Abstract
Early diagnosis of neonatal infection has proved problematic due to the inadequacy of currently available laboratory tests. Neonatal sepsis is associated with an increase in plasma-derived cytokine levels, but an increase of a single cytokine cannot identify neonatal sepsis specifically and multiple cytokine levels are required. The time constraints and relatively large volume of plasma required to measure multiple cytokines from newborn infants by conventional enzyme-linked immunosorbent assay (ELISA) techniques is prohibitive. We therefore applied cytometric bead array (CBA) technology for simultaneous measurement of multiple cytokines from a group of 18 term neonates with infection confirmed by culture and a control group. ‘Normal’ ranges were established for each cytokine from 1–7-, 8–14- and 15–21-day-old newborns. There was no significant change in the levels of cytokines from infants in different control age groups, suggesting that basal cytokine levels are unchanged in the first 3 weeks of life. In the patient groups, however, there was a significant difference in several cytokines between the different age groups. Interleukin (IL)-6, IL-10 and IL-12 were increased significantly in the 1–7-day-old patient group compared to either the 8–14 and 15–21 age group, suggesting that infection in utero is associated with increased levels of these cytokines compared to infection acquired following birth. When individual patient cytokine levels were compared to normal control reference ranges, two patients failed to show significant elevation of any cytokine tested. All other patients showed elevated levels of between one and nine cytokines tested (mean of 4·6). There was no correlation between elevated cytokine levels and types of infective organism or patient age. In conclusion, neonatal sepsis is associated with the elevation of multiple plasma cytokines. The use of CBA kits is a rapid, easy, low sample volume and sensitive method to measure multiple plasma cytokines.
Keywords: cytokines, cytometric bead array, flow cytometry, inflammatory, neonatal sepsis, Th1/Th2
INTRODUCTION
Infection remains an important cause of neonatal morbidity and mortality despite the development of broad-spectrum antibiotics and technical advances in life support therapy. Diagnosis of congenital or neonatal infection is often based on clinical signs. However, clinical symptoms of infection may not be specific, and for this reason early diagnosis is often based on the results of laboratory tests, which are currently inadequate. Serological tests and isolation of microorganisms do not give immediate results and haematological tests that are used currently to screen for the presence of infection in the newborn can be difficult to interpret. For example, leucocytosis is difficult to interpret due to the large ‘normal’ range in the newborn (8–32·5 × 103µl) and increase in neutrophil band forms is subjective, with different morphologists giving varying results.
Cytokines are an integral part of the immune response. Several studies have shown that neonatal sepsis is associated with an increase in plasma levels of inflammatory cytokines such as interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)-α, granulocyte-macrophage colony stimulating factor (G-CSF), interferon (IFN)-γ, IL-1ra and IL-1β[1–4]. One study showed that it was possible to distinguish between clinically ill (but not infected) neonates and neonates with early onset sepsis by measurement of elevated levels of several proinflammatory cytokines in preterm but not term infants [5]. However, labour delivery has also been associated with elevated levels of IL-6 compared to caesarean delivery, while fetal distress has been shown to be associated with elevated levels of IL6 and IL-8 and decreased TNF-α at birth [6]. Romagnoli et al. showed that IL-6 levels were elevated during early onset infection while IL-10 levels were raised later during the infective process [7]. In addition, Kallmam et al. showed that measurement of plasma levels of IL-6 in newborns could distinguish proven and clinical sepsis from transient tachypnoea but not from respiratory distress syndrome [8]. Therefore, it is consistent with the current literature that quantification of any single plasma cytokine will not identify neonatal infection specifically and that multiple cytokine levels are required.
