The Journal of Maternal-Fetal & Neonatal Medicine

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Role of lactoferrin in neonatal care: a systematic review Deepak Sharma, Sweta Shastri & Pradeep Sharma To cite this article: Deepak Sharma, Sweta Shastri & Pradeep Sharma (2017) Role of lactoferrin in neonatal care: a systematic review, The Journal of Maternal-Fetal & Neonatal Medicine, 30:16, 1920-1932, DOI: 10.1080/14767058.2016.1232384 To link to this article: http://dx.doi.org/10.1080/14767058.2016.1232384

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Date: 14 June 2017, At: 14:37

http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2017; 30(16): 1920–1932 ! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14767058.2016.1232384

INVITED REVIEW

Role of lactoferrin in neonatal care: a systematic review Deepak Sharma1, Sweta Shastri2, and Pradeep Sharma3 1

Consultant Neonatologist, NEOCLINIC, Plot number 3 & 4, Everest Vihar, TN Mishra Marg, Nirman Nagar, Jaipur, Rajasthan, India, 2Department of Pathology, N.K.P Salve Medical College, Nagpur, Maharashtra, India, and 3Department of Medicine, Mahatma Gandhi Medical College, Jaipur, Rajasthan, India Abstract

Keywords

Background: Lactoferrin (LF) is present in breast milk and have numerous properties including antimicrobial, antiviral, antifungal, and anticancer. Recent studies have emphasized the role of LF in neonatal care Aims and objective: To evaluate the various roles of LF in neonatal care in preterm infants. Search methods: The literature search was done for this systematic review by searching the electronic database namely Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, EMBASE, Web of Science, Scopus, Index Copernicus, African Index Medicus (AIM), Thomson Reuters (ESCI), Chemical Abstracts Service (CAS), SCIWIN (Scientific World Index), Google Scholar, Latin American and Caribbean Health Sciences Information System (LILACS), Index Medicus for the Eastern Mediterranean Region (IMEMR), Index Medicus for the South-East Asian Region (IMSEAR), Western Pacific Region Index Medicus (WPRIM), various sites for ongoing trials namely clinical trial registry (www.clinicaltrials.gov, www.controlled-trials.com, Australian and New Zealand Clinical Trials Registry (http://www.anzctr.org.au), Indian Clinical Trials Registry (http://ctri.nic.in/Clinicaltrials), and the World Health Organization (WHO) International Clinical Trials Registry, and Platform (http://www.who.int/ictrp/search/en/) and abstracts of conferences namely proceedings of Pediatric Academic Societies (American Pediatric Society, Society for Pediatric Research, and European Society for Pediatric Research). Results: Nine eligible studies were analyzed that fulfilled the inclusion criteria of the systematic review. Six duplicate publications were excluded from review. Four studies were excluded due to nonfulfillment of inclusion criteria. All of the studies had more than one outcome of interest. Four studies showed reduction in late onset sepsis (LOS), one showed reduction in invasive fungal infection (IFI), three showed significant decrease in incidence of necrotizing enterocolitis (NEC), one showed reduction in NEC scares, and two showed decrease in mortality, and one showed decrease in combined death and/or NEC. Only one study evaluated role of LF for ventilator-associated pneumonia (VAP) reduction and showed lower rate of VAP. Still the role of LF in Bronchopulmonary dysplasia (BPD) and Retinopathy of prematurity (ROP) is unclear. Conclusion: LF has shown to be promising agent for reduction of LOS and NEC. The role of LF in prevention of neonatal mortality, BPD, and ROP needs further studies. The trials that are going on around the world may be able to give reply of this question in future

Late onset sepsis, mortality, neonatal sepsis, necrotizing enterocolitis, pneumonia, preterm, lactoferrin

Introduction Description of the condition Preterm neonates have unique sets of problem, and these are known to have long-term implication on the neonatal neurodevelopmental outcome. The various problems includes neonatal sepsis (Early onset sepsis [EOS] and late onset sepsis [LOS]), Necrotizing enterocolitis (NEC), Bronchopulmonary dysplasia (BPD), Retinopathy of prematurity (ROP), Anemia of prematurity (AOP), and Osteopenia of prematurity (OOP)

Address for correspondence: Dr. Deepak Sharma, Consultant Neonatologist, NEOCLINIC, Plot number 3 & 4, Everest Vihar, TN Mishra Marg, Nirman Nagar, Jaipur, Rajasthan, India. E-mail: [email protected]

History Received 15 July 2016 Revised 22 August 2016 Accepted 31 August 2016 Published online 20 September 2016

[1–3]. Neonatal sepsis and NEC are the most common cause of neonatal mortality and morbidities in very low birth weight (VLBW) and extreme low birth weight (ELBW) infants [4]. Recent published studies showed that prematurity and neonatal sepsis are the major contributor in infant mortality [5–7]. Neonatal sepsis has been defined as the presence of bacteria in sterile body fluids that include blood, urine, cerebrospinal, peritoneal, and pleural fluids [8,9]. Many interventions have been tried for preventing neonatal sepsis and catheter-related bloodstream infections (CRBSI) [10–22]. NEC is a devastating gastrointestinal (GI) disease that occurs usually in ELBW [23,24], and its incidence is inversely proportional to gestational age at birth [25]. NEC is associated with high neonatal morbidity and mortality with mortality rate reported around 20–30% [26]. Numerous interventions have been tried in

Lactoferrin in neonatal care

DOI: 10.1080/14767058.2016.1232384

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Figure 1. Figure showing conversion of LF to LFcin and its spectrum of action. Figure copyright Deepak Sharma.

studies to reduce the incidence of NEC, but the incidence remains unchanged [27–32].

detail mechanism of action can be read from various other excellent reviews [48,49,51,52].

