A brief questionnaire for measuring alarm fatigue in nurses and physicians in intensive care units.

When exposed to hundreds of medical device alarms per day, intensive care unit (ICU) staff can develop "alarm fatigue" (i.e., desensitisation to alarms). However, no standardised way of quantifying alarm fatigue exists. We aimed to develop a brief questionnaire for measuring alarm fatigue in nurses and physicians. After developing a list of initial items based on a literature review, we conducted 15 cognitive interviews with the target group (13 nurses and two physicians) to ensure that the items are face valid and comprehensible. We then asked 32 experts on alarm fatigue to judge whether the items are suited for measuring alarm fatigue. The resulting 27 items were sent to nurses and physicians from 15 ICUs of a large German hospital. We used exploratory factor analysis to further reduce the number of items and to identify scales. A total of 585 submissions from 707 participants could be analysed (of which 14% were physicians and 64% were nurses). The simple structure of a two-factor model was achieved within three rounds. The final questionnaire (called Charité Alarm Fatigue Questionnaire; CAFQa) consists of nine items along two scales (i.e., the "alarm stress scale" and the "alarm coping scale"). The CAFQa is a brief questionnaire that allows clinical alarm researchers to quantify the alarm fatigue of nurses and physicians. It should not take more than five minutes to administer.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

Characterization of the lytic archaeal virus Drs3 infecting Methanobacterium formicicum.

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated "Drs3", as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.

Detection of rat hepatitis E virus in wild Norway rats (Rattus norvegicus) and Black rats (Rattus rattus) from 11 European countries.

Rat hepatitis E virus (HEV) is genetically only distantly related to hepeviruses found in other mammalian reservoirs and in humans. It was initially detected in Norway rats (Rattus norvegicus) from Germany, and subsequently in rats from Vietnam, the USA, Indonesia, China, Denmark and France. Here, we report on a molecular survey of Norway rats and Black rats (Rattus rattus) from 12 European countries for ratHEV and human pathogenic hepeviruses. RatHEV-specific real-time and conventional RT-PCR investigations revealed the presence of ratHEV in 63 of 508 (12.4%) rats at the majority of sites in 11 of 12 countries. In contrast, a real-time RT-PCR specific for human pathogenic HEV genotypes 1-4 and a nested broad-spectrum (NBS) RT-PCR with subsequent sequence determination did not detect any infections with these genotypes. Only in a single Norway rat from Belgium a rabbit HEV-like genotype 3 sequence was detected. Phylogenetic analysis indicated a clustering of all other novel Norway and Black rat-derived sequences with ratHEV sequences from Europe, the USA and a Black rat-derived sequence from Indonesia within the proposed ratHEV genotype 1. No difference in infection status was detected related to age, sex, rat species or density of human settlements and zoological gardens. In conclusion, our investigation shows a broad geographical distribution of ratHEV in Norway and Black rats from Europe and its presence in all settlement types investigated.

Random mutagenesis identifies factors involved in formate-dependent growth of the methanogenic archaeon Methanococcus maripaludis.

Methane is a key intermediate in the carbon cycle and biologically produced by methanogenic archaea. Most methanogens are able to conserve energy by reducing CO2 to methane using molecular hydrogen as electron donor (hydrogenotrophic methanogenesis), but several hydrogenotrophic methanogens can also use formate as electron donor for methanogenesis. Formate dehydrogenase (Fdh) oxidizes formate to CO2 and is involved in funneling reducing equivalents into the methanogenic pathway, but details on other factors relevant for formate-dependent physiology of methanogens are not available. To learn more about the factors involved in formate-dependent growth of Methanococcus maripaludis strain JJ, we used a recently developed system for random in vitro mutagenesis, which is based on a modified insect transposable element to create 2,865 chromosomal transposon mutants and screened them for impaired growth on formate. Of 12 M. maripaludis transposon-induced mutants exhibiting this phenotype, the transposon insertion sites in the chromosome were mapped. Among the genes, apparently affecting formate-dependent growth were those encoding archaeal transcription factor S, a regulator of ion transport, and carbon monoxide dehydrogenase/acetyl-CoA synthase. Interestingly, in seven of the mutants, transposons were localized in a 10.2 kb region where Fdh1, one of two Fdh isoforms in the organism, is encoded. Two transcription start sites within the 10.2 kb region could be mapped, and quantification of transcripts revealed that transposon insertion in this region diminished fdhA1 expression due to polar effects.

