Severity of Meningococcal Disease in Children and the Angiotensin-Converting Enzyme Insertion/Deletion Polymorphism

David Harding, Paul B. Baines, David Brull, Vassilis Vassiliou, Ian Ellis, Anthony Hart, Alistair P. J. Thomson, Steve E. Humphries, and Hugh E. Montgomery

Peter Dunn Neonatal Intensive Care Unit and Department of Child Health, University of Bristol, Bristol; Paediatric Intensive Care Unit, Royal Liverpool Children's Hospital, Liverpool; Centre for Cardiovascular Genetics, Department of Medicine, University College London Medical School, London; Institute of Child Health, Royal Liverpool Children's Hospital, Liverpool; and University Department of Medical Microbiology, University of Liverpool, Liverpool, United Kingdom

	ABSTRACT

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Critical illness outcome may be causally related to inflammatory response severity. Given that tissue angiotensin-converting-enzyme (ACE) regulates such responses and that the deletion (D) [rather than insertion (I)] variant of the ACE gene is associated with higher tissue ACE levels, DD genotype might be associated with a poorer outcome in a uniform infectious disease state. Illness severity (Pediatric RIsk of Mortality score, the Glasgow Meningococcal Septicaemia Prognostic Score [GMSPS], and clinical course) was recorded for consecutive white patients with meningococcal disease (n = 110, 34 DD genotype, 61 male, aged 49.4 ± 5.4 months) referred to the Royal Liverpool Children's Hospital, UK. Compared with children with  I allele, DD genotype was associated with 14% higher predicted risk of mortality (p = 0.038), higher GMSPS (DD 9.4 ± 0.5, ID/II 7.7± 0.4 [mean ± SEM], p = 0.013), greater prevalence of inotropic support (76% versus 55%, p = 0.03) and ventilation (82% versus 63%, p = 0.04), and longer Pediatric Intensive Care Unit (PICU) stay (5.8 versus 3.9, p = 0.02). DD genotype frequency was 6% (1 case) for the 18 children who did not require PICU care, 33% for the 84 PICU survivors, and 45% for those who died (p = 0.01). ACE DD is associated with increased illness severity in meningococcal disease.

	INTRODUCTION

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Keywords: ACE; polymorphism; Meningococcus

Increased activity in human tissue renin-angiotensin systems (RAS) (1, 2) including white blood cells (WBC) (3) drives a tissue proinflammatory response (4-8), whose magnitude seems causally related to poorer outcome in critical illness such as meningococcal infection (9-11). The absence (Dallele) rather than presence (I allele) of a 284 base-pair marker in the human ACE gene is associated with higher circulating ACE activity (12), and DD genotype with raised tissue ACE activity (1) (+75% in WBCs) (13). If increasing RAS activity is associated with an exaggerated inflammatory response, itself causally related to poorer outcome, then DD genotype might be associated with impaired critical illness outcome. We have explored this hypothesis.

	METHODS

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Hospital ethics committee approval was granted. Informed consent was obtained from each subject's legal guardian.

Subjects and Controls

Subjects comprised all cases of meningococcal disease admitted to the Pediatric Intensive Care Unit (PICU) of The Royal Liverpool Children's Hospital (Liverpool, UK) between March 1995 and January 1998, and those managed on its pediatric wards. All were managed using standardized protocols, with prospective data collection from admission by one trained observer (P.B.) (14). Control genotypes were determined in 841 UK healthy white children born between April 1991 and December 1992 (15, 16).

Meningococcal sepsis (with or without evidence of meningitis, below) was diagnosed by standard clinical criteria (10, 14, 17, 18) including evidence of infection (e.g., fever, raised WBC count) and purpuric rash, and absence of proven alternative infective etiology. Microbiologic confirmation was sought using blood cultures, throat swab, and meningococcal antigen analysis, and (from October 1996) polymerase chain reaction for meningococcal DNA (19).

Meningococcal meningitis was diagnosed by clinical features together with lumbar puncture (> 5 × 10⁶ polymorphonuclear cells/ml, glucose concentration  50% that of blood), and evidence of Neisseria meningitidis in cerebrospinal fluid or blood.

Illness Severity

This was quantified by Pediatric RIsk of Mortality (PRISM) (20) (previously-validated; see Refs. 21-23) and Glasgow Meningococcal Septicaemia Prognostic (GMSPS) Scoring (21) on PICU admission. Need for mechanical ventilatory support, inotropic support within 48 hours of admission, number of PICU treatment days, and survival to hospital discharge were also recorded. GMSPS and age-adjusted default PRISM score were calculated for patients not admitted to the PICU.

