Infections in patients hospitalized for fever as related to duration and other predictors at admittance.

PURPOSE: To find the underlying diagnoses of patients admitted to a medical department with symptoms of fever to ascertain whether the duration of fever, temperature readings, and common laboratory tests could give a clue to the final diagnosis. METHODS: A retrospective study of the records of 299 patients admitted to the Medical Department, Haukeland University Hospital from July 1st, 2001 until June 30th, 2004 for fever without any diagnosis suggesting the cause of the fever. RESULTS AND CONCLUSIONS: Infections were the most common diagnoses, but the proportion of patients with infections declined with increasing duration of fever before admission (63.7 % when <7 days, 45.8 % when >21 days). A group of patients on immunosuppressive therapy were all hospitalized within 1 week from the debut of fever, and their causes of fever were comparable to those of non-immunocompromised patients with fever of the same duration. With fever <7 days, patients with bacterial or viral infection had higher maximum temperatures (medians 39.2 and 38.9 °C, respectively) than those without infection (median 38.0 °C). Patients with bacterial infection had higher C-reactive protein (CRP) levels than patients in the other groups, whereas viral infection was associated with higher lymphocyte counts. The neutrophil:lymphocyte ratio was higher in patients with fever due to bacterial infections than in those with viral infections. For patients with fever for <1 week, neutrophil [area under the curve (AUC) 0.723], white blood cell (WBC, AUC 0.692), and monocyte (AUC 0.691) counts and CRP levels (AUC 0.684) were the best single indicators of bacterial infection.

Flow cytometry technique for analysing Leishmania promastigote phagocytosis by human polymorphonuclear leucocytes and monocytes.

In this study, we developed a flow cytometry technique for studying Leishmania (L.) mexicana phagocytosis by human polymorphonuclear leucocytes (PMNs) and monocytes. Leishmania promastigotes are elongated in shape and flagellated. This influences the light scatter when phagocytosis is measured by flow cytometry. Accordingly, we developed an oxidative burst method for measuring the phagocytic process. As this is an indirect marker of phagocytosis, we used confocal, light and electron microscopy to verify that promastigotes were, indeed, internalized by the phagocytes. For both PMNs and monocytes, the optimal conditions for achieving high sensitivity in flow cytometry detection were 5% pooled human serum and 15 min. incubation time. Incubations at 35, 37 and 39°C were also equally efficient for both PMNs and monocytes. Optimal parasite ratios were 10 parasites per PMN and 20 parasites per monocyte. Under these conditions, Leishmania were readily phagocytosed by human PMNs and monocytes and the effects of other influences, such as treatment, would be readily detectable. This indicated that these cells may play a role in the immune response against Leishmania.

Pandemic influenza vaccination elicits influenza-specific CD4+ Th1-cell responses in hypogammaglobulinaemic patients: four case reports.

In these case reports, we investigated pandemic influenza 2009 vaccination of primary hypogammaglobulinaemic patients. Three combined variable immunodeficiency (CVID) patients and one X-linked agammaglobulinaemia (XLA) patient were vaccinated with the pandemic vaccine A/California/7/2009 (H1N1)-like split virus (X179a) adjuvanted with the oil-in-water emulsion AS03. Subsequently, serum and peripheral blood mononuclear cells were sampled and used to measure the haemagglutination inhibition (HI) and antibody-secreting cell (ASC) responses. In addition, the IFN-γ, IL-2 and TNF-α producing CD4(+) Th1-cell response was determined as these cytokines are important indicators of cell-mediated immunity. Two of the CVID patients responded to vaccination as determined by a >4-fold rise in HI antibodies. These subjects also had influenza-specific ASC numbers, which, albeit low, were higher than prevaccination levels. In addition, vaccination induced CD4(+) Th1-cell responses in both the XLA patient and the CVID patients, although the frequency of influenza-responsive cells varied amongst the patients. These results suggest that hypogammaglobulinaemia patients can mount a CD4(+) Th1 cell-mediated response to influenza vaccination and, additionally, that influenza vaccination of some hypogammaglobulinaemia patients can produce an influenza-specific humoral immune response. The findings should be confirmed in larger clinical studies.

