The mild inflammatory response in febrile neutropenic lymphoma patients with low risk of complications is more pronounced in patients receiving tobramycin once daily compared with three times daily.

We evaluated inflammatory markers in febrile neutropenic lymphoma patients undergoing high-dose chemotherapy with autologous stem cell support. Based on MASCC scores, our patients had a low risk of serious complications and a perspective of a benign initial clinical course of the febrile neutropenia. We also studied the impact of tobramycin given once versus three times daily on these immune markers. Sixty-one patients participating in a Norwegian multicentre prospective randomized clinical trial, comparing tobramycin once daily versus three times daily, given with penicillin G to febrile neutropenic patients, constituted a clinically homogenous group. Four patients had bacteraemia, all isolates being Gram-positive. Thirty-two patients received tobramycin once daily, and 29 patients received tobramycin three times daily. Blood samples were taken at the onset of febrile neutropenia and 1-2 days later. All samples were frozen at -70 °C and analysed at the end of the clinical trial for C-reactive protein (CRP), procalcitonin (PCT), complement activation products, mannose-binding lectin (MBL) and 17 cytokines. We found a mild proinflammatory response in this series of patients. CRP was non-specifically elevated. Ten patients with decreased MBL levels showed the same mild clinical and proinflammatory response. Patients receiving tobramycin once daily showed a more pronounced proinflammatory response compared with patients receiving tobramycin three times daily. Overall, febrile neutropenic cancer patients with a benign clinical course show a mild proinflammatory immune response.

Molecular characteristics of pharyngeal and invasive emm3 Streptococcus pyogenes strains from Norway, 1988-2003.

A major virulence factor of group A streptococci (GAS) is the M protein. Strains with the M3 type are more often associated with necrotizing fasciitis (NF) and streptococcal toxic shock syndrome, and have a higher case fatality rate than strains of other M types. To better understand the epidemiology of M3 GAS strains in Norway, we analyzed 59 invasive and 69 pharyngeal isolates with respect to prophage content, allelic variation in emm3, mtsR encoding the metal transporter of Streptococcus repressor (mtsR), and sclB coding for streptococcal collagen-like protein B. The Norwegian emm3 strains were very homogeneous, mainly harboring the emm allele 3.1 and prophage profile PhiG3.01. Other prophage profiles were transient. The mutation in mtsR known to truncate the protein and result in decreased capacity to cause NF was not found in our isolates. The sclB gene usually harbored five or eight contiguous repeats of a CAAAA pentanucleotide sequence and a highly modular and variable collagen structural motif (CSM) region with 9 and 12 amino acid M3-specific conserved motif repeats distributed across the entire CSM region. Strains with 5 CAAAA repeats emerged in 1993 and these strains were associated with the increase in invasive M3 cases in the period 1993-2003.

Benzylpenicillin plus an aminoglycoside versus meropenem in neutropenic lymphoma and leukaemia patients with a suspected bacterial infection: a randomized, controlled trial.

OBJECTIVES: In Norway, initial treatment of febrile neutropenia (FN) has traditionally been benzylpenicillin plus an aminoglycoside. Internationally, FN is often treated with a broad-spectrum ß-lactam antibiotic. We aimed to compare these two regimens in a prospective, randomized, trial in patients with lymphoma or leukaemia with an expected period of neutropenia ≥7 days, and a suspected bacterial infection. METHODS: Adult neutropenic patients with lymphoma or leukaemia, and a suspected bacterial infection, were randomized for treatment with benzylpenicillin plus an aminoglycoside or meropenem. The primary endpoint was clinical success, defined as no modification of antibiotics and clinical stability 72 h after randomization. RESULTS: Among 322 randomized patients, 297 proved evaluable for analyses. Fifty-nine per cent (95% CI 51%-66%), (87/148) of the patients given benzylpenicillin plus an aminoglycoside were clinically stable, and had no antibiotic modifications 72 h after randomization, compared with 82% (95% CI 75%-87%), (122/149) of the patients given meropenem (p <0.001). When the antibiotic therapy was stopped, 24% (95% CI 18%-32%), (36/148) of the patients given benzylpenicillin plus an aminoglycoside, compared with 52% (95% CI 44%-60%), (78/149) of the patients given meropenem, had no modifications of their regimens (p <0.001). In the benzylpenicillin plus an aminoglycoside arm, the all-cause fatality within 30 days of randomization was 3.4% (95% CI 1.2%-7.9%), (5/148) of the patients, compared with 0% (95% CI 0.0%-3.0%), (0/149) of the patients in the meropenem arm (p 0.03). CONCLUSION: Clinical success was more common in FN patients randomized to meropenem compared with the patients randomized to benzylpenicillin plus an aminoglycoside. The all-cause fatality was higher among the patients given benzylpenicillin plus an aminoglycoside.

