MANUSCRIPT CITATION:

Sinha A, Gupta SS, Chellani H, Maliye C, Kumari V, Arya S, Garg BS, Gaur SD, Gaind R, Deotale V, Taywade M, Prasad MS,Thavraj V, Mukherjee A, Roy M. Role of probiotics VSL#3 in prevention of suspected sepsis in low birthweight infants in India: a randomised controlled trial. BMJ Open 2015; 5:e006564. PMID: 26163028

REVIEWED BY:

Dr Anu Thukral MD DM (Assistant Professor)
Dr M Jeeva Sankar MD DM (Assistant Professor)
Newborn Health and Knowledge Centre, WHO Collaborating Centre for training and Research in Newborn Health, Division of Neonatology
Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India

TYPE OF INVESTIGATION:

Prevention

QUESTION:

(P) In 0–2-month-old low birthweight infants, (I) does daily supplementation of probiotic (VSL#3; a mixture of 8 probiotic strains with 10 billion colony forming units) starting on third day of life and continued through 30 days (C) compared to placebo (O) reduce the risk of serious bacterial infection (PSBI)(T) in the first month of life?

METHODS:

• Design: Randomized controlled trial with stratified block-randomized design. Authors have stratified infants by birth weight (1500-2000g, 2001-2500g) and sex with allocation ratio of 1:1. A computer-generated table was used for random table generation. The random list was generated in block size of four.
• Allocation concealment: Allocation concealment was ensured by using sequentially numbered sachet packets.
• Blinding: Parents of enrolled infants, investigators and field workers were masked to group allocation; data analysis was also performed in blinded manner.
• Setting: Community settings in rural India
• Patients: All newborn infants aged 3 days (weighing 1500–2500 g), born in the hospital, residing within 20–25 km of the hospital, and not planning to shift residence for at least the next 2 months were included (number eligible for screening = 5927). The study excluded premature infants born before 34 weeks, sick infants (not otherwise defined), those with congenital malformations incompatible with life, and those with guardians not giving consent and belonging to out of study areas. Participants were enrolled by a physician in the hospital and followed up in the community for 2 months for occurrence of sepsis and other morbidities.
• Follow-up period: Follow-up visits were carried out by the fieldworker for supervising supplementation over 30 days and for detection of morbidities over 2 months.
• Intervention:
  • Intervention group: The intervention consisted of administration of the probiotic preparation VSL#3 (a mix of eight strains: Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii spp bulgaricus), at a dose of 10 billion colony forming units for 30 days, starting on the third day of life. The content of the probiotic sachet was mixed in expressed breast milk in a plastic cup and fed to the infant. A sterilised plastic cup and stirrer were provided along with the sachets.
  • Control group: A similar-looking maltodextrin preparation in the same outer packing was administered to the control group.
• Assessment: 
  • Clinical detection of neonatal sepsis was performed during visits, using the Integrated Management of Neonatal and Childhood Illnesses (IMNCI) algorithm for detection of possible serious bacterial infection (PSBI). PSBI was suggested by the presence of any of the following signs of infection: convulsions or fast breathing (60 breaths per minute or more); severe chest in-drawing or nasal flaring or grunting; 10 or more skin pustules or a large boil; axillary temperature 37.5°C or above (or feels hot to touch); temperature less than 35.4°C (or feels cold to touch); lethargic or unconscious or less than normal movements. Fieldworkers referred and accompanied sick infants to the study hospital for treatment. At the hospital, the infants were examined by a physician, blood cultures were obtained, and treatment was carried out as per the protocol of the hospital. Study staff were trained in the IMNCI algorithm and given practice on eliciting signs of neonatal sepsis. Study procedures were standardised and regular exercises were conducted so as to reduce inter-observer and intra-observer variability.
• Outcomes:
  • Primary Outcome:
    • The primary outcome was PSBI as defined by IMNCI algorithm, diagnosed by fieldworkers or physicians.
  • Secondary Outcome:
    • Overall morbidity pattern (local infection, suspected sepsis, possible serious bacterial infection, feeding problems, other morbidities etc.) in 0–2-month-old LBW infants
    • Stool colonisation pattern in 10% of subjects
    • Side effects due to the probioticVSL#3, if any.
  • On the recommendation of the DSMB, data on diagnosis of sepsis by a physician was also recorded as an amendment to the protocol.
  • Gut Colonisation Sub study
    • Stool samples from 202 (101 each in the intervention and placebo arms) enrolled infants were collected on day ‘1’, day ‘21’ and day ‘60’ to correspond to the end of follow-up. The samples were collected in sterile specimen jars (plastic containers) and transported to the lab at 4°C, and stored at −20°C. Processing was completed within 10 days to evaluate their bacterial microflora composition and enzymatic activities. Sequencing and real-time PCR were conducted on DNA samples extracted from stool specimens.
• Analysis and sample size: Previous studies reported a 17% incidence of neonatal sepsis in low birth weight neonates receiving home based newborn care (HBNC) Assuming a 10% loss to follow-up, 1340 infants were needed (670 in each group) to observe a 30% relative reduction (absolute reduction 5%) in incidence of sepsis at 5% significance with 80% power. Analyses were performed by intention to treat. Software ‘R’(V.3.0.0) was used for calculation of PSBI risk, incidence rates, confidence intervals (CIs) and incidence rate ratios. The authors used Kaplan-Meier survival analysis curves .to compare the survival curves in the probiotic and placebo arms and ‘t test’ after log conversion to compare colony counts groups in the gut colonisation substudy.
• Patient follow-up: 668 infants in the probiotic group and 672 infants in the control arm were included in analysis.

