Genotypic and phenotypic antimicrobial resistance of Irish Clostridioides difficile isolates, 2022.

OBJECTIVES: Infection with Clostridioides difficile (C. difficile) usually occurs after antibiotic treatment for other infections and can cause gastro-intestinal disorders of variable severity. C. difficile can be resistant to a wide spectrum of antimicrobials. Detection of antimicrobial resistance (AMR) is important to direct optimal treatment and surveillance of AMR patterns in the overall population. Correlation between genotypic markers and phenotypic AMR is not yet well defined. The aim for this study is to assess whether and to what extent genotypic determinants of AMR correlate with phenotypic resistance. METHODS: 99 C. difficile isolates were phenotypically characterized for resistance to eight antibiotics using Sensititre plates or E-tests. Their genomes were screened for genetic markers of resistance. Accuracy, sensitivity, specificity, positive and negative predictive values were calculated. RESULTS: We found high rates of resistance (>50%) to cefoxitin and clindamycin, intermediate rates of resistance (10% - 50%) to moxifloxacin and tetracycline and low rates of resistance (<10%) to imipenem, metronidazole, vancomycin, and rifampicin. For moxifloxacin, tetracycline, and clindamycin, we found a good correlation between genotypic and phenotypic AMR, with an overall accuracy of 96% (95% CI 90%-100%), 78% (95% CI 68%-86%) and 86% (95% CI 77%-92%) respectively. For the other five antibiotics, accurate estimates on the correlation could not be made. CONCLUSION: Our results suggest that for moxifloxacin, tetracycline and clindamycin, phenotypic resistance in C. difficile can be predicted by genetic indicators and used for public health purposes. However, for the other five antibiotics, the model is not accurate and further development is necessary.

Predicting community acquired bloodstream infection in infants using full blood count parameters and C-reactive protein; a machine learning study.

Early recognition of bloodstream infection (BSI) in infants can be difficult, as symptoms may be non-specific, and culture can take up to 48 h. As a result, many infants receive unneeded antibiotic treatment while awaiting the culture results. In this study, we aimed to develop a model that can reliably identify infants who do not have positive blood cultures (and, by extension, BSI) based on the full blood count (FBC) and C-reactive protein (CRP) values. Several models (i.e. multivariable logistic regression, linear discriminant analysis, K nearest neighbors, support vector machine, random forest model and decision tree) were trained using FBC and CRP values of 2693 infants aged 7 to 60 days with suspected BSI between 2005 and 2022 in a tertiary paediatric hospital in Dublin, Ireland. All models tested showed similar sensitivities (range 47% - 62%) and specificities (range 85%-95%). A trained decision tree and random forest model were applied to the full dataset and to a dataset containing infants with suspected BSI in 2023 and showed good segregation of a low-risk and high-risk group. Negative predictive values for these two models were high for the full dataset (> 99%) and for the 2023 dataset (> 97%), while positive predictive values were low in both dataset (4%-20%).   Conclusion: We identified several models that can predict positive blood cultures in infants with suspected BSI aged 7 to 60 days. Application of these models could prevent administration of antimicrobial treatment and burdensome diagnostics in infants who do not need them. What is Known: • Bloodstream infection (BSI) in infants cause non-specific symptoms and may be difficult to diagnose. • Results of blood cultures can take up to 48 hours. What is New: • Machine learning models can contribute to clinical decision making on BSI in infants while blood culture results are not yet known.

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model.

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection. Importance: With the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract.

Short-term and Long-term Outcomes of a Disruption and Disconnection of the Pancreatic Duct in Necrotizing Pancreatitis: A Multicenter Cohort Study in 896 Patients.