Retention of phagocytic functions in cryopreserved human monocytes.

Phagocytosis and respiratory burst activity were measured by flow cytometry in fresh and cryopreserved human monocytes, after ingestion of Escherichia coli and Staphylococcus aureus. Mononuclear leukocytes, isolated from 15 healthy donors, were divided into two portions, of which one was examined immediately and the other was cryopreserved for 3 weeks. Morphological characteristics and expression of receptors involved in phagocytosis were similar in fresh and cryopreserved monocytes. Furthermore, both internalization of bacteria and respiratory burst activity remained unchanged after cryopreservation. Transmission electron microscopy confirmed actual internalization of bacteria and not merely bacterial attachment to monocytes. Monocytes were demonstrated to retain integral cellular functions during cryopreservation. This may imply that the method has potential for use in basal and clinical trials.

Flow cytometric assay for the measurement of serum opsonins to Neisseria meningitidis serogroup B, serotype 15.

A flow cytometric phagocytosis assay has been developed for the measurement of human serum opsonins to serogroup B meningococci. Live bacteria and bacteria inactivated by heat, formalin or ethanol were labelled with fluorescein-isothiocyanate (FITC). The bacteria were opsonized with sera from patients with group B meningococcal disease and sera from healthy controls, and phagocytosis determined by combined measurements of FITC-fluorescence and forward angle light scatter. Optimal sensitivity was obtained using viable bacteria, 5% serum, 20 bacteria per leukocyte capable of phagocytosis, 7.5 min opsonization time, 5 min phagocytosis time, 37 degrees C, and continuous agitation during opsonization and phagocytosis. The opsonic activity of sera from convalescent patients was markedly higher than that of sera from patients with acute illness. Only minor day-to-day and interindividual variations were observed. The flow cytometric phagocytosis technique is a rapid and reproducible method for the measurement of serum opsonins to meningococci.

Negative selection of human monocytes using magnetic particles covered by anti-lymphocyte antibodies.

We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque gradient separated mononuclear cells and 32% from whole blood. In a lucigenin enhanced chemiluminescence assay there was no significant difference between cells separated immunomagnetically and those separated on a gradient. Nor did electron microscopy show any significant difference in morphology between such monocytes. Negative selection using magnetic polymer particles is an efficient method for the separation of monocytes with intact morphology and function as measured by chemiluminescence.

Antibiotic susceptibility of blood culture isolates of Enterobacteriaceae from six Norwegian hospitals 1991-1992.

From August 1991 to February 1992, each of the six largest hospitals throughout Norway collected 84 to 107 consecutive blood culture isolates of Enterobacteriaceae, altogether 571 isolates. The distribution of various species and genera at the different hospitals was uniform; Escherichia coli being most prevalent (57-67%), followed by Klebsiella spp. (12-18%) and Proteus mirabilis (7-11%). Twenty-one and 4% of E. coli isolates were resistant to ampicillin and cefuroxime, respectively, and 11% of Klebsiella isolates were cefuroxime resistant. Five Enterobacter isolates and one Citrobacter isolate were resistant to ceftazidime, and one Salmonella isolate was resistant to imipenem. All isolates were susceptible to ciprofloxacin and tobramycin. These results were compared with the antibiotic consumption in each hospital region. Although hospitals in the regions with the highest consumption of ampicillin tended to have a higher percentage of isolates resistant to this agent, no significant differences were found. There was no significant difference between hospitals regarding prevalence of cefuroxime-resistant isolates.

Antibiotic susceptibility of blood culture isolates of Enterobacteriaceae. A Norwegian multicenter study.

