Nosocomial transmission clusters and risk factors in Moraxella catarrhalis.

We report an objective examination of nosocomial transmission events derived from long-term (10-year) data from a single medical centre. Cluster analysis, based on the temporal proximity of genetically identical isolates of the respiratory pathogen Moraxella catarrhalis, identified 40 transmission events involving 33 of the 52 genotypes represented by multiple isolates. There was no evidence of highly transmissible or outbreak-prone genotypes. Although most clusters were small (mean size 3.6 isolates) and of short duration (median duration 25 days), clustering accounted for 38.7% of all isolates. Significant risk factors for clustering were multi-bed wards, and winter and spring season, but bacterial antibiotic resistance, manifested as the ability to produce a beta-lactamase was not a risk factor. The use of cluster analysis to identify transmission events and its application to long-term data demonstrate an approach to pathogen transmission that should find wide application beyond hospital populations.

An outbreak of infections caused by non-typeable Haemophilus influenzae in an extended care facility.

Nosocomial outbreaks of infection due to non-typeable Haemophilus influenzae (NTHi) are rarely described. There are a few published reports that suggest that elderly patients with underlying pulmonary disease are at risk and that person-to-person spread is key to disease transmission. During the summer months of 2005, we documented an outbreak of NTHi infections in a Veterans Affairs nursing home. Thirteen patients developed conjunctivitis or lower respiratory infection involving a beta-lactamase-negative biotype III NTHi isolate, with an indistinguishable SmaI macrorestriction pattern. Patients were elderly males usually with underlying cardiac and pulmonary disease. A case-control study failed to demonstrate any specific significant risk factor for NTHi infection and there was no evidence of spatial clustering of cases within the nursing home. A random throat culture survey involving nursing home patients during the outbreak showed that only one of 19 persons was colonized with NTHi. The outbreak concluded following appropriate treatment and an emphasis on universal and respiratory droplet precautions. All patients recovered and a specific inciting event for the outbreak was never defined. Literature review revealed a spectrum of responses to nosocomial NTHi infections and a lack of consensus regarding the infection control approach towards NTHi outbreaks.

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and gamma-secretase activity.

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and gamma-secretase cleavage of substrates. The structural requirements for PS incorporation into the gamma-secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and gamma-secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and gamma-secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for gamma-secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and gamma-secretase activities. Exchange of residues 95-98 of PS1 (sw95-98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95-98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog gamma-secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with gamma-secretase substrates and gamma-secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. gamma-Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for gamma-secretase that alters the ratio of amyloid-beta peptide species produced, leading to the amyloid-beta peptide aggregation that causes familial Alzheimer's disease.

A domain at the C-terminus of PS1 is required for presenilinase and gamma-secretase activities.

The structural requirements for presenilin (PS) to produce active presenilinase and gamma-secretase enzymes are poorly understood. Here we investigate the role the cytoplasmic C-terminal region of PS1 plays in PS1 activity. Deletion or addition of residues at the PS C-terminus has been reported to inhibit presenilinase endoproteolysis of PS and alter gamma-secretase activity. In this study, we use a sensitive assay in PS1/2KO MEFs to define a domain at the extreme C-terminus of PS1 that is essential for both presenilinase and gamma-secretase activities. Progressive deletion of the C-terminus demonstrated that removal of nine residues produces a PS1 molecule (458ST) that lacks both presenilinase processing and gamma-secretase cleavage of Notch and APP substrates. In contrast, removal of four or five residues had no effect (462ST, 463ST), while intermediate truncations partially inhibited PS1 activity. The 458ST mutant was unable to replace endogenous wtPS1 in HEK293 cells. Although 458ST was able to form a gamma-secretase complex, this complex was not matured, illustrated by mutant PS1 instability, lack of endoproteolysis, and little production of mature Nicastrin. These data indicate that the C-terminal end of PS1 is essential for Nicastrin trafficking and modification as well as the replacement of endogenous PS1 by PS1 transgenes.

BRO beta-lactamase alleles, antibiotic resistance and a test of the BRO-1 selective replacement hypothesis in Moraxella catarrhalis.

