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A protocol for enhancing the diagnostic accuracy and predictive validity of neonatal opioid withdrawal syndrome: The utility of non-invasive clinical markers
Clinical markers of nows
https://orcid.org/0000-0002-7388-8640
Maylott Sarah E. Conceptualization Methodology Visualization Writing – original draft Writing – review & editing 1 *
Lester Barry M. Conceptualization Funding acquisition Investigation Methodology Project administration Resources Supervision Writing – review & editing 2 3 4 5
Brown Lydia Methodology Project administration Writing – review & editing 6
Castano Ayla J. Methodology Project administration Writing – review & editing 6
Dansereau Lynne Methodology Project administration Writing – review & editing 2
Crowell Sheila E. Conceptualization Methodology Resources Writing – review & editing 7
Deboeck Pascal Conceptualization Formal analysis Methodology Writing – review & editing 2
Salisbury Amy Conceptualization Methodology Resources Supervision Writing – review & editing 5 6 8
Conradt Elisabeth Conceptualization Formal analysis Funding acquisition Investigation Methodology Resources Supervision Writing – review & editing 1
1 Department of Psychiatry & Behavioral Sciences, Duke University, Durham, NC, United States of America
2 Center for the Study of Children at Risk, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America
3 Department of Psychiatry, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America
4 Department of Pediatrics, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America
5 Women and Infants Hospital, Providence, Rhode Island, United States of America
6 Department of Psychology, University of Utah, Salt Lake City, Utah, United States of America
7 Department of Psychology, University of Oregon, Eugene, Oregon, United States of America
8 School of Nursing, Virginia Commonwealth University, Richmond, Virginia, United States of America
Metwally Ammal Mokhtar Editor
National Research Centre, EGYPT
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: sarah.maylott@duke.edu
10 9 2024
2024
19 9 e030617620 2 2023
10 6 2024
© 2024 Maylott et al
2024
Maylott et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Every 15 minutes in the US, an infant exposed to opioids is born. Approximately 50% of these newborns will develop Neonatal Opioid Withdrawal Syndrome (NOWS) within 5 days of birth. It is not known which infants will develop NOWS, therefore, the current hospital standard-of-care dictates a 96-hour observational hold. Understanding which infants will develop NOWS soon after birth could reduce hospital stays for infants who do not develop NOWS and decrease burdens on hospitals and clinicians. We propose noninvasive clinical indicators of NOWS, including newborn neurobehavior, autonomic biomarkers, prenatal substance exposures, and socioeconomic environments. The goals of this protocol are to use these indicators shortly after birth to differentiate newborns who will be diagnosed with NOWS from those who will have mild/no withdrawal, to determine if the indicators predict development at 6 and 18 months of age, and to increase NOWS diagnostic sensitivity for earlier, more accurate diagnoses.

http://dx.doi.org/10.13039/100000026 National Institute on Drug Abuse R01DA049755 Lester Barry M. http://dx.doi.org/10.13039/100000026 National Institute on Drug Abuse R01DA049755 Conradt Elisabeth This work is funded by the National Institute of Drug Abuse [R01DA049755 to BL and EC]. The funders did not and will not have a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://nida.nih.gov/. Data AvailabilityDeidentified research data will be made available upon reasonable request when the study is completed and published
Data Availability

Deidentified research data will be made available upon reasonable request when the study is completed and published
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pmcIntroduction

Substance use during pregnancy is a public health crisis. Approximately 14–22% of pregnant women fill a prescription for opioids, and close to 7% report using opioids while pregnant [1]. About half of newborns exposed to opioids in utero are diagnosed with Neonatal Opioid Withdrawal Syndrome (NOWS) [2]. These newborns become physiologically dependent while in utero and endure drug withdrawal following the cessation of exposure after delivery. Hospitalization of infants diagnosed with NOWS results in around $1.5 billion in hospital charges across a decade [3], and these infants are four times more likely to require post discharge rehospitalization than children without a NOWS diagnosis [4]. The impact of NOWS lingers beyond birth, with developmental [5] and educational deficits [6]. Presently, there are no evidence-based clinical markers of NOWS [7]. The absence of reliable and valid diagnostic measures may limit medical professionals’ ability to provide optimal care to opioid exposed newborns. We propose research-based clinical markers for identifying infants with NOWS.

Neonatal opioid withdrawal

The pathophysiology of opioid withdrawal is complex in neonates, given their rapid neurological development [8]. Opioids readily cross from the placenta into fetal circulation, with increasing transmission over gestation as the placental barrier expands and thins, enhancing blood flow to the fetus [9]. Generally, opioid exposure triggers μ-, κ-, and δ-receptors concentrated in the central nervous system and gastrointestinal tract [10]. In neonates, while δ- and κ-receptors are less developed, their μ-receptors’ density and affinity are comparable to adults [11]. Consequently, the μ-receptor binding of opioids and then the cessation binding (i.e., the cessation of opioid exposure) after birth may lead to withdrawal signs, including autonomic instability, high-pitched cry, irritability, tremors, and difficulty feeding and sleeping [12].

Challenges and potential advancements to NOWS diagnostics and clinical care

Restrictions to current diagnostic guidelines for NOWS

To guide treatment of NOWS, clinicians use observational diagnostic tools to evaluate newborn central nervous system disturbances, metabolic, vasomotor, and respiratory disturbances, and gastrointestinal disturbances [13, 14]. However, these diagnostic tools are not often thoroughly researched, psychometrically sound, and/or standardized, and the choice of diagnostic tool depends on independent hospital protocols [15]. Many criteria for diagnosis are subjective, with scoring varying not only between hospitals but also between hospital staff [7]. Unfortunately, clinicians do not have a predictive model to determine NOWS onset or severity. With the current tools, most neonates are not diagnosed with NOWS until 36 hours after birth and withdrawal signs can be delayed 5 to 7 days, depending on the opioid’s half-life [10], delaying optimal care. This “wait-and-see” approach may be unnecessary for the 20–40% of newborns who will not develop NOWS. Furthermore, the dichotomous diagnosis of NOWS ignores the variability in NOWS severity and limits the predictive validity.

Proposed advancements for NOWS diagnostic protocols

In this protocol we propose several biobehavioral markers of NOWS including the NeoNatal Neurobehavioral Scale (NNNS-II), newborn cry and sleep, heart rate variability (HRV), salivary cortisol, and epigenetic markers to advance diagnostic procedures for NOWS. We suggest that these markers will advance the current clinical protocols by 1) predicting NOWS onset and severity, 2) developing a protocol to detect risk for NOWS 24 hours after birth, 3) measuring NOWS severity outcomes as a spectrum influenced by exposure type, frequency, and co-substance use, 4) using the spectrum of NOWS severity to predict longitudinal neurodevelopment 5) and as an indicator of successful treatment (Table 1).

10.1371/journal.pone.0306176.t001 Table 1 Advancing the science of NOWS research.

Current clinical and research practices	Proposed advancement	
Not predictive of NOWS onset or severity	Establishing evidence-based clinical markers that predict onset and severity of NOWS	
NOWS is often not diagnosed until 36 hours after birth and can be delayed 5 to 7 days	These predictors could be used to develop an algorithm that detects risk for NOWS 24 hours after birth	
Utilizes a simple binary outcome in diagnosis (i.e., yes/no)	Measure NOWS severity as a spectrum of outcomes influenced by type of exposure, frequency of use, & other substance exposure	
Predicts probability of adverse neurodevelopmental outcomes from a dichotomous diagnosis	Use evidence-based clinical markers to predict longitudinal neurodevelopment outcome by a continuous distribution of NOWS severity	
Measures length of hospital stay as an indicator of a successful treatment outcome without considering other factors that impact length of stay, such as CPS (i.e., foster care) involvement	Measure NOWS severity as an indicator of successful treatment outcome	

Clinical measure 1: Newborn neurobehavior

The NNNS-II is a standardized assessment of both newborn neurologic integrity and behavioral functioning based on the original NNNS exam [16], which captures differences in exposed newborns who require pharmacological treatment from those who do not need intervention [17]. Moreover, newborn neurobehavior predicted the resolution of NOWS, suggesting that the NNNS-II can be used to identify newborns at risk for NOWS [18]. The original NNNS was also predictive of developmental outcomes through 4.5-years of age children, including behavior problems, school readiness, and IQ [19].