From May to November 1997 each of six major hospitals throughout Norway collected 72 to 104 consecutive blood culture isolates of Enterobacteriaceae, altogether 563 isolates. Escherichia coli was the predominating organism (69%), followed by Klebsiella spp. (15%), Enterobacter spp. (6%), and Proteus mirabilis (4%). The susceptibility of the isolates to ampicillin, cefuroxime, ceftazidime, imipenem, tobramycin, and ciprofloxacin was determined by the E-test. 37% and 7% of the isolates were resistant to ampicillin and cefuroxime, respectively, and 1% were resistant to ceftazidime and tobramycin. Only one isolate of P. mirabilis was imipenem resistant. All isolates were susceptible to ciprofloxacin. The prevalence of ampicillin-resistant isolates at each hospital varied from 21 to 45%, and of cefuroxime-resistant isolates from 3 to 9%. The results were compared with those of a similar study performed in 1991-1992. No significant changes in the susceptibility to the various agents could be demonstrated. The high frequency of isolates resistant to ampicillin has clearly limited the usefulness of this agent in the treatment of septicemia and other serious infections caused by Enterobacteriaceae.

Two-layer gradient separation of granulocytes for functional tests.

Two gradient separation techniques were compared with the dextran sedimentation method for the separation of granulocytes from blood. Both techniques gave adequate yield and excellent purity, and flow cytometric analysis of surface membrane markers gave no indication of subset selection during the procedure. Cells separated by the three methods behaved comparably in functional tests including random migration, chemotaxis and chemiluminescence. The gradient separation techniques are rapid and efficient methods for the preparation of near 100% pure granulocyte suspensions for functional studies.

Immunization against serogroup B meningococci. Opsonin response in vaccinees as measured by chemiluminescence.

One hundred and thirteen healthy volunteers were immunized twice (six weeks apart) with four different doses (12.5, 25, 50 and 100 micrograms, measured as protein content) of an outer membrane vesicle vaccine from a serogroup B meningococcal strain (44/76, B:15:P1.16) complexed to serogroup C meningococcal polysaccharide and/or Al(OH)3 i.e. 12 different vaccines. Serum opsonic activity against the serogroup B strain was measured using a chemiluminescence method. A significant rise in serum opsonic activity was demonstrated in 84 volunteers (74%) six weeks after the first injection and in 97 (86%) six weeks after the second. All vaccinees with low preimmunization values (less than 25 mVs) experienced a significant increase in opsonic activity. A dose-related response was most evident for the vaccines containing adjuvant, and these vaccines were associated with a maximum response six weeks after the second injection, while the vaccines without Al(OH)3 induced a peak response six weeks after the first injection. The postimmunization opsonic activity was similar to that found in convalescent sera, indicating that the vaccines may protect against serogroup B meningococcal disease.

IgG subclass antibodies to serogroup B meningococcal outer membrane antigens following infection and vaccination.

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B, serotype 15:P1.16) were quantitated by an enzyme-linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non-meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG1 and IgG3 antibodies to the class I protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgG1 and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.

Monocyte phagocytosis of opsonized Neisseria meningitidis serogroup B.

The chemiluminescence (CL) was examined when peripheral blood monocytes were incubated with opsonized Neisseria meningitidis, serogroup B, serotype 15:P1.16 or serotype 2a:P1.2. The monocytes were separated from a mononuclear cell suspension by an immunomagnetic negative selection technique using magnetic polystyrene microspheres coated with monoclonal antibodies specific for T and B lymphocytes. More than 90% of the lymphocytes were removed, yielding a suspension containing 93% monocytes. Optimal sensitivity for phagocytosis was obtained using 1% serum (10 microliters), 72 bacteria per monocyte cell, and 7.5 min opsonization and incubation time during continuous agitation at 37 degrees C. The CL was amplified by lucigenin. Preliminary experiments suggest that convalescent sera from patients with group B meningococcal disease induced increased CL responses compared to acute sera. Sera from volunteers immunized with an outer membrane complex vaccine from serogroup B, serotype 15:P1.16 or 2a:P1.2 meningococci also induced increased CL activity compared to preimmune sera. No such response was shown when a group B capsular polysaccharide vaccine was given. This response pattern was also demonstrated by a flow cytometric phagocytosis technique (FCM). Internalization of meningococci by monocytes was demonstrated by a FCM quenching technique and by transmission electron microscopy. CL and FCM represent rapid and reproducible methods for the measurement of opsonophagocytosis of meningococci by monocytes and may be performed with minute amounts of sera.