MAIN RESULTS:

A total of 668 infants were randomized to intervention group and 672 to the control group. The relative proportion of infants in the two birth weight strata – 1500 to 1999 g and 2000 to 2499g – was comparable between the intervention and control groups. The mean birth weight was 2261 g and 2263 g in the intervention and control groups, respectively. Baseline variables including sex, mother’s schooling, religion, standard of living, mode of delivery, morbidities during pregnancy, and mean standard of living index were comparable in the two groups.

Primary outcome:

1. The overall risk of PSBI was similar in the probiotic (84/688; 12.2%) and control groups (107/672; 15.9%) (RR 0.79; 95% CI 0.56 to 1.03).
2. A significant 71% reduction in the risk of PSBI in infants weighing 1500 to 1999 g (4/74 vs. 14/75; RR 0.29; 95% CI 0.10 to 0.84). Importantly this subgroup was not pre-specified.
3. A 32% reduction in the risk of PSBI among the non pre-specified subgroup of female infants was observed (36/348 vs. 53/349; RR 0.68; 95% CI 0.46 to 0.99).
4. The incidence rate per 100 days of PSBI was lower in the probiotics group (2.61/1000 days of follow-up vs. 3.40/1000 days; IRR 0.77; 95% CI 0.59 to 0.99; p=0.049).

Secondary outcomes:

1. There was no significant difference in the proportion of infants with local infection (3.0% vs. 3.4%), feeding problems (18.9% vs. 16.4%), or other morbidities (35.9% vs. 34.2%) between the two groups.
2. There was no statistical difference in absolute colony counts in the two groups on days 1, 21 and 60.
3. No side effects of VSL#3 were reported.

Post-hoc analysis

1. There was no reduction in the overall risk of physician diagnosed sepsis in the probiotic group (38/688 vs. 54/672; RR 0.71; 95% CI 0.47 to 1.06).
2. The incidence rate per 1000 days in the probiotic arm was 1.07/ 1000 days vs. 1.59/1000 days with placebo (IRR 0.67; (95% CI 0.45 to 0.99), p=0.048) for physician diagnosed sepsis. In the 1500 to 1999g weight stratum, there was no case of sepsis diagnosed by the physician versus an incidence rate of 2.40/1000 follow-up days in the placebo arm (p=0.002).

CONCLUSION:

The authors concluded that daily supplementation with probiotics VSL#3 for 30 days in low birth weight infants led to a non-significant 21% reduction in risk of neonatal sepsis. While demonstrating a ~20% treatment effect is promising, given the size of the current report it is unclear whether probiotics provide a protective effect against neonatal sepsis in the studied population. Therefore, a larger study with sufficient power is warranted to determine the true preventive effect of VSL#3 on neonatal sepsis in these infants.

