To our knowledge, this is the first population study evaluating cytokine profiles in ELBW with NS, BS and FS. Although cytokine levels and acute-phase reactants have been used as markers of neonatal sepsis, the response of these biomarkers to FS has not been systematically studied14
. In this exploratory, hypothesis generating study, we identified a panel of five inflammatory markers to be significantly associated with sepsis group in the 1st
three weeks of life on adjusted analysis - IFN-γ, IL-10, IL-18, TGF-β and TNF-α. These cytokines are potential candidate biomarkers of neonatal BS and FS that may allow early diagnosis before blood cultures become positive and that may also differentiate between BS and FS. Cytokines were not significantly different between groups on D0 suggesting that fetal inflammatory response syndrome is neither a marker for nor predisposes to neonatal sepsis and that systematic difference in cytokine profiles do not exist between infants who later acquire sepsis (BS or FS) or not. Significant group differences in cytokines in the pre-infection period suggest that changes in levels of these cytokines may precede clinical signs and symptoms and microbiologic confirmation of diagnosis. After the microbiologic diagnosis of sepsis, group differences in cytokine levels became more prominent as compared to values before microbiologic confirmation suggesting that these differences reflect a response to infection rather than conferring an inherent predisposition to infection.
In keeping with our hypothesis, IL-10 (Th-2 cytokine) was increased and TGF- β decreased in infants with FS compared to those with BS or NS. However, contrary to our hypothesis, Th-1 cytokines, IFN-γ, IL-18, and TNF-α, were increased in infants with FS. IL-1β, IL-12, IL-4, IL-5, IL-6 and IL-17 did not show any association with sepsis group in our study. Our hypotheses were based on cytokine profiles described in sepsis in experimental models and adults. Only one observational study of FS and BS in a small group of term and preterm neonates has shown significant differences in C-reactive protein (CRP) and IL-6 levels15
. Our results support the complexity of the immune response involving multiple cytokines with divergent functions and suggest that the immune response may differ in ELBW neonates.
initiator of the inflammatory response, has been reported to be increased early in neonatal sepsis. These studies included small number of patients, did not differentiate between FS and BS16-18
, were not limited to ELBW19,20
and included infants with culture negative sepsis21,22
. In our study TNF-α was significantly higher in ELBW with FS compared to BS and NS.
IL-6, one of the most studied cytokines in neonatal sepsis, increases early, but its half-life is short and its sensitivity decreases after 12 to 24 hours of infection. Most studies included small number of patients, did not differentiate between FS and BS16,18,23-25
, were not limited to ELBW19,20,26-32
and included infants with culture negative sepsis33
. In one study, IL-6 levels were significantly elevated in BS compared to FS in a cohort of term and preterm infants15
. The lack of association of IL-6 with culture proven BS and FS in our large homogenous population of ELBW infants suggests that IL-6 may not play a role in immune response to sepsis in ELBW. Alternately, imprecision in timing of collection of samples in relation to clinical suspicion of infection in our study may have led to spuriously negative results because of the short half-life of IL-6.
IL-17, the signature cytokine of TH17 cells, has important role in induction of neutrophilmediated protective immune response against extracellular bacterial or fungal pathogens such as Klebsiella pneumonia
and Candida albicans
in experimental models11,34,35
. However, in ELBW subjects of our study, IL-17 levels were not significantly different across sepsis group.
IL-18 is a pro-inflammatory cytokine that reduces polymorphonuclear cell apoptosis, potentiates IFN-γ production, and induces production of TNF-α, IL-1β, and IL-836
. IL-18 is protective against infection caused by a number of intracellular and extracellular pathogens. In a recent comprehensive study of >140 serum analytes from neonates evaluated for late-onset sepsis (including BS, FS, culture-negative clinical sepsis), IL-18 was elevated in infected vs. non-infected neonates and in FS37
. In our study as well, IL-18 emerged as a potential biomarker to differentiate FS from NS and BS.
IL-10 is an anti-inflammatory cytokine responsible for downregulation of the inflammatory process and maintaining homeostasis in vital organs36
. Elevated levels of IL-10 indicate infectious disease in neonates, and IL-10 remains elevated until 24 hours after the beginning of sepsis5,26,38
In our study, IL-10 not only discriminated between BS and NS but also between FS and BS or NS.
