articleA prospective study of the sensitivity , specificity and diagnostic performance of soluble intercellular adhesion molecule 1 , highly sensitive C-reactive protein , soluble E-selectin and serum amyloid A in the diagnosis of neonatal infection

Background: Diagnosis of neonatal infection is difficult, because of it's non-specific clinical presentation and the lack of reliable diagnostic tests. The purpose of this study was to examine the potential diagnostic value of serum soluble intercellular adhesion molecule-1 (sICAM-1), soluble E-selectin (sE-selectin), highly sensitive C-reactive protein (hsCRP) and serum amyloid A (SAA) measurements, both individually and in combination in the setting of a neonatal intensive care unit. Methods: 219 consecutive serum samples were taken from 149 infants undergoing sepsis work up in a neonatal intensive care unit. Clinical diagnosis was established in a prospective manner, blind to the results of the study measurements. Infants were classified by an experienced paediatrician as infected or not-infected, one week after presentation. Classification was based on clinical presentation, routine laboratory and radiological investigations and response to therapy. The infected group were sub-classified as (a) culture positive infection or (b) culture negative infection. sICAM-1, sE-selectin, hsCRP and SAA levels were determined from stored serum samples after diagnosis was established. Further sub-group analysis of results was undertaken according to early or late onset of infection and preterm or term status. Statistical analysis utilised Mann Whitney U test and ROC curve analysis. Results: There were significantly increased serum levels of sICAM-1, hsCRP, E selectin (p < 0.001) and SAA (p = 0.004) in infected infants compared with non-infected. ROC curve analysis indicated area under the curve values of 0.79 (sICAM1), 0.73 (hsCRP), 0.72 (sE-selectin) and 0.61 (SAA). ROC curve analysis also defined optimum diagnostic cut-off levels for each measurement. The performance characteristics of sICAM-1, hsCRP and sE-selectin included a high negative predictive value (NPV) for culture positive infection and this was enhanced by combination of all 4 measurements. Clinical subgroup analysis suggested particularly high NPV for early onset symptoms, however further studies are required to elucidate this finding. Conclusions: All four study measurements demonstrated some diagnostic value for neonatal infection however sICAM-1, hsCRP and sE-selectin demonstrated the highest NPV individually. The optimum diagnostic cut off level for hsCRP measurement in this study was much lower than currently used in routine clinical practice. Use of a combination of measurements enhanced diagnostic performance, demonstrating sensitivity of 90.3% and NPV of 91.3%. This study suggests there may be value in use of several of these markers, individually and in combination to assist in excluding neonatal infection. Further work is needed to confirm a specific role in the exclusion of early onset infection. BioMed Central © 2010 Edgar et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Edgar et al. BMC Pediatrics 2010, 10:22 http://www.biomedcentral. com/1471-2431/10/22 Page 2 of 16 Background The diagnosis of infection in preterm infants is difficult, both because of the non-specific clinical presentation and the lack of reliable diagnostic tests [1]. As a result of this uncertainty, antimicrobial chemotherapy is often commenced on the slightest clinical suspicion of infection. This approach, whilst effective in combating acute infection, increases the risks of drug side effects and the emergence of drug resistant organisms within neonatal units. Over recent years there has been great interest in the potential diagnostic value of a range of haematological, and immunological surrogate markers of infection [1,2]. The value of physiological measurements in this context has also recently been examined [3]. C reactive protein (CRP) is a component of the innate immune system and increased levels of CRP are observed early in response to severe bacterial infection. As a classical acute phase reactant, however, CRP elevation alone has insufficient specificity for diagnosis of neonatal infection. Our previous studies indicated that CRP > 6 mg/l and sICAM-1> 300 ng/ml in plasma samples collected in endotoxin free conditions [4,5] were independent predictors of infection with a high sensitivity for clinical infection (95%) and NPV of 97%[4,5]. However such sampling methods are impractical in routine practice and we subsequently demonstrated in a retrospective study, that routinely collected serum samples gave very similar mean levels of sICAM-1[6]. It has also been demonstrated, independent of those studies, that hsCRP measurement below 1 mg/l provides increased sensitivity for neonatal infection [7]. As most diagnostic laboratories do not provide hsCRP measurement, the optimum diagnostic cut off level for CRP/hsCRP