Hospital-acquired rotavirus and norovirus acute gastroenteritis in a pediatric unit, in 2014-2015.

The occurrence of hospital-acquired acute gastroenteritis (AGE) is a major concern for public health. RotavirusA (RVA) and norovirus (NoV) are common causes of viral AGE in the pediatric population, and their role in nosocomial infections has been proven, remaining poorly investigated. To investigate RVA and NoV in hospital-acquired AGE, 55 stool samples from children with nosocomial AGE were collected between May 2014 and May 2015. To evaluate virus spreading routes, 51 environmental swabs were collected from staff and patients' rooms. Stools were tested for both RVA and NoV RNA by reverse-transcription-PCR. Nucleotide sequencing and phylogenetic analysis were performed to characterize the viruses. Forty-seven of 55 cases analyzed resulted positive for RVA. The predominant genotype was G4P[8] (18/55) followed by G1P[8] (14/55). Mixed RVA infections were also detected (7/55). Twenty-two samples were positive for NoV, and GII.4 was revealed to be the predominant genotype. Seventeen samples were positive for both RVA and NoV. This study aimed to evaluate the burden of norovirus and rotavirus nosocomial AGE, contributing to identify the environment source of infections and to activate effective strategies for intervention. The reduction in nosocomial AGE cases is an important aspect, considered the worsened disease course in transplant, cancer, and intensive care unit inpatients.

Capture and concentration of viral and bacterial foodborne pathogens using apolipoprotein H.

The need for improved pathogen separation and concentration methods to reduce time-to-detection for foodborne pathogens is well recognized. Apolipoprotein H (ApoH) is an acute phase human plasma protein that has been previously shown to interact with viruses, lipopolysaccharides (LPS) and bacterial proteins. The purpose of this study was to determine if ApoH was capable of binding and efficiently capturing two representative human norovirus strains (GI.1 and GII.4), a cultivable surrogate, and four bacterial pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus). Experiments were carried out using an ApoH-conjugated magnetic bead-based capture followed by pathogen detection using nucleic acid amplification. For all three viruses studied, >10% capture efficiency (<1 Log10 loss in RT-qPCR amplifiable units) was observed. The same capture efficiencies were observed for the bacterial pathogens tested, with the exception of E. coli O157:H7 (approximately 1% capture efficiency, or 2 Log10 loss in CFU equivalents). The efficiency of the capture steps did not vary as a consequence of input target concentration or in the presence of an abundance of background microflora. A complementary plate-based capture assay showed that ApoH bound to a variety of human norovirus virus-like particles. ApoH has the potential to be a broadly reactive ligand for separating and concentrating representative foodborne pathogens, both bacteria and viruses.

Disease course and viral shedding in experimental Norwalk virus and Snow Mountain virus infection.

Norovirus is the most common cause of acute infectious gastroenteritis, causing approximately 21 million cases annually in the USA. The virus is highly contagious and resistant to decontamination, making outbreaks difficult to control. To facilitate the development of better control methods, this study characterized the viral shedding patterns in stools from subjects experimentally infected with genogroup I or II norovirus. Viral stool titers were determined by quantitative real-time RT-PCR for all stools produced in the first 7 days post-challenge and representative stools through day 35 post-challenge. The shedding titers and disease course were analyzed with respect to virus type, illness, and subject demographics. Infection with GII.2 Snow Mountain (SMV) resulted in more symptoms and a higher frequency of painful symptoms compared to GI.1 Norwalk (NV) infection. However, NV infection produced stool viral titers approximately 2 logs higher than those seen in SMV infections. Both NV and SMV were shed in stools for up to 3 weeks after the resolution of symptoms, but long shedding durations were more common in NV infections. For each challenge virus, shedding titers and patterns were not correlated with subject demographics or clinical course. This is the first study to report shedding dynamics in experimental GII norovirus infection.

Laboratory evidence of norwalk virus contamination on the hands of infected individuals.

