Lung ultrasound immediately after birth predicts the need for surfactant therapy in very- and extremely preterm infants, the DOLFIN Jr Study

Early identication of infants requiring surfactant therapy improves outcomes. We evaluated the accuracy of delivery lung ultrasound (LUS) to predict in and extremely preterm infants.


Background
Non-invasive respiratory support has replaced routine mechanical ventilation and surfactant therapy as the initial respiratory strategy for extremely premature infants with respiratory distress syndrome (RDS) [1].However, over 50% of infants eventually require surfactant and early surfactant decreases the risk of death and signi cant pulmonary injury in infants with RDS compared to delayed treatment [1][2][3][4].Infants with RDS often have oxygen requirements that increase gradually until a predetermined threshold is reached for surfactant [5].Thus, identifying infants in the delivery room who will eventually require exogenous surfactant may be bene cial.
Animal studies have shown that immediately after birth, surfactant therapy increases both the uniformity and degree of lung aeration, which increases the surface area for gas exchange [6,7].At birth, the surface area for gas exchange is initially low but increases exponentially as the lung aerates [8].Increasing the fraction of inspired oxygen (FiO 2 ) increases the partial pressure gradient for oxygen diffusion and counteracts the effect of a low surface area on oxygen exchange of the partially liquid-lled lung.Thus, a higher FiO 2 in the delivery room likely indicates poor lung aeration, whereas surfactant therapy can increase the degree of lung aeration and thereby reduce the oxygen requirement.
We aimed to characterize changes in LUS images immediately after birth for infants born at < 32 0/7 weeks gestation and evaluate the diagnostic accuracy of LUS in predicting the need for surfactant therapy.

Methods
We performed a prospective, observational study of infants born at < 32 0/7 weeks gestational age between January 2018 and December 2019.Study sites were two perinatal hospitals, Monash Medical Centre (MMC) and the Royal Women's Hospital (RWH), in Melbourne, Australia, together averaging more than 300 very and extremely preterm births per year.This study is reported in accordance with the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines [38,39].

Study Design
Infants born at < 32 0/7 weeks without an antenatal diagnosis of signi cant cardiopulmonary pathology (e.g.diaphragmatic hernia or cyanotic congenital heart malformation) were eligible for participation if a researcher was available to perform LUS scans.Antenatal parental consent was sought if time permitted at both hospitals.At MMC, infants were eligible for written, retrospective parental consent if there was insu cient time to discuss the study prior to birth.The study was approved by the human research ethics committees at both institutions and registered with the Australian New Zealand Clinical Trials Registry (ACTRN12617001256369).
At both institutions, infants were initially managed with CPAP, using pressures of 5-8cmH 2 O and FiO 2 0.3, titrated to maintain SpO 2 91-95%.Infants requiring more support to maintain SpO 2 in this range were considered for intubation and surfactant therapy or minimally invasive surfactant therapy (MIST) [37,40].Indications for intubation while on CPAP included frequent apneas requiring stimulation, regardless of FiO2 requirement.Intubation-surfactant-extubation (INSURE) was not practiced at either institution [41].At the RWH, high ow nasal cannula could be used for primary respiratory support following admission in infants ≥ 30 weeks with an FiO 2 requirement ≤ 0.3 [42].Nasal intermittent positive pressure ventilation was not used as a primary treatment strategy [43].

Data Collection
We obtained serial LUS video recordings using a GE Venue 50 ultrasound machine (GE Healthcare, Chicago, USA) and a "hockey-stick," L8-18i linear transducer with a depth of 2 cm and a gain of 60.LUS was performed at 5-10 minutes, 11-20 minutes, 1-3 hours, then every 24 hours in the rst 72 hours after birth.
The LUS transducer was placed in the infant's axillae with the notch pointed towards the infant's head.The transducer was 3.5 cm long, typically capturing images from 2-3 intercostal spaces.During the rst three timepoints, the infants were assessed in a supine position.At subsequent timepoints, the infants could be assessed in either prone or supine positions, thereby minimizing handling.The transducer was adjusted until a "bat sign" was achieved and the image was optimized for the sharpest pleural line, most aeration, and least uid retention [16,44,45].Interpretation of LUS ndings were not provided to the clinical team unless the researcher detected additional pulmonary pathology (e.g.pneumothorax, effusion, congenital defect).Mode of respiratory support, CPAP level or mean airway pressure, and FiO 2 were collected by the researcher at the time of each LUS.We also collected clinical and demographic information until discharge from hospital.
Lung Ultrasound Grading LUS videos were scored at the end of study recruitment by 2 assessors blinded to all clinical data (SB and DB) using a previously validated system that grades each side of the chest on a scale of type 0-3 (Fig. 1) [9-11, 28, 29, 46].Along this scale, type 1 (whiteout) is characterized by coalescence of "B-lines" representing retention of lung liquid/atelectasis, and type 3 is characterized by a lack of B-lines and the presence of "A-lines", representing full lung aeration.In instances of score disagreement, a third blinded assessor acted as an arbiter (OK).Unilateral type 1 or worse was considered test positive.