The assay time constraints and relatively large volume of serum/plasma required to measure multiple cytokines by conventional enzme-linked immunosorbent assay (ELISA) techniques are prohibitive in the case of neonates. We applied a faster, sensitive cytometric bead array (CBA) technology, using CBA kits from BD Pharmingen, which combines the principles of the ‘sandwich’-based immunoassay with flow cytometry for simultaneous measurement of multiple cytokines from small sample volumes. Analytical sensitivity using these kits have been shown previously to be comparable to conventional ELISA (correlation coefficient >90%) and spiked recovery experiments showed consistencies of recovery throughout the cytokine calibration range [9]. For these studies we measured Th1/Th2 cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) and inflammatory cytokines (IL-8, IL-1β, IL-6, IL-10, TNF-α and IL-12) in a group of term neonates with infection (confirmed by bacterial culture,n = 18) and a control group (n = 6) aged 1–7, 8–14 and 15–21 days.
PATIENTS AND METHODS
Reference ranges
Following institutional ethics approval and parental consent, blood was collected into EDTA. Full blood counts were determined using a Technicon H1E (Bayer Diagnostics), and blood films examined following May–Grunwald–Giemsa staining. Any sample showing features suggestive of infection was excluded from the ‘normal’ group. Reference ranges were established for each cytokine from six newborn infants at 0–7, 8–14 and 15–21 days of age. All infants tested were >35 weeks gestation when born.
Control samples
These were prepared simultaneously with the patient samples and were included with every analysis. Controls included at least one blood from an infant in which possibility of infection had been excluded. ‘Septic screening’ is routinely carried out at birth in most infants admitted to the neonatal intensive care unit at the Women's and Children's Hospital. This group was selected from infants in whom no infection was subsequently detected by blood culture or viral serology. Blood samples were transported immediately to the laboratory for processing. Plasma was separated by centrifugation (500 g for 10 min) at 4°C and stored at −80°C until analysis.
Infected infants
All infants admitted to the Women's and Children's Hospital intensive care unit with suspected infection within a 12-month period were eligible for inclusion in the study. Newborn infants were selected for inclusion in the ‘proven infected’ group on the basis of the following selection criteria: (i) organism cultured from blood or cerebrospinal fluid; (ii) urine bacterial culture or streptococcal antigen-positive; (iii) positive culture from endotracheal tube with radiological evidence of parenchymal lung involvement; and (iv) clinical and radiological evidence of necrotizing enterocolitis with supportive evidence of infection. There were 18 infants with positive bacterial infection in this group, six infants in each group at ages 0–7, 8–14 and 15–21 days.
Cytokine analysis
Th1 and Th2 cytokines including IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α were quantified simultaneously using a human Th1/Th2 cytokine cytometric bead array (CBA) kit. Inflammatory cytokines including IL-12, TNF-α, IL-10, IL-6, IL-1β and IL-8 were also quantified using an inflammatory CBA kit. The CBA kits and CBA software were provided by BD Pharmingen. These assay kits provided a mixture of six microbead populations with distinct fluorescent intensities (FL-3) and were precoated with capture antibodies specific for each cytokine. Fifty µl of plasma or the provided standard cytokines were added to the premixed microbeads in 12 mm × 75 mm Falcon tubes (BD). After the addition of 50 µl of a mixture of PE conjugated antibodies against the cytokines, the mixture was incubated for 3 h in the dark at room temperature (method varied slightly for the inflammatory CBA kit). This mixture was washed and centrifuged at 500 g for 5 min and the pellet resuspended in 300 µl of wash buffer. The FACSCalibur flow cytometer (BD Pharmingen) was calibrated with setup beads and 3000 events were acquired for each sample. Individual cytokine concentrations were indicated by their fluorescent intensities (Fl-2) and were computed using the standard reference curve of cellquest and CBA software (BD Pharmingen).
CBA inter- and intra-assay performance
To address intra-assay performance of the CBA Th1/Th2 cytokine assay, 10 replicate samples of three different levels of the Th1/Th2 standards were tested in a single assay. Interassay reproducibility was assessed by using two replicate samples of three different levels of the human standards in four separate experiments.