Clinical measure 2: Newborn cry acoustics

We can further increase objectivity of NOWS assessments through automated measures. “Abnormal” or “high-pitched” newborn crying is a primary NOWS sign, and yet, is one of the most subjective and poorly quantified [7]. Despite scoring limitations, crying is a crucial biobehavioral marker for NOWS diagnoses. In utero substance exposure increases central nervous system irritability, activates the hypothalamic-limbic system, and triggers abnormal infant crying [20]. Recording and analyzing infant crying offers a way to accurately capture and interpret atypical cry characteristics. Cry analysis relies on specific mechanisms in cry production, including threshold and latency, indicative of central nervous system reactivity, energy, dysphonation, and utterances, signifying respiratory control, and fundamental frequency, hyperphonation, formant frequencies, and mode changes, demonstrating neural control of the vocal tract [21]. Infants with in utero opioid exposure tend to have shorter cry utterances and extremely high-pitched cries, and infants co-exposed to opioids and cocaine have comparably louder and higher pitched cries [21]. High-pitched and hyperphonated cries have been reported in infants with neurologic problems [22], suggesting that infants with substance exposures, especially those with polysubstance exposures may be at increased neurological risk. Preliminary acoustic cry data showed that it was 81% accurate in predicting NOWS; moreover, acoustics were more accurate in some cases when the observational crying score was incorrectly evaluated (Unpublished data). Abnormal infant crying has also been linked to delays in cognitive and motor functioning at 24 months of age [22]. This automated assessment could help identify infants at greater risk for NOWS and subsequent neurodevelopmental disruptions, while also shortening assessment times, reducing burdens on clinical staff, and limiting subjectivity of observational judgements.

Clinical measure 3: Newborn sleep patterns

Disrupted sleep is another important indicator of NOWS, as a sign of nervous system dysregulation [23] or interruptions of autonomic regulation of arousal and movement during sleep [24]. Infants exposed to opioids in utero displayed greater wakefulness with disruptions in the amount of REM and non-REM sleep, suggesting sleep state instability [25]. Further, newborns exposed to higher doses of opioids in utero showed more disturbances in neonatal sleep, indicating that sleep patterns provide insight into the neurobehavior of newborn withdrawal [26]. Using sleep mattress recordings, we categorize disrupted sleep patterns associated with NOWS [12]. We propose to utilize these automated tools to help limit diagnostic subjectivity, improve diagnostic sensitivity, ease clinician burdens, and reduce healthcare costs [27].

Clinical measure 4: Newborn heart rate variability

Like the biobehavioral measures, biological markers, such as newborn heart rate variability (HRV), salivary cortisol, and epigenetic mechanisms also provide vital information for identifying newborns at higher risk for NOWS. HRV—the beat-by-beat fluctuations in cardiovascular activity—reflects newborns’ autonomic nervous system functioning, which controls involuntary physiological responses through the sympathetic and parasympathetic systems [28]. These systems work in opposition to create homeostasis; the sympathetic nervous system works to increase heart rate while the parasympathetic nervous system decreases heart rate [28]. Fetal HRV is often suppressed in opioid exposed infants, especially those exposed to more than one substance, reflecting a dysfunction in this balance [29, 30]. Furthermore, newborns prenatally exposed to opioids had higher heart rate, lower HRV, and more blunted respiratory sinus arrythmia—beat-to-beat variability in heart rate that occurs with respiration—than non-exposed newborns [31]. HRV was also less variable in prenatally exposed newborns diagnosed with NOWS compared to those exposed newborns not diagnosed [32]. Newborn HRV could help identify those infants at greater risk for developing severe withdrawal signs.

Clinical measure 5: Newborn salivary cortisol

Salivary cortisol is another non-invasive biological indicator that may help identify newborns at higher risk for severe NOWS signs, facilitating the detection of newborns’ dysregulated stress responses. Prenatal opioid use is often associated with elevated life stressors such as poverty, psychopathology, and poor general health, as well as exposure to violence, trauma, and/or abuse. Maternal stress can negatively impact fetal development [33]. This process can happen through a variety of mechanisms, including via heightened maternal cortisol levels in the bloodstream, through the reduced expression of placental 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts active cortisol to inactive cortisone [34], and by autonomic nervous system activation, which may diminish blood flow to the placenta, limiting fetal nutrients and oxygen [35]. Excessive exposure to maternal stress hormones during pregnancy is theorized to adversely impact fetal programming, adapting fetal physiology to the expected postnatal environment by modifying their stress response system’s (hypothalamic-pituitary-adrenal axis; HPA axis) set points [36, 37]. In utero exposure to opioids may also contribute to this fetal programming [36]. In animal models, prolonged in utero exposure to opioids suppressed opioid receptors, leading to offspring with impaired or overactive negative feedback regulation of their HPA axis [38]. In fact, newborn salivary cortisol remains elevated in infants who develop more severe withdrawal, potentially indicating HPA axis dysregulation or an altered stress response due to the severity of withdrawal. Regardless of the pathophysiology, salivary cortisol offers a measure of newborn neuroendocrine functioning that is reflective of risk for NOWS [39].

Clinical measure 6: Newborn epigenetics

Epigenetic mechanisms offer insight into the variability of NOWS diagnoses and severity. One such mechanism is DNA methylation, a process by which a methyl group attaches to a particular region on the DNA, effectively preventing transcription and reducing the likelihood that the gene will be expressed [40]. Stress response genes (COMT, NR3C1), opioid receptor genes (OPRM1, OPRK1, PNOC), and opioid-related genes (ABCB1, CYP2D6) are associated with newborn withdrawal from prenatal opioid exposure and may be useful markers of susceptibility to NOWS and pharmacotherapy [41, 42]. For example, the COMT gene has been associated with shorter hospital stays for neonates prenatally exposed to opioids. This gene is associated with the dopaminergic system and expression of this gene has been linked to increases in circulating catecholamines—a neurotransmitter that helps the body respond to stress—potentially improving stress tolerance in opioid-exposed infants [43]. Prenatal DNA methylation of NR3C1—a gene associated with glucocorticoid receptors—has been linked to greater cortisol reactivity in infancy [44], which may indicate NOWS severity. Methylation of this gene likely reduces cortisol binding sites by limiting the number of glucocorticoid receptors, increasing the amount of cortisol in the bloodstream. Along with neonatal salivary measures of cortisol, identifying the epigenetic mechanisms related to adverse prenatal environments may help pinpoint infants prenatally exposed to opioids with altered stress response systems, who are more likely to need pharmacological interventions for NOWS. Furthermore, prenatal opioid exposure is related to increased DNA methylation in opioid receptor and opioid-related genes [41, 42]. Higher levels of OPRM1 promoter methylation at transcription factor binding sites have been associated with more severe NOWS signs and an increased need for pharmacological intervention [45, 46]. This hypermethylation is theorized to downregulate OPRM1 gene expression and reduce μ-opioid receptors, resulting in neonates needing more medication to combat withdrawal [46]. Epigenetics could be a vital clinical marker of newborns’ withdrawal severity and response to pharmacotherapy [47].

Clinical measure 7: Maternal toxicology

Use of maternal hair for prenatal substance use toxicology is another key measure, providing information on the levels and types of opioid exposure and other drug metabolites across pregnancy [48]. We can determine potential risk and severity for neonatal withdrawal depending on type, timing, or amount of exposure [49]. Newborns exposed to higher doses of methadone in utero were more likely to be treated for NOWS and to spend longer in the hospital than newborns exposed to lower doses [50]; however, the effect of opioid dose on the severity of withdrawal continues to be debated. Further, newborns were treated for NOWS longer if they had a longer gestation (37–42 weeks treated for an average of 39.5 days; 33–36 weeks treated for an average of 20 days; 23–32 weeks treated for an average of 10 days). These findings may be due to the high permeability of the placenta in the late third trimester [51] or that current assessments are not geared toward assessing NOWS in preterm infants who may display differences in neurological development and, therefore, withdrawal signs at birth.

