Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/µL as determined by RT-PCR on a surrogate sample panel containing clinical samples with varying SARS-CoV-2 viral load. We benchmarked our direct RT-PCR method against a reference qPCR method in 368 nasopharyngeal swab samples, confirming a sensitivity score of 99.4% and a specificity score of 98.5% as compared to the reference method. In summary, we here describe a novel rapid direct RT-PCR method to detect SARS-CoV-2 gRNA in clinical specimens, which can be completed in significantly less time compared to conventional PCR methods making it ideal for large-scale screening applications.

High Prevalence of Clostridioides difficile Ribotype 176 in the University Hospital in Kosice.

Dysbiosis of the gut microbiota, caused by antibiotics, plays a key role in the establishment of Clostridioides difficile CD). Toxin-producing strains are involved in the pathogenesis of Clostridioides difficile infection (CDI), one of the most common hospital-acquired infections. We cultured a total of 84 C. difficile isolates from stool samples of patients hospitalized at Louis Pasteur University Hospital in Kosice, Slovakia, that were suspected of CDI and further characterized by molecular methods. The presence of genes encoding toxin A, toxin B, and binary toxin was assessed by toxin-specific PCR. CD ribotypes were detected using capillary-based electrophoresis ribotyping. A total of 96.4% of CD isolates carried genes encoding toxins A and B, and 54.8% of them were positive for the binary toxin. PCR ribotyping showed the presence of three major ribotypes: RT 176 (n = 40, 47.6%); RT 001 (n = 23, 27.4%); and RT 014 (n = 7, 8.3%). Ribotype 176 predominated among clinical CD isolates in our hospital. The proportion of RT 176 and RT 001 in four hospital departments with the highest incidence of CDI cases was very specific, pointing to local CDI outbreaks. Based on our data, previous use of antibiotics represents a significant risk factor for the development of CDI in patients over 65 years of age.

Antibiotic resistance in the invasive bacteria Escherichia coli.

OBJECTIVES: The beta-lactamases with extended spectrum of activity (ESBL) are medically one of the most important group of enzymes. Another group of beta-lactamases representing of Enterobacteriaceae is group of the AmpC-type cephalosporinases. The presented study provides identification and determination of the spectrum of resistance against different and clinically used antimicrobial drugs in the clinical isolates of Escherichia coli. METHODS: These isolates had origin in different departments of the L. Pasteur University Hospital in Kosice. The goal was the detection of beta-lactamase production with extended-spectrum effect and testing of AmpC-type cephalosporinases by several phenotypic tests in clinical isolates. MALDI-TOF MS analysis was performed on a Microflex MALDI Biotyper. Samples were positively tested for ESBL with the use of the disc diffusion method. PCR were performed with a series of primers designed for the detection of Ambler class A, B and C beta-lactamase genes. RESULTS: For all 485 isolates, we determined the production of ESBL, which we detected in 166 E. coli isolates, which represents a 34.2% prevalence of ESBL production. It is clear from the results that the prevalence of ESBL-producing E. coli out of the total number of E. coli investigated reached 34.2%. In the monitored period, we confirmed at least one resistance gene from 485 E. coli in 188 positive isolates. CONCLUSIONS: We describe a complex ESBL epidemiology. The study revealed a high rate of ESBL-producing E. coli isolates; blaTEM and blaSHV enzymes dominated in ESBL-positive E. coli isolates in the L. Pasteur University Hospital in Kosice.

Virulence, phylogenetic background and antimicrobial resistance in Escherichia coli associated with extraintestinal infections.

BACKGROUND: Escherichia coli (E. coli) is a major cause of urinary tract infections and bloodstream infections and an important agent in the resistance to antibiotics. The present study sought to determine associations between virulence, phylogenetic background and antimicrobial resistance of E. coli strains isolated from patients with extraintestinal infections. METHODS: A total of three hundred ten E. coli strains were isolated from blood, skin and soft tissue and urine. PCR methods were used to detect four main phylogenetic groups (A, B1, B2 and D) and 11 virulence genes (3 toxins, 3 adhesins, 1 siderophore, 4 capsule synthesis proteins and protectins). Standard broth microdilution test was used to determine sensitivity to 12 antimicrobial drugs. RESULTS: The most common and the most virulent phylogenetic group B2 was found in 193 (62.3%) isolates. The lowest virulence was observed among the group A. Analysis of virulence factors revealed the kpsMTII gene in 212 (68.4%), aer in 194 (62.6%) and tra in 184 (59.4%) of isolates, respectively. Multi-drug resistant (MDR) phenotype was noticed in 165 (53.2%) isolates. Lower representation of the MDR phenotype was detected in E. coli containing all groups of virulence genes and in the avirulent E. coli. CONCLUSIONS: Our study documented that E. coli associated with 3 different extraintestinal infections contain various virulence factors. Genes afa, pap, aer, neuC show significant differences among the 3 groups of the strains tested and might be the prerequisite virulence factors in bloodstream infections. Isolates containing all groups of virulence genes predominantly originate in the blood and belong to the B2 phylogenetic group. Overall, we identified significantly higher incidence of all the groups of virulence genes examined among the B2 group. Prevalence of the MDR phenotype and high levels of resistance to ampicillin, ciprofloxacin and trimetoprim/sulfamethoxazole reflect the trend observed worldwide in recent years.

