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Background: Symptoms of post-acute sequelae of COVID-19 (PASC) may improve following SARS-CoV-2 vaccination. However few prospective data that also explore the underlying biological mechanism are available. We assessed the effect of vaccination on symptomatology of participants with PASC, and compared antibody dynamics between those with and without PASC. Methods: RECoVERED is a prospective cohort study of adult patients with mild to critical COVID-19, enrolled from illness onset. Among participants with PASC, vaccinated participants were exact-matched 1:1 on age, sex, obesity status and time since illness onset to unvaccinated participants. Between matched pairs, we compared the monthly mean numbers of symptoms over a 3-month follow-up period, and, using exact logistic regression, the proportion of participants who fully recovered from PASC. Finally, we assessed the association between PACS status and rate of decay of spike- and RBD-binding IgG titers up to 9 months after illness onset using Bayesian hierarchical linear regression. Findings: Of 349 enrolled participants, 316 (90.5%) had ≥3 months of follow-up, of whom 186 (58.9%) developed PASC. Among 36 matched pairs with PASC, the mean number of symptoms reported each month during 3 months of follow-up were comparable between vaccinated and unvaccinated groups. Odds of full recovery from PASC also did not differ between matched pairs (OR 1.57 [95%CI 0.46–5.84]) within 3 months after the matched time-point. The median half-life of spike- and RBD-binding IgG levels were, in days (95%CrI), 233 (183–324) and 181 (147–230) among participants with PASC, and 170 (125–252) and 144 (113–196) among those without PASC, respectively. Interpretation: Our study found no strong evidence to suggest that vaccination improves symptoms of PASC. This was corroborated by comparable spike- and RBD-binding IgG waning trajectories between those with and without PASC, refuting any immunological basis for a therapeutic effect of vaccination on PASC.

Background Few robust longitudinal data on long-term coronavirus disease 2019 (COVID-19) symptoms are available. We evaluated symptom onset, severity and recovery across the full spectrum of disease severity, up to one year after illness onset. Methods The RECoVERED Study is a prospective cohort study based in Amsterdam, the Netherlands. Participants aged ≥18 years were enrolled following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis via the local public health service and from hospitals. Standardized symptom questionnaires were completed at enrollment, 1 week and month later, and monthly thereafter. Clinical severity was defined according to World Health Organization (WHO) criteria. Kaplan-Meier methods were used to compare time from illness onset to symptom recovery, by clinical severity. We examined determinants of time to recovery using multivariable Cox proportional hazards models. Results Between 11 May 2020 and 1 May 2021, 342 COVID-19 patients (192 [56%] male) were enrolled, of whom 99/342 (29%) had mild, 145/342 (42%) moderate, 56/342 (16%) severe, and 42/342 (12%) critical disease. The proportion of participants who reported at least 1 persistent symptom at 12 weeks after illness onset was greater in those with severe/critical disease (86.7% [95% confidence interval {CI} = 76.5–92.7%]) compared to those with mild or moderate disease (30.7% [95% CI = 21.1–40.9%] and 63.8% [95% CI = 54.8–71.5%], respectively). At 12 months after illness onset, two-fifths of participants (40.7% [95% CI = 34.2–7.1]) continued to report ≥1 symptom. Recovery was slower in female compared to male participants (adjusted hazard ratio [aHR] 0.65 [95% CI = .47–.92]) and those with a body mass index [BMI] ≥30kg/m2 compared to BMI Conclusions COVID-19 symptoms persisted for one year after illness onset, even in some individuals with mild disease. Female sex and obesity were the most important determinants of speed of recovery from symptoms.

BackgroundSemi-quantitative bacterial culture is the reference standard to diagnose urinary tract infection, but culture is time-consuming and can be unreliable if patients are receiving antibiotics. Metagenomics could increase diagnostic accuracy and speed by sequencing the microbiota and resistome directly from urine. We aimed to compare metagenomics to culture for semi-quantitative pathogen and resistome detection from urine. MethodsIn this proof-of-concept study, we prospectively included consecutive urine samples from a clinical diagnostic laboratory in Amsterdam. Urine samples were screened by DNA concentration, followed by PCR-free metagenomic sequencing of randomly selected samples with a high concentration of DNA (culture positive and negative). A diagnostic index was calculated as the product of DNA concentration and fraction of pathogen reads. We compared results with semi-quantitative culture using area under the receiver operating characteristic curve (AUROC) analyses. We used ResFinder and PointFinder for resistance gene detection and compared results to phenotypic antimicrobial susceptibility testing for six antibiotics commonly used for urinary tract infection treatment: nitrofurantoin, ciprofloxacin, fosfomycin, cotrimoxazole, ceftazidime, and ceftriaxone. FindingsWe screened 529 urine samples of which 86 were sequenced (43 culture positive and 43 culture negative). The AUROC of the DNA concentration-based screening was 0·85 (95% CI 0·81-0·89). At a cutoff value of 6·0 ng/mL, culture positivity was ruled out with a negative predictive value of 91% (95% CI 87-93; 26 of 297 samples), reducing the number of samples requiring sequencing by 56% (297 of 529 samples). The AUROC of the diagnostic index was 0·87 (95% CI 0·79-0·95). A diagnostic index cutoff value of 17·2 yielded a positive predictive value of 93% (95% CI 85-97) and a negative predictive value of 69% (55-80), correcting for a culture-positive prevalence of 66%. Gram-positive pathogens explained eight (89%) of the nine false-negative metagenomic test results. Agreement of phenotypic and genotypic antimicrobial susceptibility testing varied between 71% (22 of 31 samples) and 100% (six of six samples), depending on the antibiotic tested. InterpretationThis study provides proof-of-concept of metagenomic semi-quantitative pathogen and resistome detection for the diagnosis of urinary tract infection. The findings warrant prospective clinical validation of the value of this approach in informing patient management and care. FundingEU Horizon 2020 Research and Innovation Programme.

Previous studies have shown high acquisition risks of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) among international travelers visiting antimicrobial resistance (AMR) hotspots. Although antibiotic use and travelers’ diarrhea have shown to influence the ESBL-E acquisition risk, it remains largely unknown whether successful colonization of ESBL-E during travel is associated with the composition, functional capacity and resilience of the traveler’s microbiome. The microbiome of pre- and post-travel fecal samples from 190 international travelers visiting Africa or Asia was profiled using whole metagenome shotgun sequencing. A metagenomics species concept approach was used to determine the microbial composition, population diversity and functional capacity before travel and how it is altered longitudinally. Eleven travelers were positive for ESBL-E before travel and removed from the analysis. Neither the microbial richness (Chao1), diversity (effective Shannon) and community structure (Bray–Curtis dissimilarity) in pretravel samples nor the longitudinal change of these metrics during travel were predictive for ESBL-E acquisition. A zero-inflated two-step beta-regression model was used to determine how the longitudinal change in both prevalence and abundance of each taxon was related to ESBL acquisition. There were detected increases in both the prevalence and abundance of Citrobacter freundii and two members of the genus Bacteroides, in association with remaining uncolonized by ESBL-E. These results highlight the potential of these individual microbes as a microbial consortium to prevent the acquisition of ESBL-E. The ability to alter a person’s colonization resistance to a bacterium could be key to intervention strategies that aim to minimize the spread of MDR bacteria.

