research proposal on neonatal sepsis

Neonatal sepsis: a systematic review of core outcomes from randomised clinical trials

Affiliations.

• 1 Discipline of Paediatrics, Trinity College Dublin, The University of Dublin & Children's Hospital Ireland (CHI) at Tallaght, Dublin, Ireland.
• 2 John Stearne Medical Library, Trinity College Dublin, St. James' Hospital, Dublin, Ireland.
• 3 Trinity Translational Medicine Institute, St. James Hospital, Dublin, Ireland.
• 4 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin, Dublin, Ireland.
• 5 Department of Pediatrics and Adolescence Medicine, University Hospital of North Norway, Tromsø, Norway.
• 6 Paediatric Research Group, Faculty of Health Sciences, UiT-The Arctic University of Norway, Tromsø, Norway.
• 7 School of Nursing and Midwifery, Faculty of Health, University of Plymouth, Plymouth, UK.
• 8 Division of Woman and Baby, Department of Neonatology, Wilhelmina Children's Hospital (part of UMC Utrecht) and University of Utrecht, Utrecht, The Netherlands.
• 9 Neonatal Health and Development, Telethon Kids Institute, Perth, WA, Australia.
• 10 Neonatal Directorate, King Edward Memorial Hospital for Women, Perth, WA, Australia.
• 11 Clinic of Neonatology, Department Mother-Woman-Child, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
• 12 Paediatric Critical Care Research Group, Child Health Research Centre, University of Queensland, Brisbane, QLD, Australia.
• 13 Paediatric Intensive Care Unit, Queensland Children's Hospital, Brisbane, QLD, Australia.
• 14 Department of Pediatrics, Bern University Hospital, University of Bern, Bern, Switzerland.
• 15 Department of Neonatology, Pirogov Russian National Research Medical University, Moscow, Russia.
• 16 Department of Paediatrics, Tergooi Hospital, Blaricum, The Netherlands.
• 17 Department of Paediatrics, Amsterdam UMC, Emma Children's Hospital, University of Amsterdam, Amsterdam, The Netherlands.
• 18 Department of Neonatology, Radboud Institute for Health Sciences, Radboud University Medical Center, Amalia Children's Hospital, Nijmegen, The Netherlands.
• 19 Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden.
• 20 Department of Paediatrics, University of Florida, Gainesville, FL, USA.
• 21 Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FY, USA.
• 22 Department of Neonatology, Clinic for Paediatric Cardiology, Intensive Care and Neonatology, University Medical Centre Göttingen, Göttingen, Germany.
• 23 Neonatal Unit, Department of Obstetrics and Gynecology, Motol University Hospital and Second Faculty of Medicine, Prague, Czech Republic.
• 24 Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic.
• 25 Department of Pediatrics, Division of Neonatology, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands.
• 26 Department of Pediatrics, Women & Infants Hospital of Rhode Island, Alpert Medical School of Brown University, Providence, RI, USA.
• 27 Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Columbia University Medical Center, New York City, NY, USA.
• 28 Division of Neonatology, Edward Doisy Research Center, Saint Louis University, St. Louis, MO, USA.
• 29 Institute of Translational Medicine, University of Liverpool, Centre for Women's Health Research, Liverpool Women's Hospital, Liverpool, UK.
• 30 Department of Neonatal Medicine, School of Public Health, Faculty of Medicine, Imperial College London, Chelsea and Westminster Campus, London, UK.
• 31 Discipline of Paediatrics, Trinity College Dublin, The University of Dublin & Children's Hospital Ireland (CHI) at Tallaght, Dublin, Ireland. [email protected].
• 32 Trinity Translational Medicine Institute, St. James Hospital, Dublin, Ireland. [email protected].
• 33 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin, Dublin, Ireland. [email protected].
• 34 Department of Paediatrics, Coombe Women's and Infant's University Hospital, Dublin, Ireland. [email protected].
• 35 Department of Neonatology, CHI at Crumlin, Dublin, Ireland. [email protected].
• PMID: 34997225
• PMCID: PMC9064797
• DOI: 10.1038/s41390-021-01883-y

Background: The lack of a consensus definition of neonatal sepsis and a core outcome set (COS) proves a substantial impediment to research that influences policy and practice relevant to key stakeholders, patients and parents.

Methods: A systematic review of the literature was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. In the included studies, the described outcomes were extracted in accordance with the provisions of the Core Outcome Measures in Effectiveness Trials (COMET) handbook and registered.

Results: Among 884 abstracts identified, 90 randomised controlled trials (RCTs) were included in this review. Only 30 manuscripts explicitly stated the primary and/or secondary outcomes. A total of 88 distinct outcomes were recorded across all 90 studies included. These were then assigned to seven different domains in line with the taxonomy for classification proposed by the COMET initiative. The most frequently reported outcome was survival with 74% (n = 67) of the studies reporting an outcome within this domain.

Conclusions: This systematic review constitutes one of the initial phases in the protocol for developing a COS in neonatal sepsis. The paucity of standardised outcome reporting in neonatal sepsis hinders comparison and synthesis of data. The final phase will involve a Delphi Survey to generate a COS in neonatal sepsis by consensus recommendation.

Impact: This systematic review identified a wide variation of outcomes reported among published RCTs on the management of neonatal sepsis. The paucity of standardised outcome reporting hinders comparison and synthesis of data and future meta-analyses with conclusive recommendations on the management of neonatal sepsis are unlikely. The final phase will involve a Delphi Survey to determine a COS by consensus recommendation with input from all relevant stakeholders.

© 2022. The Author(s).

Publication types

• Systematic Review
• Research Support, Non-U.S. Gov't
• Delphi Technique
• Infant, Newborn
• Neonatal Sepsis* / diagnosis
• Neonatal Sepsis* / therapy
• Outcome Assessment, Health Care
• Randomized Controlled Trials as Topic
• Research Design*
• Treatment Outcome

Grants and funding

• MR/N008405/1/MRC_/Medical Research Council/United Kingdom

• Open access
• Published: 05 January 2023

Prevalence and determinants of early onset neonatal sepsis at two selected public referral hospitals in the Northwest Ethiopia: a cross-sectional study

• Tadesse Yirga Akalu 1 ,
• Yared Asmare Aynalem 2 ,
• Wondimeneh Shibabaw Shiferaw 2 ,
• Melaku Desta 1 ,
• Haile Amha 1 ,
• Dejen Getaneh 3 ,
• Bayachew Asmare 1 &
• Yoseph Merkeb Alamneh 4  

BMC Pediatrics volume  23 , Article number:  10 ( 2023 ) Cite this article

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Introduction

Globally, neonatal mortality is decreasing, and road maps such as the Early Newborn Action Plan set ambitious targets for 2030. Despite this, deaths in the first weeks of life continue to rise as a percentage of total child mortality. Neonatal sepsis with early onset continues to be a significant cause of death and illness. The majority of sepsis-related deaths occur in developing nations, where the prevalence and causes of newborn sepsis are yet unknown. As a result, the goal of this study was to determine the prevalence of early-onset sepsis and identify determinant factors.

A cross-sectional study was conducted on 368 study participants in referral hospitals of East and West Gojjam Zones from March 1 st to April 30 th , 2019. Study participants were selected at random using lottery method. Face-to-face interviews with index mothers for maternal variables and neonatal record review for neonatal variables were used to collect data using a structured pretested questionnaire. Data were entered into Epidata 3.1 and then exported to STATA/SE software version 14. Finally, the logistic regression model was used for analysis. Statistical significance was declared at P  < 0.05 after multivariable logistic regression.

A total of 368 newborns and their index mothers took part in this study. The mean age of the newborns was 4.69 days (± 1.93SD). Early-onset neonatal sepsis was seen in 34% of the babies. Nulliparity (AOR: 3.3, 95% CI: 1.1–9.5), duration of labor > 18 h after rupture of membranes (AOR: 11.3, 95% CI: 3.0—41.8), gestational age of 32–37 weeks (AOR: 3.2, 95% CI: 1.2—8.5), and neonates who require resuscitation at birth (AOR: 4, 95% CI: 1.4 -11.8) were all found to be significantly associated with early-onset neonatal sepsis.

Conclusion and recommendation

Early-onset neonatal sepsis was found to be high in this study. Early-onset neonatal sepsis was found to be associated with maternal, obstetric, and neonatal variables. Comprehensive prevention strategies that target the identified risk factors should be implemented right away.

Peer Review reports

A neonate, often known as a newborn infant, is a baby who is born within the first 28 days of life [ 1 ]. The phrase "neonatal sepsis" refers to the systemic response to infection in newborns within the first four weeks following delivery [ 2 ]. It is a clinical pattern that develops as a result of microbial blood infection in the first month of life [ 3 ]. Neonatal sepsis is defined as early-onset sepsis (if the onset of clinical features occurs between birth and 7 days) or late-onset neonatal sepsis (LONS) if the onset of clinical features occurs between 8 and 28 days after birth [ 4 , 5 ]. Early onset neonatal sepsis (EONS) has been defined in a variety of ways depending on the age of commencement, with bacteremia occurring as early as 7 days after birth [ 3 ]. Early onset neonatal sepsis (EONS) develops vertically from the mother and presents soon after birth [ 6 ]. EONS are caused by bacteria that infect the maternal genitourinary system, contaminating the amniotic fluid, placenta, cervix, and vaginal canal. When the amniotic membranes burst before delivery, the pathogen may ascend, causing an intra-amniotic infection. As a result, the infection can be acquired by the infant either in pregnancy or at delivery [ 7 ].

Maternal and neonatal risk factors for EONS include maternal age of 35 or less than 20 years, preterm, parity, cesarean delivery, urinary tract infection in the third trimester of pregnancy, and neonate sex, meconium stained amniotic fluid, birth asphyxia, and birth weight [ 8 , 9 ]. Procedures that change the amniotic cavity during pregnancy, such as cervical cerclage and amniocentesis, can raise the risk of intra-amniotic infection and neonatal sepsis [ 10 ]. Prematurity, low birth weight, congenital malformations, complex or instrument-assisted delivery, [ 3 ]and low APGAR scores (score of 6 at 5 min) are all associated with EONS in addition to the mother's variables [ 11 , 12 ]. Premature neonatal immune system immaturity, particularly low immunoglobulin G (IgG) levels due to diminished maternal IgG trans placental transfer, increases the risk of sepsis in preterm newborns [ 13 , 14 ].

Neonatal death is still the leading cause of death among children under the age of five [ 15 ]. Prematurity, asphyxia, and sepsis account for 87% of neonatal deaths worldwide [ 16 ]. According to World Health Organization, nearly 45% of under-five deaths accounts for neonatal death, with 75% occurring in the first seven days of life, and sepsis being the second most common cause . [ 17 ]. Neonatal death rates from EONS range from 16.7 to 40% [ 6 , 18 , 19 ]. Despite significant advancements in neonatal care, 40% of infants die with sepsis or suffer with neurodevelopmental impairment as a result of a lack of laboratory reagents to detect early-onset neonatal sepsis [ 20 ].

Because of greater Group B Streptococci treatment during pregnancy, the proportion of EONS relative to late onset sepsis (LOS) has been quickly reducing in high-income countries (HIC) [ 21 , 22 ]. Despite EONS can be treated and weakened quickly, it can also cause neonatal death in a matter of hours or days [ 23 ].Early onset neonatal sepsis is a common and serious problem for neonates, particularly preterm newborns [ 24 ].

The primary target of the national newborn and child survival plan is to reduce under-five mortality from 64 to 29 per 1000 children, infant mortality from 44 to 20 per 1000 children, and neonatal death from 28 to 11 per 1000 children by 2020 [ 25 ]. Identifying risk factors and putting in place core interventions are the keys towards preventing 415,688 and 210,234 deaths in children under the age of five and neonates, respectively [ 17 ]. As a result, quantifying the true number of cases of early-onset newborn sepsis in underdeveloped nations is extremely challenging [ 26 ]. A recent systematic review and meta-analysis in Ethiopia showed the prevalence of EONS was 75.44% and further studies were recommended [ 27 ]. Consequently, data on early onset infant sepsis is limited in the research area, which has the greatest rate of early neonatal death. As a result, the primary goal of this study was to quantify the prevalence of early-onset newborn sepsis in Northwest Ethiopia and identify associated factors. Knowledge of determinant factors related to early onset neonatal sepsis helps the clinician for early recognition and lowering death and illness.

Materials and methods

Study area and period.

The study was done from March 1 st to April 30, 2019, in public referral hospitals in the East and West Gojjam zones of Ethiopia's Amhara Region. There are only two public referral hospitals in the two zones, and both were included in the study since they provide inpatient neonatal treatment. Debre Markos Referral Hospital is located in Debre Markos, the town of East Gojjam Administrative Zone, while Felege Hiwot Referral Hospital is located in Bahir Dar, the town of Amhara regional state. Both are located in Northwest Ethiopia. According to information gathered from these hospitals' administrative offices, they offer a variety of services in the outpatient, inpatient, and operating room theatre departments. Debre Markos Referral Hospital [ 28 ] and Felege Hiwot Referral Hospital [ 29 ] serve a catchment area of around 3.5 million and 5 million people, respectively.

Study design and population

Data were collected from March 1 st to April 30 th , 2019, among neonates admitted to neonatal intensive care units using a hospital-based cross-sectional study design. The source population included all neonates who received care as inpatients or outpatients in the specified public referral hospital. During the data collecting period, all neonates admitted to neonatal intensive care units (NICUs) were included as study population. The study included neonates under the age of seven days who were admitted in the two public referral hospitals. Neonates with early discharge, incomplete charts, and died on arrival were all excluded from the study.

Sample size determination

The single population proportion formula was used to calculate the sample size. Given the prevalence of early onset sepsis, which is estimated to be 65% [ 12 ]. The level of confidence is 95%, while the margin of error is 5%. After calculating the sample size, a 5% non-response rate was added, yielding a total sample size of 368.

where: n – initial sample size

Z – standard normal value at 95% CI which is 1.96

P – Prevalence of early onset sepsis 65%

d – Possible margin of error tolerated which is 5%

Sampling techniques and procedure

The study participants were selected using a simple random sampling technique using the medical registration numbers of neonates admitted to neonatal intensive care units. For the NICUs at the two hospitals, the samples were distributed proportionally using the probability proportional to size (PPS) allocation technique (215 samples from Felege Hiwot Referral Hospital and 153 samples from Debre Markos Referral Hospital).

Operational definition

Early onset neonatal sepsis: was defined as presence of at least one of [difficulty feeding, history of convulsions, movement only when stimulated, respiratory rate of 60 or more breaths per min, severe chest retractions, or a temperature of 37.5 °C or higher or 35.5 °C or lower, Cyanosis, grunting and change in level of activity] with in the first seven days after birth [ 30 ]. Presence of any one of clinical signs and symptoms predict severe infection (based on an expert pediatrician’s assessment) and was associated with a sensitivity and specificity of 85% and 75% [ 31 ].

Data collection tool and quality control

The questionnaire was written in English and then translated into the local language. An impartial translator checked for consistency by re-translating it into English. Four diploma nurses and two BSc degree nurses were recruited as data collectors and supervisors respectively. Before the actual data collection, the data collectors and supervisors were briefed for two days on data collection procedures and study objectives. In Finote Selam Hospital, a pretest was conducted using 5% of the total sample size, which was not included in the actual sampling, and necessary tool adjustments were made. Data was collected through a pretested questionnaire administered by an interviewer. Index mothers were interviewed and neonatal records were reviewed using checklists for neonatal characteristics such as the APGAR (Activity, Pulse, Grimace, Appearance, and Respiration) score. Supervisors and investigators oversaw the data gathering technique on a daily basis to ensure the quality of the data. A review was conducted to ensure that the questionnaire was complete, and corrections were made. Prior to data entry, each questionnaire and data sheet were double-checked.

Data processing and analysis

The data were entered into the EPI-data Version 3.1 software. The data that were entered was checked and cleaned. Then, it was exported to STATA version 14 for analysis. To describe the research variables in relation to the population, descriptive statistics such as frequency, proportions, and percentages were generated and displayed in tables and graphs. To see if there were any relationships between the dependent and explanatory variables, bivariable and multivariable logistic regressions were used. To determine the relationship between the two variables, the odds ratio and p -value were calculated. To adjust for probable confounders, variables with a p -value ≤ 0.25 were entered into a multivariable logistic regression model. Finally, statistical significance was declared at a P -value of ≤ 0.05 and 95% confidence interval.

Socio-demographic characteristics of study participants

A total of 368 newborns and their index mothers took part in this study, with a 100% response rate. The mean age of neonates was 4.69 days with (± 1.93 SD), while the mean age of index mothers was 29.5 years with (± 7 SD). More over half of the index mothers (232) were between the ages of 20 and 34, and the majority of the study participants (84%) were married. When it came to where they lived, the majority (263, or 71.5%) were from urban centers. In terms of household income, 179 (48.6%) of study participants had a low socioeconomic level. In terms of gender, female neonates comprised 199 (54.08%) of the total, while male neonates comprised 169 (45.92%). (Table 1 ).

Gynecologic and obstetric characteristics of the mother

In terms of antenatal care (ANC), the majority of women 305 (82.88%) received ANC during their current pregnancy, with 238 (64.8%) receiving complete ANC. In terms of method of birth, the proportion of newborns delivered spontaneously was greater 208 (56.5%) than the number of neonates born via caesarian Sect. 40(10.9%) and instrumental 120(32.6%). Similarly, 351 (95.4%) of infants were attended by health care professionals. The proportion of neonates born to mothers whose labor lasted less than 12 h following rupture of membranes was roughly half of the study participants 179 (48.64%). However, 54 (14.67%) of women had a labor that lasted more than 18 h following the rupture of the membrane. Twenty three present of index mothers had intranatal fever. But, most of the index mothers (77%) had no history of intranatal fever besides Majority of mothers (91%, 94%) had no history of foul smelling amniotic fluid and antepuartum hemorrhage respectively. Majority of index mothers, (82%, and 91.3%) had no history of urinary tract infection (UTI) and pregnancy induced hypertension (PIH) respectively during their pregnancy (Fig.  1 ).

Gynecologic and obstetric characteristics of index mothers for the study on prevalence and determinants of Early Onset Neonatal Sepsis at two selected public referral Hospitals in the Northwest Ethiopia: 2019

Birth characteristics of neonatal variables

In terms of gestational age, the majority of the study participants 253 (68.7%) were born between 37 and 42 weeks after the last normal menstrual period. Similarly, the majority of the study participants' first and fifth minute APGAR scores were above seven, with 265 (72.01%) and 293 (79.62%) respectively. In terms of neonatal weight, more than half 228 (62.0%) were born in the normal birth weight range (2500–4000 g), although a small percentage of neonates were either overweight (> 4000 g) or very low birth weight (1500 g), accounting for 14(3.8%) and 13(3.5%), respectively. Similarly, the majority of the study participants 250 (67.9%) cried right after birth (Table 2 ).

Associated factors of early onset neonatal sepsis

This study was intended to determine the prevalence of early onset neonatal sepsis and its determinant factors. Early onset neonatal sepsis was found in 34% of newborns admitted during the study period. In the multivariable logistic regression, all variables with a P -value of less than 0.25 in bivariable analysis (Maternal age, Maternal age, Maternal parity, Duration of labor after rupture of membranes, Number digital of per-vaginal examination, Gestational age, Birth weight, Resuscitation at birth) were adjusted for nulliparity, duration of membrane rupture, gestational age, and resuscitation at birth were all found to be significantly associated with the occurrence of early onset neonatal sepsis in the adjusted multivariable logistic regression.

Maternal parity was found to be a significant risk factor for neonatal sepsis with early onset neonatal sepsis. In particular, neonates born from nulliparous mothers were three times (AOR: 3.3, 95% CI: 1.1- 9.5) more likely than those born to para one mothers to develop early onset sepsis.

The duration of labor following membrane rupture was revealed to be a major determinant in early onset neonatal sepsis. Specifically, neonates born after a labor lasting more than 18 h (AOR: 11.3, 95% confidence CI: 3.0—41.8) were eleven times (AOR: 11.3, 95% CI: 3.0—41.8) more likely to develop early onset sepsis than neonates born after a labor lasting less than 12 h. Neonatal gestational age was also found to be a major predictor of neonatal sepsis with early onset. Early onset sepsis was three times more common in neonates born between 32 and 37 weeks gestational age (AOR: 3.2, 95% CI: 1.2—8.5) than in neonates born between 37 and 42 weeks gestational age. Furthermore, resuscitation at birth was found to be an independent predictor of neonatal sepsis with early onset. When compared to neonates who were not resuscitated at birth, neonates who were resuscitated at birth were approximately four times more likely to develop early onset neonatal sepsis (AOR: 4, 95% CI: 1.4 -11.8) (Table 3 ).

The goal of this study was to determine the prevalence of early onset neonatal sepsis (EONS) and identify possible determinant factors in neonates admitted to public referral hospitals in Northwest Ethiopia. According to this finding, the prevalence of early onset neonatal sepsis was 34%, and nulliparity, duration of membrane rupture, gestational age, and resuscitation at birth were the significant independent predictors.

Early onset sepsis was found to be prevalent in 34% of research participants referred to neonatal intensive care units at the two selected referral hospitals. This conclusion was consistent with a study conducted in Dil Chora Referral Hospital, Eastern, Ethiopia [ 32 ]and South Sinai, Egypt [ 33 ] which found that the prevalence of early onset neonatal sepsis in neonatal intensive care units was 40.5% and 31.8% respectively. Early onset neonatal sepsis (EONS) was found to be higher than studies conducted in Indonesia 26.6% [ 34 ] and in India 20.9% [ 35 ], in hospitals of Wolaita Sodo Town 26.9% [ 36 ]. The primary cause of the disparity could be differences in sociodemographic factors. Women in these developed countries are thought to have a higher level of awareness and knowledge. Women in Ethiopia, on the other hand, are considered ineligible for school and financially unable to care for their newborns. The second likely explanation is that diagnostic criteria for cases of EONS varied. For example, in our study, we employed just clinical signs suggestive of sepsis, which overestimates EONS, whereas studies in developed countries, culture positive results were used to identify EONS. Other possible factors were no standardized policy for screening for infections in asymptomatic pregnant women and poor antenatal care which could result in insufficient time for maternal antibiotic coverage prior to or during labour. This finding was lower than a systematic review and meta-analysis in Ethiopia 75.4% (95% CI: 68.3, 82.6) [ 27 ], Gondar 59.6% [ 37 ], in Bishoftu General Hospital, Neonatal Intensive Care Unit, Debrezeit-Ethiopia 81.4% [ 13 ], in a tertiary center in Kathmandu, Nepal 91.39% [ 38 ] Public Hospitals of Hawassa City Administration, Southern Ethiopia,80.9% [ 39 ]. This variation could be due to unique cultural features of the population, local obstetrics and neonatal practices, socioeconomic and sexual practice, hygiene, and nutritional differences over settings [ 33 ] as well as due to clinical features for sepsis identification, study methodology, and sample size differences were observed among studies.

The occurrence of EONS was shown to be significantly associated with gestational age in this study. When compared to term neonates, neonates born less than 37 weeks were three times more likely to develop early onset sepsis. This finding was supported by studies in Indonesia [ 40 ], Mexico [ 41 ], India [ 42 ], South Africa [ 43 ], Pakistan [ 44 ], Ghana [ 45 ], Nepal [ 46 ] and China [ 47 ]. This association could be related to premature newborns' undeveloped immune systems and malfunctioning neutrophils. Furthermore, premature newborns lack complement proteins, making them prone to sepsis ascending [ 48 ]. Furthermore, premature neonates have insufficient immunoglobulin G (IgG), making them susceptible to sepsis from pathogenic microorganisms [ 49 ].

Early onset newborn sepsis was found to be significantly associated with the length of labor following membrane rupture. In particular, neonates born from women who had a labor after rupture of membrane that lasted more than 18 h were eleven times more likely to suffer with early onset sepsis than those born from women who had a labor after rupture of membrane that lasted less than 18 h. This finding was consistent with research conducted in the United States [ 50 ], Thailand [ 51 ], Tanzania [ 52 ] and India [ 42 ] that found labor time to be an independent determinant factor for early onset sepsis. Membrane rupture that lasts a long time (PROM) before delivery was associated with Chorioamnionitis which poses direct fetal risks from vertical transmission or ascending infection from vaginal flora due to the loss of a barrier, it is possible that PROM could be an independent risk factor for early onset neonatal sepsis.

Furthermore, maternal parity was found to be an independent predictor of with early onset neonatal sepsis. When compared to neonates delivered to para-one mothers, neonates from nulliparous women were three times more likely to develop early onset neonatal sepsis. A study conducted in Ghana [ 45 ], India [ 53 ], and South Africa [ 43 ] supports this finding. Null parity is commonly accompanied with a number of sepsis related factors, including prolonged labor and repeated per-vaginal digital examinations. Early onset neonatal sepsis was also observed to be associated with resuscitation at birth. When compared to neonates who were not resuscitated at birth, neonates who were resuscitated at birth were four times more likely to have early-onset sepsis. This finding was supported by research conducted in Tanzania [ 54 ], Ghana, [ 45 ] and the United States [ 50 ]. Poor practices and non-compliance with guidelines by health professionals during the process may expose the neonate to a higher risk of sepsis. These findings could be due to the fact that, if procedure of resuscitation is done forcefully, it may cause laceration to the susceptible and easily breakable mucous membrane of the neonate and serve as a route of entry for pathogens from unsterile equipment [ 5 ]. It’s going to also lead microbes into the lower air way of the newborn with an immature immune system. This is often due to the lumen of airways of the neonate is too narrow, and respiratory secretions are copious compared to older children which could predispose to easily destruction of smaller air sacs and leads to sepsis. In conclusion, the outcomes of this study reveal that the prevalence of early onset newborn sepsis was found to be considerably higher at the two public referral hospitals. In addition, maternal obstetric parameters such as maternal parity, duration of labor after membrane rupture, and neonatal variables such as gestational age of the neonate and neonatal resuscitation at birth were revealed to be risk factors for early onset neonatal sepsis. Comprehensive risk-reduction methods that target the identified determinants should be improved. As a result, health care providers working in neonatal intensive care units should follow guidelines when performing invasive procedures, improve maternal education on determinants such as prolonged rupture of membrane (PROM), and incorporate routine neonatal sepsis screening into neonatal and maternal care. To avoid early-onset newborn sepsis, neonates who have been resuscitated at delivery and those born to nulliparous should get special attention.

Strength and limitations of the study

This research has a number of advantages. First, we used primary data, with the exception of certain neonatal variables, to reduce the number of missing values. Second, it was carried out relatively over a wider research region. But, this study is not unique, because participant memory and self-report nature of determinant factors are potential limitations that could cause bias. Furthermore, sepsis cases were not identified based on culture-confirmed laboratory results. Neonates with signs and symptoms of sepsis may not be truly septic for culture, this could expose our findings to selection bias. Therefore, the true figure of EONS could more or less than the findings of this study for it’s based only on sign and symptoms.

Availability of data and materials

The data sets analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

Antenatal Care

Activity, Pulse, Grimace, Appearance, Respiration

Confidence Interval

Early Onset Neonatal Sepsis

Late Onset Neonatal Sepsis

Neonatal Intensive Care Unit

Adjusted Odds Ratio

Prolonged Rupture of Membrane

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Acknowledgements

First of all, our special thanks & deepest gratitude goes to Debre Markos University for supporting us to undertake this research work. We would also like to extend our heartfelt thanks for the study participants, data collectors and supervisor who participated in the study. We are also thankful for administrators of Hospitals, and head nurses of NICU in the respective Hospitals.

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Akalu, T.Y., Aynalem, Y.A., Shiferaw, W.S. et al. Prevalence and determinants of early onset neonatal sepsis at two selected public referral hospitals in the Northwest Ethiopia: a cross-sectional study. BMC Pediatr 23 , 10 (2023). https://doi.org/10.1186/s12887-022-03824-y

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Neonatal sepsis in a tertiary health facility in Cape Coast, Ghana

Roles Formal analysis, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing

Affiliation Department of Internal Medicine and Therapeutics, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana

Roles Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation Department of Community Medicine, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana

Roles Conceptualization, Investigation, Resources, Writing – review & editing

Affiliation Department of Pediatrics, Cape Coast Teaching Hospital, Cape Coast, Ghana

Roles Investigation, Writing – review & editing

Affiliation Department of Anaesthesia and Pain Management, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana

Roles Data curation, Writing – review & editing

Affiliation Department of Biostatistics, Cape Coast Teaching Hospital, Cape Coast, Ghana

• Joshua Panyin Craymah, 
• Derek Anamaale Tuoyire, 
• Portia Adjei-Ofori, 
• Oluwayemisi Esther Ekor, 
• Paul Aduoku Ninson, 
• Milton Henschel Kojo Armoh Ewusi

• Published: May 8, 2024
• https://doi.org/10.1371/journal.pone.0302533
• Reader Comments

Neonatal Sepsis remains a significant burden globally, accounting for over 2.5 million neonatal deaths annually, with low-and middle-income countries (LMIC) including Ghana disproportionately affected. The current study sought to ascertain the prevalence of neonatal sepsis and associated factors based on analysis of institutional records from Cape Coast Teaching Hospital (CCTH) in Ghana.

The study involved a retrospective cross-sectional review of randomly sampled medical records of 360 neonates CCTH from January 2018 to December 2021. Descriptive proportions and binary logistic regression analysis were conducted to estimate the prevalence of neonates with sepsis and associated factors.

The prevalence of neonates with sepsis over the period was estimated to be 59%, with early-onset neonatal sepsis (EONS) and late-onset neonatal sepsis (LONS) accounting for about 29% and 30%, respectively. Neonatal factors associated with sepsis were low Apgar score (AOR = 1.64; 95% CI:1.01–2.67, p = 0.047) and low birth weight (AOR = 2.54; 95% CI:1.06–6.09, p = 0.037), while maternal factors were maternal education (AOR = 2.65; 95% CI:1.04–6.7, p = 0.040), caesarean deliveries (AOR = 0.45; 95% CI:0.26–0.75, p = 0.003), maternal infection (AOR = 1.79; 95% CI:1.09–2.94, p = 0.020) and foul-smelling liquor (AOR = 1.84; 95% CI:1.09–3.07, p = 0.020).

The study underscores the need for improved routine care and assessment of newborns to prevent the onset of neonatal sepsis, with particular emphasis on the neonatal and maternal risk factors highlighted in the current study.

Citation: Craymah JP, Tuoyire DA, Adjei-Ofori P, Ekor OE, Ninson PA, Ewusi MHKA (2024) Neonatal sepsis in a tertiary health facility in Cape Coast, Ghana. PLoS ONE 19(5): e0302533. https://doi.org/10.1371/journal.pone.0302533

Editor: Abera Mersha, Arba Minch University, ETHIOPIA

Received: December 20, 2022; Accepted: April 9, 2024; Published: May 8, 2024

Copyright: © 2024 Craymah et al. 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.

Data Availability: The specific data file with variables generated for this study can be found on the Open Science Framework data repository at https://osf.io/C9YS4/ .

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The critical nature of the neonatal period (first 28 days of life) for child survival cannot be overemphasized. An estimated 2.5 million infants die within their first month of life annually, representing about half of deaths in children under 5 years of age [ 1 ]. Neonatal sepsis accounts for a significant proportion of all infection-related mortality and morbidity among infants within the neonatal period. Although the global burden of neonatal sepsis is difficult to ascertain, modelled data from the United Nations Inter-Agency Group for Child Mortality Estimation (UN IGME) suggest that 375,000 neonatal deaths resulted from sepsis across the globe in 2018 [ 2 ]. Estimates based on systematic review and meta-analyses of studies between 1979 and 2019 report the worldwide number of neonatal sepsis cases to be between 1.3 to 3.9 million annually, with deaths ranging from 400,000 to 700,000 [ 3 ].

The available evidence on the burden of neonatal sepsis point to significant disparities with low- and middle-income countries (LMICs) disproportionately affected. According to the Global Sepsis Alliance, the rate neonatal sepsis is about 40 times higher in LMICs while deaths are twice as high compared with more advanced countries [ 4 ]. The picture in sub-Saharan Africa is no different with about 35 neonatal deaths occurring per 1,000 live births [ 5 ].

Previous studies have documented a number of risk factors for neonatal sepsis which could generally be classified as maternal, neonatal and hospital care related factors [ 6 – 8 ]. Maternal factors typically involve factors which result in the transmission of infections from mother to foetus or neonate including urinary tract infection, premature rupture of membranes (PROM), chorioamnionitis, early breast feeding, place of delivery, prolonged labour cord care, mode of delivery and maternal demographics such as age [ 9 ]. Factors at the level of the neonate often relate to immunological immaturity which increases their susceptibility to infections. These include prematurity, sex, rashes, congenital abnormality, low Apgar and neonatal resuscitation [ 10 ]. Hospital related factors are generally associated with nosocomial infections from prolonged hospital admission, poor hygiene, invasive procedures, superficial infection, non-lacteal feeding among others [ 11 , 12 ].

Although prior studies on neonatal sepsis and associated factors in LMICs abound, there is limited literature on the subject in Ghana. The few prior studies [ 13 – 15 ] in Ghana have mainly been conducted within the context of the nation’s capital, Accra, with little insights from other regions. With the view to extending the discourse neonatal sepsis in Ghana, the current study sought to ascertain the prevalence of neonatal sepsis and associated factors based on analysis of institutional records from Cape Coast Teaching hospital (CCTH) in Ghana. Considering that CCTH serves as a referral tertiary facility for both the Central and Western regions of Ghana, insights from this study could be useful for the design of interventions to address the problem of neonatal sepsis in the locale and similar context.

Study setting and design

The study involved a retrospective cross-sectional review of medical records at neonatal intensive care unit (NICU) of the Cape Coast Teaching Hospital (CCTH) from January 1, 2018 to December 31, 2021. The hospital (CCTH) was established in 1998 as the Central regional hospital and later upgraded to the status of a teaching hospital for the training of various cadres of health professionals including doctors and nurses [ 16 ]. The facility is currently the largest referral centre in the central region with a 400-bed capacity and provides a variety of health care services including out-patient care, in-patient care, emergency care as well as specialist clinics.

The NICU from which data for the current study was sourced is housed within the Pediatric care ward for the management and treatment of critical neonatal disorders. The unit has a cot capacity of 20 and is equipped with four (4) incubators and six (6) phototherapy devices. An average of 80 neonates are admitted to the NICU on a monthly basis with neonatal sepsis among the top ten (10) indication for admission in the unit.

Study population and sampling

The target population for the study was neonates hospitalized at NICU of CCTH from January 1, 2018 to December 31, 2021. Neonatal sepsis was diagnosed based on laboratory investigations and the WHO Integrated Management of Neonatal and Childhood Illness (IMNCI) clinical features. The IMNCI clinical signs for diagnosis of neonatal sepsis include either fever (37.5°C) or hypothermia (35.5°C), tachypnea (60 breaths per minute), poor feeding, severe chest in-drawing, lethargy, convulsion, diminished sucking, and unconsciousness [ 17 ]. A sampling frame was constructed from the hospital’s electronic health records (EHR) system which contained data on all neonatal admissions for the period under review obtained from the biostatics unit of the hospital. The sampling frame consisted of 3,455 cases from which a minimum sample size of 360 was estimated using Yemane’s formula;

n = N / 1+N (e) 2 , where: n = sample size; N = the population size; e = the acceptable sampling error *95% confidence level and p = 0.5 are assumed [ 18 ].

Accordingly, n = 3,455/1+ 3,455(0.05) 2 = 358.49 ~ 360.

Using allocations proportional-to-admission per year, the number of cases to be sampled for each respective year under review was determined as presented in Table 1 . For instance, 670 neonatal cases admitted in the year 2018, represented about 19.4% of the 3,455 cases from 2018–2021. This translated into 70 cases sampled for the year 2018. The respective number of neonatal cases for each year were then selected based on the folder identification numbers for each neonatal case using the simple random sampling function in STATA 16.0 software.

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https://doi.org/10.1371/journal.pone.0302533.t001

Data extraction and analysis

Following the sampling of neonatal cases from the hospital’s electronic health records (EHR), a data extraction form was designed in Microsoft Excel for the extraction of relevant information for the purpose of the study. The form comprised of two (2) main sections. The first section focused on extracting socio-demographic information of the neonates and their mothers (age of mother, age of neonates, marital status, education, occupation, and residence). This was followed by the section on clinical information relating to the neonate (sex of the new-born, gestation time, Apgar score, neonatal jaundice, birth asphyxia, birthweight, neonatal resuscitation) and their mothers (mode of delivery, premature rupture of membranes (PROM), maternal infection, meconium stained liquor, chorioamnionitis, prolonged labour, foul-smelling liquor, breastfeeding, bubble continuous positive air pressure (B-CPAP), oxygen via mask, oxygen via nasal prongs), including a sub-section on the final clinical diagnosis (neonatal sepsis or otherwise).

Upon extraction, the data was transferred into STATA 16.0 software for analyses. The analyses involved the use of both descriptive and inferential statistical techniques. Descriptive analysis involved the use of frequencies and proportions to describe the various factors (socio-demographic and clinical characteristics of neonates and index mothers) the prevalence of sepsis as well as the proportional distribution of sepsis across the various factors considered in the study. With respect to inferential statistics, multivariable binary logistic regression analyses were conducted to determine the factors associated with neonatal sepsis. Statistical significance was set at P<0.05 with odds ratios used to interpret the associations found between the various factors and neonatal sepsis.

Ethical consideration

Ethical approval for the study was obtained on 14 th February, 2020 from the Institutional Review Board of the CCTH. Further, administrative approvals were granted by the management of CCTH, while biostatistics department ensured that all neonatal records were anonymized before the release of the data on for the purpose of this study.

Characteristics of neonates and index mothers

As shown in Table 2 , the mean age of neonates was 3.04±4.32 days old, with majority (78%) of them aged 0–7 days old, and more than half of neonates (62.5%) being female. The mean age of the mothers was 27.23±5.92 years, with about half (51.4%) of them aged 20–29 years. More than four-in-ten mothers (43.3%) had completed basic school, about two-thirds (63.9%) were married, 54% were employed, and over six-in-ten (66%) lived in urban areas.

https://doi.org/10.1371/journal.pone.0302533.t002

With respect to the clinical characteristics, more than half (55%) of neonates were preterm births, approximately 65% had an Apgar score lower than six (6), and 55% of them had a low birth weight (<2.5kg). About four-in-ten (42%) of the neonates had birth asphyxia and 35% of them were jaundiced or resuscitated. Majority (71%) of the index mothers delivered their neonates through spontaneous vaginal delivery (SVD), with over a quarter (27%) of them experiencing PROM and 37% having maternal infection. Again, about 25% had meconium liquor, 11% had chorioamnionitis, 42% had prolonged labour, 31% had history of foul-smelling liquor and about 42% initiated early breastfeeding. About a fifth (20%) of the neonates required bubble continuous positive airway pressure (B-CPAP), and about 27% and 23% receiving oxygen via nasal prongs and mask, respectively.

Prevalence of neonatal sepsis

The period (2018–2021) prevalence of neonatal sepsis from the sample of 360 was 59%, with early-onset neonatal sepsis (EONS) and late-onset neonatal sepsis (LONS) accounting for about 29% and 30%, respectively. These estimates translate to an average yearly prevalence of neonatal sepsis of 15%. Nonetheless, the disaggregated results as depicted in Fig 1 indicate that the highest prevalence of neonatal sepsis was observed in 2019 (65%). Further, EONS reduced over the period from 37% in 2018 to about 14% in 2021, while LONS increased from 24% to about 42% over the same period.

EONS = Early onset neonatal; LONS = Late onset neonatal sepsis.

https://doi.org/10.1371/journal.pone.0302533.g001

Neonatal sepsis by characteristics of neonates and index mothers

Table 3 presents the distribution of neonatal sepsis across the various characteristics of neonates and their index mothers. Neonatal sepsis was higher among neonates older than one week (70%) and among female neonates (60%). On the other hand, neonatal sepsis reduced as age and educational level of mother increased. For instance, neonatal sepsis reduced from 71% among mothers younger than 20 years to about 52% mothers aged 30 years or more; and from 68% among mothers without education to about 38% among those with tertiary level of education. In addition, higher proportion of sepsis was observed among neonates whose mothers were married (61%), employed, (60%) and mothers residing in rural localities (66%). The chi-squared test showed that these observed socio-demographic variations was statistically significant (p<0.05) for age of neonate, age of index mother and mother’s educational level.

https://doi.org/10.1371/journal.pone.0302533.t003

With respect to clinical characteristics of neonates, more than six-in-ten hospitalized neonates with each of the following characteristics were diagnosed with neonatal sepsis; late initiation of breastfeeding (61%), low birth weight (65%), low Apgar scores (64%), preterm (64.0%), and asphyxia (62.0%). More than half of the babies with jaundice (58%) and those who were resuscitated after birth (55%) were diagnosed with neonatal sepsis. Regarding maternal clinical characteristics, neonatal sepsis was higher in neonates whose mothers delivered via spontaneous vaginal delivery (SVD) (64%) compared with caesarean section (C/S) (47%). Neonatal sepsis was higher among babies whose mothers had infection (67%), prolonged labour (66%) and foul-smelling liquor (68%), compared with mothers without such clinical characteristics. This was the case for their counterparts whose mothers did not have a history of PROM (60%), meconium liquor (62%), and chorioamnionitis (59%). However, these clinical characteristics were statistically significantly for gestational age, Apgar score, neonatal weight, mode of delivery, maternal infection, prolonged labor, foul smelling and oxygen via mask.

Factors independently associated with neonatal sepsis

In modeling to determine factors independently associated with neonatal sepsis multivariable logistic analyses, six factors emerged, namely; educational level of mothers, mode of delivery, maternal infection, fouls smelling, neonatal weight and Apgar score ( Table 4 ). Neonates whose index mothers had no formal education (AOR = 2.65; 95% CI:1.04–6.70) had significantly higher odds of developing neonatal sepsis. In addition, the odds of a neonate developing neonatal sepsis were significantly higher for those whose mothers had an infection (AOR = 1.79, 95% CI:1.09–2.94) or foul-smelling liquor (AOR = 1.84, 95% CI:1.09–3.07), with reference to neonates whose mothers had no infection or foul-smelling liquor. On the other hand, neonate who were delivered via caesarian section (C/S) had significantly lower odds of developing neonatal sepsis 0.45 (95% CI:0.26–0.75). Regarding neonatal factors, neonates with a low birth weight (AOR = 2.54; 95% CI: 1.06–6.09) and those with a low Apgar score (AOR = 1.64; 95% CI: 1.01–2.67) had significantly higher odds of developing neonatal sepsis.

https://doi.org/10.1371/journal.pone.0302533.t004

We investigated the prevalence of neonatal sepsis and associated factors using data drawn from the NICU of the Cape Coast Teaching Hospital from 2018 to 2021. We observed that approximately 59% of neonates were diagnosed with sepsis over the period under review. Although varied rates of neonatal sepsis have been reported by different studies across LMICs tend to have higher rates than more advanced countries. Nonetheless, the prevalence in the current study is higher than reports from earlier studies conducted in other LMICs including Haiti (54.8%) [ 19 ] and Ethiopia (35%) [ 20 ]. The relative variation in the prevalence of neonatal sepsis between our study and other studies could largely be attributable to differences in study design and period of review. For instance, while some studies have applied a case-control design [ 21 ] others were based on cohort analysis [ 22 ].

Previous studies conducted in Ethiopia [ 23 ], Nigeria [ 24 ] and in Ghana [ 21 ] have reported higher prevalence of EOS compared with LOS, in contrast to our observations in this current study. This points to the possibility differences in source of infections resulting in sepsis. Neonates in prior studies might have been exposed to infections from factors related to hospital environment, while neonates from our study might have been exposed to home/community infections [ 25 , 26 ]. Indeed, this is evident in the higher LOS observed in our study for the years 2020 and 2021 following the institution of COVID-19 policies requiring the early discharge of women within 24 hours after delivery. Perhaps, this finding highlights the need to emphasize education of women on proper hygiene practices prior to discharge and during postnatal visits.

Although prior extant studies in Ghana [ 21 ] and Egypt [ 27 ] report no associated between educational level of mothers and neonatal sepsis, our study observed that neonates born to mothers without formal education had a higher propensity of developing neonatal sepsis. Uneducated mothers are less likely to be aware of or adhere to infection control procedures, and may also have less likely to spot danger signs in order to prevent sepsis due [ 28 ]. Postnatal counselling and health education on childcare practices tailored to mothers without formal education help in reducing the risk of sepsis among neonates born to such mothers.

As reported in prior studies in India [ 29 ] and Ethiopia [ 30 ], the current study found a higher probability of neonatal sepsis among neonates with a birth weight below 2.5 kilograms. Neonates with a low birth weight tend to have underdeveloped immune systems, and are therefore susceptible to vertical transmission of organisms from the mother before or during birth, as well as nosocomial infections during the course of hospital care after birth [ 30 ]. This is further supported by studies which have found low birth weight infants to have increased gram-negative pathogens and reduced gram-positive pathogen [ 31 , 32 ]. Our finding linking low Apgar score (<6) to neonatal sepsis resonates with prior studies [ 21 , 33 ]. It is known that stressful labour conditions reduce the ability of neonates to adopt to extra uterine life, thereby predisposing them to sepsis, especially in the event of immunological insult or revival from asphyxia [ 34 , 35 ]. This implies that aseptic precautions are strictly adhered to while caring for and performing procedures in infants with low birth weight or low Apgar score.

Similar to the findings of Atlaw et al [ 36 ] whiles studying neonatal sepsis in Ethiopia, we found neonates delivered via CS to have lower probability if developing sepsis. Indeed, the literature suggests an indirect pathway between CS delivery and neonatal sepsis, such as through lacerations from sharp instruments during the procedure that may serve portals of entry for microorganisms [ 37 , 38 ]. The increased risk of neonatal sepsis observed in the current study for neonates whose mothers had a history of maternal infection has similarly been reported in the literature [ 33 , 39 ]. Such neonates might have acquired sepsis through vertical transmission from their mothers who had an infection during the course of pregnancy. Women typically have a suppressed immune system during pregnancy which exposes them to infections which could be transmitted to the newborn before or during birth if not properly managed.

Neonates who were born to mothers with a history of foul-smelling liquor had greater odds of developing neonatal sepsis, with reference to those born from mothers without a history of foul-smelling liquor. Foul-smelling liquor has similarly been linked with neonatal sepsis in previous studies [ 40 , 41 ] and suggested to an indication of chorioamnionitis which results in systemic infection when neonates come in contact with it. Early detection and treatment of the mother with chorioamnionitis could reduce the baby’s chances of developing neonatal sepsis.

Limitation of the study

The study has some inherent limitations as acknowledged in the foregoing. A significant amount of the patient’s history information and biodata were missing. There was also a dearth of laboratory investigation to confirm those who indeed had sepsis. Another limitation was that the information on the LHIMS were entered by different physicians with different years of experience which might lead to information bias.

The findings of the analysis were derived from a tertiary health facility, which limits its representativeness. Thus, more longitudinal research addressing the same contributing factors and management of such illnesses for newborns across all tertiary facilities in Ghana would be more instructive.

The study highlights a high prevalence of neonatal sepsis over the period with higher rates of late onset sepsis among newborns. A number of factors both at the neonatal (low birth weight and Apgar score) and maternal (caesarean delivery, maternal infection, and foul-smelling liquor) level were found to significantly predict neonatal sepsis. These findings underscore the need for health care providers to improve their routine care and assessment of neonates to curb the incidence of neonatal sepsis. In doing so, particular emphasis should be on neonates with low birth weight and Apgar score and those born to mothers via caesarean section, or mothers with maternal infection, and foul-smelling liquor.

Acknowledgments

The authors would like to acknowledge the Cape Coast Teaching Hospital, especially department of biostatistics for their tremendous support during the conduct of this study.

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• 2. UNICEF. United Nations Inter-agency group for child mortality estimation (UN IGME). Levels and trends in child mortality report 2019, estimates developed by the United Nations Inter-Agency Group for Child Mortality Estimation. New York; 2019. [Cited 2022 Feb 15]. Available from: http://www.unicef.org/sites/default/files/2019-10/UN-IGME-child mortality-report-2019.pdf.
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Regulation of Alternative Splicing of Lipid Metabolism Genes in Sepsis-Induced Liver Damage by RNA-Binding Proteins

• Open access
• Published: 09 May 2024

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• Buzukela Abuduaini 1 ,
• Zhang Jiyuan 2 ,
• Aliya Rehati 3 ,
• Zhao Liang 4 &
• Song Yunlin 1 , 5  

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RNA binding proteins (RBPs) have the potential for transcriptional regulation in sepsis-induced liver injury, but precise functions remain unclear. Our aim is to conduct a genome-wide expression analysis of RBPs and illuminate changes in the regulation of alternative splicing in sepsis-induced liver injury. RNA-seq data on “sepsis and liver” from the publicly available NCBI data set was analyzed, and differentially expressed RBPs and alternative splicing events (ASEs) in the healthy and septic liver were identified. Co-expression analyses of sepsis-regulated RBPs and ASEs were performed. Models of sepsis were established to validate hepatic RBP gene expression patterns with different treatments. Pairwise analysis of gene expression profiles of sham, cecum ligation puncture (CLP), and CLP with dichloroacetate (CLPDCA) mice allowed 1208 differentially expressed genes (DEGs), of which 800 were up-regulated and 408 down-regulated, to be identified. DEGs were similar in both Sham and CLPDCA mice. The KEGG analysis showed that up-regulated genes as being involved in cytokine-cytokine receptor interaction and IL-17 signaling pathway and down-regulated genes in metabolic pathways. Differences in lipid metabolism–related alternative splicing events, including A3SS, were also found in CLP and CLPDCA compared with sham mice. Thirty-seven RBPs, including S100a11, Ads2, Fndc3b, Fn1, Ddx28, Car2, Cisd1, and Ptms, were differentially expressed in CLP mice and the regulated alternative splicing genes(RASG) with the RBP shown to be enriched in lipid metabolic and oxidation-reduction-related processes by GO functional analysis. In KEEG analysis the RASG mainly enriched in metabolic pathway. The models of sepsis were constructed with different treatment groups, and S100a11 expression in the CLP group found to be higher than in the sham group, a change that was reversed by DCA. The alternative splicing ratio of Srebf1 and Cers2 decreased compared with the sham group increased after DCA treatment. Abnormal profiles of gene expression and alternative splicing were associated with sepsis-induced liver injury. Unusual expression of RBPs, such as S100a11, may regulate alternative splicing of lipid metabolism–associated genes, such as Srebf1 and Cers2, in the septic liver. RBPs may constitute potential treatment targets for sepsis-induced liver injury.

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INTRODUCTION

Sepsis describes a dysregulated immune response to an infection that leads to multiple organ dysfunction. A clearer understanding of its pathophysiology is crucial for the optimization of sepsis management [ 1 ]. The metabolic and immune functions of the liver place this organ at the center of the body’s response to the systemic infection underlying sepsis, during which it participates in the bacterial clearance, production of acute-phase proteins and cytokines, and adaptation of metabolism to inflammation. It is acknowledged that sepsis-induced hepatic dysfunction aggravates the septic prognosis and is an independent predictor of mortality in the intensive care unit [ 2 ]. However, work remains to be done on mechanistic aspects of sepsis-induced liver injury to enable identification of molecular targets for diagnosis and treatment.

The current study reveals that propofol can inhibit hepatic oxidative stress, lipid peroxidation, and inflammation, which ultimately helps protect the liver from sepsis [ 3 ]. Liver LD (lipid droplet) overload is associated with increased sepsis severity and liver injury. The synthesis of hepatic LDs can be reduced by inhibiting DGAT1, which can lead to a decrease in inflammation, lipid peroxidation, and improvement in liver function [ 4 ]. During sepsis, there is a starvation response which is worsened by the rapid decline of hepatic peroxisome proliferator activated receptor alpha(PPARa) and PGC-1a levels, leading to poor mitochondria function, excess free fatty acids, lipotoxicity, and glycerol. Mice treated with the PPARa agonist pemafibrate are protected against bacterial sepsis as it improves hepatic PPARa function and reduces lipotoxicity and tissue damage [ 5 ]. Aberrations in fatty acid metabolism, including lipolysis, beta-oxidation, and lipogenesis, contribute to the pathogenesis of sepsis. Clinically, this phenomenon is addressed as steatosis in the liver after the onset of sepsis [ 6 ]. These findings confirm that dysregulated lipid metabolism is a critical factor in the hepatic pathology of sepsis and lipid metabolism in liver damage associated with sepsis.

RNA binding proteins (RBPs) regulate gene expression by binding RNA during transcription, splicing, modification, transportation, translation, and degradation. Abnormal RBP expression or altered function has suggested therapeutic targets for a number of disorders [ 7 ]. Recent studies have indicated that abnormal RBP expression has been implicated in the immune response to sepsis, and targeting has been shown to reduce inflammation and organ injury [ 8 , 9 ]. RBP dysfunction might be expected to impact splicing of downstream genes with implications for the septic liver [ 10 , 11 ]. Therefore, a systematic genome-wide analysis of abnormal RBP expression may illuminate splicing modifications specific to septic liver injury.

The GEO database was searched with the keywords “sepsis or severe sepsis or septic shock,” “RNA binding protein or RBP,” and “liver,” and GSE167127 data was selected to analyze RBP expression and alternative splicing. Relevant publications had 12 mice divided into sham operation (Sham), cecal ligation and puncture (CLP), and cecal ligation and puncture treatment with dichloroacetic acid (CLPDCA) groups. Transcriptomic analysis revealed that differentially expressed genes (DEGs) in CLP mice were reversed by dichloroacetic acid (DCA) therapy but by an unknown mechanism. DCA is used to treat lactic acidosis, inborn errors of mitochondrial metabolism, and diabetes [ 12 ]. It has been shown to improve sepsis survival in animal models [ 13 ] but effects on RBP and sepsis-induced liver injury remain unknown. The current study explored the impact of DCA treatment in reversing RBP and alternative splicing changes and attenuation of septic liver damage. Underlying mechanisms require further investigation.

MATERIAL AND METHODS

Retrieval and processing of public data.

We obtained publicly available data files from the Sequence Read Archive (SRA) and converted them to fastq format using the NCBI SRA Tool, fastq-dump, To ensure high quality reads, we used a FASTX-Toolkit to remove low-quality bases and evaluated the resulting clean reads with FastQC. The whole liver RNA-seq was conducted 30 hours after cecal ligation puncture(CLP) and the mice were euthanized 30 hours following surgery to collect liver tissue. Intraperitoneal administration of Dichloroacetate (DCA) was given 24 hours after surgery, with tissue collected 6 hours after DCA administration [30, h after surgery].

Read Alignment and Differentially Expressed Gene (DEG) Analysis

The clean reads were aligned to the mouse GRCm39-M27 genome using HISAT [ 14 ], with a tolerance of up to four mismatches. We utilized the uniquely mapped reads to determine the read count and read per kilobase of exon per million fragments mapped (RPKM) for each gene to assess its expression level. To evaluate differential expression, we employed the DEseq2 software and estimated gene dispersion, fitting the negative binomial distribution model. This allowed us to assess differential expression using either the Wald or likelihood ratio test. We represent differential expression as the fold change (FC) with false discovery rate (FDR).

Alternative Splicing Analysis

The AB Las pipeline was utilized to identify alternative splicing events (ASEs) and regulated alternative splicing events (RASEs), following previously described methods [ 15 , 16 ]. The pipeline was able to detect ten different types of ASEs from splice junction reads, including exon skipping (ES), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), intron retention (IR), mutually exclusive exons (MXE), mutually exclusive 5′UTRs (5pMXE), mutually exclusive 3′UTRs (3pMXE), cassette exon, A3SS&ES, and A5SS&ES. Fisher’s exact test was employed to determine statistical significance between pairs of samples, using alternative and model reads as input data. The RASE ratio was defined as changes to ratios of alternatively spliced reads and constitutively spliced reads between paired samples, with a threshold of ratio ≥ 0.2 and p -value ≤ 0.05 set for RASE detection. Student’s t -test was performed to evaluate the significance of the ratio alteration, with a p -value of 0.05 indicating RASEs.

Functional Enrichment Analysis

To analyze enriched terms among the differentially expressed genes (DEGs), we conducted gene ontology (GO) enrichment analysis. This involved utilizing the KOBAS 2.0 server [ 17 ] to filter significantly enriched terms. Enrichment was defined using the hypergeometric test and Benjamini-Hochberg FDR controlling procedure. Additionally, we employed Reactome ( http://reactome.org ) pathway profiling to further investigate functional enrichment of specific genes.

Co-expression Analysis

To explore the regulatory relationship between RASE and DERBPs, we calculated Pearson’s correlation coefficients (PCCs). Based on the PCCs value, we classified their relationship as positive correlated, negative correlated, and non-correlated.

Reverse Transcription qPCR Validation of DEGs and ASEs

The study used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to validate selected DEGs and ASEs in the CLP, CLPDCA, and control groups. The RT-qPCR test was performed 72 hours post cecal ligation puncture (CLP), and liver tissues were collected from the mice 72 hours after the surgery. Dichloroacetate (DCA) was administered intraperitonealy 24 hours after surgery, with tissue collected 48 hours after the DCA administration (72 hours after the surgery). Total RNA was extracted from animal samples and used to transcribe RNA into cDNA using M-MLV Reverse Transcriptase (Vazyme). The Step One Real-Time PCR System was used to perform real-time PCR with HieffTM qPCR SYBR® Green Master Mix (Low Rox Plus; YEASEN, China). The PCR protocol involved denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s. PCR amplifications were carried out in triplicate for each sample. RNA expression levels were normalized to GAPDH (Table 1 ).

Statistical Analysis

To demonstrate the grouping of samples based on the first two components, we utilized the factoextra R package to perform principal component analysis (PCA). During this analysis, we normalized the reads for each gene using Tags Per Million (TPM). We also incorporated a script named sogen to visualize next-generation sequence data and genomic annotations. For clustering, we utilized Euclidean distance and generated a heatmap in R. To compare two groups, we employed Student’s t -test. The data is presented as mean ± SD (standard deviation of the mean). The statistical differences among the groups were analyzed using the one-way ANOVA tool. The statistical significance was shown as follows: an asterisk (*) indicates p < 0.05, two asterisks (**) indicate p < 0.01, three asterisks (***) indicate p < 0.001, and four asterisks (****) indicate p < 0.0001.

Liver Gene Expression Profiles in Sham, CLP, and CLPDCA Group

A total of 1208 DEGs were identified from 4 sham, 4 CLP, and 3 CLPDCA samples, including 800 up-regulated and 408 down-regulated genes between CLP and sham (Fig. 1 a). A further 125 DEGs were identified between the CLPDCA and sham groups, including 67 up-regulated and 58 down-regulated genes. Inspection of Figure 1 a shows that treatment with DCA had partially reversed the changes in gene expression found between the sham and CLP groups. Hierarchical cluster analysis of significantly different expression patterns in different groups of samples found DEG patterns to be more similar between CLPDCA and sham groups than between CLP and sham (Fig. 1 b). Analysis of DEG overlap identified 48 co-up-regulated and 36 co-down-regulated DEGs in the CLP and CLPDCA treatment groups relative to the sham group. However, a comparison of sham and CLP groups revealed 750 up-and 372 down-regulated genes. The differences in numbers of up- and down-regulated genes support the view that treatment of CLP mice with DCA partially reversed abnormal gene expression induced by CLP (Fig. 1 c).

Gene expression profile of mouse liver tissue. a Significant DEGs in CLP and CLPDCA septic mice. b Expression heatmap of significant DEGs among CLP, CLPDCA, and sham. c Venn diagram showing overlap of DEGs in CLP and CLPDCA samples. d Bar plot of the most enriched GO biological processes of genes up-regulated in CLP. e Bar plot of the most enriched GO biological processes of genes down-regulated in CLP.

Functional analysis was performed on DEGs between CLP and sham. GO analysis showed co-up-regulated genes to be enriched in the biological processes of cell adhesion, inflammatory response, immune system processes, positive regulation of cell migration, negative regulation of external apoptotic signal pathways through death domain receptors, chemotaxis, and other functional pathways (Fig. 1 d). Co-down-regulated DEGs were enriched in oxidation-reduction and lipid metabolic processes (Fig. 1 e). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified up-regulated genes as being involved in cytokine-cytokine receptor interaction and IL-17 signaling pathway and down-regulated genes in metabolic pathways. Genes that were up-regulated in the comparison of CLP with CLPDCA were enriched in FoxO signaling and glucagon signaling pathways (supplement1).

The involvement of lipid metabolism–related genes in the impact of sepsis on liver tissue was highlighted by the outcomes of the functional analyses described above. Inflammation/immune and cell apoptotic–related genes, including cytokine-cytokine receptor interaction and the IL-17 signaling pathways, also featured prominently in the differential expression results. It may be suggested that the actions of DCA on signaling of the transcription factor, FoxO, and hormone, glucagon, are involved in reversing abnormal gene expression. Lipid metabolism was also highlighted as a process worthy of further investigation as a potential therapeutic target.

Alternative Splicing Patterns in Sham, CLP, and CLPDCA Group

AS events at nine variable splicing sites, including A3SS, A5SS, and ES, were found from RNA-Seq data. Hierarchical clustering analysis showed that the 4 CLP samples formed one cluster, and the three sham and three CLPDCA samples formed a second cluster. Thus, the variable splicing patterns of genes in CLPDCA and sham were more similar to one another than either was to CLP (Fig. 2 a). RASEs present in CLP and CLPDCA groups relative to sham were identified by t -test, and the most significant were A3SS and A5SS with the cassette exon and ES following (Fig. 2 b). The implication of the findings above is that the pattern of A3SS and A5SS splicing may be associated with gene expression in the septic liver.

Regulated alternative splicing events in septic mice. a PCA of RASE ratios with confidence ellipse for all groups. b Bar plot of RASEs in CPL versus sham liver and CLPDCA versus sham liver. c Scatter plot of enriched GO terms relating to ASEGs in CPL versus sham liver. d Scatter plot of enriched GO terms relating to ASEGs in CPLDCA versus sham liver.

Genes associated with alternative splicing events (ASEGs) were subjected to GO functional analysis. Those differentially expressed between the CLP and sham groups showed enrichment for the processes of oxidation-reduction, lipid metabolism, mitochondrial morphogenesis, and positive regulation of fatty acid biosynthesis (Fig. 2 c). Those differentially expressed between CLPDCA and CLP were enriched for protein ubiquitination, lipid metabolism, redox balance, autophagy, DNA template–dependent negative and positive regulation of transcription (Fig. 2 d). These results give further confirmation of the involvement of lipid metabolism and oxidation-reduction reactions in sepsis and also show that DCA treatment ameliorates the sepsis-dependent changes by acting on the same pathways.

Overlap analysis of DEGs and ASEGs between CLP and sham revealed 61 genes were differentially expressed between the two groups. Similar analysis of the CLPDCA and CLP groups showed only four genes with differential expression (Fig. 3 a). GO analysis of the differences between CLP and sham showed enrichment for redox processes, lipid metabolism, and regulation of transcription by RNA polymerase II (Fig. 3 b).

Regulated alternative splicing events. a Venn diagram showing the overlap of ASEGs and DEGs. b Bar plot of enriched GO terms relating to the overlapping genes in a .

A comparison of AS event ratios of the CLP and sham groups revealed that 390 events were up-regulated and 396 down-regulated. However, a similar comparison of CLPDCA with sham revealed only 43 up-regulated and 66 down-regulated variable alternative splicing events. Again, it can be seen that the pattern observed for DEGs has been repeated. Abnormal splicing in the septic liver may cause tissue damage, and treatment with DCA reverses the effect by reversing the changes in abnormal splicing.

Up- and down-regulation of differential AS events in CLPDCA tissue were 244 and 249 compared with CLP, indicating that CLPDCA reversed the abnormal gene expression profile (Fig. 4 a). Cluster analysis carried out by screening the covariant AS events with read number > 10 in at least 80% of the samples showed that the four CLP samples were clustered in one group and the 3 sham and 3 CLPDCA samples were clustered in a second group (Fig. 4 b). Up-regulation of AS events with CLP treatment may be associated with septic liver damage, and up-regulation of AS events with DCA treatment reversed the CLP effect and may indicate a protective effect on the septic liver. GO analysis of the genes involved in the AS events above was performed.

Differential AS events in the CLP- and CLPDCA-treated liver. a Venn diagram of overlapping AS events in CLP versus sham and CLPDCA versus sham liver. b Hierarchical clustering heat map of all significant ASEG ratios. The AS filter included all detectable splice junctions with at least 80% of samples having ≥ 10 splice junction supporting reads. c Bar plot of the most enriched GO biological processes of the CLP-specific ASEGs from b . d Bar plot of the most enriched GO biological processes of the CLPDCA-specific ASEGs from b .

The 10 most enriched pathways involved in biological processes relating to the AS genes identified above included triglyceride homeostasis, positive regulation of fatty acid biosynthesis, redox processes, negative regulation of mRNA splicing by spliceosomes, protein transport, lipid metabolism, negative regulation of adipocyte differentiation, lactation, and in utero embryonic development (Fig. 4 c). GO analysis of DCA-specific AS events indicated that redox processes, actin cytoskeleton, phospholipid biosynthesis, RNA processing, protein ubiquitination, autophagy, immune system processes, mitochondrion organization, ubiquitin-dependent ERAD pathways, and positive transcriptional regulation of DNA templates (Fig. 4 d).

Human RBP genes were intersected with the DEGs found above to be associated with CLP/sham and CLPDCA/CLP differences. A total of 37 RBP genes were up- or down-regulated in the CLP group compared with the sham (Fig. 5 a). Treatment with DCA reversed the expression changes of the RBP genes brought about by CLP (Fig. 5 a). Some of the RBP genes identified are likely to be involved in AS events. The 20 RBPs showing the greatest degree of up- or down-regulation in the CLP group were screened (Fig. 5 b), and a co-expression network of hub RBPs and ASEGs was constructed. RBP S100A11 was found to be likely to regulate multiple differential AS events (Fig. 5 c). GO and KEGG analyses of the 10 RBPs co-expressed most frequently with ASEGs were carried out. GO analysis showed enrichment in lipid metabolism, redox processes, drug responses, cell division, cell cycle, ion transport, proteolysis, positive and negative regulation of transcription by RNA polymerase II, and DNA-dependent negative regulation (Fig. 5 d).

Interaction network of RNA-binding proteins and alternative splicing-associated genes. a Venn diagram showing overlapping differentially expressed ASEGs and RBP genes in septic mice. b Heatmap of differentially expressed RBP genes in CLP samples. RBPs were filtered by expected fragments per kilobase of transcript per million fragments mapped (FPKM) ≥1 in 80% of samples. The color key from blue to red indicates z -score color range. c The scatter plot shows ASEGs by CLP versus sham co-expressed with differentially expressed RBP genes from b . d Enriched GO biological processes of ASEGs co-disturbed with the top 10 differentially expressed RBPs. e The co-deregulation in CLP-treated liver of AS network and RBPs (left; circle size, number of connections), AS events (middle left), ASEGs (center right), and enriched GO biological process terms of co-disturbed ASEGs and RBP genes (right).

In order to investigate the regulatory relationship between RASE and DERBPs, a co-expression analysis was conducted. Pearson’s correlation coefficients (PCCs) were calculated and used to categorize their association as positively correlated, negatively correlated, or non-correlated. Relationship pairs between DERBPs and RASE that exhibited a correlation coefficient value of at least 0.85 and a p -value of no greater than 0.01 were identified through screening. An interaction network of differentially expressed RBPs and ASEGs suggested that CLP-induced up- or down-regulation of liver RBPs could be reversed by DCA treatment, affecting splicing of downstream genes (Fig. 5 e). A KEGG analysis showed the ASEGs co-disturbed with differentially expressed RBPs enriched in metabolic pathways, drug metabolism, porphyrin and chlorophyll metabolism, steroid biosynthesis, ascorbic acid and uronate metabolism, cytochrome P450 metabolism, riboflavin metabolism, pentose and gluconate interconversion, and sulfur metabolism (Fig. 6 ).

The most enriched KEGG pathways of ASEGs co-disturbed with the top 10 differentially expressed RBPs.

Co-expression Analysis Between Sepsis-Regulated RBPs and NIR

An interaction and co-expression network was constructed to encompass all connections among RBPs, AS events, and ASEGs. The dysregulation of RBP gene expression caused by CLP was found to be reversed by DCA treatment with a likely impact on AS events (Fig. 7 a and b). Genes involved in metabolic pathways, in particular, lipid metabolism, which is subject to AS, were the focus of the investigation. The abnormally expressed RBP, S100A11, was found to affect AS of some genes encoding products involved in lipid metabolism, such as SREBF1 and CERS2 (Figs. 7 c, d and 8 ).

Abnormally expressed RBP genes with effects on lipid metabolism–associated genes. a Network showing deregulation of lipid metabolism by abnormal RBP expression. b Box plot showing expression of S100a11 in CLP, CLPDCA, and sham samples. c Box plot showing the splicing ratio profile of the Cers2 gene across 11 samples. d Visualization of junction read distribution of Cers2 gene in samples from different groups. Splice junctions are labeled with SJ read number.

Gene expression and splicing regulation of lipid metabolism–associated genes. a Box plot showing the splicing ratio profile of the Serbf1 gene across 11 samples. b Visualization of junction reads distribution of Serbf1 gene in samples from different groups. Splice junctions are labeled with SJ read number.

Analysis the Genome-Wide AS in Lipid Metabolism and Oxidation-Reduction–Related Genes

In the CLP group, the top ten alternative splicing event genes were Cers2, Tm7sf2, Crot, Pcyt2, Mgll, Sc5d, Elovl5, Akr1c6, Srebf1, and Gm44805 when compared with the Sham group. These genes were mainly enriched in metabolic lipid processes in the GO enrichment analysis of biological processes. Additionally, Cyp2d10, Tm7sf2, Paox, Uox, Sc5d, P4ha1, Haao, Akr1c6, Cp, and Bdh2 were the alternative splicing event genes that were mainly enriched in the oxidation-reduction process in the GO enrichment analysis of biological processes. The alternative splicing ratio profile of lipid metabolism and oxidation-reduction–related genes is shown in Table 2 and Fig.  9 .

Top ten node lipid metabolic and oxidation-reduction–related alternative splicing genes and relative alternative splicing events ratio in Sham, CLP, and CLPDCA group.

Furthermore, in the CLP group, the splicing ratio of Lyplal, Ppfibp1, Pnpla2, Lrps3, Pnpla6, Pnpla8, Mgll, Apoc3, and Lipc exhibits significant differences when compared to the Sham group. Additionally, the splicing ratio of P4ha1, Akr1c6, Cyp2d10, Tm7sf2, Bdh2, Gm44805, and Sc5d decreased in the CLP group compared to the Sham group. On the other hand, the Pcyt2, Uox, Paox, and Elovl5 genes showed an increase in their splicing ratio in the CLP group. The CLP group showed a significant reduction in the reads of ES in Lyplal1 compared to the Sham group. The reads of A3SS were increased in Mgll and Apoc3, and there was a significant reduction in the reads of cassette exon in Ppfibp1 and Pnpla2. as illustrated in Fig.  10 . These findings suggest that the alternative splicing of genes related to lipid metabolism and oxidation might be involved in the development of liver damage during sepsis.

