Septic shock remains a major cause of mortality among children in intensive care unit. Activation of hypothalamic-pituitary-adrenal (HPA) axis in response to stress is crucial for adaptation and maintenance of homeostasis. Studies have found a high incidence (40-65%) of adrenal dysfunction in septic shock patients [1-3]. Assessment of adrenal status in septic shock by measuring serum cortisol has prognostic and therapeutic value [4, 5].

Salivary cortisol estimation is non-invasive, easy to perform, represents the biologically active, free fractions of cortisol, and hence offers advantage over serum cortisol [6,7]. Studies on salivary cortisol in children with septic shock remain scanty. We conducted this study to assess the baseline and corticotropin stimulated salivary cortisol in children with fluid unresponsive septic shock, and its association with mortality and refractoriness of shock.

Saliva sample (2 mL) was collected either by spitting method into a plastic screw cap tube or from oral cavity using sterile cotton swab and then transferring it into the tube. The samples were centrifuged and stored frozen at -20°C until analysis (within 1-2 months). Three saliva samples from each patient were obtained, one at baseline (soon after a diagnosis of fluid unresponsive septic shock) and others after low dose ACTH (1µg intravenous Synacthen injection) stimulation at 30 and 60 min. We collected salivary samples irrespective of any specific time of the day as most critically ill patients lose the diurnal variation in their cortisol levels [11]. The mouth was cleaned at least half hour before post-stimulation sampling. No saliva sample was contaminated with blood. Salivary cortisol was estimated using the Cortisol ELISA (IBL International Germany), minimal analytical sensitivity was 0.138 nmol/L and functional sensitivity 0.828 nmol/L. Results of salivary cortisol assay were not available for deciding about use of steroids in any patient. Basal salivary cortisol values to define absolute adrenal insufficiency and hypercortisolism, were based on our own controls and another study [12]. Relative adrenal insufficiency was diagnosed if the peak increment in salivary cortisol after ACTH stimulation was <25 nmol/L (greater of the value at 30 or 60 min after stimulation minus baseline value), based on previous studies reporting total serum cortisol response to corticotrophin stimulation in septic shock [4, 13, 14]. Statistical analysis was performed using SPSS software version 16. Continuous variables were compared by t-test or Mann-Whitney test and categorical variables using chi-square or Fisher’s exact test.

The median (IQR) basal salivary cortisol level in healthy control group (n=79) was 2.6 (1.3-7.6) nmol/L. There were no significant variation in basal salivary cortisol values among healthy controls in reference to different age groups and sex. The median (IQR) basal salivary cortisol level (nmol/L) was significantly higher (P<0.001) in patients with septic shock [19.8 (7.2-42.4)] compared to healthy children [2.6 (1.3-7.6)], however, patients in septic shock group were more often undernourished (weight for age <2 SD score: 82.5% vs 46.9%). The mean age and gender distribution were similar in both the groups. Of the 51 patients with fluid refractory shock, eight had positive blood culture results.

The baseline parameters and salivary cortisol were compared between survivors and non-survivors (Table I). Absolute adrenal insufficiency was diagnosed in 8 (15.7%), and relative adrenal insufficiency in 35 (68.6%) patients. Post-stimulation salivary cortisol was higher at 30 min compared to that at 60 min, however, the difference was insignificant (P=0.715). Amongst patients who had absolute adrenal insufficiency (n=8), seven (87.5%) also demonstrated relative adrenal insufficiency (6 died); and six (75%) had catecholamine refractory shock (all died). Web Table I shows various parameters in relation to relative adrenal insufficiency.

TABLE I  Salivary Cortisol in Children with Fluid Unresponsive Septic Shock 

The difference in 30 min post-stimulation and baseline salivary cortisol (nmol/L) was significantly higher in survivors (mean±SD 39.9±70.5) compared to non-survivors (29.1±90.1), (P=0.657) and those with fluid refractory shock (mean±sd=42.7±76.1) in comparison to catecholamine refractory shock (28.7±86.4), (P=0.582).

We studied the adrenal status in children with fluid refractory septic shock using salivary cortisol measurements. We observed absolute adrenal insufficiency in 15.7% and relative insufficiency in 68.6% of these children. A good correlation between salivary cortisol and free serum cortisol has been reported among adult patients with septic shock, in morning samples [13]. Furthermore, a greater relative increase in salivary cortisol than total serum cortisol after stimulation has been observed [5]. This would be expected when the binding capacity of available cortisol-binding globulin (CBG) is exceeded above saturation point, and it could be responsible for the high variability of post-stimulatory values of total serum cortisol and also poor correlation with salivary concentrations at individual times [5]. Thus, in situations of having low CBG concentration, with its maximum saturation, and lower albumin level one would expect more rise in free cortisol level than total serum cortisol level after stimulation. Therefore, salivary cortisol can be used as surrogate of serum free cortisol.

The baseline salivary cortisol was higher in patients than control. This may be because of an initial adequate response to the stress induced by septic shock. Patients in non-survivor group had higher baseline value, and subsequent increment after stimulation was less compared to those who survived. Similarly, patients with catecholamine refractory shock had higher baseline value, and subsequent increment after stimulation was smaller compared to those who had only fluid refractory shock, though statistically insignificant. These smaller post-stimulation increments may be due to the fact that the HPA axis is already maximally stimulated, but it may be also a result of interference with the capacity of the adrenal cortex to produce glucocorticoids, and a lack of cortisol reserve [15]. We diagnosed primary adrenal insufficiency in 15.7% and relative adrenal insufficiency in 68.6% of children with fluid unresponsive septic shock, higher than reported by others [1, 14]. This may be because our children were sicker, all fluid unresponsive septic shock cases. Sarthi, et al. [14] reported relative adrenal insufficiency in 30% children with septic shock and primary insufficiency in none and there was no association with mortality but with catecholamine refractory shock. Though they used low-dose stimulation test similar to ours, they estimated serum cortisol. Although inconclusive due to small sample size, our data gives insight about incidence of absolute and relative adrenal insufficiency among fluid unresponsive septic shock children, and its association with catecholamine refractory shock and survival, which may have therapeutic implication.

Acknowledgment: We acknowledge Late Dr Y C Govil for his inputs, supervision and care for the study patients.

Contributors: SNS conceived and supervised the study, analyzed data and finalized the manuscript. He will act as guarantor. SR written the protocol, recruited patients and helped in analysis and manuscript writing. SA supervised the study and revised the manuscript. AS helped in recruiting patients and manuscript writing. VB analyzed the samples and revised the manuscript. The final manuscript was approved by all authors.

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