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BACKGROUND: Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of nosocomial infection, driven by its ability to spread between patients and persist in the hospital environment. Here, we investigated the impact of a long-established cardiothoracic hospital moving to new premises with close to 100% single-occupancy rooms on the rates of environmental contamination and infection or colonisation by VRE. METHOD: Prospective environmental surveillance for VRE was conducted at five time-points between April and November 2019, once in the original building, and four times in the new building. Flocked swabs (n=100/time-point) were used to sample bedspaces, bathrooms, computers and sluices in the critical care unit and the cardiothoracic ward. Environmental VRE was supplemented by clinical VRE isolates from the same time period, and underwent whole genome sequencing. Incidence rate ratios (IRR) of VRE infection/colonisation was determined for the one-year period before and after the hospital move, and compared to a nearby hospital. FINDINGS: In the original location, the first environmental screen found 29% VRE positivity. The following four screens in the new location showed a significant reduction in positivity (1-6%, p

BackgroundUnregulated antimicrobial use is common in both hospital and community settings of low- and middle-income countries (LMICs). However, discrete data regarding the use/misuse of antimicrobials at pharmacies in LMICs are limited. This study was conducted to understand the knowledge, attitude, and practice of pharmacy employees on antimicrobial dispensing in Nepal.MethodsWe conducted a cross-sectional survey using a structured questionnaire on 801 pharmacy employees working in community and hospital pharmacies located in Lalitpur metropolitan city (LMC) of Kathmandu, Nepal between April 2017 and March 2019.ResultsNot all respondents replied to all questions asked. A majority (92%, 738/801) of respondents agreed that the demand for non-prescription antimicrobials was common. Sixty nine percent (437/635) of participants responded that they would ask of prescription before dispensing. Suspected respiratory tract infection was the most common reason demanding for non-prescription antimicrobials, identified by 68% (535/792) of respondents. Azithromycin was the most commonly prescribed and sold antimicrobials, as reported by 46%, (361/787) and 48% (377/779) of participants respectively. A majority (87%; 696/800) of respondents agreed on antimicrobial resistance (AMR) to be a global public health threat; 54% (429/796) perceived antimicrobial misuse to be an important driver of AMR, while only 39% (315/801) replied that judicious dispensing of antimicrobials can help curb AMR.ConclusionOur study revealed that the unfounded dispensing and use of antimicrobials is prevalent among pharmacies in Kathmandu, Nepal. This overreliance on antimicrobials, notably azithromycin, may escalated the burden of AMR. We identified several drivers of inappropriate antimicrobial dispensing practice in pharmacies, which will aid public health authorities in addressing these issues. Further studies considering the role of other stakeholders, such as physicians, veterinarians, general public, and policy makers are required to obtain a more holistic perspectives on the practices of antimicrobial use so to curb the extant AMR crisis.

Background A considerable portion of the HIV pandemic is composed of people under antiretroviral therapy, many of whom get a late diagnosis. Patients starting antiretroviral therapy (ART) at a very advanced stage of HIV disease attain a low recovery of CD4 T cells. Factors associated with poor recovery are incompletely described. This study aimed at finding variables associated with CD4 T cell recovery in late-presenting HIV patients. Methods We studied a cohort of HIV+ patients initiating ART with very low basal CD4 T cell counts. We defined immune recovery as the net increase in circulating CD4 T cell counts after one year on ART. We analyzed diverse routine laboratory determinations at different times using Least Absolute Shrinkage and Selection Operator (LASSO), adaptive LASSO and Conditional Inference Random Forest. Results CD4/CD8 ratio, % CD4 T cells and CD8 T cell counts at different times were the main recovery correlates, validated by all approaches. Unexpectedly, basal hematocrit was a consistent predictor. Additionally, week 24 creatinine had a high lasso coefficient, and alkaline phosphatase had a high conditional inference random forest coefficients, although neither was verified by other tests. Conclusions CD4 T cell proportions are associated with CD4 T cell recovery, independently of cell counts. Inflammation-related variables could also affect reconstitution. These accessible variables may reflect underlying mechanisms and could improve the follow up of patients starting ART with an advanced HIV infection.

