Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing.

Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology's (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000-3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.

Prevalence of genotypic antimicrobial resistance in clinical Shiga toxin-producing Escherichia coli in Norway, 2018 to 2020.

Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe to fatal disease in humans. Antimicrobial treatment is sometimes necessary, but contraindicated due to undesirable clinical outcome. However, recent studies have shown promising outcomes following antimicrobial treatment. Before the establishment of a possible antimicrobial treatment strategy for STEC infections, the prevalence of antimicrobial resistance in STEC needs to be determined.Gap Statement. The resistance status of Norwegian clinical STEC is not known and should be assessed.Aim. We aim to characterize genotypic antimicrobial resistance determinants in clinical STEC in Norway, and determine the prevalence of genotypic resistance in order to inform possible antimicrobial treatment options for STEC infections.Methodology. We included all clinical STEC submitted to the Norwegian Reference Laboratory from March 2018 to April 2020. All samples were whole-genome sequenced and screened for genotypic antimicrobial resistance,virulence determinants and plasmid incompatibility groups. We performed phylogenetic clustering of STEC by core-genome multi-locus sequence typing, and statistical association analyses between isolate characteristics and genotypic resistance.Results. A total of 459 STEC were analysed. For 385 (83.9 %) STEC we did not identify any antimicrobial resistance determinants. Seventy-four STEC (16.1 %) harboured antimicrobial resistance determinants against one or more antimicrobial classes. The most frequent genotypic resistance was identified against aminoglycosides (10.5 %). Thirty-nine STEC (8.5 %) had a multi-drug resistance (MDR) genotype. Genotypic resistance was more prevalent in non-O157 than O157 STEC (P=0.02). A positive association was seen between genotypic resistance and the low-virulent STEC O117:H7 phylogenetic cluster (no. 14) (P<0.001). Genotypic resistance was not significantly associated to high-virulent STEC. STEC O146:H28 and isolates harbouring the plasmid replicon type IncQ1 were positively associated with MDR.Conclusion. The overall prevalence of genotypic resistance in clinical STEC in Norway is low (16.1 %). Genotypic resistance is more prevalent in non-O157 strains compared to O157 strains, and not significantly associated to high-virulent STEC. Resistance to antimicrobials suggested for treatment, especially azithromycin is low and may present an empiric treatment alternative for severe STEC infections.

Outbreak caused by the SARS-CoV-2 Omicron variant in Norway, November to December 2021.

In late November 2021, an outbreak of Omicron SARS-CoV-2 following a Christmas party with 117 attendees was detected in Oslo, Norway. We observed an attack rate of 74% and most cases developed symptoms. As at 13 December, none have been hospitalised. Most participants were 30-50 years old. Ninety-six percent of them were fully vaccinated. These findings corroborate reports that the Omicron variant may be more transmissible, and that vaccination may be less effective in preventing infection compared with Delta.

Characterization of Shiga Toxin 2a Encoding Bacteriophages Isolated From High-Virulent O145:H25 Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) may cause severe disease mainly due to the ability to produce Shiga toxins (Stx) encoded on bacteriophages. In Norway, more than 30% of the reported cases with STEC O145:H25 develop hemolytic uremic syndrome (HUS), and most cases, with known travel history, acquired the infection domestically. To describe phage characteristics associated with high virulence, we extracted the Stx2a phage sequences from eight clinical Norwegian O145:H25 STEC to conduct in-depth molecular characterization using long and short read sequencing. The Stx2a phages were annotated, characterized, and compared with previously published Stx2a phages isolated from STEC of different serotypes. The Norwegian O145:H25 Stx2a phages showed high sequence identity (>99%) with 100% coverage. The Stx2a phages were located at the integration site yciD, were approximately 45 kbp long, and harbored several virulence-associated genes, in addition to stx2a, such as nanS and nleC. We observed high sequence identity (>98%) and coverage (≥94%) between Norwegian O145:H25 Stx2a phages and publicly available Stx2a phages from O145:H25 and O145:H28 STEC, isolated from HUS cases in the USA and a hemorrhagic diarrhea case from Japan, respectively. However, low similarity was seen when comparing the Norwegian O145:H25 Stx2a phage to Stx2a phages from STEC of other serotypes. In all the Norwegian O145:H25 STEC, we identified a second phage or remnants of a phage (a shadow phage, 61 kbp) inserted at the same integration site as the Stx2a phage. The shadow phage shared similarity with the Stx2a phage, but lacked stx2a and harbored effector genes not present in the Stx2a phage. We identified a conserved Stx2a phage among the Norwegian O145:H25 STEC that shared integration site with a shadow phage in all isolates. Both phage and shadow phage harbored several virulence-associated genes that may contribute to the increased pathogenicity of O145:H25 STEC.

