Colonisation and interaction between S. epidermidis and S. aureus in the nose and throat of healthy adolescents.

Nasal colonisation with Staphylococcus aureus is a risk factor for developing nosocomial infections. It has been reported that S. epidermidis may produce a serine protease (Esp) inhibiting S. aureus biofilm formation and nasal colonisation. We aimed to analyse the correlation between S. aureus nasal and/or throat carriage and co-colonisation with S. epidermidis strains carrying esp, and the inhibitory effects of S. epidermidis culture supernatants on S. aureus biofilm formation and growth. We obtained 114 S. epidermidis isolates from the nose and 74 S. aureus from the nose and/or throat of healthy adolescents. S. aureus biofilm formation was analysed in a microtitre plate assay and the prevalence of ica, encoding biofilm formation, and esp was analysed with polymerase chain reaction (PCR). Inhibitory effects of S. epidermidis culture supernatants on S. aureus biofilm formation and growth was analysed in vitro. esp prevalence and expression was correlated with inhibitory effects. We detected biofilm formation in 45/74 (61%) S. aureus strains. The ica operon was more prevalent in isolates colonising the nose (12/15; 80%) versus isolates colonising the throat only (8/46; 17%). Almost two-thirds of S. epidermidis culture supernatants displayed high (≥ 50%) S. aureus biofilm inhibitory activity, without affecting growth. We found no correlation between the level of inhibitory activity and S. aureus colonisation. esp was ubiquitous in S. epidermidis, but esp expression did not correlate with biofilm inhibitory activity. S. epidermidis culture supernatants inhibit S. aureus biofilm formation, but do not affect bacterial growth. esp expression was not correlated with the inhibitory effects observed.

Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma.

BACKGROUND: MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation. METHODS: A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3'-untranslated region (3'UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3'UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line. RESULTS: The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells. CONCLUSION: The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.

Persistent strains of coagulase-negative staphylococci in a neonatal intensive care unit: virulence factors and invasiveness.

Coagulase-negative staphylococci (CoNS) are the major cause of nosocomial bacteraemia in neonates. The aim of this study was to investigate whether persistent strains of CoNS possess specific bacterial characteristics as compared with sporadic non-cluster isolates. In total, 180 blood culture isolates (95 contaminants and 85 invasive isolates) obtained from a single neonatal unit over a 12-year period were studied. Pulsed-field gel electrophoresis (PFGE) identified 87 persistent CoNS strains (endemic clones). The two largest PFGE clusters belonged to a single clonal complex according to multilocus sequence typing. Patients colonised or infected with endemic clones were of lower gestational age than those infected with non-cluster strains. One Staphylococcus haemolyticus cluster appeared to selectively colonise and infect the most extreme pre-term infants. Endemic clones were characterised by high levels of antibiotic resistance and biofilm formation. All 51 isolates belonging to the two largest PFGE clusters were ica operon-positive. Genes encoding Staphylococcus epidermidis surface protein B and the production of phenol-soluble modulins (PSMs) were also more prevalent among endemic clones than among non-cluster strains. However, endemic clones were not more prevalent among invasive isolates than among contaminants. These findings indicate that multiple selective factors, including antibiotic resistance, biofilm formation, surface proteins with adhesive properties, and PSMs regulated by agr, increase the ability of CoNS to persist in a hospital environment. It may be more prudent, when searching for new therapeutic targets, to focus on ubiquitous components of CoNS instead of putative virulence factors that do not clearly contribute to increased invasive capacity.

Outcome of children with high-risk acute lymphoblastic leukemia (HR-ALL): Nordic results on an intensive regimen with restricted central nervous system irradiation.

