Clinical efficacy of a next-generation sequencing gene panel for primary immunodeficiency diagnostics.

Primary immunodeficiencies (PIDs) are rare monogenic inborn errors of immunity that result in impairment of functions of the human immune system. PIDs have a broad phenotype with increased morbidity and mortality, and treatment choices are often complex. With increased accessibility of next-generation sequencing (NGS), the rate of discovery of genetic causes for PID has increased exponentially. Identification of an underlying monogenic diagnosis provides important clinical benefits for patients with the potential to alter treatments, facilitate genetic counselling, and pre-implantation diagnostics. We investigated a NGS PID panel of 242 genes within clinical care across a range of PID phenotypes. We also evaluated Phenomizer to predict causal genes from human phenotype ontology (HPO) terms. Twenty-seven participants were recruited, and a total of 15 reportable variants were identified in 48% (13/27) of the participants. The panel results had implications for treatment in 37% (10/27) of participants. Phenomizer identified the genes harbouring variants from HPO terms in 33% (9/27) of participants. This study shows the clinical efficacy that genetic testing has in the care of PID. However, it also highlights some of the disadvantages of gene panels in the rapidly moving field of PID genomics and current challenges in HPO term assignment for PID.

Co-infection as a confounder for the role of Clostridium difficile infection in children with diarrhoea: a summary of the literature.

Although Clostridium difficile is a major cause of antibiotic-associated diarrhoea in adults, the incidence and severity of C. difficile infection (CDI) in children is unclear. One complicating factor in assessing the role of CDI in children is the possibility of co-infection with other gastrointestinal pathogens. In this review, we summarise the literature concerning C. difficile co-infections in young children, in an attempt to discuss the rate of co-infections and their potential role in the severity of CDI clinical presentation. We identified 31 studies where co-infections were analysed, comprising 1,718 patients with positive C. difficile tests. The pooled percentage of reported co-infections was 20.7% (range 0-100%). Viral co-infections were most commonly reported (46%), with bacteria and parasites accounting for 14.9% and 0.01% of cases, respectively. However, the panel of co-infections tested for varied considerably among studies and 38% of stated co-infections did not have a pathogen reported. Substantial variation in how and when tests for gastrointestinal co-infections are carried out, small sample sizes and a lack of clear CDI case definitions preclude meaningful conclusions on the true rate of co-infections in this patient population. This review suggests that co-infections may be common in children with diarrhoea who tested positive for C. difficile. Given a lack of CDI case definitions, especially in young children under the age of 5 years, a broad panel of pathogens should be tested for to exclude other microbiological causes. However, the summarised poor quality of the available literature on this subject highlights a need for further studies.

Invasive Propionibacterium acnes infections in a non-selective patient cohort: clinical manifestations, management and outcome.

An increasing number of reports suggest that Propionibacterium acnes can cause serious invasive infections. Currently, only limited data exist regarding the spectrum of invasive P. acnes infections. We conducted a non-selective cohort study at a tertiary hospital in the UK over a 9-year-period (2003-2012) investigating clinical manifestations, risk factors, management, and outcome of invasive P. acnes infections. Forty-nine cases were identified; the majority were neurosurgical infections and orthopaedic infections (n = 28 and n = 15 respectively). Only 2 cases had no predisposing factors; all neurosurgical and 93.3 % of orthopaedic cases had a history of previous surgery and/or trauma. Foreign material was in situ at the infection site in 59.3 % and 80.0 % of neurosurgical and orthopaedic cases respectively. All neurosurgical and orthopaedic cases required one or more surgical interventions to treat P. acnes infection, with or without concomitant antibiotic therapy; the duration of antibiotic therapy was significantly longer in the group of orthopaedic cases (median 53 vs 19 days; p = 0.0025). All tested P. acnes isolates were susceptible to penicillin, ampicillin and chloramphenicol; only 1 was clindamycin-resistant. Neurosurgical and orthopaedic infections account for the majority of invasive P. acnes infections. Most cases have predisposing factors, including previous surgery and/or trauma; spontaneous infections are rare. Foreign material is commonly present at the site of infection, indicating that the pathogenesis of invasive P. acnes infections likely involves biofilm formation. Since invasive P. acnes infections are associated with considerable morbidity, further studies are needed to establish effective prevention and optimal treatment strategies.

Immunocompromised children and young people are at no increased risk of severe COVID-19.