Statistical analysis
Group comparisons were made using the non-parametric Mann–Whitney U-test and individual data were compared to normal reference ranges using Student's t-test for independent samples at a 95% confidence level. Pearson's rank correlation and anova were used for data comparison. The statistical analysis was performed on raw or logarithmically transformed data where appropriate.
RESULTS
Reference ranges
‘Normal’ ranges for plasma cytokine levels were established using blood samples taken from specimens received for routine postnatal investigation with haematological complete blood pictures and clinical assessment demonstrating no evidence of infection. Levels of cytokines (mean, s.d. and range) for the different control age groups are shown in Table 1. Controls are also classified by postpartum age at time of collection.
Table 1.
Plasma cytokine levels (pg/ml) for patient and control groups
| Name | ID | CAT | Weeks | Days | Th1/Th2 CBA kit | Inflammatory CBA kit | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IFN-γ | TNF-α | IL-10 | IL-5 | IL-4 | IL-2 | IL-12 | TNF-α | IL-10 | IL-6 | IL-1b | IL-8 | |||||
| P1 | 57 | c-staph | 1 | 2 | 0 | 0 | 1·2 | 1·5 | 0 | 0 | 0 | 0 | 1·6 | 0 | 2·1 | 14·6 |
| P2 | 58 | gbs | 1 | 3 | 0 | 1·4 | 1·8 | 2·2 | 1·5 | 1·4† | 21·6 | 11 | 7·1 | 0 | 89·5 | 57·5 |
| P3 | 59 | serrati | 1 | 2 | 0 | 0 | 8·4 | 7·5 | 0 | 0 | 8·7 | 0 | 26 | 9·9 | 19·4 | 380 |
| P4 | 60 | s.aure | 1 | 5 | 723 | 3·5 | 13·5 | 2·1 | 0 | 0 | 24·7 | 7·8 | 27·2 | 3080 | 0 | 861 |
| P5 | 61 | kleb | 1 | 1 | 10·3 | 8·9 | 4313·8 | 1·9 | 0 | 0 | 12·7 | 13·1 | 4763 | 4270 | 30 | 529 |
| P6 | 63 | candid | 1 | 3 | 30 | 0 | 10·9 | 0 | 0 | 0 | 31·9 | 1·9 | 33·8 | 66·4 | 36·3 | 122 |
| C1 | 62 | c | 1 | 7 | 0 | 0 | 2·1 | 1·3 | 0 | 0 | 9·6 | 0 | 7·6 | 0 | 11·5 | 47 |
| C2 | 64 | c | 1 | 5 | 1·7 | 0 | 2·5 | 1·8 | 1·2 | 0 | 0 | 0 | 7·8 | 0 | 16·4 | 73 |
| C3 | 65 | c | 1 | 4 | 4·9 | 1·5 | 2·4 | 2·1 | 1·5 | 0 | 0 | 2·6 | 2·9 | 0 | 6·5 | 67 |
| C4 | 66 | c | 1 | 6 | 2·6 | 0 | 2·3 | 1·6 | 0 | 0 | 0 | 1·5 | 3·6 | 0 | 0 | 26 |
| C5 | 67 | c | 1 | 2 | 1·2 | 1 | 1·6 | 6·7 | 1·3 | 0 | 0 | 0 | 0 | 34·1 | 10·2 | 95 |
| C6 | 68 | c | 1 | 6 | 1·7 | 1·4 | 1·4 | 1·8 | 1·2 | 0 | 0 | 0 | 0 | 0 | 0 | 54 |
| *Mean | 4·8 | 2·0 | 0·5 | 2·0 | 2·5 | 0·9 | 0 | 1·6 | 0·7 | 3·6 | 5·7 | 7·4 | 60·3 | |||
