Introduction
N-terminal pro-brain natriuretic peptide (NT-proBNP) is a low molecular
weight peptide (8.5 kDa) expressed in the ventricular myocardium
secondary to pressure and volume increases that has diuretic and
natriuretic effects and is the most used biomarker for diagnosis and
prognosis in heart failure
[1–3]. There has
been recent research interest in NT-proBNP as a reliable biomarker in
several pediatric scenarios
[4]. Increasing
evidence supports the use of serum NT-proBNP levels as a potential
biomarker of myocardial strain and disease severity for respiratory
conditions, including acute bronchiolitis
[5–8]. Serial
monitoring of serum NT-proBNP concentrations in these infants would
require multiple blood sampling through venipuncture in an otherwise
vulnerable population. NT-proBNP is a non-biologically active molecule
with no active clearance mechanisms that is removed from plasma via
passive excretion mainly by the kidney
[1,3,9,10].
The investigations in premature newborns and infants with congenital
heart diseases suggest that urine NT-proBNP determination could be
easily performed with current kit assays for serum NT-proBNP
determination
[11–14].
Therefore, urinary NT-proBNP may have the potential as a non-invasive
and reliable biomarker of severity in acute bronchiolitis that has not
yet been investigated.
This pilot study aims to determine urinary NT-proBNP in a cohort of
infants with acute bronchiolitis, analyze its correlation with serum
NT-proBNP concentrations, and explore its association with the severity
of the disease. The hypothesis was that concentrations of both serum and
urine NT-proBNP are correlated, and therefore, NT-proBNP levels would
indicate severe acute bronchiolitis by using urine instead of blood
analysis.
2. Material and methods
2.1 Design, setting and patients: This prospective
observational study was conducted between 1st October 2021 and 31st
March 2022 in the Pediatric Department of a tertiary university hospital
in Spain after the approval by the ethics committee of our institution
(approval number: 1338-N-20; October 2021). We included infants less
than one-year-old hospitalized with acute bronchiolitis of any severity.
The diagnosis and management of infants with bronchiolitis were made per
the attending physician’s discretion, following current international
recommendations
[15]. Infants
with significant congenital anomalies, including cardiac diseases,
chronic renal diseases, acute kidney injury, incomplete data, and
refusal of parental consent, were excluded from the final analysis.
2.2. Specimen collection: Time-matched urine and blood samples
were collected at the time of inclusion, always before initiation of any
inotropic or ventilatory support. If follow-up blood or urine laboratory
analysis were solicited during the episode of hospitalization at the
discretion of the attending pediatrician, new time-matched samples of
blood and urine were collected in those patients. Blood samples were
obtained by venipuncture, and urine samples were obtained by urethral
catheterization (UC) and urine bag (UB). The attending pediatrician
chose the method of urine collection according to the clinical
characteristics of the patients. The attending physician evaluated the
respiratory involvement of the included cases by the clinical severity
score of San Joan de Deu Hospital
(BROSJOD)[16] at
the time of specimen collection. The pH, pCO2, HCO3, lactate, C-reactive
protein (CRP), procalcitonin, creatinine and sodium were also determined
in these blood samples.
2.3. NT-proBNP analysis: Fresh samples (at least 2 ml)
were immediately sent for analysis, without being frozen, to our
institution’s certified clinical chemistry laboratory. Serum and urinary
concentrations of NT-proBNP were determined using a chemiluminescent
micro-particle immunoassay (CMIA), Alere NT-proBNP, for Alinity i assay
(Abbott, Spain). The intra-assay and inter-assay coefficients of
variation were 1.9% to 2.9% and 2.6% to 5.4%, respectively, with an
analytical range of 8.3 to 35 000 pg/mL. No subjects had serum and urine
NT-proBNP concentrations lower or higher than the assay linearity limit.
This equipment has not previously been found suitable for measuring
urinary NT-proBNP levels. These assays and quality controls were
performed according to the manufacturer’s recommendations. Because the
samples were not obtained in a predefined time in all patients, we
corrected the urine NT-proBNP by urine creatinine levels (urine
NT-proBNP/creatinine ratio in pg/mg) to address a potential bias caused
by different urine concentrations and to reduce inter-subject
variability. To address the limitation of the substantial age-dependency
of the levels of NT-proBNP in neonates and infants, we calculated Z-log
values (Z-score for skewed variables) adjusted for age in days as
previously reported
[17].
