Smooth Roads Ahead: Lessons From our Sick Neonate Retrieval Service.

Strategies for free transfer of sick neonates to hospitals are in place, but reports suggest suboptimal status of the same across the country. Over 7 years, our Sick Neonate Retrieval Service (SNRS) transported 165 neonates, of whom 92.1% survived. Safe, stable transportation mandates the presence of a neonatology-trained doctor and nurse in an equipped ambulance.

Retrospective identification of cell-intrinsic factors that mark pluripotency potential in rare somatic cells.

Pluripotency can be induced in somatic cells by the expression of OCT4, KLF4, SOX2, and MYC. Usually only a rare subset of cells reprogram, and the molecular characteristics of this subset remain unknown. We apply retrospective clone tracing to identify and characterize the rare human fibroblasts primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis increased the reprogramming efficiency. We provide evidence for a unified model in which cells can move into and out of the primed state over time, explaining how reprogramming appears deterministic at short timescales and stochastic at long timescales. Furthermore, inhibiting the activity of LSD1 enlarged the pool of cells that were primed for reprogramming. Thus, even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Piscis: a novel loss estimator of the F1 score enables accurate spot detection in fluorescence microscopy images via deep learning.

Single-molecule RNA fluorescence in situ hybridization (RNA FISH)-based spatial transcriptomics methods have enabled the accurate quantification of gene expression at single-cell resolution by visualizing transcripts as diffraction-limited spots. While these methods generally scale to large samples, image analysis remains challenging, often requiring manual parameter tuning. We present Piscis, a fully automatic deep learning algorithm for spot detection trained using a novel loss function, the SmoothF1 loss, that approximates the F1 score to directly penalize false positives and false negatives but remains differentiable and hence usable for training by deep learning approaches. Piscis was trained and tested on a diverse dataset composed of 358 manually annotated experimental RNA FISH images representing multiple cell types and 240 additional synthetic images. Piscis outperforms other state-of-the-art spot detection methods, enabling accurate, high-throughput analysis of RNA FISH-derived imaging data without the need for manual parameter tuning.

Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states.

The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.

Heart rate and heart rate variability following sleep deprivation in retired night shift workers and retired day workers.

Shift workers experience poor sleep and dysregulated cardiac autonomic function during sleep. However, it is unknown if this dysregulation persists into retirement, potentially accelerating the age-associated risk for adverse cardiovascular outcomes. Using sleep deprivation as a physiological challenge to cardiovascular autonomic function, we compared heart rate (HR) and high-frequency heart rate variability (HF-HRV) during baseline and recovery sleep following sleep deprivation between retired night shift and day workers. Participants were retired night shift (N = 33) and day workers (N = 37) equated on age (mean [standard deviation] = 68.0 [5.6] years), sex (47% female), race/ethnicity (86% White), and body mass index. Participants completed a 60-h lab protocol including one night of baseline polysomnography-monitored sleep, followed by 36 h of sleep deprivation and one night of recovery sleep. Continuously recorded HR was used to calculate HF-HRV. Linear mixed models compared HR and HF-HRV during non-rapid eye movement (NREM) and REM sleep between groups during baseline and recovery nights. Groups did not differ on HR or HF-HRV during NREM or REM sleep (ps > .05) and did not show differential responses to sleep deprivation. In the full sample, HR increased and HF-HRV decreased from baseline to recovery during NREM (ps < .05) and REM (ps < .01). Both groups exhibited cardiovascular autonomic changes during recovery sleep following 36 h of sleep deprivation. Sleep deprivation appears to induce cardiovascular autonomic changes that persist into recovery sleep in older adults, regardless of shift work history.

Diverse clonal fates emerge upon drug treatment of homogeneous cancer cells.

Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells1-7. Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy7-9; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.

Cardiorespiratory Adverse Events after First Vaccination in Preterm Neonates With Gestational Age ≤30 Weeks.

OBJECTIVE: To document the adverse cardiorespiratory events following first routine immunization in preterm neonates. METHODS: We retrieved records of neonates with gestational age ≤30 weeks, and included those who developed cardiorespiratory events after first vaccines before discharge. Our Unit's protocol is to administer Bacillus Calmette-Guerin (BCG), hepatitis B vaccine to those discharged at <8 weeks postnatal age. Hexavalent, BCG, pneumococcal vaccine and rotavirus vaccines are given at 8 weeks of age, if hospital stay is predicted to be longer. Unit compliance to vaccination administration at appropriate ages were also measured. RESULTS: Data of 161 neonates ≤30 weeks (17.4% <27 week) who completed care in the unit was studied. Cardio-respiratory adverse events were reported in 21(13.7%). None of these required initiation of invasive ventilation. High flow nasal cannula therapy and caffeine restart were required for these events in 14 (9.3%) and 6 (3.9%) neonates, respectively. Lower gestational age, bronchopulmonary dysplasia and sepsis were significant risk factors on univariate analysis. On multivariate analysis, continued need for respiratory support at 4 weeks of age (P=aOR 14.5 (95% CI 5-59.1) was the only independent risk factor for post-vaccination cardiorespiratory adverse events. Of 38 who were not vaccinated at recommended ages by unit policy, 25 were missed opportunities, the rest were deemed unstable for vaccinations at that age by the clinical team. CONCLUSION: Adverse cardiorespiratory events were uncommon after first vaccinations in very preterm neonates. Administering vaccines in this group before discharge would allow monitoring for these events, especially for those who require long-term respiratory support.

Retrospective identification of intrinsic factors that mark pluripotency potential in rare somatic cells.

Pluripotency can be induced in somatic cells by the expression of the four "Yamanaka" factors OCT4, KLF4, SOX2, and MYC. However, even in homogeneous conditions, usually only a rare subset of cells admit reprogramming, and the molecular characteristics of this subset remain unknown. Here, we apply retrospective clone tracing to identify and characterize the individual human fibroblast cells that are primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis led to increased reprogramming efficiency, identifying it as a barrier to reprogramming. Changing the frequency of reprogramming by inhibiting the activity of LSD1 led to an enlarging of the pool of cells that were primed for reprogramming. Our results show that even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Effect of Clinician-directed Technical Specifications on Entrance Skin Doses in Neonates.

OBJECTIVE: To compare the entrance skin doses (ESD) before and after implementation of a radiation safety policy in neonates (RSN), which focused on clinician-directed technical specifications on the digital X-ray machine. METHODS: Prospective observations included two sets of X-rays: Before (BRSN) and after (ARSN) implementation of RSN (documented indication for X-ray/expected posttest findings, settings of 40 kVp, 0.5 mAs, film-focus distance 100 cm, gonadal-shield, optimal collimation, and post-shoot image-enhancement). RESULTS: 33 and 32 X-rays were analyzed in respective groups. Mean (SD) of calculated and machine-quantified ESD (µGy/m2) was higher in BRSN group as compared to ARSN group (P <0.001). All ARSN X-rays were interpretable for expected post-test findings. CONCLUSION: Clinicians' cognizance of ability to make consequential bedside technical specifications, can reduce ESD without affecting interpretability. These single observations could have a larger impact in sick neonates, where multiple X-rays are done.

Optimizing antibiotic use in culture-negative healthcare-associated infection with a 'stop' policy: a descriptive analytical study.

BACKGROUND AND OBJECTIVES: Many sick neonates receive antibiotics for the clinical diagnosis of probable/possible sepsis. Reports suggest rampant antibiotic use in culture-negative sepsis. We introduced an antibiotic stop policy (ASP), by defining 'completed course duration of antibiotics' in the setting of culture-negative suspected healthcare-associated infection (HAI). Antibiotic overuse days (AOD) before antibiotic stop policy (BASP) and after antibiotic stop policy (AASP) were compared. METHODS: This descriptive analytical study was conducted to measure the change in AOD after implementing ASP in culture-negative HAI. We also sought to evaluate situations in which antibiotic overuse is likely (lower gestation, ventilation, central lines) and safety of the ASP, measured as not having to restart antibiotics in the week following completed course. RESULTS: A total of 126 neonates were initiated on a new antibiotic (started or changed) for suspected HAI. Of these, 43 were excluded. Patient days of 5175 and 5208 were analyzed in BASP and AASP, respectively. Implementation of an ASP reduced AOD (from 14.49 to 3.26 AOD per 1000 patient days; p value <0.01). Safety was ensured; the number of babies who had to be restarted on antibiotics within 1 week of stopping therapy was similar in both groups. All-cause mortality and relevant morbidities were comparable between groups. CONCLUSIONS: A significant decrease in AOD after the introduction of an ASP was noted, in neonates with culture-negative suspected HAI. This difference was noted even in the most vulnerable extreme preterm babies and those requiring ventilation and central lines.

Prevalence of Group B Streptococcus in pregnant women in Kerala and relation to neonatal outcomes: a prospective cross-sectional study.

BACKGROUND AND OBJECTIVES: Early onset sepsis (EOS) in neonates is a scourge that contributes to morbidity and mortality. Prominent stakeholders recommend universal screening of antenatal women for Group B Streptococcus (GBS) and intrapartum antibiotic prophylaxis (IAP) for those who are carriers. However, there are controversies. Other guidelines allow region-specific protocols due to sociodemographic, geographical and ethnic differences. We planned to analyze the prevalence of GBS rectovaginal carriage at 36-37 weeks gestation and its effect on early neonatal status. METHODS: This prospective multidisciplinary study (Obstetrics, Perinatology, Neonatology, Microbiology and Infectious diseases) was conducted in our tertiary care center between February 2020 and May 2021. RESULTS: In our study group which included 966 mothers who delivered at the hospital, 4.8% of mothers who were screened by genito-rectal swabs were positive for GBS at 36-37 weeks gestation. All these mothers were given IAP as per protocol. Other organisms detected on screening mothers were Candida and Gram-negative bacteria. None of the neonates born to these mothers required any intensive care unit admission or therapy for systemic illness. There was no difference in clinically relevant outcomes between neonates who were born to GBS-positive mothers as compared to those born to negative screen result mothers. CONCLUSIONS: GBS prevalence in our cohort was lower than most scientific reports. The neonates born to carrier mothers did not present with signs of early-onset sepsis.

Every treasured drop! Blood transfusion requirements in very preterm neonates after implementation of blood conservation strategies: an observational analytical study.

BACKGROUND: Certain morbidities are inevitable in preterm infants; the challenge lies in minimizing them. Anemia of prematurity is multifactorial. Therapy largely depends on adult red blood cell transfusions (RBCT); which inherently, are not without problems. Most literature in this respect are retrospective or evaluate individual stratagems to reduce RBCT. METHODS: This observational analytical study was planned to compare need for RBCT, before and after institution of blood conservation strategies (BCS). All those ≤30 weeks gestation at birth during two-time epochs were included (Before BCS: retrospective; After BCS: prospective). BCS constituted of delayed cord clamping (DCC), strict sampling indications, micro-sampling with point-of-care testing (MS-POCT) and adherence to RBCT thresholds. RESULTS: Of 45 enrolled neonates in each group, proportion of those requiring even 1 RBCT was significantly reduced after BCS [51.1% vs. 26.7%, p = 0.02, OR 0.35, 95%CI (0.14, 0.84)]. Calculated cumulative blood volume losses (35.3 ml vs. 21.9 ml) and loss per kilogram birth weight (35.3 ml/kg vs. 20.12 ml/kg) were significantly lower after BCS (p = 0.0036). Need for >1 RBCT, mean lowest Hb, mean maximum-hemoglobin drop, need for arterial lines were reduced. Adherence to RBCT thresholds were acceptably good in both time epochs. However, the compliance to DCC was low in both groups, identifying one area of focus with scope for massive improvement. CONCLUSIONS: Need for RBCT transfusions largely attributable to reduced blood losses for lab analysis were reduced after BCS. Installation of in-house MS-POCT seemed to be the pivotal factor. Units that care for very preterm infants must make attempts to procure MS-POCT equipment.