Description of the intervention

Why it is important to do this review

Lactoferrin (LF), also known lacto-transferrin, is an ironbinding protein, and composed of 703 amino acid sequence [33]. It is a normal component of human colostrum, milk, tears, saliva, cerebrospinal fluid, and vaginal secretions [34]. LF is converted to lactoferricin (LFcin) by the action of pepsin in stomach. LF and LFcin exert many actions including antimicrobial, antifungal, antiviral, and anticancer [35] (Figure 1). Bovine LF (BLF) that has been used mostly in all preclinical and clinical trials, has 77% amino acid homology with human LF with same N-terminal peptide [33].

LF has immersed as a new tool for prevention of neonatal sepsis and NEC. It has also shown positive effect on the prevention of other neonatal morbidities. Recently many trials have published the role of LF in prevention of neonatal sepsis and NEC. This review evaluated the role of LF in reduction of neonatal morbidities including neonatal sepsis, NEC, pneumonia, and neonatal mortality.

How the intervention might work LF has broad microbicidal activity namely antibacterial, antiviral, and antifungal (Table 1). The antibacterial property is seen against Escherichia coli [36], Staphylococcus aureus [37], Klebsiella pneumoniae, Streptococcus mutans, Pseudomonas aeruginosa [38], Haemophilus influenzae [39], Helicobacter pylori [40], Clostridium difficile [41], Shigella flexnieria [42], and other organisms. The antifungal property is against Candida albicans and yeast [43]; antiviral property is against HCV [44], HIV [45], and various other viruses; anticancer property is seen against head and neck squamous cell carcinoma [46] and breast carcinoma [47]. It also acts as antioxidant, plays role in regulation of cell growth and differentiation (intestine, bone, and hematopoietic tissues), enzymatic and signal transduction, and immunomodulation [48] (Figure 1) The various proposed mechanisms of LF antimicrobial property are cell membrane disruption, iron sequestration, disruption of cell surface expressed virulence proteins, cell membrane damage, prevention of microbial adhesion to host cells, and inhibition of formation of biofilm [49] (Table 2) (Figure 2). The antineoplastic property of LF is because of its ability to cause cell cycle arrest, promotion of apoptosis and necrosis, and antiangiogenic and antimetastatic property (Figure 3). Development of resistance against LF will require multiple simultaneous mutations in structure of LF; therefore, development of resistance is rare [50]. The

Methods The literature search was done for this systematic review by searching the following: A. Cochrane Central Register of Controlled Trials (CENTRAL) B. Electronic database searches of PubMed, EMBASE, Web of Science, Scopus, Index Copernicus, African Index Medicus (AIM), Thomson Reuters (ESCI), Chemical Abstracts Service (CAS), SCIWIN (Scientific World Index), Google Scholar, Latin American and Caribbean Health Sciences Information System (LILACS), Index Medicus for the Eastern Mediterranean Region (IMEMR), Index Medicus for the South-East Asian Region (IMSEAR), and Western Pacific Region Index Medicus (WPRIM) C. Following sites for ongoing trials: clinical trial registry (www.clinicaltrials.gov, www.controlled-trials.com, Australian and New Zealand Clinical Trials Registry (http:// www.anzctr.org.au), Indian Clinical Trials Registry (http:// ctri.nic.in/Clinicaltrials), and the World Health Organization (WHO) International Clinical Trials Registry and Platform (http://www.who.int/ictrp/search/en/) D. Abstracts of conferences: proceedings of Pediatric Academic Societies (American Pediatric Society, Society for Pediatric Research, and European Society for Pediatric Research) The terms used were ‘‘Lactoferrin,’’ ‘‘talactoferrin,’’ ‘‘neonatal sepsis’’, ‘‘necrotizing enterocolitis’’, ‘‘NEC’’, ‘‘newborn’’, ‘‘neonate’’, and ‘‘pneumonia’’ through 25 February 2016. No language restrictions were applied, and

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Table 1. Spectrum of various effects of LF. Antimicrobial A. Antibacterial 1. Pseudomonas aeruginosa 2. Haemophilus influenzae 3. E. coli 4. Helicobacter pylori 5. Clostridium difficile 6. Shigella flexnieria 7. Staphylococcus aureus 8. Streptococcus mutans 9. Streptococcus pneumoniae 10. Aggregatibacter actinomycetemcomitans 11. Yersinia enterocolitica 12. Listeria monocytogenes

B. Antifungal 1. Candida albicans 2. Yeast

C. Antiviral 1. Human immunodeficiency virus 2. Cytomegalovirus (CMV) 3. Herpes simplex virus (HSV) 4. Hepatitis C virus (HCV) 5. Rotavirus 6. Poliovirus (PV) 7. Respiratory syncytial virus (RSV) 8. Hepatitis B virus (HBV) 9. Parainfluenza virus (PIV) 10. Alphavirus 11. Hantavirus 12. Human papillomavirus (HPV) 13. Feline calicivirus (FCV) 14. Adenovirus 15. Enterovirus 71 (EV71) 16. Echovirus 6 17. Influenza A virus 18. Japanese encephalitis virus 19. Tomato yellow leaf curl virus (TYLCV)

D. Anticancer 1. Head and neck squamous cell carcinoma 2. Breast cancer 3. Colon carcinoma 4. Malignant melanoma 5. Bronchogenic carcinoma

Adopted from Sharma et al. [51]. Epub 2015 Mar 17. PubMed PMID: 25758631 with permission.