INTRODUCTION: Necrotizing pancreatitis may result in a disrupted or disconnected pancreatic duct (DPD) with the potential for long-lasting negative impact on a patient's clinical outcome. There is a lack of detailed data on the full clinical spectrum of DPD, which is critical for the development of better diagnostic and treatment strategies. METHODS: We performed a long-term post hoc analysis of a prospectively collected nationwide cohort of 896 patients with necrotizing pancreatitis (2005-2015). The median follow-up after hospital admission was 75 months (P25-P75: 41-151). Clinical outcomes of patients with and without DPD were compared using regression analyses, adjusted for potential confounders. Predictive features for DPD were explored. RESULTS: DPD was confirmed in 243 (27%) of the 896 patients and resulted in worse clinical outcomes during both the patient's initial admission and follow-up. During hospital admission, DPD was associated with an increased rate of new-onset intensive care unit admission (adjusted odds ratio [aOR] 2.52; 95% confidence interval [CI] 1.62-3.93), new-onset organ failure (aOR 2.26; 95% CI 1.45-3.55), infected necrosis (aOR 4.63; 95% CI 2.87-7.64), and pancreatic interventions (aOR 7.55; 95% CI 4.23-13.96). During long-term follow-up, DPD increased the risk of pancreatic intervention (aOR 9.71; 95% CI 5.37-18.30), recurrent pancreatitis (aOR 2.08; 95% CI 1.32-3.29), chronic pancreatitis (aOR 2.73; 95% CI 1.47-5.15), and endocrine pancreatic insufficiency (aOR 1.63; 95% CI 1.05-2.53). Central or subtotal pancreatic necrosis on computed tomography (OR 9.49; 95% CI 6.31-14.29) and a high level of serum C-reactive protein in the first 48 hours after admission (per 10-point increase, OR 1.02; 95% CI 1.00-1.03) were identified as independent predictors for developing DPD. DISCUSSION: At least 1 of every 4 patients with necrotizing pancreatitis experience DPD, which is associated with detrimental, short-term and long-term interventions, and complications. Central and subtotal pancreatic necrosis and high levels of serum C-reactive protein in the first 48 hours are independent predictors for DPD.

Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism.

Enterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells. IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

Diagnostic accuracy of rapid antigen tests in asymptomatic and presymptomatic close contacts of individuals with confirmed SARS-CoV-2 infection: cross sectional study.

OBJECTIVE: To assess the diagnostic test accuracy of two rapid antigen tests in asymptomatic and presymptomatic close contacts of people with SARS-CoV-2 infection on day 5 after exposure. DESIGN: Prospective cross sectional study. SETTING: Four public health service covid-19 test sites in the Netherlands. PARTICIPANTS: 4274 consecutively included close contacts (identified through test-and-trace programme or contact tracing app) aged 16 years or older and asymptomatic for covid-19 when requesting a test. MAIN OUTCOME MEASURES: Sensitivity, specificity, and positive and negative predictive values of Veritor System (Beckton Dickinson) and Biosensor (Roche Diagnostics) rapid antigen tests, with reverse-transcriptase polymerase chain reaction (RT-PCR) testing as reference standard. The viral load cut-off above which 95% of people with a positive RT-PCR test result were virus culture positive was used as a proxy of infectiousness. RESULTS: Of 2678 participants tested with Veritor, 233 (8.7%) had a RT-PCR confirmed SARS-CoV-2 infection of whom 149 were also detected by the rapid antigen test (sensitivity 63.9%, 95% confidence interval 57.4% to 70.1%). Of 1596 participants tested with Biosensor, 132 (8.3%) had a RT-PCR confirmed SARS-CoV-2 infection of whom 83 were detected by the rapid antigen test (sensitivity 62.9%, 54.0% to 71.1%). In those who were still asymptomatic at the time of sampling, sensitivity was 58.7% (51.1% to 66.0%) for Veritor (n=2317) and 59.4% (49.2% to 69.1%) for Biosensor (n=1414), and in those who developed symptoms were 84.2% (68.7% to 94.0%; n=219) for Veritor and 73.3% (54.1% to 87.7%; n=158) for Biosensor. When a viral load cut-off was applied for infectiouness (≥5.2 log10 SARS-CoV-2 E gene copies/mL), the overall sensitivity was 90.1% (84.2% to 94.4%) for Veritor and 86.8% (78.1% to 93.0%) for Biosensor, and 88.1% (80.5% to 93.5%) for Veritor and 85.1% (74.3% to 92.6%) for Biosensor, among those who remained asymptomatic throughout. Specificities were >99%, and positive and negative predictive values were >90% and >95%, for both rapid antigen tests in all analyses. CONCLUSIONS: The sensitivities of both rapid antigen tests in asymptomatic and presymptomatic close contacts tested on day 5 onwards after close contact with an index case were more than 60%, increasing to more than 85% after a viral load cut-off was applied as a proxy for infectiousness.

Neutralising Antibodies against Enterovirus and Parechovirus in IVIG Reflect General Circulation: A Tool for Sero-Surveillance.