OBJECTIVES: We aimed to prospectively describe the incidence and clinical spectrum of SARS-CoV-2 infection in immunocompromised paediatric patients in the UK. METHODS: From March 2020 to 2021 weekly questionnaires were sent to immunocompromised paediatric patients or their parents. Information, including symptom presentation and SARS-CoV-2 PCR test results, was collected from 1527 participants from 46 hospitals. Cross-sectional serology was investigated in February and March 2021. RESULTS: Until the end of September 2020, no cases were reported. From September 28th 2020 to March 2021 a total of 38 PCR-detected SARS-CoV-2 infections were reported. Of these, four children were admitted to hospital but none had acute severe COVID-19. Increasing age in association with immunodeficiency increased reporting of SARS-CoV-2 infection. Worsening of fever, cough, and sore throat were associated with participants reporting SARS-CoV-2 infection. Serology data included 452 unvaccinated participants. In those reporting prior positive SARS-CoV-2 PCR, there were detectable antibodies in 9 of 18 (50%). In those with no prior report of infection, antibodies were detected in 32 of 434 (7•4%). CONCLUSIONS: This study shows SARS-CoV-2 infections have occurred in immunocompromised children and young people with no increased risk of severe disease. No children died.

Improving paediatric antimicrobial stewardship in hospital-based settings: why, where and how?

BACKGROUND: Antimicrobial resistance (AMR) is being recognized as a priority by healthcare organizations across the world. However, many children are managed on IV antimicrobials in hospital with very little consideration of antimicrobial stewardship issues. OBJECTIVES: A nurse-led paediatric ambulatory outpatient parenteral antimicrobial therapy (OPAT) service, managing children with common infections being ambulated on short courses of IV antimicrobials, was introduced within Southampton Children's Hospital in January 2018. We evaluated the impact of this service in terms of the quality of antimicrobial prescribing and timing of ambulation in children presenting with common infections. METHODS: All cases managed within the service were reviewed in two separate 2 month time periods: prior to introduction of the service (September-October 2016) and then prospectively after its introduction (September-October 2018). RESULTS: A total of 96% of IV antibiotic management decisions at 48 h were deemed appropriate in 2018, compared with 75% in 2016. A total of 64% of patients were ambulated on IV antibiotics at some point during their treatment course in 2018, compared with 19% in 2016. However, a significant proportion of antimicrobial decisions made at the point of presentation to hospital remained suboptimal in 2018. CONCLUSIONS: Children are commonly managed with IV antibiotics in hospital. We demonstrate marked improvements in appropriate antimicrobial use through the introduction of a nurse-led ambulatory OPAT service. In addition, such a service can promote a greater proportion of children being ambulated from hospital, freeing up valuable inpatient beds and potentially delivering cost savings that can be used to fund such services.

Heterologous Two-Dose Vaccination with Simian Adenovirus and Poxvirus Vectors Elicits Long-Lasting Cellular Immunity to Influenza Virus A in Healthy Adults.

BACKGROUND: T-cell responses against highly conserved influenza antigens have been previously associated with protection. However, these immune responses are poorly maintained following recovery from influenza infection and are not boosted by inactivated influenza vaccines. We have previously demonstrated the safety and immunogenicity of two viral vectored vaccines, modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAdOx1 expressing conserved influenza virus antigens, nucleoprotein (NP) and matrix protein-1 (M1). We now report on the safety and long-term immunogenicity of multiple combination regimes of these vaccines in young and older adults. METHODS: We conducted a Phase I open-label, randomized, multi-center study in 49 subjects aged 18-46years and 24 subjects aged 50years or over. Following vaccination, adverse events were recorded and the kinetics of the T cell response determined at multiple time points for up to 18months. FINDINGS: Both vaccines were well tolerated. A two dose heterologous vaccination regimen significantly increased the magnitude of pre-existing T-cell responses to NP and M1 after both doses in young and older adults. The fold-increase and peak immune responses after a single MVA-NP+M1 vaccination was significantly higher compared to ChAdOx1 NP+M1. In a mixed regression model, T-cell responses over 18months were significantly higher following the two dose vaccination regimen of MVA/ChAdOx1 NP+M1. INTERPRETATION: A two dose heterologous vaccination regimen of MVA/ChAdOx1 NP+M1 was safe and immunogenic in young and older adults, offering a promising vaccination strategy for inducing long-term broadly cross-reactive protection against influenza A. FUNDING SOURCE: Medical Research Council UK, NIHR BMRC Oxford.