Background Since their emergence in Indonesia in 2005, 200 human infections with clade 2.1 highly pathogenic avian influenza A/H5N1 virus have been reported, associated with exceptionally high mortality (84%) compared to regions affected by other genetic clades of this virus. To provide potential clues towards understanding this high mortality, detailed clinical virological analyses were performed in specimens from 180 H5N1 patients, representing 90% of all Indonesian patients and 20% of reported H5N1-infected patients globally. Methods H5N1 RNA was quantified in available upper- and lower-respiratory tract specimens as well as fecal and blood samples from 180 patients with confirmed infection between 2005 and 2017. Mutations in the neuraminidase and M2 genes that confer resistance to oseltamivir and adamantanes were assessed. Fatal and nonfatal cases were compared. Results High viral RNA loads in nasal and pharyngeal specimens were associated with fatal outcome. Mortality increased over time during the study period, which correlated with increasing viral RNA loads on admission. Furthermore, the prevalence of amantadine resistance–conferring M2 mutations increased over time, and viral loads were higher in patients infected with viruses that harbored these mutations. Compared to observations from other regions, viral RNA was detected more frequently in feces (80%) and particularly in blood (85%), and antiviral responses to oseltamivir appeared less pronounced. Conclusions These observations confirm the association of viral load with outcome of human H5N1 infections and suggest potential differences in virulence and antiviral responses to oseltamivir that may explain the exceptionally high mortality related to clade 2.1 H5N1 infections in Indonesia., Detailed virological analyses of 180 Indonesian patients infected with avian influenza A/H5N1 virus point to potential differences in virulence and antiviral responses to oseltamivir that may explain the exceptionally high mortality related to these infections in Indonesia compared to other regions.

BackgroundSingle-dose baloxavir rapidly reduces influenza virus titers and symptoms in patients with uncomplicated influenza, but viruses with reduced in vitro susceptibility due to amino acid substitutions at position 38 of polymerase acidic protein (PA/I38X) sometimes emerge.MethodsWe evaluated the kinetics, risk factors, and effects on clinical and virologic outcomes of emergence of PA/I38X-substituted viruses.ResultsViruses containing PA/I38X substitutions were identified 3–9 days after baloxavir treatment in 9.7% (36/370) of patients, of whom 85.3% had transient virus titer rises. Median time to sustained cessation of infectious virus detection was 192, 48, and 96 hours in the baloxavir recipients with PA/I38X-substituted viruses, without PA/I38X-substituted viruses, and placebo recipients, respectively. The corresponding median times to alleviation of symptoms were 63.1, 51.0, and 80.2 hours, respectively. After day 5, symptom increases occurred in 11.5%, 8.0%, and 13.0%, respectively, and in 8.9% of oseltamivir recipients. Variant virus emergence was associated with lower baseline neutralizing antibody titers.ConclusionsThe emergence of viruses with PA/I38X substitutions following baloxavir treatment was associated with transient rises in infectious virus titers, prolongation of virus detectability, initial delay in symptom alleviation, and uncommonly with symptom rebound. The potential transmissibility of PA/I38X-substituted viruses requires careful study.Clinical Trial RegistrationNCT02954354.

We describe the lessons learned during a SARS-CoV-2 variant-of-concern Alpha outbreak investigation at a normal care unit in a university hospital in Amsterdam in December 2020. The outbreak consisted of nine nurses and two roomed-in patient family members. (attack rate 18%). One nurse tested positive with a phylogenetically distinct variant, after a documented infection 83 days prior. Three key points were taken from this investigation. First, it was controlled by adherence to existing guidelines, despite increased transmissibility of the variant. Second, viral sequencing can inform transmission cluster inference, but the epidemiological context is essential to draw appropriate conclusions. Third, reinfections with Alpha variants can occur rapidly after primary infection.

Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similarly to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants of concern (VOCs) in a set of sera from patients infected with viral sequence-confirmed VOCs. Infections with D614G or Alpha strains induced the broadest immunity, whereas individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 were antigenically most distinct from D614G, associated with immune escape, and possibly will require vaccine updates to ensure vaccine effectiveness.

Altered pharmacokinetics (PK) of hydrophilic antibiotics in critically ill patients is common, with possible consequences for efficacy and resistance. We aimed to describe ceftazidime population PK in critically ill patients with a proven or suspected Pseudomonas aeruginosa infection and to establish optimal dosing. Blood samples were collected for ceftazidime concentration measurement. A population PK model was constructed, and probability of target attainment (PTA) was assessed for targets 100% T &gt, MIC and 100% T &gt, 4 × MIC in the first 24 h. Ninety-six patients yielded 368 ceftazidime concentrations. In a one-compartment model, variability in ceftazidime clearance (CL) showed association with CVVH. For patients not receiving CVVH, variability in ceftazidime CL was 103.4% and showed positive associations with creatinine clearance and with the comorbidities hematologic malignancy, trauma or head injury, explaining 65.2% of variability. For patients treated for at least 24 h and assuming a worst-case MIC of 8 mg/L, PTA was 77% for 100% T &gt, MIC and 14% for 100% T &gt, 4 × MIC. Patients receiving loading doses before continuous infusion demonstrated higher PTA than patients who did not (100% T &gt, MIC: 95% (n = 65) vs. 13% (n = 15), p &lt, 0.001 and 100% T &gt, 4 × MIC: 20% vs. 0%, p = 0.058). The considerable IIV in ceftazidime PK in ICU patients could largely be explained by renal function, CVVH use and several comorbidities. Critically ill patients are at risk for underexposure to ceftazidime when empirically aiming for the breakpoint MIC for P. aeruginosa. A loading dose is recommended.

A Ugandan child with an unexplained encephalitis was investigated using viral metagenomics. Several sequences from all segments of a novel orthobunyavirus were found. The S-segment, used for typing, showed 41% amino acid diversity to its closest relative. The virus was named Ntwetwe virus, after the hometown of the patient.

Background and objectives Treatment guidelines do not recommend antibiotic use for acute respiratory infections (ARI), except for streptococcal pharyngitis/tonsillitis and pneumonia. However, antibiotics are prescribed frequently for children with ARI, often in absence of evidence for bacterial infection. The objectives of this study were 1) to assess the appropriateness of antibiotic prescriptions for mild ARI in paediatric outpatients in relation to available guidelines and detected pathogens, 2) to assess antibiotic use on presentation using questionnaires and detection in urine 3) to assess the carriage rates and proportions of resistant intestinal Enterobacteriaceae before, during and after consultation. Materials and methods Patients were prospectively enrolled in Children’s Hospital 1, Ho Chi Minh City, Vietnam and diagnoses, prescribed therapy and outcome were recorded on first visit and on follow-up after 7 days. Respiratory bacterial and viral pathogens were detected using molecular assays. Antibiotic use before presentation was assessed using questionnaires and urine HPLC. The impact of antibiotic usage on intestinal Enterobacteriaceae was assessed with semi-quantitative culture on agar with and without antibiotics on presentation and after 7 and 28 days. Results A total of 563 patients were enrolled between February 2009 and February 2010. Antibiotics were prescribed for all except 2 of 563 patients. The majority were 2nd and 3rd generation oral cephalosporins and amoxicillin with or without clavulanic acid. Respiratory viruses were detected in respiratory specimens of 72.5% of patients. Antibiotic use was considered inappropriate in 90.1% and 67.5%, based on guidelines and detected pathogens, respectively. On presentation parents reported antibiotic use for 22% of patients, 41% of parents did not know and 37% denied antibiotic use. Among these three groups, six commonly used antibiotics were detected with HPLC in patients’ urine in 49%, 40% and 14%, respectively. Temporary selection of 3rd generation cephalosporin resistant intestinal Enterobacteriaceae during antibiotic use was observed, with co-selection of resistance to aminoglycosides and fluoroquinolones. Conclusions We report overuse and overprescription of antibiotics for uncomplicated ARI with selection of resistant intestinal Enterobacteriaceae, posing a risk for community transmission and persistence in a setting of a highly granular healthcare system and unrestricted access to antibiotics through private pharmacies. Registration This study was registered at the International Standard Randomised Controlled Trials Number registry under number ISRCTN32862422: http://www.isrctn.com/ISRCTN32862422

Summary: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy. Methods: This prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course. Findings: Our cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41–78). Median disease course (time from onset of symptoms to death) was 22 days (range 5–44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5–44]; ten patients with neutrophilic plugs, 21 days [5–44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets.. Interpretation: In patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19. Funding: Amsterdam UMC Corona Research Fund.

Background Laboratory testing for respiratory tract infections (RTIs) is not routinely performed in primary care. Their etiology is usually unknown, and although RTIs are mainly of viral origin, the contribution of different respiratory viruses is uncertain. Our study aims to increase our insight into the epidemiology of respiratory viruses in primary care and to evaluate the accuracy of the general practitioner's (GP's) clinical diagnosis of influenza virus infection. Methods We prospectively recruited patients who presented with RTI symptoms at a primary care facility in Amsterdam, the Netherlands, during the 2015-2016 winter season. Demographic and clinical characteristics of patients were summarized using a questionnaire. Nasopharyngeal swabs were collected and tested with a multiplex polymerase chain reaction assay detecting 14 respiratory viruses. Results One or more respiratory viruses were present in 42.5% of the patients (n = 353). The most frequently detected viruses were rhinovirus (11.6%), human coronavirus (8.8%), and influenza A virus (7.6%). Sensitivity of GP's clinical diagnosis for influenza virus infection was 52.6% and specificity was 78.3%. Conclusions Despite the use of a sensitive polymerase chain reaction, a respiratory virus could be detected in less than half of the patients visiting the GP with RTI symptoms. It is difficult to clinically distinguish influenza from other causes of RTIs. Correct etiological diagnosis of RTIs is needed because it contributes to differential diagnosis, might give direction to development of specific antiviral therapies and vaccines, reduce unnecessary prescription of antibiotics, and clarifies the clinical spectrum of the different respiratory viruses.

Secondary bacterial pneumonia is a frequent complication of influenza, associated with high morbidity and mortality. We hypothesized that treatment with neutralizing influenza A antibody AT10_002 protects against severe secondary pneumococcal infection in a mouse model of influenza A infection. Influenza A (H3N2) virus-infected male C57Bl6 mice were treated intravenously with either AT10_002 or a control 2 days postinfection. Seven days later, both groups were infected with Streptococcus pneumoniae and killed 18 hours later. Mice receiving AT10_002 showed less loss of bodyweight compared with controls (+1% vs -12%, P < .001), lower viral loads in bronchoalveolar lavage fluids (BALFs) (7 vs 194 RNA copies per µL; P < .001), and reduced bacterial outgrowth in lung homogenates (3.3 × 101 vs 2.5 × 105 colony-forming units per mg; P < .001). The treatment group showed lower pulmonary wet weights, lower cell counts, and lower protein levels in BALF compared with controls. Treatment with AT10_002 was associated with lower levels of tumor necrosis factor-α, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (KC), and interferon-γ in BALF and lower IL-6 and KC in lung homogenates. Treatment with anti-influenza antibody AT10_002 is associated with reduced weight loss, viral load, bacterial outgrowth, and lung injury in a murine model of secondary pneumococcal pneumonia following influenza infection.

Supplemental Digital Content is available in the text., Background: Human parechoviruses (HPeVs) are common pathogens in young children, and in the Netherlands, HPeV1, HPeV3 and HPeV4 are the most frequently detected genotypes. HPeV3 in particular has been associated with severe disease in young infants below 3 months of age while the other genotypes more often infect older children and elicit mild symptoms. We investigated if maternal neutralizing antibodies (nAbs) against HPeV1, HPeV3 and HPeV4 protect young Dutch infants from severe disease related to HPeV infection. Methods: We conducted a prospective case–control study of Dutch mother–infant pairs. Thirty-eight HPeV-infected infants and their mothers were included as cases, and 65 HPeV-negative children and their mothers as controls. Results: In control infants, we observed nAb seropositivity rates of 41.4%, 33.3% and 27.6%, with median nAb titers of 1:16, 1:12 and 1:8, against HPeV1, HPeV3 and HPeV4, respectively. In control mothers, nAb seropositivity rates were 84.6%, 55.4% and 60.0% with median nAb titers of 1:128, 1:32 and 1:45 against HPeV1, HPeV3 and HPeV4, respectively. The HPeV3 nAb seroprevalence was significantly lower in HPeV3-infected infants and their mothers (0.0% with P < 0.05 and 10.0% with P < 0.001, respectively). In contrast, no differences in nAb seroprevalence against HPeV1 or HPeV4 could be detected between case and control infants or mothers. Conclusions: Our results suggest that young Dutch infants are protected against severe disease related to HPeV1 and HPeV4 by maternal nAbs, but less so against HPeV3 explaining the distinct age distributions and disease severity profiles of children infected with these HPeV genotypes.

Background: Detecting SARS-CoV-2 antibodies may help to diagnose COVID-19. Head-to-head validation of different types of immunoassays in well-characterized cohorts of hospitalized patients remains needed. Methods: We validated three chemiluminescence immunoassays (CLIAs) (Liaison, Elecsys, and Abbott) and one single molecule array assay (SIMOA) (Quanterix) for automated analyzers, one rapid immunoassay RIA (AllTest), and one ELISA (Wantai) in parallel in first samples from 126 PCR confirmed COVID-19 hospitalized patients and 158 pre-COVID-19 patients. Specificity of the AllTest was also tested in 106 patients with confirmed parasitic and dengue virus infections. Specificity of the Wantai assay was not tested due to limitations in sample volumes. Results: Overall sensitivity in first samples was 70.6 % for the Liaison, 71.4 % for the Elecsys, 75.4 % for the Abbott, 70.6 % for the Quanterix, 77.8 % for the AllTest, and 88.9 % for the Wantai assay, respectively. Sensitivity was between 77.4 % (Liaison) and 94.0 % (Wantai) after 10 dpso. No false positive results were observed for the Elecsys and Abbott assays. Specificity was 91.1 % for the Quanterix, 96.2 % for the Liaison, and 98.1 % for the AllTest assay, respectively. Conclusion: We conclude that low sensitivity of all immunoassays limits their use early after onset of illness in diagnosing COVID-19 in hospitalized patients. After 10 dpso, the Wantai ELISA has a relatively high sensitivity, followed by the point-of-care AllTest RIA that compares favorably with automated analyzer immunoassays.