BACKGROUND: Improvement in outcome of childhood high-risk (HR) ALL was sought with a very intensive Nordic protocol leaving most patients without CNS-RT. METHODS: A total of 426 consecutive children entered the NOPHO-92 HR-ALL program. HR criteria included WBC > or =50 x 10(9)/L, CNS or testicular involvement, T-cell, lymphomatous features, t(9;22), t(4;11), or slow response. Of these, 152 children had very high risk (VHR) with special definitions. CNS consolidation was based on high-dose MTX (8 g/m2) and ARA-C (12 g/m2) alternating. VHR patients also received cranial RT. RESULTS: The 9-year EFS was 61 +/- 3%, OS 74 +/- 2%, and EFS for T-ALL 62 +/- 4%. Cumulative incidence of isolated CNS relapse was 4.7 +/- 1%, and CNS relapse in total 9.9 +/- 2%. Poor prognostic factors were WBC > or =200 x 10(9)/L and a very slow response. CONCLUSIONS: HR-ALL was successfully treated on the NOPHO-92 regimen, with a relatively low CNS relapse rate for non-irradiated children. WBC > or =200 x 10(9)/L and very slow response emerged as strong poor prognostic factors.

Rapid PCR detection of the methicillin resistance gene, mecA, on the hands of medical and non-medical personnel and healthy children and on surfaces in a neonatal intensive care unit.

The hands of medical personnel are the chief vectors for transmission of antibiotic-resistant bacteria and probably serve as an important reservoir for antibiotic resistance genes in hospitals. In this survey we examined different reservoirs of the methicillin resistance gene, mecA, using a simplified PCR method. Samples (n = 151) were taken from the hands of medical and non-medical personnel and healthy children and from surfaces in a neonatal intensive care unit (NICU). We also performed sampling from 4 different body sites in 5 of the medical personnel. Fifteen out of 16 nurses (94%) from the ICU carried the mecA gene on their hands, whereas only 35% of the paediatric nurses were mecA-positive. Of all medical personnel, 44% carried the mecA gene on their hands. There was a significant difference (p < 0.015) between medical and non-medical personnel in terms of the carriage rate of mecA. Four samples from surfaces in a NICU--2 ventilators, 1 bench and 1 telephone--were positive for mecA. Our results are comparable with those from previous studies on reservoirs of methicillin-resistant coagulase-negative staphylococci using conventional culture techniques.

Rapid detection of the methicillin-resistance gene, mecA, in coagulase-negative Staphylococci.

Phenotypical methods are routinely used to detect methicillin resistance in Staphylococci. These methods are time-consuming and there are difficulties in detecting all resistant strains carrying the mecA gene. We detected methicillin-resistant Staphylococci in biological samples by PCR amplification of mecA, without the time-consuming step of identifying a bacterial isolate. Methicillin-resistant Staphylococci isolates were also detected by screening on agar supplemented with oxacillin. The biological samples were collected from the hands of 17 healthcare workers at the Department of Paediatrics at the University Hospital of Tromsø. mecA was amplified in 12 of the 17 samples. The gene was verified by DNA sequencing of the PCR amplicon. Using the phenotypical method, methicillin-resistant Staphylococci were isolated from 6 of the samples. In all 6 of these samples, mecA was amplified by PCR. We conclude that PCR is a sensitive and specific method for detecting methicillin resistance in Staphylococci. The PCR detection of mecA is rapid, fairly simple and can easily be assimilated into the routines of a clinical microbiological laboratory.

Human polyomavirus BK (BKV) and neuroblastoma: mechanisms of oncogenic action and possible strategy for novel treatment.

BACKGROUND: We reported previously that nearly all human neuroblastomas analyzed contain and express genomic DNA sequences deriving from the human polyomavirus BK (BKV) [Flaegstad et al.: Cancer Res 59:1160-1163, 1999]. PROCEDURE: Here we show that the BKV large T antigen is expressed and bound to p53 in neuroblastoma cells and that this interference compromises the tumor suppressor function of p53. RESULTS: Treatment of neuroblastoma cells with large T antigen antisense constructs relocated active p53 to the nucleus. The relocation event was accompanied by enhanced p21(waf1/cip1) expression as well as induced apoptosis. CONCLUSIONS: Continuous antisense oligonucleotide treatment of nude rats with human neuroblastoma xenografts resulted in a significant but incomplete reduction of tumor growth compared to rats treated with saline.

[Invasive pneumococcal infections in children from Troms, Nordland and Sor-Trondelag 1980-95]. / Invasive pneumokokkinfeksjoner hos barn fra Troms, Nordland og Sør-Trøndelag 1980-95.