Table 2. Mechanism of action of LF.

 1. Microbicidal actions of LF A. Disruption of cell membrane B. Iron sequestration C. Prevention of biofilm formation D. Proteolysis of virulence factors E. Blocks bacterial adhesion to host cells by binding to glycosaminoglycans F. Initiates ‘‘anoikis’’ in which cells containing viable bacteria undergo apoptosis G. Enhances the growth of the normal commensal bifidogenic microflora in the gut 2. Anticancer action A. Cell cycle arrest B. Promotes apoptosis C. Antiangiogenesis D. Antimetastasis E. Immune modulation F. Promotes necrosis Adopted from Sharma et al. [51]. Epub 2015 Mar 17. PubMed PMID: 25758631 with permission.



Used in preterm (537 completed weeks of gestation) neonates (528 days) Neonatal sepsis, NEC, mortality, or pneumonia as primary or secondary outcomes

Exclusion criteria   

LF intervention in term neonates or infants Type of studies other than RCT Having primary or secondary outcomes other than NEC, sepsis, mortality, or pneumonia.

Types of interventions Oral or local application of LF at any dosage or duration used in neonates compared with placebo or no intervention. Selection of studies

we searched randomized and quasi-randomized trials identified by reviewing the abstract. Inclusion criteria  

Randomized controlled trial that have used LF Both oral and local application of LF

All relevant article abstracts were screened by two independent reviewers. When eligibility for inclusion was unclear from the abstract, the full text was screened. Two reviewers then abstracted data from all articles meeting the inclusion criteria. We selected all trial registries, abstracts, and information provided by the sponsor or main investigator and discarded duplicated information.

DOI: 10.1080/14767058.2016.1232384

Lactoferrin in neonatal care

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Figure 2. Figure showing mechanism and spectrum of antimicrobial property. Figure copyright Deepak Sharma.

Figure 3. Figure showing mechanism of anticancer property. Figure copyright Deepak Sharma.

Data extraction and management We collected data of the trial methodology using the ‘‘PICO’’ format (population, intervention, control, and outcome) of the unpublished and published results.

excluded from the systematic review [62–66]. Four studies did not meet the inclusion criteria, hence were excluded from the systematic review [67–70] (Figure 4) (Tables 3 and 4). Lactoferrin and late onset sepsis (LOS)

Assessment of risk of bias All the selected studies were analyzed for bias namely bias in sequence generation, allocation concealment, blinding of participants, personnel, outcome assessors, incomplete outcome data, selective outcome reporting, and other source of bias. All the relevant data of the included studies were studied, and the neonatal outcomes included were neonatal sepsis, NEC, mortality, and pneumonia.

Results Description of studies This systematic review included nine eligible studies fulfilling the inclusion criteria [53–61]. Six duplicate publications were

Manzoni et al. showed significant reduction in incidence of LOS in the BLF and BLF plus Lactobacillus rhamnosus GG (LGG) groups (9/153 [5.9%] and 7/151 [4.6%], respectively) when compared to the placebo group (29/168 [17.3%]) (risk ratio [RR], 0.34; 95% confidence interval [CI], 0.17–0.70; p ¼ 0.002 for BLF versus control and RR, 0.27; 95% CI, 0.12– 0.60; p50.001 for BLF plus LGG versus control). When stratification was done for birth weight, the decrease in LOS was significant in ELBW neonates (RR, 0.31; 95% CI, 0.14–0.70; p ¼ 0.002 for BLF versus control and RR, 0.30; 95% CI, 0.13–0.69; p ¼ 0.002 for BLF plus LGG versus control), whereas it was not significant in neonates weighing 1001–1500 g (RR, 0.46; 95% CI, 0.12–1.74; p ¼ 0.34 for BLF versus control and RR, 0.16; 95% CI, 0.02–1.27; p ¼ 0.07 for

D. Sharma et al.

Figure 4. PRISMA 2009 flow diagram for this systematic review.

J Matern Fetal Neonatal Med, 2017; 30(16): 1920–1932

Identification

1924

Records identified through database searching (n = 19)

Additional records identified through other sources (n = 0)

Eligibility

Screening

Records after duplicates removed (n = 13)

Records screened (n = 13)

Full-text articles assessed for eligibility (n = 13)

Records excluded (n = 0)

Full-text articles excluded, with reasons (n = 4)

Included

Studies included in qualitative synthesis (n = 09)

BLF plus LGG versus control). Analysis of the combined treatment groups (BLF and BLF + LGG) versus the control group showed a significant reduction of LOS in treated infants (16/304 [5.3%] versus 29/168 [17.3%]) (RR, 0.26; 95% CI, 0.14–0.50; p50.001). The decrease was significant both in ELBW neonates (12/107 [11.2%] versus 22/60 [36.7%]; p50.001) and in neonates weighing 1001–1500 g (4/197 [2.0%] versus 7/108 [6.5%]; p ¼ 0.05) [53]. Stefanescu et al. did not reported any significant reduction in the sepsis episode in the intervention arm (Biotene group 1/20 versus control group 1/21) (p ¼ 0.71) [55]. Sherman et al. showed equal rate of bacteremia in their study population (6.7% rate for each) but overall, placebo-treated infants were two times more likely to develop a primary outcome (Odds Ratio [OR] ¼ 1.6). When these infants were formula fed, infants receiving placebo were four times more likely to develop a primary outcome (OR ¼ 3.9) [56]. Akin et al. showed significant difference in number of sepsis attacks per 1000 patient days. In the placebo group, the sepsis attack rate was 17.3 sepsis/1000 patient days, whereas in intervention arm, the rate was 4.4 sepsis/1000 patient days (p ¼ 0.007) [57]. Ochoa et al. showed that LOS occurred less frequently in the LF group than in the control group. In the intention-to-treat analysis, the cumulative LOS incidence in the LF group was 12.6% versus 22.1% in the placebo group. For the VLBW neonates, the sepsis rates in the LF group were 20.0% versus 37.5% in the placebo group, showing 46% reduction in sepsis [59]. Kaur et al. showed significant reduction in the incidence of first episode of culture-proven LOS in the BLF than in the placebo group (2/63 [3.2%] in BLF versus 9/67 [13.4%] in placebo; RR, 0.211; 95% CI: 0.044–1.019; p ¼ 0.036). There was also significant reduction in incidence of probable sepsis

