Neonatal Saliva Research Articles

Ultrasensitive detection of six sepsis-associated proteins in neonatal saliva

Sepsis is a life-threatening condition that affects millions of newborns every year. Current diagnostic practices require painful, often repetitive, blood collections to isolate and culture the causative microorganism. Definitive return of results can take up to 5 days, exposing newborns to potentially unnecessary antibiotics. Developing a more rapid, non-invasive assessment of infectious status based upon host immune response provides an alternative diagnostic approach to infection screening. However, additional blood collection for multiplex quantification of inflammatory biomarkers in neonates remains prohibitive. Alternatively, saliva may serve as an informative biofluid, though detection of biologically relevant proteins in saliva remains challenging, with analyte concentrations 100 to 1000 times lower compared to blood. To address this challenge, we developed a panel of six ultra-sensitive single-molecule array (Simoa) assays for inflammatory biomarkers known to be associated with neonatal sepsis: serum amyloid A1 (SAA1), lipopolysaccharide-binding protein (LBP), chemokines CCL20, CXCL6, CXCL12; and the adipokine resistin. When these assays were tested on 40 neonatal salivary samples, we found that CXCL6 and CCL20 were significantly elevated in infected and septic neonates compared to those uninfected. The small volumes of fluid used (~10 μL saliva) and limited concentrations of these analytes in saliva (pg/mL) underscore the importance of ultra-sensitive measurements in the development of non-invasive diagnostics and reinforces the value of neonatal saliva as a viable sample matrix for monitoring infection. We hypothesize these assays could be useful in future diagnostic tests to discriminate between infected and uninfected neonates.

Validation of Gene Expression Patterns for Oral Feeding Readiness: Transcriptional Analysis of Set of Genes in Neonatal Salivary Samples.

Neonatal health assessment is crucial for detecting and intervening in various disorders. Traditional gene expression analysis methods often require invasive procedures during sample collection, which may not be feasible or ideal for preterm infants. In recent years, saliva has emerged as a promising noninvasive biofluid for assessing gene expression. Another trend that has been growing is the use of "omics" technologies such as transcriptomics in the analysis of gene expression. The costs for carrying out these analyses and the difficulty of analysis make the detection of candidate genes necessary. These genes act as biomarkers for the maturation stages of the oral feeding issue. Salivary samples (n = 225) were prospectively collected from 45 preterm (<34 gestational age) infants from five predefined feeding stages and submitted to RT-qPCR. A better description of the targeted genes and results from RT-qPCR analyses were included. The six genes previously identified as predictive of feeding success were tested. The genes are AMPK, FOXP2, WNT3, NPHP4, NPY2R, and PLXNA1, along with two reference genes: GAPDH and 18S. RT-qPCR amplification enabled the analysis of the gene expression of AMPK, FOXP2, WNT3, NPHP4, NPY2R, and PLXNA1 in neonatal saliva. Expression results were correlated with the feeding status during sample collection. In summary, the genes AMPK, FOXP2, WNT3, NPHP4, NPY2R, and PLXNA1 play critical roles in regulating oral feeding and the development of premature infants. Understanding the influence of these genes can provide valuable insights for improving nutritional care and support the development of these vulnerable babies. Evidence suggests that saliva-based gene expression analysis in newborns holds great promise for early detection and monitoring of disease and understanding developmental processes. More research and standardization of protocols are needed to fully explore the potential of saliva as a noninvasive biomarker in neonatal care.

Salivary Therapeutic Monitoring of Buprenorphine in Neonates After Maternal Sublingual Dosing Guided by Physiologically Based Pharmacokinetic Modeling.