Non-polio enteroviruses (NPEV) and parechoviruses (PeV) are widespread pathogens that cause significant morbidity. Surveillance is based on culturing or genotyping of virus strains found in clinical samples. Sero-surveillance, by measuring neutralising antibodies (nAb) through virus neutralisation assays (VNA), could provide additional information as it offers a more comprehensive overview of exposure to circulating types in the general population. In our study we evaluated Intravenous immunoglobulins (IVIG) to generate sero-surveillance data. We performed VNA of nineteen NPEV and PeV with Dutch IVIG batches from two different time points (2010 and 2017) and an IVIG batch from Vietnam (2011). We compared our findings with geno- and sero-surveillance data and evaluated changes over time and between the two countries. Our findings show a good correlation with what is known from geno-surveillance data. The highest nAb titres were found against strains from Enterovirus B, while we did not observe nAb titres against strains belonging to Enterovirus C. In conclusion, we demonstrated that sero-surveillance by means of IVIG can be used to obtain insight into circulation of EV and PeV genotypes. This is of particular interest for public health, to evaluate changes over time and population susceptibility to emerging genotypes.

World-Wide Prevalence and Genotype Distribution of Enteroviruses.

Enteroviruses (EVs) are highly prevalent viruses world-wide, causing a wide range of diseases in both children and adults. Insight in the global prevalence of EVs is important to define their clinical significance and total disease burden, and assists in making therapeutic decisions. While many studies have been conducted to describe epidemiology of EVs in specific (sub)populations and patient cohorts, little effort has been made to aggregate the available evidence. In the current study, we conducted a search in the PubMed and Embase (Ovid) databases to identify articles reporting EV prevalence and type distribution. We summarized the findings of 153 included studies. We found that EVs are highly prevalent viruses in all continents. Enterovirus B was the most detected species worldwide, while the other species showed continent-specific differences, with Enterovirus C more detected in Africa and Enterovirus A more detected in Asia. Echovirus 30 was by far the most detected type, especially in studies conducted in Europe. EV types in species Enterovirus B-including echovirus 30-were often detected in patient groups with neurological infections and in cerebrospinal fluid, while Enterovirus C types were often found in stool samples.

Eligibility for Liver Transplantation in Patients with Perihilar Cholangiocarcinoma.

BACKGROUND: Liver transplantation (LT) has been performed in a select group of patients presenting with unresectable or primary sclerosing cholangitis (PSC)-associated perihilar cholangiocarcinoma (pCCA) in the Mayo Clinic with a reported 5-year overall survival (OS) of 53% on intention-to-treat analysis. The objective of this study was to estimate eligibility for LT in a cohort of pCCA patients in two tertiary referral centers. METHODS: Patients diagnosed with pCCA between 2002 and 2014 were included from two tertiary referral centers in the Netherlands. The selection criteria used by the Mayo Clinic were retrospectively applied to determine the proportion of patients that would have been eligible for LT. RESULTS: A total of 732 consecutive patients with pCCA were identified, of whom 24 (4%) had PSC-associated pCCA. Overall, 154 patients had resectable disease on imaging and 335 patients were ineligible for LT because of lymph node or distant metastases. An age limit of 70 years led to the exclusion of 50 patients who would otherwise be eligible for LT. After applying the Mayo Clinic criteria, only 34 patients (5%) were potentially eligible for LT. Median survival from diagnosis for these 34 patients was 13 months (95% CI 3-23). CONCLUSION: Only 5% of all patients presenting with pCCA were potentially eligible for LT under the Mayo criteria. Without transplantation, a median OS of about 1 year was observed.

Recombination Analysis of Non-Poliovirus Members of the Enterovirus C Species; Restriction of Recombination Events to Members of the Same 3DPol Cluster.

Enteroviruses (EVs) are highly prevalent viruses worldwide. Recombination is known to occur frequently in EVs belonging to species Enterovirus A, Enterovirus B, and Enterovirus C. Although many recombinant vaccine-derived poliovirus (VDPV) strains have been reported, our knowledge on recombination in non-polio EVs in the species Enterovirus C is limited. Here, we combined a dataset consisting of 11 newly generated full-length Enterovirus C sequences and 180 publicly available sequences to study recombination dynamics in non-polio EVs. To identify recombination patterns, maximum likelihood phylogenetic trees of different genomic regions were constructed, and segregation analyses were performed. Recombination was observed between members of the same 3DPol cluster, but was rarely observed between members of different clusters. We hypothesize that this restriction may have arisen through their different compartmentalization in respiratory and enteric tracts related to differences in cellular tropisms so that the opportunity to recombine may not be available.

Parechovirus A prevalence in adults in The Netherlands.