When treating critically ill patients with gentamicin for severe infection, peak concentrations (C-max) determine clinical efficacy and trough concentrations (C-min) determine toxicity. Despite administration of body weight-standardised starting doses, a wide range of Cmax is generally observed. Furthermore, in therapeutic drug monitoring, several measures of renal function are used to predict appropriate Cmin and gentamicin dosing intervals, but the most accurate predictor is not known. This study aimed to quantify the impact of several patient parameters on gentamicin Cmax values and to determine which measure of renal function best predicts gentamicin clearance (CL). Clinical data and serum gentamicin levels were retrospectively collected from all critically ill patients treated with gentamicin at our intensive care unit between 1 January and 30 June 2011. Data were analysed using non-linear mixed-effects modelling (NONMEM v.7.1.2). A two-compartmental model was developed based on 303 gentamicin concentration-time data from 44 critically ill patients. Serum albumin levels explained 25% of interindividual variability in the volume of distribution (V-d). Creatinine clearance calculated from the creatinine concentration in a 6-h urine portion (CalcCL(Cr)) resulted in acceptable estimation of gentamicin CL, whilst serum creatinine (SCr) and creatinine clearance estimated by the Cockcroft-Gault formula (CGCL(Cr)) overestimated gentamicin CL and therefore underestimated C-min. In conclusion, low albumin concentrations resulted in a larger Vd and lower Cmax of gentamicin. These results suggest that use of a higher gentamicin starting dose in critically ill patients with hypoalbuminaemia may prevent underdosing. Urinary CalcCLCr is a better predictor of C-min than SCr or CGCL(Cr). (C) 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved

Key Points This meta-analysis evaluates the diagnostic accuracy of rapid tests for the detection of respiratory viruses. Sensitivity of tests varied considerably, but specificity was high. Although newly developed tests seem more sensitive, high quality evaluations of these tests are lacking., Rapid diagnosis of respiratory virus infections contributes to patient care. This systematic review evaluates the diagnostic accuracy of rapid tests for the detection of respiratory viruses. We searched Medline and EMBASE for studies evaluating these tests against polymerase chain reaction as the reference standard. Of 179 studies included, 134 evaluated rapid tests for influenza viruses, 32 for respiratory syncytial virus (RSV), and 13 for other respiratory viruses. We used the bivariate random effects model for quantitative meta-analysis of the results. Most tests detected only influenza viruses or RSV. Summary sensitivity and specificity estimates of tests for influenza were 61.1% and 98.9%. For RSV, summary sensitivity was 75.3%, and specificity, 98.7%. We assessed the quality of studies using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) checklist. Because of incomplete reporting, the risk of bias was often unclear. Despite their intended use at the point of care, 26.3% of tests were evaluated in a laboratory setting. Although newly developed tests seem more sensitive, high-quality evaluations of these tests are lacking.

IntroductionSuspected urinary tract infection (UTI) ranks among the most common reasons for antibiotic use in nursing homes. However, diagnosing UTI in this setting is challenging because UTI often presents with non-specific symptomatology. Moreover asymptomatic bacteriuria is common in elderly, which complicates attribution of causality to detection of bacteria in urine. These diagnostic challenges contribute to overuse of antibiotics and emergence of antimicrobial resistance in nursing homes. Given the diagnostic challenges, there is a need for point-of-care (POC) diagnostic tests to support clinical rules for diagnosing UTI. Procalcitonin (PCT) and C reactive protein (CRP) are inflammatory blood markers that have been proven useful to support diagnosis and monitoring of (bacterial) respiratory tract infections and sepsis. While limited studies suggest their usefulness in supporting UTI diagnosis, their utility has not been studied in elderly populations for this purpose.Methods and analysisIn a 24-month matched prospective study, ‘PROGRESS’ will assess and compare the sensitivity of rapid POC measurements of blood CRP and PCT levels to support clinical rules for diagnosing UTI in nursing home residents. The primary outcome measure is sensitivity of the POC tests to identify patients with true UTI based on the predefined definition, as derived from receiver operating curves.Ethics and disseminationThis study will be conducted in accordance with Good Clinical Practice guidelines and the principles of the Declaration of Helsinki. The study protocol is approved by the Medical Ethical Committee of Amsterdam UMC location VUmc with reference number 2017.350 and National Central Committee on Research involving Human Subjects with reference number NL62067.029.17.Trial registration numberNTR6467.

Background Viral bronchiolitis is the most common cause of respiratory failure requiring invasive ventilation in young children. Bacterial co-infections may complicate and prolong paediatric intensive care unit (PICU) stay. Data on prevalence, type of pathogens and its association with disease severity are limited though. These data are especially important as bacterial co-infections may be treated using antibiotics and could reduce disease severity and duration of PICU stay. We investigated prevalence of bacterial co-infection and its association with disease severity and PICU stay. Methods Retrospective cohort study of the prevalence and type of bacterial co-infections in ventilated children performed in a 14-bed tertiary care PICU in The Netherlands. Children less than 2 years of age admitted between December 2006 and November 2014 with a diagnosis of bronchiolitis and requiring invasive mechanical ventilation were included. Tracheal aspirates (TA) and broncho-alveolar lavages (BAL) were cultured and scored based on the quantity of bacteria colony forming units (CFU) as: co-infection (TA > 10^5/BAL > 10^4 CFU), low bacterial growth (TA ^5/BAL ^4 CFU), or negative (no growth). Duration of mechanical ventilation and PICU stay were collected using medical records and compared against the presence of co-infection using univariate and multivariate analysis. Results Of 167 included children 63 (37.7%) had a bacterial co-infection and 67 (40.1%) low bacterial growth. Co-infections occurred within 48 h from intubation in 52 out 63 (82.5%) co-infections. H.influenza (40.0%), S.pneumoniae (27.1%), M.catarrhalis (22.4%), and S.aureus (7.1%) were the most common pathogens. PICU stay and mechanical ventilation lasted longer in children with co-infections than children with negative cultures (9.1 vs 7.7 days, p = 0.04 and 8.1vs 6.5 days, p = 0.02). Conclusions In this large study, bacterial co-infections occurred in more than a third of children requiring invasive ventilation for bronchiolitis and were associated with longer PICU stay and mechanical ventilation. These findings support a clinical trial of antibiotics to test whether antibiotics can reduce duration of PICU stay.

Background Recognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months. Methods Hierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors. Results Of the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4–5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward. Conclusion AFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management.

The appropriate use of microbiological investigations is an important cornerstone of antibiotic stewardship programmes, but receives relatively limited attention. This study aimed to identify influencing factors in performing microbiological diagnostic tests and to assess the need for a clinical guideline. We performed a qualitative (focus group) and quantitative (online questionnaire survey) study among medical specialists and residents to identify physicians’ considerations in performing microbiological diagnostic tests and to assess the need for a diagnostic guideline. The questionnaire consisted of 14 statements, divided into three categories: knowledge, influencing factors and presence of guidelines. The questionnaire was sent to physicians of the departments of internal medicine, intensive care, paediatrics and pulmonology in five hospitals in the Netherlands. Sub-analyses for medical specialists versus residents and for paediatric versus non-paediatric departments were performed. We included 187 completed questionnaires in our analyses. The physicians reported having adequate knowledge on methods, time-to-result and accuracy, but inadequate knowledge on costs of the tests. Patients’ clinical condition, comorbidity, local guidelines and accuracy of tests were appraised as the four most important influencing factors to perform tests. Over 70% (132/187) of physicians reported being interested in a guideline for microbiological diagnostic testing. Fifteen physicians (8.0%) provided additional comments. This study identifies the influencing factors to microbiological testing and shows the demand for a clinical guideline among physicians. Importance: Microbiological diagnostic tests are an important cornerstone within antibiotic stewardship programmes [1–5]. These programmes aim to ameliorate the appropriate use of antibiotics and thus improve clinical outcomes of infectious diseases, whilst reducing the emergence of antimicrobial resistance [6]. However, inappropriate microbiological testing is a widely recognised problem [7–12], and influencing factors to testing have not been studied in the past. Our research shows the demand for a clinical guideline among physicians, and it identifies their influencing factors to testing. These results can be used to create a clinical guideline for microbiological diagnostic testing, thus supporting antibiotic stewardship programmes and reducing antimicrobial resistance.