Analysis
In the absence of data allowing formal sample size calculation, we pre-speci ed a convenience sample of 50 participants for this observational study.Means and standard deviations with Student's t-test for comparisons are reported for normally distributed continuous variables.Medians with interquartile ranges with Mann Whitney U test for continuous variables or Wilcoxon signed rank test for ordinal variables, are reported when the distribution was skewed.The scores of the right and left side of the chest of each infant at each timepoint were summed and analyzed using a Friedman's test to determine changes in LUS over time.Three investigators, blinded to the infant's clinical condition, independently assigned LUS scores for 100 images.Interobserver agreement was tested using Kendall's W coe cient of concordance.We used Receiver Operating Characteristic (ROC) analysis to determine and compare the accuracy of LUS performed on infants receiving non-invasive ventilation at 5-10 minutes, 11-20 minutes, and 1-3 hours, and of FiO 2 at the time of each LUS, to predict ST using the DeLong method [47].
We used Youden's method to determine the optimum cut-off score for LUS and threshold for FiO 2 [48].For the selected cut-off values, we report test characteristics in terms of sensitivity, speci city, positive predictive and negative predictive values with corresponding 95% con dence intervals (CIs).
We used the pROC package in R version 3.6.2(R Foundation, Vienna, Austria) and MedCalc for Windows, version 15.0 (MedCalc Software, Ostend, Belgium) for analysis [49].Other statistical analyses were performed using IBM SPSS Statistics 21.0 (SPSS, Inc, Chicago, USA).Statistical signi cance was de ned as p < 0.05.

Results
Fifty-two infants < 32 0/7 weeks postmenstrual age were enrolled.Demographic and clinical details are shown in Table 1.We included 463 video recordings for analysis with no exclusions due to image quality.There were no pneumothoraces, pleural effusions, or congenital lung malformations.Infants were stable throughout all studies with no signi cant changes in temperature or oxygen requirement immediately following LUS assessments.Kendall's W coe cient of concordance con rmed excellent inter-rater reliability between three assessors (W = 0.96, p < 0.001), which was consistent with previous studies [28,29].No LUS images were obtained for 2 infants at the 5-10 minute timepoint due to the absence of an investigator (Fig. 2).Twenty-two infants were managed with non-invasive respiratory support and did not receive surfactant therapy.Ten additional infants did not require intubation but received surfactant therapy via MIST.Twenty infants were intubated by 72 hours after birth, all received surfactant.
Lung ultrasound versus fraction of inspired oxygen to predict surfactant Diagnostic accuracy evaluation was performed independently for each study timepoint for all infants until they were intubated, upon which subsequent assessments did not contribute to the diagnostic evaluation (Fig. 2).The optimal thresholds for predicting ST across timepoints were type 1 on at least one side of the chest (type 1/2) for LUS, and FiO 2 of 0.3.Using ROC analysis of diagnostic ability (Fig. 3), we found that LUS had an area under the curve (AUC) at 5-10 minutes, 11-20 minutes, and 1-3 hours of 0.78 (95% CI 0.66-0.90),0.76 (95% CI 0.65-0.88)and 0.86 (95% CI 0.75-0.97)respectively.FiO 2 produced lower AUCs of 0.45 (95% CI 0.29-0.62),0.60 (95% CI 0.44-0.76)and 0.81 (95% CI 0.68-0.95) at the same three points after birth respectively.There was a statistically signi cant difference in AUC for LUS compared to FiO 2 at the rst (5-10 minute) timepoint (p = 0.001), but not at the 11-20 minute (p = 0.088) or 1-3 hour timepoints (p = 0.473).False positives and false negatives to predict surfactant: LUS and FiO 2 Eleven infants with an FiO 2 requirement > 0.3 in the delivery room did not receive surfactant therapy and were considered false positives for FiO 2 requirement.Ten of the 11 (91%) infants had LUS type 2/2 or better and were correctly identi ed by LUS as not needing surfactant therapy.There were 20 infants with an FiO 2 requirement < 0.3 in the delivery room who eventually received surfactant therapy and were considered false negatives for FiO 2 requirement.Fourteen of the 20 (70%) infants had LUS type 1/2 or worse and were correctly identi ed by LUS as needing surfactant therapy.
Eighty-ve and 95% of infants who did not receive ST had LUS 2/2 or better at 5-10 and 11-20 minutes respectively.Three infants with type 2/2 at the 1-3 hour assessment who received ST were 23-27 weeks (Fig. 4).Two were intubated for apneas with a low FiO 2 requirement and received surfactant after intubation.The third received ST via MIST.No infant ≥ 28 weeks with type 2/2 or better after neonatal intensive care unit (NICU) admission received ST.All infants born at 23-27 6/7 weeks with type 1/2 LUS or worse received ST.There were 3 infants with type 1/2 or worse in the delivery room who did not receive ST and were considered false positives.All three infants were ≥ 30 weeks and ≥ 1400 g at birth.
Twenty-six infants (50%) had lower LUS grades on subsequent assessments compared to their previous LUS score, either in the delivery room (11-20 minutes, 6 infants) or on admission to the NICU (1-3 hours, 20 infants).We have termed this "backsliding" [29].Infants with backsliding while on CPAP had a longer duration between rupture of membranes and birth (p = 0.046), otherwise, there were no signi cant predictive patient characteristics for backsliding while on CPAP (Table 3).Infants with backsliding while on CPAP were more likely to require intubation than infants on CPAP who did not have backsliding (p = 0.01).Seven of the eight (88%) infants intubated in the delivery room had backsliding following intubation, prior to the administration of surfactant in the NICU.Two of these infants had backsliding twice, seen at 11-20 minutes while on CPAP and, again, after intubation, at the 1-3 hour timepoint.
There were 10 infants that had a grade of 0.5 on at least one side of the chest, at a minimum of one timepoint.All 10 infants received surfactant therapy and 9/10 were intubated.No grades of 0.5 were seen at 5-10 minutes.In 4 infants, type 0.5 was observed only after intubation including the only infant with type 0.5 at 11-20 minutes.
Response to surfactant therapy and lung ultrasound Thirty infants received surfactant.Two infants were intubated prior to the rst LUS assessment.The rst dose was given at a median time of 2.5 hours (IQR 2-3.4) with median FiO 2 of 36.5% (IQR 28-49%).
Adding the LUS score of both sides of the chest together, the median LUS score prior to receiving surfactant while on CPAP was 2 (IQR 2-2), which improved to 3 (IQR 2-4, p = 0.001) at a median of 2.5 hours (IQR 0.5-21) after surfactant was given.Supplemental oxygen decreased to 25% (IQR 21-28, p = 0.001) after surfactant therapy.Five out of thirty infants had backsliding after surfactant therapy was given.Two of these infants had received MIST and showed backsliding while on CPAP, backsliding was seen in the other 3 after intubation.