Human parechoviruses (HPeV) of the species Parechovirus A are highly prevalent disease-causing pathogens in children worldwide. HPeVs are capable of causing severe disease in adults as well, but the prevalence in adults may be much lower. The aim of our present study was to determine the prevalence of HPeV in clinical samples from adults sent in for diagnostic procedures in a tertiary hospital in the Netherlands. From a total of 10,645 samples obtained from 6175 patients, 20 samples from 11 patients (0.18%) tested positive for HPeV by RT-PCR. Two patients were positive for HPeV-1, two for HPeV-3, and one for HPeV-6. Six HPeVs could not be typed. Eight of the 11 HPeV-positive patients were immunocompromised. Due to comorbidity, we were unable to attribute the patients' clinical symptoms to the HPeV infection. The HPeV prevalence in adults found in this study is low compared to HPeV prevalence in children. This may be largely explained by the high seropositivity rates in adults, although there could be other mechanisms involved.

Parechovirus A Pathogenesis and the Enigma of Genotype A-3.

ParechovirusA is a species in the Parechovirus genus within the Picornaviridae family that can cause severe disease in children. Relatively little is known on ParechovirusA epidemiology and pathogenesis. This review aims to explore the ParechovirusA literature and highlight the differences between ParechovirusA genotypes from a pathogenesis standpoint. In particular, the curious case of Parechovirus-A3 and the genotype-specific disease association will be discussed. Finally, a brief outlook on ParechovirusA research is provided.

High frequency and diversity of parechovirus A in a cohort of Malawian children.

Parechoviruses (PeVs) are highly prevalent viruses worldwide. Over the last decades, several studies have been published on PeV epidemiology in Europe, Asia and North America, while information on other continents is lacking. The aim of this study was to describe PeV circulation in a cohort of children in Malawi, Africa. A total of 749 stool samples obtained from Malawian children aged 6 to 60 months were tested for the presence of PeV by real-time PCR. We performed typing by phylogenetic and Basic Local Alignment Search Tool (BLAST) analysis. PeV was found in 57% of stool samples. Age was significantly associated with PeV positivity (p = 0.01). Typing by phylogenetic analysis resulted in 15 different types, while BLAST typing resulted in 14 different types and several indeterminate strains. In total, six strains showed inconsistencies in typing between the two methods. One strain, P02-4058, remained untypable by all methods, but appeared to belong to the recently reclassified PeV-A19 genotype. PeV-A1, -A2 and -A3 were the most prevalent types (26.8%, 13.8% and 9.8%, respectively). Both the prevalence and genetic diversity found in our study were remarkably high. Our data provide an important contribution to the scarce data available on PeV epidemiology in Africa.

Seroepidemiology of Parechovirus A3 Neutralizing Antibodies, Australia, the Netherlands, and United States.

Recent parechovirus A3 (PeV-A3) outbreaks in Australia suggest lower population immunity compared with regions that have endemic PeV-A3 circulation. A serosurvey among populations in the Netherlands, the United States, and Australia before and after the 2013 Australia outbreak showed high PeV-A3 neutralizing antibody prevalence across all regions and time periods, indicating widespread circulation.

High frequency of Polio-like Enterovirus C strains with differential clustering of CVA-13 and EV-C99 subgenotypes in a cohort of Malawian children.

Enteroviruses (EVs) are among the most commonly detected viruses infecting humans worldwide. Although the prevalence of EVs is widely studied, the status of EV prevalence in sub-Saharan Africa remains largely unknown. The objective of our present study was therefore to increase our knowledge on EV circulation in sub-Saharan Africa. We obtained 749 fecal samples from a cross-sectional study conducted on Malawian children aged 6 to 60 months. We tested the samples for the presence of EVs using real time PCR, and typed the positive samples based on partial viral protein 1 (VP1) sequences. A large proportion of the samples was EV positive (89.9%). 12.9% of the typed samples belonged to EV species A (EV-A), 48.6% to species B (EV-B) and 38.5% to species C (EV-C). More than half of the EV-C strains (53%) belonged to subgroup C containing, among others, Poliovirus (PV) 1-3. The serotype most frequently isolated in our study was CVA-13, followed by EV-C99. The strains of CVA-13 showed a vast genetic diversity, possibly representing a new cluster, 'F'. The majority of the EV-C99 strains grouped together as cluster B. In conclusion, this study showed a vast circulation of EVs among Malawian children, with an EV prevalence of 89.9%. Identification of prevalences for species EV-C comparable to our study (38.5%) have only previously been reported in sub-Saharan Africa, and EV-C is rarely found outside of this region. The data found in this study are an important contribution to our current knowledge of EV epidemiology within sub-Saharan Africa.