Human norovirus (NoV) outbreak investigations suggest that the hands of infected individuals play an important role in NoV transmission. However, there is no experimental evidence documenting the likelihood and degree of NoV contamination on hands. As part of a clinical trial designed to evaluate the efficacy of high-pressure processing for Norwalk virus (NV) inactivation in oysters, 159 hand rinse samples were collected from 6 infected and 6 uninfected subjects. NV was concentrated from the samples by polyethylene glycol precipitation, followed by RNA extraction using an automated guanidinium isothiocyanate-silica method. NV RNA was detected and quantified using multiple NV-specific reverse transcription-quantitative PCR (RT-qPCR) assays. A total of 25.4% (18/71) of the hand rinse samples collected from 6 infected volunteers were presumptively positive for NV, with an average of 3.86 log10 genomic equivalent copies (GEC) per hand. Dot blot hybridization of PCR products obtained using a different primer set, and DNA sequencing of selected amplicons, provided further confirmation of the presence of NV in the hand rinses. NV contamination was also detected in two hand rinse samples obtained from one uninfected subject. These findings provide definitive evidence of NV contamination on the hands of infected subjects observed under controlled clinical research conditions. Such data support the need for better hand hygiene strategies to prevent NoV transmission.

Epidemiology of norovirus gastroenteritis outbreaks in two primary schools in a city in eastern China.

We describe the epidemiology of 2 outbreaks of norovirus (GII) gastroenteritis in elementary schools in a city in eastern China using data from field investigations, pathogen testing, and face-to-face interviews. The transmission shows a point source type. In a case-control study, we identified airborne and person-to-person transmission as the source of the outbreaks.

Norovirus infectivity in humans and persistence in water.

To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log(10) reduction throughout 427 days and a significant 1.10-log(10) reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.).

Quantification of Norwalk virus inocula: Comparison of endpoint titration and real-time reverse transcription-PCR methods.

Human noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis. In order to fully characterize features such as persistence and infectious dose, precise quantification of virus concentration is necessary. The purpose of this study was to compare two methods [endpoint titration RT-PCR and quantitative RT-PCR (RT-qPCR)] with respect to quantification of Norwalk virus (NV) in inocula made from purified stock suspensions of human fecal specimens. A full-length NV RNA transcript was developed to facilitate quantification using RT-qPCR and provided log linear detection in the range of 49-4.9 x 10(4) genome equivalent copies (GEC) per reaction. Endpoint titration RT-PCR was used to estimate PCR detection units, and RT-qPCR was used to estimate genome copies in two NV inocula (8fIIa and 8fIIb) used in previous human challenge studies. Overall, RT-qPCR was 1.1-1.6 log(10) more sensitive (lower detection limit) than endpoint titration RT-PCR when the same RNA release method, PCR primers and thermocycle program were used. These findings have important implications for many experimental interpretations, not the least of which is estimating the median infectious dose in human challenge studies.

Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples.

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

Environmental health aspects of drinking water-borne outbreak due to karst flooding: case study.

Climate change may increase the incidence of waterborne diseases due to extreme rainfall events, and consequent microbiological contamination of the water source and supply. As a result of the complexity of the pathways from the surface to the consumer, it is difficult to detect an association between rainfall and human disease. The water supply of a Hungarian city, Miskolc (174,000 inhabitant), is mainly based on karstic water, a vulnerable underground water body. A large amount of precipitation fell on the catchment area of the karstic water source, causing an unusually strong karstic water flow and flooding, and subsequent microbiological contamination. The presence of several potential sources of contamination in the protective zone of the karstic water source should be emphasized. The water supplier was unprepared to treat the risk of waterborne outbreak caused by an extreme weather event. Public health intervention and hygienic measures were taken in line with epidemiological actions, focusing on the protection of consumers by providing safe drinking water. The contamination was identified, and measures were taken for risk reduction and prevention. This case study underlines the increasing importance of preparedness for extreme water events in order to protect the karstic water sources and to avoid waterborne outbreaks.

Effectiveness of liquid soap and hand sanitizer against Norwalk virus on contaminated hands.

Disinfection is an essential measure for interrupting human norovirus (HuNoV) transmission, but it is difficult to evaluate the efficacy of disinfectants due to the absence of a practicable cell culture system for these viruses. The purpose of this study was to screen sodium hypochlorite and ethanol for efficacy against Norwalk virus (NV) and expand the studies to evaluate the efficacy of antibacterial liquid soap and alcohol-based hand sanitizer for the inactivation of NV on human finger pads. Samples were tested by real-time reverse transcription-quantitative PCR (RT-qPCR) both with and without a prior RNase treatment. In suspension assay, sodium hypochlorite concentrations of >or=160 ppm effectively eliminated RT-qPCR detection signal, while ethanol, regardless of concentration, was relatively ineffective, giving at most a 0.5 log(10) reduction in genomic copies of NV cDNA. Using the American Society for Testing and Materials (ASTM) standard finger pad method and a modification thereof (with rubbing), we observed the greatest reduction in genomic copies of NV cDNA with the antibacterial liquid soap treatment (0.67 to 1.20 log(10) reduction) and water rinse only (0.58 to 1.58 log(10) reduction). The alcohol-based hand sanitizer was relatively ineffective, reducing the genomic copies of NV cDNA by only 0.14 to 0.34 log(10) compared to baseline. Although the concentrations of genomic copies of NV cDNA were consistently lower on finger pad eluates pretreated with RNase compared to those without prior RNase treatment, these differences were not statistically significant. Despite the promise of alcohol-based sanitizers for the control of pathogen transmission, they may be relatively ineffective against the HuNoV, reinforcing the need to develop and evaluate new products against this important group of viruses.