OBJECTIVES: The hypothesis that BRO-1 selectively replaced the BRO-2 isoform of the Moraxella catarrhalis BRO beta-lactamase was tested by examining the temporal distribution, antibiotic resistance and epidemiological characteristics of isolates from a long-term collection at a single locale. METHODS: A rapid, one-step PCR assay conducted on 354 isolates spanning 1984-1994 distinguished bro alleles in over 97% of the beta-lactamase-producing isolates. Probes of dot blots were used to distinguish PCR failure from non-beta-lactamase-mediated penicillin resistance. RESULTS: BRO-2 isolates comprised 0-10% of the population per year with no evidence of a decline over time. All beta-lactamase producers exceeded the clinical threshold for penicillin resistance. Bimodality of penicillin MICs for beta-lactamase producers was caused by variation within BRO-1 rather than differences between BRO-1 and BRO-2. Non-beta-lactamase factors also confer resistance to penicillin and may contribute to the BRO-1 bimodality. The 13 BRO-2 isolates were associated with diverse genotypes within which there was evidence of epidemiologically linked clusters. The exclusive association of BRO-2 with four unrelated genotypes suggested maintenance of BRO-2 by recurrent mutation or horizontal exchange. CONCLUSIONS: The relative rarity of BRO-2 throughout the study, the absence of a declining temporal trend, and genetic diversity within BRO-2 all failed to support the hypothesis that BRO-2 was more common in the past and has been selectively replaced by BRO-1.

A comparative evaluation of loading times and exposures for permanent prostate brachytherapy.

The loading of needles for loose seed implantation of the prostate gland results requires a significant amount of effort and some radiation exposure to members of the medical staff. This study was performed to quantify the time spent and exposure levels associated with implant preparation, as well as to investigate any improvement in the time or exposure burden due to the introduction of a new loading device. The movements and radiation exposures for two single, highly experienced dosimetrists were monitored for ten conventionally loaded iodine implant cases. These same cases were reloaded with dummy sources using the sleeved system to determine time savings, if any. Two of these ten cases were then loaded with live sources using the sleeved system to determine relative exposure to the loading staff between the two methods. The results were then analyzed to generate per-seed and per-needle loading time and exposure burdens. Formulas are presented that may be used to determine the average time to load implants and the resultant staff exposure, both with the conventional technique and with the sleeved method. On the average, it takes an experienced loader 48 min to prepare an implant for the operating room, receiving a hand dose of about 10 mrem and a whole body dose of about 1 mrem. The sleeved system reduced these values by at least half. The time and exposure burden associated with the preparation of iodine loose seed implants has been characterized. The use of the sleeved needles resulted in significant time and exposure reductions for the medical staff.

Genetic trends in a population evolving antibiotic resistance.

The evolution of antibiotic resistance provides a well-documented, rapid, and recent example of a selection driven process that has occurred in many bacterial species. An exhaustive collection of Moraxella catarrhalis that spans a transition to chromosomally encoded penicillin resistance was used to analyze genetic changes accompanying the transition. The population was characterized by high haplotypic diversity with 148 distinct haplotypes among 372 isolates tested at three genomic regions. The power of a temporally stratified sample from a single population was highlighted by the finding of high genetic diversity throughout the transition to resistance, population numbers that remained high over time, and no evidence of departures from neutrality in the allele frequency spectra throughout the transition. The direct temporal analysis documented the persistence, antibiotic status, and haplotypic identity of strains undergoing apparent clonal expansions. Several haplotypes that were beta-lactamase nonproducers in early samples converted to producers in later years. Maintenance of genetic diversity and haplotype conversions from sensitive to resistant supported the hypothesis that penicillin resistance determinants spread to a diverse array of strains via horizontal exchange. Genetic differentiation between sample years, estimated by F(ST), was increasing at a rate that could cause complete haplotype turnover in less than 150 years. Widespread linkage disequilibrium among sites within one locus (copB) suggested recent mutation followed by clonal expansion. Nonrandom associations between haplotypes and resistance phenotypes provided further evidence of clonal expansion for some haplotypes. Nevertheless, the population structure was far from clonal as evidenced by a relatively low frequency of disequilibria both within sites at a second locus (M46) as well as between loci. The haplotype-antibiotic resistance association that was accompanied by gradual haplotype turnover is consistent with a hypothesis of genetic drift at marker loci with directional selection at the resistance locus.

The epidemiology of mupirocin resistance among methicillin-resistant Staphylococcus aureus at a Veterans' Affairs hospital.