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COMMENTARY

Low birth weight (LBW) is an important indirect cause of neonatal mortality and infections. Considering nearly 30% prevalence of LBW in India and nearly one-third of these neonates dying due to sepsis, even a modest decline in the incidence of sepsis due to a preventive intervention could potentially avert thousands of neonatal deaths.¹

Amongst the immunotherapeutic agents for sepsis prevention probiotics have attracted both interest and debate in the past decade ^2,3 with many institutions using probiotics routinely.⁴ A review of the level of evidence for probiotics in preterm infants suggests no side effects and long-term outcomes are reassuring.⁵ The concept of probiotics being helpful in prevention of sepsis seems interesting and novel since few interventions have been proven to be effective in preventing sepsis in LBW infants, apart from exclusive breastfeeding and hand hygiene. ^6,7

This study aimed to estimate reduction in incidence of suspected sepsis in 0–2 month-old LBW infants with a daily supplementation of probiotic VSL#3(10 billion colony-forming units over a period of 30 days). This intervention, if proven to be efficacious, could be an important public health intervention for prevention of neonatal infections. The study showed no significant reduction in the risk of suspected sepsis (PSBI) diagnosed by the fieldworker. In the un-pre-specified subgroup (weighing 1500 to 1999g), the reduction in risk of PSBI was statistically significant.

The present study was a community-based randomized trial from with high rates of follow up and good adherence to treatment in both groups. There are however a few important limitations which need to be considered before presuming a possible beneficial role of probiotic administration in LBW infants for prevention of sepsis. First, the findings of statistically significant reduction in PSBI in the subgroup weighing 1500 to 1999g might be a chance finding because of the small numbers involved. Second, suspected sepsis in itself is not an objective outcome. The significant difference in the subgroup analysis makes it imperative to interpret the results with caution. Third, there was no statistical difference in the colony counts in the two groups on days 1, 21, 60. The authors mention the absence of an interaction effect between the treatment and the birth weight group which is not clear. Fourth, the intervention initiated on day 3 to 7 cannot address the risk of early onset sepsis (EOS), an important cause of morbidity and mortality in LBW neonates. Hence, it may be incorrect to interpret this modest decline in the incidence of sepsis being attributed to due to preventive intervention with probiotics.

To conclude, this study suggests possible modest beneficial effect in the subgroup of infants weighing 1500 to 1999 g. The results underscore the need of a larger study with sufficient power to conclusively evaluate the role of probiotics among LBW infants in a population at high risk of mortality from sepsis. Assuming the overall mortality in low birth weight infants to be 20% and the addition of probiotics shall decrease this rate by an absolute value 5% (to 15%), nearly 945 infants will be required in each group (assuming study power to be 80%)

References

1. Million Death Study C, Bassani DG, Kumar R, et al. Causes of neonatal and child mortality in India: a nationally representative mortality survey. Lancet 2010;376:1853-60.
2. Lin HC, Su BH, Chen AC, et al. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics 2005;115:1-4.
3. Hoyos AB. Reduced incidence of necrotizing enterocolitis associated with enteral administration of Lactobacillus acidophilus and Bifidobacterium infantis to neonates in an intensive care unit. Int J Infect Dis 1999;3:197-202.
4. Deshpande GC, Rao SC, Keil AD, Patole SK. Evidence-based guidelines for use of probiotics in preterm neonates. BMC Med 2011;9:92.
5. Mihatsch WA, Vossbeck S, Eikmanns B, Hoegel J, Pohlandt F. Effect of Bifidobacterium lactis on the incidence of nosocomial infections in very-low-birth-weight infants: a randomized controlled trial. Neonatology 2010;98:156-63.
6. Ohlsson A, Lacy JB. Intravenous immunoglobulin for preventing infection in preterm and/or low birth weight infants. Cochrane Database Syst Rev 2013:CD000361.
7. Pammi M, Abrams SA. Oral lactoferrin for the prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2015:CD007137.