Simone Naylor

Severity of Meningococcal Disease Associated with Genomic Bacterial Load

BACKGROUND Diagnostic polymerase chain reaction (PCR) detection of Neisseria meningitidis has enabled accurate quantification of the bacterial load in patients with meningococcal disease. METHODS Quantification of the N. meningitidis DNA level by real time-PCR was conducted on whole-blood samples obtained from patients presenting with meningococcal disease to hospitals throughout England and Wales over a 3-year period. Levels were correlated with clinical outcome, infecting serogroup, and host factors including, interleukin-1 genotype (IL-1). RESULTS Bacterial loads were available for 1045 patients and were not associated with the age of the patient, delay in sample submission, or administration of antibiotics prior to admission. The median log bacterial load was higher in 95 patients who died (5.29 log(10)copies/mL; interquartile range, 4.41-6.30 log(10)copies/mL) than in 950 patients who survived (3.79 log(10)copies/mL; interquartile range, 2.87-4.71 log(10)copies/mL). Logistic regression revealed that age (odds ratio, 1.04 per 1-year increase in age) and bacterial load (odds ratio, 2.04 per log(10)-copies/mL increase) had a statistically significant effect on the risk of death. Infection with N. meningitidis serogroup C was associated with increased risk of death and an increased bacterial load. Also associated with a higher bacterial load were prolonged hospitalization (duration, >10 days); digit, limb, or soft-tissue loss; and requirement of hemodialysis. Carriage of IL-1RN(+2018) was associated with increased mortality (odds ratio, 2.14; P=.07) but not with a higher bacterial load. CONCLUSIONS In meningococcal disease, bacterial load is associated with likelihood of death, development of permanent disease sequelae, and prolonged hospitalization. The bacterial load was relatively higher in patients infected with N. meningitidis serogroup C than in those infected with other serogroups. The effects of age and IL-1 genotype on mortality are independent of a high genomic bacterial load.

Variation within Genes Encoding Interleukin-1 and the Interleukin-1 Receptor Antagonist Influence the Severity of Meningococcal Disease