Vaccine effectiveness against infection with the Delta (B.1.617.2) variant, Norway, April to August 2021.

Some variants of SARS-CoV-2 are associated with increased transmissibility, increased disease severity or decreased vaccine effectiveness (VE). In this population-based cohort study (n = 4,204,859), the Delta variant was identified in 5,430 (0.13%) individuals, of whom 84 were admitted to hospital. VE against laboratory confirmed infection with the Delta variant was 22.4% among partly vaccinated (95% confidence interval (CI): 17.0-27.4) and 64.6% (95% CI: 60.6-68.2) among fully vaccinated individuals, compared with 54.5% (95% CI: 50.4-58.3) and 84.4% (95%CI: 81.8-86.5) against the Alpha variant.

AMR-Diag: Neural network based genotype-to-phenotype prediction of resistance towards ß-lactams in Escherichia coli and Klebsiella pneumoniae.

Antibiotic resistance poses a major threat to public health. More effective ways of the antibiotic prescription are needed to delay the spread of antibiotic resistance. Employment of sequencing technologies coupled with the use of trained neural network algorithms for genotype-to-phenotype prediction will reduce the time needed for antibiotic susceptibility profile identification from days to hours. In this work, we have sequenced and phenotypically characterized 171 clinical isolates of Escherichia coli and Klebsiella pneumoniae from Norway and India. Based on the data, we have created neural networks to predict susceptibility for ampicillin, 3rd generation cephalosporins and carbapenems. All networks were trained on unassembled data, enabling prediction within minutes after the sequencing information becomes available. Moreover, they can be used both on Illumina and MinION generated data and do not require high genome coverage for phenotype prediction. We cross-checked our networks with previously published algorithms for genotype-to-phenotype prediction and their corresponding datasets. Besides, we also created an ensemble of networks trained on different datasets, which improved the cross-dataset prediction compared to a single network. Additionally, we have used data from direct sequencing of spiked blood cultures and found that AMR-Diag networks, coupled with MinION sequencing, can predict bacterial species, resistome, and phenotype as fast as 1-8 h from the sequencing start. To our knowledge, this is the first study for genotype-to-phenotype prediction: (1) employing a neural network method; (2) using data from more than one sequencing platform; and (3) utilizing sequence data from spiked blood cultures.

Pre-screening and preventive quarantine likely explains the low SARS-CoV-2 prevalence among Norwegian conscripts.

Objective: We aim to discuss whether preventive quarantine can mitigate the spread of Covid-19 during the pandemic. Design: We did a cross-sectional, observational study design in a mass-screening program in the enrolment to the Norwegian military during April 19-28th 2020 (COVID-NOR-MIL). Subjects: 1170 presumptively healthy young Norwegian conscripts. Setting: A structured interview encouraged the coming conscripts to a self-imposed preventive quarantine the last two weeks before enrolment. Main outcome measures: All conscripts underwent a PCR-based test with nasopharyngeal swabs at the day of enrolment. Results: Only two tested positive. The study discusses the predictive value of the RT-PCR test and the risk of false positive and false negative results, particularly when using the test in a low-prevalent cohort, even if the test properties of sensitivity and specificity is almost 100%. Further, the study discusses the challenge of whether a positive SARS-CoV-2 PCR-test represent viable and contagious virus or only viral remnants. Conclusion: The adherence to self-imposed preventive quarantine is a challenge and is a subject to further research. Implications: We want to draw the attention to the potential value of a thorough pre-screening processes and self-imposed preventive quarantine to minimize the potential spread of SARS-Cov-2.

Severe acute respiratory syndrome coronavirus 2 prevalence in 1170 asymptomatic Norwegian conscripts.

BACKGROUND: Accurate estimates of SARS-CoV-2 infection in different population groups are important for the health authorities. In Norway, public infection control measures have successfully curbed the pandemic. However, military training and service are incompatible with these measures; therefore extended infection control measures were implemented in the Norwegian Armed Forces. We aimed to describe these measures, discuss their value, and investigate the polymerase chain reaction (PCR) prevalence and seroprevalence of SARS-CoV-2, as well as changes in antibody titer levels over the 6-week military training period in a young, asymptomatic population of conscripts. METHODS: In April 2020, 1170 healthy conscripts (median age 20 years) enrolled in military training. Extended infection control measures included a pre-enrollment telephone interview, self-imposed quarantine, questionnaires, and serial SARS-CoV-2 testing. At enrollment, questionnaires were used to collect information on symptoms, and SARS-CoV-2 rapid antibody testing was conducted. Serial SARS-CoV-2 PCR and serology testing were used to estimate the prevalence of confirmed SARS-CoV-2 and monitor titer levels at enrollment, and 3 and 6 weeks thereafter. RESULTS: At enrollment, only 0.2% of conscripts were SARS-CoV-2 PCR-positive, and seroprevalence was 0.6%. Serological titer levels increased nearly 5-fold over the 6-week observation period. Eighteen conscripts reported mild respiratory symptoms during the 2 weeks prior to enrollment (all were PCR-negative; one was serology-positive), whereas 17 conscripts reported respiratory symptoms and nine had fever at enrollment (all were PCR- and serology-negative). CONCLUSIONS: The prevalence of SARS-CoV-2 was less than 1% in our sample of healthy Norwegian conscripts. Testing of asymptomatic conscripts seems of no value in times of low COVID-19 prevalence. SARS-CoV-2 antibody titer levels increased substantially over time in conscripts with mild symptoms.

