Frank Stuber

A genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-alpha concentrations and outcome of patients with severe sepsis

OBJECTIVES To determine the allele frequency and genotype distribution of a bi-allelic tumor necrosis factor (TNF) gene polymorphism and plasma TNF-alpha concentrations in postoperative intensive care unit (ICU) patients suffering from severe sepsis. DESIGN Prospective, consecutive entry study of patients with severe sepsis in a postoperative ICU. SETTING University hospital. PATIENTS Forty patients with diagnosis of severe sepsis, admitted to the ICU between June 1993 and December 1994. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS A 782 basepairs fragment of genomic DNA, including the polymorphic site of the restriction enzyme Ncol within the TNF locus, was amplified by means of polymerase chain reaction. The genotype of each patient was determined after Ncol digestion of the amplified product and subsequent agarose gel electrophoresis. Reading the size of the resulting DNA bands from the agarose gel demonstrated the genotype, as defined by the two alleles TNFB1 and TNFB2. Serial blood samples were drawn every sixth hour during the first 48 hrs and every 12th hour thereafter, for < or = 96 hrs after diagnosis. TNF-alpha plasma concentrations were detected by an enzyme-linked immunosorbent assay. Assessment of organ dysfunction was performed by calculating a Multiple Organ Failure score. The overall allele frequency (TNFB1 0.35; TNFB2 0.65) and genotype distribution (TNFB1 homozygotes 10%; TNFB1/TNFB2 heterozygotes 48%; TNFB2 homozygotes 42%) in 40 patients with severe sepsis were comparable with those values found in normal individuals. Development of multiple organ failure occurred in 33 (82.5%) of 40 patients, whereas 23 (57.5%) of 40 patients did not survive. In contrast to the overall allele frequency, nonsurvivors showed a significantly higher prevalence of the allele TNFB2(p < .005). Patients homozygous for the allele TNFB2 demonstrated a higher mortality rate than heterozygous (TNFB1/TNFB2) patients (p = .0022). In addition, patients with TNFB2 homozygotes displayed higher circulating TNF-alpha concentrations as well as higher Multiple Organ Failure scores compared with heterozygous (TNFB1/TNFB2) patients. CONCLUSIONS The bi-allelic Ncol polymorphism within the TNF locus is a genomic marker for patients with increased TNF-alpha response and poor prognosis in severe sepsis. The amount of TNF released in situations of severe infection and sepsis appears to be influenced genetically. TNFB2 homozygous individuals displaying increased circulating TNF plasma concentrations combined with high mortality rate may be included in future studies testing anti-TNF strategies in severe sepsis.

Effects of mechanical ventilation on release of cytokines into systemic circulation in patients with normal pulmonary function.

BackgroundMechanical ventilation with high tidal volumes (VT) in contrast to mechanical ventilation with low VT has been shown to increase plasma levels of proinflammatory and antiinflammatory mediators in patients with acute lung injury. The authors hypothesized that, in patients without previous lung injury, a conventional potentially injurious ventilatory strategy with high VT and zero end-expiratory pressure (ZEEP) will not cause a cytokine release into systemic circulation. MethodsA total of 39 patients with American Society of Anesthesiologists physical status I–II and without signs of systemic infection scheduled for elective surgery with general anesthesia were randomized to receive mechanical ventilation with either (1) VT = 15 ml/kg ideal body weight on ZEEP, (2) VT = 6 ml/kg ideal body weight on ZEEP, or (3) VT = 6 ml/kg ideal body weight on positive end-expiratory pressure of 10 cm H2O. Plasma levels of proinflammatory and antiinflammatory mediators tumor necrosis factor, interleukin (IL)-6, IL-10, and IL-1 receptor antagonist were determined before and 1 h after the initiation of mechanical ventilation. ResultsPlasma levels of all cytokines remained low in all settings. IL-6, tumor necrosis factor, and IL-1 receptor antagonist did not change significantly after 1 h of mechanical ventilation. IL-10 was below the detection limit (10 pg/ml) in 35 of 39 patients. There were no differences between groups. ConclusionsInitiation of mechanical ventilation for 1 h in patients without previous lung injury caused no consistent changes in plasma levels of studied mediators. Mechanical ventilation with high VT on ZEEP did not result in higher cytokine levels compared with lung-protective ventilatory strategies. Previous lunge damage seems to be mandatory to cause an increase in plasma cytokines after 1 h of high VT mechanical ventilation.

Gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specific genetic predisposition to sepsis.

ObjectivesTo determine whether the genotype frequencies of the five bi-allelic polymorphisms in the bactericidal/permeability increasing protein (BPI) (Lys216 → Glu;Pst I polymorphism in intron 5; silent mutation G545 → C) and the lipopolysaccharide binding protein (LBP) (Cys98 → Gly; Pro436 → Leu) are associated with the incidence and lethality of sepsis. DesignCase control study of patients with sepsis. SettingIntensive care units within university hospitals. PatientsA total of 204 patients diagnosed with sepsis and 250 healthy blood donors. InterventionsNone. Measurements and Main Results Short DNA fragments containing the polymorphic sites of the LBP and BPI locus were amplified by the polymerase chain reaction or mismatched polymerase chain reaction. The individual polymorphisms were determined with the appropriate restriction enzyme digestions and subsequent agarose gel electrophoresis. The presence of LBP genotypes with the less frequent Gly98 allele was found to be associated with sepsis (p < .02) in male patients, but not in females. Patients which were homozygote for either of the rare Gly98 (n = 6) and/or Leu436 (n = 5) LBP alleles, furthermore, exclusively were nonsurvivors of sepsis. The genotype frequencies in the BPI gene did not differ between patients and control individuals. ConclusionsOur findings suggest that common polymorphisms in the gene for LBP in combination with male gender are associated with an increased risk for the development of sepsis and, furthermore, may be linked to an unfavorable outcome. These data support the important immunomodulatory role of LBP in Gram-negative sepsis and suggest that genetic testing may be helpful for the identification of patients with an unfavorable response to Gram-negative infection.

Utility of a Commercially Available Multiplex Real-Time PCR Assay To Detect Bacterial and Fungal Pathogens in Febrile Neutropenia

ABSTRACT Infection is the main treatment-related cause of mortality in cancer patients. Rapid and accurate diagnosis to facilitate specific therapy of febrile neutropenia is therefore urgently warranted. Here, we evaluated a commercial PCR-based kit to detect the DNA of 20 different pathogens (SeptiFast) in the setting of febrile neutropenia after chemotherapy. Seven hundred eighty-four serum samples of 119 febrile neutropenic episodes (FNEs) in 70 patients with hematological malignancies were analyzed and compared with clinical, microbiological, and biochemical findings. In the antibiotic-naïve setting, bacteremia was diagnosed in 34 FNEs and 11 of them yielded the same result in the PCR. Seventy-three FNEs were negative in both systems, leading to an overall agreement in 84 of 119 FNEs (71%). During antibiotic therapy, positivity in blood culture occurred only in 3% of cases, but the PCR yielded a positive result in 15% of cases. In six cases the PCR during antibiotic treatment detected a new pathogen repetitively; this was accompanied by a significant rise in procalcitonin levels, suggestive of a true detection of infection. All patients with probable invasive fungal infection (IFI; n = 3) according to the standards of the European Organization for Research and Treatment of Cancer had a positive PCR result for Aspergillus fumigatus; in contrast there was only one positive result for Aspergillus fumigatus in an episode without signs and symptoms of IFI. Our results demonstrate that the SeptiFast kit cannot replace blood cultures in the diagnostic workup of FNEs. However, it might be helpful in situations where blood cultures remain negative (e.g., during antimicrobial therapy or in IFI).

Respiratory Depression with Tramadol in a Patient with Renal Impairment and CYP2D6 Gene Duplication

We observed opioid-related respiratory depression in a patient receiving tramadol via patient-controlled analgesia. Predisposing factors were the patients genetic background and renal impairment. Complete recovery occurred after naloxone administration, thus confirming opioid intoxication. Analysis of the patients genotype revealed a CYP2D6 gene duplication resulting in ultra-rapid metabolism of tramadol to its active metabolite (+)O-desmethyltramadol. Concomitant renal impairment resulting in decreased metabolite clearance enhanced opioid toxicity. This genetic CYP2D6 variant is particularly common in specific ethnic populations and should be a future diagnostic target whenever administration of tramadol or codeine is anticipated, as both drugs are subject to a comparable CYP2D6-dependent metabolism.

The hypothalamic-pituitary-adrenal axis of patients with severe sepsis: altered response to corticotropin-releasing hormone.

