Validation of quantitative real-time polymerase chain reaction for detection of Legionella pneumophila in hospital water networks.

BACKGROUND: Rapid monitoring of Legionella pneumophila (Lp) is essential to reduce the risk of Legionnaires' disease in healthcare facilities. However, culture results take at least eight days, delaying the implementation of corrective measures. Here, we assessed the performance of a qPCR method and determined qPCR action thresholds for the detection of Lp in hospital hot water networks (HWNs). METHODS: Hot water samples (N = 459) were collected from a hospital HWN. Lp were quantified using iQ-Check® Quanti real-time PCR Quantification kits (Bio-Rad) and the results were compared with those of culture. qPCR thresholds corresponding to the culture action thresholds of 10 and 1000 cfu/L were determined on a training dataset and validated on an independent dataset. RESULTS: Lp concentrations measured by culture and qPCR were correlated for both the training dataset (Spearman's correlation coefficient ρ = 0.687, P<0.0001) and the validation dataset (ρ = 0.661, P<0.0001). Lp qPCR positivity thresholds corresponding to culture action thresholds of 10 cfu/L was 91 genome units (gu) per litre (sensitivity, 86.4%; negative predictive value - NPV, 93.3%) and that corresponding to culture action thresholds of 1000 cfu/L was 1048 gu/L (sensitivity, 100%; NPV, 100%). CONCLUSION: Detection of Lp by qPCR could be implemented with confidence in hospitals as a complement to culture in the monitoring strategy to speed up the implementation of corrective measures.

Bacterial contamination of organ graft preservation solution and infection after transplantation.

Infectious complications represent a major cause of morbidity and mortality in patients with organ transplantation. Contamination of preservation solution (PS) can lead to life-threatening complications in the recipients. For a 3-year period, we investigated the bacterial contamination of both PSs and graft fragments, recipient infections, and explored the link between them. In total 137 organs were transplanted, and 131 organ and perfusate cultures out of 426 tested (30.8%) gave a positive bacterial culture, mainly with coagulase-negative staphylococci. Overall, 80 recipients out of 137 (58.4%) had at least 1 infection during the 4-month post-graft surveillance period. Twelve recipients had an infection with the same bacterial species that was recovered in the corresponding graft. However, based on pulsed-field gel electrophoresis typing results, only 1 case was very likely cross-transmitted via the transplantation.

Comparison of pulsed-field gel electrophoresis and whole-genome-sequencing-based typing confirms the accuracy of pulsed-field gel electrophoresis for the investigation of local Pseudomonas aeruginosa outbreaks.

AIM: To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model. METHODS: We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline. FINDINGS: PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates without evident epidemiological links grouped by PFGE were separated by cgMLST (16/65 isolates) differentiating cities, suggesting that PFGE should be kept for the investigation of local outbreaks. Importantly, hypermutator isolates still shared the pulsotype with their parents (16/65 isolates), whereas they were not recognized by cgMLST. This shows that PFGE was less affected than WGS-based typing by the accelerated genetic drift that occurs in epidemic P. aeruginosa. CONCLUSIONS: although WGS-based typing has logically become the new reference standard, we show here that the PFGE can be used with confidence for the investigation of local outbreaks caused by P. aeruginosa.

mcr-1 is borne by highly diverse Escherichia coli isolates since 2004 in food-producing animals in Europe.

OBJECTIVES: In November 2015, a plasmid-mediated colistin resistance, MCR-1, was described in animals, food and humans in China, and it was considered as a potential emerging threat to public health. Therefore, we screened for the mcr-1 gene a European collection of colistin-resistant Escherichia coli (n=218) and Salmonella spp. (n=74) isolated from diseased food-producing animals between 2004 and 2014 and characterized the mcr-1-positive clones. METHODS: Screening for mcr-1 gene was performed by PCR on isolates for which inhibition diameter was <15 mm around a 50 µg disk of colistin. Positive E. coli isolates were then characterized by phylogrouping, multilocus sequence typing and pulsed-field gel electrophoresis typing. Antibiotic susceptibility was determined by disk diffusion testing or by broth microdilution. RESULTS: Among the collection, 42 E. coli and three Salmonella spp. were positive for mcr-1, with continuous detection since 2004 mainly from bovine and swine digestive infections. Most of the mcr-1-positive strains were resistant to amoxicillin and cotrimoxazole but remained susceptible to cephalosporins, carbapenems and piperacillin/tazobactam. All but one isolate were resistant to colistin, with a minimum inhibitory concentration of >2 mg/L. Most of the mcr-1-positive E. coli belonged to the phylogroup A with two prevalent clonal complexes, CC10 and CC165, in which sequence type 10 and sequence type 100 were overrepresented and pulsed-field gel electrophoresis typing revealed a high diversity of pulsotypes. CONCLUSIONS: MCR-1 was detected yearly in European food-producing animal since 2004 with a high diversity of pulsotypes supporting the dissemination of mcr-1 via plasmids.

