Surfactant Deficiency in Infants with Severe Acute Viral Bronchiolitis Francine Hartmann, PT, MSc1, Humberto Holmer Fiori, MD, PhD1, Pedro Celiny Ramos Garcia, MD, PhD1, Jefferson Piva, PhD2, and Renato Machado Fiori, MD, PhD1 Objectives To evaluate surfactant content and function through the lamellar body count (LBC) and stable microbubble test (SMT) in mechanically ventilated infants with severe acute viral bronchiolitis.

Study design Controlled cross-sectional study of 32 infants receiving mechanical ventilation: 16 with a diagnosis of acute viral bronchiolitis and 16 with normal lungs. Tracheal fluid was collected and LBC was performed in an automated cell counter. Samples were kept frozen and thawed for testing. At the time of analysis, samples were diluted in a dithiothreitol solution, vortexed for 10 seconds, and aspirated by the cell counter. SMT was performed using the Pattle technique. Results In the bronchiolitis group, the median (IQR) LBC was significantly lower than in the control group: 130 000 (61 250-362 250) vs 518 000 (180 250-896 000) lamellar bodies/mL; P = .003. Median (IQR) SMT values were also significantly lower in the bronchiolitis group: 10 (2-13) vs 400 (261-615) microbubbles/mm2; P < .001. Conclusions Infants with acute viral bronchiolitis have reduced surfactant content and function. We speculate that these simple tests may be useful to identify infants with bronchiolitis who would benefit from surfactant replacement therapy. (J Pediatr 2014;-:---).

A

cute viral bronchiolitis is the most common lower respiratory tract infection of infants and children, and a common cause of hospitalization among children #3 years of age. It is mainly caused by the respiratory syncytial virus, which accounts for 50%-80% of cases, but other viral etiologies occur. The condition is seasonal, and causes inflammation and obstruction of the lower airways.1-4 It is speculated that bronchiolitis is associated with surfactant deficiency. Studies have reported changes in surfactant proteins and in surface tension measured with the pulsating bubble surfactometer or by the click test in tracheal aspirates from children with bronchiolitis.5-7 In studies that tested the administration of exogenous surfactant, the predominant effects were a significant improvement in oxygenation, duration of mechanical ventilation, and length of pediatric intensive care unit (PICU) stay.8-10 Assessment of surfactant production and function in neonates has been performed by means of the lamellar body count (LBC) and stable microbubble test (SMT), which have yielded good accuracy for the diagnosis of respiratory distress syndrome (RDS).11-13 The objective of the present study was to assess surfactant content and function by means of the LBC and SMT tests in mechanically ventilated infants with severe acute viral bronchiolitis.

Methods This study was conducted in the PICU of Hospital S~ao Lucas, the teaching hospital of Pontifıcia Universidade Cat olica do Rio Grande do Sul, Porto Alegre, Brazil, from June 2011 through March 2013. The study was approved by the institutional Research Ethics Committee and informed consent for participation was obtained from 1 of the parents or legal guardians of each patient. The study sample included infants (age #12 months) admitted to the PICU with acute viral bronchiolitis receiving mechanical ventilation (bronchiolitis group). Diagnosis was established by the attending physician on the basis of the following clinical findings: first episode of acute-onset expiratory wheezing and signs of viral respiratory illness, such as nasal discharge, cough, and fever, accompanied by breathing difficulty.14 The control group consisted of age-matched patients (age #12 months) who were on mechanical ventilation for postoperative management or other clinical conditions, regardless of whether they were admitted to the PICU, and who did not have any acute or chronic cardiopulmonary conditions. The purpose of this control

DTT LB LBC PICU RDS SMB SMT

Dithiothreitol Lamellar body Lamellar body count Pediatric intensive care unit Respiratory distress syndrome Stable microbubble Stable microbubble test

From the 1Department of Pediatrics, Pontifıcia  lica do Rio Grande do Sul (PUCRS); Universidade Cato and 2Department of Pediatrics, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2014.02.030

