BDA hospital dentists survey 2017.

In 2016 the BDA undertook to investigate the demographics and distribution of its hospital members alongside their morale and motivation. This is the first time the BDA had undertaken such a survey and it is the first time any workforce research into the Hospital Dental Services (HDS) has been published by any party. Subsequent freedom of information requests have suggested that BDA HDS member data is representative of the overall HDS workforce. Of particular note is the distribution of some of the 'smaller' specialties with some seemingly only existing at dental school level alongside morale and motivation levels in relation to other dental roles.

Trapping volume control in optical tweezers using cylindrical vector beams.

We present the result of an investigation into the optical trapping of spherical microparticles using laser beams with a spatially inhomogeneous polarization direction [cylindrical vector beams (CVBs)]. We perform three-dimensional tracking of the Brownian fluctuations in the position of a trapped particle and extract the trap spring constants. We characterize the trap geometry by the aspect ratio of spring constants in the directions transverse and parallel to the beam propagation direction and evaluate this figure of merit as a function of polarization angle. We show that the additional degree of freedom present in CVBs allows us to control the optical trap strength and geometry by adjusting only the polarization of the trapping beam. Experimental results are compared with a theoretical model of optical trapping using CVBs derived from electromagnetic scattering theory in the T-matrix framework.

An optimized procedure for fluorescence-activated cell sorting (FACS) isolation of autonomic neural progenitors from visceral organs of fetal mice.

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine and TH is also present in a subset of adult pelvic ganglia neurons . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

The number of amino acid residues mismatches correlates with flow cytometry crossmatching results in high PRA renal patients.

Highly sensitized renal transplant candidates present a group at high risk for acute and chronic rejection. The probability of finding compatible donors for these recipients is significantly lower in comparison to those who have low PRA values. As a consequence, these patients spend longer time on the waiting list and become tethered to dialysis. The results of final cross match (XM) are critical for making a decision about whether such a candidate receives an organ or not. The degree of donor and recipient HLA compatibility predicts the results of XM. The goal of this study was to expand a variety of acceptable HLA-AB mismatches (MM) for high PRA kidney recipients using the HLAMATCHMAKER algorithm. This strategy focuses on the fine structural features of HLA polymorphism comprising amino acid residues or triplets (AAT), which are located in alpha-helical coils of HLA molecules and are available to antibodies. We analyzed serum samples from thirty-nine highly alloimmunized recipients (PRA > or = 85%). The level of sensitization was detected using FlowPRA Class I Screening Test. This group of transplant candidates included thirteen recipients who demonstrated negative results of final T/B FCXM and twenty-six, who were FCXM positive. The application of the HLAMATCHMAKER algorithm based on the HLA class I donor and recipient typing allowed us to detect the total number of AATMM as well as the number of immunogenic AAT in both FCXM negative and FCXM positive groups of recipients. Significantly greater numbers of both total and highly immunogenic AATMM have been emerged in the group of FCXM positive patients. Furthermore, the results of this analysis have shown a high degree of probability of positive FCXM if the number of highly immunogenic AATMM was > or = 1 (chi(2) = 22.9 Yate's correction; p = 0.000001). We did not observe overlapping between antibody specificity and permissible HLA-AB MM detected using the HLAMATCHMAKER strategy. Thus, the number of highly immunogenic AATMM can serve as a reliable predictive value for final FCXM results in highly sensitized renal transplant candidates. The HLAMATCHMAKER algorithm appears to be the proper strategy to find donors for high PRA recipients.

Clinical characteristics of Chlamydia pneumoniae infection as the sole cause of community-acquired pneumonia.

The clinical characteristics of 26 patients with community-acquired pneumonia due to Chlamydia pneumoniae as the only identified pathogen who required hospitalization were evaluated. Most patients (18) had reinfection based on serological results. The mean age of the patients was 55 years (38 years, patients with primary infection; 63 years, patients with reinfection), and the gender representation was equal. Generally, illness was mild and associated with limited temperature elevation and nonspecific symptoms. The presence of comorbid illnesses and the requirement of supplemental oxygen therapy were the most common criteria for hospital admission.

