Study objectives and design This is a nested case-control study that comprised 495 neonates(473 infants in control group and 22 infants with lung diseases in case group, confirmed retrospectively) in the NICU of the Obstetrics & Gynecology Hospital of Fudan University, Shanghai, China, from 1 January 2020 to 1 April 2020.
All infants delivered in the obstetric department were routinely transferred to NICU observation ward for termporarily monitoring(no more than 6 hours) in case of potential diseases. During the study period, these infants enrolled consequtively no matter they with or without respiratory symptoms and some of them were excluded as following criteria: ①absence of complete and qualified clinic data or ultrasound images; ②with cardiac issues that is diagnosed after admission to NICU. As our pilot studies showed that some infants with previously considered pathological LUS image patterns(mentioned in the introduction) were confirmed to be healthy, we made every infant enrolled in this study received LUS inspection to acquire all possible kinds of patterns in healthy infants. The images were collected with a newly designed scanning protocol(seen as following) at a predetermined time (0.5 hours, 1 hour, 2 hours, 4 hours, or 6 hours after birth,).
Because the diagnosis of most respiratory diseases of neonates are based on CXR that generally might be done only when infants have severe respiratory difficulty, determining healthy infants shortly after birth is difficult. So we adopted the nested case-control design that collecting data of all participants right first and decided the case and control groups after all patients were diagnosed.
Scanning protocol Lung ultrasound was routinely performed at bedside using a Sparq Ultrasound System (Philips Healthcare, Andover, MA) equipped with a 3–13 MHz linear array transducer and concurrently reported using a reporting template within the ICU electronic patient record. To acquire a constant (between different inspectors and different inspections) and comprehensive description of the neonates’ lungs, a new scanning protocol was designed and applied. We improved the conventional scanning protocol[13] in which the probe scans continually over 6 lung regions to a new protocol in which the probe scans at 20 predetermined points (shown in Fig. S1).
Defining RDS, TTN, congenital pneumonia, pneumothorax and healthy infants
RDS was defined in two ways: using a combination of chest radiography (Berlin-CXR)[14] and the PS application threshold recommended by the European Consensus Guidelines[15].
TTN is a clinical diagnosis and is supported by findings from chest radiographs, such as increased lung volumes with flat diaphragms and mild cardiomegaly [16].
Congenital pneumonia was diagnosed based on comprehensive evidence[17] from complete blood counts, C-reactive proteins, cultures for main types of pathogens (listed in the reference), as well as findings on CXR.
PTX was mainly confirmed by clinical features, LUS, and closed thoracic drainage. LUS, which is believed to have higher sensitivity than CRX[18, 19].
These diagnosis are made by the experienced neonatology specilist in this study team. To acquire the X-ray evidence mentioned above, suspected patients were routinely inspected by a technician, and the conclusions were drawn by a junior doctor and verified by a senior doctor from the radiology department.
Healthy infants
After excluding the diseases mentioned above, infants were regarded as healthy and confirmed on 3-day follow-up. However, regarding mild TTN that can be a physiologic diagnosis needs no further medical care and hard to differentiate, we classified these infants into control group so that the conclusion of this study is pratical.
Low-risk and high-risk image patterns
Previous studies have indicated that "A-line"[4], "small amounts, and a large amount of B-line"[20](defined as "coalescence B line" in this reference) is normal patterns for neonates, and has regarded "compact B-line", "dense B-line"(or defined as "white lung" in these references), "consolidation" as abnormal patterns[4, 2, 21, 12, 22]. However, in our pilot study, either supposed normal or abnormal patterns can be seen both in healthy infants and patients. To make our conclusion, to which physicians can make a definite and timely decision according, practical, we regarded the patterns as high-risk and low-risk instead of simply naming as "normal" or "abnormal".
To clarify different B-line patterns(shown in Fig. 3) and its various significance for lung diseases, especially the "large amount of DB"(low risk) and "compact B-line"(high risk), "dense B-line"(high risk), we characterized the low-risk patterns of B-lines(distributed B-lines) as"can be discriminated against each other". This can be very useful when assessing neonates on dynamic LUS according to our experience.
Statistical analysis
Data was shown as frequencies or percentages and as the means and standard deviations or medians and interquartile ranges according to distribution. Differences between the groups were compared by the chi-squared or Fisher’s exact test for categorical variables and Student’s t-test or Mann-Whitney U test for continuous variables, depending on the distribution. Sensitivity, specificity, LR, PPV and NPV were calculated to evaluate the predictive value of LUS patterns. A nominal 2-sided probability value < 0.05 was considered to indicate statistical significance. All of the calculations were performed using SPSS 23.0 (SPSS Inc. Chicago, IL).