Maureen Dawson

Development and Evaluation of a Tetraplex Flow Cytometric Assay for Quantitation of Serum Antibodies to Neisseria meningitidis Serogroups A, C, Y, and W-135

ABSTRACT A rapid and simple method for the simultaneous quantitation of serum immunoglobulin G (IgG) antibodies specific for Neisseria meningitidis serogroups A, C, Y, and W-135 was developed and evaluated. Four bead sets were generated, each conjugated with one of the meningococcal capsular polysaccharides (A, C, Y, or W-135) and serologically assessed by the use of antimeningococcal international reference sera. Cross-reactivity studies demonstrated no inhibition between monoplex and multiplex assays, and the assay was linear over a 24-fold serum dilution range. Inhibition studies demonstrated that the assay is specific, with <25% heterologous inhibition occurring. The assay was also found to have low intra- and interassay variations and limits of detection ≤650 pg/ml. A comparison of the meningococcal bead assay with the standardized meningococcal enzyme-linked immunosorbent assay showed a good correlation between the IgG concentrations obtained by each assay. The tetraplex assay has the potential to be an important addition to the serologic evaluation of meningococcal capsular polysaccharide conjugate vaccines.

Effect of sequence variation in meningococcal PorA outer membrane protein on the effectiveness of a hexavalent PorA outer membrane vesicle vaccine

Though meningococcal serogroup C conjugate vaccines have been introduced into the UK infant immunisation schedule, there is currently no vaccine solution for serogroup B disease. PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate. A hexavalent PorA outer membrane vesicle (OMV) vaccine has been evaluated in phase I and II trials with promising results. This vaccine contains six different PorA OMPs each representing a different serosubtype. However, considerable sequence variation occurs in the variable regions (VRs) encoding these serosubtypes. By using recombinant P1.5,10 PorA variants we have demonstrated that the killing of this particular serosubtype combination was due mainly to the induction of antibody to the VR2 (P1.10) epitope region, and that after three or four doses of vaccine there was a significant reduction in the killing of variants P1.10a (three doses, p<0.0001; four doses, p = 0.003) and P1.10f (three doses, p<0.0001; four doses, p = 0.002), as compared to responses to the P1.10 strain, when the P1.10 serosubtype was used as the immunogen. Since large numbers of serosubtype variants are known to exist, this finding may have implications for the use of PorA as a meningococcal serogroup B vaccine.

Avidity maturation following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine in UK infants

To date, there are no data assessing the utility of avidity indices as a surrogate marker for the induction of immunological memory following meningococcal serogroup B outer membrane vesicle (OMV) vaccination. We studied infants who had been immunized with three doses of a recombinant hexavalent PorA OMV vaccine at ages 2-4 months, together with a fourth dose at age 12-18 months. A control group had received a single dose of the same vaccine at age 12-18 months. As previously reported, serum bactericidal antibody (SBA) titres increased after each of the first three doses, with a significant increase observed from 6 months post third dose to 1 month post fourth dose. The geometric mean avidity indices (GMAI), against strain H44/76 OMVs, increased from 1 month post first dose to 1 month post third dose. Significant increases in GMAI were observed at 6 months post third dose and again following the fourth dose. At 32-42 months of age, though the SBA titres had returned to post first dose levels, the GMAI remained elevated. No increase in avidity was observed in the control group. Antibody avidity indices are useful laboratory markers for the priming of immunological memory following vaccination with meningococcal serogroup B OMV vaccines.

A survey into toxic shock syndrome (TSS) in UK burns units

Toxic shock syndrome (TSS) is a rare complication of a Staphylococcus aureus infection and is primarily seen in children with small burns. The true incidence of TSS in burns patients is not known and the number of presumptive cases rarely reported. This survey was undertaken to determine if the incidence of TSS in children with burns could be related to the type of dressing used to cover the wound. A questionnaire was compiled and sent to the Senior Nurse in charge of each of the UK burns units. General information on the number of admissions, age of the patient, cause of injury and burn wound management was sought. An 81% response was obtained after two mailshots and follow up telephone calls. Seventy percent (23/33) of units which answered the survey nursed children. Of these, eight units had either not encountered TSS previously or not had a case within the past two years. These units were small, admitting a maximum of 50 patients each year. Of the units where TSS was encountered, approximately 2.5% of children admitted showed symptoms of TSS. Of the units who nursed both adults and children, seven units had seen TSS in burned adult patients which has not been reported in the literature. Of the eight units where TSS had not been recently encountered, four routinely administered prophylactic antibiotics to prevent infection whereas routine administration of antibiotics occurred in only two of the 15 units where TSS was seen. Although wound management procedures differed slightly there were many similarities. These included wound cleaning with normal saline, covering with either silver sulphadiazine (1%) or povidone iodine (10%), depending upon the infection status, and dressing with a paraffin tulle, gauze and crepe bandages. No association between the management of the burn wound and subsequent development of TSS could be established.