Effects of antimicrobials on Shiga toxin production in high-virulent Shiga toxin-producing Escherichia coli.

PURPOSE: Antimicrobial treatment of Shiga toxin-producing Escherichia coli (STEC) infections is controversial because antimicrobials may stimulate Shiga toxin (Stx) production, and thereby increase the risk of developing haemolytic uremic syndrome (HUS). Previous in vitro studies have shown this mainly in infections caused by STEC serotype O157:H7. The aim of this study was to investigate induction of Stx transcription and production in different serotypes of STEC isolated from severely ill patients, following their exposure in vitro to six different classes of antimicrobials. METHODS: We investigated Stx transcription and production in 12 high-virulent STEC strains, all carrying the stx2a gene, of six different serotypes following their exposure to six classes of antimicrobials. Liquid cultures of the STEC strains were incubated with sub-inhibitory concentrations of the antimicrobials. We used reverse-transcription quantitative PCR to measure the relative expression of Stx2a mRNA and an enzyme-linked immunosorbent assay to quantify Stx production. RESULTS: In general the antibiotics tested showed only minor effects on transcriptional levels of Stx2a. Ciprofloxacin caused an increase of Stx production in all but two strains, while gentamicin, meropenem and azithromycin did not induce Stx production in any of the STEC strains examined. STEC O104:H4 was the serotype that in greatest extent responded to antimicrobial exposure with an increase of stx2a transcription and Stx production. CONCLUSION: Gentamicin, meropenem and azithromycin exposure did not result in elevated Stx production. We recommend that this finding is investigated further in the search for candidates for future antimicrobial treatment of STEC.

Asymptomatic COVID-19 with ambiguous test results. / Asymptomatisk covid-19 med usikre testresultater.

BACKGROUND: Testing for SARS-CoV-2 using polymerase chain reaction (PCR) and SARS-CoV-2 antibody tests is a significant part of the effort to combat the COVID-19 pandemic. Mass testing of healthy individuals raises several issues, however, and the results can be challenging to interpret. CASE PRESENTATION: A healthy 19-year-old man entered the military after two weeks of quarantine. The recruit had no respiratory symptoms or fever before, during or after his enrolment, and no history of SARS-CoV-2 exposure. At enrolment, he had a positive rapid test and a venous blood sample showed antibodies against SARS-CoV-2. PCR tests of specimens obtained from the upper respiratory tract were negative at enrolment and at week three, but were positive at week six. INTERPRETATION: The overall assessment of all the tests indicates a probable asymptomatic infection. This case report illustrates the challenge of interpreting screening results in asymptomatic individuals.

Status of COVID-19 vaccine development. / Status for vaksineutvikling mot covid-19.

The pandemic caused by SARS-CoV-2 has created a global humanitarian and economic crisis for which there is currently no solution in sight. Much hope has therefore been pinned on a vaccine that can protect against the disease COVID-19. As of August 2020, the World Health Organization has registered 173 vaccine candidates as being in development. Six candidates have entered phase 3 trials, and the first results from these are expected in the autumn.

Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing.

Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1-2 days for identification of bacterial agent and an additional 2-3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnostic approach with the ability to rapidly identify pathogens and AMR-encoding genes. Here we have used nanopore sequencing of bacterial DNA extracted from positive blood cultures for identification of pathogens, detection of plasmids and AMR-encoding genes. To our knowledge, this is the first study to gather the above-mentioned information from nanopore sequencing and conduct a comprehensive analysis for diagnostic purposes in real-time. Identification of pathogens was possible after 10 minutes of sequencing and all predefined AMR-encoding genes and plasmids from monoculture experiments were detected within one hour using raw nanopore sequencing data. Furthermore, we demonstrate the correct identification of plasmids and blaCTX-M subtypes using de novo assembled nanopore contigs. Results from this study hold great promise for future applications in clinical microbiology and for health care surveillance purposes.

Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins.

Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity.

Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli.

Enterotoxigenic Escherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

Characterization of immunological cross-reactivity between enterotoxigenic Escherichia coli heat-stable toxin and human guanylin and uroguanylin.

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.

Heat-stable enterotoxin of enterotoxigenic Escherichia coli as a vaccine target.

Enterotoxigenic Escherichia coli (ETEC) is responsible for 280 million to 400 million episodes of diarrhea and about 380,000 deaths annually. Epidemiological data suggest that ETEC strains which secrete heat-stable toxin (ST), alone or in combination with heat-labile toxin (LT), induce the most severe disease among children in developing countries. This makes ST an attractive target for inclusion in an ETEC vaccine. ST is released upon colonization of the small intestine and activates the guanylate cyclase C receptor, causing profuse diarrhea. To generate a successful toxoid, ST must be made immunogenic and nontoxic. Due to its small size, ST is nonimmunogenic in its natural form but becomes immunogenic when coupled to an appropriate large-molecular-weight carrier. This has been successfully achieved with several carriers, using either chemical conjugation or recombinant fusion techniques. Coupling of ST to a carrier may reduce toxicity, but further reduction by mutagenesis is desired to obtain a safe vaccine. More than 30 ST mutants with effects on toxicity have been reported. Some of these mutants, however, have lost the ability to elicit neutralizing immune responses to the native toxin. Due to the small size of ST, separating toxicity from antigenicity is a particular challenge that must be met. Another obstacle to vaccine development is possible cross-reactivity between anti-ST antibodies and the endogenous ligands guanylin and uroguanylin, caused by structural similarity to ST. Here we review the molecular and biological properties of ST and discuss strategies for developing an ETEC vaccine that incorporates immunogenic and nontoxic derivatives of the ST toxin.

Febrile neutropenia in children with cancer: a retrospective Norwegian multicentre study of clinical and microbiological outcome.

Our objective was to describe clinical and laboratory characteristics, treatment and outcome among Norwegian children with cancer suffering from chemotherapy-induced febrile neutropenia (FN). We retrospectively reviewed data on paediatric FN episodes in 7 Norwegian hospitals during a 2.5-y period. A total of 236 episodes of FN occurred in 95 children. Acute lymphoblastic leukaemia was the most common diagnosis (49 patients). Blood cultures yielded growth in 39 episodes (17%). Primary empirical antibiotic regimens could be assigned to 2 main groups: 1) benzylpenicillin or ampicillin and an aminoglycoside (58%) or 2) a regimen based on third-generation cephalosporins (42%). There were no statistically significant differences in outcome between the 2 regimens in terms of need to change initial antibiotic treatment, d of fever or maximum C-reactive protein values. One infection-related death (fungal septicaemia) occurred during the study period. We conclude that incidence of septicaemia and clinical outcome is similar to recent international trials on paediatric FN, but antibiotic treatment in Norway differs from international guidelines. However, patients in our study were successfully and safely treated, irrespective of the primary empirical antibiotic regimen.

Magnetic resonance imaging of patients with increased blood pressure and altered blood pressure response to exercise after coarctation repair.

OBJECTIVE: Patients successfully operated for coarctation of the aorta are frequently subjected to altered blood pressure (BP) at rest and BP response during exercise. The relationship between these variables and blood flow, peak velocity, restenosis and other morphological features of the thoracic aorta as revealed by magnetic resonance imaging (MRI) was evaluated. DESIGN: Fifty-one patients subjected to coarctectomy of the aorta were examined by MRI. In addition, a control group of 23 healthy volunteers was evaluated. Morphology of the aorta was demonstrated with both ECG-triggered SE imaging and gadolinium-enhanced MR aortography. Flow-weighted MRI was applied for quantitative flow and velocity measurements. RESULTS: Structural alteration of the aorta was more commonly seen in those patients having increased BP at rest or altered BP response during exercise than those with a normal BP profile. The luminal diameter of the narrowest site of the aorta was decreased in all patient groups. Accordingly, the peak velocity at the corresponding site was significantly (p < 0.01) increased. However, blood flow was significantly (p < 0.01) decreased among those patients with normal BP profile compared with the other patient groups as well as the controls. CONCLUSION: Other structural changes than restenosis may contribute as well to the altered BP profile of patients subjected to coarctectomy. Reduced blood flow appears to correlate with normal BP profile, whereas the peak velocity measurements that are obtained by MRI are not able to differentiate between the patient groups. The comprehensive and reliable data obtained by non-invasive techniques, i.e. MRI and Doppler, may replace catheterization when deciding the need for intervention.