Trends in drug resistance, serotypes, and molecular types of Streptococcus pneumoniae colonizing preschool-age children attending day care centers in Lisbon, Portugal: a summary of 4 years of annual surveillance

Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.     

T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection

In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). In this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. In this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed.     

Correlation of P-selectin and lipoprotein(a), and other lipid parameters in preeclampsia

Preeclampsia is a pregnancy specific disorder and is thought to be associated with generalized endothelial dysfunction. P-selectin, an adhesion molecule, mediates the interaction of monocytes, platelets, and endothelial cells. Increased P-selectin levels and altered lipid and lipoprotein metabolism were reported in preeclampsia and during pregnancy. In order to investigate the relationship between serum P-selectin and lipoprotein(a), and other lipid parameters, 28 preeclampsia [13 severe (group I) and 15 mild preeclampsia (group II), 15 healthy pregnant (group III) and 20 non-pregnant (group IV)] women were investigated. Serum P-selectin, lipoprotein(a), total cholesterol, triglyceride, and high density lipoprotein cholesterol were measured and low-density lipoprotein cholesterol was derived. Serum P-selectin concentrations were consistently and significantly higher in the severe preeclampsia group than in the mild preeclampsia, healthy pregnancy, and non-pregnant control groups (P<0.0001, for all). The mild preeclampsia group also had increased serum P-selectin concentrations compared with the healthy pregnancy group and non-pregnant controls (P<0.05 and P<0.0001, respectively). Serum P-selectin and lipoprotein(a) levels revealed a significant and linear increase with the severity of preeclampsia. There were also significant (in groups I and II) and borderline (in groups III and IV) correlations between P-selectin and total cholesterol. The present study suggests that P-selectin may be an additional risk marker for preeclampsia, and may be useful in distinguishing women with mild and severe preeclampsia and normal pregnancy. Received: 9 November 2001 / Accepted: 6 February 2002     

Dipolar sources of the early scalp somatosensory evoked potentials to upper limb stimulation Effect of increasing stimulus rates

Brain electrical source analysis (BESA) of the scalp electroencephalographic activity is well adapted to distinguish neighbouring cerebral generators precisely. Therefore, we performed dipolar source modelling in scalp medium nerve somatosensory evoked potentials (SEPs) recorded at 1.5-Hz stimulation rate, where all the early components should be identifiable. We built a four-dipole model, which was issued from the grand average, and applied it also to recordings from single individuals. Our model included a dipole at the base of the skull and three other perirolandic dipoles. The first of the latter dipoles was tangentially oriented and was active at the same latencies as the N20/P20 potential and, with opposite polarity, the P24/N24 response. The second perirolandic dipole showed an initial peak of activity slightly earlier than that of the N20/P20 dipolar source and, later, it was active at the same latency as the central P22 potential. Lastly, the third perirolandic dipole exaplaining the fronto-central N30 potential scalp distribution was constantly more posterior than the first one. In order to evaluate the effect of an increasing repetition frequency on the activity of SEP dipolar sources, we applied the model built from 1.5-Hz SEPs to traces recorded at 3-Hz and 10-Hz repetition rates. We found that the 10-Hz stimulus frequency reduced selectively the later of the two activity phases of the first perirolandic dipole. The decrement in strength of this dipolar source can be explained if we assume that: (a) the later activity of the first perirolandic dipole can represent the inhibitory phase of a “primary response”; (b) two different clusters of cells generate the opposite activities of the tangential perirolandic dipole. An additional finding in our model was that two different perirolandic dipoles contribute to the centro-parietal N20 potential generation. Received: 5 August 1997 / Accepted: 26 November 1997     

Evaluation of three commercially available hepatitis C virus antibody detection assays under the conditions of a clinical virology laboratory.