Studies included in quantitative synthesis (systematic review) (n = 09)

in the BLF-treated group (4/63 [6.3%]) when compared with placebo (14/67 [20.9%]); RR 0.257; 95% CI: 0.08–0.828; p ¼ 0.016). The incidence of any LOS showed a significant reduction with BLF supplementation when compared with placebo (6/63 (9.5%) versus 23/67 [34.3%], RR, 0.201; 95% CI: 0.076–0.537; p ¼ 0.001) [60]. Barrington et al. did not report any difference in LOS, at least one LOS episode (RR,0.82; 95% CI: 0.27–2.54); total number of LOS episodes (RR, 0.62; 95% CI: 0.21– 1.82); and LOS episodes/1000 patient days receiving study intervention (RR,0.62; 95% CI: 0.23–1.70) [61]. Lactoferrin and fungal sepsis Manzoni et al. showed that fungal colonization rates were similar in the three arms of the study. However, IFI occurred less frequently in the treatment groups (0/153 [0%] with BLF and 2/151 [1.3%] with BLF plus LGG, versus 9/168 [5.4%] with control; p ¼ 0.004 (significant) and p ¼ 0.07 [nonsignificant], respectively). The rate of progression from colonization to IFI was 0/27 (0%) with BLF and 2/25 (8.0%) with BLF plus LGG, versus 8/31 (25.8%) with control (p ¼ 0.005 and p ¼ 0.16, respectively) [53]. Manzoni et al. reported that the incidence of fungal colonization (atleast one site) was comparable (17.6%, 16.6%, and 18.5% in BLF, BLF plus LGG, and control group, respectively; p ¼ 0.89 [BLF] and 0.77 [BLF plus LGG]). There was significant reduction in total IFIs in BLF and BLF plus LGG (0.7% (1/153) and 2.0% (3/151), respectively) compared with control (7.7% [13/168]; p ¼ 0.002 [BLF] and 0.02 [BLF plus LGG]). This difference was also seen in both ELBW (0.9% [BLF] and 5.6% [BLF plus LGG], versus 15.0% [control]) and in 1001–1500-gram

Bovine lactoferrin Supplementation for prevention of late onset sepsis in very low birth-weight neonates: a randomized trial. Exclusion criteria Parental consent lacking/ refused Ongoing antifungal prophylaxis Early onset sepsis (before the third day of life) Liver failure

Bovine Lactoferrin Prevents Invasive Fungal Infections (IFI) in Very Low Birth Weight Infants: A Randomized Controlled Trial Secondary analysis of 2009 study Exclusion criteria Ongoing antifungal prophylaxis

A pilot study of Biotene oral BalanceÕ gel for oral care in mechanically ventilated preterm neonates. Inclusion criteria ELBW with gestational age 28 weeks Receipt of mechanical ventilation of at least 3 days in the first week of life and in the

Manzoni et al. (2012) Italy [54]

Stefanescu et al. (2013) USA [55]

Title

Manzoni et al. (2009) Italy [53]

Serial number

Lactoferrin in neonatal care (continued )

Primary outcome Evaluate the feasibility of Biotene OralBalanceÕ gel in extremely preterm infants treated with mechanical ventilation Rate of recruitment and retention of study participants Frequency of protocol compliance and adverse effects Prevalence of VAP among study

Control group (n ¼ 168) received placebo (2 mL of a 5% glucose solution). Neonates not feeding in the first 48 h received the drug(s) or placebo by Orogastric tube.

Neonates received orally administered Bovine Lactoferrin (BLF) (100 mg/day) alone (n ¼ 153), BLF plus Lactobacillus rhamnosus GG(LGG) (6  109 colony forming units/ day) (n ¼ 151). Treatment lasted 6 (birth weight 51000 g) or 4 (birth weight 1001– 1500 g) weeks, unless neonates were discharged earlier. Drugs and placebo were administered orally, once a day. Intervention group (n ¼ 20) received oral care with Biotene OralBalanceÕ gel from the time of enrollment to the time of final extubation while mechanically ventilated. Oral care was performed every 4 hourly using sterile foam tip swabs 472 premature neonates with birth weight 5 1500 g were enrolled within the first 3 days of life

Timed oral care with sterile water (n ¼ 21). Oral care was performed every 4 hourly using sterile foam tip swabs with sterile water from 2 ml single use twist-tip vials. Oral care consisted of hygiene of the buccal mucosa,

Primary objective Evaluate the effectiveness of BLF alone or BLF plus LGG in the prevention of the first episode of LOS of bacterial or fungal origin. Secondary objective Incidence of gram-positive/gramnegative bacterial and fungal sepsis Mortality prior to discharge (overall and sepsis attributable) Incidence of urinary tract infections Fungal colonization Progression from fungal colonization to invasive fungal infection (IFI) Stage 2 or greater NEC Threshold ROP Severe (grade 3–4) IVH BPD Alteration of liver function Adverse effects or intolerance Primary outcome Incidence rates of fungal colonization and IFI Secondary outcome Intensity of fungal colonization Rate of progression to infection in colonized infants Frequencies of single fungal species in all groups Mortality related to IFI