Herd immunity induced by locally circulating strains could provide protection against introduction of new strains., Outbreaks of human enterovirus 71 (EV-71) in Asia are related to high illness and death rates among children. To gain insight into the potential threat for the population of Europe, we determined the neutralizing activity in intravenous immunoglobulin (IVIg) batches and individual serum samples from donors in the Netherlands against EV-71 strains isolated in Europe and in Asia. All IVIg batches and 41%, 79%, and 65% of serum samples from children ≤5 years of age, women of childbearing age, and HIV-positive men, respectively, showed high neutralizing activity against a Dutch C1 strain, confirming widespread circulation of EV-71 in the Netherlands. Asian B3–4 and C4 strains were efficiently cross-neutralized, predicting possible protection against extensive circulation and associated outbreaks of those types in Europe. However, C2 and C5 strains that had few mutations in the capsid region consistently escaped neutralization, emphasizing the importance of monitoring antigenic diversity among circulating EV-71 strains.

Human parechoviruses (HPeVs), a poorly studied genus within the Picornaviridae family, are classified into 19 genotypes of which HPeV1 and HPeV3 are the most often detected. HPeV1 VP1 C terminus contains an arginine-glycine-aspartic acid (RGD) motif and has been shown to depend on the host cell surface αV integrins (αV ITGs) and heparan sulfate (HS) for entry. HPeV3 lacks this motif and the receptors remain unknown. HPeVs can be detected in patient nasopharyngeal and stool samples, and infection is presumed to occur after respiratory or gastro-intestinal transmission. HPeV pathogenesis is poorly understood as there are no animal models and previous studies have been conducted in immortalized monolayer cell cultures which do not adequately represent the characteristics of human tissues. To bridge this gap, we determined the polarity of infection, replication kinetics, and cell tropism of HPeV1 and HPeV3 in the well-differentiated human airway epithelial (HAE) model. We found the HAE cultures to be permissive for HPeVs. Both HPeV genotypes infected the HAE preferentially from the basolateral surface while the progeny virus was shed toward the apical side. Confocal microscopy revealed the target cell type to be the p63+ basal cells for both viruses, αV ITG and HS blocking had no effect on the replication of either virus, and transcriptional profiling suggested that HPeV3 infection induced stronger immune activation than HPeV1. Genotype-specific host responses may contribute to the differences in pathogenesis and clinical outcomes associated with HPeV1 and HPeV3.

Artemisinin resistance is rapidly rising in Southeast Asia and may spread to African countries, where efficacy estimates are currently still excellent. Extensive monitoring of parasite clearance dynamics after treatment is needed to determine whether responsiveness to artemisinin-based combination therapies (ACT) is changing in Africa. In this study, Kenyan children with uncomplicated falciparum malaria were randomly assigned to pyronaridine-artesunate (PA) or artemether-lumefantrine (AL) treatment. Parasite clearance was evaluated over 7 days following the start of treatment by quantitative polymerase chain reaction (qPCR) and direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA), a simplified molecular malaria diagnostic. Residual parasitemia at day 7 was detected by qPCR in 37.1% (26/70) of AL-treated children and in 46.1% (35/76) of PA-treated participants (P = 0.275). Direct-on-blood PCR nucleic acid lateral flow immunoassay detected residual parasites at day 7 in 33.3% (23/69) and 30.3% (23/76) of AL and PA-treated participants, respectively (P = 0.692). qPCR-determined parasitemia at day 7 was associated with increased prevalence and density of gametocytes at baseline (P = 0.014 and P = 0.003, for prevalence and density, respectively) and during follow-up (P = 0.007 and P = 0.011, respectively, at day 7). A positive db-PCR-NALFIA outcome at day 7 was associated with treatment failure (odds ratio [OR]: 3.410, 95% confidence interval [CI]: 1.513-7.689, P = 0.003), but this association was not found for qPCR (OR: 0.701, 95% CI: 0.312-1.578, P = 0.391). Both qPCR and db-PCR-NALFIA detected substantial residual submicroscopic parasitemia after microscopically successful PA and AL treatment and can be useful tools to monitor parasite clearance. To predict treatment outcome, db-PCR-NALFIA may be more suitable than qPCR.

Background Influenza constitutes a leading cause of morbidity and mortality worldwide. There is limited information about the aetiology of infection presenting clinically as influenza in hospitalised adults and children in South-East Asia. Such data are important for future management of respiratory infections. Objectives To describe the aetiology of infection presenting clinically as influenza in those hospitalised in South-East Asia. Methods Respiratory specimens archived from July 2008 to June 2009 from patients hospitalised with suspected influenza from Indonesia, Thailand and Vietnam were tested for respiratory viruses and atypical bacteria by polymerase chain reaction. Results A total of 1222 patients’ samples were tested. Of 1222, 776 patients (63·5%) were under the age of 5. Viruses detected included rhinoviruses in 229 of 1222 patients (18·7%), bocaviruses in 200 (16·4%), respiratory syncytial viruses in 144 (11·8%), parainfluenza viruses in 140 (11·5%; PIV1: 32; PIV2: 12; PIV3: 71; PIV4: 25), adenovirus in 102 (8·4%), influenza viruses in 93 (7·6%; influenza A: 77; influenza B: 16) and coronaviruses in 23 (1·8%; OC43: 14; E229: 9). Bacterial pathogens were Mycoplasma pneumoniae (n = 33, 2·7%), Chlamydophila psittaci (n = 2), C. pneumoniae (n = 1), Bordetella pertussis (n = 1) and Legionella pneumophila (n = 2). Overall, in-hospital case fatality rate was 29 of 1222 (2·4%). Conclusion Respiratory viruses were the most commonly detected pathogens in patients hospitalised with a clinical suspicion of influenza. Rhinovirus was the most frequently detected virus, and M. pneumoniae, the most common atypical bacterium. The low number of detected influenza viruses demonstrates a low benefit for empirical oseltamivir therapy, unless during an influenza outbreak.

Plasmid-mediated transferable colistin resistance encoded by the mcr-1 gene was described in Escherichia coli and Klebsiella pneumoniae isolates from pigs and chicken at a prevalence of around 20%, and in clinical isolates from human beings at a prevalence of around 1% in China.1 The prevalence of the mcr-1 gene in Enterobacteriaceae in other countries and in the community is unknown. [...]

International audience; Acute meningoencephalitis (AME) is associated with considerable morbidity and mortality in children in developing countries. Clinical specimens were collected from children presenting with AME at two Cambodian paediatric hospitals to determine the major aetiologies associated with AME in the country. Cerebrospinal fluid (CSF) and blood samples were screened by molecular and cell culture methods for a range of pathogens previously associated with AME in the region. CSF and serum (acute and convalescent) were screened for antibodies to arboviruses such as Japanese encephalitis virus (JEV), dengue virus (DENV), and chikungunya virus (CHIKV). From July 2010 through December 2013, 1160 children (one month to 15 years of age) presenting with AME to two major paediatric hospitals were enroled into the study. Pathogens associated with AME were identified using molecular diagnostics, cell culture and serology. According to a diagnostic algorithm, a confirmed or highly probable aetiologic agent was detected in 35.0% (n=406) of AME cases, with a further 9.2% (total: 44.2%, n=513) aetiologies defined as suspected. JEV (24.4%, n=283) was the most commonly identified pathogen followed by Orientia tsutsugamushi (4.7%, n=55), DENV (4.6%, n=53), enteroviruses (3.5%, n=41), CHIKV (2.0%, n=23) and Streptococcus pneumoniae (1.6%, n=19). The majority of aetiologies identified for paediatric AME in Cambodia were vaccine preventable and/or treatable with appropriate antimicrobials.