Context Prognoses of patients with meningococcal disease vary widely. Patients with severe disease may have unusually high levels of proinflammatory cytokines, such as interleukin-1. The release of such cytokines may be genetically determined. Contribution This genotype study of 1106 British patients with meningococcal disease found that carrying the common allele of one of the interleukin-1 genes (IL1B) was associated with increased likelihood of survival. Among patients carrying this allele, those who also carried the rare allele of another interleukin-1 gene (IL1RN) had a decreased likelihood of survival. Implications Genotype at the interleukin-1 gene cluster influences likelihood of survival from meningococcal disease. The Editors In the sepsis syndrome, patients with severe manifestations exhibit maladaptive proinflammatory cytokine release with unusually high plasma concentrations of cytokines (1). Meningococcal disease is an example of gram-negative sepsis in which the prognosis varies widely among patients. The mortality rate of meningococcal disease ranges from 5% to 20%, depending on whether patients present with predominant features of meningitis or septicemia (2); in patients with purpura fulminans, the mortality rate can be as high as 50% (3). The host cytokine response in meningococcal disease is genetically determined (4); lipopolysaccharide-stimulated peripheral monocytes derived from first-degree relatives of patients who have died of meningococcal disease exhibit low tumor necrosis factor (TNF) and high interleukin (IL)-10 release compared with monocytes derived from first-degree relatives of survivors. Interleukin-1, together with TNF, is a central, early, and highly inflammatory mediator of the innate response to bacterial challenge (5). Unusually high levels of IL-1 and its endogenous antagonist IL-1 receptor antagonist (IL-1ra) can be detected in patients with meningococcal disease, particularly those with severe manifestations (6). The IL-1 gene cluster on chromosome 2q contains three related genes within a 430-kilobase region (IL1A, IL1B, and IL1RN), which encode IL-1, IL-1, and IL-1ra (Figure 1) (7, 8). A single nucleotide polymorphism (SNP) is present within IL1B 511 nucleotides upstream of the transcriptional start site (IL1B[511]), which is in 99.5% linkage disequilibrium with another at position (31). This SNP at (31) affects the transcription-initiation complex of IL1B (9, 10). Also, the IL1RN gene contains a variable-number tandem repeat (VNTR) (denoted as IL1RN*2), which is in linkage disequilibrium with several polymorphisms in IL1RN, including one at position +2018 within exon 2 (11, 12) (Glossary). In the general white population, the frequency of the rarer alleles of the SNPs is 33% at IL1B(511) and 28% at IL1RN(+2018) (9, 11). In a previous study that we conducted, a relatively small number of patients with meningococcal disease had genotyping at various loci across the IL-1 gene cluster (13). We showed that the genotype at IL1B(511) had some association with likelihood of death from meningococcal disease; IL1RN(+2018) possibly contributed to the effect. We now examine DNA derived from a separate and much larger group of patients with microbiologically confirmed meningococcal disease to retest the association with these two SNPs. Figure 1. The interleukin-1 ( IL-1 ) gene cluster. kb IL1A IL1B IL1RN IL1B IL1B IL1RN VNTR Methods Patients The Meningococcal Reference Unit (MRU) for England and Wales offers a service for polymerase chain reaction (PCR) detection of Neisseria meningitidis in blood and cerebrospinal fluid (CSF) samples from patients with suspected meningococcal disease in addition to culture confirmation of disease. From July 1998 to November 1999, we archived all whole blood samples from patients who were subsequently confirmed to have meningococcal disease, either by culture or by PCR detection of N. meningitidis in blood or CSF. Demographic and clinical information relating to the samples was obtained from the submitting clinical team and confirmed by the Consultant in Communicable Disease Control of the relevant District Health Authority. To estimate socioeconomic status of patients, each was assigned a Townsend deprivation score on the basis of his or her residential post code, if available. This score is derived from four variables measured in the 1991 United Kingdom National Census: proportion of economically active residents unemployed, households without a car, households not owner occupied, and households overcrowded (14). Each enumeration district typically comprises a neighborhood of 200 to 400 people. A Townsend deprivation score of high positive indicates low economic status, and a negative score indicates high economic status. After clinical information had been collated, we coded samples so that patients could no longer be identified. DNA from 839 northern English blood donors (age range, 18 to 65 years) was used as a noninfected control sample. Blood donors in the United Kingdom are unpaid volunteers. The Ethics Committees of the Public Health Laboratory Service for England and Wales and of the South Sheffield Health District reviewed the study. The final protocols approved by the committees were adhered to throughout the study. Laboratory Methods DNA was extracted from whole blood samples by standard methods. We probed for two SNPs: IL1B(511) and IL1RN(+2018) using a validated 5 nuclease assay (TaqMan probe) allelic discrimination test (Applied Biosystems, Foster City, California). Probes, primer sequences, and cycling conditions have been previously published (12). We indicate the frequent allele of each gene variant as 1 and the rarer allele as 2, as is standard nomenclature. Homozygosity is indicated as 1/1 or 2/2, and heterozygosity is indicated as 1/2. Carriage of one particular allele is indicated, for example, as 2+ for the rarer allele, meaning that patients were either 2/2 or 1/2; lack of carriage is denoted as 1 or 2. Statistical Analysis The chi-square test was used for preliminary analysis of carriage of a specific allele, unless otherwise stated. Logistic regression analysis was used to elucidate interactions between genotypes, age, infecting serogroup, and Townsend deprivation score. Stata software, version 6 (Stata Corp., College Station, Texas), was used for statistical analyses. Role of the Funding Source The funding source had no role in the design, conduct, or reporting of the study or in the decision to submit the manuscript for publication. Results Patients and Microbiological and Demographic Data During the study period, 4620 cases of meningococcal disease occurring throughout England and Wales were notified to the Office for National Statistics on the basis of clinical or laboratory diagnosis; 3502 cases were verified by the MRU by culture or PCR detection of N. meningitidis in blood or CSF (Figure 2). During the same period, 2807 whole blood samples were received by the MRU for PCR detection of N. meningitidis because of suspected meningococcal infection. Among this group, we identified 1106 patients with meningococcal disease confirmed by culture or PCR (39% confirmed by culture, 25% by PCR, and 36% by both culture and PCR). Of the DNA samples, 91 were from patients who died and 1015 were from patients who survived. The mean (SD) age of patients who died (22.2 2.5 years; median, 15 years [range, 0 to 82 years]) was significantly higher than that of patients who survived (12.2 0.5 years; median, 6 years [range, 0 to 84 years]) (P = 0.002; MannWhitney U test) (Table 1). Children between 4 and 11 years of age made up 18.2% of all cases but only 6.6% of all deaths. In contrast, 3.5% of cases were in adults between 60 and 84 years of age, but this group made up 12.1% of all deaths within this cohort. Figure 2. The Meningococcal Reference Unit confirms the identity of all Neisseria meningitidis isolates recovered from blood and cerebrospinal fluid at hospitals throughout England and Wales and also receives blood samples for polymerase chain reaction ( PCR N. meningitidis. N. meningitidis Table 1. Characteristics of Patients Who Survived and Those Who Died Postal codes were available for 729 patients who survived and 75 patients who died. These were used to calculate a Townsend deprivation score for each patient (Table 1), which revealed a wide range of patient socioeconomic status. Table 1 also shows the serogroups of N. meningitidis causing disease in this cohort. As expected for a sample collected in England and Wales during this period, most patients in whom a serogroup was identified were infected with serogroup B; most of the remaining patients were infected with serogroup C. Among patients who died, 52% were infected with serogroup C; in contrast, 33% of survivors were infected with this serogroup. Effect of Interleukin Genotype on Likelihood of Infection Table 2 shows the genotypes of patients with meningococcal disease who died and those who survived, as well as genotypes of blood donor controls. Most of the whole blood samples were successfully genotyped for both markers. Table 2. Genotype Distribution forIL1B(511) andIL1RN(+2018) Chi-square analysis indicated a significant overall relationship between the distributions of both IL1B(511) and IL1RN(+2018) among patients who survived, patients who died, and blood donor controls (Table 2). However, when the genotypes of all patients with meningococcal disease (patients who survived and died combined) were compared with the genotypes of blood donor controls, the representation of IL1B(511) (P = 0.11) and of IL1RN(+2018) (P = 0.15) did not differ. This suggested that these polymorphisms are equally represented in the general population and in patients with meningococcal disease and that they do not influence the likelihood of disease. In contrast, preliminary analysis suggested some effect at both markers on likelihood of death among patients with meningococcal disease. E