Brotes por norovirus en residencias y centros sanitarios de Cataluña / Norovirus outbreaks in hospitals and nursing homes in Catalonia, Spain

Fundamentos. La baja dosis infectiva y las múltiples víasde transmisión facilitan la presentación de brotes de norovirus.El objetivo fue comparar la incidencia de brotes por norovirusen hospitales y residencias en Cataluña.Métodos. Se realizó un estudio descriptivo de la serie debrotes de norovirus en el período del 15/10/2004 al 30/10/2005.Se rellenó una encuesta epidemiológica para cada brote. Lasvariables fueron: número de expuestos, enfermos, mecanismo detransmisión, ámbito (centros sanitarios o residencias), regiónsanitaria, mes del año, y duración. Mediante técnicas de PCR seinvestigó la presencia de norovirus en muestras clínicas. Se calculóla incidencia en cada centro y la incidencia anual de brotespor centros. Las diferencias se determinaron con la prueba de c2y la t de Student con un grado de significación (p) inferior a 0,05.Resultados. Se detectaron 17 brotes, 6 en centros sanitariosy 11 en residencias. La tasa de ataque global fue del33,4% (652/1951) y fue ligeramente superior en las residencias(35,2%) que en los centros sanitarios (31,4%).El 94,1% (16/17) de los brotes se produjeron por transmisiónpersona a persona y sólo el 5,9% (1/17) por alimentos. Lamedia de días entre el primer y último caso del brote fue de11,4 (DE = 6,9).La duración media de los síntomas fue de 2,39 días(SD=1,6) y también fue superior en los pacientes hospitalizados2,63 (SD=1,7) en comparación a los pacientes de residencias1,97 (SD=1,7) (p < 0,0001).Conclusiones. Norovirus es responsable de un númeroimportante de brotes por transmisión persona a persona. Se debeprotocolizar su control para reducir su número y su duración(AU)

Background. The low infectious dose and multipletransmission routes favour the appearance of norovirusoutbreaks. The objective of this study was to compare theincidence of norovirus outbreaks in hospitals and nursinghomes in Catalonia.Methods. A descriptive study of norovirus outbreaksbetween 15/10/2004 and 30/10/2005 was carried out. Anepidemiological survey was completed for each outbreak.Norovirus in clinical samples was determined by PCRtechniques. The incidence in each centre and the annualincidence of outbreaks by centre were calculated. Differenceswere calculated using the chi-square test and the Student’s ttest, taking a p value of > 0.05 as significant.Results. Seventeen outbreaks (6 in hospitals and 11 innursing homes) were detected. The global attack rate was33.4% (652/1951) and was slightly higher in nursing homes(35.2%) than in hospitals (31.4%).A total of 94.1% (16/17) of outbreaks were caused byperson-to-person transmission and only 5.9% (1/17) by foods.The mean number of days between the first and last case was11.4 (SD = 6.9).The mean duration of symptoms was 2.39 days (SD=1.6),and was higher hospitals, 2.63 (SD=1.7), than in nursinghomes, 1.97 (SD=1.7) (p < 0.0001).Conclusions. Norovirus is responsible for a large numberof outbreaks due to person-to-person transmission. Controlshould be standardized to reduce the number and duration ofoutbreaks(AU)

[Rapid detection of norwalk GII with reverse transcription loop-mediated isothermal amplification].

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.

Norwalk virus shedding after experimental human infection.

Noroviruses are the most common cause of viral gastroenteritis in the United States. To determine the magnitude and duration of virus shedding in feces, we evaluated persons who had been experimentally infected with Norwalk virus. Of 16 persons, clinical gastroenteritis (watery diarrhea and/or vomiting) developed in 11; symptomatic illness lasted 1-2 days. Virus shedding was first detected by reverse transcription-PCR (RT-PCR) 18 hours after participant inoculation and lasted a median of 28 days after inoculation (range 13-56 days). The median peak amount of virus shedding was 95 x 10(9) (range 0.5-1,640 x 10(9)) genomic copies/g feces as measured by quantitative RT-PCR. Virus shedding was first detected by antigen ELISA approximately 33 hours (median 42 hours) after inoculation and lasted 10 days (median 7 days) after inoculation. Understanding of the relevance of prolonged fecal norovirus excretion must await the development of sensitive methods to measure virus infectivity.