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Quantitation of IgG subclass antibody responses after immunization with a group B meningococcal outer membrane vesicle vaccine, using monoclonal mouse-human chimeric antibodies as standards.

An ELISA method was developed to quantitate gravimetrically (microgram/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1/3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

Aminoethyl-isothiourea inhibits the increase in plasma endothelin-1 caused by serogroup A streptococci and prolongs survival in rat peritoneal sepsis.

To elucidate the possible roles of nitric oxide (NO), endothelin-1 (ET-1), and reactive oxygen species (ROS) in the pathophysiology of serogroup A streptococcal (GAS) peritoneal sepsis, we investigated the effects of aminoethylisothiourea (AE-ITU), an inducible NO synthase (iNOS) inhibitor, and a ROS scavenger, and the ET-1 receptor antagonist bosentan. In rats, live GAS inocula, 3 x 10(8) and 1 x 10(9) cfu/kg, entailed a 24-h mortality of 10% and 90%, respectively. GAS caused increases in tissue iNOS activity (9 h), in serum nitrite/nitrate (9-24 h), and in intracellular leukocyte ROS levels (3-6 h). These changes were all prevented by the pre-treatment with AE-ITU. A novel finding was that AE-ITU also prevented the GAS-induced marked increase in plasma ET-1 at 6 h. Short-term (7-h) survival was improved by both AE-ITU and by bosentan. The mechanism(s) for the beneficial effects of AE-ITU may possibly be a combined mode of action; iNOS inhibition, ROS scavenging, and inhibition of the increase in plasma ET-1 caused by GAS.

Changed expression of leukocyte adhesion molecules and increased production of reactive oxygen species caused by Streptococcus pyogenes in human whole blood.

To elucidate the innate immune responses to group A streptococci (GAS) important in the pathophysiology of sepsis, flow cytometric techniques were applied to study the effects of live and heat-inactivated GAS, including their particulate and soluble components, on the expression of leukocyte adhesion molecules CD11b (Mac-1) and CD62L (L-selectin), and leukocyte production of reactive oxygen species (ROS) in human whole blood. GAS caused marked time- and concentration-dependent increases in CD11b and ROS, while CD62L was downregulated. Live and heat-inactivated GAS induced similar changes in leukocyte adhesion molecules, whereas ROS production induced by heat-inactivated GAS (and its particulate fraction) was 4 (2.5)-fold higher than with live GAS. Leukocyte nitric oxide production (24 h) was not enhanced. Although GAS proved a more potent inducer of ROS production, leukocyte responses to GAS were similar to those reported for lipolysaccharides, indicating that Gram-positive and Gram-negative bacteria activate common pathways in the inflammatory response. High ROS production may contribute to tissue damage caused by GAS.

Serogroup determination of Neisseria meningitidis by whole-cell ELISA, dot-blotting and agglutination.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

Strain differentiation of Neisseria meningitidis by small-fragment restriction endonuclease analysis (SF-REA).

Following an outbreak involving 3 cases of serogroup B meningococcal disease in a rural part of Western Norway, 2 clinical and 99 carrier isolates of Neisseria meningitidis were examined by small-fragment restriction endonuclease analysis (SF-REA) using EcoRI, to determine its potential for strain differentiation. SF-REA characterized all isolates and provided reproducible results with acceptable inter-clonal differentiation. The results of SF-REA correlated well with those of serological typing and were used to determine clonal diversity and prevalence of invasive strains among the carrier isolates. SF-REA was also useful in demonstrating acquisition of a new carrier strain after eradication of the initial strain by ofloxacin. Thirty-one different restriction patterns/pattern complexes were recognized among the 101 isolates. The two clinical isolates had identical restriction patterns and showed > or = 90% similarity to those of six carrier isolates. In three out of six apparent treatment failures, successful eradication of the original strain by ofloxacin was demonstrated by SF-REA. SF-REA proved valuable in strain differentiation of Neisseria meningitidis, complemented serology, and characterized all isolates which could not be typified by serology.

Low occurrence of antibiotic resistance in Escherichia coli and staphylococci isolated from blood cultures in two Norwegian hospitals in 1991-92 and 1995-96.

