Probiotics prevent NEC in rats too!

Mark Underwood has been very active in the investigations of probiotics and their effects on the neonatal bowel. His new publication is a fascinating exploration of what happens in a rat model of NEC when they receive bifidobacterium infantis, also known as bifidobacterium longum subspecies infantis. (I don’t pretend to understand how bacteriologists decide if 2 strains are the same species or subspecies, it all seems somewhat arcane to me; I sort of get Gram stains but after that…).

One of the first good things about this study is the opening line of the abstract ‘

Probiotics decrease the risk of necrotising enterocolitis

No ifs, ands, or buts; I like it.

The introduction is a brief  resumé of the lines of evidence in animal models regarding the efficacy of probiotics for prevention of NEC.

The most promising preventative approaches to date include provision of human milk and probiotics (4,5). The observations that many Enterobacteriaceae out-compete commensal organisms in the inflamed intestine by utilizing an alternative respiratory pathway (6), together with the recent descriptions of a bloom of Enterobacteriaceae associated with NEC (7) shed new light on a possible central role of the intestinal microbiota in this disease.

The neonatal rat model of NEC is an invaluable experimental tool for examining the pathogenesis of NEC and potential mechanisms of protection (8,9,10). The strength of this model is its inclusion of stressors and enteral feeding, both of which are factors associated with human NEC. In the rat model, the stressors include separation from the dam, tube feeding, hypoxia, hypothermia, and enteral nourishment with bovine-based rat milk substitute (11).

Previous studies with this model have demonstrated a protective effect of probiotic Bifidobacterium bifidum with decreased NEC, decreased apoptosis, and decreased inflammation (8,9,10). Mouse and piglet studies have demonstrated alterations of the intestinal microbiota in NEC (12,13)

The references are all listed at the end of this post for those interested.

These investigators studied 3 groups of neonatal rats. They either got donor breast milk (that is they were fed by surrogate rat mummies) or they received artificial formula, or they received artificial formula and probiotics. The animals in the 3 groups were exposed to asphyxia and cold stress, 1 minute of breathing 100% nitrogen and 10 minutes of being put in the fridge (at 4 degrees). None of the breast-milk fed baby ratlings developed NEC, but 80% of the formula fed ones did. Giving B. infantis decreased this to about 36%. There was a decreased expression of a number of pro-inflammatory mediators in their intestines and less histologic injury also. Interestingly they weren’t able to find a lot of bifidobacteria in the caecum of the animals, or to note any consistent difference in the microbiomes between those which got bifidos and those which did not.

Apart from the demonstration of gut protection and NEC prevention and reduced inflammation with probiotics, this study also illustrates what we don’t yet understand about probiotics. In our work, which we haven’t yet finished analyzing to publish, in preterm babies who all received probiotics, only a tiny proportion of fecal organisms were the probiotics. Nevertheless they have clinical effects. Work like this new study will help us to understand, and eventually to select the most promising organisms for future clinical trials.

4. Underwood MA. Human milk for the premature infant. Pediatr Clin North Am2013;60:189207.

5. Deshpande G, Rao S, Patole S, Bulsara M. Updated meta-analysis of probiotics for preventing necrotizing enterocolitis in preterm neonates. Pediatrics 2010;125:92130.

6. Winter SE, Winter MG, Xavier MN, et al. Host-derived nitrate boosts growth of E. coli in the inflamed gut. Science 2013;339:70811.

7. Mai V, Young CM, Ukhanova M, et al. Fecal microbiota in premature infants prior to necrotizing enterocolitis. PLoS One 2011;6:e20647.

8. Khailova L, Dvorak K, Arganbright KM, et al. Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol2009;297:G9409.

9. Khailova L, Mount Patrick SK, Arganbright KM, Halpern MD, Kinouchi T, Dvorak BBifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2010;299:G111827.

