Development of oseltamivir and zanamivir resistance in influenza A(H1N1)pdm09 virus, Denmark, 2014.

Antiviral treatment of immunocompromised patients with prolonged influenza virus infection can lead to multidrug resistance. This study reveals the selection of antiviral resistance mutations in influenza A(H1N1)pdm09 virus in an immunocompromised patient during a 6-month period. The patient was treated with two courses of oseltamivir (5 days and 2 months, respectively), with the first course starting at symptom onset, and subsequently zanamivir (2 months and 10 days, respectively). Respiratory samples were investigated by Sanger and next generation sequencing (NGS) and, for NGS data, low-frequency-variant-detection analysis was performed. Neuraminidase-inhibition tests were conducted for samples isolated in Madin-Darby canine kidney cells. In a sample collected 15 days after the end of the first treatment with oseltamivir (Day 20 post-symptom onset), oseltamivir resistance was detected (mutation H275Y with 60.3% frequency by NGS). Day 149 when the patient had almost completed the second zanamivir treatment, mixes of the following resistance mutations were detected; H275Y(65.1%), I223R(9.2%), and E119G(89.6%), accompanied by additional mutations, showing a more complex viral population in the long-term treated patient. Two samples obtained on Day 151 from bronchoalveolar lavage (BAL) and nasopharyngeal swab, respectively, showed different mutation profiles, with a higher frequency of antiviral resistance mutations in BAL. The results emphasise the importance of timely antiviral resistance testing both for treatment of individual patients as well as for preventive measures to control the development and transmission of antiviral resistant viruses.

Increased concentrations of the soluble mannose receptor in serum from patients with pneumococcal bacteraemia, and prediction of survival.

BACKGROUND: The soluble mannose receptor (sMR) is a new serum marker of macrophage activation. The aim of the present study was to investigate sMR as a prognostic marker in patients with invasive pneumococcal disease (IPD), and compare it to other inflammatory biomarkers. METHODS: Samples from 128 patients with IPD were collected at the time of first positive blood culture and analysed using an in-house sMR assay. Clinical data were retrieved from patient files. The main outcome investigated was in-hospital mortality. RESULTS: The median sMR concentration in the entire group of patients was 0.77 mg/L. There was a significant difference in sMR concentration between patients below (n = 92, sMR = 0.82 mg/L) or above (n = 36, sMR = 0.73 mg/L) the age of 75 (p < 0.001). In the entire group there was a significant difference in sMR concentrations between survivors (n = 107, sMR = 0.72 mg/L) and non-survivors (n = 21, sMR = 1.38 mg/L), but for patients 75 years or older this difference was not statistically significant. For prediction of survival sMR seemed most promising (area under the receiver operating characteristic curve (AUC) = 0.79) compared with sCD163 (AUC = 0.70) and CRP (AUC = 0.73). In patients 75 years or older the AUC was lower for all three markers (sMR = 0.56, sCD163 = 0.38, CRP = 0.66). CONCLUSIONS: The results of this study designate sMR as a potential new biomarker in infectious disease. Additionally, it emphasizes the importance of research into macrophage malfunction in elderly patients.