OBJECTIVE: To describe the clinical and molecular epidemiology of mupirocin-resistant (MR) and mupirocin-susceptible (MS) methicillin-resistant Staphylococcus aureus (MRSA) at a Veterans' Affairs hospital and to assess risk factors associated with the acquisition of MR MRSA. DESIGN: All clinical MRSA isolates for the period October 1990 through March 1995 underwent susceptibility testing to mupirocin. Mupirocin resistance trends were measured, and MS MRSA and MR MRSA isolates underwent typing by pulsed-field gel electrophoresis (PFGE). A retrospective case-control study was conducted to evaluate risk factors for having MR versus MS MRSA. SETTING: The James H. Quillen Veterans' Affairs Medical Center in Mountain Home, Tennessee, included a 324-bed acute-care hospital, a 120-bed nursing home, and a 525-bed domiciliary. Colonizations and infections with MRSA were endemic, and mupirocin ointment was commonly used. PATIENTS: Inpatients and outpatients at the facility. RESULTS: MS MRSA was recovered from 506 patients and MR MRSA from 126. Among MR MRSA isolates, 58% showed low-level mupirocin resistance (minimum inhibitory concentration [MIC] > or = 4 to 256 microg/mL), and 42% showed high-level mupirocin resistance (MIC > or = 512 microg/mL). A significant increase (P=.002) in the number of high-level MR isolates occurred during the 1993 to 1995 period. A case-control study showed that presence of a decubitus ulcer correlated with high-level resistant isolates (P<.05). The distribution of PFGE patterns did not differ for MR and MS MRSA CONCLUSIONS: Use of mupirocin ointment in a program aimed at managing endemic MRSA infection or colonization resulted in a significant increase in the recovery of high-level MR MRSA isolates. These isolates appeared to emerge from our existing MRSA pool. A case-control study provided few clues concerning patients likely to harbor MR MRSA. We confirmed the position that the extended use of mupirocin ointment should be avoided in settings where MRSA is endemic.

Long-term trends in susceptibility of Moraxella catarrhalis: a population analysis.

A retrospective, population analysis of antimicrobial susceptibility patterns was performed on Moraxella catarrhalis isolates recovered from a single medical centre to detect temporal trends and infer potential mechanisms of reduced susceptibility. The duration of this study, June 1984 to July 1994, encompassed the period during which the frequency of beta-lactamase production expanded from 30 to 96% in the population. MICs of penicillin G, cefamandole, ceftriaxone, amoxycillin/clavulanate, imipenem, clarithromycin, tetracycline, ciprofloxacin and trimethoprim/sulphamethoxazole for a representative sample of 375 isolates were determined. Analyses were conducted to test for variation in susceptibility among isolates, correlations of susceptibility levels among different antimicrobial agents, and temporal patterns in susceptibility. All antimicrobials except clarithromycin displayed significant differences among isolates within years, and mean MICs of all antimicrobial agents except tetracycline and clarithromycin varied significantly between years. Temporal trends to a reduction in susceptibility were detected to four of five beta-lactam antimicrobials (all except cefamandole). Significant correlations in MICs were uncovered among all pairs of four beta-lactam antimicrobials in both producers and non-producers of beta-lactamase. In contrast, cefamandole MICs were correlated only with ceftriaxone and penicillin, and these were limited to beta-lactam producing isolates; cefamandole and amoxycillin/clavulanate showed a correlation limited to non-producing isolates. For some antimicrobials, trends toward decreasing susceptibility may have been caused by an increased proportion of beta-lactamase producing isolates in the population, but the observation of significant decreases in susceptibility limited to beta-lactamase-producing isolates suggests that the underlying factors were different forms of beta-lactamase, beta-lactamase-dependent modifiers and/or additional factors.

Normal human right and left ventricular mass, systolic function, and gender differences by cine magnetic resonance imaging.

Our objective was to establish normal ranges of left and right ventricular mass and function with cine magnetic resonance imaging (MRI) and to determine gender differences. Seventy-five healthy subjects (age range 8-55, mean 28 yr) were studied with cine MRI. Ten dogs were imaged for autopsy validation with a mean difference between actual and MRI-determined mass of 0.2 A +/- 8.4 g. Intraobserver and interobserver variability and interstudy variability were 5-6%. All parameters were significantly different between males and females except ejection fraction and the left ventricular mass to end-diastolic volume ratio. Agreement with published autopsy series, including gender differences, was excellent. This study presents normative MRI data that can be used for comparing individual patients and for further study of right and left ventricular interaction.

Person-to-person transmission of Kingella kingae among day care center attendees.