Nasopharyngeal Bacterial Carriage in the Conjugate Vaccine Era with a Focus on Pneumococci.

Seven-valent pneumococcal conjugate vaccine (PCV7) was included in the UK national immunisation program in 2006, and this was replaced by thirteen-valent PCV in 2010. During this time, the carriage of vaccine-type Streptococcus pneumoniae decreased but pneumococcal carriage remained stable due to increases in non-vaccine-type S. pneumoniae. Carriage studies have been undertaken in various countries to monitor vaccine-type replacement and to help predict the serotypes, which may cause invasive disease. There has been less focus on how conjugate vaccines indirectly affect colonization of other nasopharyngeal bacteria. If the nasopharynx is treated as a niche, then bacterial dynamics are accepted to occur. Alterations in these dynamics have been shown due to seasonal changes, antibiotic use, and sibling/day care interaction. It has been shown that, following PCV7 introduction, an eradication of pneumococcal vaccine types has resulted in increases in the abundance of other respiratory pathogens including Haemophilus influenzae and Staphylococcus aureus. These changes are difficult to attribute to PCV7 introduction alone and these studies do not account for further changes due to PCV13 implementation. This review aims to describe nasopharyngeal cocarriage of respiratory pathogens in the PCV era.

Evaluation of swabbing methods for estimating the prevalence of bacterial carriage in the upper respiratory tract: a cross sectional study.

OBJECTIVES: Bacterial carriage in the upper respiratory tract is usually asymptomatic but can lead to respiratory tract infection (RTI), meningitis and septicaemia. We aimed to provide a baseline measure of Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae and Neisseria meningitidis carriage within the community. Self-swabbing and healthcare professional (HCP) swabbing were compared. DESIGN: Cross-sectional study. SETTING: Individuals registered at 20 general practitioner practices within the Wessex Primary Care Research Network South West, UK. PARTICIPANTS: 10,448 individuals were invited to participate; 5394 within a self-swabbing group and 5054 within a HCP swabbing group. Self-swabbing invitees included 2405 individuals aged 0-4 years and 3349 individuals aged ≥5 years. HCP swabbing invitees included 1908 individuals aged 0-4 years and 3146 individuals aged ≥5 years. RESULTS: 1574 (15.1%) individuals participated, 1260 (23.4%, 95% CI 22.3% to 24.5%) undertaking self-swabbing and 314 (6.2%, 95% CI 5.5% to 6.9%) undertaking HCP-led swabbing. Participation was lower in young children and more deprived practice locations. Swab positivity rates were 34.8% (95% CI 32.2% to 37.4%) for self-taken nose swabs (NS), 19% (95% CI 16.8% to 21.2%) for self-taken whole mouth swabs (WMS), 25.2% (95% CI 20.4% to 30%) for nasopharyngeal swabs (NPS) and 33.4% (95% CI 28.2% to 38.6%) for HCP-taken WMS. Carriage rates of S. aureus were highest in NS (21.3%). S. pneumoniae carriage was highest in NS (11%) and NPS (7.4%). M. catarrhalis carriage was highest in HCP-taken WMS (28.8%). H. influenzae and P. aeruginosa carriage were similar between swab types. N. meningitidis was not detected in any swab. Age and recent RTI affected carriage of S. pneumoniae and H. influenzae. Participant costs were lower for self-swabbing (£41.21) versus HCP swabbing (£69.66). CONCLUSIONS: Higher participation and lower costs of self-swabbing as well as sensitivity of self-swabbing favour this method for use in large population-based respiratory carriage studies.

Experience with pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) in children and adults.

