Neurally adjusted ventilatory assist for children on veno-venous ECMO.

NAVA may improve veno-venous ECMO weaning in children. This is a retrospective small series, describing for the first time proof-of-principle for the use of NAVA in children on VV ECMO. Six patients (age 1-48 months) needed veno-venous ECMO. Controlled conventional ventilation was replaced with assisted ventilation as soon as lung compliance improved, and could trigger initiation and termination of ventilation. NAVA was then initiated when diaphragmatic electrical activity (EAdi) allowed for triggering. NAVA was possible in all patients. Proportionate to EAdi (1.8-26 µV), initial peak inspiratory pressures ranged from 21 to 34 cm H2O, and the tidal volume (Vt) from 3 to 7 ml/kg. During weaning, peak pressures increased proportionally to EAdi increase (5.2-41 µV), with tidal volumes ranging from 6.6 to 8.6 ml/kg. ECMO was weaned after a median time of 1.75 days on NAVA. Following ECMO weaning, the median duration of mechanical ventilation, and intensive care unit stay were 4.5 days, and 13.5 days, respectively. Survival to hospital discharge was 100%. In conclusion, combining NAVA to ECMO in paediatric respiratory failure is safe and feasible, and may help in a smoother ECMO weaning, since NAVA allows the patient to drive the ventilator and regulate Vt according to needs.

A multiparametric PCR-based tool for fast detection and identification of spore-forming bacteria in food.

The presence of psychrotrophic or highly thermoresistant spore-forming bacteria in food and feedstuff responsible for food poisoning and spoilage raises major safety and economical issues. The aim of this study was to evaluate the performances of a ready-to-use PCR assay (alternative method) in comparison with the standard microbiological plating method regarding spore-forming bacteria detection in food samples. An overnight sample enrichment was selected to increase sporeformer diversity recovery, spore germination, bacterial growth and favour DNA extraction. A total of 180 sporeformer isolates representing 38 different species and 8 genera were tested in the PCR assays. Inclusivity and exclusivity results ensured specific detection and identification of the majority of targeted genera and species. Validation studies carried on artificially contaminated food samples showed detection of the inoculated contaminants in most cases, with increased detection limit for the alternative method which enabled detection with up 1 spore of B. cereus in 25 g food sample. Using naturally contaminated food samples, standard method comforted the alternative method. In a number of cases, the alternative method was able to identify species not detected with the standard method. In addition, identification and discrimination between the B. cereus group members was possible. Thus, associated to a key element, i.e., the enrichment step, the developed multiparametric PCR-based assays reported in this study provide a fast, sensitive and reliable detection and identification tool for mostly encountered spore-forming food contaminants.

A PCR-based method for monitoring Legionella pneumophila in water samples detects viable but noncultivable legionellae that can recover their cultivability.

Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 10(6) genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 10(5) and 10(2) metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.

Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems.

We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.

An immunomagnetic separation-reverse transcription polymerase chain reaction (IMS-RT-PCR) test for sensitive and rapid detection of viable waterborne Cryptosporidium parvum.

The public health problem posed by the waterborne parasite Cryptosporidium parvum incited the water supply industry to develop very accurate analytical tools able to assess the presence of viable oocysts in drinking water. In this study, we report the development of a viability assay for C. parvum oocysts based on immunomagnetic separation and reverse transcription polymerase chain reaction (IMS-RT-PCR). The detection limit of the IMS-RT-PCR assay, which targets the hsp70 heat shock-induced mRNA, was in the range of ten viable oocysts per 100-l tap water samples. Purified Cryptosporidium parvum oocysts were exposed to heating, freezing and three chemical disinfection treatments namely, chlorination, chlorine dioxide treatment and ozonation under conventional doses used in water treatment plants, then detected by IMS-PCR and IMS-RT-PCR. The results obtained by IMS-PCR showed that none of the treatments had an effect on oocyst detection. The inactivation of oocysts by boiling resulted in no RT-PCR signal. Chlorine as well as chlorine dioxide did not influence oocyst viability as determined by IMS-RT-PCR. Ozone more effectively inactivated oocysts. The IMS-RT-PCR assay in conjunction with IMS-PCR marks the development of a combined detection and viability test which can be used for drinking water quality control as well as for reliable evaluation of treatment efficiency.