The aim of this study was to investigate the antibiotic resistance rates of major bacterial pathogens causing bloodstream infections in two very different types of hospital in Norway. We examined all Escherichia coli and staphylococci (330 isolates) causing bloodstream infections from one general county hospital and one specialist national cancer hospital during the periods 1991-92 and 1995-96. Minimal inhibitory concentrations (MICs) were determined using the E-test. E. coli and staphylococci constituted 46.7% of all isolates from bloodstream infections in the two hospitals. Overall, E. coli isolates were resistant to amoxicillin (21%), trimethoprim (21%), doxycycline (20%) and trimethoprim-sulphamethoxazole (17%), while Staphylococcus aureus strains were resistant to benzylpenicillin (66%). No methicillin-resistant S. aureus was detected. Coagulase-negative staphylococci were often multiresistant, but remained fully sensitive to vancomycin. For a few antibiotics, significantly more resistance was found in the specialist hospital. In our material we found no significant increase in resistance between 1991-92 and 1995-96. In conclusion, antimicrobial resistance still remains low in important bacterial pathogens causing bloodstream infections in Norway.

Serotyping and subtyping of Neisseria meningitidis isolates by co-agglutination, dot-blotting and ELISA.

Typing of meningococci with a panel of serotype and subtype specific monoclonal antibodies (MAbs) was compared in co-agglutination, dot-blotting and ELISA tests. Twenty reference strains, 50 case isolates and 133 throat isolates from healthy carriers were studied. The typing results with dot-blotting and ELISA were identical, whereas co-agglutination gave different results for three case and 24 carrier strains. The distribution of serotypes and subtypes among the strains is reported. The combination of the subtypes P1.1 and P1.15 in a serotype 15 patient strain was observed. With one case strain and 15 carrier strains, neither serotype nor subtype could be determined. Non-typable and non-subtypable isolates were further characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Co-agglutination is useful for typing small numbers of strains with a few MAbs, but less suitable for large-scale typing than the other two methods. Dot-blotting needs less expensive equipment, smaller volumes of antibodies and fewer manipulations than ELISA.

Antibodies to meningococcal class 1 outer-membrane protein and its variable regions in patients with systemic meningococcal disease.

Antibodies to the meningococcal serosubtype-specific P1.7,16 protein and its variable regions (VR) were analysed in 28 convalescent sera drawn 8-36 months after systemic meningococcal disease by immunoblotting and enzyme immunoassay (EIA) methods. EIA antigens were the meningococcal P1.7,16 protein, produced in Bacillus subtilis, and peptides covering its VR1 (P1.7 region) and VR2 (P1.16 region) inserted into a bacterial penicillinase protein. In the immunoblotting method, three meningococcal reference strains were used; they expressed either the P1.7,16 protein, or only its VR1 or VR2 epitopes in their class 1 proteins. Both methods showed a strong IgG response in four sera to P1.7,16 and VR2, but not to VR1; 18 sera had no or weak anti-class 1 protein activity. The six remaining sera were positive only on blots. The VR2-specific sera had 30-fold higher bactericidal activity than those with negligible P1.7,16 responses. Previous vaccination of the patients with a B:15:P1.7,16 meningococcal vaccine was associated with a strong anti-P1.7,16 and anti-VR2 booster response that declined with time. The subtype-specific antibody activity in some sera indicated colonisation after disease by meningococci with class 1 proteins different from the strain that had caused disease.

Small-fragment restriction endonuclease analysis in epidemiological mapping of group A streptococci.

The usefulness of small-fragment restriction endonuclease digest analysis (SF-REA) of group A streptococcal DNA with EcoRI, as a supplement to the more conventional T serotyping, was assessed for epidemiological characterisation. One hundred and thirty-five clinical isolates from 1988-1990 were examined. SF-REA provided characteristic fingerprints of all isolates, whereas eight isolates were non-typable by T serotyping. Generally, there was a striking correlation between the results obtained with the two techniques. Furthermore, SF-REA reliably classified the eight T-non-typable isolates and occasionally revealed subgroups within the T serotypes. In addition, SF-REA was useful for the clarification of discrepancies between serotyping results from two different reference laboratories. No obvious correlation was observed between the DNA fingerprints and the clinical manifestations of infection or the geographical origin of the group A streptococcal isolates. SF-REA is a valuable supplement to T typing in epidemiological studies and frequently appears to be a more efficient tool for strain differentiation.

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection.

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.

Outer membrane vesicles from Neisseria meningitidis: effects on tissue factor and plasminogen activator inhibitor-2 production in human monocytes.