Plasma YKL-40 and all-cause mortality in patients with chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is hallmarked by inflammatory processes and a progressive decline of lung function. YKL-40 is a potential biomarker of inflammation and mortality in patients suffering from inflammatory lung disease, but its prognostic value in patients with COPD remains unknown. We investigated whether high plasma YKL-40 was associated with increased mortality in patients with moderate to very severe COPD. METHODS: Four hundred and ninety-three patients with moderate to very severe COPD were followed prospectively for up to 10 years. Patients were divided into two groups according to plasma YKL-40: concentration higher than the 75th percentile for age-matched healthy subjects (i.e. high levels) and normal levels. Outcome was overall survival (OS) and was evaluated in uni- and multivariate proportional hazards Cox regression analyses and adjusted for factors affecting mortality. RESULTS: Median plasma YKL-40 was increased in patients with COPD (81 ng/ml, p < 0.001) compared to healthy subjects (40 ng/ml). Patients with high plasma YKL-40 had a hazard ratio (HR) of 1.42 (95% CI: 1.15-1.75, p = 0.001) for all-cause mortality. Multivariate analysis showed that YKL-40 (HR 1.38; 95% CI: 1.11-1.72, p = 0.004), age (HR 1.05; 95% CI: 1.03-1.06, p < 0.0001), Severe COPD (HR 1.35; 95 CI: 1.03-1.76, p = 0.03) very severe COPD (HR 2.19; 95% CI: 1.60 - 2.99 < 0.0001), neutrophil granulocyte count (HR 1.05; 95% CI: 1.01-1.08, p = 0.01), and a smoking history of > 40 years (HR 1.38; 95% CI: 1.11-1.71, p = 0.003) were independent prognostic markers of OS. CONCLUSION: High plasmaYKL-40 is associated with increased mortality in patients with moderate to very severe COPD, suggesting a role for YKL-40 as a potential biomarker of mortality in this patient group.

Calprotectin--a marker of mortality in COPD? Results from a prospective cohort study.

Calprotectin comprises more than 45% of the cytosolic content of neutrophil granulocytes. Because pathogenesis, disease activity and disease progression in COPD are believed to be partly dependent of neutrophil driven inflammation we decided to investigate whether plasma level of calprotectin (p-calprotectin) was associated with all-cause mortality in patients with COPD. We measured p-calprotectin in blood samples from 460 patients with moderate to very severe COPD in stable phase. Patients were stratified into three groups according to p-calprotectin level. Outcome measure was all-cause mortality. Analyses were adjusted for factors known to influence mortality using a Cox regression analysis. We found a time dependent correlation between p-calprotectin levels and mortality during the first 5 years of follow-up. Increasing levels of p-calprotectin were associated with concomitant increases in mortality from HR 1.56 (CI 95%: 1.03 -2.38) at calprotectin between 100 -200 ng/ml to HR 2.02 (CI 95%: 1.27-3.19) at calprotectin >200 ng/ml. P-calprotectin could be a useful marker of all-cause mortality in patients suffering from moderate to very severe COPD.

Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain.

BACKGROUND: Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly respond to selection pressures, such as those imposed by the immunological host response and antiviral therapy. We have applied deep sequencing to characterize influenza intra-host variation in a transmission chain consisting of three cases due to oseltamivir-sensitive viruses, and one derived oseltamivir-resistant case. METHODS: Following detection of the A(H1N1)pdm09 infections, we deep-sequenced the complete NA gene from two of the oseltamivir-sensitive virus-infected cases, and all eight gene segments of the viruses causing the remaining two cases. RESULTS: No evidence for the resistance-causing mutation (resulting in NA H275Y substitution) was observed in the oseltamivir-sensitive cases. Furthermore, deep sequencing revealed a subpopulation of oseltamivir-sensitive viruses in the case carrying resistant viruses. We detected higher levels of intra-host variation in the case carrying oseltamivir-resistant viruses than in those infected with oseltamivir-sensitive viruses. CONCLUSIONS: Oseltamivir-resistance was only detected after prophylaxis with oseltamivir, suggesting that the mutation was selected for as a result of antiviral intervention. The persisting oseltamivir-sensitive virus population in the case carrying resistant viruses suggests either that a small proportion survive the treatment, or that the oseltamivir-sensitive virus rapidly re-establishes itself in the virus population after the bottleneck. Moreover, the increased intra-host variation in the oseltamivir-resistant case is consistent with the hypothesis that the population diversity of a RNA virus can increase rapidly following a population bottleneck.

Serum vitamin D in patients with chronic obstructive lung disease does not correlate with mortality--results from a 10-year prospective cohort study.