Identification and application of these evidence-based predictors of NOWS could advance clinical care of newborns with prenatal opioid and other substance exposures, leading to a standardized screening routine for all newborns at risk of developing NOWS, and even flag newborns not initially identified as opioid-exposed.

Developmental consequences of prenatal opioid exposure

The implications of in utero opioid exposure extend beyond birth [52], with exposed infants displaying lower cognitive functioning than their non-exposed peers, a divergence that appears magnified as children age [53, 54]. These children show cognitive and language impairments around 2 months [55], inhibitory control difficulties at 2 years [56], and externalizing and internalizing behaviors, social issues, attention problems [54], and lower executive functioning at 4.5 years of age [57]. Extant literature focuses on developmental differences in those infants exposed to opioids in utero and non-exposed infants. However, differences between exposed infants diagnosed with NOWS and exposed infants not diagnosed with NOWS could highlight important risk and resiliency factors [58]. Furthermore, few studies consider adverse socioeconomic circumstances, maternal psychopathology, or polysubstance use [59], which may impact both prenatal and postnatal environments [36]. The postnatal environment plays a substantial role in infant development, and yet, is understudied. However, large-scale studies, such as the Maternal Lifestyle Study and The Infant Development, Environment, and Lifestyle (IDEAL) Study have found that early adversity is a strong determinant of child problem behavior, partially explaining the relationship between prenatal substance exposure and developmental outcomes [60, 61].

Current study

Our study protocol has three aims. First, to determine NOWS onset and severity using a battery of sensitive clinical markers (Fig 1). Second, to evaluate the predictive validity of these clinical markers on neurodevelopmental outcomes at 6 and 18 months and test whether NOWS severity predicts neurodevelopment impairment. Third, to increase NOWS clinical diagnostic sensitivity and objectivity for earlier, more accurate diagnoses.

10.1371/journal.pone.0306176.g001 Fig 1 Conceptual model for Aim 1 (top) and Aim 2 (bottom).

Methods

This study was approved by the Institutional Review Board at the University of Utah on September 14th, 2019 (00124221) and Women and Infants Hospital on February 13th, 2020 (1479081–3). Written consent was obtained for all participants. Enrollment and data collection began in 2019 and will be completed in 2025.

Study population

We collect data from 312 newborns with prenatal opioid and other substance exposure across two sites, Women and Infants Hospital of Rhode Island and the University of Utah Hospital. We collect follow-up data at 6 and 18 months of age. Both sites recruit participants prenatally or shortly after birth (Table B in S1 Text). Birthing parents are over 18 years old and identified to have used opioids during pregnancy via medical records. Newborns are medically stable and include singleton and twin pregnancies, term and preterm infants, and breast- and bottle-fed infants. Newborns with congenital abnormalities, genetic syndromes, metabolic disturbances, or serious medical illnesses, such as sepsis, asphyxia, seizures, or respiratory failure are excluded. Newborns are also excluded if they are unable to take oral medications or if their caregiver is unable to provide informed consent (Table A in S1 Text).

Newborn (Time 1)

Procedure

We collect data approximately 24–48 hours after birth, prior to treatment for NOWS. The birthing parent/legal guardian provides informed consent and is asked to fill out questionnaires assessing maternal sociodemographic risk and psychopathology. Newborn birth characteristics are extracted from medical records.

For the newborn assessments, we begin by collecting the first of three saliva samples using a small cotton swab. Then we measure infant heartrate, placing three electrodes on the upper (negative) and lower (positive) chest around the infant’s heart, and on the right lower side of the chest (neutral) and are left on the infant for the full newborn visit. For the next 10 minutes, infant heartrate is monitored. Next, we assess neurobehavior with the NNNS-II, which lasts about 15 minutes. The cry recording begins during the NNNS-II, is stopped after the assessment concludes, and then is restarted to record continuously for the length of hospital stay. Caregivers and hospital staff are asked to remain quiet while newborn cries are recorded. After the NNNS-II, we measure infant heartrate again for 10 minutes. We collect the second and third saliva samples 20- and 30-minutes post-NNNS-II assessment, respectively, and the buccal sample. ECG leads and wires are removed post newborn assessments, so that we cause no additional stress during the exams. Finally, we collect maternal hair samples for toxicology analyses. If the hair is too short or birthing parents refuse to provide the hair sample, the missing data is tracked but participants are still included in the study.

We also record the number of primary caregiver changes over the course of the study, starting at birth. This occurs when there is a change in custody. The child’s primary caregiver at birth is typically the biological mother. Caregiver transitions occur when the child is placed with a foster parent, adoptive parent, or other caregiver (e.g., family member). Reasons for caregiver transitions include child protective services involvement (e.g., neglect, abuse, substance use), incarceration of caregiver, death of caregiver, adoption, and/or reunification, among others. Caregiver changes may be an indicator of problem behavior [62, 63]. Next, we set up a passive sleep sensor and cry recorder on the newborn’s bassinet/pram. Newborn sleep and cry are checked daily and recorded continuously throughout infants’ stay in the hospital beginning with the start of the NNNS newborn assessment.

Maternal measures

Maternal sociodemographic risk. Maternal sociodemographic risk is comprised of socioeconomic status and financial insecurity. The birthing parent reports socioeconomic factors with a brief demographic survey and financial insecurity with the Family Resource Scale [64], which measures whether the family has income for basic needs, time for self, and time for family. The family resource scale has high test-retest reliability and predictive validity [64].

Maternal psychopathology. Maternal psychopathology is measured with the Patient Health Questionnaire-9 (PHQ-9), four Patient-Reported Outcomes and Measurement (PROMIS) surveys from the NIH Toolbox, the Difficulties in Emotion Regulation-Short Form (DERS-SF), the Borderline Symptoms Checklist, “My Inner Feelings” (BSL-23), and the Connor-Davison Resilience Scale (CD-RISC). The PHQ-9 [65] is a 9-item self-report measure of depression with good psychometric properties. The PROMIS assesses anger (5-items), anxiety (8-items), depression (8-items), and emotional support (4-items) [66]. The 18-item DERS-SF [67] is an assessment of emotion dysregulation, with strong psychometric properties. The BSL-23 is a self-rating measure of borderline symptomatology, with good psychometric properties [68]. Lastly, the Connor-Davidson Resilience Scale (CD-RISC-10) is 10 items, each rated on a 5-point scale (0–4), with higher scores reflecting greater resilience [69].

Maternal trauma. Maternal history of trauma is assessed from the Adverse Childhood Experiences (ACEs) [70] and the Benevolent Childhood Experiences (BCEs) [71]. We use 19-items from the ACEs measure to retrospectively assess the possibility of maltreatment in childhood and as a teenager. In contrast, the BCEs is a 10-item self-report measure of positive childhood experiences relating to perceived safety and security, positive quality of life, and interpersonal support.

Prenatal substance exposure. Opioids and other substance exposure are measured via confirmatory toxicology analysis from the birthing parent’s hair [48] by Omega Laboratories Inc. (www.omegalabs.net). Opioids and metabolites in the blood are deposited in the growing hair, which provides a historical marker of drug exposure. Therefore, we require 9 cm of hair (third trimester: 3cm closest to the scalp, second trimester: >3–6 cm from the scalp, first trimester: >6–9 cm from the scalp). We collect a lock of hair from multiple sections of the crown area—to minimize noticeability—as close as possible to the scalp and approximately the diameter of a drinking straw (e.g., 6mm or 90–120 strands of hair). We also ascertain if hair has been treated in the last 9 months, which may affect specimen integrity.

Maternal alcohol use cannot be measured via maternal hair, so it is assessed via the Substance Use Inventory (SUI; i.e., Weiss et al. [Unpublished]) [72], a self-report measure of quantity and frequency of substance use that was adapted from Timeline Follow-back interview, with high reliability for alcohol use [73]. We also abstract relevant substance use data from the medical record.