Opioid use disorder (OUD) during pregnancy is associated with high mortality rates and neonatal opioid withdrawal syndrome (NOWS). Buprenorphine, an opioid, is used to treat OUD and NOWS. Buprenorphine active metabolite (norbuprenorphine) can cross the placenta and cause neonatal respiratory depression (EC 50 = 35 ng/mL) at high brain extracellular fluid (bECF) levels. Neonatal therapeutic drug monitoring using saliva decreases the likelihood of distress and infections associated with frequent blood sampling. An adult physiologically based pharmacokinetic model for buprenorphine and norbuprenorphine after intravenous and sublingual administration was constructed, vetted, and scaled to newborn and pregnant populations. The pregnancy model predicted that buprenorphine and norbuprenorphine doses would be transplacentally transferred to the newborns. The newborn physiologically based pharmacokinetic model was used to estimate the buprenorphine and norbuprenorphine levels in newborn plasma, bECF, and saliva after these doses. After maternal sublingual administration of buprenorphine (4 mg/d), the estimated plasma concentrations of buprenorphine and norbuprenorphine in newborns exceeded the toxicity thresholds for 8 and 24 hours, respectively. However, the norbuprenorphine bECF levels were lower than the respiratory depression threshold. Furthermore, the salivary buprenorphine threshold levels in newborns for buprenorphine analgesia, norbuprenorphine analgesia, and norbuprenorphine hypoventilation were observed to be 22, 2, and 162 ng/mL. Using neonatal saliva for buprenorphine therapeutic drug monitoring can facilitate newborn safety during the maternal treatment of OUD using sublingual buprenorphine. Nevertheless, the suitability of using adult values of respiratory depression EC 50 for newborns must be confirmed.

Salivary therapeutic monitoring of methadone toxicity in neonates after transplacental transfer from parturient mothers treated with oral methadone guided by PBPK modeling

Opioid use disorders (OUD) during pregnancy are related to neonatal opioid withdrawal syndrome (NOWS). R,S-methadone used to treat OUD and NOWS can penetrate the placenta. High neonatal brain extracellular fluid (bECF) levels of R,S-methadone can induce respiratory depression in newborns. The purpose of this work was to estimate neonatal bECF and saliva levels to establish the neonatal R,S-methadone salivary thresholds for respiratory depression after maternal oral dosing despite the sparse data in pregnancy and newborn populations. An adult physiologically-based pharmacokinetic (PBPK) model for R,S-methadone after intravenous and oral administration was constructed, vetted, and scaled to newborn and pregnancy populations. The pregnancy model predicted the R-methadone and S-methadone doses transplacentally transferred to newborns. Then, the newborn PBPK model was used to estimate newborn exposure after such doses. After maternal oral dosing of R,S-methadone (43.8 mg/day), the neonatal plasma levels were below the respiratory depression threshold. Further, the bECF levels were above the analgesia threshold for more than 96 h. The salivary thresholds for the analgesic effects of R-methadone, S-methadone, and R,S-methadone were estimated herein at 1.7, 43, and 16 ng/mL, respectively. Moreover, the salivary thresholds for the respiratory depression of R-methadone and R,S-methadone were estimated at 58 and 173 ng/mL, respectively. Using neonatal salivary monitoring of methadone can be useful in ensuring newborns' safety during maternal OUD treatment.

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Cumulative stress, PTSD, and emotion dysregulation during pregnancy and epigenetic age acceleration in Hispanic mothers and their newborn infants

ABSTRACT Pregnancy can exacerbate or prompt the onset of stress-related disorders, such as post-traumatic stress disorder (PTSD). PTSD is associated with heightened stress responsivity and emotional dysregulation, as well as increased risk of chronic disorders and mortality. Further, maternal PTSD is associated with gestational epigenetic age acceleration in newborns, implicating the prenatal period as a developmental time period for the transmission of effects across generations. Here, we evaluated the associations between PTSD symptoms, maternal epigenetic age acceleration, and infant gestational epigenetic age acceleration in 89 maternal-neonatal dyads. Trauma-related experiences and PTSD symptoms in mothers were assessed during the third trimester of pregnancy. The MethylationEPIC array was used to generate DNA methylation data from maternal and neonatal saliva samples collected within 24 h of infant birth. Maternal epigenetic age acceleration was calculated using Horvath’s multi-tissue clock, PhenoAge and GrimAge. Gestational epigenetic age was estimated using the Haftorn clock. Maternal cumulative past-year stress (GrimAge: p = 3.23e-04, PhenoAge: p = 9.92e-03), PTSD symptoms (GrimAge: p = 0.019), and difficulties in emotion regulation (GrimAge: p = 0.028) were associated with accelerated epigenetic age in mothers. Maternal PTSD symptoms were associated with lower gestational epigenetic age acceleration in neonates (p = 0.032). Overall, our results suggest that maternal cumulative past-year stress exposure and trauma-related symptoms may increase the risk for age-related problems in mothers and developmental problems in their newborns.

