First outbreak of nosocomial Legionella infection in term neonates caused by a cold mist ultrasonic humidifier.

BACKGROUND: To date, all descriptions of legionellosis in neonates have emerged from a small number of isolated case reports in newborns with unusually severe pneumonia. In December 2008, a large outbreak of Legionella infection occurred in term neonates in Cyprus, providing new information on the epidemiological and clinical features of Legionellosis in this age group. METHODS: An environmental investigation was performed at a small private hospital where the infected neonates were delivered. The medical records of the infected neonates were retrospectively reviewed to obtain clinical data on presentation, complications, and course of disease. RESULTS: Nine of the 32 (28%) newborns who were exposed to the contaminated source at the private nursery were infected with Legionella. Six subjects had pulmonary infiltrates, but in 3 cases there were no abnormal radiological findings and clinical presentation was mild. In 4 neonates, pulmonary infiltrates at presentation were bilateral and extensive and 3 died, conferring a mortality rate of 50% in subjects with pulmonary infiltrates and an overall mortality of 33.3%. Legionella pneumophila serogroup 3 was recovered in neonatal biological samples, although in some patients there was implication of a second strain, serogroup 1. It was determined that the neonates were infected while in the nursery at the private hospital by aerosol produced by a recently installed cold-mist humidifier that was filled with contaminated water. CONCLUSIONS: Use of humidifiers in nursery units must be avoided as the risk of disseminating Legionella in neonates is very high. In neonates legionellosis should be suspected when signs of infection first appear and take an unusual course, even when no pulmonary infiltrates appear.

A membrane-based quantitative carrier test to assess the virucidal activity of disinfectants and persistence of viruses on porous fomites.

A membrane-based quantitative carrier test method to assess the virucidal activity of disinfectants and the persistence of viruses on fomites under different environmental conditions is described. The method is based on the inactivation of the virus adsorbed to cellulose ester membranes followed by the direct enumeration of the viruses surviving the treatment without the need of an elution step. The method was suitable for four different human enteroviruses tested. Experiments comparing the infectivity loss of human enteroviruses in suspension or adsorbed to the filters after treatment with chlorine and glutaraldehyde showed that the human enteroviruses tested suffered significantly greater log10 reductions when suspended than when adsorbed. Significant differences in the effect of the disinfectants on the various human enteroviruses tested were also observed. Moreover, the procedure allowed determining the inactivation of viruses on fomites under different environmental conditions. Low temperatures and high relative humidities favored the survival of human enteroviruses. Also, viruses adsorbed to the membranes retained their infectivity frozen at -70 degrees C for more than 1 year, thus providing the possibility of preparing very simple reference materials for testing virucidal activities of antiseptics and disinfectants.

Counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters as a simple method for counting viruses in raw sewage and sewage effluents.

Using the VIRMETADEN (acronym derived from virus adsorption enumeration) method to count cytopathogenic viruses adsorbed to cellulose nitrate membrane filters from prefiltered and decontaminated sewage samples was shown to be feasible. The numbers of naturally occurring enteroviruses recovered by the VIRMETADEN method were significantly higher than those obtained by standard plaque assay. For prefiltration of sewage samples, low protein binding polyvinylidene fluoride (PVDF) membranes of 0.65 and 5 microm pore size proved to be at least as good as prefilters used previously, both in the volume that could be filtered and in the low virus retention. Regarding decontamination, 0.22 microm pore size PVDF membranes were as good as chloroform treatment. Alternatively, decontamination with 0.05% phenol followed by filtration through 0.65 microm PVDF membrane filters proved to be an excellent method for clarifying and decontaminating the sample prior to enumerating viruses using the VIRMETADEN method. It allows good recovery and greater decontaminated sample volumes. The application of VIRMETADEN to decontaminated raw sewage and secondary effluent gave recoveries of approximately 100% of vaccinal polioviruses seeded in sewage. The advantage of this approach is that the same method is applicable to sewage affluents and effluents with a simple and fast method, which implies lower material and labour costs.

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking.

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.