Control group (n ¼ 168) received placebo (2 mL of a 5% glucose solution). Neonates not feeding in the first 48 h received the drug(s) or placebo by Orogastric tube

Neonates received orally administered Bovine lactoferrin (BLF) (100 mg/day) alone (n ¼ 153), BLF plus Lactobacillus rhamnosus GG(LGG) (6  109 colony forming units/ day) (n ¼ 151). Treatment lasted 6 (birth weight 51000 g) or 4 (birth weight 1001– 1500 g) weeks, unless neonates were discharged earlier. Drugs and placebo were administered orally, once a day

472 premature neonates with birth weight 5 1500 g were enrolled within the first 3 days of life

41 neonates born before 28 weeks of gestation and mechanically ventilated between 7 and 10 postnatal days.

Outcome

Control group

Intervention group

Population

Table 3. The various studies included in the systematic review. All the data have been entered in PICO form (population, intervention, control, and outcome).

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Oral lactoferrin to prevent nosocomial sepsis and NEC of premature neonates and effect on T-regulatory cells. Inclusion criteria Inborn Birth weight less than 1500 g Having a gestational age of less than 32 weeks at birth. Exclusion criteria Parental consent lacking/ refused Having severe congenital anomalies or severe perinatal asphyxia Expiring before allocation to any group within the first 72 h Bovine lactoferrin supplementation for prevention of necrotizing enterocolitis in VLBW neonates: a randomized clinical trial Continuation of the study published in 2009 to get enough power to properly assess the effects of BLF on prevention of NEC. Inclusion criteria All VLBW neonates (inborn and outborn) admitted to a participant NICU prior to 48 h of life

Akin et al. (2014) Turkey [57]

Intervention group (n ¼ 25) received 200 mg LF daily till discharge. Intervention in either group was done after the baby reached 20 mL/kg/ day feeding volume and was continued throughout the hospitalization period. Both placebo or BLF was diluted in prepared milk or formula to maintain blinding

Neonates received orally administered Bovine Lactoferrin (BLF) (100 mg/day) alone (n ¼ 247), BLF plus Lactobacillus rhamnosus GG(LGG) (6  109 colony forming units/day) (n ¼ 238) from birth until day 30 of life (day 45 for neonates less than 1000 g at birth). Drug and placebo administration began on the third

743 VLBW neonates

with Biotene OralBalanceÕ gel from 2-ml single use twist-tip vials. Oral care consisted of hygiene of the buccal mucosa, tongue, and areas around ETT tube. Intervention arm (n ¼ 60) received Enteral TLF from the 1st to 29th d of life with dose of 150 mg/ kg every 12 hourly

Intervention group

50 neonates either VLBW or born before 32 weeks

120 preterm infants with birth weights (BW) of 750– 1500 g

Population

Primary outcomes Bacteremia Meningitis Pneumonia Urinary tract infection NEC Secondary outcomes Sepsis syndrome NEC scares Primary objective Evaluate the tolerability and effectiveness of BLF in the prevention of nosocomial sepsis, NEC, and mortality in either VLBW or infants born before 32 weeks. Secondary objective To see the effect of maturation and BLF on T-regulatory cells (Treg) levels of BLF-treated infants at birth and discharge in the context of hypothetic BLF interaction with Treg

Primary outcome  Stage 2 NEC Death-and/or  Stage 2 NEC prior to discharge

Control arm infants (n ¼ 60) were given enteral placebo (P) from the 1st to 29th d of life; dose was 150 mg/kg every 12 hourly

Control group (n ¼ 258) received placebo

Control group (n ¼ 25) received placebo (2 ml saline) Third group healthy neonates (n ¼ 16) were formed among healthy term neonates for comparison between healthy term neonates and former BLF or placebo treated, VLBW infants at time of discharge

participants. Secondary outcome Effect on tracheal aspirate Gram stain and culture

Outcome

tongue, and areas around ETT tube.

Control group

D. Sharma et al.

Manzoni et al. (2014) Italy and New Zealand [58]

A clinical trial of Talactoferrin (TLF) to reduce infection in very preterm infants: safety and efficacy

interval between days 7 and 10 of life.

Title

Sherman et al. (2013) USA [56]

Serial number

Table 3. Continued

1926 J Matern Fetal Neonatal Med, 2017; 30(16): 1920–1932

Randomized Controlled Trial of Lactoferrin for Prevention of Sepsis in Peruvian Neonates Less than 2500 g Exclusion criteria Neonates with underlying gastrointestinal problems that prevent oral intake Predisposing conditions that profoundly affect growth and development (chromosomal abnormalities, structural brain anomalies, etc.) Family history of cow milk allergy Neonates that lived far from Lima Neonates whose parents declined to participate Efficacy of bovine lactoferrin supplementation in preventing late onset sepsis in low birth weight neonates: randomised placebo controlled clinical trial Inclusion criteria Inborn neonates 5 2000 gram Asymptomatic at birth Admitted to NICU in the first 12 h of birth with no maternal risk factors for sepsis Exclusion criteria Presence of congenital anomalies Severe birth asphyxia (Apgar score 5 3 at 5 min of life or cord blood pH 5 7) History of maternal chorioamnionitis Suspected congenital infections Family history of cow milk allergy The Lacuna Trial: a doubleblind randomized controlled 79 infants were enrolled within the first 48 h of age, if they

121 low birth weight (less than 2000 grams) neonates within 12 h of birth

190 infants with birth weight between 500 and 2500 g within first 72 h of life

40 infants were randomized to receive milk (either

Bovine Lactoferrin [BLF] (n ¼ 59) was supplemented daily from first to 28th day of life. Neonates between 1000 and 1249 g received a daily dose of 100 mg (80– 100 mg/kg/day) of BLF, those between 1250 and 1499 g received 150 mg (100–120 mg/kg/day), while those weighing 1500–1749 g and 1750– 1999 g received 200 mg (114–133 mg/kg/ day) and 250 mg (125– 142 mg/kg/day), respectively. The contents were dissolved in milk and administered

day of life with 1 daily dose; all doses including placebo were diluted in prepared milk so as to maintain blinding. Neonates not feeding in the first 48 h received the drug(s) or placebo by Orogastric tube BLF was given enterally at 200 mg/day in 3 divided doses over the first 4 weeks of life to 95 infants. Capsules containing LF or placebo were opened and mixed with whatever the neonates were taking orally or by tube at that time (breast milk, infant formula, or dextrose) Intervention was given as soon as the patient started receiving any amount of oral or tube feedings

Primary outcome Feeding tolerance (continued )

Primary outcome Incidence of culture-proven (blood or CSF) bacterial and fungal sepsis After 72 h of life, during the hospital stay or during follow up for 4 weeks. Secondary outcome Incidence of probable LOS Incidence of any LOS and sepsisattributable mortality

Control group (n ¼ 62) received placebo (100 mg Glucon D) daily from first to 28th day of life.

39 infants received milk (either maternal breast

Primary outcome Risk of first episode of LOS clinically defined sepsis (culture-proven sepsis and culture-negative clinical infection) within 4 weeks (28 days) from enrollment. Secondary outcomes Frequency of culture-proven sepsis Pathogen-specific LOS NEC Duration of hospitalization Overall mortality rate Infection-related mortality Frequency of adverse events Treatment intolerance Control group received placebo (maltodextrin) enterally at 200 mg/day in 3 divided doses over the first 4 weeks of life (n ¼ 95)

Lactoferrin in neonatal care

Barrington et al. (2016) Canada [61]

Kaur et al. (2015) India [60]

Ochoa et al. (2015) Peru [59]

No administration of any Probiotic product Informed parental consent had been released

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J Matern Fetal Neonatal Med, 2017; 30(16): 1920–1932

BLF: bovine lactoferrin; BPD: bronchopulmonary dysplasia; CSF: cerebrospinal fluid: IFI: invasive fungal infection; IVH: intraventricular hemorrhage; LOS: late onset sepsis; LBW: low birth weight; LGG: Lactobacillus rhamnosus GG; NEC: necrotizing enterocolitis; VLBW: very low birth weight; ROP: retinopathy of prematurity; TLF: talactoferrin.

milk or preterm formula) with no additives maternal breast milk or preterm formula) mixed with 100 mg per day of BLF once daily pilot trial of lactoferrin supplementation in the very preterm infant Inclusion criteria Infants 5 31 weeks of age Inborn Admitted before 24 h of age Exclusion criteria Infants with proven or suspected gastrointestinal anomalies or serious cardiac anomalies Moribund and not expected to survive

had not yet fed or had received milk for 5 24 h

Control group Intervention group Population Title Serial number

Table 3. Continued

Secondary outcome LOS Death and the combined variable of death or LOS NEC (stage 2 or worse) Duration of total parenteral nutrition Number of times placed nil by mouth Growth variables at discharge ROP BPD

D. Sharma et al.

Outcome

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weight infants (0% and 0% versus 3.7%). The progression rate colonization-infection was significantly lower in the BLF groups: 3.7% (BLF) and 12% (BLF plus LGG), versus 41.9% (control); p ¼ 0.001 (BLF) and p ¼ 0.02 (BLF plus LGG) [54]. Kaur et al. did not show any significant reduction in fungal sepsis in the intervention arm when compared with control arm (LF 0/63 (0%) versus control 1/67 (1.5%), p ¼ 0.330) [60]. Lactoferrin and NEC Manzoni et al. showed that NEC stage  2 occurred less frequently in group with BLF plus LGG (0/151 [0%]) versus control (10/168 [6.0%]) (p ¼ 0.002) but not with BLF (3/153 [1.9%]) versus control (10/168 [6.0%]) (p ¼ 0.09) [53]. Stefanescu et al. showed no effect on NEC incidence with oral LF application (Biotene group (3/20 [15%] versus control (3/21 [14%]) (p ¼ 0.95) [55]. Sherman et al. did not reported separately the incidence of reduction of NEC in the primary outcome, although NEC scares (defined as clinical scenario that involved a cessation of enteral feedings and initiation of antibiotics based on gastric residuals, occult or gross blood in the stool, abdominal distention, and radiographs showing dilated bowel loops and an abnormal bowel gas pattern, but without a sentinel loop or pneumatosis intestinalis) were seen in six Talactoferrin (TLF) fed and eight placebo-treated infants with OR of 1.4 [56]. Akin et al. showed significant reduction in NEC  2 [BLF group (0/22) versus control group (5/25)] (p ¼ 0.05) [57]. Manzoni et al. showed in their largest trial that NEC was significantly lower in groups BLF and BLF + LGG (5/247 [2.0%] and 0/238 [0%], respectively) than in controls (14/258 [5.4%]) (RR ¼ 0.37; 95% CI: 0.136–1.005; p ¼ 0.055 for BLF versus control [nonsignificant]; RR ¼ 0.00; p50.001 for BLF + LGG versus control [significant]) [58]. Barrington et al. did not report any significant difference in feeding tolerance, that was defined as time to achieve full feeds (140 ml/kg/day) and NEC (LF 4/40 versus Control 4/39) (RR 0.47, 95CI: 0.04– 5.45) [61]. Lactoferrin and neonatal mortality Manzoni et al. showed significant reduction in sepsisattributable mortality in the two treatment groups (0/153 [0%] with BLF and 1/151 [0.7%] with BLF plus LGG, versus 8/168 [4.8%] with control; p ¼ 0.008 and p ¼ 0.04, respectively), although there was significant difference was seen in overall mortality [53]. Manzoni et al. did not report any significant reduction in neonatal mortality attributable to IFI in both BLF (0/153 [0%] versus 2/168 [1.2%]; p ¼ 0.50) and BLF plus LGG group (0/151 [0%] versus 2/168 [1.2%]; p ¼ 0.50) [54]. Stefanescu et al. showed no effect on neonatal mortality in intervention group (Biotene group 2/20 [10%] versus control 3/21 [14%]) (p ¼ 0.68) [55]. Akin et al. showed no difference on neonatal mortality with LF supplementation (LF 0/22 versus Control 1/25) (p ¼ 1) [57]. Manzoni et al. showed that the incidence of death-and/or NEC was significantly lower in both treatment arms (4.0% and 3.8% in BLF and BLF + LGG versus 10.1% in control; RR ¼ 0.39; 95% CI: 0.19–0.80; p ¼ 0.008; RR ¼ 0.37; 95% CI: 0.18–0.77; p ¼ 0.006, respectively) [58]. Ochoa et al. did not find any difference in sepsis-related mortality (LF 4/95 [4.2%] versus

22 VLBW enrolled from July 2012 to January 2013 received oral BLF 100 mg/day (neonates 1000 g were treated for 6 weeks and neonates more than 1000 grams to less than 1500 grams were treated for 4 weeks)

127 VLBW infants

Bovine lactoferrin (BLF) supplementation for the prevention of clinical and laboratory-confirmed LOS in VLBW: a single-center study

Vecchiato et al. (2013) Italy [70]

BLF: bovine lactoferrin; LOS: late onset sepsis; VLBW: very low birth weight.

155 infants received routine cow’s milk-based infant formula from 14–365 days of age

165 infants received investigational formulas with BLF at 0.6 g/L and 160 infants received investigational formulas with BLF containing 1.0 g/L from 14–365 days of age

480 Healthy 12- to 16-dayold infants were randomized to 3 groups

Growth and tolerance of formula with lactoferrin in infants through one year of age: doubleblind, randomized, controlled trial

Johnston et al. (2015) USA [69]

105 VLBW enrolled from 2005 to 2007 taken as historical control

278 children received 500 mg twice a day maltodextrin as placebo (diluted in 25 mL of water)

277 children received 500 mg twice a day of BLF or placebo (diluted in 25 mL of water)

555 children enrolled at 12– 18 months and followed for 6 months with daily home visits

Randomized double-blind controlled trial of bovine lactoferrin for prevention of diarrhea in children

Ochoa et al. (2013) Peru [68]

Not a RCT and did not fulfilled the inclusion criteria of systematic review

Infants were term and the investigator did not assess pre-specified neonatal outcomes of systematic review

Study participants not neonates

Majority of enrolled infants were beyond the neonatal period. Investigator did not assess pre-specified neonatal outcomes of systematic review

Primary outcome Impact of long-term feeding of bovine lactoferrin– enhanced formula on growth, hematologic and immune parameters, and the evaluation of common childhood illnesses in term or nearterm healthy infants Primary outcome Diarrhea incidence during the 6-month intervention. Secondary outcome HFA (Height for age) and WFH (Weight for height) z-scores Longitudinal prevalence of diarrhea Duration of diarrhea Severity of diarrhea Dehydration Prevalence of loose stools. Primary outcome Weight growth rate from 14–120 days of age. Secondary outcome Anthropometry Tolerance measures Medically confirmed adverse events through 365 days of age Primary outcome Incidence of clinical (C-LOS) and laboratoryconfirmed LOS (LC-LOS) 26 infants received powdered Similac with iron formula (3 mg/L elemental iron) without added bovine lactoferrin (102 mg/L bovine lactoferrin)

26 infants received powdered Similac with iron formula (3 mg/L elemental iron) either with bovine lactoferrin containing 850 mg/L bovine lactoferrin

79 healthy infants with age of 0 to 4 weeks, born at 34 weeks of gestation and 2000 g, and strictly bottle fed. 27 infants had dropout

Reason for exclusion

Outcome

Control group

Intervention group

Population

A Double-Blind, PlaceboControlled, Pilot Study of Bovine Lactoferrin Supplementation in Bottle-fed Infants

Title

King et al. (2007) the Netherlands [67]

Serial number

Table 4. The various studies excluded from the systematic review. All the data have been entered in PICO form (population, intervention, control, and outcome).