Clinical Value of Serial Quantitative Analysis of Cytomegalovirus DNA in Blood and Saliva Over the First 24Months of Life in Congenital Infection: The French Cymepedia Cohort.

To evaluate cytomegalovirus (CMV) viral load dynamics in blood and saliva during the first 2years of life in symptomatic and asymptomatic infected infants and to identify whether these kinetics could have practical clinical implications. The Cymepedia cohort prospectively included 256 congenitally infected neonates followed for 2years. Whole blood and saliva were collected at inclusion and months 4 and 12, and saliva at months 18 and 24. Real-time CMV polymerase chain reaction (PCR) was performed, results expressed as log10 IU/mL in blood and in copies per milliliter in saliva. Viral load in saliva progressively decreased from 7.5 log10 at birth to 3.3 log10 at month 24. CMV PCR in saliva was positive in 100% and 96% of infants at 6 and 12months, respectively. In the first month of life, neonatal saliva viral load of less than 5 log10 was related to a late CMV transplacental passage. Detection in blood was positive in 92% of neonates (147/159) in the first month of life. No viral load threshold values in blood or saliva could be associated with a high risk of sequelae. Neonatal blood viral load of less than 3 log10 IU/mL had a 100% negative predictive value for long-term sequelae. Viral loads in blood and saliva by CMV PCR testing in congenital infection fall over the first 24months. In this study of infants affected mainly after primary maternal infection during pregnancy, all salivary samples were positive in the first 6months of life and sequelae were not seen in infants with neonatal blood viral load of less than 3 log10 IU/mL.

Maternal–fetal stress and DNA methylation signatures in neonatal saliva: an epigenome-wide association study

BackgroundMaternal stress before, during and after pregnancy has profound effects on the development and lifelong function of the infant’s neurocognitive development. We hypothesized that the programming of the central nervous system (CNS), hypothalamic–pituitary–adrenal (HPA) axis and autonomic nervous system (ANS) induced by prenatal stress (PS) is reflected in electrophysiological and epigenetic biomarkers. In this study, we aimed to find noninvasive epigenetic biomarkers of PS in the newborn salivary DNA.ResultsA total of 728 pregnant women were screened for stress exposure using Cohen Perceived Stress Scale (PSS), 164 women were enrolled, and 114 dyads were analyzed. Prenatal Distress Questionnaire (PDQ) was also administered to assess specific pregnancy worries. Transabdominal fetal electrocardiograms (taECG) were recorded to derive coupling between maternal and fetal heart rates resulting in a ‘Fetal Stress Index’ (FSI). Upon delivery, we collected maternal hair strands for cortisol measurements and newborn’s saliva for epigenetic analyses. DNA was extracted from saliva samples, and DNA methylation was measured using EPIC BeadChip array (850 k CpG sites). Linear regression was used to identify associations between PSS/PDQ/FSI/Cortisol and DNA methylation. We found epigenome-wide significant associations for 5 CpG with PDQ and cortisol at FDR < 5%. Three CpGs were annotated to genes (Illumina Gene annotation file): YAP1, TOMM20 and CSMD1, and two CpGs were located approximately lay at 50 kb from SSBP4 and SCAMP1. In addition, two differentiated methylation regions (DMR) related to maternal stress measures PDQ and cortisol were found: DAXX and ARL4D.ConclusionsGenes annotated to these CpGs were found to be involved in secretion and transportation, nuclear signaling, Hippo signaling pathways, apoptosis, intracellular trafficking and neuronal signaling. Moreover, some CpGs are annotated to genes related to autism, post-traumatic stress disorder (PTSD) and schizophrenia. However, our results should be viewed as hypothesis generating until replicated in a larger sample. Early assessment of such noninvasive PS biomarkers will allow timelier detection of babies at risk and a more effective allocation of resources for early intervention programs to improve child development. A biomarker-guided early intervention strategy is the first step in the prevention of future health problems, reducing their personal and societal impact.