No previous studies have investigated levels of TGF-β, a bipolar cytokine that can both trigger and inhibit the immune system, in neonatal sepsis39
. In the present study, ELBW with sepsis had lower levels of TGF-β on D14 compared to infants with NS in the pre-infection period. In the post-infection period, TGF-β levels were significantly different on D14-21 between FS and BS or NS and between BS and NS.
On ROC analysis, three cytokines demonstrated moderate to high accuracy in discriminating between FS and NS or BS after microbiologic confirmation of diagnosis despite the fact that DBS were obtained at pre-defined postnatal ages and not timed in relation to 1st positive blood culture. These robust trends suggest that these cytokines may be biomarkers of neonatal FS and BS and need to be validated in prospective studies with blood samples obtained in relation to time of clinical suspicion of infection and 1st positive blood culture.
The majority of clinical characteristics that were strongly associated with sepsis group in our study have been previously reported1
. Center difference was a significant covariate in most of the analyses. This could be related to differing ethnic populations, proportions of inborn/outborn infant, clinical practices, or other unidentified factors. Wide variation in the incidence of invasive fungal infections between NICUs has been shown in multiple publications3,40
. Importantly, even after controlling for these covariates, the association between FS and five cytokines remained significant suggesting their role in the pathogenesis of FS. Postnatal time trends and GA differences for cytokines have been described by our group previously41
This is the 1st
population based study to compare cytokine concentrations in the 1st
three weeks of life in ELBW with FS, BS or NS. Two small studies has previously explored the cytokine signature of host response to different microbiologic agents in neonates with elevated IL-18 and CRP and lower IL-6 levels being hallmarks of fungal infections15,37
. Strengths of our study include a large sample size; prospectively collected data permitting a thorough investigation of the association of sepsis group and cytokine response at pre-defined time points, standardized collection of repeated, timed blood samples for three weeks starting from birth; assay of multiple cytokines from small volumes of blood using the Luminex assay; and multivariate adjustment for control of clinical variables.
Despite the important findings of this study, there are limitations. Although infants with BS represented a pure group with no positive fungal blood/CSF cultures; 68.5% of infants with FS also had positive blood/CSF bacterial cultures before or after the 1st
episode of FS. Positive blood cultures for bacteria during the period of fungal infection are characteristic of the natural history of disease of invasive fungal infections in ELBW1
. Contamination of FS group with BS should have under-estimated differences between the two groups; in spite of that we were able to detect significant differences between groups for five cytokines suggesting that these reflect true differences in cytokine response in BS and FS. Secondly, blood samples were timed in relation to postnatal age and not to clinical suspicion of infection. It has been shown that cytokine levels decline rapidly after onset of infection. Therefore DBS obtained at specified postnatal ages may have occurred at different time intervals before or after onset of infection. Despite this imprecision, we obtained consistent and significant predictive values for three cytokines at 14-21 days of age, which is close to the median age of onset of confirmed sepsis, suggesting dramatic effects. Although the study design of collection of protocol driven samples at pre-defined postnatal ages in this hypothesis-generating secondary study was not ideal to assess response to infection, it did allow evaluation of cytokines levels prior to diagnosis of sepsis which may not be possible to assess in a study design where sample collection is dictated by clinical suspicion of infection. Though these results may reflect true differences between FS, BS and NS because of the robust trends and biologic plausibility, they need to be validated in carefully designed large longitudinal cohort studies with prospective blood collection in relation to sepsis evaluation. Thirdly, it is difficult to distinguish between primary and secondary mediators in the cytokines described. Additionally, circulating cytokine concentrations may not reflect their biologic activity. Although we included 11 biomarkers, other markers (procalcitonin, CRP) that have been shown to be useful for the early detection of neonatal sepsis were not studied5,37
. Lastly, we did not correct for multiple comparisons since the posthoc subgroup analysis in this study is exploratory in nature and hypothesis generating. Therefore, reliance should be placed on the observed effect size rather than on statistical significance testing as a basis for decision making42
In conclusion, in this prospective study, IL-10, IL-18, TGF-β and TNF-α were found to be the most promising indicators of FS vs. BS or NS in ELBW. These findings offer insight into immune regulation in ELBW with sepsis, the prospect of rapid differentiation of neonatal BS and FS, early institution of targeted antimicrobial therapy and development of new treatments for this disease by enhancing the production of cytokines that confer protection. These novel findings will require validation in larger rigorously designed prospective studies.