BACKGROUND: Recent studies have found vitamin D (25-OHD) deficiency and insufficiency to be common among patients with COPD. Serum level of 25-OHD seems to correlate to pulmonary function, COPD disease staging, and increased susceptibility to respiratory infections. We wanted to investigate whether vitamin D deficiency or insufficiency was associated with mortality rate in patients suffering from advanced COPD. METHODS: 25-OHD serum levels were measured in 462 patients suffering from moderate to very severe COPD. Patients were stratified into three groups according to serum levels of 25-OHD. Outcome measure was mortality in a 10 year follow-up period. Kaplan-Meier curves (KM) were plotted and mortality hazard ratios (HR) were calculated using Cox Proportional Hazard regression (Cox PH). RESULTS: Serum 25-OHD deficiency and insufficiency were prevalent. We were unable to demonstrate any association between baseline serum levels of 25-OHD and mortality rate. We found an association between mortality and age [HR 1.05 (CI 95%: 1.03-1.06)], Charlson score [HR 1.49 (CI 95%: 1.06-2.09)], increasing neutrophil count [HR 1.05 (CI 95%: 1.02-1.09)], severe [HR 1.41 (CI 95%: 1.06-1.86)]/very severe COPD [HR 2.19 (CI 95%: 1.58-3.02)] and a smoking history of more than 40 pack years [HR 1.27 (CI 95%: 1.02-1.70)]. CONCLUSIONS: Serum level of 25-OHD does not seem to be associated with mortality rate, suggesting no or only a minor role of 25-OHD in disease progression in patients with moderate to very severe COPD.

[Increasing antibiotic resistance: the prognostic impact has yet to be determined]. / Usikker prognostisk betydning af stigende antibiotikaresistens.

The rising prevalence of multiresistant bacteria has become a major clinical concern. Yet, the prognostic impact is not well elucidated. A growing number of studies have found associations between acquired resistance, inappropriate therapy and mortality. The key underlying factors seem to be delay of appropriate antibiotic therapy and possibly use of more toxic therapy. However, methodological issues including control group selection and adjustment for potential confounders must be taken into consideration when assessing outcome studies for patients infected with resistant microorganisms.

Incidence and prevalence of hospital-acquired infections in a cohort of patients admitted to medical departments.

INTRODUCTION: Hospital-acquired infections (HAI) are a significant cause of morbidity and mortality. Only point prevalence analyses of HAI have been recorded in Denmark. The aim of this study was to investigate the incidence and prevalence of HAI in patients admitted to departments of internal medicine. MATERIAL AND METHODS: The study involved seven departments and was designed as a cohort study based on reviews of medical records. Except for patients who had previously been admitted within the preceding 30 days, the study included all patients admitted for more than 48 hours during the 45-day study period. HAI was defined according to the criteria established by the Center for Disease Control and Prevention, USA. RESULTS: The incidence of HAI was 1.7 (62/3,568) per 100 days at risk (95% confidence interval (CI) 1.4-2.2), while the total prevalence of HAI was 9.7% (345/3,568) (95% CI 8.7-10.6). Exposure to bladder catheter was associated with an increased risk of urinary tract infection, incidence rate ratio 4.9; (95% CI 1.8-11.5). For the initial 14 days of hospitalization, the incidence of HAI was independent, while the prevalence increased linearly with duration of admittance. CONCLUSION: The incidence of HAI was relatively constant during the initial 14-day-period of hospitalization, suggesting that shortening the period will have no major impact on the incidence of HAI. The prevalence was 9.7%, which is in line with results from prior studies.

Detection of non-DeltaGT NCF-1 mutations in chronic granulomatous disease.

AIMS: Chronic granulomatous disease (CGD) is a rare inherited disorder caused by mutations in the subunits of the NADPH oxidase complex, leaving phagocytes unable to produce superoxide and thereby unable to kill invading microorganisms. A subgroup of CGD patients (approximately 20%) is reported to have mutations in NCF-1 encoding p47-phox, which is part of the cytosolic component of NADPH oxidase. More than 94% of these patients share the same mutation, a 2 bp GT deletion in the GTGT dinucleotide repeat in the start of exon 2. The presence of two pseudogenes more than 98% homologous to the functional NCF-1 has complicated the identification of other mutations in the gene. The aim of this study was to find a general technique for detection of non-GT deletion mutations in the coding region of NCF-1. RESULTS: A technique involving GeneScan analysis followed by amplification of cDNA with intact dinucleotide repeat was set up and used to identify a novel mutation in exon 7 of NCF-1 in a patient with autosomal recessive CGD, explaining the disease by changing a UGG codon to a premature UGA STOP codon. CONCLUSION: The method is generally applicable for the detection of NCF-1 mutations in patients with suspected CGD.