The simultaneous occurrence of human norovirus and hepatitis E virus in a Norway rat (Rattus norvegicus).

Wild rats can be reservoirs and vectors for several human pathogens. An initial RT-PCR screening of the intestinal contents of Norway rats trapped in the sewer system of Copenhagen, Denmark, for caliciviruses revealed the presence of a human norovirus in one of 11 rodents. Subsequent phylogenetic analysis of the ~4.0-kb 3'-terminus of the norovirus genome resulted in the identification of a recombinant GI.b/GI.6 strain. The simultaneous detection of hepatitis E virus-like particles in the feces of this rat by transmission electron microscopy was confirmed by RT-PCR and sequence determination, resulting in the identification of a novel rat hepatitis E virus.

Discovery and genetic characterization of novel caliciviruses in German and Dutch poultry.

Caliciviruses (CV) were identified in the intestinal contents of five chickens and one turkey from various regions in Germany between 2009 and 2011 by degenerate reverse transcription PCR. The full 7,656-nt-long genomic sequence of the turkey CV L11043 was determined. Partial nucleotide sequences were determined for nine chicken strains. Phylogenetic analysis based on partial deduced amino acid sequences of the protease and RNA polymerase and the complete VP1 capsid sequence identified two distinct clusters of avian CVs, the first of which contained chicken CVs that were closely related to strains found in German chickens in Bavaria and that had been proposed to form a novel CV genus (proposed name: Bavovirus). In contrast, the turkey CV strain L11043 and three chicken CV strains formed a genetically distinct second cluster. Distance analysis suggested that the strains of the second cluster may represent members of two distinct genogroups of another novel CV genus (proposed name: Nacovirus). Based on the newly obtained sequence information, two real-time RT-PCR assays were developed and used to identify bavovirus and nacovirus in pooled intestinal contents from 24 chicken farms in Germany and the Netherlands. Of these, 20 (83 %) were positive for bavovirus, 11 (46 %) were positive for nacovirus, and nine (38 %) were positive for both bavovirus and nacovirus. Attempts were made to propagate chicken and turkey CVs from both the bavovirus and nacovirus clusters in primary chicken cecal cells, embryonal liver cells and fibroblast cells, but these attempts were not successful.

Genetic characterization of a novel calicivirus from a chicken.

We describe the identification and genetic characterization of a novel enteric calicivirus, detected by transmission electron microscopy and RT-PCR in two clinically normal chickens and in a chicken with runting and stunting syndrome from different flocks in southern Germany. Positive findings were confirmed by sequencing. The complete nucleotide sequence and genome organization of one strain (Bavaria/04V0021) was determined. The genome of the Bavaria virus is 7,908 nt long and contains two coding open reading frames. Phylogenetic analysis of the deduced partial 2C helicase/NTPase, 3C cysteine protease, RNA-dependent RNA polymerase and complete VP1 capsid protein amino acid sequences showed that the virus is genetically related to but distinct from sapoviruses and lagoviruses. Morphologically, the Bavaria virus particles are 37-42 nm in diameter and exhibit characteristic cup-shaped surface depressions.

Viral multiplex quantitative PCR assays for tracking sources of fecal contamination.

Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected humans, pigs, cattle, sheep, deer, and goats, and for the detection of F+ RNA bacteriophage genogroups I to IV, which are associated with human and animal wastes. The sensitivity of this viral toolbox (VTB) was tested against 10-fold dilution series of DNA plasmids that carry the target sequences of the respective viruses and was shown to detect at least 10 plasmid copies for each assay. A panel of human and animal enteric and respiratory viruses showed these assays to be highly sensitive and specific to their respective targets. The VTB was used to detect viruses in fecal and environmental samples, including raw sewage and biosolids from municipal sewage treatment plants, abattoir sewage, and fecally contaminated shellfish and river water, which were likely to contain animal or human viruses.