Discussion
This is the rst delivery room study to assess the utility of LUS and report on the evolution of LUS score after birth in very-and extremely preterm infants.We found that LUS is feasible to perform, has good diagnostic accuracy for surfactant therapy using a single axillary view, and can track the progress of lung aeration over the rst few hours after birth.
Previous studies have shown that LUS performed upon admission to neonatal intensive care can predict the need for surfactant therapy [10,22,33,34].A recently published trial suggests that using LUS after NICU admission to guide surfactant therapy decreased the time to receive surfactant and increased the clinical response to surfactant versus using an FiO 2 threshold in infants < 32 weeks [36].Our study suggests that delivery room LUS may identify infants requiring surfactant therapy even sooner after birth, while also widening the diagnostic capability to include the signi cant proportion of infants who are intubated and/or receive surfactant therapy prior to NICU admission.
Lung ultrasound changes consistent with RDS may precede clinical signs which may not appear until several hours after birth.In the delivery room, an FiO 2 threshold of 0.3 produced more false positives (11 infants) and false negatives (20 infants) than LUS.LUS correctly identi ed that 10/11 of the FiO 2 false positives would not need surfactant and that 14/20 of the FiO 2 false negatives would need surfactant, including 100% of the extremely preterm infants.
Furthermore, we found that surfactant therapy signi cantly improved the LUS grading indicating better lung aeration.This is consistent with the observation that surfactant therapy greatly increases the uniformity of lung aeration and the distribution of ventilation in preterm rabbits [6].As such, rapid identi cation of poor lung aeration using LUS may facilitate targeted surfactant in the delivery room to infants likely to bene t most from the treatment.
The time related changes in LUS grading and the relatively high incidence of backsliding is particularly interesting and provides further evidence that lung aeration is not a unidirectional process.Indeed, it has been well documented in animal studies that airways can re ll with liquid, particularly in the immature lung in the absence of an end-expiratory pressure [8].In keeping with this, duration of rupture of membranes was shorter in the group with backsliding on CPAP compared to infants with no backsliding on CPAP in a univariate comparison of risk factors.As such, the nding that a high proportion (88%) of infants displayed backsliding following intubation in the delivery room, suggests that an increase in positive end-expiratory pressure may have reversed backsliding in infants being managed with invasive ventilation [54].Thus, further studies are warranted to determine whether LUS has utility for determining optimal PEEP levels in intubated and ventilated infants.Similarly, 21 of the 26 infants demonstrating backsliding were initially managed on CPAP, which raises the question as to whether the introduction of CPAP or higher CPAP levels may have reversed the backsliding.LUS may be able to provide critical feedback in real time to optimize respiratory management in neonates and should be the aim of future studies [54].
The nding that overall LUS grading was lowest at 1-3hours after birth, even compared with 5-10 minutes after birth, raises several very important questions.In particular, as spontaneous breathing is primarily responsible for lung aeration after birth and the clearance of liquid that re-renters the airways between breaths, backsliding at 1-3 hours after birth may indicate breathing fatigue leading to airway re ooding and/or atelectasis.Indeed, as infants with backsliding while on CPAP were more likely to be intubated, backsliding may provide an early signal that an infant's respiratory drive is unable to maintain lung aeration or that distending pressure from the CPAP was insu cient.We feel backsliding warrants consideration in future studies and validation by other investigators.
Like backsliding, LUS type 0.5 was seen most commonly in intubated infants.Type 0.5 was previously observed in term infants as they initiated breathing after birth and is theorized to represent atelectasis [28].Atelectasis may be caused by more severe RDS, a poorly positioned endotracheal tube, or loss of functional residual capacity in infants who receive muscle relaxants prior to intubation [55,56].
Our study has several limitations, including the observational design, a small cohort, and a clinically important variation in gestational age.Nevertheless, the fact that over 50% of infants were < 28 weeks gestation allows generalization of our ndings to extremely preterm infants.A further strength was that LUS score was graded blind to the clinical course.It will be important to validate the accuracy and reliability of our ndings when LUS is performed in real-time at the bedside, alongside clinical information by a wider range of clinicians.Type 1 is associated with respiratory distress syndrome.Type 0.5 and type 0 are associated with partially aerated lungs and liquid lled lungs, respectively.These images have been seen transiently as infants initiate breathing after birth, prior to establishment of the pleural line.Type 0.5 depicts the pleural line with a patchy appearance with poor de nition, consisting of speckled hyperechoic areas mixed with hypoechoic lung tissue.Type 0 has been seen prior to the initiation of breathing and establishment of the pleural line.This is a true US image (no air artifacts) as the US beam passes exclusively through liquid and is described as lung hepatization.
Distribution of lung ultrasound grades at each study timepoint for infants who did and did not subsequently receive surfactant.Top panel shows all infants, middle and lower panels show data within gestational age subgroups.In two infants, the researcher was unable to acquire LUS images at 5-10 minutes after birth.ST: surfactant replacement therapy, 1/2: type 1 or worse on at least one side of the chest, 2/2: type 2 or better on both sides of the chest.