Lipopolysaccharide-containing outer membrane vesicles (OMV-LPS) which are spontaneously released from Neisseria meningitidis during logarithmic growth were studied for their ability to induce procoagulant (tissue factor), profibrinolytic (urokinase-type plasminogen activator) and antifibrinolytic (plasminogen activator inhibitor-2) factors in purified human monocytes. Cell-associated tissue factor was 5.0-fold (n=5) increased, peaking after 8 h, in the presence of OMV-LPS (1 microg/ml, final concentration). Plasminogen activator inhibitor-2 release from monocytes was maximal after 24 h OMV-LPS (1 microg/ml) stimulation and 13.7-fold (n=5) increased compared to controls; whereas urokinase-type plasminogen activator antigen in culture medium remained uninfluenced by OMV-LPS. In conclusion, these OMV-induced imbalances favor fibrin deposition in the monocyte microenvironment and is probably of great importance in the development of disseminated intravascular coagulation, microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.

Antibiotic resistance of pneumococci in Norway.

A collection of 178 pneumococcal isolates found in Norway during the period 1987-1994 were tested for their susceptibility to benzylpenicillin, macrolides (azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin), fluoroquinolones (ciprofloxacin, sparfloxacin), imipenem, chloramphenicol, and vancomycin by a standard agar dilution procedure. To benzylpenicillin, two strains (1%) showed resistance and 14 strains (8%) intermediate susceptibility. Towards erythromycin, eight strains (4%) showed resistance and four strains (2%) intermediate susceptibility. Cross-resistance was demonstrated among the macrolides. Among the fluoroquinolones, intermediate susceptibility occurred with 42% of the isolates for sparfioxacin and 90% for ciprofloxacin; to the latter 5.1% proved resistant. The sum of intermediate and highly resistant isolates was 53% for chloramphenicol. Both penicillin-resistant strains were isolated during the last 2 years of collection and came from patients of non-Norwegian ethnic background. Imported strains appeared over represented among the strains resistant to penicillin and macrolides. Only imipenem and vancomycin showed full susceptibility for all pneumococci tested. An over representation of serogroup 6 strains was apparent among the strains with intermediate susceptibility and high resistance to benzylpenicillin. It is apparent that high-level resistance has, not so far, become a difficult problem in Norway. Nevertheless, the situation requires monitoring of the resistance level, particularly in meningitis and septic patients, and certainly in patients who cntail a higher than usual possibility of acquiring pneumococci from pools of resistant strains outside Norway (visitors, immigrants and recent returness from abroad).

Antibiotic sensitivity still prevails in Norwegian blood culture isolates.

We describe the antimicrobial susceptibility of bacteraemia isolates from Norway. From March 1998 to February 1999, four university hospitals covering all parts of Norway collected their first 10 isolates each month. Minimal inhibitory concentrations were determined for: Enterobacteriaceae (n=192), staphylococci (n=89) and Streptococcus pneumoniae (n=69) using the Etest. NCCLS breakpoints were used. About 20% of all blood culture isolates in Norway in this period were investigated. Compared with countries outside Scandinavia antibiotic sensitivity still prevails. Only minor differences in resistance were found between participating hospitals, between hospital departments and between hospital- and community-acquired pathogens. The prudent use of antibiotics in Norway may contribute to the fact that antibiotic resistance still remains low in the most common bacterial pathogens causing bloodstream infections.

Meningitis outbreaks and vaccination strategy.

Three outbreaks of meningitis caused by Neisseria meningitidis serogroup A (subgroup III) are described: Niger (1991), Burundi (1992), and Guinea (1993). These outbreaks showed unusual characteristics: a shorter inter-epidemic interval (Niger), unusual geographical location outside the meningitis belt (Burundi and Guinea), and high age-specific attack rates in all age groups (Burundi and Guinea). Mass immunization campaigns mobilized considerable human and financial means (US $322,000 and 3000 person-days of work for health personnel to immunize 629,000 people in Guinea). The vaccination coverage was over 80% in densely populated areas (Burundi and urban Guinea), but below 50% in less populated areas (24/27 and 26/30 sub-districts in Niger and Guinea, respectively). The preventive fraction (proportion of cases prevented by vaccination) was substantial in Guinea (35% for a vaccine efficacy of 85%) and was higher where the campaign was initiated earlier. An 'alert' threshold indicating the onset of an epidemic of 15/100,000 cases in one week showed good sensitivity (94%), specificity (98%) and positive predictive value (89%) in Burundi, permitting quick decision making outside the meningitis belt. These 3 meningococcal meningitis outbreaks show the need for epidemic emergency preparedness and for vigilance on the whole African continent.