| s.d. | 1·79 | 1·65 | 0·73 | 0·45 | 2·05 | 0·70 | 0 | 3·9 | 1·10 | 3·46 | 13·92 | 6·57 | 23·68 | |||
| Range | 1–7·4 | 0–5·5 | 0–2·9 | 1·7–3·5 | 0–6·6 | 0–2·3 | >0 | 0–9·6 | 0–2·9 | 0–11 | 0–32 | 0–21 | 13–108 | |||
| P7 | 69 | c-staph | 2 | 8 | 16·4 | 0 | 3·7 | 19 | 1·1 | 3·3 | 0 | 0 | 7·2 | 0 | 13·4 | 80 |
| P8 | 70 | c-staph | 2 | 10 | 2·4 | 1·4 | 4·3 | 18·5 | 0 | 0 | 0 | 0 | 6·5 | 182 | 0 | 360 |
| P9 | 71 | ecoli | 2 | 8 | 1·9 | 0 | 0 | 3·8 | 0 | 0 | 20·2 | 0 | 0 | 0 | 45·7 | 62 |
| P10 | 72 | c-staph | 2 | 10 | 0 | 1·2 | 497·3 | 1·7 | 0 | 0 | 0 | 0 | 425 | 0 | 29 | 0 |
| P11 | 73 | mycop | 2 | 9 | 0 | 0 | 1·6 | 0 | 0 | 0 | 0 | 0 | 10·4 | 0 | 8·1 | 45 |
| P12 | 74 | candid | 2 | 11 | 8·3 | 1·8 | 70·6 | 0 | 0 | 0 | 0 | 5·8 | 243 | 100 | 14·6 | 3594 |
| C7 | 75 | c | 2 | 9 | 0 | 0 | 1·8 | 4·5 | 0 | 0 | 0 | 0 | 5·2 | 0 | 4·2 | 62 |
| C8 | 86 | c | 3 | 13 | 6·4 | 0 | 5·9 | 2·8 | 0 | 0 | 0 | 0 | 14 | 23·7 | 7 | 106 |
| C9 | 77 | c | 2 | 10 | 0 | 0 | 2 | 1·7 | 1·9 | 1·2 | 0 | 0 | 0 | 0 | 0 | 22 |
| C10 | 89 | c | 2 | 14 | 5·5 | 0 | 1·4 | 0 | 2·2 | 0 | 0 | 0 | 9 | 22·8 | 0 | 96 |
| C11 | 79 | c | 2 | 13 | 0 | 1·3 | 0 | 0 | 1·6 | 0 | 0 | 2·9 | 4 | 5·4 | 17 | 129 |
| C12 | 80 | c | 2 | 9 | 0 | 0 | 2·6 | 4 | 1·2 | 1·4 | 0 | 5·8 | 8 | 0 | 13 | 61 |
| *Mean | 11·3 | 2·0 | 0·2 | 2·3 | 2·2 | 1·1 | 0·4 | 0 | 1·4 | 6·7 | 8·6 | 6·9 | 79·3 | |||
| s.d. | 2·25 | 3·08 | 0·53 | 1·97 | 1·94 | 0·95 | 0·70 | 0 | 2·43 | 4·79 | 11·50 | 6·95 | 38·41 | |||
| Range | 7–16·1 | 0–8 | 0–1·2 | 0–6·3 | 0–6 | 0–3 | 0–1·7 | >0 | 0–6·3 | 0–16 | 0–32 | 0–20 | 2–156 | |||
| P13 | 81 | candid | 3 | 17 | 0 | 0 | 2·1 | 0 | 0 | 0 | 141 | 0 | 0 | 16 | 155 | 97 |
| P14 | 82 | candid | 3 | 16 | 0 | 0 | 2·6 | 5·1 | 1·7 | 0 | 0 | 0 | 9·5 | 0 | 12·1 | 162 |
| P15 | 84 | c-staph | 3 | 15 | 251·7 | 1·9 | 188·1 | 0 | 0 | 1·1 | 0 | 4·5 | 325 | 167·3 | 4·2 | 105 |
| P16 | 85 | gbs | 3 | 19 | 9 | 2·2 | 4·2 | 1·3 | 1·2 | 2·3 | 0 | 9·7 | 0 | 15 | 67·8 | 150 |
| P17 | 76 | unident | 3 | 18 | 19 | 0 | 13·8 | 1·9 | 1·2 | 0 | 8·5 | 1·4 | 34·8 | 39·7 | 23 | 78 |
| P18 | 78 | unident | 3 | 16 | 2·7 | 0 | 12·4 | 7 | 1·2 | 0 | 4·3 | 0 | 35 | 64·4 | 14·5 | 186 |
| C13 | 83 | c | 3 | 20 | 3·7 | 0 | 1·9 | 1·7 | 1·1 | 0 | 0 | 0 | 0 | 26·5 | 0 | 142 |
| C14 | 86 | c | 3 | 16 | 6·4 | 0 | 5·9 | 2·8 | 0 | 0 | 0 | 0 | 14 | 23·7 | 7 | 106 |