Detection of chlamydiosis in a shipment of pet birds, leading to recognition of an outbreak of clinically mild psittacosis in humans.

Avian chlamydiosis was detected in a shipment of > 700 pet birds from a Florida bird distributor that were sold to nine Atlanta-area pet stores in August 1995. Respiratory illness among persons who had recently acquired birds from this shipment was reported to local public health officials. The attack rate of acute respiratory illness was 10.7% among persons in households exposed to birds from the implicated flock vs. 1.8% among control households (odds ratio, 6.60; 95% confidence interval, 1.39-31.2). Illness and serological evidence of infection in the absence of symptoms were more common among persons in households with recently purchased birds that were sick or that had died and among persons who had had direct contact with the birds. Clinical psittacosis or serological evidence of Chlamydia psittaci infection was found in 30.7% of households with birds from the infected flock. Mild illnesses and asymptomatic infections in exposed persons were unusual features of this outbreak.

Chlamydia DNA extraction for use in PCR: stability and sensitivity in detection.

We evaluated multiple procedures for extracting chlamydial DNA from specimens for detection in PCR tests. Commercial kits and an in-house method were tested for their sensitivity and utility. Quantifiable chlamydial elementary bodies (EB) were used for spiking buffy coats from EDTA-collected blood. EBs of Chlamydia pneumoniae at 2,500 and 25 EB/ml were used as specimens for DNA extraction using seven different procedures. These included either columns (3 procedures), centrifugation (1), glass (1), or patented extraction matrices (2), coupled with either alcohol precipitation (6) or heat-detergent treatment (1). Five procedures required 10-40 minutes manipulation; two required 2-5 hours. PCR results for DNA extracts using chlamydial 16S genus primers were generally more intensely positive with denser bands on electrophoresis gels for the higher concentrations of EB (up to 4+ for stained product on gels) than was PCR with lower EB concentrations (up to 2+). Further, the incidence of procedures with positive results was: 5 of 7 for chlamydial genus primers with 5 EB vs. 6 of 7 with 500 EB. Maximal sensitivity for one of the extractions was in the range of 2.5-5.0 EB/ml of test specimen with 4 of 5 replicates being positive with EB controls or extracts. Extracts were stable up to 2+ weeks at 4 degrees C and were effective in multiplexing with fluorescent-tagged primers. Taking into consideration the time factor and sensitivities, the two procedures with extraction matrices are favored for routine laboratory use.

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

Utility of complement fixation and microimmunofluorescence assays for detecting serologic responses in patients with clinically diagnosed psittacosis.

The serodiagnosis of human psittacosis was considerably improved by a microimmunofluorescence (MIF) assay that uses selected strains of Chlamydia psittaci, C. pneumoniae, and C. trachomatis as antigens. The 78 patients examined in the study were clinically diagnosed as having psittacosis on the basis of compatible clinical symptoms following exposure to sick birds. The conventional complement fixation (CF) test identified 36 patients, or 46% (36 of 78) of the total, as positive. Antibody responses to C. psittaci were demonstrated by the MIF test in all 36 CF-positive patients. The MIF test also detected antibody responses to C. psittaci in 12 patients (15% of the total) whose sera were negative or anticomplementary in the CF test. Seven patients, or 9% (7 of 78) of the total, were identified by the MIF test as having C. pneumoniae infections. About 30% of the study patients (23 of 78) showed no serologic evidence of either C. psittaci or C. pneumoniae infection by both the CF and the MIF tests. Four distinctive serologic reaction patterns were observed in the study patients. Recognition of these reaction patterns and judicious corroboration of serologic responses to the chlamydial species by the MIF test with epidemiologic and clinical information will increase the efficiency and accuracy of serodiagnosis for human psittacosis.

Radiographic appearance of Chlamydia pneumoniae (TWAR strain) respiratory infections. CBPIS Study Group. Community-based Pneumonia Incidence Study.

