Genetic relatedness within serotypes of penicillin-susceptible Streptococcus pneumoniae isolates

The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.     

Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Pneumococcal carriage in children in The Netherlands: a molecular epidemiological study

In 1999, Engelen and coworkers investigated colonization in Amsterdam among 259 children attending 16 day-care centers (DCCs) and among 276 children who did not attend day-care centers (NDCCs). A 1.6- to 3.4-fold increased risk for nasopharyngeal colonization was observed in children attending DCCs compared with NDCC children, while no difference in antibiotic resistance was found between groups. The serotype and genotype distributions of 305 nasopharyngeal Streptococcus pneumoniae isolates of the latter study were investigated. The predominant serotypes in both the DCC and the NDCC groups included 19F (19 and 18%, respectively), 6B (14 and 16%, respectively), 6A (13 and 7%, respectively), 23F (9 and 7%, respectively), and 9V (7 and 7%, respectively). The theoretical vaccine coverage of the 7-valent conjugate vaccine was 59% for the DCC children and 56% for the NDCC group. Genetic analysis of the pneumococcal isolates revealed 75% clustering among pneumococci isolated from DCC attendees versus 50% among the NDCC children. The average pneumococcal cluster size in the DCC group was 3.8 and 4.6 isolates for two respective sample dates (range, 2 to 13 isolates per cluster), while the average cluster size for the NDCC group was 3.0 (range, 2 to 6 isolates per cluster). Similar to observations made in other countries, these results indicate a higher risk for horizontal spread of pneumococci in Dutch DCCs than in the general population. This study emphasizes the importance of molecular epidemiological monitoring before, during, and after implementation of pneumococcal conjugate vaccination in national vaccination programs for children.     

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.     

Surgical pathology of 104 tricuspid valves (2000–2005) with classic right-sided Ebstein''s malformation

BACKGROUND: Ebstein's anomaly has been described extensively in autopsy material. However, there have been no large surgical pathology series of this malformation. OBJECTIVE: To review clinical and surgical pathologic features of a large number of cases of Ebstein's anomaly from a single institution. METHODS: Review of medical histories, surgical reports, and surgical pathology reports at the Mayo Clinic (2000-2005). RESULTS: Among 104 patients, the mean age was 31 years (2 months-79 years), and 57% were female. Common ECG abnormalities included right bundle branch block (58%), first-degree heart block (31%), preexcitation (18%), and nonspecific intraventricular conduction delay/block (15%). Moreover, 74% had inter-atrial communication, 13% mitral valve prolapse, and 5% bicuspid aortic valve. Clinically, all had tricuspid regurgitation (severe in 74%), and 17% of anterior leaflets were fenestrated. No tricuspid valve was calcified. Surgically, tricuspid tissue was removed during replacement in 99% and repair in 1%. The anterior tricuspid leaflet was resected in 98%, and its length was 0.81-9.3 cm/m2 body surface area (mean, 3.3). Characteristically, leaflets were large and had irregular shapes and numerous short cordal or direct myocardial insertions. One tricuspid valve had two papillary fibroelastomas. None had clinical or pathologic evidence of active or healed endocarditis. CONCLUSIONS: Among patients with Ebstein's malformation, tricuspid valve tissue almost exclusively was removed during valve replacement and represented the anterior leaflet. Valve tissue was generally large, irregularly shaped, and associated with insertion of short cords or myocardial stumps. Interestingly, although appreciably deformed, Ebstein valves were not associated with infective endocarditis.     

Relative importance of C4 binding protein in the modulation of the classical pathway C3 convertase in patients with systemic lupus erythematosus

Serum concentrations of C1q, C4, C4 binding protein (C4bp), C3 and C2 haemolytic activity have been measured in 110 samples from 20 patients with systemic lupus erythematosus (SLE). Significant reductions in comparison to normal levels were found in the mean serum concentrations of C4, C3 and C4bp as well as C2 haemolytic activities. For patients serum concentrations of C4 correlated with C2 haemolytic activities (r = 0.91) and C4bp (r = 0.79); the C2 haemolytic levels correlated with the concentration of C4b (r = 0.72). It is concluded that serum concentrations of the complement components C4 and C2, which are the constituents of the classical pathway C3 convertase, are regulated by C4bp in vivo. Further metabolic studies are required to determine the causes of decreased serum concentrations of C4bp in patients with SLE.     

Biomorphometric analysis of human prostatic carcinoma by using three-dimensional computer models

Advances in the detection of carcinoma of the prostate during the last 15 years have accounted for a sharp increase and then an abrupt decrease in the incidence of the disease. A more recent decline in its mortality rates has been variously interpreted as either the success of early detection and improved treatment or lead-time bias. The recently reported Prostate Cancer Prevention Trial had an overall detection rate that approached the 30%-40% prevalence rates reported in autopsy series in which men died of other causes. However, the prognostic information that can be obtained from prostate cancer found on biopsy is limited. Three-dimensional computer modeling is one technique that allows multiple studies on "immortal" prostates to test methods of biopsy sampling accuracy and to assist in the determination of the disease's severity. Computer modeling can assess detection rates and assesses tumor multifocality and heterogeneity. It can provide a more accurate representation of tumor volume, aiding in therapeutic decision making, and can assess sampling errors of various biopsy methods. It has been shown to be superior to wire-frame technique by immortalizing the original shape and dimensions of the surgically excised prostate gland. Moreover, our 3-dimensional computer modeling system improves upon other systems: It is more than a simple extension of the planimetric technique, and it is able to demarcate clearly the boundaries of Gleason grades just 1 grade apart.     

Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis.

One hundred fifty-three Mycobacterium bovis strains from cattle, various animal species from zoos and wild parks, and humans were analyzed for three different genetic markers for use in the epidemiology of bovine tuberculosis. M. bovis strains isolated from cattle were found to carry a single IS6110 element, whereas the majority of strains from other animals such as antelopes, monkeys, and seals harbored multiple IS6110 elements, suggesting that the reservoirs in cattle and wild animals are separated. Because the single IS6110 element in cattle strains is located at the same chromosomal position, strain differentiation by insertion sequence fingerprinting was hampered. Therefore, we investigated the usefulness of the direct repeat and polymorphic GC-rich repeat elements for strain differentiation. Both markers allowed sufficient strain discrimination for epidemiological purposes. Evidence is presented that in Argentina, most human M. bovis infections are due to transmission from cattle, whereas M. bovis infections among humans in the Netherlands are mainly contracted from animals other than cattle. Various outbreaks of M. bovis among animals and humans are described, including a small one which likely involved transmission from human to human.