Invasive pneumococcal infections may be severe. We have examined epidemiology, risk factors and outcome of these infections. In the years 1980-95, 76 children below the age of 15 with invasive pneumococcal infections were admitted to the hospitals in the counties of Troms, Nordland and Sør-Trøndelag in Norway. The incidence rate in children 0-2 years old was 10.3 cases per 100,000 persons per year, and 1.8 in children 3-14 years. Of the patients 24 had meningitis and 52 bacteraemia. All bacteriological isolates were sensitive to benzylpenicillin. Seven patients died and five developed sequelae. Thirty-one of the children had risk factors prior to the infection. Children with hypo- or hyperventilation at the time of arrival, and/or impaired circulation fared worse than those with normal findings. Children with underlying risk factors have a much higher frequency of invasive pneumococcal infections than other children. Patients who had impaired circulation or ventilation on admission have a bad prognosis for healthy survival.

[Antibiotic treatment of acute otitis in children]. / Antibiotikabehandling av akutt ørebetennelse hos barn.

Treatment of acute otitis is one of the most common reasons for prescribing antibiotics for children. Recent studies have shown no, or only a small, effect of antibiotic treatment of this condition. We examined the files from the City of Tromsø's Emergency Department, which catchment area includes about 12,300 children under the age of 15. During the period from March 1997 to May 1998, 784 children presented with acute otitis, 91.5% of whom received antibiotic treatment. The most frequently drug used drug was penicillin V. Even if there are no or only a small effect of antibiotic treatment of this condition, most children receive such treatment. This study will give baseline data for new studies aimed at reducing the use of antibiotics.

A possible contributory role of BK virus infection in neuroblastoma development.

The tumor suppressor protein p53 is aberrantly localized to the cytoplasm of neuroblastoma cells, compromising the suppressor function of this protein. Such tumors are experimentally induced in transgenic mice expressing the large tumor (T) antigen of polyomaviruses. The oncogenic mechanisms of T antigen include complex formation with, and inactivation of, the tumor suppressor protein p53. Samples from 18 human neuroblastomas and five normal human adrenal glands were examined. BK virus DNA was detected in all neuroblastomas and none of five normal adrenal glands by PCR. Using DNA in situ hybridization, polyomaviral DNA was found in the tumor cells of 17 of 18 neuroblastomas, but in none of five adrenal medullas. Expression of the large T antigen was detected in the tumor cells of 16 of 18 neuroblastomas, but in none of the five adrenal medullas. By double immunostaining BK virus T antigen and p53 was colocalized to the cytoplasm of the tumor cells. Immunoprecipitation revealed binding between the two proteins. The presence and expression of BK virus in neuroblastomas, but not in normal adrenal medulla, and colocalization and binding to p53, suggest that this virus may play a contributory role in the development of this neoplasm.

Primary BK virus (BKV) infection due to possible BKV transmission during bone marrow transplantation is not the major cause of hemorrhagic cystitis in transplanted children.

In allogeneic bone marrow transplanted (BMT) patients BK virus (BKV) reactivation has been associated with haemorrhagic cystitis (HC). However, it is far from obvious which patients will develop HC, since BKV, a human polyomavirus, is ubiquitious and infects children at an early age. To investigate if a primary BKV infection, as such or possibly due to transmission of BKV by the marrow graft during BMT, was correlated to the development of HC, 45 children were followed for possible BKV seroconversion and development of HC at different time points after BMT. Serum samples were collected from the 45 allogeneic BMT children and their donors before transplantation, and from the patients at 3, 6 and 12 months after BMT. These sera were analysed for the presence of specific antibodies towards BKV by hemagglutination inhibition (HAI) and by IgG- and IgM-class specific enzyme linked immunosorbent (ELISA) assays. Twelve of the 45 BMT children had a documented episode of HC or hematuria. All patients and 98% of the donors were HAI positive before BMT, while with ELISA 87% of the patients and 84% of the donors were positive. Moreover, most HC and hematuria children (11/12) were seropositive with both assays before BMT, making it impossible to investigate possible BKV transmission through the bone marrow graft during BMT by serology. Still, serological changes such as ELISA seroconversion, IgM antibodies and/or HAI titer increases were significantly (p=0.016) more common in patients with HC (58%) than without HC (24%), but these changes occured mainly after HC symptomatology had already resolved. However, there was a near significant difference (p=0.053) in BKV seroprevalence by ELISA among the donors of patients with HC or hematuria (67%) as compared to the donors (91%) of patients without HC.