BACKGROUND: An estimated 3.2 million patients annually develop healthcare-associated infections (HCAIs) in Europe alone amid the major challenge of increasing antimicrobial resistance. Critically ill children warrant specific evaluation because of differences in epidemiology, causative organisms and infection sites. OBJECTIVES: To examine the prevalence and antimicrobial susceptibility patterns of three types of HCAI in critically ill children and determine the effect on their disease course. MATERIALS AND METHODS: Retrospective cohort review of critically ill children admitted to a general paediatric ICU (PICU) at a regional academic tertiary referral centre over a 3 year period. RESULTS: There were 1930 admissions with a median age of 38 months. Children with HCAIs had a higher incidence of comorbidities (74% versus 24%) and a longer median length of stay (8 days versus 3 days). We identified 26 positive isolates (blood, lower respiratory and urine) taken 48 h or more after admission. The combined incidence was 1.34%. Hospital-acquired pneumonia accounted for 58% of HCAIs, urinary tract infections for 31% and bloodstream infections for 11%. The majority (61.5%) of HCAIs were caused by Gram-negative organisms. Seven isolates were resistant to antimicrobials used to treat HCAI. All of these were Gram-negative organisms (Pseudomonas aeruginosa, Klebsiella oxytoca and Escherichia coli). CONCLUSIONS: These data revealed a low incidence of HCAIs, 27% of which were resistant Gram-negative organisms. Critically ill children with HCAIs were more likely to have comorbidities and an increased length of stay. These factors may increasingly impact on PICU bed availability, an already limited resource.

Background Typhoid fever remains a significant cause of morbidity and mortality in Asia and Africa. The emergence of azithromycin resistance in South Asia is concerning, as azithromycin is one of the last effective oral drugs for treating typhoid. Objectives To describe the molecular mechanism and phylogenetics of azithromycin-resistant (AzithR) Salmonella Typhi isolates from Patan Hospital, Kathmandu, Nepal. Methods Whole-genome sequences of three AzithR S. Typhi isolates (MIC >256 mg/L) were analysed and compared with a global collection to investigate the azithromycin resistance mechanism and phylogenetic structure. Clinical information is reported for one of the three patients infected with AzithR S. Typhi. Results The three AzithR isolates belonged to the H58 lineage and were genetically identical; they were distantly related to contemporaneous S. Typhi from Nepal and AzithR S. Typhi recently described in Bangladesh. Azithromycin resistance was mediated by a non-synonymous mutation in the acrB gene (R717L). The three AzithR isolates showed reduced susceptibility to ciprofloxacin (double mutation in the gyrA: S83F and D87G), and were susceptible to ampicillin, chloramphenicol and co-trimoxazole. Clinical information from one patient suggested non-response to azithromycin treatment. Conclusions This is the first molecular description of AzithR S. Typhi in Nepal. These organisms showed no phylogenetic link to AzithR S. Typhi in Bangladesh. Our data suggest that increasing use of azithromycin may pose a strong selective pressure driving the emergence of AzithR S. Typhi in South Asia. Further investigations are needed to evaluate treatment responses to azithromycin, predict evolutionary trajectories, and track the transmission of these organisms.

Background. Hypervirulent Klebsiella pneumoniae causes severe disseminated infections, typically with hepatic and central nervous system involvement including endophthalmitis. Case Presentation. We report a fatal case of an undocumented Chinese migrant in her 60s who presented to the emergency department with abdominal pain, lethargy, and headache over the preceding two weeks. She had a new diagnosis of diabetes mellitus on admission. Computed tomography scan of the thorax, abdomen, and pelvis showed bilateral pneumonia with liver abscesses. The patient was treated with empirical broad-spectrum antibiotics before K. pneumoniae was isolated from cerebrospinal fluid and blood cultures. The isolate was further characterised as a ST23 (ST: sequence type), serotype K1 hypervirulent strain using Nanopore sequencing. Despite admission to the intensive care unit, the patient died within 48 hrs of admission. Conclusions. This case demonstrates the need for increased awareness of hypervirulent K. pneumoniae, even in countries where it occurs infrequently. Novel, rapid, sequencing technologies can support diagnosis in unusual presentations.