Fifty Kingella kingae organisms, isolated from tonsillar cultures of day care center attendees during an 11-month period, and 60 isolates derived from epidemiologically unrelated individuals, including 19 isolates from respiratory carriers and 41 isolates from patients with invasive infections, were typed by immunoblotting, pulsed-field gel electrophoresis, and ribotyping. One strain, defined by unique immunoblotting, pulsed-field gel electrophoresis, and ribotyping patterns, represented 14 day care isolates (28%) and was frequently isolated during the first half of the follow-up period; a second strain represented 23 (46%) isolates and prevailed during the last 5 months. Children frequently carried the same strain continuously or intermittently for weeks or months, when it was replaced by a new strain. Epidemiologically unrelated organisms showed greater variability, and no strain represented >5% of isolates. The present results support person-to-person transmission of K. kingae among young children in the day care setting.

Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method.

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

Right ventricular performance and mass by use of cine MRI late after atrial repair of transposition of the great arteries.

BACKGROUND: The long-term adaptation of the right ventricle after atrial repair of transposition of the great arteries (TGA) remains a subject of major concern. Cine magnetic resonance imaging (MRI), with its tomographic capabilities, allows unique quantitative evaluation of both right and left ventricular function and mass. Our purpose was to use MRI and an age-matched normal population to examine the typical late adaptation of the right and left ventricles after atrial repair of TGA. METHODS AND RESULTS: Cine MRI was used to study ventricular function and mass in 22 patients after atrial repair of TGA. Images were obtained in short-axis sections from base to apex to derive normalized right and left ventricular mass (RVM and LVM, g/m2), interventricular septal mass (IVSM, g/m2), RV and LV end-diastolic volumes (EDV, mL/m2), and ejection fractions (EF). Results 8 to 23 years after repair were compared with analysis of 24 age- and sex-matched normal volunteers and revealed markedly elevated RVM, decreased LVM and IVSM, normal RV size, and only mildly depressed RVEF. Only 1 of 22 patients had clinical RV dysfunction, and this patient had increased RVM. CONCLUSIONS: Cine MRI allows quantitative evaluation of both RV and LV mass and function late after atrial repair of TGA. Longitudinal studies that include these measurements should prove useful in determining the mechanism of late RV failure in these patients. On the basis of these early data, inadequate hypertrophy does not appear to be the cause of late dysfunction in this patient group.

MRI complements standard assessment of right ventricular function after lung transplantation.

BACKGROUND: Changes in right ventricular mass and ejection fraction after single-lung transplantation for pulmonary hypertension are poorly understood. METHODS: To complement functional data provided by echocardiography, radionuclide ventriculography, and right heart catheterization, magnetic resonance imaging was used to assess right ventricular function in 5 single-lung transplant recipients with preoperative pulmonary hypertension and right ventricular dysfunction (right ventricular ejection fraction, 0.21 +/- 0.09). The right and left ventricular mass, ejection fraction, and mass ratio (left ventricular mass/right ventricular mass) were calculated from the magnetic resonance images. RESULTS: The mean pulmonary artery pressure fell from 72 +/- 18 to 21 +/- 8 mm Hg after transplantation. At 3 months after transplantation both the left ventricular and right ventricular ejection fractions approached normal values, as shown by both radionuclide ventriculography and magnetic resonance imaging, but the right ventricular mass remained abnormally high with slightly low mass ratios. By 1 year both the left ventricular and right ventricular masses had regressed to normal with near-normal mass ratios. CONCLUSIONS: Right ventricular performance returns to nearly normal early after transplantation, but the right ventricular mass regresses over a more prolonged time. Cine magnetic resonance imaging provides a noninvasive means of assessing changes in right ventricular function and mass after lung transplantation.

Molecular analysis of bacterial pathogens in otitis media with effusion.