10. Underwood MA, Kananurak A, Coursodon CF, et al. Bifidobacterium bifidum in a rat model of necrotizing enterocolitis: antimicrobial peptide and protein responses. Pediatr Res2012;71:54651.

11. Dvorak K, Coursodon-Boyiddle CF, Snarrenberg CL, Kananurak A, Underwood MA, Dvorak B. Helicobacter hepaticus increases intestinal injury in a rat model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2013;305:G58592.

12. Carlisle EM, Poroyko V, Caplan MS, Alverdy JA, Liu D. Gram negative bacteria are associated with the early stages of necrotizing enterocolitis. PLoS One 2011;6:e18084.

13. Azcarate-Peril MA, Foster DM, Cadenas MB, et al. Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria. Gut Microbes 2011;2:23443.

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The voice of Parents, now in print.

Katharina Staub and other parents have written and published an article in Acta Paediatrica. ‘ Our child is not just a gestational age‘.

In the NICU babies are often referred to by how many weeks they spent in the uterus, so X is a 24 weeker, Y is a 23 weeker, etc. Worse, decisions are made about whether or not to institute or continue life sustaining interventions based on estimates of completed weeks of gestational age, when other factors are just as important, and babies may not even be offered active intervention as a result of a sometimes very limited analysis; which gives an overwhelming influence to the estimated (or often, guesstimated) gestational age.

I have already discussed this article in a post, as it was already available on-line, but as it has now appeared in the print edition I thought I would give another mention to this article, one of too few which have really given a voice to parents.

Posted in Neonatal Research, The CPS antenatal counselling statement | Tagged | 1 Comment

Can Paracetamol (acetaminophen) close the ductus arteriosus?

A new publication by Afif El-Khuffash and his colleagues (El-Khuffash A et al. Efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies. Pediatr Res. 2014;76(3):238-44) describes a retrospective review of paracetamol use in newborn infants, and an in vitro study using mouse ductal tissue. The clinical part of the paper shows that with a short course of oral paracetamol there was no response, with a longer course of oral paracetamol there were some responses, and with intravenous paracetamol for between 2 and 6 days there were many PDA closures, and others that tended to constrict. The numbers are very small but suggestive.

Afif also reports the effects of paracetamol and indomethacin on preterm and term mouse ductal tissue. Paracetamol had no effect on the preterm, and lesser effect on the term, compared to indomethacin. With high enough concentrations of paracetamol there were indeed constrictive responses to paracetamol.

This all suggests that it is indeed possible that paracetamol might be effective. But I won’t be expecting miraculous results.

This is also the suggestion of an article from Israel (Nadir E, Kassem E, Foldi S, Hochberg A, Feldman M. Paracetamol treatment of patent ductus arteriosus in preterm infants. J Perinatol. 2014;34(10):748-9), 7 extremely preterm infants with large PDA were treated, they either had failed ibuprofen or had a contra-indication. Most closed without surgery. Again suggestive but no more than that.

Presumably there are placebo controlled trials coming, I hope so.

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Probiotics: so what about those ELBW babies?

After a comment to my previous post about probiotics, I wanted to clarify what I said about the ELBW baby, that is, the newborn with a birth weight below 1001 grams.

It is true that there are few studies that have reported outcomes separately under 1kg, I referred to the 3 articles quoted by the authors of the commentary. In fact one other article reported a study which was exclusively in ELBW, that is Al-Hosni et al. They had 50 patients per group and 2 cases of NEC per group.

So the data for babies who are known to be less than 1 kg looks like this


However, in many of the other studies there were large proportions of the babies who were under 1 kg.