Newborn measures

Neurobehavioral assessment. The NeoNatal Neurobehavioral Scale (NNNS-II) is a standardized comprehensive evaluation of the neurobehavioral performance of newborns [16]. NNNS-II summary scores are described in Table D in S1 Text.

Cry assessment. The cry assessment is measured via recording of an infant’s cry—a sequence of utterances and silences. An utterance is a contained vocal output, either voiced (generated via vocal vibrations) or unvoiced (due to frication or tension in the vocal tract). The infant cry recording and analysis system uses state-of-the-art Cepstral analysis (Fig 2) to extract acoustic parameters from cry recordings [74]. The cry analysis system classifies utterances or silence (time between utterances). Acoustic parameters are calculated for each sound segment (Table 2). The cry analysis system is unique from other speech analyzers since it applies current digital signal processing techniques and is specifically tailored to newborn acoustic data which are sensitive to the developing vocal tract and oral cavity.

10.1371/journal.pone.0306176.g002 Fig 2 Examples of digital spectrogram and cry recorder.

Red bands indicate individual cry bursts/utterances that contain pitch characteristics implicated in NOWS.

10.1371/journal.pone.0306176.t002 Table 2 Cry variables.

Category	Output	
Timing Variables	Measures of utterance duration, categorization of long & short utterances (+/-500 milliseconds), latency to cry, and inter-utterance intervals	
Energy Variables	Measures of loudness of cry overall and within 6 frequency bands, including mean, min/max, & SD	
Pitch Measures	Mean, min/max, SD, and amplitude of F0 for each utterance, and output modeling of the shape and complexity of pitch contours across the utterance	
Hyper-Pitch	Mean, min/max, SD, and amplitude of F0 detected above 1kHertz	
Formants (F1;F2)	Description of frequency centered at the first resonance of F0 (mean, min/max, SD, amplitude)	
Voicing	Number of times that reflexes on one side of the infant’s body are stronger/weaker relative to the other side	
Fricatives	Number of observed infant stress/abstinence signs across organ systems	

Sleep. We assess newborn sleep in the hospital with the Emfit QS sleep recordings from the Emfit passive under-mattress sensor (https://emfit.com/). The Emfit measures total recovery, integrated recovery, whole night heart-rate-variability RMSSD, heart and breathing rates, sleep score, sleep time, nervous system balance, the three sleep classes classification including REM, resting heart rate, tossing & turning, movement activity.

Heartrate. Newborn heartrate is monitored with a MindWare Technologies Mobile Impedance Cardiograph (https://mindwaretech.com/) and a Surface Pro tablet (https://www.microsoft.com/en-us/surface) with Biolab software.

Cortisol. Saliva is collected with SalivaBio Infant’s Swabs from Salimetrics to measure infant cortisol levels and stored in Swab Storage Tubes from Salimetrics (https://salimetrics.com/collection-method/infant-swab-device/) in a -80°C freezer until saliva is shipped to the University of Trier, Germany to be assayed.

Epigenetics. Buccal cell swabs are collected with an Isohelix SK-1S/MS-01 Buccal Swabs cotton swab on the inside of the infant’s cheek to examine epigenetics. Buccal cell samples are stored in a -80°C freezer until they are ready to be shipped to Emory University, Georgia for processing.

NOWS diagnosis and severity. NOWS diagnosis is assessed using the Modified Finnegan Neonatal Abstinence Scoring Tool [12], the Neonatal Withdrawal Inventory (NWI) [75] or the Eat, Sleep, Console (ESC) [76]. The diagnosis of NOWS is made when the Finnegan or NWI score reach a predefined threshold.

NOWS severity is measured using the length of NOWS treatment, maximum dose of treatment medication, and necessity for additional medications to treat NOWS, such as phenobarbital.

6 months (Time 2)

Procedure

For the 6-month visit, we ask the primary caregiver to complete a series of questionnaires assessing maternal sociodemographic risk, psychopathology, and trauma, as well as infant crying, sleeping, feeding, and temperament measures. Next, we assess fine and gross motor development with the Peabody Developmental Motor Scales. We also obtain childcare information and medical history from the medical records to document the number and severity of medical illnesses. Finally, we collect a buccal sample and standard pediatric anthropometry (i.e., weight, length, head circumference).

Maternal measures

Maternal sociodemographic risk, psychopathology, and trauma surveys are repeated from the birth visit (see Newborn Maternal Measures). If the birthing parent is not available, the primary caregiver reports these measures to best reflect the environment of the child.

Infant measures. Infant Motor Development. We measure infant motor development with the Patterson Medical Peabody Developmental Motor Scales—Second Edition (PDMS-2) [77]. The PDMS-2 has sound psychometric properties and is used from birth to 5 years of age to assess gross and fine motor skills.

Infant crying problems: Frequency, intensity, and duration of infant cries are assessed via caregiver-report on a 14-item cry assessment [78].

Infant sleep: We assess infant sleep quality and patterns using the Brief Infant Sleep Questionnaire-Revised (BISQ-R), which is a reliable and valid parent-report questionnaire that assesses infants sleep onset time, sleep latency, and number of night-wakings [79]. It has been validated against actigraphy and sleep diaries and has high test-rest reliability [80].

Infant feeding: Caregivers fill out a 3-item questionnaire measuring breastfeeding practices since the birth [81]. We also use the Feeding Matters Infant and Child Feeding Questionnaire (ICFQ), an evidence-based tool which assesses children’s feeding habits to identify feeding disorders. The ICFQ is psychometrically sound and has 6 core items that may be used to identify those infants that need pediatric feeding referrals [82].

Infant temperament: We assess infant temperament with the Infant Behavior Questionnaire-revised (IBQ) [83]. The IBQ has established psychometric support for measuring temperament related to activity level, smiling and laughter, distress and latency to novel stimuli, distress to limitations, soothability, and duration of orienting.

18 months (Time 3)

Procedure

For the final visit, the primary caregiver is asked to complete a series of questionnaires examining maternal sociodemographic risk, psychopathology, and trauma, as well as toddler language development, crying, sleeping, feeding, and temperament measures. We also obtain childcare information and medical history from the medical records to document the number and severity of medical illnesses. We use the Bayley Scales of Infant and Toddler Development IV to examine cognitive, language, and motor outcomes, and we measure toddler executive functioning skills with the Wand task, the A-not-B task, and the Hide the Pots task. Lastly, we collect a buccal sample from the toddler and collect anthropometrics (length, weight, head circumference).

Maternal measures

Maternal sociodemographic risk, psychopathology, and trauma surveys are repeated from the birth and 6-month visit (see Newborn and 6-month Maternal Measures). If the birthing parent is not available, the primary caregiver reports these measures to best reflect the environment of the child.

Toddler measures

Neurodevelopmental outcomes. We use the Bayley Scales of Infant and Toddler Development IV to assess cognitive, motor, and language outcomes [84]. The Bayley is an optimal neurodevelopmental assessment in early childhood given its strong reliability and moderately strong predictive validity [85].

Toddler problem behavior. We measure toddler problem behavior with the Infant and Toddler Social and Emotional Assessment (ITSEA) [86]. The ITSEA is a measure of internalizing and externalizing child behavior, as well as dysregulation and competence. It has test-retest and interrater reliability and strong construct validity in children age 12–36 months [87].

Executive functioning. We assess toddler executive functioning with the Glitter Wand task [88], the A-not-B task [89], and the Hide the Pots task [90]. All tasks measure key components of executive functioning including set shifting or shifting between rules, inhibitory control, and working memory.

Expressive and receptive language. We assess toddler language with the Bayley scales and maternal report of toddler receptive and expressive language development using the MacArthur Communicative Development Inventories [91] which has strong reliability and validity.

Toddler feeding. We measured toddler feeding problems with the ICFQ (For psychometrics see Infant Feeding under 6-month Infant Measures) for 15- to 24- month infants. For a full list of measures see Table 3.

10.1371/journal.pone.0306176.t003 Table 3 Protocol measures.