Detection of Escherichia coli sequence type 131 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: implications for infection control policies?

BACKGROUND: Sequence type 131 (ST131) is a predominant lineage among extraintestinal pathogenic Escherichia coli. It plays a major role in the worldwide dissemination of extended-spectrum ß-lactamase (ESBL)-producing E. coli. The ST131 pandemic is mainly the result of clonal expansion of the single well-adapted subclone H30-Rx, which is acquired in hospitals more frequently than other ESBL-producing E. coli clones. AIM: To develop a rapid method using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify ST131 for infection control purposes. METHODS: Peak biomarkers of ST131 were identified from the mass spectrum profiles of 109 E. coli isolates (including 50 ST131 isolates). FINDINGS: The models accurately identified ST131 isolates from mass spectrum profiles obtained with and without protein extraction. CONCLUSIONS: The rapid identification of ST131 isolates with MALDI-TOF MS can be easily implemented in the laboratory, and could help to target infection control measures in patients carrying multi-drug-resistant E. coli that are more likely to spread.

Hospital cross-transmission of extended-spectrum ß-lactamase producing Escherichia coli and Klebsiella pneumoniae.

OBJECTIVES: We had for objective to measure the incidence and the clonal diversity of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum ß-lactamases (ESBL) in order to assess the role of patient stay in amplification of the phenomenon, in our teaching hospital. MATERIAL AND METHODS: We measured the quarterly incidence rates of E. coli and K. pneumoniae producing or not producing ESBL in clinical samples between 1999 and 2010. The incidence of ESBL-producing isolates was season-adjusted. We determined the pulsotype of and identified the ESBL in all non-redundant strains isolated between 2009 and 2010. RESULTS: The incidence for 1000 hospitalization days increased from 0.00 to 0.44 for ESBL-producing E. coli, from 0.012 to 0.24 for ESBL-producing K. pneumoniae, from 1999 to 2010. Fifty-three different clones of E. coli were identified among the 61 genotyped isolates. The 28 K. pneumoniae isolates genotyped clustered into 11 different clones, among which one major epidemic clone that included 18 isolates. Respectively 66 and 75% of E. coli and K. pneumoniae isolates produced a CTX-M group 1 ESBL. CONCLUSION: The hospital seems to play a different role in the amplification of ESBL according to the producing species (K. pneumoniae or E. coli). ESBL-producing E. coli seem to have a limited cross-transmission within the hospital and seem to be added to non-producers. Conversely, ESBL-producing K. pneumoniae seem to be cross-transmitted within the hospital and to replace non-producers.

[Simulation of lung motions using an artificial neural network]. / Utilisation d'un réseau de neurones artificiels pour la simulation des mouvements pulmonaires.

PURPOSE: A way to improve the accuracy of lung radiotherapy for a patient is to get a better understanding of its lung motion. Indeed, thanks to this knowledge it becomes possible to follow the displacements of the clinical target volume (CTV) induced by the lung breathing. This paper presents a feasibility study of an original method to simulate the positions of points in patient's lung at all breathing phases. PATIENTS AND METHODS: This method, based on an artificial neural network, allowed learning the lung motion on real cases and then to simulate it for new patients for which only the beginning and the end breathing data are known. The neural network learning set is made up of more than 600 points. These points, shared out on three patients and gathered on a specific lung area, were plotted by a MD. RESULTS: The first results are promising: an average accuracy of 1mm is obtained for a spatial resolution of 1 × 1 × 2.5mm(3). CONCLUSION: We have demonstrated that it is possible to simulate lung motion with accuracy using an artificial neural network. As future work we plan to improve the accuracy of our method with the addition of new patient data and a coverage of the whole lungs.

Development of a new CBR-based platform for human contamination emergency situations.

In the case of a radiological emergency situation, involving accidental human exposure, it is necessary to establish as soon as possible a dosimetry evaluation. In most cases, this evaluation is based on numerical representations and models of the victims. Unfortunately, personalised and realistic human representations are often unavailable for the exposed subjects. Hence, existing models like the 'Reference Man' representative of the average male individual are used. However, the accuracy of the treatment depends on the similarity of the phantom to the victim. The EquiVox platform (Research of Equivalent Voxel phantom) developed in this work uses the case-based reasoning principles to retrieve, from a set of existing phantoms, the most adapted one to represent the victim. This paper introduces the EquiVox platform and gives the example of in vivo lung monitoring optimisation to prove its efficiency in choosing the right model. It also presents the artificial neural network tools being developed to adapt the model to the victim.