| C15 | 87 | c | 3 | 19 | 0 | 0 | 0 | 1·1 | 0 | 0 | 0 | 3·4 | 5 | 0 | 13·4 | 38 |
| C16 | 88 | c | 3 | 17 | 0 | 1·1 | 3·2 | 3·6 | 0 | 0 | 0 | 2·4 | 13 | 0 | 11·4 | 136 |
| C17 | 89 | c | 3 | 15 | 5·5 | 0 | 1·4 | 0 | 2·2 | 0 | 0 | 0 | 9 | 22·8 | 0 | 96 |
| C18 | 90 | c | 3 | 18 | 2·6 | 0 | 0 | 2·7 | 0 | 0 | 0 | 0 | 6 | 0 | 17 | 12 |
| *Mean | 17·5 | 3·0 | 0·2 | 2·1 | 2·0 | 0·6 | 0 | 0 | 0·9 | 7·8 | 12·1 | 8·1 | 88·3 | |||
| s.d. | 1·87 | 2·70 | 0·44 | 2·23 | 1·31 | 0·92 | 0 | 0 | 1·53 | 5·27 | 13·38 | 7·08 | 52·69 | |||
| Range | 14–21 | 0–8·4 | 0–1 | 0–6·4 | 0–3·6 | 0–2·4 | >0 | >0 | 0–4 | 0–18 | 0–39 | 0–22 | 0–193 | |||
Gbs, group B streptococcus: s.aureus, Staphylococcus aureus: c-staph, coagulase negative staphylococcus: ecoli, Escherichia coli: ser, Serratia marscecens: klebs, Klebsiella: mycoplas, Mycoplasma pneumoniae: unident, unidentified organisms.
Mean, standard deviation and range for cytokine levels for control groups aged 0–7, 8–14 and 15–21 days.
Bold type indicates significantly increased level of cytokine in infected group compared to control group. C (control), P (patient).
Types of infective organisms
A variety of organisms was isolated from the patient group (see Table 1). Blood cultures from five patients were positive for coagulase negative staphylococcus, four for Candida albicans, two for group B streptococcus, one for Staphylococcus aureus, one for Escherichia coli, one for Serratia marscecens, one for Klebsiella and one for Mycoplasma pneumoniae. In two patients no microorganisms were isolated; however, both patients were judged to be genuinely septic using the selection criteria for infected infants (see Patients and methods).
Overall cytokine analysis
Analysis of individual cytokine levels for the control group showed no significant difference between the infant age groups at 0–7, 8–14 and 15–21 days (Mann–Whitney, P > 0·05).
Cytokines TNF-α and IL-10 were measured with both Th1/Th2 and inflammatory CBA kits. Although individual TNF-α and IL-10 levels appeared somewhat different when measured using the Th1/Th2 and inflammatory CBA kits, there was no significant difference between data for the control group obtained with the different kits (P > 0·05).
Infants with confirmed infection
Depending on the age of the patient, we observed a significant difference in the relative levels of certain cytokines.