Assessments	Tools	Time	
Birth	6m	18m	
Maternal Sociodemographic	Family Resource Scale	√	√	√	
Demo; SES = income, education, and occupation	√	√	√	
Maternal Psychopathology	Patient Health Questionnaire–9	√			
Patient-Reported Outcomes and Measurement Information System	√	√	√	
Borderline Symptoms Checklist	√	√	√	
Difficulties in Emotion Regulation-Short Form	√	√	√	
Connor-Davidson Resilience Scale	√			
Maternal Trauma History	Adverse Childhood Experiences Questionnaire	√			
Benevolent Childhood Experiences Scale	√	√	√	
NOWS Severity	Medical Record Abstraction: Length of treatment, maximum dose, additional NOWS medications		√	√	
Number of Caregiver Transitions	Caregiver transitions	√	√	√	
Infant Epigenetics	Buccal	√	√	√	
Opioid and Substance Exposure	Hair toxicology	√			
Substance Use Inventory	√	√	√	
NOWS Diagnosis	Medical Record Abstraction	√			
Newborn birth characteristics	Medical Record Abstraction	√			
Newborn Neurobehavior	NNNS-II	√			
Cortisol	Saliva	√x3			
Infant Heart Rate	Heart Rate Variability/ECG/Mindware	√x2			
Infant Cry	Cry recording	√			
Cry Checklist		√		
Infant Sleep	EMFIT QS sleep sensor	√			
Brief Infant Sleep Questionnaire–Revised		√	√	
Infant Feeding	Infant and Child Feeding Questionnaire		√	√	
Infant breastfeeding questionnaire		√		
Crying, Sleeping, & Feeding	Follow-up & pediatric medical history interview		√	√	
Neurodevelopmental Outcomes	Peabody Developmental Motor Scales–2nd Ed.		√		
Bayley Scales of Infant and Toddler Development–IV			√	
Infant Temperament	Infant Behavior Questionnaire		√	√	
Toddler Problem Behavior	Infant Toddler Social and Emotional Assessment			√	
Expressive & Receptive Language	MacArthur-Bates Communicative Developmental Inventories			√	
Executive Functioning	Wand Task			√	
A not B Task			√	
Hide the Pots Task			√	

Infant temperament. We assess infant temperament with the Infant Behavior Questionnaire-revised (IBQ) [83]. For more information see Infant Temperament in 6-month Infant Measures.

Infant sleep. We assess infant sleep quality and patterns using the Brief Infant Sleep Questionnaire-Revised (BISQ-R). For more information see Infant Sleep in 6-month Infant Measures.

Infant crying problems. Frequency, intensity, and duration of infant cries are assessed via caregiver-report on a 14-item cry assessment [78].

Data analyses

Aim 1

Assess infant neurobehavior, newborn autonomic biomarkers, prenatal opioid use, and sociodemographics as predictors of NOWS onset and severity

We use generalized linear models to examine whether impaired newborn neurobehavior, abnormal cry, sleep, cortisol, HRV, epigenetics, prenatal opioid exposure as measured in maternal hair, and/or low socioeconomic status are predictive of NOWS diagnosis and severity. We control for site, maternal age, maternal psychopathology, gestational age, birth weight, newborn medical complications, and newborn sex. To control for several confounding variables, we use logistic regression to estimate the probability of NOWS diagnosis given the confounders; the resulting propensity scores are used as a covariate adjustment in subsequent analyses. As the occurrence of NOWS cannot be randomized, the use of propensity scores controls for imbalances across subject characteristics and allows for inferences to better approximate the findings of randomized clinical trials [92]. We also run models to determine if effects differ based on biological sex.

Aim 2

Evaluate the predictive validity of these clinical markers and test whether NOWS severity predicts neurodevelopment impairment at 6 and 18 months

We use generalized linear models with a normally distributed outcome to examine impairment in neurodevelopment later in infancy and toddlerhood. Multiple regressions are run independently predicting social, emotional, developmental, and executive functioning outcomes at the 6- and 18-month assessments. The propensity scores from Aim 1 are utilized to control for confounding variables, and the number of caregiver transitions. We model main effects of the proposed predictors and interactions with NOWS diagnosis, and models to determine sex differences. The same consolidated measures are used from Aim 1, and subsequent exploratory analyses of consolidated measure components are evaluated using leave-one-out cross-validation.

Aim 3

Gauge the accuracy and feasibility of implementing proposed newborn autonomic biomarkers as NOWS diagnostic assessments

We examine the diagnostic accuracy of our newborn autonomic biomarkers compared to the current diagnostic measures, the Finnegan, NWI, and ESC. We assess the sensitivity, specificity, and predictive value of each proposed newborn autonomic marker [93, 94] and potential combinations thereof.

Discussion

We propose to advance NOWS standard-of-care by developing a diagnostic assessment based on replicable and reliable clinical markers indicative of infants’ neurobehavioral outcomes. We aim to distinguish these early clinical markers of NOWS risk that are detectable in the first 24 hours after birth, using standardized tools that are routine care in some hospitals (NNNS-II), automated assessments that could be easily disseminated into well baby nurseries or NICUs (infant cry analyzer, sleep sensor), autonomic biomarkers (HRV, salivary cortisol, epigenetics), and maternal measures (hair toxicology, sociodemographic information).

This study protocol is not without limitations. Confounding factors like maternal stress—due to poverty, psychopathology, and poor general health, as well as exposure to violence, trauma, and/or abuse—may complicate the interpretations of our findings. For example, while fetal and newborn heart rate variability have been linked to opioid exposure [30, 31], greater endorsements of prenatal stress have also been associated with reduced fetal heart rate variability [95]. Similarly maternal stress may also play a role in infant cortisol levels [33, 36]. Therefore, we measure maternal sociodemographic risk, psychopathology, and trauma history in this protocol to account for potential confounding. Further, the COMT and NR3C1 gene methylation are related to both infant stress and opioid exposure, which may confound findings. Site-level differences may also complicate findings; therefore, we account for site within each analysis. The current study protocol recruits from a hard to retain population, and the length of the study and the number of study measures increase the complexity of the study, which may make recruitment and retention less feasible. However, to combat this limitation, we implement an evidence-based recruitment and retention plan for high-risk families (See Tables B & C in S1 Text).

Early identification of NOWS could lead to more precise treatment, which could reduce severity, contribute to more timely hospital discharges, decrease hospital costs, and improve care by eliminating disruptions inherent in a hospital setting. With this protocol, we could improve our capacity to care for this patient population.

Supporting information

S1 Text (DOCX)

We thank the families who continue to generously donate their time to participate in our study. Thank you to the dedicated staff research assistants.

10.1371/journal.pone.0306176.r001
Decision Letter 0
Metwally Ammal Mokhtar Academic Editor
© 2024 Ammal Mokhtar Metwally
2024
Ammal Mokhtar Metwally
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
30 Oct 2023

PONE-D-23-04246Enhancing the diagnostic accuracy and predictive validity of neonatal opioid withdrawal syndrome: The utility of non-invasive clinical markersPLOS ONE

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[Note: HTML markup is below. Please do not edit.]

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Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: On line 37, the word "consent" was left out and should be added.

Paragraph beginning line 79 - the authors should clarify that the mu-receptor binding, and then lack of, is primarily responsible for the symptoms of withdrawal as described; individuals not familiar with this area may not make that connection.

Line 95 - May want to change the wording "evidenced-based". Given the recent ongoing studies evaluating the assessment of infants with NOWS, this is now becoming evidenced-based. Would delete that phrase.

Line 106 authors state NNNS-II; later it is called NNNS. Please be consistent.

The authors report that infants exposed to both opioids and cocaine have louder and higher pitched cries (line 133). How can cry be used in infants with co-exposures? Is this a limitation?

The authors report that maternal stress can negatively impact fetal development, and thus impact the cortisol levels. How does the maternal stress play into the impact on HRV? That should be considered when assessing the HRV and may limit the applicability of HRV in certain populations. Similarly, including maternal stressors as a confounding factor in the cortisol measurements is important.

In assessing the COMT gene - has this also been assessed in individuals with higher stress levels to ensure that the increase in truly in relation to the opiate exposure?