Genetic Polymorphism of the Binding Domain of Surfactant Protein-A2 Increases Susceptibility to Meningococcal Disease

BACKGROUND Meningococcal disease occurs after colonization of the nasopharynx with Neisseria meningitidis. Surfactant protein (SP)-A and SP-D are pattern-recognition molecules of the respiratory tract that activate inflammatory and phagocytic defences after binding to microbial sugars. Variation in the genes of the surfactant proteins affects the expression and function of these molecules. METHODS Allele frequencies of SP-A1, SP-A2, and SP-D were determined by polymerase chain reaction in 303 patients with microbiologically proven meningococcal disease, including 18 patients who died, and 222 healthy control subjects. RESULTS Homozygosity of allele 1A1 of SP-A2 increased the risk of meningococcal disease (odds ratio [OR], 7.4; 95% confidence interval [CI], 1.3-42.4); carriage of 1A5 reduced the risk (OR, 0.3; 95% CI, 0.1-0.97). An analysis of the multiple single-nucleotide polymorphisms in SP-A demonstrated that homozygosity for alleles encoding lysine (in 1A1) rather than glutamine (in 1A5) at amino acid 223 in the carbohydrate recognition domain was associated with an increased risk of meningococcal disease (OR, 6.7; 95% CI, 1.4-31.5). Carriage of alleles encoding lysine at residue 223 was found in 61% of patients who died, compared with 35% of those who survived (OR adjusted for age, 2.9; 95% CI, 1.1-7.7). Genetic variation of SP-A1 and SP-D was not associated with meningococcal disease. CONCLUSIONS Gene polymorphism resulting in the substitution of glutamine with lysine at residue 223 in the carbohydrate recognition domain of SP-A2 increases susceptibility to meningococcal disease, as well as the risk of death.

