The value of virus serology in epidemiological studies of acute otitis media in children.

BACKGROUND: Acute otitis media (AOM) is a major health problem in young children. There is a general conception that AOM is a bacterial disease but with the availability of sensitive diagnostic methods, it has gradually become evident that viruses play an important role in the pathogenesis of AOM. Paired blood samples are seldom taken from infants although valuable information could be obtained by serological methods. During the recent Finnish Otitis Media (FinOM) Cohort Study, in addition to nasopharyngeal aspirates (NPA) and middle ear fluids (MEF), paired acute and convalescent serum samples were collected from children with AOM. OBJECTIVES: To establish the diagnostic value of serological methods in etiological and epidemiological studies of AOM. STUDY DESIGN: A complete set of NPA, MEF, and paired sera was collected during 447 events of AOM experienced by 179 children between 2 months and 2 years of age. Antigens of respiratory syncytial virus (RSV), adenoviruses, influenza A and B, and parainfluenza types 1-3 in NPAs and MEFs were detected by time-resolved fluoroimmunoassay (TR-FIA), and antibody titers were determined by complement fixation test (CFT) or by enzyme immunoassay. RESULTS: A total of 163 virus-positive events were identified. Of those, only 34 were positive by TR-FIA and by serology. From 48 events a positive result was obtained only by TR-FIA and from 81 only by serology. CONCLUSION: Although serological methods are usually of little use in clinical practice, epidemiological studies clearly gain value if serology is included. The number of virus-positive findings dramatically increased by including serological tests in the diagnostic work-up of these AOM events.     

Evaluation of an internally controlled real-time polymerase chain reaction assay targeting the groEL gene for the detection of Bartonella spp. DNA in patients with suspected cat-scratch disease

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10–100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other bacteria, including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory.     

Heterogeneous molecular mechanisms underlie attenuated familial adenomatous polyposis

PurposeFamilial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases.MethodsWe studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3–100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls.ResultsOne patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency.ConclusionAttenuated polyposis patients without “conventional” APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.     

Comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci

Objective: To compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (GRE) from clinical fecal specimens: kanamycin-aesculin-azide (KAA) agar; bile-aesculin-polymixin (BAP) agar; aztreonam-amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar.
Methods: Fecal specimens from 125 patients were inoculated onto each medium. Media were examined for enterococci after incubation for up to 48 h. Enterococci were identified to species level, and glycopeptide phenotypes were determined by measuring minimum inhibitory concentrations of vancomycin and teicoplanin.
Results: GRE were isolated from 44/125 samples. Enterococcus faecalis and Enterococcus faecium isolates, expressing glycopeptide resistance of the VanA or VanB phenotypes, were recovered from 27/33 (82%) specimens on BAP medium, 26/33 (79%) on KAA medium, and 21/33 (64%) on CBN and CBAA media. Enterococcus gallinarum and Enterococcus casseliflavus isolates expressing low-level glycopeptide resistance (VanC phenotype) were recovered from 14/15 (93%) specimens on CBAA medium, 7/15 (47%) on KAA and CBN media, and 6/15 (40%) on BAP medium.
Conclusions: The media tested in this study, with the exception of CBN medium, detected at least 75% of patients colonized by GRE. Further development of BAP, CBAA and KAA media is warranted to improve growth and selectivity.     

Cloning and characterization of the cDNA encoding the HA protein of a hemagglutination-defective measles virus strain

cDNA clones corresponding to the mRNA for the hemagglutinin of the hemagglutination-defective strain AK-1 of measles virus were isolated and characterized. Compared with the prototype Edmonstron strain, 60 nucleotide substitutions that resulted in 18 amino acid changes were detected. An additional potential N-linked glycosylation site was added by point mutation, which was supported by the observation that the hemagglutinin of the AK-1 strain was stained more heavily after NaDodSO₄PAGE and periodic acid-Schiff (PAS) staining than the Edmonston strain. Computer-assisted analysis revealed that three reverse turns in the secondary structure had disappeared in the hemagglutinin of the AK-1 strain. Moreover, one of these structural changes occurred in the closely glycosylated region at amino acid residues 168–240, which appeared to be a biologically important functional domain. The isoelectric point calculated from the predicted amino acid sequence became about 1 pH unit more basic in the AK-1 strain than the Edmonston strain. This present study is the first sequence analysis of the hemagglutinin gene in a hemagglutination-defective strain of the measles virus.     

Prevalence and risk factors for Chlamydia trachomatis infection in adolescent females and young women in central Brazil

In order to determine the prevalence and risk factors for Chlamydia trachomatis infection in adolescent females and young women in central Brazil, 296 subjects attending two public health services were evaluated. The overall prevalence of C. trachomatis infection, as determined using polymerase chain reaction, was 19.6% (95% confidence interval [CI], 15.3–24.7). In multivariate analysis, young age (odds ratio [OR]_adjusted 2.32, 95%CI 1.1–4.8, p<0.05) and having 2–3 (OR_adjusted 3.41, 95%CI 1.6–6.3, p<0.05) or ≥4 sexual partners in life (OR_adjusted 3.10, 95%CI 1.1–6.3, p<0.05) were factors significantly associated with chlamydial infection. In conclusion, the prevalence of C. trachomatis infection was high in the studied population and risk factors were related to age and sexual behavior.     

Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003

Phylogenetic analysis of 45 enterovirus 71 (EV71) isolates for 6 years in Yamagata, Japan, clarified that the annual outbreak of hand-foot-and-mouth disease was due to four genetically distinct subgenogroups, including a novel "B5." Our results suggest that the importation of EV71 from surrounding countries has had a major epidemiological impact on the local community used in our study.     

Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.

The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease.