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.

Effect of Early Parent Participation Program on Physiological Stability in Preterm Infants: A Randomized Controlled Trial.

OBJECTIVE: This research aimed to study the impact of early parent participation program (EPPP) for preterm infants in neonatal intensive care unit (NICU) on physiological instability, breastmilk feeding rates, and discharge timing. STUDY DESIGN: Families of 147 infants born between 28 and 33 weeks' gestation were randomized at birth to EPPP group or conventional care (CC). Families in the EPPP group were trained soon after admission by using a structured education program and encouraged to spend more time with their baby. Soon after enrolment (day of life 1 to 2), they would sequentially participate in daily NICU care processes such as orogastric tube feeding, nesting, oil massages, diaper changes, and daily weight checks. Families in the CC group would undergo the same after their infant was off parenteral nutrition and respiratory support. Proportion of infants having physiological instability (significant apnea, feeding intolerance, or needing investigation for sepsis) in two groups was compared. RESULTS: There was a significant reduction in the proportion of infants with physiological instability (feeding intolerance) in the EPPP group (relative risk = 0.70 [0.52-0.94], p = 0.016). Infants in EPPP group had a trend toward higher breastmilk feeding rates at discharge (66 vs. 51%, p = 0.076). CONCLUSION: Very early parent participation was feasible in the NICU and led to decrease in physiological instability in preterm infants. KEY POINTS: · Family-integrated care is beneficial; however, it is often started later in the NICU course.. · This trial showed that very early involvement of family in NICU care processes is feasible and safe.. · Structured parent participation started very early improves physiological stability in preterm infants (mainly tolerance to feeds)..

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.

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.

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.

Iron Status of the Moderate and Late Preterm Infant: A Prospective Cohort Study.

Guidelines on micronutrient supplementation in moderate to late preterm infants (MLP) are mostly extrapolated from those for smaller preterms, largely due to lack of systematic studies on physiological status in this special group of infants. Actual practices vary widely. We prospectively studied iron status by measurement of serum ferritin (SF) and haematological indices at 4 months corrected age in infants born between 32 and 36 weeks gestation (MLP), after they received 2 mg/kg/day oral iron from 6 weeks of postnatal age. Proportion of MLP having normal iron status (iron replete), i.e., neither iron deficiency (ID) nor iron excess was measured. ID anaemia, growth and development, risk factors for ID were also analysed. Of the 82 infants studied, 78% babies were late preterm. Seventy-four (90.3%) were iron replete (no deficiency or excess) at 4 months. High variability in SF levels (minimum of 9.8 to maximum of 252.2 µg/l) with median (IQR) of 57.45 µg/l (37.02-98.85) was noted in the entire cohort; and also within those who were iron deficient with median (IQR) of 17.50 µg/l (11.70-18.90). There was no difference in haematological indices of ID infants when compared to those with normal iron status. Inspite of oral iron supplementation with reasonable compliance, 8.5% MLP were iron deficient at 4 months corrected age. The high variability noted in SF levels could justify the need for monitoring iron status in this group of preterm infants. This could quintessentially aid individualization of iron supplementation advice.

Exclusive Breast Milk vs. Hybrid Milk Feeding for Preterm Babies-A Randomized Controlled Trial Comparing Time to Full Feeds.

When breastmilk is insufficient to meet planned feed volumes, neonatologists need to continue parenteral nutrition (PN) or use formula. This trial conducted at a tertiary care unit in South India between August 2014 and April 2016 compared time to full feeds in preterms fed 'mother's milk alone(MM)' vs. 'hybrid feed-mother's milk supplemented with formula(HF)'. We also compared time to regain birth weights, duration of PN, feed intolerance, Necrotizing Enterocolitis stage 2 or more, all-cause mortality, Extrauterine growth restriction, Healthcare associated infections, exclusive breast milk feeding rates at discharge, Retinopathy of prematurity requiring laser therapy, abnormal neurosonogram and oxygen dependency at 28 days. Neonates between 27 and 32 weeks were randomized into MM/HF when breast milk was insufficient. HF received formula to reach targeted feed volumes. MM received more PN to meet fluid requirements. 54 babies were analyzed in MM and 58 in HF. Time to full feeds were similar-MM (14.1 ± 4 days); HF (13.5 ± 4 days), p = 0.45. Exclusive breast milk feeding rates at discharge were higher in MM when compared to HF (74% vs. 51%). Other secondary outcomes were similar between groups. When mother's milk is unavailable in sufficient quantities, preterm babies may receive hybrid feeds. (Clinical trials registry of India no. REF/2016/02/006622).

Euthermia in Stable Preterm Babies: 'Cocooning' for Warmth! - A Randomized Controlled Trial.