The authors conclude in lines 217-220 that infants with a longer gestation have worse NOWS. This should be reconsidered. Currently, the assessments that are used are focused on the term infant and what is expected at that gestational age. This then leads to the inability to accurately assess preterm infants for NOWS, given that they have very different neurological development at birth and during the period of expected withdrawal. I would not conclude that term infants have worsex withdrawal, but rather that we are not able to assess withdrawal in preterm accurately.

Line 262 - states that data is collected 24 hours of life, when infant is pre-symptomatic. I would take out pre-symptomatic, as some infants do develop withdrawal symptoms at that time, especially when they are exposed to opioids with short half-lives.

What are "caregiver transitions"? This should be defined.

Does the removal of the HRV monitor result in stress in the infant that could impact the samples collected for cortisol / gene measurement?

Please include citations to support line 283 - the family resource scale has high test-retest reliability and predictive validity.

If the pregnant individual does not have long enough hair, are they excluded from the study?

Were all infants included in the study singleton? Term? Breastfed or bottle fed?

What is the timing of this study (later it is stated 2020)? Why was Bayley III used instead of IV? The Bayley III would have been outdated at that time.

The authors say "Infant measures" when describing the 18 month visit. The term infant is no longer applicable and should be changed.

If the cry analyzer is used in an open bay NICU, how accurate is it? Will it pick up the noise of surrounding infants?

Are there results of this protocol? There is a lot of description of the protocol, with almost no discussion and no results. The title implies that there should be results given. If this is meant to be a protocol paper, that should be clearly stated in the text rather than simply stated as the article type. The discussion should also include more points about confounding factors that may impact the assessments, strengths and weaknesses of the approach, etc.

Reviewer #2: I read with inerest the paper by Mylott and colleagues about a possible protocol for the early identification of NOWS, aiming to provide a more targeted approach to neonates ar risk, allowing an early identification of NOWS and also of those infants that will not develop this problem. moreover the later timepoints will allow to evaluate the neurodeveoplment of this particular population, that is evidently at risk for neurodevelopment impairment.

The suggested protocol enclosed multiple approaches to target from different sides the problem, analysing different aspects of the same issue.

The proposal is surely interesting and complete, and the results of its application will consequently be awaited.

The main concern about this approach is obviously the complexity, that requires many different approaches and is consequently time-consuming and makes it less feasible in a setting with less resources. Authors should adress this aspect.

**********

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Reviewer #2: No

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10.1371/journal.pone.0306176.r002
Author response to Decision Letter 0
Submission Version1
5 Jan 2024

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When submitting your revision, we need you to address these additional requirements.

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2. Please include “Protocol” in the manuscript title.

Authors: “Protocol” was added to the title: A protocol for enhancing the diagnostic accuracy and predictive validity of neonatal opioid withdrawal syndrome: The utility of non-invasive clinical markers

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

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We will update your Data Availability statement on your behalf to reflect the information you provide.

4. We note that you have referenced (Unpublished) om page 28, which has currently not yet been accepted for publication. Please remove this from your References and amend this to state in the body of your manuscript: (ie “Bewick et al. [Unpublished]”) as detailed online in our guide for authors

http://journals.plos.org/plosone/s/submission-guidelines#loc-reference-style

Authors: We added i.e., Weiss et al. [Unpublished] to the body of the text.

Additional Editor Comments:

The manuscript is interested meanwhile, the reviewers have raised a number of points which we believe would improve the manuscript and may allow a revised version to be published in PLOS one.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: On line 37, the word "consent" was left out and should be added.

Authors: We thank the reviewer for pointing out this error. We added “consent” to line 37.

Paragraph beginning line 79 - the authors should clarify that the mu-receptor binding, and then lack of, is primarily responsible for the symptoms of withdrawal as described; individuals not familiar with this area may not make that connection.

Authors: We now clarify this connection for the reader:

“Consequently, the μ-receptor binding of opioids and then the cessation binding (i.e., the cessation of opioid exposure) after birth may lead to withdrawal signs, including autonomic instability, high-pitched cry, irritability, tremors, and difficulty feeding and sleeping (12).”

Line 95 - May want to change the wording "evidenced-based". Given the recent ongoing studies evaluating the assessment of infants with NOWS, this is now becoming evidenced-based. Would delete that phrase.

Authors: We changed the wording “evidenced-based” on line 95 to describe the limitations of the current clinical assessments in more detail:

“However, these diagnostic tools are not often thoroughly researched, psychometrically sound, and/or standardized, and the choice of diagnostic tool depends on independent hospital protocols (15).”

Line 106 authors state NNNS-II; later it is called NNNS. Please be consistent.

Authors: We now refer to the NeoNatal Neurobehavioral Scale as the NNNS-II, based off the original NNNS exam.

The authors report that infants exposed to both opioids and cocaine have louder and higher pitched cries (line 133). How can cry be used in infants with co-exposures? Is this a limitation?

Authors: We discuss the implications of exposures and co-exposures for infant cry measures:

“Infants with in utero opioid exposure tend to have shorter cry utterances and extremely high-pitched cries, and infants co-exposed to opioids and cocaine have comparably louder and higher pitched cries (20). High-pitched and hyperphonated cries have been reported in infants with neurologic problems (21), suggesting that infants with substance exposures, especially those with polysubstance exposures may be at increased neurological risk.”

The authors report that maternal stress can negatively impact fetal development, and thus impact the cortisol levels. How does the maternal stress play into the impact on HRV? That should be considered when assessing the HRV and may limit the applicability of HRV in certain populations.

Similarly, including maternal stressors as a confounding factor in the cortisol measurements is important.

Authors: We thank the reviewer for this feedback. We added a limitations section to discuss maternal stress as a confounding variable:

“This study is not without limitations. Confounding factors like maternal stress—due to poverty, psychopathology, and poor general health, as well as exposure to violence, trauma, and/or abuse—may complicate the interpretations of our findings. For example, while fetal and newborn heart rate variability have been linked to opioid exposure (30,31), greater endorsements of prenatal stress have also been associated with reduced fetal heart rate variability (92). Similarly maternal stress may also play a role in infant cortisol levels (33,36). Therefore, we measure maternal sociodemographic risk, psychopathology, and trauma history in this protocol to account for potential confounding.”

In assessing the COMT gene - has this also been assessed in individuals with higher stress levels to ensure that the increase in truly in relation to the opiate exposure?

Authors: The COMT gene has been assessed in individuals with higher stress exposure. At present it is unclear whether associations between opioid exposure and COMT gene is due to opioid exposure or related exposures such as stress. We included this as a limitation:

“Further, the COMT and NR3C1 gene methylation are also related to both infant stress and opioid exposure, which may confound findings.”

The authors conclude in lines 217-220 that infants with a longer gestation have worse NOWS. This should be reconsidered. Currently, the assessments that are used are focused on the term infant and what is expected at that gestational age. This then leads to the inability to accurately assess preterm infants for NOWS, given that they have very different neurological development at birth and during the period of expected withdrawal. I would not conclude that term infants have worse withdrawal, but rather that we are not able to assess withdrawal in preterm accurately.

Authors: We thank the reviewer for this suggestion. We added this an explanation for late term infants’ likelihood of being treated for NOWS:

“These findings may be due to the high permeability of the placenta in the late third trimester (50) or that current assessments are not geared toward assessing NOWS in preterm infants who may display differences in neurological development and, therefore, withdrawal signs at birth.”

Line 262 - states that data is collected 24 hours of life, when infant is pre-symptomatic. I would take out pre-symptomatic, as some infants do develop withdrawal symptoms at that time, especially when they are exposed to opioids with short half-lives.

Authors: We thank the reviewer for highlighting this point. We removed “pre-symptomatic” as a descriptive as some of our infants may show symptoms prior to the 24-hour mark.

What are "caregiver transitions"? This should be defined.

Authors: We now define caregiver transitions:

“We record the number of primary caregiver changes over the course of the study, starting at birth. This occurs when there is a change in custody. The child’s primary caregiver at birth is the biological mother. Caregiver transitions occur when the child is placed with a foster parent, adoptive parent, or other caregiver (e.g., family member). Reasons for caregiver transitions include DCFS involvement (e.g., neglect, abuse, substance use), incarceration of caregiver, death of caregiver, adoption, and/or reunification, among others.”