Detection of diphtheria antitoxin by four different methods.

OBJECTIVE: To investigate the reliability of the different methods used in Norway and Russia for detection of diphtheria antitoxin. METHODS: One hundred and twenty-two sera were selected among Russian serum samples previously collected for seroepidemiologic studies of diphtheria antitoxin. The sera were selected to cover the total antitoxin range and were analyzed by four different antidiphtheria toxin assays: an in vitro toxin neutralization test using Vero cells (in vitro NT), an in vivo neutralization test using rabbit skin inoculation (in vivo NT), an indirect enzyme immunoassay (EIA) and a passive hemagglutination assay (PHA). The results were expressed according to the international standard as: not protected (<0.01 IU/mL), relatively protected (0.01-0.1 IU/mL) or protected (≥0.1 IU/mL). The sensitivity, specificity and inter-rater agreement (K or Kw) of each method were related to the in vitro NT selected as the reference method. RESULTS: The in vivo NT test corresponded very well with the in vitro NT in its ability to differentiate between protection/relative protection and no protection (sensitivity 97%, specificity 87% and K=0.84). The EIA test showed a high sensitivity (96%), but since many sera were categorized as protected rather than not protected, the specificity (30%) and inter-rater agreement (K=0.29) were low. The PHA test had a very high specificity (100%) but a low sensitivity (86%). CONCLUSIONS: The agreement between the two neutralization tests was high. If none of the neutralization assays is routinely available, the PHA test can be used to predict the need for vaccination on an individual basis but should not be used for seroepidemiologic studies, since the protection rate for diphtheria would be falsely too low, due to the lower sensitivity. The indirect EIA test used in this study should not be used routinely.

Effects of polycyclic aromatic compounds on the drought tolerance of Folsomia fimetaria (Collembola, Isotomidae).

Drought tolerance (survival at 98.2% relative humidity and 20 degress C) was investigated for adult individuals of the springtail Folsomia fimetaria after three weeks of exposure to different polycyclic aromatic compounds (PACs). Seven PACs were investigated (acridine, dibenzofuran, carbazole, fluorene, dibenzothiophene, fluoranthene, and pyrene), with each substance used at several exposure concentrations. Sublethal concentrations of dibenzothiophene, fluorene, fluoranthene, and pyrene caused a dose-related decrease in drought tolerance in exposed adults, whereas no such relationship was found for acridine, dibenzofuran, and carbazole. The organisms used in the drought tolerance study were taken directly from standard ecotoxicity tests that had just been completed, hence the concentrations causing subsequent effects on drought tolerance and those affecting survival and reproduction in the ecotoxicity tests could be compared. Some of the tested substances significantly reduced the drought tolerance of F. fimetaria at concentrations that had little effect on survival, indicating a synergistic effect of the two stressors. However, drought tolerance was affected at concentrations below the threshold value for toxicity in standard tests (no-observed-effect concentration for reproduction) only for dibenzothiophene. In the field, soil organisms exposed to contaminants also face multiple environmental stressors such as drought and cold. Because the reduced drought tolerance is a side effect of chemical exposure, such effects should be considered when extrapolating from laboratory to field conditions.

Freeze induced glucose accumulation in the enchytraeid, Fredericia ratzeli from Greenland.

The tolerance of freezing and associated accumulation of cryoprotectants was studied in an Arctic population of the enchytraeid Fredericia ratzeli. At -3 and -5 degrees C specimens readily froze when slowly cooled in contact with moist soil. A small fraction of the animals (10-20 %) survived internal ice formation under these circumstances. Frozen specimens had elevated glucose concentrations as compared to unfrozen control animals acclimated at 0 degrees C. In a fraction of the animals, equal to the fraction surviving freezing, a high concentration of glucose was detected. The highest values amounted to ca. 150 microg mg(-1) dry weight, corresponding to ca. 270 mmoles L(-1). It is argued that the physiology of freeze tolerance in this enchytraeid resembles the physiology described for freeze tolerant earthworms and frogs.