Streptococcus pneumoniae-related infections are a major cause of morbidity and mortality in people of all ages worldwide. Pneumococcal vaccine development started in 1911 with a whole cell vaccine and more recently multivalent plain polysaccharide and polysaccharide conjugate vaccines have been developed. The recent vaccines rely on capsular polysaccharide antigens to induce serotype-specific immune responses. We summarize here the presentations on pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) given during the integrated symposium organized and funded by Pfizer International Operations during the 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) 31 March to 3 April 2012, London, UK. A dramatic reduction in the incidence of invasive pneumococcal diseases (IPD) due to vaccine serotypes (VST-IPD) has been reported since the introduction of a hepta-valent pneumococcal conjugate vaccine (PCV7). An indirect (herd) effect has been demonstrated to be associated with PCV7 infant vaccination programmes, with many studies reporting reductions in VST-IPD in populations that are not eligible for PCV7 vaccination. Since 2010, a 13-valent pneumococcal conjugate vaccine (PCV13) has been introduced into national immunization programmes and results from early surveillance suggest that this vaccine also has an impact on the serotypes unique to PCV13, as well as continuing to protect against the PCV7 serotypes. Data from a passive surveillance system in Europe in 2009, for instance, showed that the highest incidence of IPD remains in those aged >65 years and in children <5 years. PCV13 has now been licensed for vaccination of adults >50 years based on safety and immunogenicity data; an efficacy trial is being conducted. Regardless of previous pneumococcal vaccination status, if the use of 23-valent polysaccharide is considered appropriate, it is recommended to give PCV13 first. Novel immunization strategies remain the only practical means to reduce significantly the remaining global mortality and morbidity due to S. pneumoniae in adults.

Sampling methods for the study of pneumococcal carriage: a systematic review.

Streptococcus pneumoniae is an important pathogen worldwide. Accurate sampling of S. pneumoniae carriage is central to surveillance studies before and following conjugate vaccination programmes to combat pneumococcal disease. Any bias introduced during sampling will affect downstream recovery and typing. Many variables exist for the method of collection and initial processing, which can make inter-laboratory or international comparisons of data complex. In February 2003, a World Health Organisation working group published a standard method for the detection of pneumococcal carriage for vaccine trials to reduce or eliminate variability. We sought to describe the variables associated with the sampling of S. pneumoniae from collection to storage in the context of the methods recommended by the WHO and those used in pneumococcal carriage studies since its publication. A search of published literature in the online PubMed database was performed on the 1st June 2012, to identify published studies that collected pneumococcal carriage isolates, conducted after the publication of the WHO standard method. After undertaking a systematic analysis of the literature, we show that a number of differences in pneumococcal sampling protocol continue to exist between studies since the WHO publication. The majority of studies sample from the nasopharynx, but the choice of swab and swab transport media is more variable between studies. At present there is insufficient experimental data that supports the optimal sensitivity of any standard method. This may have contributed to incomplete adoption of the primary stages of the WHO detection protocol, alongside pragmatic or logistical issues associated with study design. Consequently studies may not provide a true estimate of pneumococcal carriage. Optimal sampling of carriage could lead to improvements in downstream analysis and the evaluation of pneumococcal vaccine impact and extrapolation to pneumococcal disease control therefore further in depth comparisons would be of value.

Continued control of pneumococcal disease in the UK - the impact of vaccination.

Streptococcus pneumoniae, also known as the pneumococcus, is an important cause of morbidity and mortality in the developed and developing world. Pneumococcal conjugate vaccines were first introduced for routine use in the USA in 2000, although the seven-valent pneumococcal conjugate vaccine (PCV7) was not introduced into the UK's routine childhood immunization programme until September 2006. After its introduction, a marked decrease in the incidence of pneumococcal disease was observed, both in the vaccinated and unvaccinated UK populations. However, pneumococci are highly diverse and serotype prevalence is dynamic. Conversely, PCV7 targets only a limited number of capsular types, which appears to confer a limited lifespan to the observed beneficial effects. Shifts in serotype distribution have been detected for both non-invasive and invasive disease reported since PCV7 introduction, both in the UK and elsewhere. The pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV, Synflorix; GlaxoSmithKline) and 13-valent pneumococcal conjugate vaccine (PCV13, Prevenar 13; Pfizer) have been newly licensed. The potential coverage of the 10- and 13-valent conjugate vaccines has also altered alongside serotype shifts. Nonetheless, the mechanism of how PCV7 has influenced serotype shift is not clear-cut as the epidemiology of serotype prevalence is complex. Other factors also influence prevalence and incidence of pneumococcal carriage and disease, such as pneumococcal diversity, levels of antibiotic use and the presence of risk groups. Continued surveillance and identification of factors influencing serotype distribution are essential to allow rational vaccine design, implementation and continued effective control of pneumococcal disease.

Identification of novel pneumolysin alleles from paediatric carriage isolates of Streptococcus pneumoniae.