2.4. Research endpoints: The primary outcome of this
study was the relationship between serum and urine NT-proBNP levels. The
secondary outcome was the development of severe acute bronchiolitis. We
selected the longer length of stay (LOS) hospitalization, the need of
PICU admission for ventilatory support (invasive or non-invasive) and
the longer duration of ventilatory support as clinical outcomes
indicative of severe bronchiolitis.
2.5. Statistics: Mean ± standard deviation (SD)
(median and 25th–75th percentiles (IQR) where appropriate) and
proportions were reported for continuous and categorical variables,
respectively. Since the serum NT-proBNP and urinary NT-proBNP/creatinine
ratio concentrations exhibited skewed distributions, the log-10
transformed (log10) values were used in the statistical analysis to
stabilize variances and used this value in the statistical analysis. As
the Z-log-NT-proBNP presented a normal distribution, the serum
Z-log-NT-proBNP value was directly used in the statistical analysis. The
relationship between urine NT-proBNP concentrations and all blood
parameters (including serum NT-proBNP) were assessed using Pearson’s
correlation coefficient and linear regression analysis, where the
strength of correlation was evaluated by the squared correlation
coefficient (R-squared). All time matched samples were used (n=36
samples from 17 patients) for this correlation analysis. To explore the
association of urine NT-proBNP levels with clinical outcomes indicative
of severity, we used only the first sample obtained from each patient
before starting any inotropic or ventilatory support (n=17) to avoid
possible influences of these treatments on the value of urine NT-proBNP.
The Mann-Whitney U test and Spearman correlations were utilized to
assess associations of log-10-urine NT-proBNP/creatinine ratio levels
with categorical and continuous variables respectively. Because of the
potential for type I error due to multiple comparisons, findings for
analyses should be interpreted as exploratory. A sample size estimation
was not performed due to the exploratory nature of our study. All tests
were two-sided, and a p-value of < 0.05 was considered
statistically significant. We used Stata v.16 software (StataCorp,
College Station, Texas).
3. Results
Twenty patients were initially assessed for enrollment, and 3 cases were
excluded due to incomplete laboratory data. Therefore, 17 infants
(median age 68 (36-91) days; 11 (65%) male sex) with 36 time-matched
samples were included in the final analysis. Two samples were determined
at the emergency room in 2 infants that did not require hospitalization,
18 samples were determined in 9 cases at the pediatric ward, and 16
samples were determined in 6 patients that required PICU admission and
ventilatory support. Echocardiography was performed only in the patient
that required inotropic support, showing signs of severe biventricular
dysfunction secondary to the respiratory involvement that recovered
progressively until complete spontaneous normalization, with no final
diagnosis of any primary cardiac disease. Table 1 shows the baseline
characteristics and clinical outcomes of the 17 cases included.
In the 36 samples obtained, the median concentrations of serum NT-proBNP
resulted significantly higher than those of urine NT-proBNP (1246
(470-2158) pg/ml vs 300 (114-1192) pg/ml; p<0.001). The mean
log-10-serum NT-proBNP values were 3 (0.52) pg/ml. The mean serum
Z-log-NT-proBNP was 1.41 (0.91), with 11 (30%) samples showing raised
serum NT-proBNP (Z-log > 1.96). The median urine
NT-proBNP/creatinine ratio was 15.3 (4.8-66.6) pg/mg, and the mean
log-(10)-urine NT-proBNP/Creatinine ratio was 1.26 (0.67) pg/mg. The
log-(10)-urine NT-proBNP/Creatinine ratio was positively and strongly
correlated with the log-10-serum-NT-proBNP concentrations (r = 0.790;
p<0.001). This correlation resulted improved when using the
serum Z-log-NT-proBNP values (r = 0.867; p<0.001). The scatter
plots for these relationships are shown in Figure 1 and fitted by the
following linear equations: 1) log-10-serum-NT-proBNP = 2.27 + 0.616 x
log10-urine NT-proBNP/Creatinine ratio (R-squared coefficient = 0.624; p
< 0.001); and 2) Serum Z-log-NT-proBNP = -0.07 + 1.17 x
log10-urine NT-proBNP/Creatinine ratio (R-squared coefficient = 0.751; p
< 0.001). The log-10-urine NT-proBNP/creatinine ratio showed
also a moderate positive correlation with CRP, procalcitonin and pCO2
levels, and a moderate negative correlation with the weight (Table 2).
