Aim: To evaluate the accuracy of two PCR-based techniques for detecting SARS-CoV-2 variant Alpha (B.1.1.7). Materials & methods: A multicenter prospective cohort with 1137 positive specimens from Slovenia was studied. A mutation-based assay (rTEST-COVID-19 qPCR B.1.1.7 assay) and amplification curve pattern analysis of the Allplex SARS-CoV-2 assay were compared with whole-genome sequencing. Results: SARS-CoV-2 variant Alpha was detected in 155 samples (13.6%). Sensitivity and specificity were 98.1 and 98.0%, respectively, for the rTEST-COVID-19 qPCR B.1.1.7 assay and 97.4 and 97.5%, respectively, for amplification curve pattern analysis. Conclusion: The good analytical performance of both methods was confirmed for the preliminary identification of SARS-CoV-2 variant Alpha. This cost-effective principle for screening SARS-CoV-2 populations is also applicable to other emerging variants and may help to conserve some whole-genome sequencing resources.

Background: Clostridium difficile is an important cause of nosocomial diarrhoea. Strains are further differentiated into PCR ribotypes, and some ribotypes (e.g. 027) are often associated with outbreaks, increased disease severity and increased mortality. Here we describe the diversity of C. difficile among human isolates in Slovenia. Methods: All eight microbiological diagnostic laboratories providing C. difficile diagnostics in Slovenia have participated. Isolates from two month-winter period were collected and ribotyped. The following data were also collected from the laboratories: number of all tested samples, number of all positive samples, and patient age and gender. Results: In a two-month period, altogether 860 samples were tested for C. difficile in all participating laboratories. Of those, 154 (17.9 %) samples from 12.5 patients were positive. The percentage of positive samples in different laboratories ranged from 13.3 to 43.2 %. Two out of eight laboratories did not have positive samples. C. difficile strains (n= 149) were grouped into 35 ribotypes. However, 57.7% of all strains belonged only to two PCR ribotypes (027 and 014/020). PCR ribotype 027 was not present in Slovenia until 2010, but was in this study the most prevalent PCR ribotype and present mainly in the northeast region. Conclusions: There is a substantial diversity of C. difficile ribotypes in Slovenia. A high prevalence of ribotype 027 and a high percentage of positive samples in some laboratories indicate the presence of C. difficile outbreaks.

Background : Clostridium difficile is an important cause of nosocomial diarrhoea. Strains are further differentiated into PCR ribotypes, and some ribotypes (e.g. 027) are often associated with outbreaks, increased disease severity and increased mortality. Here we describe the diversity of C. difficile among human isolates in Slovenia. Methods : All eight microbiological diagnostic laboratories providing C. difficile diagnostics in Slovenia have participated. Isolates from two–month- winter period were collected and ribotyped. The following data were also collected from the laboratories: number of all tested samples, number of all positive samples, and patient age and gender. Results : In a two-month period, altogether 860 samples were tested for C. difficile in all participating laboratories. Of those, 154 (17.9 %) samples from 125 patients were positive. The percentage of positive samples in different laboratories ranged from 13.3 to 43.2 %. Two out of eight laboratories did not have positive samples. C. difficile strains (n= 149) were grouped into 35 ribotypes. However, 57.7 % of all strains belonged only to two PCR ribotypes (027 and 014/020). PCR ribotype 027 was not present in Slovenia until 2010, but was in this study the most prevalent PCR ribotype and present mainly in the northeast region. Conclusions : There is a substantial diversity of C. difficile ribotypes in Slovenia. A high prevalence of ribotype 027 and a high percentage of positive samples in some laboratories indicate the presence of C. difficile outbreaks.

BACKGROUND Shiga toxin-producing E. coli or Vero cytotoxin-producing E. coli (VTEC) are characterised by the ability to produce either one or both cytotoxins referred to as Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). VTEC infection may result in life-threatening conditions such as haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Due to different methods of monitoring and identification of these bacteria in recent years, the existing data on reported cases of VTEC infections probably do not reflect reality. Our study of VTEC strains isolated in different regions of Slovenia, showed serogroups, major virulence factors and specific epidemiological data that can serve as a basis for further laboratory and epidemiological surveillance of VTEC infections. METHODS A total of 66 VTEC strains, isolated from stool samples of patients with diarrhoea from the year 1993 to 2009, were collected at NIPH (National Institute of Public Health). The data of patient’s age and gender, onset of illness and clinical manifestation of disease were gathered. The serogroups of isolated strains were determined with antisera following manufacturer’s instructions. The ability to produce verocytotoxins was tested using the reversed passive latex agglutination method. The presence of genes for intimin (eae), enterohaemolysin (ehxA) and verotoxins (vtx1 and vtx2) were determined by polymerase chain reaction (PCR). RESULTS Infection with VTEC was encountered throughout the year, but most people were ill in the summer and autumn months. More than half of patients (57.6 %) were younger than five years. Collected VTEC strains belonged to serogroups O17, O26, O91, O103, O111, O113, O126, O128, O145, O148 and O157 (the most frequent were O157 and O26). A high percentage of VTEC strains showed the presence of intimin (86.4 %) and enterohaemolysin (86.4 %) genes. The gene for vtx1 was found in 22.7 % of strains, the vtx2 in 57.6 % of strains, while the presence of both genes was determined in 19.7 % of strains. The presence of the vtx2 gene was determined in all strains associated with HUS and TTP, most of them possessed the eae and ehxA genes too. These patients were mostly older people and young children. CONCLUSIONS Most infections with VTEC occurred in the warmer months of the year, most patient were small children. The severity of VTEC infection is determined by several factors such as the E. coli serogroup, the type of Shiga toxin produced and presence of other virulence genes. The most common serogroups among the study strains were O157 and O26. VTEC O26 has been the most commonly isolated serogroup in recent years, nevertheless more and more different serological groups began to emerge. In all strains associated with HUS and TTP, the vtx2 gene was determined. Further typing of verocytotoxin encoding genes will contribute to assess the risk for complications with VTEC infection. The study provides insights about the age of the patients, seasonal distribution of disease, serogroups and genotypes of the agent.