Study Entry Criteria (birth weight, g) Mean birth weight of probiotic group
Bin-Nun <1500 1152
Braga 750-1500 1194
Dani <1500 (or <33wk) 1325
Fernandez-Carrocera <1500 1090
Kitajima <1500 1026
Lin 2005 <1500 1104
Lin 2008 <1500 1029
Manzoni <1500 1212
Mihatsch <1500 (and <30wk) 856
Rougé <1500 (and <32 wk) 1115
Samanta <1500 ( and <32 wk) 1172
Sari <1500 (or <33 wk) 1231

Some of the other studies have included larger or more mature babies, Costalos for example excluded babies under 28 weeks, and Stratiki babies under 27 weeks. Ren excluded infants under 1 kg, and two other Chinese studies that I don’t have access to the original manuscript (Di and Ke) included babies under 32 weeks.

If we redo the meta-analysis with only those studies that were restricted to the very low birth weight, (excluding even Dani and Sari above) there are over 3,600 babies in the trials, all under 1500 grams, with an average birth weight below 1200 grams in all of the studies.

In other words there are substantial numbers of babies under 1001 grams in these studies, probably around a third of them, with probably, given the birth weight distribution of NEC, at least half of the cases of NEC. (In the CNN database over the last few years about 60 to 66% of NEC cases among the VLBW babies were among those who were under 1001g)

Here is the Forest Plot:

As you can see the effect on NEC is significant p<0.00001, with little heterogeneity, and a relative risk of 0.45.

And the Funnel plot of the same data:

Is there any other therapy in the NICU where we demand separate evidence for the ELBW when it is clearly proven to be effective in the VLBW (which includes a large number of ELBWs)?

I can’t think of any, nor of any other therapy tested in RCTs which only enrolled VLBW infants and which have included 3,600 VLBW babies.

The demand for specific studies in ELBW babies is bogus. Babies over 1 kg get NEC also, so why would anyone exclude them from trials of NEC prophylaxis?

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The Time for a Confirmative Necrotizing Enterocolitis Probiotics Prevention Trial in the Extremely Low Birth Weight Infant in North America Is Well Past

What on earth are these people waiting for?

A number of authors from Toronto and elsewhere have written a ridiculous editorial (entitled ‘The Time for a Confirmative Necrotizing Enterocolitis Probiotics Prevention Trial in the Extremely Low Birth Weight Infant in North America Is Now!’) which basically suggests that North American babies should be randomized to placebo in a trial to prove that North american babies have the same decrease in NEC as babies in the rest of the world. It recommends, in essence, that we should lie to parents about the proven benefits and safety of probiotics in order to do a North American trial, because, I guess Canadian and US babies are somehow different, even though they die of NEC just like everyone else.

Lets dissect their arguments.

1. Breast milk is proven to prevent NEC

2. Probiotics have not been adequately studied in ELBW babies (weights under 1 kg).

3. Previous studies have methodologic limitations

1. If we apply the same criteria to donor breast milk as they do to probiotics, this statement is evidently false. There are 3 informative studies in the systematic review by Bill McGuire that they quote, (the 4th study had no cases of NEC). One of them, Steve Gross’s study from 1983 excluded infants under 27 weeks, and excluded growth retarded infants, it was a single center study with about 20 patients in each of 3 groups, and there is no pre-specified sample size in the publication. Tyson’s single center, un-blinded, study, only randomized infants at 10 days of age if they were stable and extubated, they included 81 VLBW babies and had 3 cases of NEC (all in formula fed babies). Alan Lucas randomized 159 babies in a multicenter trial (initially reported in 1984 with the NEC results reported in 1990) , there was a non-significant reduction in NEC with donor breast milk compared to preterm enriched formula. Only 20 of the babies had a birth weight below 1200 grams, and there is no explicit calculation of sample size reported.

Even if we were to accept that a systematic review of 3 small trials from over 30 years ago can give us reliable information about the efficacy of an intervention among our current babies, and even if we are to ignore the potential inflation of significance when a few small trials are meta-analyzed, and if we ignore the fact that these trials have more deficiencies than the trials of probiotics; we still end up with a reduction of NEC which is barely significant, the upper limit of the 95% CI is 0.99.