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group was to establish a normal reference range for lamellar body (LB) and stable microbubble (SMB) counts. Data on demographics, ventilation settings at the time of tracheal aspirate collection, arterial blood gases, and markers of severity and clinical course—such as the pediatric index of mortality, PaO2/fraction of inspired oxygen ratio, highest peak inspiratory pressure, duration of vasoactive drug use, and length of stay—were collected from patients in the bronchiolitis group. Furthermore, the most recent imaging studies of the lungs before tracheal aspirate collection were examined by a radiologist with particular experience in pediatric imaging. Radioimmunoassays were performed for viruses in tracheal aspirate specimens per routine PICU protocols. In both the bronchiolitis and the control groups, all treatment-related decisions were entirely at the discretion of the providers responsible for patient care. Tracheal aspirate samples were obtained by a staff physical therapist per routine institutional protocols.13 In short, mechanical respiration was briefly interrupted and a 0.5-mL aliquot of saline solution was instilled via the endotracheal tube. The patient was then reconnected to the ventilator. After a few respiratory cycles, the ventilator was disconnected again and a catheter was gently advanced through the endotracheal tube until resistance was felt. The tube was suctioned as the probe was gently withdrawn, and the resulting tracheal aspirate was collected into a closed suction system. Samples were frozen and kept at 20 C for 48-96 hours. After thawing at room temperature, the SMT was performed by means of the adapted technique by Pattle et al.13,15 All counting procedures were performed by an experienced examiner who was blinded to sample provenance. LBC was performed in the hematology laboratory of the Hospital S~ao Lucas, the teaching hospital of Pontifıcia Universidade Cat olica do Rio Grande do Sul. To measure SMT, an aliquot of 40 mL of tracheal aspirate was suctioned into a Pasteur pipette (BRAND GMBH + CO KG, Wertheimer, Germany) with an 11.5 cm stem and 1 mm diameter and placed on a count chamber (Neubauer Improved Bright-Line, Optik Labor, Germany) without the slide cover. With the pipette held vertically, its tip almost touching the counting chamber, the aliquot was suctioned in and quickly expelled out 20 times. It was then expelled over the counting chamber, which was immediately inverted and placed under a binocular microscope, forming a hanging drop. After 4 minutes, the count area was examined with a magnification of 10 10, and the microbubbles—bubbles smaller than 15 mm—were counted. Five of the 25 squares forming 1 mm2 were counted (1 square on each quadrant and the central square). If less than 200 SMBs were present per mm2 (SMB/mm2), the entire mm2 was considered. The measurements could not be duplicated because of the limited volume of the samples. LBC was performed with an automated cell counter Sysmex XT-1800i cell counter (Sysmex Corporation, Kobe, Japan) in the hematology laboratory at Hospital S~ao Lucas. Tracheal samples (25-50 mL) were diluted in a liquefying agent (dithiotreitol) without centrifugation. To perform 2

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the LBC, a 10 mg/mL solution of dithiothreitol (DTT; Invitrogen Corporation, Carlsbad, California) in distilled water was prepared in advance and kept frozen at 20 C in Eppendorf tubes until use. Samples were placed in a test tube containing the DTT solution at a ratio of 1 part tracheal aspirate to 6 parts DTT. This sample was vortexed for 10 seconds, and the material was aspirated by the automated Sysmex XT1800i cell counter (Sysmex Corporation). LBC was performed using the platelet channel. All results obtained were multiplied by 7 to correct for dilution. After specimen collection, patients with acute viral bronchiolitis were followed until PICU discharge or hospital day 28. Statistical Analyses The minimum sample size was calculated as 10 patients per group, for an alpha level of 5%, a statistical power of 80%, and expected rates of the factor of interest (surfactant deficiency) of 70% in the exposure group and 10% in the control group. Asymmetrically distributed data were expressed as medians and IQRs. The Mann–Whitney U and c2 tests were used for comparison of continuous and categorical variables respectively. Spearman rank correlation coefficients were used to test for correlation between LBC and SMT results and the ventilator settings and vasoactive drugs duration.