OBJECTIVE: To determine if the polymerase chain reaction (PCR) can detect bacterial DNA in pediatric middle ear effusions that are sterile by standard cultural methods. DESIGN: Single-center, blinded, comparative study of diagnostic assays. The PCR-based detection systems for Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae were designed and validated using a battery of DNAs obtained from cultured bacteria. Chronic middle ear effusion specimens were collected and comparatively analyzed by culture and the PCR. SETTING: Tertiary care pediatric hospital. PATIENTS: A total of 97 middle ear effusions were collected from pediatric outpatients at Children's Hospital of Pittsburgh (Pa) during myringotomy and tube placement for chronic otitis media with effusion (duration > 3 months). All patients had failed multiple courses of antimicrobial therapy and were diagnosed by a combination of validated otoscopy and tympanograms. MAIN OUTCOME MEASURE: Differences in the percentage of positive test results between PCR-based assays and culture for M catarrhalis, H influenzae, and S pneumoniae. RESULTS: Of the 97 specimens of otitis media with effusion, 28 (28.9%) tested positive by both culture and PCR for M catarrhalis, H influenzae, or S pneumoniae. An additional 47 specimens (48%) were PCR positive/culture negative for these three bacterial species. Thus, 75 (77.3%) of the 97 specimens tested PCR positive for one or more of the three test organisms. The minimum number of bacterial genomic equivalents present in the average culture-negative ear was estimated to be greater than 10(4) based on dilutional experiments. CONCLUSIONS: The PCR-based assay systems can detect the presence of bacterial DNA in a significant percentage of culturally sterile middle ear effusions. While this finding is not proof of an active bacterial infectious process, the large number of bacterial genomic equivalents present in the ears is suggestive of an active process.

Transposition of the Responder element (Rsp) of the Segregation distorter system (SD) to the X chromosome in Drosophila melanogaster.

In order to test whether the meiotic drive system Segregation distorter (SD) can operate on the X chromosome to exclude it from functional sperm, we have transposed the Responder locus (Rsp) to this element. This was accomplished by inducing detachments of a compound-X chromosome in females carrying a Y chromosome bearing a Rsps allele. Six Responder-sensitive-bearing X chromosomes, with kappa values ranging from 0.90 to 1.00, were established as permanent lines. Two of these have been characterized more extensively with respect to various parameters affecting meiotic drive. SD males with a Responder-sensitive X chromosome produce almost exclusively male embryos, while those with a Rsp-Y chromosome produce almost exclusively female embryos. This provides a genetic system of great potential utility for the study of early sex-specific differentiation events as it allows the collection of large numbers of embryos of a given sex.

Liquid chromatographic determination of carbamazepine in tablets.

A simple and rapid liquid chromatographic method is described for the qualitative and quantitative determination of carbamazepine in tablet composites and individual tablets, using the internal standard technique. Analyses were performed on a C-18 reverse-phase column with tetrahydrofuran-methanol-water (8 + 37 + 55) as the mobile phase. A linear relationship was obtained between detector responses at 254 nm and amounts of carbamazepine injected ranging from 0.2 to 1.7 micrograms. The coefficient of variation for 10 consecutive injections of a standard preparation was 0.4%. Recoveries of carbamazepine from 100 and 200 mg tablets averaged 101.4 and 99.7%, respectively. Assay results for commercial tablets analyzed by the proposed method agreed favorably with those obtained by the method of USP XXI. The assay results for individual tablets indicated that deviations from the average value and the range of individual values are much wider with the compendial method than with the proposed method.

Characterization of a new monoclonal antibody to a cell surface antigen on colorectal cancer and fetal gut tissues.

Murine hybridoma were raised against the human colon carcinoma cell line CL-187. One clone was found to secrete a monoclonal antibody (ND-1) that recognizes a large external antigen (LEA) on human colon carcinoma cells. With indirect immunofluorescence on formaldehyde-fixed cells, more than 90% of the human colorectal carcinoma cell lines tested expressed LEA. Almost all of the 46 human noncolorectal and nonhuman cell lines tested did not express LEA, including cancer cell lines from other endodermally derived tissues. Staining of frozen sections from human colorectal tumors, noncolorectal tumors, normal adult, and normal fetal tissues showed expression of the antigen on colorectal cancer tissue, fetal colon, and fetal biliary epithelium. LEA can also be detected in the serum and ascites of colorectal cancer patients. Double indirect immunofluorescence with rabbit anti-carcinoembryonic antigen (CEA) antibody and ND-1 monoclonal antibody on a human colorectal carcinoma cell line showed that LEA is distinct from CEA. Physicochemical analysis of LEA showed that it has a large molecular weight, is resistant to extraction from the cell surface, and that sialic acid is an important component of the antigenic site. Because of the specificity for colorectal cancer tissue along with certain biochemical properties, LEA appears to be unique when compared with other tumor-associated antigens. Further research is needed to define the clinical usefulness of LEA in either the diagnosis or treatment of colorectal carcinoma.

Familial paroxysmal dystonic choreoathetosis: a neurologic disorder simulating psychiatric illness.

A brother and sister with familial paroxysmal choreoathetosis are presented, and the relevant literature is reviewed.