Steroid metabolite cross-reactivity leading to diagnostic dilemma in a male infant with salt-wasting crisis: A case report

INTRODUCTION

Salt-wasting crisis in the newborn period can be caused by aldosterone deficiency, as seen in congenital adrenal hypoplasia and congenital adrenal hyperplasia (CAH), or by aldosterone resistance, as seen in pseudohypoaldosteronism (PHA).^[1,2] Salt-wasting CAH is the severe form of CAH, characterized by both cortisol and aldosterone deficiency, leading to life-threatening adrenal crisis, if untreated. Whereas, PHA can be inherited and permanent due to mineralocorticoid (MC) receptor dysfunction, or transient, secondary to renal resistance to aldosterone caused by acute pyelonephritis or obstructive renal anomalies.^[3] As management of these two conditions differs, it is important to make the correct diagnosis to prevent long-term morbidity and mortality in affected neonates and infants. We describe a male infant who presented with salt-wasting crisis and highlight the diagnostic challenges encountered in establishing the correct diagnosis.

CASE REPORT

A 2-month-old male infant, born at term to a primigravida mother out of a non-consanguineous union with a birth weight of 3.6 kg, presented with poor weight gain, recurrent vomiting (2–3 episodes/day), and loose stools (3–4 episodes/day) for 3 weeks. He had an uneventful perinatal period and was on breastfeeds and supplemental formula milk. For 2 days before presentation, he refused to feed, had difficulty in breathing, and was lethargic. There was no history of fever. At admission, the infant was lethargic, clinically dehydrated, and afebrile. He had tachycardia (heart rate: 148 bpm), feeble peripheral pulses, delayed capillary refill time, normal blood pressure, and tachypnea (respiratory rate: 72/min) with acidotic breathing. His weight was 3.9 kg. There were no obvious dysmorphic features or congenital anomalies. Respiratory, cardiac, and abdominal examination was otherwise unremarkable. He had normal male genitalia with a well-developed scrotum with rugosities, both testes in the scrotal sac measuring 3 mL, stretched penile length of 3.3 cm, and urethral meatus at the tip of the penis (external masculinization score was 12/12). There was no obvious hyperpigmentation. Initial investigations revealed severe hyponatremia (110 mmol/L, reference range: 135–145 mmol/L), hyperkalemia (5.2 mmol/L, reference range: 3.5–5.0 mmol/L), and metabolic acidosis (pH: 7.048, reference range: 7.35–7.45; bicarbonate: 6 mmol/L, reference range: 22–26 mmol/L). Blood glucose was normal (75 mg/dL). Renal function was abnormal, with elevated urea (133.6 mg/dL, reference range: 7–20 mg/dL) and creatinine (2.3 mg/dL, reference range: 0.2–0.8 mg/dL). Further workup for salt-wasting crisis revealed significantly elevated serum cortisol (>62 µg/dL, reference range: 5–25 µg/dL), plasma aldosterone (>100 pg/mL, reference range: 1.7–23 pg/mL), and plasma renin (>500 µIU/mL, reference range - supine: 2.8–40 µIU/mL), which were measured using chemiluminescence immunoassay (CLIA) method. The 17-hydroxyprogesterone (17OHP), measured using the enzyme-linked immunoassay method, was also elevated (49.5 ng/mL, reference range: <2 ng/mL). The C-reactive protein was high (50 mg/L, normal: <5 mg/L). Ultrasonography of the abdomen revealed bilateral hydronephrosis and internal echoes in the urinary bladder, and normally sized adrenals. A complete urine examination showed 8–10 pus cells, negative for nitrites and leukocytes [Table 1].

Failure to thrive and salt-wasting crisis were clearly evident in this infant at admission. The moderate elevation of 17OHP suggested the possibility of salt-wasting CAH. However, the very high aldosterone and cortisol levels in the presence of hydronephrosis, pyuria, and elevated CRP made the treating team consider urosepsis with associated transient PHA and treat accordingly.

