model

Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and cholinergic nicotinic receptor subunit alpha2 [Chrna2]), two general markers for motor neurons (NeuN and vesicular acethylcholine transporter [VAChT]), and two markers for fast motor neurons (Chondrolectin and calcitonin‐related polypeptide alpha [Calca]), we analyzed the survival and connectivity of these cells during disease progression in the Sod1^G93A mouse model. Most calbindin‐immunoreactive (IR) Renshaw cells survive to end stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end stage (Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN‐IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in diminished control of motor neuron firing. J. Comp. Neurol. 521:1449–1469, 2013. © 2012 Wiley Periodicals, Inc.     

Thrombolysis for stroke caused by infective endocarditis: an illustrative case and review of the literature

Infective endocarditis represents a classical contra-indication to thrombolysis for acute ischemic stroke due to a potential increased risk of intracranial hemorrhage. However, some case reports have suggested safety and potential efficacy of intravenous or intra-arterial thrombolysis in stroke related to infective endocarditis. We present a case of ischemic stroke related to infective endocarditis who was treated with intravenous tissue plasminogen activator within the first 3 h of symptoms onset and subsequently developed symptomatic multifocal intracerebral hemorrhages, and summarize currently available data on this issue.     

Trends in prevalence and characteristics of post-neonatal cerebral palsy cases: A European registry-based study

The present paper aims to analyze trends over time in prevalence of cerebral palsy of post-neonatal origin, to investigate whether changes are similar according to severity and to describe the disability profile by etiology. Post-neonatal cases, birth years 1976 to 1998, were identified from the Surveillance of Cerebral Palsy in Europe collaboration (19 population-based registries). A recognized causal event occurring between 28 days and 24 months of age was considered to define the cases. Trends in prevalence were explored using graphical methods (Lowess and Cusum control chart) and modeled with negative binomial regressions. Over the study period, 404 cases were identified as post-neonatal cases (5.5% of the total). Mean prevalence rate was 1.20 per 10,000 live births (95% CI [1.08–1.31]). A significant downward trend was observed (p = 0.001), with an accentuated decrease in the 1990s. The prevalence of severe cases which account for around one third of the total also significantly decreased over time (p < 0.001). In 46% of cases, an infectious aetiology was reported; the corresponding prevalence significantly decreased since 1989. No significant decrease was observed for the rate of cases due to a vascular episode or of traumatic origin. Our results emphasize the need of large population-based surveillance systems to reliably monitor trends in prevalence in rare subgroups of children like those with acquired cerebral palsy. The decrease of the overall prevalence as well as those of the most severe cases may be partly due to public health actions targeted to prevent such events.