DNA methylation in relation to gestational age and brain dysmaturation in preterm infants.

Preterm birth is associated with dysconnectivity of structural brain networks and is a leading cause of neurocognitive impairment in childhood. Variation in DNA methylation is associated with early exposure to extrauterine life but there has been little research exploring its relationship with brain development. Using genome-wide DNA methylation data from the saliva of 258 neonates, we investigated the impact of gestational age on the methylome and performed functional analysis to identify enriched gene sets from probes that contributed to differentially methylated probes or regions. We tested the hypothesis that variation in DNA methylation could underpin the association between low gestational age at birth and atypical brain development by linking differentially methylated probes with measures of white matter connectivity derived from diffusion MRI metrics: peak width skeletonized mean diffusivity, peak width skeletonized fractional anisotropy and peak width skeletonized neurite density index. Gestational age at birth was associated with widespread differential methylation at term equivalent age, with genome-wide significant associations observed for 8870 CpG probes (P < 3.6 × 10−8) and 1767 differentially methylated regions. Functional analysis identified 14 enriched gene ontology terms pertaining to cell–cell contacts and cell–extracellular matrix contacts. Principal component analysis of probes with genome-wide significance revealed a first principal component that explained 23.5% of the variance in DNA methylation, and this was negatively associated with gestational age at birth. The first principal component was associated with peak width of skeletonized mean diffusivity (β = 0.349, P = 8.37 × 10−10) and peak width skeletonized neurite density index (β = 0.364, P = 4.15 × 10−5), but not with peak width skeletonized fraction anisotropy (β = −0.035, P = 0.510); these relationships mirrored the imaging metrics’ associations with gestational age at birth. Low gestational age at birth has a profound and widely distributed effect on the neonatal saliva methylome that is apparent at term equivalent age. Enriched gene ontology terms related to cell–cell contacts reveal pathways that could mediate the effect of early life environmental exposures on development. Finally, associations between differential DNA methylation and image markers of white matter tract microstructure suggest that variation in DNA methylation may provide a link between preterm birth and the dysconnectivity of developing brain networks that characterizes atypical brain development in preterm infants.

Sample stability for congenital cytomegalovirus assay using Alethia after prolonged storage at different temperatures

Congenital cytomegalovirus (cCMV) infection is a leading non-genetic cause of sensorineural hearing. Alethia CMV assay is proved to be accurate, simple and appears suitable for CMV testing using neonatal saliva. However, long storage of swab samples is not validated across different time periods and storage temperature. This study verifies the effects of storage time and temperature in detection of CMV from saliva samples using molecular assay developed by Alethia.

Literature Review and an Italian Hospital Experience about Post-Natal CMV Infection Acquired by Breast-Feeding in Very Low and/or Extremely Low Birth Weight Infants.

Breastfeeding is recommended for all neonates due to a known variety of beneficial effects, but infants can be infected by cell-associated bacteria and viruses from breast milk, such as cytomegalovirus (CMV). The majority of CMV-seropositive breastfeeding women have a viral, self-restricted reactivation, can shed the virus in the milk for about 12 weeks after delivery, and can transmit the infection to their offspring. Post-natal CMV-infected term infants are mainly asymptomatic, while very low birth weight (VLBW, <1500 g) and extremely low birth weight (ELBW, <1000 g) infants may present with severe disease, short-term sequelae ranging from abnormalities in laboratory indexes to sepsis-like syndrome, and long-term sequelae such as developmental problems. Thus, the use of thermally treated maternal milk for VLBW/ELBW infants may be indicated to prevent/reduce the risk of CMV transmission. Different techniques, with varying efficacy in eradicating CMV and maintaining the activity of biological compounds in milk are available: long/short pasteurization, freeze-thawing, the use of microwaves, and ultraviolet-C irradiation. In our NICU, the use of maternal raw milk is always strongly recommended for term/preterm infants, but to reduce risk of CMV transmission, freeze-thawing mother’s own milk is used in neonates with GA ≤ 30 weeks or/and weight ≤ 1000 g, usually regardless of serological maternal condition, as CMV screening is not routinely offered to pregnant women and the milk of seroimmune mothers is not evaluated for CMV reactivation, as its rate is similar to seroprevalence. Over the last 4 years, we had 10 VLBW/ELBW newborns in our NICU with late-onset sepsis and negative cultures. In these cases, the research of CMV DNA in neonatal urine or saliva, for the diagnosis of post-natal symptomatic infection (once congenital transmission has been excluded) may be useful and not invasive. The take-home message we would like to share is that acquired CMV infection should be considered in VLBW/ELBW infants breastfed by seropositive mothers and presenting severe symptoms—particularly sepsis with negative cultures. This could allow pediatricians to make better-quality diagnoses, perform supportive therapy, provide antiviral treatment if needed, or establish a “pre-emptive” therapy for these high-risk neonates.