IL-12 was increased significantly in the 1–7-day-old (week 1) patient group compared to either the 8–14 (week 2) and 15–21 (week 3) age group (P = 0·021 and P = 0·039, respectively) (Table 2). IL-10 was increased significantly in the 1–7-day-old patient group compared to either the 8–14 and 15–21 age group (P = 0·047 and P = 0·040, respectively). IL-6 was significantly increased in the 1–7-day-old patient group compared to either the 8–14 and 15–21 age group (P = 0·039 and P = 0·041, respectively).
Table 2.
Differences in plasma cytokine levels for different groups
| Cytokine | Group A | Group B | Probability |
|---|---|---|---|
| IL-12 | Week 1 patients* | Week 2 patients | P = 0·021 |
| IL-12 | Week 1 patients* | Week 3 patients | P = 0·039 |
| IL-10 | Week 1 patients* | Week 2 patients | P = 0·047 |
| IL-10 | Week 1 patients* | Week 3 patients | P = 0·040 |
| IL-6 | Week 1 patients* | Week 2 patients | P = 0·039 |
| IL-6 | Week 1 patients* | Week 3 patients | P = 0·041 |
| IL-10 | Week 1 patients* | Week 1 controls | P = 0·047 |
| IL-12 | Week 1 patients* | Week 1 controls | P = 0·021 |
| IL-6 | Week 1 patients* | Week 1 controls | P = 0·039 |
Cytokine increased significantly in this group.
When individual patient cytokine levels were compared to normal control reference ranges, two patients failed to show any significant elevation of any cytokine tested, patients P1 and P11 aged 2 and 9 days old, respectively (see Table 1). All other patients showed elevated levels of between one and nine cytokines tested for (mean of 4·6).
Levels of IL-10 were elevated above control in nine patients. IFN-γ, IL-12, IL-6 and IL-1β were elevated in eight patients. IL-8 and TNF-α were elevated in six patients. IL-5 and IL-2 were elevated in four patients. IL-4 was not elevated in any patient. There was no correlation between elevated cytokine levels and types of infective organism or patient age, nor was there any correlation between cytokine levels for each patient age group.
There was no significant difference between the results obtained by Th1/Th2 and inflammatory CBA kits for TNF-α and IL-10 levels (P > 0·05). The 1–7-day-old patient age group showed 32/60 individual elevated cytokine levels, while the 8–14 and 15–21 age groups showed 16/60 and 25/60 raised cytokine levels, respectively.
CBA inter- and intra-assay performance
The intra-assay analysis showed an average coefficient of variation (CV) <6% for all six cytokine standards tested. Interassay analysis of 10 replicate samples of three different levels of the Th1/Th2 cytokine standards showed CV ≤10%. For example, the intra-assay average percentage CV and interassay percentage CV for IFN-γ was 3·7 and 8%, respectively, TNF-α (4 and 6·7%), IL-10 (2·3 and 5·7), IL-5 (4·7 and 6·7%), IL-4 (4 and 5·3%), IL-2 (3 and 7·3%).
DISCUSSION
This study demonstrates that neonatal infection in the first 3 weeks of life is associated with elevated levels of multiple plasma cytokines. Sixteen of 18 patients showed elevation of cytokines when both cytokine panels were utilized. The most informative single cytokine, IL-10, was increased in nine of 18 (50%) infected patients. The results from the current study suggest that if a single CBA kit was composed of the most frequently elevated cytokines, it may have the same predictive value for neonatal infection as the two kits combined. Elevation of a single cytokine using a combination of IFN-γ, IL-12, TNF-α, IL-10, IL-6 and IL-1β may identify 89% of patients with infection and save considerably on time and reagent costs. To improve the specificity of the analysis of multiple cytokines for neonatal infection, a control group of age-matched neonates with inflammation, but without infection, would be required.