BACKGROUND: Most studies evaluating antibody detection assays are conducted on samples from healthy blood donors but not on samples of hospitalized patients which can show non-specific reactions. OBJECTIVES: To compare the performance of three commercial automated assays for the detection of hepatitis C virus (HCV) antibodies, Monolisa anti-HCV Plus version 2, Axsym anti-HCV 3.0 and Vitros anti-HCV, on a population of hospitalized patients. STUDY DESIGN: The specificity of the assays was prospectively evaluated in 2020 routine serum samples. In order to assign the serostatus of each sample, those giving positive or discordant results were further tested by three immunoblots and by RT-PCR (Roche). Moreover, the sensitivity was evaluated on eight commercial HCV seroconversion panels. RESULTS: The Monolisa, Axsym and Vitros assays showed specificities of 99.64%, 99.12% and 99.33%, respectively. Concerning the sensitivity, among 49 samples, the number of positive results was 21, 24 and 24 for the Monolisa, Axsym and Vitros kits, respectively. The differences were not statistically significant at an alpha risk of 5%. CONCLUSIONS: All assays appeared to be reliable for routine screening, but there were a surprising number of indeterminate samples that could not be resolved by confirmatory tests.     

Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: the impact of two linked polymorphisms

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms. Here, we examine the impact of the mutations and polymorphisms identified in the LIG4 syndrome patients. Examination of recombinant mutant proteins shows that the severity of the clinical features correlates with the level of residual ligase activity. The polymorphisms decrease the activity of DNA ligase IV by approximately 2-fold. When combined with the otherwise mild R278H mutation, the activity is reduced to a level similar to other LIG4 patients who display immunodeficiency and developmental delay. This demonstrates how coupling of a mutation and polymorphism can have a marked impact on protein function and provides an example where a polymorphism may have influenced clinical outcome. Analysis of additional mutational changes in LIG4 syndrome (R580X, R814X and G469E) have led to the identification of a nuclear localization signal in DNA ligase IV and sites impacting upon DNA ligase IV adenylation.     

A novel amperometric biosensor based on artichoke (Cynara scolymus L.) tissue homogenate immobilized in gelatin for hydrogen peroxide detection

A biosensor for specific determination of hydrogen peroxide was developed by using homogenized artichoke (Cynara scolymus L.) tissue in combination with a dissolved oxygen probe and applied in determination of hydrogen peroxide in milk samples. Artichoke tissue, which has catalase activity, was immobilized with gelatine by means of glutaraldehyde and fixed on a pretreated teflon membrane. The electrode response was maximum when 0.05 M phosphate buffer was used at pH 7.0 and at 30 degrees C. Upon addition of hydrogen peroxide, the electrode gives a linear response in a concentration range of 5.0-50 x 10(-5) M with a response time of 3 min. The method was also applied to the determination of hydrogen peroxide in milk samples.     

Prediction of AVM obliteration after stereotactic radiotherapy using radiobiological modelling

This study was carried out in order to derive the radiobiological parameters of the dose-response relation for the obliteration of arteriovenous malformation (AVM) following single fraction stereotactic radiotherapy. Furthermore, the accuracy by which the linear Poisson model predicts the probability of obliteration and how the haemorrhage history, location and volume of the AVM influence its radiosensitivity are investigated. The study patient material consists of 85 patients who received radiation for AVM therapy. Radiation-induced AVM obliterations were assessed on the basis of post-irradiation angiographies and other radiological findings. For each patient the dose delivered to the clinical target volume and the clinical treatment outcome were available. These data were used in a maximum likelihood analysis to calculate the best estimates of the parameters of the linear Poisson model. The uncertainties of these parameters were also calculated and their individual influence on the dose-response curve was studied. AVM radiosensitivity was assumed to be the same for all the patients. The radiobiological model used was proved suitable for predicting the treatment outcome pattern of the studied patient material. The radiobiological parameters of the model were calculated for different AVM locations, bleeding histories and AVM sizes. The range of parameter variability had considerable effect on the dose-response curve of AVM. The correlation between the dosimetric data and their corresponding clinical effect could be accurately modelled using the linear Poisson model. The derived response parameters can be introduced into the clinical routine with the calculated accuracy assuming the same methodology in target definition and delineation. The known volume dependence of AVM radiosensitivity was confirmed. Moreover, a trend relating AVM location with its radiosensitivity was observed.     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.