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.

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.

Analysis of integrated virological and epidemiological reports of norovirus outbreaks collected within the Foodborne Viruses in Europe network from 1 July 2001 to 30 June 2006.

The Foodborne Viruses in Europe network has developed integrated epidemiological and virological outbreak reporting with aggregation and sharing of data through a joint database. We analyzed data from reported outbreaks of norovirus (NoV)-caused gastroenteritis from 13 European countries (July 2001 to July 2006) for trends in time and indications of different epidemiology of genotypes and variants. Of the 13 countries participating in this surveillance network, 11 were capable of collecting integrated epidemiological and virological surveillance data and 10 countries reported outbreaks throughout the entire period. Large differences in the numbers and rates of reported outbreaks per country were observed, reflecting the differences in the focus and coverage of national surveillance systems. GII.4 strains predominated throughout the 5-year surveillance period, but the proportion of outbreaks associated with GII.4 rose remarkably during years in which NoV activity was particularly high. Spring and summer peaks indicated the emergence of genetically distinct variants within GII.4 across Europe and were followed by increased NoV activity during the 2002-2003 and 2004-2005 winter seasons. GII.4 viruses predominated in health care settings and in person-to-person transmission. The consecutive emergence of new GII.4 variants is highly indicative of immune-driven selection. Their predominance in health care settings suggests properties that facilitate transmission in settings with a high concentration of people such as higher virus loads in excreta or a higher incidence of vomiting. Understanding the mechanisms driving the changes in epidemiology and clinical impact of these rapidly evolving RNA viruses is essential to design effective intervention and prevention measures.

Data quality of 5 years of central norovirus outbreak reporting in the European Network for food-borne viruses.

BACKGROUND: The food-borne viruses in Europe (FBVE) network database was established in 1999 to monitor trends in outbreaks of gastroenteritis due to noroviruses (NoVs), to identify major transmission routes of NoV infections within and between participating countries and to detect diffuse international food-borne outbreaks. METHODS: We reviewed the total of 9430 NoV outbreak reports from 13 countries with date of onset between 1 January 2002 and 1 January 2007 for representativeness, completeness and timeliness against these objectives. RESULTS: Rates of reporting ranged from a yearly average of 1.8 in 2003 to 11.6 in 2006. Completeness of reporting of an agreed minimum dataset improved over the years, both for epidemiological and virological data. For the 10 countries that provided integrated (epidemiological AND virological) reporting over the 5-year period, the completeness of the minimum dataset rose from 15% in 2003 to 48% in 2006. Two countries have not been able to combine both data types due to the structure of the national surveillance system (England and Wales and Germany). Timeliness of reporting (median days between the onset of an outbreak and the date of reporting to the FBVE database) differed greatly between countries, but gradually improved to 47 days in 2006. CONCLUSION: The outbreaks reported to the FBVE reflect the lack of standardization of surveillance systems across Europe, making direct comparison of data between countries difficult. However, trends in reported outbreaks per country, distribution of NoV genotypes, and detection of diffuse international outbreaks were used as background data in acute questions about NoV illness and the changing genotype distribution during the 5-year period, shown to be of added value. Integrated reporting is essential for these objectives, but could be limited to sentinel countries with surveillance systems that allow this integration. For successful intervention in case of diffuse international outbreaks, completeness and timeliness of reporting would need to be improved and expanded to countries that presently do not participate.

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.

Viral abundance and a high proportion of lysogens suggest that viruses are important members of the microbial community in the Gulf of Trieste.

Epifluorescence microscopy and transmission electron microscopy were applied to study virioplankton community in the Gulf of Trieste (northern Adriatic Sea). The total viral abundance was in a range between 2.5 x 10(9)/L and 2.9 x 10(10)/L and was positively correlated with trophic status of the environment. Viruslike particles were significantly correlated with bacterial abundance in all samples studied. Correlations with other physicochemical or biological parameters were not significant. The data suggest that, because of the substantial fraction of tailed viruses present (26%), bacteriophages are an important component of the virioplankton community in the Gulf of Trieste. The abundance of viruslike particles in the seawater changed at hour intervals in a range from 1.3 x 10(9)/L to 5.1 x 10(9)/L. A significant fraction (71%) of the bacterial isolates was inducible in vitro by mitomycin C, and a high occurrence (51%) of lysogenic isolates with more than one phage morphotype present in the lysate was detected. The presence of lysogenic bacteria in the seawater was confirmed in situ with a mitomycin C induction experiment on the natural bacterial population. Results suggest that virioplankton is an abundant component of the microbial community in the Gulf of Trieste.

Viral abundance and a high proportion of lysogens suggest that viruses are important members of the microbial community in the Gulf of Trieste.

Epifluorescence microscopy and transmission electron microscopy were applied to study virioplankton community in the Gulf of Trieste (northern Adriatic Sea). The total viral abundance was in a range between 2.5 x 10(9)/L and 2.9 x 10(10)/L and was positively correlated with trophic status of the environment. Viruslike particles were significantly correlated with bacterial abundance in all samples studied. Correlations with other physicochemical or biological parameters were not significant. The data suggest that, because of the substantial fraction of tailed viruses present (26%), bacteriophages are an important component of the virioplankton community in the Gulf of Trieste. The abundance of viruslike particles in the seawater changed at hour intervals in a range from 1.3 x 10(9)/L to 5.1 x 10(9)/L. A significant fraction (71%) of the bacterial isolates was inducible in vitro by mitomycin C, and a high occurrence (51%) of lysogenic isolates with more than one phage morphotype present in the lysate was detected. The presence of lysogenic bacteria in the seawater was confirmed in situ with a mitomycin C induction experiment on the natural bacterial population. Results suggest that virioplankton is an abundant component of the microbial community in the Gulf of Trieste.

Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies.

Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.

Incidence of Blastocystis hominis in patients with diarrhoea.

We studied the occurrence of the parasite Blastocystis hominis in 1066 stool specimens from patients with diarrhoea, and investigated the relationship between the presence of B. hominis in the faeces and the age of patients. The parasite was recovered from 3.7% samples, but as the sole species of micro-organism in the stool it was recovered from 1% samples. There was no statistically significant difference in the number of B. hominis-positive stools between the younger and the older patients (P < 0.25), yet in the latter, B. hominis was more frequently identified as the only species of micro-organism as compared with the younger group (P < 0.005). The presence of B. hominis in faecal samples of patients with diarrhoea harbouring no other intestinal pathogens suggests an aetiology that should receive more attention in Slovenia.