Sepsis represents a major contributor to neonatal mortality particularly among premature infants (24) with mortality rates as high as 40% in VLBW(25). Adaption of universal intrapartum antibiotic prophylaxis (IAP) has resulted in 80% reduction of early-onset sepsis cases, but incidence of late-onset sepsis is significantly increased in modern NICU due to increased survival of extreme preemies with many invasive strategies to support their life(26)(4). Frequent screening for late-onset sepsis, with any change in their clinical status, is an essential strategy for early diagnosis and management of infection as early as possible (27). Clinical diagnosis is not feasible due to the non-specific nature of sepsis manifestations (28). Although blood culture is the cornerstone for sepsis diagnosis, it is associated with several drawbacks that renders it not suitable for rapid diagnosis or guiding antibiotic- starting decision. Critically ill septic premature infants may succumb before the result of culture is available(16). Also, the high rate of false negative results is another important challenge for the use of blood culture solely for sepsis diagnosis (29). In our study only 37% (102/274) of evaluated cohort for late-onset sepsis (LOS) had positive culture, similarly Betty and Inderpreet reported 51% culture positive rate among clinically presumed septic neonates(30). The risk of morbidities and mortality due to delayed or untreated infection versus the short and long-term hazards of frequent un-needed exposure to antibiotics, justify the pressed need for accurate and rapid test for differentiating septic from non-septic symptomatic premature infant.
In the current research we hypothesized that, as hepcidin is a well-known first line defender that rapidly increases to militate against pathogenic invasion (16), (17), its measurement can point to the septic etiology of non-specific manifestations of neonate. Coincide with our assumption, statistically significant higher serum hepcidin (Hep-S) values were recorded for culture- proven septic premature infants in our cohort as compared with their non-septic asymptomatic peers. Similarly, Wu and his team affirmed that infection is a potent inducer of hepcidin production in VLBW as it increases four folds in newborn with proven infection (9). Other researchers examined the usefulness of hepcidin level's measurement in cord blood and in serum as a biomarker of early onset sepsis and described it as valid predictor (7)(31). Serum hepcidin (Hep-S) was dramatically elevated in infants who are bacteremic or even treated for presumed sepsis but with negative culture in previous report (18). Infection and inflammation enhance IL-6 production which in turn induces hepatic hepcidin production to combat pathogenic invasion. Also, autocrine synthesis of hepcidin, can more selectively activate the appropriate cell type namely macrophages and monocyte that can fine tune the response to infection (32). Hepcidin fights pathogen either by depriving it from iron (33) or by direct antimicrobial activity against various pathogen(34). In follow-up of our septic cohort, serum hepcidin (Hep-S) were significantly lower after seven days of antibiotic therapy (convalescent sample) as compared with the acute sample, denoting the parallel dynamics of hepcidin with infection and inflammation. It is worthy note that 2 mechanically ventilated extreme premature infants in our cohort showed progressive worsening of their clinical condition despite several trials of antibiotics, both of them had progressive increase in acute phase reactants and also in serum and urinary hepcidin and they died few days after taking convalescent samples. Their cultures came positive for multidrug resistant klebsiella pneumonia. This finding is a supporting evidence for the prognostic value of hepcidin, nevertheless, this issue needs to be thoroughly investigated. We excluded the anemic and recently transfused preemies to evaluate the changes of hepcidin with variable stages of infection without other interfering factors especially those related to iron hemostasis. Receiver-operator curve for hepcidin showed AUC of 0.93 that indicates high discriminating ability of serum hepcidin concentration in differentiating septic from non-septic infants. As compared with several commonly used acute phase reactant such as CRP and procalcitonin which have good negative predictive values (NPV) but low positive predictive values (PPV) to diagnose cases of sepsis correctly (35), hepcidin showed high diagnostic profile. A cut-off value of ≥94.8ng/ml of S. hepcidin showed sensitivity (88%), specificity (94%), PPV (95%) and NPV (84%) respectively with accurately diagnosing 90.2% of presenting cases as septic or not. Close to our findings, hepcidin was reported to correctly diagnosing sepsis among both term and preterm infants by 91% at cut-off of ≥92ng/ml (9). Acute serum hepcidin values showed weak positive correlation with CRP levels (r=0.2 p=0.03). Despite the fact that, production of both of them are IL-6 dependent, (32)(36), lack of strong correlation between both of them may be explained by the different time course of their production. CRP is delayed for 48 hours to peak and its half-life time is approximately 24 hours(37), while hepcidin peaks in few hours (6 hours) (14).