Genetic analysis of capsular status of meningococcal carrier isolates

The meningococcal capsule is the primary virulence factor with systemic isolates requiring full expression of the capsule but with capability to down-regulate the capsule in order to invade. The meningococcal capsular operon is composed of a number of genes that are involved in capsular synthesis and transport. Differences in capsular synthesis genes may allow discrimination between meningococcal serogroups whereas absence of genes for either synthesis or transport imply that the meningococcus is unencapsulated. Although mechanisms such as slipped-strand mispairing and acquisition of insertion sequences have been demonstrated to be involved in regulation of capsular expression, few studies have addressed the mechanisms of capsular expression in carrier isolates. Following a community-based intervention programme for an outbreak of meningococcal disease, we collected meningococcal carrier isolates from the intervention area and control areas. We undertook genetic analysis of the capsular operon and the mechanisms of capsular regulation, together with an investigation of the potential of capsular genes to identify the genogroup of non-serogroupable isolates. Use of the siaD gene allowed the discrimination of 30/89 (34%) non-serogroupable isolates into B, C, W135 and Y with a siaA gene PCR permitting the characterization of a further 6 isolates whose capsules contained sialic acid. Slipped-strand mispairing was evident in only 4 of 13 genogroupable B isolates and the insertion sequence IS1301 was found in 2 of 36 siaA-positive isolates. Of 51 non-genogroupable isolates 25 (49%) were shown to be ctrA negative. There was a higher percentage of ctrA-positive isolates (P<0.001) amongst meningococcal strains obtained from those sampled in non-intervention schools than those sampled at intervention schools. The ctrA-negative isolates warrant further investigation of their genotypic organization since such avirulent strains may be important in conferring natural protection against invasive disease. We found that after mass antibiotic prophylaxis, recolonization occurs preferentially with non-pathogenic meningococcal strains. This as implications for assessment of the benefits of mass antibiotic and vaccination programmes for outbreak control. Previously expressed concerns of increased risk due to removal of protective ora may have been overstated.

Improved methods of detection of meningococcal DNA from oropharyngeal swabs from cases and contacts of meningococcal disease

In the UK the increasing use of pre-admission parenteral antibiotic therapy in meningococcal disease has lessened the value of routine cultures as a tool to confirm diagnosis, and laboratory confirmation of invasive meningococcal infection is achieved increasingly by non-culture, nucleic acid amplification methods. The purpose of this study was to evaluate a DNA extraction and meningococcal-specific DNA amplification methodology for detection of meningococci from oropharyngeal swabs. One hundred and six swabs from suspected or confirmed cases of meningococcal disease, and 94 swabs from contacts of meningococcal disease cases were examined. Of laboratory-confirmed cases, 38/65 (58.5%) yielded a positive oropharyngeal swab PCR result and 5/24 (20.8%) swabs from suspected but laboratory-unconfirmed cases were PCR positive. No significant differences in PCR positivity rates were found between the types of swab transport systems utilized, but transport time to the testing laboratory was found to affect PCR positivity (P < 0.05). Application of meningococcus-specific PCR to oropharyngeal swabs, in addition to routine culture of swabs, can provide valuable epidemiological information as well as case confirmation for contact management. PCR amplification of meningococcal PCR from oropharyngeal swabs will also increase the ascertainment in swabbing surveys carried out as part of meningococcal disease outbreak investigation and management.

The Effect of Temperature on Viability of Carbon- and Nitrogen-Starved Escherichia coli

Escherichia coli was grown in a defined medium at optimum temperature and then transferred to each of five different starvation regimes at 5°C, 20°C, or 37°C, for 1000 hours. Cells were maintained with growth-limiting amounts of carbon or nitrogen, or without either or both nutrients. Bacterial cell viability was assessed by dilution plating, the reduction of 2(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT), direct viable counts (DVC), and microcolony development. The recoverability of cells on solid medium declined most rapidly, and to the greatest extent in most cases, in cultures maintained at 37°C. Only nitrogen-starved cells maintained at 5°C became completely nonculturable. The reduction of INT consistently indicated higher numbers of viable cells compared to the other methods in all cultures. The viabilities of carbon- and nitrogen-limited cells, assessed by all methods, were similar to one another at each of the temperatures. Viability was lowest at 37°C. Nutrient-downshifted cells also followed a temperature-dependent pattern of survival with viability lowest at 37°C. Morphological differences were noted at different temperatures but were most obvious for nitrogen-starved cells at 37°C, which increased in length.

The Effectiveness of Lecture-Integrated, Web-Supported Case Studies in Large Group Teaching

Abstract The effectiveness of lecture-integrated and web-supported case studies in supporting a large and academically diverse group of undergraduate students was evaluated in the present study. Case studies and resource (web)-based learning were incorporated as two complementary interactive learning strategies into the traditional curriculum. A truncated version of a case study, with links to websites containing background/further information was uploaded on the university’s virtual learning environment (WebCT), to prompt students to answer questions. The overall aim was to support all students by encouraging self-directed learning. Our results show that most students who participated in the present study, irrespective of academic background, found both strategies useful in supporting the lecture and in providing them with the necessary background knowledge. Students who accessed web-links achieved significantly higher test scores in Immunology and in the module as a whole, than those who did not, irrespective of their course of study. This did not relate to A level grade in Biology and/or Chemistry. Findings from this study encourage the wider implementation of such complementary strategies to support large student groups with divergent prior knowledge.