Funder: Action Medical Research; Grant(s), Funder: Academy of Medical Sciences; Grant(s), Funder: NIHR Cambridge Biomedical Research Centre; Grant(s), Funder: Gates Cambridge Trust; Grant(s), Background: Broad-spectrum antimicrobial therapy is a key driver of antimicrobial resistance. Here, we aimed to review indications for antimicrobial therapy, determine the proportion of suspected bacterial infections that are confirmed by culture, and assess the time taken for microbiology test results to become available in the paediatric intensive care unit (PICU). Methods: A single-centre prospective observational cohort study of 100 consecutive general PICU admissions from 30 October 2019 to 19 February 2020. Data were collected from the hospital medical record and entered into a study database prior to statistical analysis using standard methods. Results: Of all episodes of suspected infection, 22% of lower respiratory tract infection, 43% of bloodstream and 0% of central nervous system infection were associated with growth on microbiology culture. 90% of children received antimicrobial therapy. Hospital-acquired infection occurred less commonly than primary infection, but an organism was grown in a greater proportion (64%) of cultures. Final laboratory reports for negative cultures were issued at a median of 120.3 hours for blood cultures and 55.5 hours for endotracheal tube aspirate cultures. Conclusions: Despite most critically children receiving antimicrobial therapy, infection was often not microbiologically confirmed. Novel molecular diagnostics may improve rationalisation of treatment in this population.

Chronic active Epstein Barr Virus (CAEBV) is a rare condition occurring in previously healthy individuals associated with persistent EBV viraemia, fever, lymphadenopathy and hepatosplenomegaly. Viral deletions have been found in CAEBV and other lymphomas. However, it is unclear how stable these deletions are, whether they are present in different sites and how they evolve overtime. We sequenced fourteen longitudinal blood samples from three European CAEBV patients and compared with CAEBV saliva samples and other sequences from EBV-related conditions. We observed large EBV deletions in blood, but not saliva from CAEBV patients. Deletions were stable over time but were lost following successful treatment. Our results are consistent with the likelihood that certain deletions in the virus from CAEBV patients are associated with the evolution and persistence of haematological clones. We propose that the loss of deletions following successful treatment should be investigated as a potential biomarker to aid CAEBV management.

Pneumonia, an inflammatory infiltrate of the alveolar airspace, is commonly triggered by bacterial infection of the lungs, or less commonly by viral or fungal infection. It remains the commonest infective reason for admission to Intensive Care as well as being the most common secondary infection acquired whilst in the Intensive Care Unit. It presents a significant global burden of disease and is especially prevalent in low- and middle-income countries. The major categories of pneumonia encountered by the Intensive Care clinician are community-acquired, ventilator-acquired, non-ventilator hospital-acquired and pneumonia in the immunocompromised patient. An appreciation of the type of pneumonia a patient has developed is critical to its effective treatment. Pneumonia is the commonest precipitant of acute respiratory distress syndrome (ARDS) and clinicians should be mindful that the evidence-base surrounding ARDS will, in large part, apply to severe pneumonia. The causative organisms which lead to pneumonia vary depending on the site of acquisition (community or hospital-acquired), the immune status of the patient and the presence of intercurrent medications including antibiotics. Current standard microbiological testing is seldom able to give a rapid answer as to which microorganisms are present and causing infection. Therefore, empirical therapy guided by a knowledge of local microbial flora and resistance patterns is the recommended course of action. This approach risks the over-treatment of pneumonia with unnecessarily broad-spectrum agents which bring with them the problems of antibiotic-associated harm. Novel rapid diagnostic tests aimed at both the pathogen and the host response hold promise in the rationalisation and appropriate targeting of antimicrobial therapy. At present neither scoring systems nor diagnostic tests are able to accurately risk stratify a patient’s need for intensive care admission. Beyond antibiotic therapy, a number of adjuvant therapies have been trialled in pneumonia although none have yet made it into widespread clinical use. Corticosteroids are recommended in some cases of community-acquired pneumonia, but their role in the patient with severe community-acquired pneumonia in ICU remains uncertain whilst they are a risk factor for the development of hospital and ventilator-acquired pneumonia. Immuno-stimulation has not yet translated from small scale clinical trials into clinical use. Supportive management includes lung protective ventilation, and those interventions proven to improve outcomes in ARDS. This review will give an overview of the epidemiology of severe pneumonia, the microbiological causes and diagnostic strategies. It will then turn to management, including antimicrobial therapy, role of adjuvant therapies, respiratory support and prevention of complications.

Background: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. Methods: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. Results: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4+ T cell (Treg), effector T cell (Teff) CD4+ and CD8+ subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69+ and Ki-67+ effector memory Teffs were followed by the emergence of memory CD8+ Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. Conclusions: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.

Background: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. Methods: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. Results: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4+ T cell (Treg), effector T cell (Teff) CD4+ and CD8+ subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69+ and Ki-67+ effector memory Teffs were followed by the emergence of memory CD8+ Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. Conclusions: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.