Objectives: Urinary tract infections (UTIs) are a common reason for empirical treatment with broad-spectrum antibiotics worldwide. However, population-based antimicrobial resistance (AMR) prevalence data to inform empirical treatment choice are lacking in many regions, because of limited surveillance capacity. We aimed to assess the prevalence of AMR to commonly used antimicrobial drugs in Escherichia coli and Klebsiella pneumoniae isolated from patients with community- or healthcare-associated UTIs on two islands of Indonesia. Methods: We performed a cross-sectional patient-based study in public and private hospitals and clinics between April 2014 and May 2015. We screened patients for symptoms of UTIs and through urine dipstick analysis. Urine culture and susceptibility testing were supported by telemicrobiology and interactive virtual laboratory rounds. Surveillance data were entered in forms on mobile phones. Results: Of 3424 eligible patients, 3380 (98.7%) were included in the final analysis, and yielded 840 positive cultures and antimicrobial susceptibility data for 657 E. coli and K. pneumoniae isolates. Fosfomycin was the single oral treatment option with resistance prevalence

Background Treatment of blood samples from hemorrhagic fever virus (HFV)-infected patients with 0.1% detergents has been recommended for virus inactivation and subsequent safe laboratory testing. However, data on virus inactivation by this procedure are lacking. Here we show the effect of this procedure on diagnostic test results and infectious Ebola virus (EBOV) titers. Methods Serum and whole-blood samples were treated with 0.1% or 1% sodium dodecyl sulfate (SDS) or 0.1% Triton X-100 and assayed for clinical chemistry and malaria antigen detection. Infectious EBOV titers were determined in SDS-treated plasma and whole blood from EBOV-infected nonhuman primates (NHPs). Infectious titers of EBOV or herpes simplex virus type 1 (HSV-1) in detergents-treated cell culture medium containing various serum concentrations were determined. Results Laboratory test results were not affected by 0.1% detergent treatment of blood samples, in contrast with 1% SDS treatment. However, 0.1% detergent treatment did not inactivate EBOV in blood samples from infected NHPs. Experiments with cell culture medium showed that virus inactivation by detergents is annulled at physiological serum concentrations. Conclusions Treatment of blood samples with 0.1% SDS or Triton X-100 does not inactivate EBOV. Inactivation protocols for HFV should be validated with serum and whole blood.

Background: Pregnant women are a high-risk group for Plasmodium falciparum infections, which may result in maternal anaemia and low birth weight newborns, among other adverse birth outcomes. Intermittent preventive treatment with sulfadoxine–pyrimethamine during pregnancy (IPTp-SP) is widely implemented to prevent these negative effects of malaria. However, resistance against SP by P. falciparum may decrease efficacy of IPTp-SP. Combinations of point mutations in the dhps (codons A437, K540) and dhfr genes (codons N51, C59, S108) of P. falciparum are associated with SP resistance. In this study the prevalence of SP resistance mutations was determined among P. falciparum found in pregnant women and the general population (GP) from Nanoro, Burkina Faso and the association of IPTp-SP dosing and other variables with mutations was studied.Methods: Blood spots on filter papers were collected from pregnant women at their first antenatal care visit (ANC booking) and at delivery, from an ongoing trial and from the GP in a cross-sectional survey. The dhps and dhfr genes were amplified by nested PCR and products were sequenced to identify mutations conferring resistance (ANC booking, n = 400; delivery, n = 223; GP, n = 400). Prevalence was estimated with generalized estimating equations and for multivariate analyses mixed effects logistic regression was used.Results: The prevalence of the triple dhfr mutation was high, and significantly higher in the GP and at delivery than at ANC booking, but it did not affect birth weight. Furthermore, quintuple mutations (triple dhfr and double dhps mutations) were found for the first time in Burkina Faso. IPTp-SP did not significantly affect the occurrence of any of the mutations, but high transmission season was associated with increased mutation prevalence in delivery samples. It is unclear why the prevalence of mutations was higher in the GP than in pregnant women at ANC booking.Conclusion: The high number of mutants and the presence of quintuple mutants in Burkina Faso confirm concerns about the efficacy of IPTp-SP in the near future. Other drug combinations to tackle malaria in pregnancy should, therefore, be explored. An increase in mutation prevalence due to IPTp-SP dosing could not be confirmed.

Background. Seasonal influenzacauses >200 000 annual hospitalizations in the United States. Current antiviral treatment options are limited to oral or inhaled agents. There is an urgent unmet need for intravenous antiviral treatments. Methods. Patients hospitalized with suspected influenzawere randomized to 5-day treatment with intravenous peramivir (600 mg once daily) or placebo; all received the institution’s standard of care (SOC) treatment. Time to clinical resolution and change in viral shedding in nasopharyngeal specimens were the primary and key secondary end points. Results. Influenza infection was confirmed in 338 of 405 enrolled patients. At the time of a preplanned interim analysis, the primary efficacy analysis population comprised 121 patients who did not receive a concurrent neuraminidase inhibitoras part of the SOC.The median (95% confidence interval) time to clinical resolution was 42.5 (34.0‐57.9) hours forperamivirversus 49.5 (40.0‐61.9)hours forplacebo(P=.97). A largertreatmenteffectwas observedin patients withhistoryof symptoms

Suggested citation for this article: Chen H, Wang Y, Liu W, Zhang J, Dong B, Fan X, et al. Serologic survey of pandemic (H1N1) 2009 virus, Guangxi Province, China [letter]. Emerg Infect Dis [serial on the Internet]. 2009 Nov [date cited]. Available from http://www.cdc.gov/EID/content/15/11/1849.htm

Objectives: Influenza household transmission studies are required to guide prevention strategies but most passively recruit index cases that seek healthcare. We investigated A(H1N1)pdm09 transmission in a household-based cohort during 2009. Methods: Health-workers visited 270 households weekly, and collected swabs from influenza-like-illness cases. If A(H1N1)pdm09 was RT-PCR-confirmed, all household members had symptoms assessed and swabs collected daily for 10-15 days. Viral RNA was quantified and sequenced and serology performed on pre-pandemic sera. Results: Index cases were detected in 20 households containing 81 people. 98.5% lacked A(H1N1)pdm09 neutralizing antibodies in pre-pandemic sera. Eleven (18.6%, 95% CI 10.7-30.4%) of 59 contacts were infected. Virus genetic diversity within households was negligible and less than between households. Index and secondary cases were distributed between mothers, daughters and sons, and had similar virus-RNA shedding and symptom dynamics. Fathers were rarely infected. Five secondary cases (45%) had no apparent symptoms and three shed virus before symptoms. Secondary infection was associated with index case wet cough (OR 1.56, 95% CI 1.22-1.99). Conclusions: In this cohort of A(H1N1)pdm09 susceptible persons, virus sequencing was capable of discriminating household from community transmission. Household transmission involved mothers and children but rarely fathers. Asymptomatic or pre-symptomatic shedding was common. © 2014 The Authors.

Objectives: To explore clinical and virological characteristics and describe the epidemiology of dengue in patients who presented with acute undifferentiated fever (AUF) at primary health centers (PHC) in Binh Thuan Province, Vietnam. Methods: A prospective observational study was conducted from 2001 to 2006 to study the aetiology in AUF patients. Demographic and clinical information was obtained, and dengue polymerase chain reaction (RT-PCR) and serology were performed on a random selection of patients. Results: Three hundred fifty-one serologically confirmed dengue patients including 68 primary and 283 secondary infections were included in this study. In 25% (86/351) dengue virus (DENV) was detected by RT-PCR among which 32 DENV-1, 16 DENV-2, 1 DENV-3 and 37 DENV-4 were identified. The predominant dengue serotype varied by year with seasonal fluctuation: DENV-4 in 2001-2002, DENV-1 and DENV-2 from 2003 to 2006. Primary dengue was more common in children. Higher viraemia levels (P = 0.010) were found in primary infections compared to secondary infections. DENV-1 infected patients had higher viraemia levels than DENV-2 (P = 0.003) and DENV-4 (P

Please cite this paper as: Friesema et al. (2012). Course of pandemic influenza A(H1N1) 2009 virus infection in Dutch patients. Influenza and Other Respiratory Viruses 6(3), e16–e20. The clinical dynamics of influenza A(H1N1) 2009 infections in 61 laboratory-confirmed Dutch cases were examined. An episode lasted a median of 7·5 days of which 2 days included fever. Respiratory symptoms resolved slowly, while systemic symptoms peaked early in the episode and disappeared quickly. Severity of each symptom was rated highest in the first few days. Furthermore, diarrhoea was negatively associated with viral load, but not with faecal excretion of influenza virus. Cases with comorbidities appeared to have higher viral loads than the cases without, suggesting a less effective immune response. These results complement information obtained through traditional surveillance.

Background Hemorrhagic fever-like illness caused by a novel Bunyavirus, Huaiyangshan virus (HYSV, also known as Severe Fever with Thrombocytopenia virus [SFTSV] and Fever, Thrombocytopenia and Leukopenia Syndrome [FTLS]), has recently been described in China. Methods Patients with laboratory-confirmed HYSV infection who were admitted to Union Hospital or Zhongnan Hospital between April 2010 and October 2010 were included in this study. Clinical and routine laboratory data were collected and blood, throat swab, urine, or feces were obtained when possible. Viral RNA was quantified by real-time reverse-transcriptase polymerase chain reaction. Blood levels of a range of cytokines, chemokines, and acute phase proteins were assayed. Results A total of 49 patients with hemorrhagic fever caused by HYSV were included; 8 (16.3%) patients died. A fatal outcome was associated with high viral RNA load in blood at admission, as well as higher serum liver transaminase levels, more pronounced coagulation disturbances (activated partial thromboplastin time, thrombin time), and higher levels of acute phase proteins (phospholipase A, fibrinogen, hepcidin), cytokines (interleukin [IL]-6, IL-10, interferon-γ), and chemokines (IL-8, monocyte chemotactic protein 1, macrophage inflammatory protein 1b). The levels of these host parameters correlated with viral RNA levels. Blood viral RNA levels gradually declined over 3-4 weeks after illness onset, accompanied by resolution of symptoms and laboratory abnormalities. Viral RNA was also detectable in throat, urine, and fecal specimens of a substantial proportion of patients, including all fatal cases assayed. CONCLUSIONS. Viral replication and host immune responses play an important role in determining the severity and clinical outcome in patients with infection by HYSV.

The XIIth International Symposium on Respiratory Viral Infections was convened by The Macrae Group (New York, NY, USA) in Taipei, Taiwan on 11-14 March 2010. This annual symposium provides a forum to discuss recent advances in respiratory virus research in an interdisciplinary fashion with specialists ranging from basic virology, epidemiology, vaccines, antivirals and management strategies. The 2010 symposium provided a detailed examination of lessons learned from the 2009 influenza pandemic with a common theme, emphasized by many speakers, being the importance of international collaboration and cooperation in responding to new infectious disease threats.

Background Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, like culture and antigen detection. However, comprehensive data on sensitivity, specificity and performance of the multiplex PCR compared to the single target PCR's is limited for most published respiratory multiplex real time PCR assays. Objectives Development and extensive analysis of an internally controlled multiplex real time rt-PCR for detection of respiratory viruses. Study design The assay was validated in comparison to single-target PCR's using plasmid targets and prospectively collected nasopharyngeal aspirates. Results Using plasmid targets the multiplex format was found to be as least as sensitive and specific as the single-target PCR and no competition was observed when different targets were present at different amounts in one tube. Clinical validation showed high concordance for all viruses tested except for samples with low levels of enterovirus. Conclusion This multiplex showed excellent specificities for all 14 respiratory viruses and sensitivity was high except for clinical samples with low levels of enterovirus.

Antiviral resistance in influenza may not only develop during treatment but also sometimes transmit widely to replace susceptible strains in the absence of drug pressure. This transmission is exemplified by the global spread of adamantane-resistant A(H3N2) viruses since 2003, oseltamivir-resistant seasonal A(H1N1) viruses since 2007, and adamantane-resistant pandemic A(H1N1) viruses in 2009, events that emphasize the unpredictability of influenza viruses and the increasing challenges of clinical management of influenza, especially given the current paucity of therapeutic choices. Three articles in this issue of The Journal of Infectious Diseases extend these observations, describing the plasticity of seasonal A(H1N1) viruses in developing dual adamantane-oseltamivir resistance [1], the effects on drug susceptibility and viral fitness of various neuraminidase (NA) mutations in pandemic A(H1N1) viruses [2], and the risk of nosocomial spread of oseltamivir-resistant pandemic A(H1N1) virus [3]. Together, these findings illustrate that single reassortment events or mutations can lead to the emergence of transmissible variants of pandemic 2009 or seasonal A(H1N1) viruses unresponsive to most, if not all, of our currently available drugs. This raises multiple questions regarding patient management and preparations for future influenza outbreaks.

Influenza infection results in substantial morbidity and mortality in hospitalized patients, including those who are immunocompromised or pregnant. Antiviral therapy likely provides considerable benefit to these patients, but few studies have been successfully conducted in these high-risk populations, and no drugs are specifically licensed for treating these subgroups. One of the key challenges facing novel antiviral drug development for influenza is determining the appropriate efficacy end points that would enable rapid regulatory approval for drug use in seriously ill patients, for whom risk-benefit assessments differ from those with uncomplicated illness. All available antiviral drugs currently affect viral replication, and respiratory tract viral titers correlate with both symptoms and measures of host inflammatory responses, including cytokine and chemokine expression that are likely responsible for many of the clinical symptoms. Consequently, we outline the evidence to support the use of primary virological end points in studies of antiviral agents involving patients who are hospitalized with severe influenza or those who are at high risk of severe and life-threatening disease.

BACKGROUND: Novel serological methods provide alternative options for sero-diagnosis, sero-epidemiology and for determining evidence of naturally acquired or vaccine induced immunity. Micro-neutralization tests are currently the gold standard for serological studies of highly pathogenic avian influenza in mammalian species but require handling live virus in a biosafety level (BSL) 3 environment. We previously reported the use of H5 pseudotyped lentiviral particles (H5pp) as an alternative to micro-neutralization tests in a BSL-2 setting (Nefkens et al., 2007). OBJECTIVE: To optimize and evaluate this newly developed H5pp assay on relevant clinical specimens. STUDY DESIGN: We optimise and evaluate the performance of the H5pp assay using well-characterized sera from humans with confirmed H5N1 disease or controls. RESULTS: The H5pp assay is a reliable serological method for the detection and quantification of neutralizing antibody to H5-viruses. CONCLUSION: H5pp provide a reliable and safe alternative for sero-diagnosis and sero-epidemiology of H5N1 infections in a BSL-2 setting

In recent years, there has been an increase in outbreaks of highly pathogenic avian influenza (HPAI) in poultry. Occasionally, these outbreaks have resulted in transmission of influenza viruses to humans and other mammals, with symptoms ranging from conjunctivitis to pneumonia and death. Here the current, knowledge of the determinants of pathogenicity of HPAI viruses in mammals is summarized. It is becoming apparent that common mechanisms exist across influenza A virus strains and subtypes, through which influenza viruses adapt to mammals and gain or loose pathogenicity. (c) 2008 Elsevier Ltd. All rights reserved.