Molecular detection of norovirus in sheep and pigs in New Zealand farms.

Human norovirus (NoV) is reportedly the major cause of non-bacterial gastroenteritis outbreaks worldwide and is commonly associated with water- and food-borne transmission via the faecal-oral route. Aside from humans, norovirus has been detected in pigs, cattle and mice. The close relatedness of some human and animal noroviruses has raised concerns about potential zoonotic transmission. Our laboratory recently reported the development of a multiplex real-time RT-PCR for the detection and genotyping of norovirus of genogroups I-III. Here we report a study of 56 faecal specimens from pigs and sheep that were collected and screened for noroviruses using this assay. Norovirus was found in 2/23 (9%) of porcine specimens (all were genogroup II) and in 8/33 (24%) of ovine specimens (all were genogroup III). Samples tested positive for norovirus were verified by conventional RT-PCR with different primer sets. Genomes of representative porcine and ovine norovirus strains underwent partial sequence analysis (343 and 2045 bases, respectively). This is the first report describing norovirus in sheep.

Detection and characterization of F+ RNA bacteriophages in water and shellfish: application of a multiplex real-time reverse transcription PCR.

Genotyping of F+ RNA bacteriophages has been used to distinguish between human and animal contributions to contaminated water and food. There are four genetically distinct genogroups of F+ RNA bacteriophages. Genogroups I and IV predominate in animal wastes and genogroups II and III in wastes of human origin. In this study, a multiplex real-time RT-PCR-based method was developed to detect and genotype F+ RNA bacteriophages. The assay was shown to be broadly reactive against a wide spectrum of F+ RNA bacteriophage strains, including MS2, GA, Q beta, MX1, SP and FI, and was able to detect and genotype F+ RNA bacteriophages in shellfish and river water. The assay is highly sensitive, with detection limits <10 PFU/reaction and <10 copies/reaction of the target sequences carried in plasmids, respectively. The applications of this assay include F+ RNA semi-quantitation and microbial source tracking.

Gastroenteritis outbreak caused by waterborne norovirus at a New Zealand ski resort.

In July 2006, public health services investigated an outbreak of acute gastroenteritis among staff and visitors of a popular ski resort in southern New Zealand. The source of the outbreak was a drinking water supply contaminated by human sewage. The virological component of the investigation played a major role in confirming the source of the outbreak. Drinking water, source stream water, and 31 fecal specimens from gastroenteritis outbreak cases were analyzed for the presence of norovirus (NoV). Water samples were concentrated by ultrafiltration, and real-time reverse transcription-PCR (RT-PCR) was used for rapid detection of NoV from both water and fecal samples. The implicated NoV strain was further characterized by DNA sequencing. NoV genogroup GI/5 was identified in water samples and linked case fecal specimens, providing clear evidence of the predominant pathogen and route of exposure. A retrospective cohort study demonstrated that staff who consumed drinking water from the resort supply were twice as likely to have gastroenteritis than those who did not. This is the first time that an outbreak of gastroenteritis in New Zealand has been conclusively linked to NoV detected in a community water supply. To our knowledge, this is the first report of the use of ultrafiltration combined with quantitative real-time RT-PCR and DNA sequencing for investigation of a waterborne NoV outbreak.

Sensitive multiplex real-time reverse transcription-PCR assay for the detection of human and animal noroviruses in clinical and environmental samples.

In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishes between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three genogroups and the first real-time RT-PCR-based method developed for the detection of bovine noroviruses. The assay was shown to be broadly reactive against a wide spectrum of norovirus genotypes, including GI/1 through GI/7, GII/1 through GII/8, GII/10, GII/12, and GII/17, in different matrices (including fecal specimens, treated and raw sewage, source water, and treated drinking water). The assay is highly sensitive, detecting low copy numbers of plasmids that carry the target sequence. A new bovine norovirus, Bo/NLV/Norsewood/2006/NZL, was identified by this assay and was further genetically characterized. The results implicate a broad range of possible applications, including clinical diagnostics, tracing of fecal contaminants, and due to its sensitivity and broad reactivity, environmental studies.