Regarding the association of initial urinary NT-proBNP values with
severity in these cohort (n=17), we found that
log-10-NT-proBNP/Creatinine ratio was higher at the time of hospital
admission in those infants that required PICU admission with ventilatory
support compared with those without this management (1.85 (1.16-2.44)
pg/mg vs 0.63 (0.45-0.84) pg/mg); p<0.001) (Figure 2), and
resulted positively and strongly correlated with the duration of the
ventilatory support (rho=0.76; p<0.001) and the LOS
hospitalization (rho=0.84; p<0.001).
4. Discussion
4.1. Main findings: This pilot study showed the feasibility of
analyzing urinary NT-proBNP in young infants with acute bronchiolitis
with the Alere NT-proBNP, for Alinity i assay. We observed a strong
positive association between serum and urinary NT-proBNP concentrations
in this setting, with both parameters being higher in patients who
developed more severe disease requiring longer hospitalizations.
4.2. Feasibility of urinary NT-proBNP in acute bronchiolitis:Several studies have demonstrated that NT-proBNP levels are detectable
in the urine of preterm newborns and infants with congenital heart
diseases (CHD). These investigations have reported significant
associations between elevated urinary NT-proBNP concentrations and
neonatal morbidities such as hemodynamically significant persistent
ductus arteriosus, retinopathy of prematurity, and bronchopulmonary
dysplasia with pulmonary hypertension
[12,14,18,19].
Urinary NT-proBNP also seems promising as a screening tool for
congenital heart diseases in newborns and has shown potential to
differentiate simple and complex CHD
[11,20].
Recently, urinary-NT-proBNP has been helpful in the pediatric ambulatory
setting to assess heart failure in children with congenital heart
diseases when combined with clinical scores
[21]. Like these
populations, obtaining blood samples for testing in acute bronchiolitis
is technically challenging for the healthcare provider and stressful and
painful for the patient, especially in cases where repeated tests are
needed for monitoring evolution. Evidence shows that it is possible to
collect urine non-invasively, efficiently, and quickly, especially in
children under three months of age, who constitute most of the
hospitalized population with bronchiolitis
[22,23]. With
this study, we point out that urinary NT-proBNP can be adequately
analyzed with the same laboratory kit as serum NT-proBNP (Alere i) in
acute bronchiolitis. Therefore, using urine samples could be beneficial
in these patients as it would replace the need for stressful blood
sampling to measure NT-proBNP, even more, if repeated samples for
monitoring evolution are required.
4.3. Urinary NT-proBNP as a surrogate of serum NT-proBNP:Several previous studies in adults demonstrate that NT-proBNP levels are
detectable in the urine of patients with heart failure with a good
correlation with plasma NT-proBNP levels in matched measurements
[24,25]. The
evidence on whether urinary NT-proBNP can replace serum NT-proBNP as a
biomarker in pediatrics is scarce. In 2011, Kurihara et al. measured the
serum and urinary NT-proBNP levels in 36 samples from 9 neonates aged
0–25 days and reported a significant correlation with an R-squared
coefficient of 0.548 between those variables
[26]. Recently,
Muller et al. studied the correlation between plasma and urine NT-proBNP
in 83 children undergoing cardiac surgery using age-adjusted values for
age and creatinine correction as we did. Notably, they also observed a
significant strong positive correlation between the two parameters (r =
0.78 preoperatively and 0.87 postoperatively; p<0.001)
[11]. Another
small sized study (n=33) by these authors also showed an excellent
correlation between plasma and urine NT-proBNP levels in 33 children
with CHD (r=0.902)
[26]. Our results
are consistent with previous evidence, suggesting that urinary NT-proBNP
concentrations could be used to surrogate serum levels in the acute
bronchiolitis setting.
4.4 . Association of urine NT-proBNP with severity:There is increasing evidence supporting the role of NT-proBNP as a
biomarker for myocardial strain in infants with severe bronchiolitis.