Technical Considerations and Protocol Optimization for Neonatal Salivary Biomarker Discovery and Analysis.

Non-invasive techniques to monitor and diagnose neonates, particularly those born prematurely, are a long-sought out goal of Newborn Medicine. In recent years, technical advances, combined with increased assay sensitivity, have permitted the high-throughput analysis of multiple biomarkers simultaneously from a single sample source. Multiplexed transcriptomic and proteomic platforms, along with more comprehensive assays such as RNASeq, allow for interrogation of ongoing physiology and pathology in unprecedented ways. In the fragile neonatal population, saliva is an ideal biofluid to assess clinical status serially and offers many advantages over more invasively obtained blood samples. Importantly, saliva samples are amenable to analysis on emerging proteomic and transcriptomic platforms, even at quantitatively limited volumes. However, biomarker targets are often degraded in human saliva, and as a mixed source biofluid containing both human and microbial targets, saliva presents unique challenges for the investigator. Here, we provide insight into technical considerations and protocol optimizations developed in our laboratory to quantify and discover neonatal salivary biomarkers with improved reproducibility and reliability. We will detail insights learned from years of experimentation on neonatal saliva within our laboratory ranging from salivary collection techniques to processing to downstream analyses, highlighting the need for consistency in approach and a global understanding of both the potential benefits and limitations of neonatal salivary biomarker analyses. Importantly, we will highlight the need for robust and stringent research in this population to provide the field with standardized approaches and workflows to impact neonatal care successfully.

Revisiting short- and long-term outcome after fetal first-trimester primary cytomegalovirus infection in relation to prenatal imaging findings.

To determine the short- and long-term outcome of pregnancies with proven first-trimester fetal cytomegalovirus (CMV) infection in a large prospective cohort. This was a prospective cohort study of pregnancies with documented primary maternal CMV infection in the first trimester and evidence of fetal infection, referred for further evaluation between January 2011 and January 2018. Maternal serological diagnosis of primary CMV infection was documented by seroconversion. Vertical CMV transmission was identified by amniocentesis with polymerase chain reaction (PCR) for the CMV genome. After birth, fetal infection was re-tested by PCR in neonatal urine or saliva samples. All patients underwent serial prenatal ultrasound scans and fetal magnetic resonance imaging (MRI) at 32-33 weeks' gestation. All neonates underwent ocular fundus examination, an ultrasound brain scan and hearing evaluation, and were followed periodically for a median of 2 years (range, 6 months to 10 years). Follow-up information was obtained from hospital charts and by telephone interviews with parents. The CMV-associated outcomes assessed were sensorineural hearing loss (SNHL), neurodevelopmental abnormality, composite clinical outcome (including SNHL and neurodevelopmental abnormality) and composite outcome (additionally including termination of pregnancy (TOP)). The association between prenatal ultrasound or MRI findings and abnormal outcome was assessed. Primary CMV infection in the first trimester occurred in 123 patients. The rate of an abnormal ultrasound finding was 30.9%, and the rate of an abnormal MRI finding was 30.1% overall and 14.1% in the subgroup of patients with normal ultrasound. Of the 85 patients with normal ultrasound, 12 had an abnormal MRI finding, of whom five (5.9%) had true anatomical findings. Fifteen patients decided to terminate the pregnancy owing to abnormal prenatal findings on either ultrasound or MRI. Overall, the rate of CMV-associated postnatal and childhood sequelae was 27.8%, with a rate of 16.7% for SNHL and 11.1% for neurodevelopmental abnormalities, mostly slight motor or verbal delay. Approximately half of the cases with CMV-associated sequelae did not have any abnormal prenatal imaging findings. Abnormal prenatal findings on ultrasound were not associated significantly with SNHL, neurodevelopmental delay or composite clinical outcome (P = 0.084, 0.109 and 0.176, respectively), but they were associated with the composite outcome including TOP (P < 0.001). We identified a non-significant trend for a higher rate of SNHL in the group with abnormal ultrasound than in those with normal ultrasound. For abnormal MRI findings, we found a correlation only with neurodevelopmental abnormality and composite outcome (P = 0.014 and P < 0.001, respectively). The risk of childhood sequelae after first-trimester fetal CMV infection is most often associated with abnormal prenatal imaging findings. However, normal imaging does not rule out the development of SNHL and minor neurodevelopmental abnormalities. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