BACKGROUND: Streptococcus suis infection is an emerging zoonosis in Asia. We determined the detailed epidemiological, clinical, and microbiological characteristics of S. suis meningitis in adults. METHODS: We prospectively studied 450 patients with suspected bacterial meningitis. Four hundred thirty-five (96.7%) of the patients participated in a trial to determine the effect of adjunctive dexamethasone treatment. For patients with S. suis infection, bacterial DNA load at hospital admission and during treatment was analyzed in cerebrospinal fluid specimens using quantitative real-time polymerase chain reaction. S. suis strains were characterized using pulsed-field gel electrophoresis and multilocus sequence typing. Putative virulence factors, including extracellular protein factor, suilysin, and muramidase released protein, were detected using polymerase chain reaction and Western blot assay. Predictors of outcome were identified using logistic regression analysis. RESULTS: S. suis was the most common pathogen and was detected in 151 (33.6%) of the patients. Fifty (33.1%) of these 151 patients reported exposure to pigs or pork. Mortality was low (2.6%; 4 of 151 patients died), but mild to severe hearing loss occurred in 93 (66.4%) of 140 patients. Severe deafness at hospital discharge was associated with age >50 years (odds ratio, 3.65; 95% confidence interval, 1.15-11.6), a strain carrying the epf gene (odds ratio, 3.42; 95% confidence interval, 1.02-11.4), and dexamethasone therapy (odds ratio, 0.23; 95% confidence interval, 0.06-0.78) but was not associated with cerebrospinal fluid bacterial DNA load. Bacterial DNA was still detectable in 58 (63%) of 92 cerebrospinal fluid samples after 6-10 days of antimicrobial treatment. Ninety-one of 92 S. suis strains had serotype 2. Thirty-three (36%) of these epidemiologically unrelated strains belonged to 1 pulsed-field gel electrophoresis cluster of multilocus sequence type 1, indicating clonal spread. CONCLUSION: S. suis serotype 2 is the most frequent cause of bacterial meningitis in adults in southern Vietnam and is associated with substantial morbidity attributable to hearing loss.

Pandemic influenza virus has its origins in avian influenza viruses. The highly pathogenic avian influenza virus subtype H5N1 is already panzootic in poultry, with attendant economic consequences. It continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. Therefore, H5N1 virus has rightly received attention as a potential pandemic threat. However, it is noted that the pandemics of 1957 and 1968 did not arise from highly pathogenic influenza viruses, and the next pandemic may well arise from a low-pathogenicity virus. The rationale for particular concern about an H5N1 pandemic is not its inevitability but its potential severity. An H5N1 pandemic is an event of low probability but one of high human health impact and poses a predicament for public health. Here, we review the ecology and evolution of highly pathogenic avian influenza H5N1 viruses, assess the pandemic risk, and address aspects of human H5N1 disease in relation to its epidemiology, clinical presentation, pathogenesis, diagnosis, and management. Copyright © 2007, American Society for Microbiology. All Rights Reserved., link_to_OA_fulltext

Background Human respiratory syncytial virus (RSV) is an important community and nosocomial pathogen in developed countries but data regarding the importance of RSV in developing countries are relatively scarce. Methods During a 1-year surveillance study in 2010, we took serial samples from children admitted to the Emergency Unit of the Respiratory Ward of Children's Hospital 1 in Ho Chi Minh City, Vietnam. RSV was detected within 72 hours of admission to the ward in 26% (376/1439; RSV A: n = 320; RSV B: n = 54; and RSV A and B: n = 2). Among those negative in the first 72 hours after admission, 6·6% (25/377) acquired nosocomial RSV infection during hospitalization (RSV A: n = 22; and RSV B: n = 3). Results Children with nosocomial RSV infection were younger (P = 0·001) and had a longer duration of hospitalization (P

Background: Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Toll-like-receptors (TLRs) are important for the recognition of the causative agent Mycobacterium tuberculosis. Negative regulation of TLRs is necessary to control deleterious inflammatory damage, but could provide a means of immune evasion by M. tuberculosis as well. Methods: To obtain insight in the extent of expression of inhibitory regulators of immunity in patients with active TB, peripheral-blood-mononuclear-cells (PBMCs) and plasma were obtained from 54 TB patients and 29 healthy blood donors from Chittagong, Bangladesh. Bilateral alveolar macrophages were obtained from an infected versus a contralateral normal lung segment of 9 patients. Statistical analyses were performed using Mann-Whitney U and Wilcoxon matched pairs testing. Correlations were calculated using the Spearman rho test. Results: PBMCs harvested from TB patients demonstrated increased mRNA expression of IL-1-receptor-associated-kinase-M, suppressor-of-cytokine-signalling-3 and Toll-interacting-protein. Flow cytometry revealed enhanced expression of IL-1-receptor-like-1 (ST2) on lymphocytes. Plasma soluble ST2 was elevated in patients with TB and correlated with established TB biomarkers, most strongly with soluble interleukin-2 receptor subunit a and interleukin-8. Alveolar macrophage mRNA expression of negative TLR regulators did not differ between the infected and contralateral lung side. Conclusion: These results show enhanced expression of distinct negative regulators of innate immunity in PBMCs of patients with TB and identify plasma soluble ST2 as a potential novel biomarker for TB disease activity

OBJECTIVE High rates of hepatitis C virus (HCV) reinfections among HIV-infected men who have sex with men (MSM) following clearance of a primary infection suggest absence of protective immunity. Here, we investigated the incidence of HCV super and reinfections in 85 HIV-infected MSM with incident HCV infection. DESIGN AND METHODS Serial sequencing of a fragment of NS5B and the HCV envelope was used to longitudinally characterize the virus. If the primary genotype was still present at the most recent viremic time point, as indicated by the NS5B sequence analysis, serial envelope 2/hypervariable region 1 (E2/HRV1) sequence analysis was performed to distinguish a new infection with the same genotype (clade switch) from intrahost evolution. Incidence rate and cumulative incidence of secondary infections were estimated, and the effect of the primary genotype (1a versus non1) on the risk of acquiring a second infection with the same genotype was determined using Cox proportional-hazards analysis. RESULTS Among 85 patients with a median follow-up of 4.8 years, incidence rate of secondary infections was 5.39 cases/100 person-years (95% confidence interval 3.34-8.26). Cumulative incidence of genotype switches was markedly higher than the cumulative incidence of clade switches (26.7 versus 4.8% at 5 years, respectively). In patients with HCV-1a as primary infection, the risk for acquiring another HCV-1a infection was reduced compared to those with a primary non-HCV-1a subsequently acquiring HCV-1a (hazard ratio 0.25, 95% confidence interval 0.07-0.93). CONCLUSION Risk of acquiring a secondary infection with the primary genotype was strikingly reduced compared with the risk of acquiring a secondary infection with a different genotype.