Pneumolysin (Ply) is a major virulence factor of Streptococcus pneumoniae and is produced by all known clinical isolates of pneumococci. Pneumolysin toxoids are being considered as vaccine candidates. We investigated the diversity of pneumolysin among 194 nasopharyngeal pneumococci characterized by serotyping and multilocus sequence typing (MLST). Eight Ply protein alleles were identified, four of which were novel. The 4 novel alleles varied at 10 different amino acid positions, from a total of 147, 3 of these substitutions have been previously reported in different combinations. The protein allele correlated closely with MLST. It is critical that the presence of pneumolysin variants is considered with regards to the potential use of Ply in future vaccine formulations, as variation in Ply amino acid sequence may influence the immunogenicity of vaccines based on the presence of an individual Ply allele.

Safety and immunogenicity of AS03B adjuvanted split virion versus non-adjuvanted whole virion H1N1 influenza vaccine in UK children aged 6 months-12 years: open label, randomised, parallel group, multicentre study.

OBJECTIVES: To compare the safety, reactogenicity, and immunogenicity of an adjuvanted split virion H1N1 vaccine and a non-adjuvanted whole virion vaccine used in the pandemic immunisation programme in the United Kingdom. DESIGN: Open label, randomised, parallel group, phase II study. SETTING: Five UK centres (Oxford, Southampton, Bristol, Exeter, and London). PARTICIPANTS: Children aged 6 months to less than 13 years for whom a parent or guardian had provided written informed consent and who were able to comply with study procedures were eligible. Those with laboratory confirmed pandemic H1N1 influenza or clinically diagnosed disease meriting antiviral treatment, allergy to egg or any other vaccine components, or coagulation defects, or who were severely immunocompromised or had recently received blood products were excluded. Children were grouped by age: 6 months-<3 years (younger group) and 3-<13 years (older group). Recruitment was by media advertising and direct mailing. Recruitment visits were attended by 949 participants, of whom 943 were enrolled and 937 included in the per protocol analysis. INTERVENTIONS: Participants were randomised 1:1 to receive AS03(B) (tocopherol based oil in water emulsion) adjuvanted split virion vaccine derived from egg culture or non-adjuvanted whole virion vaccine derived from cell culture. Both were given as two doses 21 days apart. Reactogenicity data were collected for one week after immunisation by diary card. Serum samples were collected at baseline and after the second dose. MAIN OUTCOME MEASURES: Primary reactogenicity end points were frequency and severity of fever, tenderness, swelling, and erythema after vaccination. Immunogenicity was measured by microneutralisation and haemagglutination inhibition assays. The primary immunogenicity objective was a comparison between vaccines of the percentage of participants showing seroconversion by the microneutralisation assay (fourfold rise to a titre of >or=1:40 from before vaccination to three weeks after the second dose). RESULTS: Seroconversion rates were higher after the adjuvanted split virion vaccine than after the whole virion vaccine, most notably in the youngest children (163 of 166 participants with paired serum samples (98.2%, 95% confidence interval 94.8% to 99.6%) v 157 of 196 (80.1%, 73.8% to 85.5%), P<0.001) in children under 3 years and 226 of 228 (99.1%, 96.9% to 99.9%) v 95.9%, 92.4% to 98.1%, P=0.03) in those over 3 years). The adjuvanted split virion vaccine was more reactogenic than the whole virion vaccine, with more frequent systemic reactions and severe local reactions in children aged over 5 years after dose one (13 (7.2%, 3.9% to 12%) v 2 (1.1%, 0.1% to 3.9%), P<0.001) and dose two (15 (8.5%, 4.8% to 13.7%) v 2 (1.1%, 0.1% to 4.1%), P<0.002) and after dose two in those under 5 years (15 (5.9%, 3.3% to 9.6%) v 0 (0.0%, 0% to 1.4%), P<0.001). Dose two of the adjuvanted split virion vaccine was more reactogenic than dose one, especially for fever >or=38 masculineC in those aged under 5 (24 (8.9%, 5.8% to 12.9%) v 57 (22.4%, 17.5% to 28.1%), P<0.001). CONCLUSIONS: In this first direct comparison of an AS03(B) adjuvanted split virion versus whole virion non-adjuvanted H1N1 vaccine, the adjuvanted vaccine, while more reactogenic, was more immunogenic and, importantly, achieved high seroconversion rates in children aged less than 3 years. This indicates the potential for improved immunogenicity of influenza vaccines in this age group. TRIAL REGISTRATION: Clinical trials.gov NCT00980850; ISRCTN89141709.

Disseminated intravascular coagulation and purpura fulminans secondary to infection.