Is there a gender difference in red blood cell thiopurine methyltransferase activity in healthy children?

We have examined red blood cell (RBC) thiopurine methyltransferase (TPMT) activity in a healthy population sample of Norwegian children, age 1-10 years. Boys had mean RBC TPMT activity of 11.1 +/- 2.0 U (n = 87) vs. 10.6 +/- 2.2 U (n = 71) in girls, the difference was not significant (P = 0.3). Age was negatively correlated to RBC TPMT activity (rs = -0.2, P = 0.01). As boys with acute lymphoblastic leukemia (ALL) tolerate more 6-mercaptopurine (6-MP) than girls and have a higher risk of relapse, we have searched for pharmacokinetic causes of these gender differences. The gender difference in 6-MP tolerance and clinical outcome in children with ALL cannot be explained by the minor and nonsignificant higher RBC TPMT activity in boys compared to girls.

Factors associated with fatal outcome in childhood meningococcal disease.

The purpose of this study was to identify factors associated with a fatal outcome in children with meningococcal disease and to design a new clinical scoring system. We reviewed the charts of all 137 children with meningococcal disease admitted alive to the University Hospital, Tromsø, during the years 1977-92. Twelve of the children died (8.7%). On admission the following clinical signs were significantly associated with poor outcome: peripheral vasoconstriction, cyanosis, extensive petechiae, hypotension, altered consciousness, hyperventilation and absence of neck rigidity. The laboratory parameters low pH, low base excess, thrombocytopenia, low Trombotest and leukopenia were also associated with later death. Multiple logistic regression was performed to examine the independent effect of each variable. Cyanosis, peripheral vasoconstriction and base excess < -10 mmol/l or pH < 7.35 were significantly associated with a fatal outcome. A clinical scoring system based on the extent of petechiae, the presence of peripheral vasoconstriction, hyperventilation and/or cyanosis, the absence of neck rigidity and impairment of consciousness is proposed. Twenty-nine patients received > or = 3.5 points, of whom 12 died and 12 survived. None of the patients who died had less than 3.5 points. The clinical scoring system is based solely on clinical signs. It can be done rapidly and performs well in identifying children who might benefit from early intensive care.

Prognostic factors in childhood bacterial meningitis.

The purpose of this study was to identify possible risk factors associated with a poor prognosis in childhood bacterial meningitis. We also analysed the influence of duration of symptoms and prehospital antibiotic therapy on outcome. Ninety-two children aged 1 month to 13.8 years were included, of whom 4 died (4.3%) and 14 (15.2%) experienced permanent neurological sequelae. Hearing impairment was the most frequent sequela and was strongly associated with the length of history. Multiple logistic regression revealed duration of symptoms > 48 h, pre-hospital seizures, peripheral vasoconstriction, < 1000 x 10(6)/l leucocytes in cerebrospinal fluid and temperature < or = 38.0 degrees C on admission as risk factors independently associated with later death or sequelae. There was no association between pre-hospital oral or parenteral antibiotic therapy and outcome. These risk factors may be of value in selecting patients for more intensive therapy and in identifying possible candidates for new treatment strategies.

Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients.

Our understanding of important stages in the pathogenesis of the human polyomavirus BK virus (BKV) and JC virus (JCV) infections is limited. In this context, nasopharyngeal aspirates from 201 children with respiratory diseases and saliva from 60 human immunodeficiency virus type 1-infected adults and 10 healthy adult controls were collected and analyzed for the presence of BKV and JCV DNA by PCR. Neither BKV nor JCV DNA was detected in the saliva specimens. We demonstrated BKV DNA, but no infectious BKV, in 2 of 201 nasopharyngeal aspirates. Each sample contained one unique rearranged noncoding control region variant of BKV. The results indicate that (i) BKV and JCV are not regularly associated with respiratory infections in children requiring hospitalization, (ii) nasopharyngeal cells are not an important site for primary replication of human polyomavirus BKV and JCV, and (iii) the salivary glands and oropharyngeal cells seem not to be involved in BKV and JCV persistence. We propose that for the polyomaviruses BKV and JCV the alimentary tract should be considered as a portal of entrance to the human organism.

BK and JC viruses in human immunodeficiency virus type 1-infected persons: prevalence, excretion, viremia, and viral regulatory regions.

Human polyomavirus BK (BKV) and JC (JCV) infections were examined in persons infected with human immunodeficiency virus type 1 (HIV-1). High frequencies of BKV (24%) and JCV viruria (16%) were detected by polymerase chain reaction (PCR). BKV viruria was not found in an immunocompetent control group, in contrast to a frequency of JCV viruria of 20%. The degree of HIV-induced immunodeficiency did not influence the prevalence of BKV viruria, in contrast to cytomegalovirus viruria, suggesting BKV reactivation is an early manifestation in HIV infection as well as a temporal sequence of opportunistic infections. BKV DNA but not JCV DNA was detected in peripheral blood mononuclear cells (PBMC) in 2 of 42 subjects by a sensitive nested PCR. Sequencing of viral noncoding control regions (NCCRs) revealed predominantly archetypal and TU type BKV NCCRs but only archetypal JCV NCCRs. A new, naturally occurring BKV NCCR variant was detected in 1 urine specimen and 2 PBMC samples, indicating a stable and biologically significant rearrangement. Serum levels of BKV antibodies do not seem to be diagnostically useful in HIV-infected persons.

Antibodies to dsDNA are produced during primary BK virus infection in man, indicating that anti-dsDNA antibodies may be related to virus replication in vivo.

Experimental immunizations with both the Polyomavirus BK and with the isolated viral genomic dsDNA regularly induce antibodies with a relative affinity for BK virus dsDNA. In the present study we demonstrate that the anti-dsDNA responses to BK virus in experimental animals also appear during natural BK virus infection in man. Fifty-nine children were examined over time for serological signs of primary BK virus infection. Of eight children found to undergo primary infection with BK virus, anti-BK dsDNA antibodies appeared in all. In 4 of the 8 patients the antibodies cross-reacted significantly with mammalian dsDNA, and weak cross-reactions were also noted in at least three other patients. The antibodies resembled those induced in the experimental model with regard to their relative affinity for BK dsDNA. In contrast, most, but not all, anti-dsDNA antibodies from 10 SLE patients cross-reacted extensively with dsDNA from viral and mammalian origin. Thus, a dsDNA virus like BK virus may provoke immunological intolerance to dsDNA, but, with qualities different from those produced during SLE. The present observations demonstrate that induction of anti-dsDNA antibodies is not restricted to experimental immunization of animals, but does also take place in humans during naturally acquired BK virus infection. The relevance of this model for the spontaneous production of anti-dsDNA antibodies is discussed.

[Haemophilus influenzae infections in children]. / Haemophilus influenzae-infeksjoner hos barn.

Haemophilus influenzae may cause serious infections, especially in small children. During the period 1980-90, 45 children with systemic H influenzae infections were admitted to the University Clinic, Tromsø. 17 patients had epiglottitis in the same period, but are not included in this material. One of the four septicemia patients and two of the 35 children with meningitis died, giving an overall lethality of 4.8%. Five of the patients with meningitis experienced neurological sequelae (14% of the patients with meningitis). Two of the systemic isolates of H influenzae were resistant to ampicillin because of beta-lactamase production, but all the isolates were susceptible to chloramphenicol and 3rd generation cephalosporins.

[Treatment of bacterial meningitis in children]. / Behandling av bakteriell meningitt hos barn.

The treatment of children with bacterial meningitis includes many aspects, such as fluid and antibacterial therapy, treatment of convulsions, and prophylactic use of dexamethasone. This report focuses in particular on the prophylactic use of dexamethasone together with cefalosporines in order to reduce inflammation and neurological sequelae in this disease. Probably this approach is now justified in the treatment of meningitis caused by Haemophilus influenzae. Modern principles for fluid therapy and treatment of increased intracranial pressure are also discussed.