• Institution of a conglomerate of blood conservation strategies (BCS) is an effective strategy to reduce red blood cell transfusion requirements in very preterm infants. • The need for multiple transfusions, calculated cumulative blood volume losses, number of venous samples drawn are also reduced with BCS. • The most important component of BCS is the availability of micro-sampling-point-of-care-testing technology. This facility will benefit centers which care for these high-risk infants.

Incidence of Metabolic Bone Disease After Implementation of Bone Protective Nutritional Strategies: A Prospective Cohort Study.

BACKGROUND: Metabolic bone disease (MBD) is a morbidity of multifactorial etiology with a high incidence in very preterm infants. We planned to study the incidence of MBD after implementation of bone health focussed nutritional strategy (BNS) in those <30 weeks gestation at birth. METHODS: This prospective cohort study including preterm newborns (<30 weeks) who received nutrition that incorporated (a) Early initiation of intravenous potassium phosphate; (b) Early enteral supplementation with multicomponent human milk fortifier at enteral feed tolerance of 40 mL/kg/day feeds itself; and (c) Weekly phosphorus measurements with optimization of enteral intakes. Incidence of MBD at 4 weeks of postnatal age and beyond were analyzed. Other relevant safety and clinical outcomes were measured. RESULTS: Of the 67 included neonates receiving BNS, 20.9% were classified as MBD. There was a low rate of hyper-phosphatemia (4.5%) and hyperkalemia (2.9%). Full enteral feeds were achieved by median (IQR) of 6 (5,7) postnatal days. CONCLUSION: In preterm newborns (24-30 weeks) MBD incidence was 20.9% after BNS was implemented. Intravenous potassium salt of phosphorus and early use of HMF were safe and feasible.

Microvillus Inclusion Disease: A Rare Mutation of STX3 in Exon 9 Causing Fatal Congenital Diarrheal Disease.

Inherited diarrheal disorders cause serious morbidity resulting in dependence on intensive care and parenteral nutrition. Microvillus inclusion disease (MVID) has been classically described and results from mutations in the gene coding myosin Vb, which is responsible for enterocyte polarization. Newer reports of mutations resulting in truncated syntaxin 3 (STX3) and Munc18-2 (STXBP2) proteins have been elucidated as causative. To date, five cases of STX3 abnormalities resulting in MVID have been described. We report an infant who presented with congenital diarrhea and was determined to have a rare mutation of STX3. This new finding would be beneficial in future functional genotype-phenotype correlation studies.

Endotracheal Aspirate and Ventilator-Associated Pneumonia in Neonates: Revisiting an Age-Old Debate.

OBJECTIVES: To explore the utility of endotracheal aspirates (ETA) for analyzing microbiological yield, incidence, risk factors for VAP, and clinically relevant outcomes. METHODS: Ventilated neonates suspected to have VAP were studied prospectively; they were classified as "VAP" or "No VAP" based on a predefined combination of clinical, radiological, and laboratory criteria. The microbiological yield from blood and ETA cultures was analyzed. RESULTS: Of 165 neonates who were ventilated for > 48 h, 65 were suspected of having VAP. Thirty-six (22.9%) were classified as VAP. Microbiological agents could be identified in 31 cases (86.1%) by ETA/blood cultures. Acinetobacter sp was the common organism identified. Duration of ventilation, and a higher number of reintubations before suspicion of VAP were significant risk factors for VAP. Positive ETA culture was associated with a greater duration of oxygen therapy and ventilation days after suspicion of VAP. CONCLUSIONS: The commonest culture yield from ETA in those suspected to have VAP was gram-negative bacilli. Duration of ventilation and reintubations were identified as significant risk factors for VAP. These are potentially modifiable factors. Positive ETA culture was associated with longer needs for respiratory supports. Negative ETA culture might encourage clinicians to stop antibiotics. TRIAL REGISTRATION: Clinical Trials Registry of India No. CTRI/2019/03/017912,  www.ctri.nic.in.