The GMSP score is a score derived from binary assessment of the presence or absence of set criteria. Although no other reported measures of disease severity directly constitutes a part of the GMSPS, it should be noted that some components of the GMSPS (such as presence of coma, perceived deterioration over 1 hour, hypotension, and base deficit) may relate indirectly to other recorded variables (e.g., need for ventilation or inotropes).

Genotyping

DNA was extracted and ACE genotype determined by staff blind to clinical data, using 3-primer polymerase chain reaction amplification from 1-ml samples in EDTA or Lithium heparin tubes, as previously described (24).

Statistical Analysis

Analysis used SPSS (v9.0; SPSS Inc., Chicago, IL). As tissue ACE is elevated only amongst those of DD genotype (1, 13), data were compared between DD and ID+II genotypes. Categorical data were analyzed by chi square, and continuous data by Student's t and Mann- Whitney U tests where appropriate.

	RESULTS

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Of 142 consecutive cases referred to the PICU, parental consent for genotyping was granted for 113, each of whose genotype was recorded. Factors such as parental consent are unlikely to be genotype-dependent, and lack of such bias is supported by a similar genotype distribution to that reported in other European samples and our controls. Further, clinical and demographic data did not differ between those included and those for whom no genotype was available.

110 subjects were white, whose ACE genotype distribution (25 [23%] of II, 51 [46%] of ID, and 34 [31%] of DD genotype) demonstrated Hardy-Weinberg Equilibrium and did not differ from controls (203 [24.1%] DD, 433 [51.5%] ID, 205 [24.4%] II). Demographic data (61 [55%] male, aged 49.4 ± 5.4 months, 60 [55%] referred from other hospitals; 55 [50%] with meningococcal septicemia, 6 [5%] with meningitis alone, and 49 [45%] with both; 70 [64%] with microbiologic confirmation) did not differ from the 32 excluded, and were independent of genotype. All 18 managed on medical wards had a microbiologically confirmed diagnosis, of which 16 had age-adjusted PRISM score and GMSPS calculated from one data set on referral. Meanwhile, GMSPS was determined in all 92 (including one who died in the accident and emergency department) warranting PICU admission, and PRISM score in 91.

Data for those of ID and II genotypes did not differ. When compared with those with  I allele (Table 1), those of DD genotype had higher predicted risk of mortality (p = 0.01); worse GMSP Score (p = 0.014); more frequent GMSP Score of  8 (p = 0.01), need for inotropes (p = 0.034) and ventilation (p = 0.044); longer PICU stay (p = 0.021); and (nonsignificantly) decreased ratio between arterial partial pressure of oxygen and inspired oxygen fraction (PaO2/FiO2; p = 0.09) (Table 1). Of those not requiring PICU care, 1 (6%) was of DD genotype. Of PICU survivors, 33% (28 of 84) were of DD genotype, whereas 45% (5 of 11) of those who died were of DD genotype (p = 0.013; Figure 1).

View this table: [in this window] [in a new window]   TABLE 1  MARKERS OF DISEASE SEVERITY AND OUTCOME AMONG 110 WHITE CHILDREN WITH MENINGOCOCCAL DISEASE

View larger version (31K): [in this window] [in a new window]   Figure 1.   Frequency of the ACE DD genotype related to clinical course amongst 110 white children with meningococcal disease. "No PICU" refers to those considered too well to warrant admission to PICU. The relationship of DD genotype to clinical course was statistically significant (p = 0.013; see text for details).

	DISCUSSION

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Angiotensin-converting enzyme DD genotype is associated with worse illness severity and clinical course in children with meningococcal disease. This is not confounded by genetic association with clinical presentation nor risk of contracting meningococcal disease (given similar genotype distributions and D-allele frequency amongst those with meningitis, septicemia, or both, and between patients and controls). If some cases were not referred for PICU assessment, this would only have impacted on our study if such failure were genotype-dependent. The similar genotype/allele frequencies in management groups and in controls makes this unlikely. Nor would such referral bias affect the genotype dependence of outcome amongst those who were so referred.