The baby was treated with intravenous fluid resuscitation, salt replacement with 3% saline, and corrective measures for hyperkalemia (intravenous calcium gluconate). Antibiotics were started for urosepsis after sending blood and urine cultures. Nevertheless, with continued requirement for sodium supplementation, and negative urine and blood cultures, we proceeded to do synthetic adrenocorticotropic hormone (ACTH) stimulation test using intravenous Synacthen, which showed significantly elevated 17OHP levels—370 ng/mL (1121 nmol/L), and 495 ng/mL (1499 nmol/L) (normal: <10 ng/mL, <30 nmol/L) at 30 min, and at 60 min respectively, confirming the diagnosis of salt-wasting CAH. The child was initially started on injectable hydrocortisone (HC) in stress doses (50 mg/m²/day), which was then gradually tapered to a maintenance oral dose of 15 mg/m²/day in three divided doses. Oral fludrocortisone (FC) 100 µg once a day and oral salt solution 2 g/day were added. The salt solution was freshly prepared (strength: 1 g/5 mL) using table salt and distilled water and was administered in 4–6 divided doses over 24 h by mixing with breastmilk or infant formula.

Targeted gene sequencing of CYP21A2 gene using next-generation sequencing method detected homozygous variation in intron 2 (c.293-13 C>G) along with heterozygous variations in 5’ UTR (c.-126C>T, c.-113G>A, c.-110T>C, c.-103A>G), exon 1 (c.92C>T) and exon 3 (c.332_339del), exon4 (c.518T>A), exon 6 (c.710T>A, c.713T>A, c.719T>A), exon 7 (c.844G>T, c.923dup), and in exon 8 (c.955C>T and 1069C>T). CAH in this child was caused by both homozygous and compound heterozygous pathogenic variations in the CYP21A2 gene.

DISCUSSION

Salt-wasting crisis in a neonate can be caused by adrenal insufficiency due to CAH or classic CAH, or due to PHA. CAH due to 21-hydroxylase deficiency affects cortisol and aldosterone production, leading to adrenal insufficiency. The salt-wasting form can present early with vomiting, dehydration, failure to thrive, and potentially life-threatening shock. Diagnosis is often made at birth in female newborns due to associated virilization of the genitalia. However, it can be delayed in boys and can result in life-threatening acute adrenal crisis, especially where universal newborn screening is not available.^[1,4,5] The most common form of CAH is caused by 21-hydroxylase deficiency resulting from pathogenic variants of CYP21A2 gene.^[6] PHA can be inherited in autosomal dominant or recessive forms due to a nonfunctional MC receptor gene, which regulates the epithelial sodium channel in the kidneys, or transient, secondary to acute pyelonephritis with or without urinary tract obstruction.^[2,7] There have been many published reports of PHA misdiagnosed as CAH due to the typical salt-wasting presentation.^[8,9] However, the other way around, with CAH being mistaken for PHA, was reported only a few times so far. The significantly elevated cortisol, aldosterone, and renin levels in association with obstructive uropathy-like picture in a male infant with normal genitalia led us to consider PHA as the primary diagnosis initially.

Although hypocortisolemia and low aldosterone levels are expected, high levels of serum cortisol and aldosterone can be observed in salt-wasting CAH due to cross-reactivity between different steroid metabolites when conventional laboratory techniques, such as CLIA, are used. Steroid compounds, such as 17OHP, testosterone, and dehydroepiandrosterone sulfate, at highly elevated levels can result in cross-reactivity with aldosterone assessment. Similarly, the elevated 21-deoxycortisol has significant cross-reactivity with cortisol estimation.^[10-12] Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for steroid hormone assessment can help avoid these pitfalls.^[11,13,14] However, LC-MS/MS is not quickly accessible at our center and, when accessible, has a turnaround time of 7–10 days, limiting its utility in guiding the initial treatment decisions regarding HC and MC replacement. In our child, we were able to make the correct diagnosis of CAH by performing the ACTH stimulation test and later confirmed 21-hydroxylase deficiency with the help of targeted genetic testing. It is crucial to make an accurate diagnosis in an infant with salt-wasting for appropriate long-term management and prevention of future adrenal crises. While PHA management is mostly centered around fluid management and salt replacement, CAH needs the timely initiation of glucocorticoid and MC replacement, which continues lifelong, along with the life-saving stress dosing during intercurrent illness. Education of caregivers regarding adrenal crisis prevention is vital and can only occur with an accurate diagnosis. Genetic counseling is recommended for families with affected children.

CONCLUSION

This case highlights that salt-wasting CAH can mimic PHA, particularly in male infants without genital ambiguity and with urinary tract abnormalities, and that CLIA -based assays may yield spuriously elevated aldosterone and cortisol levels in CAH due to steroid metabolite cross-reactivity. ACTH-stimulated 17OHP testing remains a critical diagnostic tool enabling the timely initiation of appropriate treatment, while targeted genetic testing confirms the diagnosis and facilitates genetic counseling for affected families.