The tumor necrosis factor polymorphism TNF (-308) is associated with susceptibility to meningococcal sepsis, but not with lethality.

Objective: To determine whether the promoter polymorphism tumor necrosis factor (TNF) (−308) is associated with susceptibility to or death from meningococcal sepsis. Design, Setting, Patients, and Participants: Association study involving 1321 patients with microbiologically proven invasive meningococcal disease presenting to hospitals throughout United Kingdom during 1998–2001, among whom 134 died. Controls were derived from 1280 northern English blood donors. Measurements: DNA from patients and controls was genotyped at TNF (−308). After analysis, DNA was subsequently genotyped at eight other markers in strong linkage disequilibrium with TNF (−308); these markers were I&kgr;BL (−62), BAT3, LST1, NOTCH4 (+1297), NOTCH4 (+3061), CCHCR1 (+436), CCHCR1 (+2271), and LT&agr;. To confirm functional relevance of TNF (−308) in the context of meningococcal disease, TNF secretion by, and TNF messenger RNA expression of macrophages derived from volunteers with known TNF (−308) genotype after exposure to Neisseria meningitidis were measured. Main Results: Among cases of meningococcal disease, likelihood of death was shown to be influenced by the age of the affected individual and also with the infecting serogroup, but was not influenced by genotype at TNF (−308) or the other linked markers. However, patients with meningococcal disease, irrespective of whether they died, were more likely to be homozygous for the rare allele at TNF (−308) (odds ratio = 1.93, 95% confidence interval 1.08–3.46), and less likely to be heterozygous for this marker (odds ratio = 0.79, 95% confidence interval 0.64–0.97), compared with the control cohort. There was no association of susceptibility to disease with the other markers studied. Macrophages derived from volunteers homozygous for the rare allele at TNF (−308) expressed higher levels of TNF messenger RNA and secreted higher concentrations of TNF compared with common homozygotes after exposure to N. meningitidis. Conclusions: Genotype at TNF (−308) modifies cellular TNF secretion in response to N. meningitidis and may influence susceptibility to meningococcal disease, but does not influence the likelihood of death after infection.

Serum Antibody Kinetics following Nasal or Parenteral Challenge with Meningococcal Polysaccharide in Healthy Adults

ABSTRACT Limited data are available on the kinetics of meningococcal serogroup C (MenC)-specific antibody responses following parenteral or nasal challenge in those who have received prior MenC vaccination (polysaccharide or conjugate). Young adults who had previously received either meningococcal A/C polysaccharide (MACP) or MenC conjugate (MCC) vaccine or naïve subjects were challenged with MACP via one of two routes, nasal or parenteral. Blood samples were taken prevaccination and on days 1 to 4 and day 10 postvaccination. MenC serum bactericidal antibody (SBA) and MenC-specific IgG were measured. Following parenteral challenge, MenC SBA and IgG responses were seen to occur between 4 and 7 days postchallenge. A lower proportion of subjects responded following nasal challenge, with naïve subjects showing little change in SBA geometric mean titer (GMT) and IgG geometric mean concentration (GMC) over the 10 days following challenge. Increases in SBA GMTs were seen between 4 and 7 days after nasal challenge in those who had received prior MCC and between 7 and 10 days in those who had received prior MACP, and the responses in the prior-MACP group were of lower magnitude than the responses of the prior-MCC group. The data presented here indicate that, following MCC vaccination, memory has been induced at the mucosal level, and these subjects were able to respond with increases in SBA levels. These results demonstrate that the speed of response (primary or secondary) to challenge with MenC polysaccharide via the nasal or parenteral route does not differ and support concerns that immunological memory alone is too slow to provide protection.