Mortality was nearly 2-fold higher (15% versus 8%, p = 0.27) amongst DD genotype versus II+ID groups. A larger sample size may be required to demonstrate genotype dependence, given that mortality is low overall (10%). That this is so is supported by the fact that predicted mortality was also doubled in those of DD genotype (30% versus 16%; p = 0.01), and that DD genotype is associated with diverse measures of illness severity normally associated with mortality. Finally, lack of absolute microbiologic proof of meningococcal infection in all subjects (leading to possible heterogeneity of the inflammatory stimulus) would be expected only to have weakened the DD genotype association identified.

The influence of single-gene polymorphisms depends upon their genetic (and hence racial) context. It is thus important in small candidate gene-association studies such as this to restrict primary analysis to those of one race. Secondary analysis may be performed to attempt to implicate findings to mixed populations. In fact, only 3 of the 113 cases genotyped (one ID, two II) were not white (1 of Bangladeshi, 1 of Pakistani, and 1 of west African origin), and inclusion of these three in no way alters the strength of any of the associations reported. However, such minor racial diversity does not allow extension of these observations to other racial groups, in whom confirmatory data should be sought.

Among those admitted to hospital, ACE genotype is associated with disease severity. However, it might be suggested that admission is itself dependent on genotypeeither because those of DD genotype die before referral or because those of II genotype are not sufficiently clinically unwell to be detected. Such admission bias seems unlikely, however, given that genotype distribution is in Hardy-Weinberg equilibrium, is similar to that previously reported in other UK adult and pediatric population samples, and is similar to that in our control sample. That the association of DD genotype with clinical outcome is not mediated through an association with clinical presentation is further suggested by the similar genotype distributions among those in each clinical category (meningitis, septicemia, or both) and similar distributions of disease state across genotype classes. However, it would perhaps not be surprising if meningococcal disease were more readily identified (in milder cases) among those of DD genotype, nor if a more florid meningococcal pattern of disease might be evident in such patients. Such issues can only be addressed in larger studies.

Although the higher DD genotype frequency occurs to some degree at the expense of the ID genotype (which falls from 77.7% to 40.5% and thence to 36.4% among those not needing PICU care, those admitted to PICU and surviving, and those who died, respectively), there remains a real rise in frequency of the D allele itself (being 44%, 54%, and 63% in each of the groups, respectively).

Inflammatory response severity is associated with poorer outcome in meningococcal disease. Both may be influenced by polymorphic variation in cytokine genes (9-11) such as those for IL-10 (9), TNF- (9, 10), and IL-1 (11), and ACE genotype may similarly exert its effects through inflammatory modulation. Increased ACE expression seen with monocyte activation/maturation (3) drives autocrine cytokine synthesis (5) and paracrine tissue inflammatory responses (4, 6, 7, 25). Inflammation itself stimulates ACE expression in adjacent cells (26), driving monocyte recruitment in a positive feedback loop (4, 27, 28). Thus, local ACE is proinflammatory in diverse tissues including brain (8), kidney (25, 29), lung (30), vasculature (31-33), heart (34), and eye (35). ACE may also influence the course of meningococcal disease, however, through other aspects of immune (36, 37), metabolic (38-43), or endocrine responses in critical illness (44).

Further studies are evidently required to confirm these observations, to explore the mechanisms underlying them, and to extend them to other forms of critical and infectious disease. Nonetheless, these data may have implications for clinical medical practice. ACE genotype may contribute to the risk stratification of patients and to the targeting of appropriate care. In addition, the way may ultimately be paved for the potential novel use of ACE inhibitors in the management of such patients. Indeed, it is already recognized that a (plaque) anti-inflammatory action may underlie the reduced rate of stroke and myocardial infarction associated with ACE inhibition (45), the therapeutic benefits of ACE inhibition in the treatment of scleroderma crisis (46), and the similarly improved outcome in patients undergoing cardiac surgery (47). The wider use of ACE inhibitors as anti-inflammatory agents amongst the critically ill may yet be seen.

	Footnotes

Correspondence and requests for reprints should be addressed to Hugh Montgomery, M.D., Centre for Cardiovascular Genetics, Department of Medicine, University College London Medical School, Rayne Bldg., 5 University St., London WC1E 6JJ, UK. E-mail: h.montgomery{at}ucl.ac.uk

(Received in original form August 21, 2001 and accepted in revised form January 18, 2002).

Acknowledgments: The authors thank the staff of the PICU at the Royal Liverpool Children's Hospital, J. Golding, M. Pembrey, and The ALSPAC Team.

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