Evaluation of rectal swab use for the determination of enteric pathogens: a prospective study of diarrhoea in adults.

OBJECTIVES: A stool sample is the sample of choice for microbiological testing of enteric pathogens causing diarrhoea, but a rectal swab can be a more practical alternative. A prospective observational study was performed to evaluate the diagnostic performance of flocked rectal swab specimens using the syndromic molecular approach to determine the aetiology of diarrhoea in adults. METHODS: We compared the performance of rectal swabs with stool samples as the reference standard in determining viral, bacterial and protozoal pathogens using real-time multiplex PCR as well as standard stool culture. Paired samples of stool and rectal swab specimens were collected from 304 adult patients with diarrhoea, presented at the Department of Infectious Diseases, University Medical Centre Ljubljana, between June 2016 and August 2017. RESULTS: Overall sensitivity of rectal swab samples in the syndromic molecular approach was 83.2% (95% CI 77.2%-88.1%). Pathogen group-specific analysis of rectal swabs showed sensitivity of 65.6% (95% CI 52.7%-77.1%) for viruses and 57.1% (95% CI 28.9%-82.3%) for parasites. For bacteria, sensitivity was 86.5% (95% CI 79.5%-91.8%) when PCR was performed and 61.4% (95% CI 52.4%-69.9%) when culture for bacteria was performed. Mean threshold cycle (Ct) values for most pathogens were higher in rectal swab specimens than in stool specimens. CONCLUSIONS: Our results indicate that rectal swabs can be used in the diagnosis of diarrhoea in adults when stool specimens are not available or when rapid aetiological determination is needed. However, rectal swabs should be analysed using a molecular approach. The mean Ct value for most pathogens is higher in rectal swab specimens than in stool specimens.

Methacrylate monolith chromatography as a tool for waterborne virus removal.

Enteric viruses are commonly present in environmental waters and represent the major cause of waterborne infections and outbreaks. Since traditional wastewater treatments fail to remove enteric viruses in the water purification process, they are released daily into environmental waters. Monolithic supports have enabled chromatography to enter the field of virology. They have been successfully used in virus purification and concentration. In this work quaternary amine (QA) methacrylate monoliths were exploited to remove enteric viruses from wastewater treatment plant effluent. Expectedly, chromatographic processing of such a complex medium was troublesome, even for monoliths, characterized by extremely large pore dimensions. This problem was solved by introducing a pre-step chromatography using hydroxyl (OH) methacrylate monoliths. This way, molecules, that would hinder virus binding to the anion-exchanger monolith, were removed. As a result, the OH pre-column reduced backpressure increase on the subsequent anion-exchanger column, and increased both QA column binding capacity and life time. Wastewater effluent samples were successfully purified from five waterborne enteric viruses (rotavirus, norovirus genogroup I and II, astrovirus, sapovirus), below the detection limit of RT-qPCR. The breakthrough of the rotavirus binding capacity was not reached for concentrations that significantly exceeded those expected in effluent waters. The obtained results confirm that methacrylate monoliths can be a valuable tool for simultaneous removal of different waterborne viruses from contaminated water sources.

Whole genome sequence analysis of bovine G6P[11] rotavirus strain found in a child with gastroenteritis.

During the rotavirus strain surveillance in Slovenia, G6P[11] bovine rotavirus strain was detected in a 5 months old boy with gastroenteritis. The strain was enrolled in a whole genome sequence analysis to determine its genome segment composition and genetic characteristics. Genotype composition for the whole genome was G6-P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3, reflecting similarities with bovine rotavirus strains. The bovine origin of the strain was confirmed in all genome segments, showing the highest nucleotide identity with bovine rotavirus strains and clustering of the RVA/Human-wt/SVN/SI-R56/07/2007/G6P[11] together with bovine rotavirus strains in phylogenetic analysis. This is the first bovine G6P[11] rotavirus strain with the whole genome analysis and the first report on rotavirus G6P[11] genotype detected in humans.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

The emergence of rotavirus genotype G9 in hospitalised children in Slovenia.

BACKGROUND: Rotavirus G9 genotype was thought to be the fifth most common genotype circulating amongst the population. In previous studies in Slovenia, only G1, G3 and G4 genotypes were detected. OBJECTIVES: To determine G and P genotypes of rotaviruses causing dehydrating gastroenteritis in children hospitalised at the University Medical Centre Ljubljana during the winter season 2001-2002. Some data obtained in previous years are included, too. STUDY DESIGN: For the G and P genotypes determination, we selected 99 of the total of 565 rotavirus positive samples. RT-PCR was carried out for G gene or partial P gene amplification. The RT-PCR product was used as a template for multiplex nested PCR using genotype-specific primers. In untypable samples, a sequence analysis of a short segment of G or P gene was performed. From the period before July 2001, 183 stool samples were examined using the same methods. RESULTS: Genotype G1 was determined in 37, G4 in 6, and G9 in 28 samples out of 99. Only one sample showed a mixed infection with G1G4 genotype specifics. Following the sequence analysis of the short segment of G gene in 11 G9 genotypes, 2 different clusters of G9 genotype were determined. All samples had the same P genotype--P[8]. G9 genotype had not been detected prior to July 2001. CONCLUSION: Rotavirus G9 genotype emerged in Slovenia in the year 2001. Two different clusters were determined which have to be further characterised in detail.

X-ray high-resolution vascular network imaging.

This paper presents the first application of high-resolution X-ray synchrotron tomography to the imaging of large microvascular networks in biological tissue samples. This technique offers the opportunity of analysing the full three-dimensional vascular network from the micrometre to the millimetre scale. This paper presents the specific sample preparation method and the X-ray imaging procedure. Either barium or iron was injected as contrast agent in the vascular network. The impact of the composition and concentration of the injected solution on the X-ray synchrotron tomography images has been studied. Two imaging modes, attenuation and phase contrast, are compared. Synchrotron high-resolution computed tomography offers new prospects in the three-dimensional imaging of in situ biological vascular networks.