PURPOSE: To report the spectrum of radiographic findings associated with a new respiratory pathogen: Chlamydia pneumoniae (TWAR strain). MATERIALS AND METHODS: Radiographs of 55 adult patients hospitalized with serologic evidence of C pneumoniae were retrospectively reviewed. RESULTS: On the basis of serologic criteria, two types of acute respiratory infection are possible: primary (first exposure) infections and recurrent, acute infection in a previously exposed individual. In the primary group, alveolar opacities (65%) with a unilateral distribution (71%) were most common at admission. Cavitary disease and hilar or mediastinal lymphadenopathy were uncommon. Small to medium-sized pleural effusions were common in both primary and recurrent groups during hospitalization. Also, both groups tended to progress to bilateral, mixed, interstitial and alveolar changes during the course of infection. CONCLUSION: Different radiographic patterns exist for the two types of acute C pneumoniae infection.

Efficient culture of Chlamydia pneumoniae with cell lines derived from the human respiratory tract.

Two established cell lines, H 292 and HEp-2, originating from the human respiratory tract, were found to be significantly more efficient and practical than the currently used HeLa 229 cells for growth of Chlamydia pneumoniae. Six strains of C. pneumoniae recently isolated from patients with respiratory ailments were used as test cultures. The H 292 and HEp-2 cells yielded much higher inclusion counts for all the test strains than did HeLa 229 cells. When they were compared with each other, H 292 cells yielded more inclusions than did HEp-2 cells, and the differences were statistically significant in 10 of 18 test sets. A simple system with these two cell lines appeared to be very efficient for culturing C. pneumoniae. It does not require treatment of tissue cells with DEAE-dextran before infection, and it may eliminate the need for serial subpassages of specimens to increase culture sensitivity. Monolayers of these cells remained intact and viable in the Chlamydia growth medium so that reinfection could take place, resulting in greatly increased inclusion counts for specimens containing few infectious units. This system may make it more practical for laboratories to culture for C. pneumoniae for treatment of infections and outbreak intervention and will facilitate studies on this recently recognized pathogen.

Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction.

Two major antigens of Bordetella pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (PT), were efficiently purified from culture filtrate by exploiting their relative hydrophobicities and differences in affinity to sialic acid-containing protein. High yields of FHA (40 to 80 mg/liter) and PT (8 to 16 mg/liter) were first produced by growing the bacteria in the modified CL medium. The FHA and PT in the culture filtrate were adsorbed onto butyl-Sepharose by hydrophobic interaction at appropriately high ionic strength. Elution of the antigens was effected by decreasing their hydrophobicities with a buffer of low ionic strength. FHA was then separated from PT with an affinity column of fetuin-Sepharose. The fraction passing through the column contained purified FHA, and the fetuin-bound PT was eluted with buffered MgCl2. The FHA and PT purified by these steps were electrophoretically and serologically identical to the reference purified FHA and PT preparations. Approximately 16 to 32 mg of purified FHA and 4 to 8 mg of purified PT were obtained from 1 liter of culture filtrate. The described procedure for making FHA and PT antigens from B. pertussis for serologic and immunologic use is very simple, efficient, and reproducible.

Preparation of filamentous hemagglutinin from Bordetella pertussis and assay for serum antibodies to filamentous hemagglutinin and pertussis toxin for clinical and public health laboratories.

A procedure that is sufficiently simple and economical for use in clinical and public health laboratories for producing and purifying filamentous hemagglutinin (FHA) and determining antibodies to this major antigen of Bordetella pertussis in serum is described. High yields of FHA (40 to 80 mg/liter) were obtained in the supernatant by cultivating B. pertussis in modified CL medium. The FHA antigen was separated from pertussis toxin (PT) and other antigens by chromatography on hydroxylapatite. Removal of residual PT activity in the FHA fraction was effected by affinity absorption of PT with Fetuin immobilized to Sepharose 4B. The FHA was used as the antigen for determining titers of immunoglobulin G (IgG), IgA, and IgM to FHA in sera of patients with pertussis by an improved enzyme-linked immunosorbent assay. Development of the interfering background color commonly observed in conventional FHA enzyme-linked immunosorbent assay procedures was eliminated by washing the reaction wells with a buffer of high ionic strength before adding the peroxidase conjugates. In the absence of nonspecific background color, the reaction endpoints were easy to read. The FHA prepared by the procedure described was identical to a reference preparation of purified FHA in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and serological specificity assays. High yields of FHA were obtained from all four strains of B. pertussis tested in this study, indicating that the procedure for enhanced production of FHA may be generally applicable to other strains of B. pertussis. Results from tests of 50 serum specimens with clinical information on pertussis for FHA and PT antibodies by the assay procedures described exemplified the usefulness and caveats of serodiagnosis for pertussis.