Splicing ratio profile of specific lipid metabolism–related genes in Sham, CLP, and CLPDCA.

Validation of Co-expressed RBPs and ASEs

We observed abnormal alternative splicing of genes related to lipid metabolism in the liver tissues of septic animal model. These genes include Cers2, Tm7sf2, Crot, Pcyt2, Mgll, Sc5d, Elovl5, Akr1c6, Srebf1, Gm44805, Lylal1, Pnpla, Lyplal1, Ppfibp1, Pnpla2, Lrp3, Pnpla6, Apoc3, Pnpla8, Mgll, Apoc3, and Lipc. Dysregulated expression of cers2 and srebf1 has been reported in sepsis, and they are considered hub genes of lipid metabolism. However, there is a lack of evidence on the expression and alternative splicing of other lipid metabolism–related genes in sepsis. To address this, Considering the evidence for the genes and alternative splicing linked to lipid metabolism in sepsis, we validated only cers2 and srebf1 in a septic animal model. S100a11 expression levels were significantly greater in the CLP group than in the sham group, although DCA eliminated this difference. Furthermore, the alternative splicing ratio of Srebf1 and Cers2 was reduced compared with the sham group and increased after DCA treatment. These results suggested that S100a11 is relevant to the pathogenesis of sepsis-induced liver damage. Its mechanism may be related to Srebf1 and Cers2 alternative splicing, regulated by S100a11 (Fig. 11 ).

Validation of RASEs and RBP. a Box plot showing expression status of S100a11 in CLP, CLPDCA, and Sham samples by qPCR validation. b Box plot showing splicing ratio profile of the Cers2 splicing event by qPCR validation. c Box plot showing the splicing ratio profile of the Serbf1 splicing event by qPCR validation. Data are shown as mean ± SD, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Transcriptomic sequencing (RNA-seq) has been used to reveal molecular phenotypic changes underlying physiological conditions and disease progression [ 18 ]. Variations in gene expression are acknowledged to affect physiological parameters. The current study analyzed RNA-seq data from liver tissue in an animal model of sepsis and found altered gene expression and varying alternative splicing associated with sepsis progression. DCA treatment was found to reverse the aberrant gene expression and ameliorate liver injury [ 19 ]. DCA activates pyruvate dehydrogenase, affecting oxidative metabolism [ 20 , 21 ] and attenuated lactic acidosis during orthotopic liver transplantation (OLT) [ 22 ] producing a hepato-protective effect [ 23 ]. DCA also protected Nnt−/− mice from developing high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) which may be due to the reactivation of pyruvate dehydrogenase, restoring the capacity of the pyruvate-supported liver mitochondria to manage peroxide [ 24 ]. Thus, the protective effect of DCA against sepsis-induced liver injury may rely on metabolic reprogramming.

The GO functional analysis showed up-regulation of genes involved in immune/inflammatory response, apoptosis, and cell migration, whereas down-regulated genes tended to be associated with redox reactions and lipid metabolism. The current study’s findings agree with previous reports in indicating that variable gene expression accompanies disease progression. Lipid metabolism has been associated with the development of sepsis [ 25 ] and consequent liver damage [ 26 ]. However, mechanistic connections between liver injury and sepsis progression remain to be elucidated.

RBPs are involved in liver inflammation and lipid homeostasis, and the expression of immune-associated RBPs is a biomarker for predicting the targeted therapeutic response of liver cancer and patient survival [ 27 ]. The current study found that CLP-induced abnormal expression of RBPs, such as S100a11, ads2, Fndc3b, Fn1, Ddx28, Car2, Cisd1, and Ptms, and DCA treatment reversed these effects. Sepsis-related RBPs were found to regulate alternative splicing of downstream genes involved in lipid metabolism. Previous studies have reported that the liver RBP, HuR, regulates lipid homeostasis in response to a high-fat diet [ 28 ], and HuR promoted miRNA-mediated up-regulation of NFI-A protein expression in Myeloid-derived suppressor cells (MDSCs) and enhanced resistance to uncontrolled infection in septic mice [ 29 ]. HuR deficiency leads to inflammation and fibrosis of the liver [ 30 ]. The cold-inducible RBP, CIRP, induces inflammatory responses in hemorrhagic shock and sepsis [ 31 ] and activation of splenic T cells dependent on the TLR4 [ 32 ]. Thus, targeting of RBPs, such as with anti-peptides of CIRP, reduced sepsis-induced inflammation and organ damage in septic mice [ 8 ]. Thus, RBPs are attractive candidates for therapeutic targeting with essential functions in liver immunity, metabolic diseases, and sepsis.

RBPs regulate the development of a number of liver diseases through variable splicing, which is otherwise a source of protein diversity [ 33 , 34 ]. Degradation of the RBP, SRSF3, led to abnormal splicing in the liver, promoting disease progression [ 35 ]. The dysregulation of AS associated with sepsis means that RBPs may be potential targets for the treatment of septic liver injury. Abnormal splicing of acidic sphingomyelinase 1 (SMPD1) mRNA is known to result in altered enzyme activity and an impact on the development of sepsis [ 10 ]. Abnormal alternative splicing of the myosin phosphatase gene resulted in reduced enzyme activity, oxidative stress, and altered NO vasodilator reserve in the early and late stages of the mouse model of LPS-induced sepsis, affecting disease progression [ 36 ]. The current findings demonstrate that DCA reversed abnormal alternative splicing events in the liver tissue in sepsis with ASEGs enriched in lipid metabolism and oxidation-reduction–related genes. Disordered lipid metabolism is known to affect alternative splicing of mRNA [ 37 , 38 , 39 ]. In summary, altered RBP function may lead to AS abnormalities associated with liver injury in sepsis, and RBPs may be a therapeutic target.

The S100a11 calcium–binding member of the S100 family is up-regulated during sepsis [ 40 ] and promotes liver steatosis via the RAGE-mediated AKT-mTOR signaling pathway [ 41 ] and foxo1-mediated autophagy and lipogenesis [ 42 ]. Based on the available evidence, it appears that S100a11 has the potential to significantly affect the activity of SREBF1, which is a vital transcription factor involved in liver lipid metabolism. It has been observed that SREBF1 can enhance lipid synthesis while reducing lipid degradation, leading to the accumulation of lipids in the liver and improper regulation of autophagy. These findings indicate that S100a11 may have a crucial role to play in the regulation of liver lipid metabolism through its impact on SREBF1 [ 43 , 44 ]. Indeed, KDM1A-mediated attenuation of SREBF1 activity underlies suppression of de novo lipogenesis by oxidative stress [ 45 ]. Moreover, Down-regulation of PPARG and SREBF1 in response to PER2 silencing highlights the importance of circadian clock signaling for lipogenesis regulation [ 46 ].

CerS2 maintains normal cell division through the MAD2‐MKLP2‐CPC axis [ 47 ] but down-regulation of CerS2 resulted in LC (long-chain) ceramide accumulation and growth arrest, unaccompanied by apoptosis [ 48 ]. Ceramide synthase is known to be enhanced in LPS-mediated septic shock in Cers2-deficient mice [ 49 ], and inhibition of ceramide synthesis prevented diabetes, steatosis, and cardiovascular disease in rodents [ 50 ]. The current study found SREBF1/CERS2 to be predicted targets of S100a11. In conclusion, lipid metabolism appears to be involved in sepsis-induced liver injury. RBP dysfunction disturbs alternative splicing of lipid metabolism genes, such as SREBF1/CerS2, indicating RBPs as possible therapeutic targets for sepsis-induced liver injury. Further investigations are required to elucidate the mechanisms involved.

We have identified several areas that require improvement in our study. To investigate sepsis liver tissue, we systematically analyzed RNA-seq data from document number one and established a sepsis animal model. We validated the gene expression of S100a11, srebf1, and cers2 using RT-qPCR, but it would be more beneficial to perform additional tests such as immuno-histochemistry and Western blot to detect protein expression levels in the animal model. It is also necessary to continuously and dynamically observe the changes in S100a11, srebf1, cers2, and liver injury markers in the animal model and conduct correlation analysis. Additionally, RNA-binding protein immuno-precipitation experiments would be helpful to elucidate further the intrinsic connections between S100a11, srebf1, cers2, and the pathogenesis of sepsis liver injury.

The liver is a vital organ for metabolism and immunity, and it plays a critical role in sepsis development. Our study focused on septic liver tissue in mice and revealed numerous differentially expressed genes, alternative splicing events, and RNA-binding proteins with abnormal expression. Through bioinformatics analysis, we identified the relationship between abnormal RNA-binding proteins and variably spliced events and found that RNA binding proteins like S100A11 can indirectly impact septic liver injury by regulating downstream genes associated with lipid metabolism, such as SREBF1 and CERS2. These discoveries offer valuable insight into the function and mechanism of RNA-binding proteins in sepsis, and they could lead to the identification of new therapeutic targets for septic liver injury.

AVAILABILITY OF DATA AND MATERIAL

The published article and its additional information files contain all of the data generated or analyzed during this investigation. The datasets supporting the findings of this study are available in the NCBI Gene Expression Omnibus and can be accessed using the GEO series accession number (GSE167127).

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ACKNOWLEDGEMENTS

The authors express their gratitude to Mr. Li Ning and Mr. Chao Cheng of the ABLife bioBig Data Institute for their insightful conversations. We are grateful to EditSprings ( https://www.editsprings.cn ) for their professional language services.

This work was supported by the National Natural Science Foundation of China (Grant No:82360381) and the State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asia High Incidence Disease Found (Grant No: SKL-HIDCA-2021-JH13), and the Youth Medical Science and Technology Talents Research Foundation of Autonomous Regional Health Commission (Grant No:WJWY-202336).

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Aliya Rehati

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Buzukela Abuduaini designed the project and Song Yunlin supervised it.Buzukela Abuduaini and Zhang Jiyuan conducted the experiments. The data was analyzed by Buzukela Abuduaini, Aliya Rehati, and Zhao Liang. Buzukela Abuduaini wrote the manuscript. All authors thoroughly analyzed the results and confidently approved the final version of the manuscript.

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Abuduaini, B., Jiyuan, Z., Rehati, A. et al. Regulation of Alternative Splicing of Lipid Metabolism Genes in Sepsis-Induced Liver Damage by RNA-Binding Proteins. Inflammation (2024). https://doi.org/10.1007/s10753-024-02017-2

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Received : 07 February 2024

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Published : 09 May 2024

DOI : https://doi.org/10.1007/s10753-024-02017-2

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