Inactivation of norovirus by chlorine disinfection of water.

In an effort to validate previous research suggesting remarkable resistance of norovirus to free chlorine disinfection, we characterized the disinfection response of purified and dispersed Norwalk virus (NV) by bench-scale free chlorine disinfection using RT-PCR for virus assays. The inactivation of NV by two doses of free chlorine (1 and 5mg/L) at pH 6 and 5 degrees C based on two RT-PCR assays was similar to that of coliphage MS2, but much faster than that of poliovirus 1. Despite the underestimation of virus inactivation by RT-PCR assays, the predicted CT values for NV based on RT-PCR assays are lower than the ones for most other important waterborne viruses and the CT guidelines for chlorine disinfection of viruses under the Surface Water Treatment Rule by the United States Environmental Protection Agency. Overall, the results of this study indicate that NV is not highly resistant to free chlorine disinfection as suggested by previous research and it is likely that NV contamination of drinking water can be controlled by adequate free chlorine disinfection practices with provision of proper pre-treatment processes before chlorination.

Histo-blood group antigen assay for detecting noroviruses in water.

We evaluated a novel, magnetic-bead-based histo-blood group antigen assay for the recovery of low numbers of norovirus particles. Using this assay, with Norwalk virus seeded in environmental waters as a model, we were able to recover 30 to 300 genomic copies of the virus.

Norwalk virus: how infectious is it?

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in Norwalk virus infectivity, as well as the degree of virus aggregation, consistent with independent (electron microscopic) observations. Explicit modeling of viral aggregation allows us to express virus infectivity per single infectious unit (particle). Comparison of a primary and a secondary inoculum showed that passage through a human host does not change Norwalk virus infectivity. We estimate the average probability of infection for a single Norwalk virus particle to be close to 0.5, exceeding that reported for any other virus studied to date. Infected subjects had a dose-dependent probability of becoming ill, ranging from 0.1 (at a dose of 10(3) NV genomes) to 0.7 (at 10(8) virus genomes). A norovirus dose response model is important for understanding its transmission and essential for development of a quantitative risk model. Norwalk virus is a valuable model system to study virulence because genetic factors are known for both complete and partial protection; the latter can be quantitatively described as heterogeneity in dose response models.

Transmission events within outbreaks of gastroenteritis determined through analysis of nucleotide sequences of the P2 domain of genogroup II noroviruses.

Tracking the spread of noroviruses during outbreaks of gastroenteritis is hampered by the lack of sequence diversity in those regions of the genome chosen for virus detection and characterization. Sequence analysis of regions of the genes encoding the RNA-dependent RNA polymerase and the S domain of the capsid does not provide sufficient discrimination between genotypically related strains of different outbreaks. However, analysis of sequences derived from the region encoding the P2 domain showed 100% similarity among strains from the same outbreak and <100% similarity among strains of different outbreaks. The prolonged nature of some hospital outbreaks, links between hospitals, and the introduction of multiple strains of a single genotype associated with an outbreak aboard a cruise ship were determined using this method. This provides a powerful tool for tracking outbreak strains and the subsequent analysis and validation of interventions in a background of multiple introductions of virus strains of the same genotype or genetic cluster.

Virological investigation on aerosol from waste depuration plants.

Aerosol from activated mud decontamination plants used for the treatment of urban sewage can represent a vehicle for bacteria, virus and fungi. As a result, they become an infective hazard for plant personnel, the general population residing in the surrounding area and the occasional visitor. The present investigation focuses on the identification of enteric-type viruses in this kind of aerosol. The following methods were employed on 214 samples collected in the 1999-2000 period: cell culture (BGM, RD, Hep-2), electron microscopy, and polymerase chain reaction (PCR). Cytopathic effect was mild in 180 samples, and severe in 14, upon their first passage in culture. Virus identification was based on positivity to both electron microscopy (EM) and PCR. Thus, one positive sample was recognized to be of enteric-type virus and two positive samples were recognized as reovirus-type. All samples were negative for Norwalk-type virus or HAV. There was considerable discrepancy between electron microscopy and PCR concerning the number of enteric-type viruses recognized. A possible explanation is contamination with animal-type enterovirus.