This brief review shows that the data supporting the idea that donor pasteurized breast milk is preferable to preterm formula for the prevention of NEC are quite limited, of relatively low quality, from a tiny number of trials enrolling even tinier numbers of ELBW infants.  There is far better data from recent high quality trials that probiotics are effective and safe: the message of this published commentary should have been that the time is ripe for an RCT of banked human milk in ELBW babies.

2. They state that probiotics have not been shown to work in the ELBW. This is another mis-statement: In Proprems there were 14 cases of NEC among 239 controls and 10 cases among 235 probiotic treated infants. In Lin’s study there were 7 cases among 78 controls, and 4 cases among 102 probiotic receiving infants. So in both studies there was less NEC among the ELBW infants. Rougé’s small study only had 3 cases of NEC in total, so not likely to be different in the under 1 kg subgroup, and in fact they don’t report NEC under 1 kg, as opposed to over. The other studies have not reported clearly the outcomes among babies under 1 kg. Which does not mean that probiotics have not been shown to work under 1 kg. Any more than the lack of reporting by hair colour means that they don’t work in redheads. One reasonable response to this situation, if you really wanted to, would be an Individual Patient Data Meta-analysis to examine efficacy under 1 kg.

But why would probiotics work in infants over 1kg and not under 1 kg? Even if there were some reason for believing that to be the case, surely that would mean that it is now essential to give probiotics to babies over 1kg?

3. Studies have methodologic limitations? well yes, some of them do, but the major limitation that Mihatsch note in their systematic review, the source of this criticism, is that they did not report how they arrived at the sample size. Neither do the Breast Milk Banking studies that they endorse, nor many other studies reported without the CONSORT guidelines.

I hope that the authors are consistent about their desire to have more evidence, and only high quality evidence. With standards like the ones they suggest they should stop using antenatal steroids, never intubate a baby, and never use inotropes or give fluid boluses.

They end this with the following:

We argue, therefore, that now is the time to conduct in the North American setting, a high quality confirmative NEC prevention trial using probiotics in at-risk ELBW infants. Evidence arising from such a trial will provide neonatologists based in the US and Canada with new evidence that has high potential for changing clinical practice and improving the health outcomes for the vulnerable, extremely premature newborn. Equally important and a prerequisite for the introduction of probiotics in NICUs in North America is a quality-based formulation of product from reliable and dependable sources in the private sector.

I argue, therefore, that this is drivel. Neonatologists based in the US and Canada already have plenty of evidence to change practice, even if some of it comes from weird places like Taiwan, Israel, Italy, Australia and New Zealand. Babies in North American NICUs are dying and losing their bowel, and developing associated neuro-developmental impairment, because of a partially preventable condition. The Australian trial used a product which comes from New Jersey. Parents have the right to know the data and to make their own choices.

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Not neonatology, not directly at least

I am a constant follower of the Science-based medicine blogs.  They have several excellent writers including Mark Crislip who coined a widely quoted phrase about so-called integrative medicine, where science based medicine is mixed with all sorts of woo; acupuncture, homeopathy and the like. The phrase goes like this: ‘if you mix cow pie with apple pie, it does not make the cow pie taste better; it makes the apple pie worse’.

His latest post is great, discussing why otherwise rational doctors can be so seriously misled when it comes to evaluating the effects of ineffective therapies such as those mentioned above.

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Buenos Aires

On my way home from a great, but too short, trip to Buenos Aires. Spoke at a conference there. I had an afternoon off and was able to take a long walking tour, including taking the Avenida de Mayo. Where I took this picture of one view from the Plaza de Mayo, where there are many political demonstrations, including an on-going display by the veterans of the Malvinas War It shows some examples of the varied architecture of the city. See if you can tell what time it was when I took the photo.



If you can see in high enough resolution, you will note that all 3 clocks have different times (and none of them were even close!)

Thanks to Gonzalo and a great group of friends and colleagues for organizing an excellent conference. See you all again soon.

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