Does the removal of the HRV monitor result in stress in the infant that could impact the samples collected for cortisol / gene measurement?

Authors: We now clarify that the removal of the HRV monitor does not impact the samples collected as we remove the ECG leads and wires after the saliva and buccal samples have been collected:

“ECG leads and wires are removed post newborn assessments, so that we cause no additional stress during the exams.”

Please include citations to support line 283 - the family resource scale has high test-retest reliability and predictive validity.

Authors: We thank the authors for pointing out this discrepancy. We report the citation to verify reliability and predictive validity of the Family Resource Scale

61. Dunst CJ, Leet HE. Family Resource Scale. Child Care Health Dev [Internet]. 1987; Available from: https://doi.org/10.1037/t33262-000

If the pregnant individual does not have long enough hair, are they excluded from the study?

Authors: We retained all participants even if they were not able to provide hair samples:

“Finally, we collect maternal hair samples for toxicology analyses. If the hair is too short or birthing parents refuse to provide the hair sample, the missing data is tracked but participants are still included in the study.”

Were all infants included in the study singleton? Term? Breastfed or bottle fed?

Authors: Our sample is not limited to singleton term infants and infants can be breast or bottle fed. We clarify this in the study population section:

“Newborns are medically stable and include singleton and twin pregnancies, term and preterm infants, and breast- and bottle-fed infants.”

What is the timing of this study (later it is stated 2020)? Why was Bayley III used instead of IV? The Bayley III would have been outdated at that time.

Authors: We thank the reviewer for highlighting this error. The study officially began in 2021 and the Bayley IV is used. We updated the appropriate sections in the paper.

The authors say "Infant measures" when describing the 18 month visit. The term infant is no longer applicable and should be changed.

Authors: We now refer to “Infant measures” at 18-months as “Toddler measures.”

If the cry analyzer is used in an open bay NICU, how accurate is it? Will it pick up the noise of surrounding infants?

Authors: We collect cry data in open bay NICUs and Intermediate Care Nurseries. After recordings are uploaded, they are screened and filtered for ambient noise so that only the target infant cries are included in analyses.

Are there results of this protocol? There is a lot of description of the protocol, with almost no discussion and no results. The title implies that there should be results given. If this is meant to be a protocol paper, that should be clearly stated in the text rather than simply stated as the article type.

Authors: We updated the title to include “protocol” to clarify there are no findings discussed within the manuscript.

The discussion should also include more points about confounding factors that may impact the assessments, strengths and weaknesses of the approach, etc.

Authors: We thank the reviewer for the feedback. We added potential confounding factors and discussed their potential impact on the study (page 22 & 23).

Reviewer #2: I read with inerest the paper by Mylott and colleagues about a possible protocol for the early identification of NOWS, aiming to provide a more targeted approach to neonates ar risk, allowing an early identification of NOWS and also of those infants tha

Attachment Submitted filename: Review_12.20.23.docx

10.1371/journal.pone.0306176.r003
Decision Letter 1
Metwally Ammal Mokhtar Academic Editor
© 2024 Ammal Mokhtar Metwally
2024
Ammal Mokhtar Metwally
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
29 Feb 2024

PONE-D-23-04246R1A protocol for enhancing the diagnostic accuracy and predictive validity of neonatal opioid withdrawal syndrome: The utility of non-invasive clinical markersPLOS ONE

Dear Dr. Maylott,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Ammal Mokhtar Metwally, Ph.D (MD)

Academic Editor

PLOS ONE

Additional Editor Comments:

The manuscript is interested meanwhile, the reviewers have raised a number of points which we believe would improve the manuscript and may allow a revised version to be published in PLOS one.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Under clinical measure 2: citation should be added to line 138-139: "preliminary acoustic cry data showed that it was 81% accurate in predicting NOWS."

Lines 150-151: While the higher doses of opioid exposure show more disturbance in sleep, higher doses of opioid exposure are not necessarily linked to more severe withdrawal in the infant. Please revise this sentence.

Please revise lines 221-224. Why would an infant with a longer gestation spend longer time in the hospital compared to an infant born at a shorter gestation (ie - 23 weeks gestation). Also, the correlation between dose exposure and severity of withdrawal continues to be debated.

Methods - when is the cry recording started? The methods states it continues during the NNNS-II but doesn't say the start. Does the infant keep the HRV leads on during the NNNS-II assessment? Is this felt to have any impact on the results of the NNNS?

Was IRB approval obtained - this was found later in the manuscript at the end of the methods? At one point consented is mentioned, but IRB approval is not included. Please move the IRB information, with the date range of enrollment (not given) to the beginning of the methods.

Line 297 - "caregiver changes may be an indicator of problem behavior." Do you have evidence to support this is true in this population? This is a high risk population, and so caregiver changes may occur due to caregiver indications. Consider removing this sentence.

What was the timing of enrollment in the study? I ask given that Finnegan was used to assess withdrawal, and ESC is becoming the gold standard approach for assessment.

If the caregiver at the 6 month visit was not the birthing caregiver, what data was collected (if any)?

Consider changing maternal measures to birthing caregiver measures

Under the 6 month infant measures, please remove those items collected at 18 months, at that makes it confusing to readers. Include those items under the 18 month infant measures.

If the caregiver at the 18 month visit was not the birthing caregiver, what data was collected (if any)?

Consider changing maternal measures to birthing caregiver measures

Reviewer #3: It is a carefully done piece of work. I am extremely satisfied with the content, structure, and scientific approach of this study protocol. I firmly believe that it is a great work with great public health significance.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0306176.r004
Author response to Decision Letter 1
Submission Version2
30 Apr 2024

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #3: Yes

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #3: Yes

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #3: Yes

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

Under clinical measure 2: citation should be added to line 138-139: "preliminary acoustic cry data showed that it was 81% accurate in predicting NOWS."

Authors: We thank the reviewer for pointing out this discrepancy. We reference preliminary data that is unpublished, so we added “(Unpublished data)” at the conclusion of the sentence:

“Preliminary acoustic cry data showed that it was 81% accurate in predicting NOWS; moreover, acoustics were more accurate in some cases when the observational crying score was incorrectly evaluated (Unpublished data).”

Lines 150-151: While the higher doses of opioid exposure show more disturbance in sleep, higher doses of opioid exposure are not necessarily linked to more severe withdrawal in the infant. Please revise this sentence.

Authors: We appreciate the feedback and agree that higher doses of opioid exposure do not necessarily result in more severe withdrawal; therefore, we changed “severity” to “neurobehavior” as outlined below:

“Further, newborns exposed to higher doses of opioids in utero showed more disturbances in neonatal sleep, indicating that sleep patterns provide insight into the neurobehavior of newborn withdrawal (26).”

Please revise lines 221-224. Why would an infant with a longer gestation spend longer time in the hospital compared to an infant born at a shorter gestation (ie - 23 weeks gestation). Also, the correlation between dose exposure and severity of withdrawal continues to be debated.

Authors: We agree that drawing conclusions from this literature may be difficult; however, we feel that it is important information to outline in the background. We have taken steps to clarify that hospital stay was associated with higher doses of opioid treatment, but that length of NOWS treatment was associated with gestational age. In other words, a 23-week preterm infant may be in the hospital longer, but may undergo treatment for NOWS for a shorter period than infants with longer gestations. We provide a theory on why this may be occurring. We also acknowledge that dose and severity of withdrawal is a topic that continues to be debated.

“Newborns exposed to higher doses of methadone in utero were more likely to be treated for NOWS and to spend longer in the hospital than newborns exposed to lower doses (50); however, the effect of opioid dose on the severity of withdrawal continues to be debated. Further, newborns were treated for NOWS longer if they had a longer gestation (37-42 weeks treated for an average of 39.5 days; 33-36 weeks treated for an average of 20 days; 23-32 weeks treated for an average of 10 days). These findings may be due to the high permeability of the placenta in the late third trimester (51) or that current assessments are not geared toward assessing NOWS in preterm infants who may display differences in neurological development and, therefore, withdrawal signs at birth.”