The effect of antibiotic therapy on Salivary Catalase kinetic parameters in neonatal at risk of Sepsis

Introduction: This study describes the effect of antibiotic therapy on salivary catalase kinetic parameters (CAT) at neonatal at risk of sepsis.&#x0D; Methods: Study is conducted from February – June 2015. Salivary samples are obtained from 20 neonates (5 normal-healthy neonates and 15 neonates at risk of sepsis) at Ulin General Hospital, Banjarmasin, Indonesia. The samples were placed in four different groups: P0 as control group (saliva + hydrogen peroxide/H2O2), P1 (saliva + 2 mg Ampicillin + H2O2), P2 (saliva + 0.2 mg Gentamicin + H2O2), P3 (saliva + 2 mg Ampicillin + 0.2 mg Gentamicin + H2O2). The solution is incubated at 37oC and 40oC before catalase (CAT) activity is measured. Catalase activity is measured by using a spectrophotometer at 240 nm. Kinetic parameters are measured at different concentrations of H2O2 and temperature. Kinetic parameters are represented by the Michaelis-Menten constant (Km) and the maximum reaction speed (Vmax) obtained through the Lineweaver-Burk curve plot.&#x0D; Results: The Km of Catalase on the saliva of neonates at risk of sepsis treated with antibiotics (4.37, 1.84, 0.12, and 0.23, 3.74, 1.5, for P1, P2, P3 respectively) was lower than the control group (17.61 and 12.54), both at 37ºC and 40ºC. Similarly, Vmax of the neonates at risk of sepsis treated with antibiotics (0.46, 0.34, 0.04 and 0.07, 0.20, 0.24) was lower than the control group (1.47 and 0.53) at 37ºC and 40ºC.&#x0D; Conclusion: The study shows that the Catalase activity at the saliva of newborns at risk of sepsis treated with antibiotic was lower than the control group.

The Utility of Speech-Language Biomarkers to Predict Oral Feeding Outcomes in the Premature Newborn.

Purpose Successful oral feeding and speech emergence are dependent upon the coordination of shared oral muscles and facial nerves. We aimed to determine if the speech-associated genes, forkhead box P2 (FOXP2), contactin-associated protein-like 2 (CNTNAP2), glutamate receptor, ionotropic, N-methyl D-aspartate 2A (GRIN2A), and neurexin 1, were detectable in neonatal saliva and could predict feeding outcomes in premature newborns. Method In this prospective, observational, preliminary study, saliva collected from 51 premature infants (gestational ages: 30-34 6/7 weeks) at different stages of oral feeding development underwent gene expression analysis. Binary (+/-) expression profiles were explored and examined in relation to days to achieve full oral feeds. Results GRIN2A and neurexin 1 rarely amplified in neonatal saliva and were not informative. Infants who amplified FOXP2 but not CNTNAP2 at the start of oral feeds achieved oral feeding success 3.20 (95% CI [-2.5, 8.9]) days sooner than other gene combinations. Conclusions FOXP2 and CNTNAP2 may be informative in predicting oral feeding outcomes in newborns. Salivary analysis at the start of oral feeding trials may inform feeding outcomes in this population and warrants further investigation.