Recent work shows that serum NT-proBNP levels are associated with
echocardiographic signs of pulmonary hypertension and subclinical
myocardial dysfunction. It could help screen patients with a worse
clinical evolution when used in conjunction with clinical scores
[5,27,28]. In
acute bronchiolitis, the airway obstruction and inflammation would
affect pulmonary vascular tone increasing the right ventricular
afterload
[8,29,30],
leading to an increased release of NT-proBNP. This could be an
explanation for the significant association observed between elevated
urinary NT-proBNP concentrations and increased pCO2, CRP, and
procalcitonin levels at any time of hospitalization in our patients. As
the significant stimuli that upregulate the NT-proBNP synthesis from the
ventricular myocardium are conditions of sustained ventricular blood
volume and pressure overload, the enhancement of NT-proBNP synthesis and
secretion would be more significant in those infants with acute
bronchiolitis with a more severe respiratory impairment that will need
PICU admission and mechanical ventilation. Noteworthy, increased values
of urinary NT-proBNP at early stages of hospitalization and before
initiation of inotropic or ventilatory support were higher in those
infants requiring PICU admission and resulted strongly associated with
longer respiratory support and LOS hospitalization, our clinical
outcomes indicative of severe bronchiolitis in this study. We found only
one previous work evaluating the utility of urinary NT-proBNP in acute
bronchiolitis. Çullas-İlarslan et al. designed a prospective
non-randomized study that included 160 patients diagnosed with lower
respiratory tract infection
[31]. They also
demonstrated the feasibility of analyzing urine NT-proBNP in this
setting, but contrary to our observations, they did not find differences
between severity groups. However, these authors included a very
heterogeneous population with mixed bronchiolitis and pneumonia cases
and a wide age range from 0 to 6 years. This fact, joined with the
different laboratory kit assays used to measure NT-proBNP, could explain
the differences between studies. In our study, urine and serum
concentration of NT-proBNP resulted strongly and positively correlated.
Therefore, it is not surprising that urine NT-proBNP presented similar
results for clinical outcomes of severity in acute bronchiolitis than
those previously reported for serum NT-proBNP, pointing out the
potential value of the measurement of urinary NT-proBNP as a
non-invasive tool to assess severity in acute bronchiolitis. A growing
body of evidence shows the applicability and benefits of non-invasive
complementary exams such as cardiopulmonary ultrasound to evaluate the
severity of acute bronchiolitis in previously healthy infants
[32–34]. Our
findings suggest that urinary NT-proBNP measurements may be another
useful non-invasive tool for this, overall, in settings where ultrasound
expertise is not available. As there are no standard values for urine
NT-proBNP concentrations and we were not powered for cut-off
estimations, we could not establish a reliably cut-off point for
outcomes in this study.
4.5. Limitations : The small sample size and exploratory nature
of this study that included mostly moderate to severe cases (35%
required ventilatory support) of acute bronchiolitis preclude the
generalization of our results. The diagnostic performance of NT-proBNP
assays in urine may be assay-specific, necessitating validation of
biomarker performance on an assay-by-assay basis
[35,36].
Therefore, our results would not be fully comparable with studies using
a different assay for NT-proBNP measurement. Although we acknowledge
that the Alere NT-proBNP for Alinity i assay has not been previously
validated to determine urinary NT-proBNP, our correlation analysis
suggests that the urinary levels provided by this assay adequately
reflect NT-proBNP serum levels. Finally, routine echocardiographic
evaluation was not performed unless suspicion of heart disease or
failure was raised. Therefore, data about the cardiac status were not
systematically recorded, and we could miss the diagnosis of simple CHD.
We believe that this limitation does not alter our results as all
patients were discharged without the need for any specific cardiac
treatment. Despite these limitations, our results are strengthened by
the methodology used, overcoming the major application limitation in
pediatrics, the strong age dependence. We also controlled the variations
of urinary concentration of NT-proBNP across the day with correction by
urinary creatinine, as samples were taken at different times for each
patient. Therefore, our results are promising and encourage us to
continue the study by recruiting more patients.
5. Conclusions
The present study demonstrated a strong positive correlation between the
serum and urinary concentrations of NT-proBNP in a small cohort of
infants with acute bronchiolitis. We further confirmed a significant
association of elevated urinary NT-proBNP concentrations with clinical
outcomes indicative of severity in this setting. These findings suggest
that the measurement of urinary NT-proBNP concentrations could be a good
and reliable surrogate for serum NT-proBNP levels and highlight the
potential value of the measurement of urinary NT-proBNP as a
non-invasive tool to assess severity in acute bronchiolitis. Due to the
limitations of our study, further research on urine NT-proBNP
measurement in the setting of acute bronchiolitis is warranted to
clarify its potential role as a non-invasive biomarker.