Results The sample was comprised of 32 patients: 16 in the bronchiolitis group and 16 in the control group (Table). There were no differences between the 2 groups regarding weight, age, or sex. Nine (56.3%) patients in the bronchiolitis group were positive for respiratory syncytial virus, 2 (12.5%) were positive for another virus, and no viruses could be detected in the remaining 5 (31.3%), in whom diagnosis was made on a clinical basis. In the control group, 13 patients (81.3%) were postabdominal surgery status, 1 (6.3%) was postneurosurgery status, 1 (6.3%) was admitted to the PICU for refractory seizures, and 1 (6.3%) had a metabolic disorder. In both groups, most patients (62.5% in the bronchiolitis group and 81.2% in the control group) were born full-term, whereas the remainder were born premature. As expected, peak inspiratory pressure and positive end-expiratory pressure at the time of tracheal aspirate collection were higher in the bronchiolitis group than in the control group (Table). Median (IQR) LBCs were significantly lower in the bronchiolitis group than in the control group: 130 000 (61 250362 250) vs 518 000 (180 250-896 000) LBs/mL, P = .003 (Figure 1). Likewise, SMB counts were also significantly lower in the bronchiolitis group than in the control group: 10 (2-13) vs 400 (261-615) SMB/mm2, P < .001 (Figure 2). Of the 16 patients with bronchiolitis, 10 (62.5%) had LBCs below the 25th percentile (180 250/mL) observed in the control group. Furthermore, all patients with bronchiolitis had SMB counts below the 25th percentile (261 SMB/mm2) for the control group. Hartmann et al

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ORIGINAL ARTICLES

Table. Patient characteristics

Demographic data Age (mo) Weight (g) Ventilator measurements PIP (cm H2O) FiO2 RR (bpm) PEEP (cm H2O) Blood gases pH PaO2 (mm Hg) PaCO2 (mm Hg) HCO3 (mEq/L) SaO2 (%) BE Severity and clinical course PIM (%) PaO2/FiO2 (mm Hg) >PIP (cm H2O) Duration of vasoactive drug use (d) Duration of mechanical ventilation (d) Length of stay (d)

Bronchiolitis group N = 16 median (IQR)

Control group N = 16 median (IQR)

3.5 (2-5) 4850 (3925-6150)

3.5 (2-9) 4167 (3174-8375)

.607 .749

34 (30.3-37.5) 0.3 (0.3-0.39) 18 (16-20) 5 (5-7)

22 (20-25) 0.3 (0.25-0.37) 20 (14-29) 5 (4-5)

PIP, highest peak inspiratory pressure; BE, base excess; FiO2, fraction of inspired oxygen; HCO3, bicarbonate; PEEP, positive end-expiratory pressure; PIM, pediatric index of mortality; PIP, peak inspiratory pressure; RR, respiratory rate; SaO2, oxygen saturation. *Eight patients.

There was no correlation between LBC and microbubble counts and pediatric index of mortality, time on ventilation, ventilation measurements, and length of stay. All patients survived and were discharged before 28 days. The radiologic findings of all patients in the bronchiolitis group were deemed consistent with acute viral bronchiolitis by the examining radiologist.

Discussion In clinical practice, most patients with acute viral bronchiolitis have an uneventful clinical course. However, a small percentage of these patients develop severe respiratory failure requiring mechanical ventilation.16 The present study found abnormal SMT and LBCs in the tracheal aspirates of mechanically ventilated infants with a clinical diagnosis of severe bronchiolitis. The low LB and SMB counts of babies with bronchiolitis as compared with those of age-matched controls suggest a deficiency in surfactant function. Reductions in surfactant protein A and disaturated phosphatidylcholine have been previously reported in infants with bronchiolitis. This deficiency of surfactant function may contribute to small airway obstruction and collapse.17 Furthermore, increased mucus production in the airways may also contribute to obstruction, especially as surfactant depletion leads to increased mucus viscosity and may limit mucus clearance.18 Five patients with bronchiolitis had their tracheal aspirate collected in the first day, 6 in the second day, and 5 in the