Performance of the Alethia CMV Assay for Detection of Cytomegalovirus by Use of Neonatal Saliva Swabs.

Congenital cytomegalovirus (cCMV) infection is a major cause of childhood hearing loss and neurodevelopmental delay. Identification of newborns with cCMV infection allows provision of beneficial interventions. However, most infants with cCMV infection have subclinical infection and go undiagnosed. Thus, expanded neonatal CMV testing is increasingly recommended. Saliva is an attractive sample type for CMV testing of newborns, because it is easier to collect than urine and more sensitive for CMV detection than dried blood spots. We evaluated the Alethia CMV assay, a rapid, easy-to-use loop-mediated isothermal amplification method for qualitative detection of CMV DNA in neonatal saliva samples. Saliva swabs were collected prospectively from newborns <21 days old and tested by the Alethia assay according to the manufacturer's instructions. Archived saliva swabs from newborns with cCMV infection were also tested retrospectively. A composite reference method (CRM; two validated PCR assays followed by bidirectional sequencing of amplicons) was performed on all samples as the reference standard comparator. Of 1,480 prospectively collected saliva swabs, 1,472 (99.5%) were negative by both the Alethia assay and CRM, 5 (0.34%) were positive by both the Alethia assay and CRM, and 3 (0.20%) were positive only by the Alethia assay. All 34 (100%) archived swabs from newborns with cCMV infection were positive by both the CRM and the Alethia assay. Overall, the Alethia assay showed 100% and 99.8% positive and negative agreement with the CRM, respectively. The Alethia CMV assay is an accurate method for identifying neonates with cCMV infection and, given its simplicity, appears suitable for CMV testing using neonatal saliva outside a reference laboratory, including remote and resource-limited settings.

Evaluation of the Prevalence of Congenital Cytomegalovirus Infection and its Clinical Outcomes in Neonates Born in Vali-e-Asr Hospital of Birjand, Iran

Background Cytomegalovirus (CMV) has been known as the most common cause for congenital infections worldwide which can lead to death in fetus and neonates as well as neuropsychiatric deficits. The aim of this study was to determine the prevalence of congenital CMV infection in newly born neonates and to evaluate the medical outcomes. Materials and Methods In this cross-sectional study, 868 neonates were selected using unconditional random sampling in 2017. Neonatal saliva was given on the first or second day of birth using a Dacron swab and tested by PCR for the presence of CMV DNA. All infants with positive CMV infection went through further tests and examinations to evaluate the clinical outcomes. Results: 787 (90.67%) and 81 (9.33%) births were term and preterm respectively. The PCR test was positive results only in 14 term neonates (1.61%). Thus, the prevalence of CMV infection in term neonates (n=14, 1.61%) was higher than that of preterm infants (n=0), although there was no statistically significant difference (P>0.05). The most common abnormalities were neutropenia (50%, n=4) followed by anemia (37.5%, n=3). Conclusion The prevalence of CMV infection in this study (1.61%) was within the global range and there was no association between CMV infection and birth weight, infant gender, and as well ae neonatal type. The frequency of symptomatic neonates at birth in this study was higher than the average global range, but almost the same as in developing countries.

Streptococcus mutans detection in saliva and colostrum samples

ABSTRACTObjective To detect Streptococcus mutans in colostrum and saliva of neonates and compare with its detection in saliva of mothers.Methods Forty-three healthy women, full-term gestations with no complications, submitted to elective Cesarean section, and their newborns were included in the study. Samples were investigated by polymerase chain reaction to detect S. mutans in genetic material from the samples.Results Approximately 16% of colostrum samples showed S. mutans , but not correlated with the presence of the bacteria in both samples of saliva. S. mutans was detected in 49 and 30% of saliva samples of mothers and neonates, respectively. There was a positive correlation in S. mutans detection between types of saliva. The number of maternal samples of saliva with detectable S. mutans was smaller in women receiving dental treatment during pregnancy. Tooth brushing, three times a day, influenced the detection of S. mutans in both the saliva and the colostrum.Conclusion Although maternal saliva may present S. mutans , few samples of colostrum present the bacteria. The presence of bacteria in neonate saliva may be related to contact before birth. Dental treatment and hygiene habits seem to influence the detection of S. mutans in samples of maternal saliva and colostrum.