High mobility group box-1 protein in patients with suspected community-acquired infections and sepsis: a prospective study.

INTRODUCTION: Sepsis is a serious condition with a significant morbidity and mortality. New insight into the immunopathogenesis of sepsis could promote the development of new strategies for diagnosis and therapy. High mobility group box-1 protein (HMGB1) has been known for many years as a nuclear chromosomal protein. Its role as a pro-inflammatory cytokine in sepsis and rheumatoid arthritis has been described recently. The aim of our study was to evaluate HMGB1 as a molecular marker in patients with community-acquired infections. METHODS: Patients suspected of having infections/sepsis and admitted to a department of internal medicine were included in the study in a prospective manner. Demographic data, comorbidity, routine biochemistry, microbiological data, infection focus, severity score, and mortality on day 28 were recorded. Plasma and serum were sampled at the time of admission. HMGB1 levels were measured with a commercially available enzyme-linked immunosorbent assay (ELISA). Procalcitonin levels were measured with a TRACE (time-resolved amplified cryptate emission) assay. Lipopolysaccharide-binding protein and interleukin-6 were measured with a chemiluminiscent immunometric assay. Soluble haemoglobin scavenger receptor (sCD163) levels were measured with an in-house ELISA. RESULTS: One hundred and ninety-four patients were included in the study. Levels of HMGB1 are presented as medians and interquartile ranges: healthy controls (0.77 ng/ml, 0.6 to 1.46), non-infected patients (1.54 ng/ml, 0.79 to 2.88), infected patients without systemic inflammatory response syndrome (2.41 ng/ml, 0.63 to 3.44), patients with sepsis (2.24 ng/ml, 1.30 to 3.75), and patients with severe sepsis (2.18 ng/ml, 0.91 to 3.85). In a receiver operator characteristic curve analysis discriminating between non-infected patients and all infected patients, the area under the curve for HMGB1 was 0.59 (P < 0.0001). HMGB1 correlated only weakly to levels of white blood cell count, neutrophils, C-reactive protein, interleukin-6, procalcitonin, and lipopolysaccharide-binding protein (P < 0.001). HMGB1 did not correlate to sCD163. CONCLUSION: In a cohort of patients with suspected community-acquired infections and sepsis, HMGB1 levels were statistically significantly higher in patients compared to the healthy controls. There was no statistically significant difference between the infected and the non-infected patients. Levels of HMGB1 correlated only very weakly to other pro-inflammatory markers and did not correlate to the anti-inflammatory marker sCD163.

Macrophage serum markers in pneumococcal bacteremia: Prediction of survival by soluble CD163.

OBJECTIVE: Soluble CD163 (sCD163) is a new macrophage-specific serum marker. This study investigated sCD163 and other markers of macrophage activation (neopterin, ferritin, transcobalamin, and soluble urokinase plasminogen activator receptor [suPAR]) as prognostic factors in patients with pneumococcal bacteremia. DESIGN: Observational cohort study. SETTING: Five university hospitals in Denmark. PATIENTS: A total of 133 patients with Streptococcus pneumoniae bacteremia (positive blood culture) and 133 age- and gender-matched controls. INTERVENTIONS: Samples were collected for biochemical analyses at the time of first positive blood culture. MEASUREMENTS AND MAIN RESULTS: sCD163 was highly correlated with other macrophage markers and was significantly elevated (median [25-75 percentiles], 4.6 mg/L [2.8-8.9]) compared with healthy controls (2.7 mg/L [2.1-3.3], p < .0001). Increased levels were observed in patients who needed intensive care (hemodialysis, p = .0011; hypotension, p = .0014; mechanical ventilation, p = .0019). Significantly lower levels of sCD163, ferritin, transcobalamin, and suPAR (but not C-reactive protein) were measured in patients > or =75 yrs. In patients <75 yrs, all macrophage markers were increased in patients who died from their infection compared with survivors, whereas no change was observed in any of the markers in the very old age. At cutoff levels of 9.5 mg/L (sCD163) and 1650 nmol/L (C-reactive protein), the relative risk for fatal outcome in patients <75 yrs was 10.1 (95% confidence interval 3.4-31.0) and 7.0 (95% confidence interval 2.4-21.6) for sCD163 and C-reactive protein, respectively. In a multivariate logistic regression model for patients <75 yrs, ferritin, transcobalamin, neopterin, and suPAR contained no significant information on the probability of survival when sCD163 and CRP were known (p = .25). CONCLUSIONS: Macrophage marker response in pneumococcal bacteremia was compromised in old age. In patients <75 yrs old, sCD163 was superior to other markers, including C-reactive protein, in predicting fatal disease outcome.