HIV control in postpartum mothers: a turbulent time.

We conducted an audit looking at the management of HIV-positive women in the postpartum period. We found that of the women with a previous AIDS-defining condition or a CD4 count <350 cells/µL, 83% were correctly continued on antiretroviral therapy (ART) and 84.1% of these had good virological control. ART was correctly stopped in 100% of women who had always had a CD4 count >500 cells/µL. A significant finding from our audit was that all of the women who had poor virological control or stopped ART against medical advice had social issues or self-reported depression. The main recommendation was to extend the pregnancy multidisciplinary team (MDT) meeting to include the 12-month postpartum period to offer support to women to try to improve treatment outcomes.

Bacterial Genomic Detection Within Cerebrospinal Fluid of Patients With Meningococcal Disease Is Influenced by Microbial and Host Characteristics

Among 384 patients with confirmed meningococcal disease, the likelihood of detecting Neisseria meningitidis DNA in cerebrospinal fluid (CSF) increased with age, serogroup B infection, and prehospitalization antibiotic treatment. Plasma and CSF genomic bacterial loads of non-B N. meningitidis serogroups correlated significantly. Serogroup B-infected patients with genotype TNF2 (-308A) had significantly higher CSF bacterial loads.

How does specialist nursing contribute to HIV service delivery across England

This study aimed to examine what specialist nursing contributes to HIV service delivery across England and how it could be optimised. A three part multi-method qualitative study was undertaken, involving (1) interviews with 19 stakeholders representing professional or service user groups; (2) interviews with nurse/physician pairs from 21 HIV services; and (3) case studies involving site visits to five services. A framework analysis approach was used to manage and analyse the data. There was substantial variability in specialist nursing roles and the extent of role development. Most hospital-based HIV nurses (13/19) were running nurse-led clinics, primarily for stable patients with almost half (6/13) also managing more complex patients. Role development was supported by non-medical prescribing, a robust governance framework and appropriate workload allocation. The availability and organisation of community HIV nursing provision determined how services supported vulnerable patients to keep them engaged in care. Four service models were identified. The study showed that there is scope for providing a greater proportion of routine care through nurse-led clinics. HIV community nursing can influence health outcomes for vulnerable patients, but provision is variable. With limited financial resources, services may need to decide how to deploy their specialist nurses for best effect.

A workforce in jeopardy - identifying the challenges of ensuring a sustainable advanced HIV nursing workforce.

Background HIV services in England face substantial challenges arising from financial pressures and changes to commissioning. A sustainable HIV specialist nursing workforce will be vital to enable them to respond to those challenges. Aims This paper examines the current workforce situation in HIV services across the country. Methods This mixed-method study involved semi-structured interviews with 19 key stakeholders and with 44 nurses/physicians from 21 purposively selected HIV services across England. Data were interpreted using a framework analysis approach. Results ‘Building a career in HIV nursing’ identified problems associated with retention and recruitment. Changes in commissioning are disrupting common career routes from sexual health to HIV nursing, and a perceived lack of a clear career pathway was seen as a barrier to recruitment. ‘Developing a specialist workforce’ explored the professional development of the current workforce, which was hampered by poor access to funding or study time for advanced study and the absence of an HIV-specific advanced nursing qualification. Conclusions The HIV nursing workforce, which provides an increasing proportion of HIV care, is facing serious recruitment and retention challenges. A strategic approach to workforce development and training is essential to overcome systemic barriers and secure the next generation of skilled practitioners.