Investigation of congenital CMV infection with the presence of CMV DNA in saliva samples of new born babies

Cytomegalovirus (CMV), is the most common cause among congenital infections and is the most seen etiology in long-term sensorineural hearing loss (SNHL) and neurological impairment. Congenital CMV infection (CCMV) was reported in 0.15-2.2% of live-borne neonates in studies from different countries. A significant proportion of infected infants are asymptomatic after birth and might only be detected by routine screening methods during the new born period. The aim of this study was to screen the saliva of live-born neonates with areal-time PCR based method for the detection of CCMV in our hospital. Saliva samples collected in half an hour after birth by dry dacron swabs and were evaluated for CMV DNA (Rt-PCR, Abbott Molecular USA) from 1000 babies born in Ege University Faculty of Medicine Hospital Obstetrics Clinic between October 2015-October 2017. For the confirmation of CCMV, saliva positive newborns were evaluated with the same method for CMV DNA from their urine or blood within 21 days. All newborns were screened for sensorineural hearing tests. Subjects were 497 girls (49.7%) and 503 boys (50.3%), with a mean weight of 3116.8 g and mean of 37.61 birth week. CMV DNA was positive in the saliva of 16 newborns (1.6%). Fourteen newborns were weakly positive for CMV DNA in their saliva and were not confirmed for CCMV infection. Congenital CMV was confirmed in only two (0.2%) with the CMV DNA results in urine and/or blood samples. One of the two newborns with CCMV was symptomatic and had a neurosensorial hearing loss. The other one was asymptomatic. Saliva samples, taken immediately after birth with a noninvasive and easy method for the detection of CMV DNA is very important for diagnosis of CCMV. Positive samples should be confirmed with CMV DNA in urine or blood samples of these newborns. In this study, detection of positivity in saliva samples that were confirmed with other samples of our newborn population for CCMV was 0.2%. The specific diagnosis for CCMV in newborns with a noninvasive and easy collecting sample is important to avoid sequelae and for public health concerns.

Salivary IgG levels in neonatal calves and its association to serum IgG: an observational pilot study.

The diagnosis of inadequate transfer of colostrum immunoglobulin G (IgG) to calf serum, often known as failure of passive transfer (<10 g/L IgG1 at 24 to 48 h), necessitates blood sampling from the calf and in some instances the presence of a veterinarian. Sampling saliva is both less invasive and easy for the producer. Previous research has shown that quantification of saliva IgG is possible in juvenile and adult cattle. The objectives of this observational pilot study were to investigate whether IgG can be quantified in neonatal calf saliva, if it is correlated to serum IgG concentrations, and if the indirect quantification of saliva IgG is achievable by use of a digital refractometer. Paired blood and saliva samples were collected from 20 healthy dairy calves aged 1 to 3 d. In these samples, IgG was quantified directly with single radial immunodiffusion and indirectly by use of a digital refractometer indicating Brix % (a subsample of n = 12 saliva samples). A strong positive correlation (r = 0.7, P < 0.001) between saliva IgG (mean ± SD; 0.2 ± 0.11 g/L) and serum IgG (32.1 ± 11.94 g/L) was found. Saliva IgG ranged from the lowest detectable value, 0.1 g/L (n = 6 samples) to 0.6 g/L. Saliva Brix (1.2 ± 0.69%) was not significantly correlated to serum IgG (n = 12, r = 0.43, P = 0.155); however, it was significantly correlated to saliva IgG (n = 12, r = 0.7, P = 0.018) and Brix in serum (n = 12, r = 0.7, P = 0.013). We conclude that IgG was quantifiable in most of the saliva samples. For saliva IgG to be of any value with regards to detecting failure of passive transfer, future studies should investigate methods that can detect IgG <0.1 g/L. The results indicate that saliva IgG can be used to predict serum IgG at levels above 10 g/L, which may warrant further exploration of the use of saliva in the surveillance of failure of passive transfer. The results of the current pilot study did not support the potential usage of a Brix % refractometer to quantify saliva IgG.