ObjectiveTo investigate the functional integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with severe sepsis by stimulating with corticotropin-releasing hormone (CRH). DesignProspective observational study in consecutive intensive care unit patients with severe sepsis. SettingSurgical intensive care unit and outpatient department of endocrinology in a university hospital. PatientsThe study included 20 patients with the diagnosis of severe sepsis; six critically ill, nonseptic patients after major surgery; ten patients with primary adrenal insufficiency; ten patients with anterior pituitary insufficiency; and ten individuals without clinical signs of HPA axis disturbance. InterventionsCRH tests were performed with an intravenous bolus injection of 100 &mgr;g of human CRH. Measurements and Main Results We studied the functional integrity of the HPA axis in patients with severe sepsis by performing the CRH test. In addition, during the period of severe sepsis, we repeatedly measured basal plasma concentrations of adrenocorticotropin hormone (ACTH) and cortisol. The mean basal plasma cortisol concentration was decreased significantly in nonsurvivors with severe sepsis (288.8 ± 29.1 [sem] nmol/L) compared with survivors (468.1± 18.6 nmol/L;p < .01). By calculating the ACTH/cortisol indices, we found no evidence for adrenal insufficiency in patients with severe sepsis. The mean ACTH/cortisol indices of nonsurvivors with severe sepsis (0.02 ± 0.008) and survivors (0.01 ± 0.002) were significantly lower compared with the index of patients with primary adrenal insufficiency (6.8 ± 1.0;p < .001). In contrast, in nonsurvivors with severe sepsis, the plasma cortisol response to CRH stimulation was impaired compared with survivors: The mean basal cortisol concentration within the CRH test was 269.4 ± 39.8 nmol/L in nonsurvivors compared with 470.8 ± 48.4 nmol/L in survivors and increased to a peak value of 421.6 ± 72.6 nmol/L in nonsurvivors and 680.7 ± 43.8 nmol/L in survivors (p < .02). However, the change in plasma cortisol, expressed as mean ± sem and calculated by subtracting the basal cortisol from the peak cortisol after CRH stimulation, was not significantly different in survivors with severe sepsis (243.5 ± 36.1, range 111.0–524.0 nmol/L, n = 15) compared with nonsurvivors (161.0 ± 38.9, range 42.0–245.0 nmol/L, n = 5;p > .05). ConclusionsWe found lower basal plasma cortisol concentrations in nonsurvivors compared with survivors of severe sepsis. In addition, the plasma cortisol response to a single CRH stimulation was impaired in nonsurvivors compared with survivors. Reduced responses to CRH stimulation may reflect a state of endocrinologic organ dysfunction in severe sepsis.

Practice and perception—A nationwide survey of therapy habits in sepsis*

Objective:To simultaneously determine perceived vs. practiced adherence to recommended interventions for the treatment of severe sepsis or septic shock. Design:One-day cross-sectional survey. Setting:Representative sample of German intensive care units stratified by hospital size. Patients:Adult patients with severe sepsis or septic shock. Interventions:None. Measurements and Main Results:Practice recommendations were selected by German Sepsis Competence Network (SepNet) investigators. External intensivists visited intensive care units randomly chosen and asked the responsible intensive care unit director how often these recommendations were used. Responses “always” and “frequently” were combined to depict perceived adherence. Thereafter patient files were audited. Three hundred sixty-six patients on 214 intensive care units fulfilled the criteria and received full support. One hundred fifty-two patients had acute lung injury or acute respiratory distress syndrome. Low-tidal volume ventilation ≤6 mL/kg/predicted body weight was documented in 2.6% of these patients. A total of 17.1% patients had tidal volume between 6 and 8 mL/kg predicted body weight and 80.3% >8 mL/kg predicted body weight. Mean tidal volume was 10.0 ± 2.4 mL/kg predicted body weight. Perceived adherence to low-tidal volume ventilation was 79.9%. Euglycemia (4.4–6.1 mmol/L) was documented in 6.2% of 355 patients. A total of 33.8% of patients had blood glucose levels ≤8.3 mmol/L and 66.2% were hyperglycemic (blood glucose >8.3 mmol/L). Among 207 patients receiving insulin therapy, 1.9% were euglycemic, 20.8% had blood glucose levels ≤8.3 mmol/L, and 1.0% were hypoglycemic. Overall, mean maximal glucose level was 10.0 ± 3.6 mmol/L. Perceived adherence to strict glycemic control was 65.9%. Although perceived adherence to recommendations was higher in academic and larger hospitals, actual practice was not significantly influenced by hospital size or university affiliation. Conclusions:This representative survey shows that current therapy of severe sepsis in German intensive care units complies poorly with practice recommendations. Intensive care unit directors perceive adherence to be higher than it actually is. Implementation strategies involving all intensive care unit staff are needed to overcome this gap between current evidence-based knowledge, practice, and perception.