Figure 1. Box plot of LBC (LBC/mL) in infants in the bronchiolitis and control groups.

third. The results were very similar in the 3 days: LB 263 666/mL, SMB 8/mm2 in the first day; LB 269 500/mL, SMB 11/mm2 on the second day; and LB 236 250/mL, SMB 14/mm2 on the third day. These data suggest that ventilation does not significantly affect LBC and microbubble count. Data on the efficacy of exogenous surfactant administration in patients with severe bronchiolitis are scarce. A recent Cochrane Review found only 3 studies of sufficient quality for inclusion in a meta-analysis, with a total of 79 patients.19 In all 3 studies, surfactant administration was associated with reductions in duration of mechanical ventilation and length of PICU stay, as well as with transient improvement in blood gas measurements.8-10 In 2 of these studies, the dose of surfactant administered was only 50 mg/kg—far below the recommended dose for correction of pulmonary immaturity and RDS in preterm neonates. None of the studies assessed endogenous surfactant production or function. In the present study, SMB counts were low among the infants with bronchiolitis, but there was significant withingroup variation. Conversely, a large proportion of infants in the bronchiolitis group had LBCs within the range found in the control group. This variability provides evidence of the discriminatory potential of these tests for identification of patients who may have more significant changes in the surfactant system. Unlike previous studies, this investigation employed 2 tests that assess 2 different aspects of the surfactant system in tracheal secretions: the LBC, which assesses the presence of surfactant in the form secreted by alveolar cells, and the SMT, which indirectly evaluates surfactant function. Both tests are quantitative, rapid, and very inexpensive, and may, thus, be appropriate for routine clinical use.12,13 Unlike RDS, which requires a very quick answer because surfactant should be given as early as possible, bronchiolitis usually

Surfactant Deficiency in Infants with Severe Acute Viral Bronchiolitis

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in patients with acute viral bronchiolitis and comparing outcomes with pretreatment LBC and SMT findings. n Submitted for publication Oct 29, 2013; last revision received Jan 14, 2014; accepted Feb 12, 2014.

References

Figure 2. Box plot of SMB counts (SMB/mm2) in infants in the bronchiolitis and control groups.

does not require immediate results, which means tests can be performed in a laboratory setting. There are no data on the cutoff point that defines severe disease in patients with bronchiolitis. Based on data on RDS of prematurity, the microbubbles in bronchiolitis were considerably below the cutoff point for RDS, which was associated with severe surfactant deficiency.12,13 LBs were also low, and most bronchiolitis patient counts were below the cutoff point for RDS. Exogenous surfactant therapy was not administered in this study. Future investigations should ascertain whether an association exists between test results indicative of significant surfactant deficiency and clinical response to surfactant therapy. One limitation of this study is that, despite standardization of the method for tracheal suction, there is an expected variability of dilution due to the different amounts of saline and tracheal fluid retrieved. The microbubble test is operatordependent and has some intrinsic variability. The difference in microbubble counts between groups is great enough to make us believe the variability found does not significantly affect the results. In the LBC, there is some variability too, even though a machine performs the counting, because the dilution of the samples may not be the same. However, the collection method used herein is probably among the most feasible for routine clinical use. In conclusion, the present study demonstrated significant changes in the surfactant system, as demonstrated by LBCs and by the SMT, among mechanically ventilated infants with acute viral bronchiolitis. We speculate that these 2 tests are able to predict which patients with this condition can benefit most from exogenous surfactant therapy. The utility of these tests in severe bronchiolitis can only be established by assessing clinical outcomes after surfactant administration