Very preterm babies, after their initial need for rigorous supports, remain in intensive care units for maintaining euthermia. We compared proportion of 'hypothermia OR hyperthermia episodes(HHE)'; physiological instability events; and weight gain in stable preterm babies between 29 and 32 weeks nursed in Cocoon warmer (CW) vs. servo-controlled Radiant warmer(RW) in the intervals between kangaroo mother care. Sixty-six babies were randomized to CW and 59 to RW; number of temperature recordings over 24 h in CW were 1417 and 1271 in RW. HHE were comparable in RW (4.64%) and CW (5.15%); RR 1.1(0.79-1.55), p = 0.6. The combined incidence of physiological instability events was less in CW than RW [(RR 0.49 (0.25-0.97), p = 0.06]. Mean weight gain was similar, being 13.4 ± 6.1 g/day in CW and 12.8 ± 4.9 g/day in RW (p = 0.55). CW was comparable to RW in thermoregulation of hospitalized stable preterm babies.

Monotherapy with amikacin or piperacillin-tazobactum empirically in neonates at risk for early-onset sepsis: a randomized controlled trial.

BACKGROUND OF THE STUDY: Neonates at risk for early-onset sepsis are started on antibiotics empirically. Antibiotic resistance to conventionally used antibiotics is increasingly being reported. Antenatal maternal antibiotic exposure in this setting contributes to low yield on blood culture drawn at birth, limiting the planning of antibiotics based on culture reports. A head-to-head comparison for selecting the appropriate antibiotic is one strategy. OBJECTIVES: To compare monotherapy with amikacin against piperacillin-tazobactum as an empirical therapy in neonates at risk for early-onset sepsis. DESIGN: Randomized open-label controlled trial with stratification and block randomization. SETTINGS: Tertiary care neonatal unit in India PARTICIPANTS: All consecutive inborn neonates delivered between 01 May 2009 and 30 April 2011 who were ≥28 week gestation and/or ≥1000 g birth weight with risk factors for early-onset sepsis. INTERVENTION: Randomized to receive either amikacin or piperacillin-tazobactum, after stratifying as asymptomatic or symptomatic within 1 h of birth. PRIMARY OUTCOME: Incidence of treatment failure to the allocated antibiotic defined as blood culture isolate reported resistant to the allocated antibiotic or progression of the illness, necessitating a change of antibiotic. RESULTS: Of 204 eligible cases, 187 were enrolled. Seventeen babies were excluded. A total of 128 neonates were stratified as asymptomatic and 59 as symptomatic. In all, 64 of the asymptomatic cases received amikacin and 64 received piperacillin-tazobactum, while 29 symptomatic babies received amikacin and 30 received piperacillin-tazobactum. Five babies had blood culture-positive sepsis, and 28 babies had strong suspicion of sepsis. There was no difference in the treatment failure in the amikacin group (3 of 93; 3.2%) compared with piperacillin-tazobactum group (2 of 94; 2.1%) (p > 0.01) and no difference in the incidence of second infection, fungal sepsis and all-cause mortality at day 7 and 28 between the two study groups (p > 0.01). CONCLUSIONS: Monotherapy with amikacin as an empirical antibiotic did not result in a higher incidence of treatment failure in neonates at risk for early-onset sepsis as compared with piperacillin-tazobactum. Both antibiotics were effective in management of babies with early-onset sepsis.

Serum Ferritin Levels in Very Preterm Infants Receiving Erythrocyte Transfusions: A Retrospective Study.

Very preterm infants often need red blood cell transfusions (RBCT) during intensive care and are at risk of iron overload. This study reviewed the records of 65 very preterm neonates who required at least one RBCT to ascertain the iron status using serum ferritin levels at 4-6 weeks age before oral iron was commenced. High serum ferritin level was found in 52.3% (n = 34) neonates. Number of RBCT were significantly and independently associated with iron excess (P < 0.001). Increased ferritin noted following transfusions in neonatal period can have implications for determining the appropriate time for starting iron supplementation in this subgroup of neonates.

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.

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.

Reducing extrauterine growth restriction in very preterm neonates: A before-after intervention study.

BACKGROUND: Focus on preterm nutrition strategies is imperative. Extrauterine growth restriction (EUGR) is a clinically relevant, but seemingly elusive consequence, often used to benchmark and compare outcomes. METHODS: This before-after observational study was designed to study the effect of a multipronged updated "nutrition care bundle" in very preterm infants on rate of EUGR compared with a cohort from a previous period. Eligible participants were neonates born at <32 weeks' gestation who completed care in the unit; a retrospective group from a previous period and a prospective cohort after implementation of the bundle were included. The bundle constituted of three key areas: (1) aggressive parenteral nutrition with high-dose amino acids and lipids from day 1, (2) "rapid-escalation" enteral feed regimens including earlier introduction of human milk fortifier (at 40-ml/kg/day feeds), and (3) colostrum mouth paint and structured oromotor stimulation to promote oral feeding. EUGR was defined as a z score difference of >-1 in weight for postmenstrual age (PMA) at discharge and at birth. RESULTS: Data of 116 infants were retrieved for the retrospective group; 103 infants were included in the prospective group. EUGR was reduced from 71% to 58% (P = 0.039) after implementation of the bundle. Infants in the prospective group achieved full oral feeds at earlier PMA (P < 0.001) and were discharged at earlier PMA (P = 0.002). CONCLUSIONS: The proportion of neonates with EUGR was reduced significantly after implementation of the revised nutrition care bundle. Achievement of full oral feeds and discharge readiness were earlier in the prospective group.

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.

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.

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.

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.

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.

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.