Interactions between environmental pollution and cold tolerance of soil invertebrates: a neglected field of research.

The adaptations to climatic stress of invertebrates have been extensively studied and the underlying physiology is relatively well understood. It is therefore possible to predict critical lower temperature and moisture limits at which a species will suffer increased mortality. Environmental pollution can reduce the tolerance to climatic stress considerably but it is not known how significant these synergistic effects are in polluted areas. Results are presented showing examples of synergism between toxic and climatic stress. The physiological mechanisms involved in the synergistic effects are discussed.

Bacterial findings in infected hip joint replacements in patients with rheumatoid arthritis and osteoarthritis: a study of 318 revisions for infection reported to the norwegian arthroplasty register.

High rates of Staphylococcus aureus are reported in prosthetic joint infection (PJI) in rheumatoid arthritis (RA). RA patients are considered to have a high risk of infection with bacteria of potentially oral or dental origin. One thousand four hundred forty-three revisions for infection were reported to the Norwegian Arthroplasty Register (NAR) from 1987 to 2007. For this study 269 infection episodes in 255 OA patients served as control group. In the NAR we identified 49 infection episodes in 37 RA patients from 1987 to 2009. The RA patients were, on average, 10 years younger than the OA patients and there were more females (70% versus 54%). We found no differences in the bacterial findings in RA and OA. A tendency towards a higher frequency of Staphylococcus aureus (18% versus 11%) causing PJI was found in the RA patients compared to OA. There were no bacteria of potential odontogenic origin found in the RA patients, while we found 4% in OA. The bacteria identified in revisions for infection in THRs in patients with RA did not significantly differ from those in OA. Bacteria of oral or dental origin were not found in infected hip joint replacements in RA.

[Antibiotic treatment of infectious endocarditis]. / Antibiotikabehandling ved infeksiøs endokarditt.

BACKGROUND: Infective endocarditis is a septic condition in which the heart valves and the endocardial surfaces are infected, causing bacteraemia. In Norway, infective endocarditis is the most common infectious disease of the heart and accounts for approximately one case per 1,000 hospital admissions. The mortality rate is 15-40%. MATERIAL AND METHODS: This article presents a review of antimicrobial treatment of infective endocarditis, based on relevant literature and the author's clinical experience. Prophylactic treatment is not described. RESULTS AND INTERPRETATION: Early start of antimicrobial treatment is extremely important in order to avoid destruction of the heart valves and should always be started as soon as endocarditis is suspected and blood cultures have been drawn. The patient must be treated with bactericidal antibiotics in high doses given intravenously for four to eight weeks, depending on the aetiologic agents and the response. Only antibiotics that have been proven effective against endocarditis should be used. The minimal inhibitory concentration (MIC) should be determined for all antibiotics used.

Bacterial contamination of drops and dropper tips of in-use multidose eye drop bottles.

The type and frequency of bacterial contamination of 638 in-use multidose eye drop bottles are reported. By dripping, bacteria were recovered from 82 bottles (12.9%). Bacterial growth considered to be clinically and microbiologically significant were found in 12 cases (1.9%). The swabbings from 38 of 180 dropper tips (21.1%) yielded bacteria, while none of the pipette aspirates from 196 bottles were positive. The flora obtained by dripping and swabbing was very similar to the normal conjunctival and skin flora. It is suggested that microorganisms isolated by dripping often originate from contaminated dropper tips. The frequency of contaminated drops did not increase with increasing duration of use of the bottles. Repeated examinations and inoculation studies indicated that the solutions were self-sterilizing. This indicates that multidose eye drop bottles preserved and dispensed as in the present study may be used for more than the usual 4 weeks without increasing the risk of ocular infection.