New, practical approach to detecting antibody to pertussis toxin for public health and clinical laboratories.

A new, practical method for determining antibody to pertussis toxin (PT) by enzyme-linked immunosorbent assay for public health and clinical laboratories is possible because of recent advances in understanding the pathobiology of Bordetella pertussis and the physicochemical properties of PT. The new approach does not require the use of highly purified PT antigen, which is difficult and expensive for most laboratories to obtain. Moreover, it employs only reagents that are commercially readily available. The method combines the purification of PT antigen and an assay for PT antibody into one process. It depends on (i) growth of B. pertussis in a simple, defined medium to obtain a PT-rich supernatant with little contamination of cellular antigens; (ii) a simple, one-step concentration of PT in the culture supernatant with Affi-Gel Blue (Bio-Rad Laboratories, Richmond, Calif.); and (iii) specific adsorption of PT as the test antigen to microtiter wells coated with fetuin for the enzyme-linked immunosorbent assay. The procedure is sensitive and specific for PT antibody. It is technically simple, reproducible, and can be performed in a modestly equipped laboratory.

Strain characterization and grouping of Campylobacter jejuni and Campylobacter coli by interaction with lectins.

Strains of Campylobacter jejuni and Campylobacter coli were characterized and grouped by their distinct reaction patterns with lectins. Heating of the Campylobacter cultures to 100 degrees C and holding for 30 to 60 min greatly enhanced their reactivity with lectins and permitted the grouping of all but 3 of 155 cultures tested in this study without interference of autoagglutination and other nonspecific activities. The lectin reaction patterns of the heated cultures were stable and reproducible. They were strain specific and independent of the heat-stable antigenic types. The lectin-reactive sites of C. jejuni and C. coli may be useful as additional markers for strain characterization. Based on these observations, a simple slide agglutination procedure is described for differentiating strains of C. jejuni and C. coli by their interaction with a selected group of commercially available lectins.

Interaction of Campylobacter jejuni and Campylobacter coli with lectins and blood group antibodies.

Lectins and blood group antibodies were used to probe the surface structures of Campylobacter jejuni and Campylobacter coli. Of the 29 strains tested, there were distinct reaction patterns. The lectin-reactive and blood group antibody-reactive sites on the bacterial surface were distinguishable from the heat-stable (lipopolysaccharide) antigenic determinants. The interactions were strain specific. The reactive sites were stable with respect to culture media and passage and may be useful as additional markers for strain characterization.

Typing of heat-stable and heat-labile antigens of Campylobacter jejuni and Campylobacter coli by coagglutination.

A coagglutination system has been devised for typing heat-stable and heat-labile antigens of Campylobacter jejuni and C. coli. The use of protein A-positive Staphylococcus aureus cells carrying Campylobacter sp. serotype antibody and the treatment of Campylobacter sp. cells with DNase in the antigen suspension permitted rapid and specific coagglutination of rough (autoagglutinable) as well as smooth cultures. Cells of S. aureus were sensitized with Campylobacter sp. serotype antisera. Four to five types of sensitized S. aureus cells were pooled. A strain of Campylobacter sp. was first tested with the pools and then typed with the individual reagents of the reactive pool. After the described procedures, 68 serotype strains tested blindly as unknowns were correctly typed according to their heat-stable or heat-labile antigens. The two most commonly used typing schemes which are based separately on the heat-stable or the heat-labile antigens as assayed by passive hemagglutination and slide agglutination, respectively, can be utilized simultaneously in the coagglutination system for strain characterization. The coagglutination system is simple, yields results rapidly, conserves typing reagents, and offers the flexibility of formulating the pools of reagents according to the experimental design or the prevalence of serotypes in a geographic location. It should be a practical system for the typing of Campylobacter spp. in public health or clinical laboratories.