Procalcitonin, lipopolysaccharide-binding protein, interleukin-6 and C-reactive protein in community-acquired infections and sepsis: a prospective study.

INTRODUCTION: Clinicians are in need of better diagnostic markers in diagnosing infections and sepsis. We studied the ability of procalcitonin, lipopolysaccharide-binding protein, IL-6 and C-reactive protein to identify patients with infection and sepsis. METHODS: Plasma and serum samples were obtained on admission from patients with suspected community-acquired infections and sepsis. Procalcitonin was measured with a time-resolved amplified cryptate emission technology assay. Lipopolysaccharide-binding protein and IL-6 were measured with a chemiluminescent immunometric assay. RESULTS: Of 194 included patients, 106 had either infection without systemic inflammatory response syndrome or sepsis. Infected patients had significantly elevated levels of procalcitonin, lipopolysaccharide-binding protein, C-reactive protein and IL-6 compared with noninfected patients (P < 0.001). In a receiver-operating characteristic curve analysis, C-reactive protein and IL-6 performed best in distinguishing between noninfected and infected patients, with an area under the curve larger than 0.82 (P < 0.05). IL-6, lipopolysaccharide-binding protein and C-reactive protein performed best in distinguishing between systemic inflammatory response syndrome and sepsis, with an area under the curve larger than 0.84 (P < 0.01). Procalcitonin performed best in distinguishing between sepsis and severe sepsis, with an area under the curve of 0.74 (P < 0.01). CONCLUSION: C-reactive protein, IL-6 and lipopolysaccharide-binding protein appear to be superior to procalcitonin as diagnostic markers for infection and sepsis in patients admitted to a Department of Internal Medicine. Procalcitonin appears to be superior as a severity marker.

Severe Actinomyces israelii infection involving the entire spinal cord.

A case of severe spinal infection with Actinomyces israelii is presented. A 38-y-old male was admitted with symptoms of infection and neurological symptoms. Magnetic resonance imaging showed extensive affection involving the entire spinal cord. Cultures of the cerebrospinal fluid revealed Actinomyces israelii. The patient was treated with long term penicillin G followed by amoxicillin. The patient recovered with severe neurological sequelae.

[Staphylococcus aureus infections--the clinical picture and treatment]. / Infektioner med Staphylococcus aureus--klinik og behandling.

Infections caused by Staphylococcus aureus comprise relatively benign local skin infections, as well as serious generalised conditions. In Denmark, more than 85% of all S. aureus isolates are found resistant to penicillin, whereas resistance to methicillin is rare (< 1%) and therefore one of the penicillinase-stable penicillins is still the drug of choice. Dicloxacillin is usually chosen, because of its superior penicillinase stability. Infections caused by methicillin resistant strains may be treated with different combinations of macrolides, fusidic acid, aminoglycosides, fluoroquinolones, and rifampicin, but vancomycin is generally used. Newer drugs like the oxazolidinones and streptogramins are effective against methicillin-resistant strains and will be available in Denmark within a short time. Antibiotic treatment, however, is most often only a supplement to surgical intervention. This paper deals with the clinical picture and treatment of some common S. aureus infections.