Infection is one of the commonest causes of disseminated intravascular coagulation (DIC). DIC is a complex disorder that results from an imbalance of the pro- and anticoagulant regulatory pathways. This chapter will explain the cellular and molecular basis of the disorder and consider the rationale behind current and experimental treatment strategies.

Coagulation in severe sepsis: a central role for thrombomodulin and activated protein C.

OBJECTIVES: To review the mechanisms that cause coagulation abnormalities in sepsis, focusing on the interaction between the vascular endothelium and the circulating coagulation factors, particularly the role of the protein C pathway and thrombomodulin. DATA SOURCES/STUDY SELECTION: Published research abstracts and review articles on the experimental and clinical investigation of the pathophysiology of disseminated intravascular coagulation in sepsis. DATA EXTRACTION AND SYNTHESIS: The data provide increasing evidence that the coagulopathy seen in sepsis is a result of a complex imbalance of pro- and anticoagulant pathways. Whereas previous research has largely studied events in the plasma, it is now apparent that reactions on cell surfaces such as the vascular endothelium are important in the control of the regulatory pathways. CONCLUSIONS: The plasma components of the protein C pathway are down-regulated in sepsis. Decreased thrombomodulin expression may cause defective function of the endothelial component of this pathway in septic patients. Treatments must be designed to overcome any functional defect.

Pathophysiology of meningococcal meningitis and septicaemia.

Neisseria meningitidis is remarkable for the diversity of interactions that the bacterium has with the human host, ranging from asymptomatic nasopharyngeal colonisation affecting virtually all members of the population; through focal infections of the meninges, joints, or eye; to the devastating and often fatal syndrome of meningococcal septic shock and purpura fulminans.

Dysfunction of endothelial protein C activation in severe meningococcal sepsis.

BACKGROUND: Impairment of the protein C anticoagulation pathway is critical to the thrombosis associated with sepsis and to the development of purpura fulminans in meningococcemia. We studied the expression of thrombomodulin and the endothelial protein C receptor in the dermal microvasculature of children with severe meningococcemia and purpuric or petechial lesions. METHODS: We assessed the integrity of the endothelium and the expression of thrombomodulin and the endothelial protein C receptor in biopsy specimens of purpuric lesions from 21 children with meningococcal sepsis (median age, 41 months), as compared with control skin-biopsy specimens. RESULTS: The expression of endothelial thrombomodulin and of the endothelial protein C receptor was lower in the patients with meningococcal sepsis than in the controls, both in vessels with thrombosis and in vessels without thrombosis. On electron microscopical examination, the endothelial cells were generally intact in both thrombosed and nonthrombosed vessels. Plasma thrombomodulin levels in the children with meningococcal sepsis (median, 6.4 ng per liter) were higher than those in the controls (median, 3.6 ng per liter; P=0.002). Plasma levels, protein C antigen, protein S antigen, and antithrombin antigen were lower than those in the controls. In two patients treated with unactivated protein C concentrate, activated protein C was undetectable at the time of admission, and plasma levels remained low. CONCLUSIONS: In severe meningococcal sepsis, protein C activation is impaired, a finding consistent with down-regulation of the endothelial thrombomodulin-endothelial protein C receptor pathway.

Analysis of pathogen-host cell interactions in purpura fulminans: expression of capsule, type IV pili, and PorA by Neisseria meningitidis in vivo.

The pattern of meningococcal surface structure expression in different microenvironments following bloodstream invasion in vivo is not known. We used immunohistochemistry to determine the expression of capsule, type IV pili, and PorA by meningococci residing in the skin lesions of children with purpura fulminans. All the skin biopsy samples showed evidence of thrombosis and, frequently, a perivascular inflammatory cell infiltrate consisting of neutrophils (elastase positive) and monocytes/macrophages (CD68 positive). Modified Gram staining revealed 20 to over 100 gram-negative diplococci in each 4-microm-thick section, usually grouped into microcolonies. Immunoperoxidase staining demonstrated that the invading meningococci expressed PorA, capsule, and type IV pilin. Expression of these antigens was not restricted to any particular environment and was found in association with meningococci located in leukocytes, small blood vessels, and the dermal interstitium. Confocal laser scanning microscopy demonstrated coexpression of pilin and capsule by numerous microcolonies. However, there was some discordance in capsule and pilin expression within the microcolonies, suggesting phase variation. The strategy employed in this study will be helpful in investigating invasive bacterial diseases where antigenic and phase variation has a significant impact on virulence and on vaccine design.