The volume of plasma and time required to measure 10 different plasma cytokines from newborns is prohibitive by conventional ELISA-based methods. This is the first report of the use of CBA kits to quantify cytokines in the plasma of term neonates and shows that it is an excellent technique to measure multiple anti- and proinflammatory cytokine levels. While there was some variability noted between individual TNF-α and IL-10 levels using both kits, there was excellent inter- and intra-assay performance using the Th1/Th2 CBA kit and no significant difference between ‘normal’ ranges obtained for these cytokines using either kit. Methodology was slightly different for Th1/Th2 and Inflammatory CBA kits, which may account for some variability in values obtained.
Several other studies have shown that neonatal sepsis is associated with increases in plasma cytokine levels such as IL-6, IL-8 and TNF-α, G-CSF, IFN-γ, IL-1ra and IL-1β[1,2,4]. We have now shown that neonatal sepsis is associated with significantly increased levels of IL-10, IL-12 and IL-6 in newborns aged 1–7 days and increased levels of IL-10 and IL-12 in neonates aged 15–21 days. However, as with other studies these data refer to overall group data and not all individual patients have significantly elevated levels of these individual cytokines.
No previous studies have identified increased IL-12 levels in the plasma of newborns with infection. The present study shows that elevation of this cytokine and IL-6, IFN-γ and IL-1β are associated with neonatal infection and may be more sensitive than IL-8, a cytokine that has reportedly been increased in several studies [2,4]. However, much larger numbers are required to clarify any statistical significance between individual cytokine sensitivity for neonatal infection with other confounding conditions such as inflammatory diseases.
IFN-γ has not been reported previously to be elevated in the plasma of neonates with infection, although one study showed an increase in IFN-γ- and IL-4- producing cord blood T cells in intrauterine infection [3]. The present study shows that levels of IFN-γ in plasma were elevated in 50% of newborns with infection, while IL-4 was not elevated in any patient. We have shown that cord blood T cell IFN-γ production in vitro was reduced significantly compared to adults [10]; however, our present study indicates that newborn T cells can up-regulate this cytokine significantly following intrauterine infection. The absence of detectable IL-4 is not related to assay sensitivity as CBA kits are at least as sensitive as ELISA (2·5 µg/ml). IFN-γ and IL-10 were both elevated in 50% of infected patients, suggesting a possible functional immunoregulatory mechanism. A previous study showed a correlation between IL-6 and IL-10 plasma cytokine levels in 45 preterm neonates with sepsis [7]. Although IL-10 and IL-6 levels were raised in 56% and 50% of patients, respectively, there was no statistical correlation between these cytokine levels and much larger numbers would be required to evaluate this.
Monitoring elevation of two plasma cytokines has been shown to be of prognostic significance during the course of neonatal infection [3,7], indicating that measurement of multiple cytokines using CBA technology may lead to a further improvement in patient prognosis. We are currently undertaking a larger patient study to evaluate the use of these kits to identify infection and possibly monitor patient prognosis in preterm, term and infants up to 3 months of age.
It is known that certain diseases predispose infants to later disease. Infections with respiratory syncitial virus and other respiratory viruses have been associated with a dysregulated cytokine response and development of childhood asthma [11,12]. A dysregulated cytokine response in individual infected neonates may serve as a warning of later sequelae. Our recent finding of a lack of up-regulation of Th1 phenotype in a newborn that died subsequently with B. pertussis infection [13] implies that Th1 cytokines are required to respond effectively to this organism. Patients with this disease who fail to up-regulate Th1 cytokines may be targeted for more intensive therapy.
In conclusion, neonatal sepsis is associated with the elevation of multiple plasma cytokines. The use of CBA kits is a rapid, easy, low sample volume and sensitive method to measure multiple plasma cytokines.
Acknowledgments
We would like to thank clinical research nurses, Ros Lontis and Louise Goodchild, for organizing patient enrolment in this study. We thank Lori Covington for critical review of the manuscript. This study was supported by a Perinatal Pathology Research Grant from the Women's and Children's Hospital.
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