Genetic factors in pain and its treatment.

Purpose of review Genomic variations influencing basal pain sensitivity, the likelihood of developing chronic pain diseases as well as the response to pharmacotherapy of pain are currently under investigation Here, we review examples of promising approaches to diagnose genetic predisposition from recently published studies. Recent findings Candidate genes such as those for catechol-O-methyltransferase, melanocortin-1 receptor, guanosine triphosphate cyclohydrolase and μ-opioid receptor have been intensively investigated, and associations were found with sensitivity to pain as well as with analgesic requirements in states of acute and chronic pain. In contrast, the impact of genetic variants of drug-metabolizing enzymes on the response to pharmacotherapy is generally well described. Polymorphisms of the cytochrome P450 enzymes influence the analgesic efficacy of codeine, tramadol, tricyclic antidepressants and nonsteroidal antiinflammatory drugs. Together with further candidate genes, they are major targets of ongoing research in order to identify associations between an individuals genetic profile and drug response (pharmacogenetics). Summary The article reviews recent studies on genetic variables influencing pain and pharmacotherapy. Examples of promising candidate genes have been intensively studied during recent years. Although the number of subjects investigated is often small, published data and current advances in genotyping and study design suggest valid and clinically relevant results to date and even more in the future.

Improved detection of blood stream pathogens by real-time PCR in severe sepsis.

ObjectiveEvaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures.DesignDual-center cohort study.SettingSurgical ICU of two university hospitals.Patients and participantsOne hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included.InterventionsNone.Measurements and resultsPCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93.ConclusionsOur cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical setting.

Analysis of two human leukocyte antigen-linked polymorphic heat shock protein 70 genes in patients with severe sepsis.

OBJECTIVE To determine whether the genotype and allelic frequencies of two human leukocyte antigen-linked bi-allelic 70-kilodalton heat shock protein (HSP70) gene polymorphisms are associated with susceptibility to and outcome of severe sepsis. Furthermore, we investigated a possible linkage between HSP70 gene polymorphisms and the previously reported and mortality-related tumor necrosis factor-beta (TNF-beta) NcoI gene polymorphism. DESIGN Consecutive entry study of patients with severe sepsis. SETTING Surgical intensive care unit in a university hospital. PATIENTS Eighty-seven patients with a diagnosis of severe sepsis. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS We studied two bi-allelic polymorphisms within the coding region of the constitutively expressed HSP70-HOM C/T, and the stress-inducible HSP70-2 G/A in patients with severe sepsis. The HSP70-HOM Ncol, HSP70-2 Pstl, and TNF-beta NcoI polymorphisms were identified by means of the polymerase chain reaction followed by restriction analysis of the polymerase chain reaction product. No significant differences in genotype and allelic frequencies were observed for both HSP70 gene polymorphisms between the 87 patients and the 110 healthy Caucasians serving as the control group. In addition, no differences in genotype and allelic frequencies between surviving and nonsurviving patients were detected. The allelic frequencies in the group of nonsurvivors were 0.8 for the HSP70-HOM C allele and 0.2 for the HSP70-HOM T allele vs. 0.87 and 0.13 for the survivors (p > .05). The frequency for the HSP70-2 G allele was 0.36 and 0.64 for the HSP70-2 A allele in the group of nonsurvivors vs. 0.41 and 0.59 for the survivors (p > .05). Analysis of a possible linkage between HSP70 and TNF-beta genotypes resulted in a significant association (odds ratio, 4.15; p < .01) of the HSP70-2 A/A homozygous genotype and the TNFB2/B2 homozygous genotype, which is reported to be a genomic marker for a poor prognosis in severe sepsis. CONCLUSIONS Our data show that the bi-allelic NcoI and PstI polymorphisms within the HSP70-HOM and HSP70-2 locus, respectively, are associated with neither susceptibility to nor outcome of severe sepsis. Moreover, we found a linkage between HSP70-2 A homozygotes and the previously reported and mortality-related homozygous genotype, TNFB2/B2, in patients suffering from severe sepsis.