DOI: 10.1080/14767058.2016.1232384

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Control 2/95 [2.1%]) (p ¼ 0.682) [68]. Kaur et al. showed significant reduction in the sepsis-attributable mortality in intervention arm (0/63 [0%] versus 5/67 [7.5%]; p ¼ 0.027) [60]. Barrington et al. did not report any significant difference in neonatal mortality (LF 4/40 versus Control 4/39) (RR 0.97; 95% CI, 0.23– 4.19) [61]. Lactoferrin and pneumonia We identified only one study that has evaluated the role in prevention of ventilator-associated pneumonia (VAP). Stefanescu et al. showed lower rate of VAP in the Biotene group, although the difference was not statistically significant (9/1000 ventilator-days versus 17/1000 ventilator-days in the sterile water group, respectively; p ¼ 0.16) [55]. Lactoferrin and retinopathy of prematurity (ROP) In the study by Manzoni et al., threshold ROP requiring surgery occurred less frequently in BLF group when compared to control arm (6/153 [3.9%] versus 19/168 [11.3%]) (p ¼ 0.02), although this difference was not seen in BLF plus LGG group (13/151 [8.6%] versus 19/168 [11.3%]) (p ¼ 0.46) [53]. Stefanescu et al. showed no difference in ROP requiring laser in the intervention group (Biotene group 3/20 [15%] versus control 5/21 [24%]) (p ¼ 0.48) [55]. Barrington et al. did not report any significant difference in ROP stage 2 (LF 7/40 versus Control 8/39) (RR 0.82; 95% CI, 0.27–2.54) [61]. Lactoferrin and bronchopulmonary dysplasia (BPD) Manzoni et al. did not find any significant reduction in BPD in BLF (4/153 [2.6%] versus 6/168 [3.6%]) (p ¼ 0.75) and BLF plus LGG group (4/151 [2.7%] versus 6/168 [3.6%]) (p ¼ 0.75), when compared to control arm [53]. Stefanescu et al. showed no difference in BPD/death in the intervention group (Biotene group 17/20 [85%] versus control 15/21 [71%]) (p ¼ 0.29) [55]. Sherman et al. showed reduction in duration of oxygen therapy by 50% in TLF-treated subjects, and BPD was seen in 7 TLF-treated infants and 10 infants in placebo group (OR ¼ 1.5) [56]. Barrington et al. did not report any significant reduction in BPD (LF [12/40] versus Control [11/39]) (RR 1.09; 95% CI: 0.41– 2.88) [61].

Discussion In this systematic review, we included nine RCT that have evaluated the role of LF in neonates. One study from the included studies was secondary analysis [54] of the first study by Manzoni et al. [53]. Seven studies evaluated the role of LF for reduction in LOS in preterm neonates. Four studies showed reduction in LOS in the intervention arm [53,57,60,68], whereas three studies were not able to show any beneficial effect over reduction of LOS [55,56,61]. Hence, we conclude that LF has role for reduction in incidence of LOS. Two studies evaluated the role of LF in reduction of IFI [54,60], although positive result was shown by only one study [54], thereby needing further more trials to show the role of LF in reduction of IFI. The role of LF in reduction of NEC was evaluated by six studies, three studies showed significant reduction in incidence of NEC [53,57,58], one study showed reduction in NEC scares [56], and two studies showed no effect [55,61]. LF use has also

J Matern Fetal Neonatal Med, 2017; 30(16): 1920–1932

been tried to reduce the neonatal mortality. This outcome was evaluated in seven studies, two studies showed decrease in mortality in intervention arm [53,60], one study showed decrease in combined death and/or NEC [58], and four studies showed no effect. Only one study evaluated role of LF in VAP reduction and showed lower rate of VAP in the Biotene group [55]. The role of LF in reduction on BPD and ROP is unclear and needs more trials for evaluating its role. The largest studies have been conducted by Manzoni et al. in Italy and New Zealand [53,54,58], and the latest study conducted by him showed the role of BLF in reduction of NEC and neonatal mortality [58]. There are many clinical trials that are going on in various countries, and the results of these trials will strengthen the evidence for use of LF in neonatal care. The various ongoing trials are: (1) Enteral LactoFerrin In Neonates (ELFIN); ISRCTN88261002, United Kingdom [71]. (2) Oral lactoferrin supplementation for prevention of sepsis in preterm neonate; NCT01821989, Egypt [72]. (3) Lactoferrin Infant Feeding Trial (LIFT) to prevent sepsis and death in preterm infants; ACTRN12611000247976, Australia [73]. (4) Study of talactoferrin oral Solution for nosocomial infection in preterm infants; NCT00854633, USA [74]. (5) Supplementation with lactoferrin in preterm newborns (lactoprenew); NCT01172236, Italy [75]. (6) Effect of prebiotic or lactoferrin supplementation in formula on the gut flora of preterm infants ISRCTN71737811, the Netherlands [76]. (7) Lactoferrin for prevention of sepsis in infants (NEOLACTO); NCT01525316 [77]. Strength of the review   

Maximum number of completed studies (nine) have been included Have taken consideration of all possible effect of LF in neonatal care Included all undergoing registered trials

Conclusion LF has shown to be promising agent for reduction of late onset sepsis and necrotizing enterocolitis. The role of LF in prevention of neonatal mortality, bronchopulmonary dysplasia and retinopathy of prematurity needs further studies. The trials that are going on around the world may be able to give reply of this question in future. The Cochrane review published in 2015, that included four studies, concluded that LF causes reduction in LOS and NEC incidence in preterm infants [78].

Acknowledgements To our parents who have always inspired us for hard work.

Declaration of interest The authors declared the absence of any competing interests.

Ethics (and consent to participate) Not applicable as this is systematic review.

DOI: 10.1080/14767058.2016.1232384

Consent to publish Not applicable.

Authors’ contributions D.S., S.S., and P.S. drafted the manuscript. D.S. and S.S. participated in the design of the study and performed the statistical analysis. D.S. and P.S. conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. The authors did not receive funds for this study.

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