Distribution and antifungal susceptibility of yeasts isolates from intensive care unit patients.

Yeasts frequently colonize non-sterile sites in the body. The aim of the study was to determine distribution in clinical samples and antifungal susceptibility to five antifungals. From January 2013 through June 2015, 800 isolates were obtained from intensive care unit patients. Candida albicans (58.9%), Candida glabrata (20.4%), Candida krusei (8.6%), and Candida parapsilosis (3.6%) were the leading species. Majority of the C. albicans isolates were susceptible to the fluconazole. Elevated voriconazole minimal inhibitory concentrations (MICs) were observed in isolates exhibiting high fluconazole MICs, most frequently in C. glabrata. Isolates with echinocandins MICs suggesting reduced susceptibility were only sporadic cases with the exception of Trichosporon spp. The amphotericin B MICs were slightly higher for some C. krusei.

Enterohemorrhagic Escherichia coli O26:H11/H-: a new virulent clone emerges in Europe.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 causes diarrhea and hemolytic uremic syndrome (HUS). Strains harboring the stx1a gene prevail, but strains with stx2a as the sole Shiga toxin-encoding gene are now emerging. The traits and virulence of the latter set of strains are unknown. We correlated stx genotypes of 272 EHEC O26 strains isolated in 7 European countries between 1996 and 2012 with disease phenotypes. We determined phylogeny, clonal structure, and plasmid gene profiles of the isolates and portray geographic and temporal distribution of the different subgroups. METHODS: The stx genotypes and plasmid genes were identified using polymerase chain reaction, phylogeny was assigned using multilocus sequence typing, and clonal relatedness was established using pulsed-field gel electrophoresis. RESULTS: Of the 272 EHEC O26 isolates, 107 (39.3%), 139 (51.1%), and 26 (9.6%) possessed stx1a, stx2a, or both genes, respectively. Strains harboring stx2a only were significantly associated with HUS (odds ratio, 14.2; 95% confidence interval, 7.9-25.6; P < .001) compared to other stx genotypes. The stx2a-harboring strains consist of 2 phylogenetically distinct groups defined by sequence type (ST) 21 and ST29. The ST29 strains are highly conserved and correspond by plasmid genes to the new virulent clone of EHEC O26 that emerged in Germany in the 1990s. This new clone occurred in 6 of the 7 countries and represented approximately 50% of all stx2a-harboring EHEC O26 strains isolated between 1996 and 2012. CONCLUSIONS: A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.

Involvement of virulence properties and antibiotic resistance in Escherichia coli strains causing pyelonephritis in children.

A total of 145 Escherichia coli strains causing pyelonephritis in children were investigated for the prevalence of genes encoding the following virulence factors (VFs): P fimbria (67.6 %), S fimbria (53.8 %), AFA adhesins (2.8 %), cytotoxic necrotizing factor 1 (37.9 %), α-hemolysin (41.4 %), and aerobactin (71.7 %). One hundred and thirty-six (93.8 %) isolates harbored at least one of the virulence genes detected in the present study. Statistically significant co-occurrent presence of two VF genes was found for α-hly-cnf1, α-hly-sfa, cnf1-sfa (p<0.001), and α-hly-pap (p=0.001). Twenty-six profiles of VF genes were detected in this study. The combinations of aer-pap and aer-pap-sfa-α-hly-cnf1 were presented with the highest frequency-both of them in 28 isolates (19.3 %). All E. coli strains included in the study were susceptible to meropenem, amikacin, and tobramycin; the highest frequency resistance was found toward ampicillin (43.4 %), piperacillin (31.7 %), tetracycline (15.9 %), and trimethoprim/sulfamethoxazole (11.7 %). The resistance to the other tested antimicrobial drugs did not exceed 3 % incidence. Overall, 55.9 % strains were susceptible to all tested anti-infective agents. Antimicrobial resistance of E. coli strains toward trimethoprim/sulfamethoxazole statistically significantly correlated with the presence of α-hly (p<0.001), sfa (p<0.01), and cnf1 (p<0.05).