Salmonella is an important zoonotic pathogen in animals and humans. In the European Union, Salmonella enterica subspecies enterica serovar Enteritidis (serovar Enteritidis) is one of the serovars most frequently associated with human illness. The most important food vehicles responsible for the infection are eggs and egg products. We describe two serovar Enteritidis outbreaks on account of consumption of contaminated eggs. The first outbreak due to vanilla cream served as dessert in a restaurant involved 36 persons. As the eggs used in preparing the vanilla cream were no longer available for examination, an indirect epidemiological link between the infected laying hen flock and humans was demonstrated by testing the faeces and dust samples from the relevant laying hen flock. In the second outbreak, two persons developed a severe form of salmonellosis after having consumed fried eggs. A sample of eggs taken from the same laying hen flock as the eggs consumed by the two patients tested positive for serovar Enteritidis.Isolates from both the outbreaks were subjected to molecular typing for the assessment of genetic relatedness. Pulsed-field gel electrophoresis (PFGE) revealed that the profiles of the majority of isolates from the same outbreak were indistinguishable and should therefore be considered to represent the same strain.This is the first molecular epidemiological investigation of serovar Enteritidis outbreaks in Slovenia that involved the public health and veterinary authorities and as such set a good example of collaboration of different national services.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) differs from healthcare-associated MRSA (HA-MRSA) in its molecular and microbiological characteristics. MATERIALS AND METHODS: Six Slovenian regional public health institutes and the National Institute of Public Health took part in monitoring CA-MRSA infections. S. aureus isolates resistant to oxacillin and susceptible to ≥ two of the four antibiotics ciprofloxacin, erythromycin, clindamycin or gentamicin were defined as CA-MRSA and further analyzed. The presence of the gene for Panton–Valentine leukocidin (PVL) was confirmed using PCR, the type of staphylococcal cassette chromosome (SCCmec) using multiplex PCR, and macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 31 strains from 31 patients were analyzed during a period of 21 months: 23 specimens were sent from hospitals, six from primary care, two from a long-term care facility. All 31 isolates contained the gene mecA. Sixteen (51.6%) isolates were identified as SCCmec type IV, three isolates were PVL positive. Using PFGE, the CA-MRSA strains were classified into 15 similarity groups. Results of antibiotic susceptibility showed there were five resistance types among the 31 strains. Simultaneous resistance against ciprofloxacin and gentamicin was often associated with the presence of SCCmec type I, strongly resembling HA-MRSA. CONCLUSIONS: PVL-positive strains of CA-MRSA have been isolated in Slovenia only rarely. We will continue to monitor strains of MRSA in order to obtain the complete microbiological and epidemiological features.

background Rotaviruses are the major cause of acute watery diarrhea in children up to 5 years of age. In 2007 and 2008 an extensive rotavirus molecular epidemiology study was conducted in Slovenia in order to provide information on rotavirus molecular epidemiology in our country. This study is part of the EuroRotaNet, European rotavirus surveillance study. Methods A total of 823 stool samples were collected from children with acute gastroenteritis in seven out of nine healthcare regions in Slovenia. The total RNA was extracted and first amplification of VP7 and VP4 genes was performed in RT-PCR. The RT-PCR product was used for genotyping in multiplex-nested PCR. Untypable strains were included in sequence analysis of VP7 and VP4 genes. Results Genotype distribution similar to that in other European countries was observed. G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8] were the most prevalent genotypes in 2007 and 2008, except of G3P[8], not detected in 2008. Relatively high rate of possible zoonotic strains was detected in 2007 (2.5%). The emerging unusual strain G10P[14] was the only potential zoonotic strain detected in 2007 and 2008. conclusions After the vaccine introduction in Slovenia in 2007, no specific changes in molecular epidemiology of rotaviruses was observed. This finding was expected since rotavirus vaccine coverage in 2007 in Slovenia was very low. Genotype G1P[8] remains the most prevalent genotype. The rotavirus strain surveillance in Slovenia should be carried on to allow monitoring the spread of some unusual rotavirus genotypes, like G10. The G10 strains should be tracked especially in vaccinated children as no data on vaccine efficiency against infection with G10 strains was presented till now.