remains a matter of debate. Eselectin is a cell adhesion molecule expressed and secreted by activated endothelial cells and is a component of an adhesion cascade that leads to leukocyte and platelet accumulation at sites of inflammation, infection, and/ or injury. Levels of soluble E-selectin (sEselectin) have also been previously documented in neonatal sepsis[8]. Serum Amyloid A (SAA) is also an acute phase protein which has been shown to be of potential diagnostic value in neonatal infection [9]. In this study, we sought to confirm and extend our earlier findings, utilising blood samples drawn in a prospective study, at the time of acute clinical deterioration and prior to antibiotic administration. We chose this time to reflect the moment at which clinicians would require additional laboratory information to guide their decision regarding whether or not, to prescribe antibiotics and were particularly interested to establish whether the high negative predictive values previously obtained were reproduced. Clinical diagnosis was established, by an experienced paediatrician looking after the infants, blinded to the results of the test measurements. The incorporation of serum amyloid A, sE-selectin and low level hsCRP (< 1 mg/l) in the study, was to establish whether these measurements would enhance diagnostic performance. Specifically, low level hsCRP measurement was included to ascertain whether a new (lower) diagnostic cut off level would be appropriate in this age group rather than the more commonly used level of < 6 mg/l. We report here the outcome of this study, in particular the performance characteristics of each study measurement regarding the diagnosis of neonatal infection. Methods Premature infants undergoing neonatal intensive care in the Department of Neonatal Medicine, Queen's Medical Centre, Nottingham, England were studied prospectively. This is a regional referral centre based in a University Teaching Hospital which receives infants both from the hospital's obstetric unit and surrounding district general hospitals. At the time of study it received approximately 400 admissions per year. Infants were recruited consecutively, as they developed acute clinical deterioration. At the time of enrolment into the study, an enrolment form was completed indicating the infant's age, underlying diagnosis, symptoms indicative of acute deterioration, investigations undertaken and therapy (if any) commenced. A peripheral venous blood sample (0.5-1.0 ml) was taken into a plain bottle prior to the administration of antibiotics and transported routinely to the laboratory. Serum was then separated and stored at -30°C until later batch analysis. There were no special storage/handling requirements. Venous blood samples were also taken at the time of routine venepuncture from a number of infants undergoing intensive care with no acute signs of deterioration, as controls. One week after deterioration, the routinely available investigative results and subsequent clinical course were reviewed (by VG & DE). Investigations commonly used included e.g. full blood count, differential white count, Creactive protein (>10 mg/L), urine, blood or swab culture, chest or abdominal X-ray and lumbar puncture Infants were classified as infected or non-infected. Clinical outcome beyond one week was not recorded. * Correspondence: david.edgar@belfasttrust.hscni. net 1 Regional Immunology Service, Royal Hospitals, The Belfast Trust, & School of Medicine, Dentistry & Biomedical Sciences, Queen's University Belfast, Northern Ireland Full list of author information is available at the end of the article Edgar et al. BMC Pediatrics 2010, 10:22 http://www.biomedcentral. com/1471-2431/10/22 Page 3 of 16 Infants were regarded as not infected if (i) their clinical deterioration was transient (ii) a specific non infective cause for the deterioration was identified (iii) there was no haematological, microbiological or radiological evidence of infection and (iv) administration of antibiotics did not result in clinical improvement. The infected group were sub-classified as (a) culture positive infection or (b) culture negative infection, based on the identification of any positive microbiological results from blood, urine, skin, CSF or other cultures. The culture negative infection subgroup included infants in whom there was either haematological or radiological evidence of infection or those who responded significantly to administration of antibiotics. Study measurements (including hsCRP) were not available to the investigators when allocating infants into their groups. A standardised "panel" of investigations was not used for all infants; rather, investigations were tailored to each individual presentation by the clinician in charge. The investigations undertaken for each infant were recorded. Table 1: Gestational age & diagnosis requiring care in NICU Diagnosis Infected (n = 74) Not Infected (n = 114) Controls (n = 27) Prematurity/LBW/VLBW/I