Serum hepcidin levels at initial evaluation (acute) and after one week of treatment (convalescent) displayed significant positive correlation with corresponding urinary hepcidin values (r=0.42, r= 0.35 respectively). This finding may answer our question "Could we find body fluid to diagnose and follow up sick preemies, other than blood?" to avoid frequent phlebotomy and its hazards. As hepcidin is excreted and can be accurately quantified in urine sample by same analytic method (ELISA) as serum hepcidin, and with this positive correlation, urinary hepcidin may replace serum hepcidin in initial diagnosis and follow up of septic preemies, particularly if frequent screening is needed during their NICU stay. Hep-U is significantly higher among clinically and laboratory proven sepsis cases as compared with non-septic infants in our series, and its level is significantly reduced with improved cases after 7 days of treatment. All previous data support the idea that, similar to Hep-S, urinary hepcidin is dramatically increased in response to IL-6 infusion(14) and its quantification can help in rapid, accurate diagnosis of sepsis in premature infants. In harmony with our data, researchers in pediatrics and adult documented the positive serum and urinary hepcidin correlation (38) (20). Simultaneous adult samples usually show better correlation than those in premature as urine sample may be delayed until involuntary void in urine bag after taking serum sample. As hepcidin follows diurnal variation(20), such delay may impair correlation.
Area under the curve of urinary hepcidin receiver operator curve is 0.87, indicating good discriminating power of measuring urinary hepcidin concentration between septic and non-septic. At cut-off value of ≥ 264 ng/mg of urinary hepcidin/urinary creatinine showed sensitivity (85%), specificity (90%), PPV (92.5%) and NPV (81%) respectively with accurately diagnosing 84.5% of presenting cases as septic or not. Urinary hepcidin was normalized for urinary creatinine, and expressed as ng/mg, to adjust for variable urine dilution. Diagnostic profile for urinary hepcidin is reasonable, with less diagnostic power than those of S. hepcidin but still with accepted accuracy in sepsis diagnosis. Up to our knowledge, no previous study was performed to evaluate the role of urinary hepcidin in sepsis diagnosis either in children or neonates. However, Muller and colleagues reported positive correlation between Hep-S and Hep-U and between both of them with iron indices(38). Accordingly, Researchers were hoping to use Hep-U as diagnostic and prognostic biomarker for iron deficiency anemia, and their results were promising(39). Another advantage of using Hep-U as diagnostic biomarker for N.sepsis, added to its non-invasive nature, is the less impact of diurnal variation on its level(40)
Although hepcidin has been discovered as early as 2000, its clinical application was delayed due to the expensive, cumbersome and invasive nature of its measuring modalities like hepcidin mRNA expression in liver biopsy sample. In 2008, enzyme linked immunosorbent assay for human hepcidin is developed that can accurately, easily and reproducibly determine physiologic and pathologic changes in hepcidin in variable body fluids(20).
Significant proportion of our cases were late preterm or near-term infants (39 infants, 32%) with gestational age 35-37 weeks. Usually those infants are apparently well with safe pulmonary status, thermal control and nutritional abilities, but indeed they significantly have issues of prematurity like poor immunity, increased risk of feeding problems, and injurious hyperbilirubinemia(41). These criteria necessitate their close follow-up to avoid such hazards.
Leading isolated causative pathogen among our septic preemies is gram negative organisms [Klebsilla pneumonia (47%) & Pseudomonas (6%), Citrobacter (6%] followed by several subtypes of staphylococcus. Previous study from other Egyptian children hospital, showed the same results (42). On the other hand, coagulase negative staphylococcus is the most common isolates of late-onset sepsis of premature in several developed countries (53%-78%) and (35%-47%) in series from developing countries. The predominant causative pathogen is affected by the geographic areas, and level of health care service, particularly adherence to infection control strategies(43)
All the previous data conclude that hepcidin concentration in different body fluid can function as promising accurate and rapid surrogate test, with blood culture, that guide empiric antibiotics –starting decision or withholding it safely until the culture results is ready in symptomatic presumed septic preemies. Urinary hepcidin has advantages over serum hepcidin as; it is non-invasive, no hazards of phlebotomy, and less variable throughout the day.
Our study has some limitation; first, small number of involved premature infants in case group as inclusion criteria was strict to involve only culture- proven cases who presented almost one third of screened cases. Second, the dual function of hepcidin as master iron regulator plus its role in innate immunity forced us to exclude all anemic or recently transfused cases to prevent bias due to disturbed iron status. Majority of extreme premature infants suffer from anemia or receive transfusions somewhere during their NICU stay and thus hepcidin may not be the ideal biomarker for their screening. To widespread its use, especially urinary hepcidin, wide-scale multicenter study should be performed that involve anemic, transfused, and culture negative cases to determine diagnostic cut-off of hepcidin in different situations.