Figures Figure 1
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Table 1
Demographic and clinical information based on gestational age at birth.

Table 2 :
Test characteristics at each study timepoint to independently predict surfactant therapy.Test positive for lung ultrasound (LUS) 1/2 is type 1 grade or worse (i.e.grade 0 or 0.5) on either side of the chest.Test positive for LUS 1/1 is type 1 or worse on both sides of the chest.Results are presented as test characteristics with 95% con dence intervals.Min = minutes, hr = hours, FiO 2 = fraction of inspired oxygen.

Table 3
Comparisons, outcomes, and test characteristics of infants on CPAP with backsliding versus without backsliding the 1-3 hour assessment.Infants diagnosed with chronic lung disease were receiving respiratory support (positive pressure or supplemental oxygen) ≥ 36 weeks corrected gestational age.
Future studies are needed to investigate whether adjustments in respiratory support, for example increased positive end expiratory pressure and/or early targeted surfactant, change the appearance of LUS in very preterm infants with signs of poor lung aeration.LUS may be able to optimize individual patient care by enabling clinicians to improve both the timeliness and appropriate targeting of interventions.Such work would establish the basis for larger trials investigating clinically important outcomes for preterm infants managed using LUS as an auxiliary tool in the delivery room.ConclusionsLung ultrasound in the delivery room is a speci c predictor of failure of non-invasive respiratory support, especially in extremely preterm infants.Earlier detection of infants needing an increase in CPAP pressure or surfactant may improve clinical outcomes, but larger studies are needed.At Monash Medical Centre, infants were eligible for written, retrospective parental consent if there was insu cient time to discuss the study prior to birth.
DeclarationsEthics approval and consent to participateThe was approved by the human research ethics committees at both the Royal Women's Hospital, Melbourne, Victoria (reference number 16/35), and Monash Medical Centre, Clayton, Victoria (reference number RES-18-0000-693A).At both study centres antenatal parental consent was sought prior to birth where possible.