Methods - when is the cry recording started? The methods states it continues during the NNNS-II but doesn't say the start. Does the infant keep the HRV leads on during the NNNS-II assessment? Is this felt to have any impact on the results of the NNNS?

Authors: The cry recording starts at the beginning of the NNNS-II assessment. We stop the recording at the end of the NNNS-II and then restart the cry recorder. We leave the cry recorder on for the remaining duration of the infant’s hospital stay. This results in distinct recordings of the infant’s cry during the NNNS-II and after the NNNS-II.

“The cry recording begins during the NNNS-II, is stopped after the assessment concludes, and then is restarted to record continuously for the length of hospital stay.”

The HRV leads are left on the infant’s chest during the NNNS-II assessment to avoid additional stress to the infant prior to the NNNS-II. The HRV leads are not felt to have any impact on the results of the NNNS-II.

“Then we measure infant heartrate, placing three electrodes on the upper (negative) and lower (positive) chest around the infant’s heart, and on the right lower side of the chest (neutral) and are left on the infant for the full newborn visit.”

Was IRB approval obtained - this was found later in the manuscript at the end of the methods? At one point consented is mentioned, but IRB approval is not included. Please move the IRB information, with the date range of enrollment (not given) to the beginning of the methods.

Authors: We moved the IRB information to the beginning of the Methods sections and included the dates of enrollment/data collection.

“This study was approved by the Institutional Review Board at the University of Utah on September 14th, 2019 (00124221) and Women and Infants Hospital on February 13th, 2020 (1479081-3). Written consent was obtained for all participants. Enrollment and data collection began in 2019 and will be completed in 2025.”

Line 297 - "caregiver changes may be an indicator of problem behavior." Do you have evidence to support this is true in this population? This is a high risk population, and so caregiver changes may occur due to caregiver indications. Consider removing this sentence.

Authors: We thank the reviewer for the feedback. Given the robust evidence that caregiver changes are related to increased risk for problems behavior, we theorized that caregiver changes may have a detrimental impact on development. We now cite this literature.

Fisher PA, Stoolmiller M, Mannering AM, Takahashi A, Chamberlain P. Foster placement disruptions associated with problem behavior: Mitigating a threshold effect. J Consult Clin Psychol. 2011;79(4):481–7.

Bada HS, Langer J, Twomey J, Bursi C, Lagasse L, Bauer CR, et al. Importance of Stability of Early Living Arrangements on Behavior Outcomes of Children With and Without Prenatal Drug Exposure. J Dev Behav Pediatr [Internet]. 2008 Jun [cited 2024 Apr 14];29(3):173. Available from: https://journals.lww.com/jrnldbp/fulltext/2008/06000/importance_of_stability_of_early_living.5.aspx?casa_token=686Fig2bOS4AAAAA:aVWtBIgzo_M0AtjbgFr_b8

What was the timing of enrollment in the study? I ask given that Finnegan was used to assess withdrawal, and ESC is becoming the gold standard approach for assessment.

Authors: The enrollment of the study began in 2019. Some participants are assessed with the Eat, Sleep, Console (ESC), as the University of Utah Hospital implemented this assessment in the maternal newborn unit in 2023; however, the NWI is still used in other units (e.g., NICU). We now cite and report use of the ESC.

If the caregiver at the 6 month visit was not the birthing caregiver, what data was collected (if any)? Consider changing maternal measures to birthing caregiver measures.

Authors: We appreciate the reviewer for highlighting this point. If the caregiver at the 6-month visit is not the birthing parent, we collect all the same self-report measures from the primary caregiver; however, they complete a different version of the infant feeding survey (Infant Feeding CG), which has modified questions regarding breastfeeding.

“If the birthing parent is not available, the primary caregiver reports these measures to best reflect the environment of the child.”

Under the 6 month infant measures, please remove those items collected at 18 months, at that makes it confusing to readers. Include those items under the 18 month infant measures.

Authors: We apologize for the confusion. We assessed several measures at both 6 and 18 months, we know provide in depth information for each measure at the first age it is used and then list the measure at the remaining ages it is used, while referencing the previous section.

If the caregiver at the 18 month visit was not the birthing caregiver, what data was collected (if any)? Consider changing maternal measures to birthing caregiver measures

If the caregiver at the 18-month visit is not the birthing parent, we collect all the same self-report measures from the primary caregiver.

“If the birthing parent is not available, the primary caregiver reports these measures to best reflect the environment of the child.”

Reviewer #3: It is a carefully done piece of work. I am extremely satisfied with the content, structure, and scientific approach of this study protocol. I firmly believe that it is a great work with great public health significance.

Authors: We appreciate the reviewer’s time and the positive feedback.

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment Submitted filename: Review_2_4.14.24.docx

10.1371/journal.pone.0306176.r005
Decision Letter 2
Metwally Ammal Mokhtar Academic Editor
© 2024 Ammal Mokhtar Metwally
2024
Ammal Mokhtar Metwally
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
12 Jun 2024

A protocol for enhancing the diagnostic accuracy and predictive validity of neonatal opioid withdrawal syndrome: The utility of non-invasive clinical markers

PONE-D-23-04246R2

Dear Dr. Maylott,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ammal Mokhtar Metwally, Ph.D (MD)

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the responsiveness by the authors and the changes made to the manuscript. No further revisions required at this time.

Reviewer #3: Well-designed, analyzed and presented study with a great positive impact for scientific world. It is high quality study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Jessie Maxwell, MD, MBA

Reviewer #3: No

**********

10.1371/journal.pone.0306176.r006
Acceptance letter
Metwally Ammal Mokhtar Academic Editor
© 2024 Ammal Mokhtar Metwally
2024
Ammal Mokhtar Metwally
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
17 Jul 2024

PONE-D-23-04246R2

PLOS ONE

Dear Dr. Maylott,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Ammal Mokhtar Metwally

Academic Editor

PLOS ONE
==== Refs
References

1 Ko J. Y. et al. , “Vital Signs: Prescription Opioid Pain Reliever Use During Pregnancy—34 U.S. Jurisdictions, 2019,” Morb. Mortal. Wkly. Rep., vol. 69 , no. 28 , pp. 897–903, Jul. 2020, doi: 10.15585/mmwr.mm6928a1 32673301
2 Reddy U. M. , Davis J. M. , Ren Z. , Greene M. F. , and for the Opioid Use in Pregnancy, Neonatal Abstinence Syndrome, and Childhood Outcomes Workshop Invited Speakers, “Opioid Use in Pregnancy, Neonatal Abstinence Syndrome, and Childhood Outcomes,” Obstet. Gynecol., vol. 130 , no. 1 , pp. 10–28, Jul. 2017, doi: 10.1097/AOG.0000000000002054 28594753
3 Patrick S. W. , Davis M. M. , Lehmann C. U. , and Cooper W. O. , “Increasing incidence and geographic distribution of neonatal abstinence syndrome: United States 2009 to 2012,” J. Perinatol., vol. 35 , no. 8 , pp. 650–655, Aug. 2015, doi: 10.1038/jp.2015.36 25927272
4 Liu G. , Kong L. , Leslie D. L. , and Corr T. E. , “A Longitudinal Healthcare Use Profile of Children with a History of Neonatal Abstinence Syndrome,” J. Pediatr., vol. 204 , pp. 111–117.e1, Jan. 2019, doi: 10.1016/j.jpeds.2018.08.032 30270164
5 Bier J. B. , Finger A. S. , Bier B. A. , Johnson T. A. , and Coyle M. G. , “Growth and developmental outcome of infants with in-utero exposure to methadone vs buprenorphine,” J. Perinatol., vol. 35 , no. 8 , pp. 656–659, Aug. 2015, doi: 10.1038/jp.2015.22 25836317
6 Oei J. L. et al. , “Neonatal Abstinence Syndrome and High School Performance,” Pediatrics, vol. 139 , no. 2 , p. e20162651, Feb. 2017, doi: 10.1542/peds.2016-2651 28093465
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