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1. Barreira ER, Precioso AR, Bousso A. Pulmonary surfactant in respiratory syncytial virus bronchiolitis: The role in pathogenesis and clinical implications. Pediatr Pulmonol 2011;46:415-20. 2. Carvalho WB, Johnston C, Fonseca MC. Acute bronchiolitis, an updated review. Rev Assoc Med Bras 2007;53:182-8. 3. Hervas D, Reina J, Yanez A, del Valle JM, Figuerola J, Hervas JA. Epidemiology of hospitalization for acute bronchiolitis in children: differences between RSV and non-RSV bronchiolitis. Eur J Clin Microbiol Infect Dis 2012;31:1975-81. 4. Walker C, Danby S, Turner S. Impact of a bronchiolitis clinical care pathway on treatment and hospital stay. Eur J Pediatr 2012;171:827-32. 5. Dargaville PA, South M, McDougall PN. Surfactant abnormalities in infants with severe viral bronchiolitis. Arch Dis Child 1996;75:133-6. 6. Kerr MH, Paton JY. Surfactant protein levels in severe respiratory syncytial virus infection. Am J Respir Crit Care Med 1999;159:1115-8. 7. Skelton R, Holland P, Darowski M, Chetcuti PA, Morgan LW, Harwood JL. Abnormal surfactant composition and activity in severe bronchiolitis. Acta Paediatr 1999;88:942-6. 8. Luchetti M, Casiraghi G, Valsecchi R, Galassini E, Marraro G. Porcinederived surfactant treatment of severe bronchiolitis. Acta Anaesthesiol Scand 1998;42:805-10. 9. Luchetti M, Ferrero F, Gallini C, Natale A, Pigna A, Tortorolo L, et al. Multicenter, randomized, controlled study of porcine surfactant in severe respiratory syncytial virus-induced respiratory failure. Pediatr Crit Care Med 2002;3:261-8. 10. Tibby SM, Hatherill M, Wright SM, Wilson P, Postle AD, Murdoch IA. Exogenous surfactant supplementation in infants with respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med 2000;162:1251-6. 11. Daniel IW, Fiori HH, Piva JP, Munhoz TP, Nectoux AV, Fiori RM. Lamellar body count and stable microbubble test on gastric aspirates from preterm infants for the diagnosis of respiratory distress syndrome. Neonatology 2010;98:150-5. 12. Eckert Seitz E, Fiori HH, Luz JH, Fiori RM. Stable microbubble test on tracheal aspirate for the diagnosis of respiratory distress syndrome. Biol Neonate 2005;87:140-4. 13. Vieira AC, Fiori HH, Garcia PC, Piva JP, Munhoz TP, Fiori RM. Lamellar body count and stable microbubble test on tracheal aspirates from infants for the diagnosis of respiratory distress syndrome. Pediatr Crit Care Med 2012;13:178-82. 14. Prais D, Schonfeld T, Amir J , Israeli Respiratory Syncytial Virus Monitoring Group. Admission to the intensive care unit for respiratory syncytial virus bronchiolitis: a national survey before palivizumab use. Pediatrics 2003;112:548-52. 15. Pattle RE, Kratzing CC, Parkinson CE, Graves L, Robertson RD, Robards GJ, et al. Maturity of fetal lungs tested by production of stable microbubbles in amniotic fluid. Br J Obstet Gynaecol 1979;86:615-22. 16. Eber E. Treatment of acute viral bronchiolitis. Open Microbiol J 2011;5: 159-64. 17. Enhorning G, Duffy LC, Welliver RC. Pulmonary surfactant maintains patency of conducting airways in the rat. Am J Respir Crit Care Med 1995;151:554-6. 18. Rubin BK, Ramirez O, King M. Mucus rheology and transport in neonatal respiratory distress syndrome and the effect of surfactant therapy. Chest 1992;101:1080-5. 19. Ventre K, Haroon M, Davison C. Surfactant therapy for bronchiolitis in critically ill infants. Cochrane Database Syst Rev 2006;CD005150.

Hartmann et al

Surfactant deficiency in infants with severe acute viral bronchiolitis.

To evaluate surfactant content and function through the lamellar body count (LBC) and stable microbubble test (SMT) in mechanically ventilated infants...
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