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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background  Multiple studies document that probiotics are effective in treating infectious diarrhoea in children. Lactobacillus rhamnosus GG is the most extensively studied but effectiveness of other strains has been poorly examined.

Aim  To determine whether L. rhamnosus strains (573L/1; 573L/2; 573L/3) (Lakcid L, Biomed, Lublin, Poland) would be effective in shortening infectious diarrhoea.

Methods  In a randomized, double-blind, placebo-controlled trial, 87 children (age range: 2 months to 6 years) with infectious diarrhoea were administered Lakcid L at a dose 1.2 × 1010 CFU or placebo, twice daily, for 5 days. Primary outcome measure was the duration of diarrhoea. Secondary measures were duration of parenteral rehydration, adverse events, and gastrointestinal tract colonization by administered strains.

Results  In an intention to treat analysis of 87 children, the mean duration of diarrhoea in the treated group: 84 ± 56 h; placebo: 96 ± 72 h (P = 0.36). In rotavirus infection: 76 ± 35 h vs. 115 ± 67 h (P = 0.03), respectively. Duration of parenteral rehydration: 15 ± 14 h vs. 38 ± 33 h (P = 0.006). Gut colonization by administered strains was 80% and 41% at five and 14 days, respectively. No adverse events were noted.

Conclusions  Administration of L. rhamnosus strains shortens the duration of rotaviral diarrhoea in children but not of diarrhoea of any aetiology. Intervention shortens the time of intravenous rehydration.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background

Multiple studies documented that probiotics are effective in treating infectious diarrhoea (ID) in children and already four meta-analyses have been published to evaluate this therapeutic option for ID.1–4Lactobacillus rhamnosus GG has been the most extensively studied of probiotic strains, where it showed consistent effect in reducing the duration of diarrhoea.5 The clinical effects of other probiotic strains like Saccharomyces boulardii or Lactobacillus reuteri in treating ID are relatively less documented.6–8 However, the positive effect of Lactobacillus GG in ID cannot be extrapolated absolutely to other probiotic strains or to patients with causes of ID other than rotavirus9 and thus each individual probiotic formulation should be checked for its effectiveness with separate clinical trials.

The cumulative frequency of hospitalization because of ID in infants and children in Poland is estimated at 5% and an average hospital stay of 9 days.10 Infectious diarrhoea creates a substantial epidemiological challenge, especially for this age group. It is conceivable that the routine use of probiotics for children with ID might result in shortening the duration of diarrhoea and in reducing its related health costs. The objective of the present study was to determine whether a combination of three probiotic L. rhamnosus strains (573L/1; 573L/2; 573L/3), administered orally, would shorten acute diarrhoea of any aetiology and especially rotaviral diarrhoea in infants and children.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Participants

We enrolled children from 2 months to 6 years of age with acute diarrhoea treated either at the pediatric ward or at the out-patient department. Diarrhoea was defined as three or more bowel movements per day of stools that are looser than normal and may contain blood, pus or mucus, for more than 1 but less than 5 days. Exclusion criteria were the following: organic gut disease, underlying chronic disease, immuno-suppressive condition or treatment and exclusively breastfed infants. The study protocol was approved by the Jagiellonian University Ethical Committee (Decision No. KBET/90/L/2001).

Interventions

On admission when informed consent was received from parents, children were weighed and examined for dehydration. The patients were rehydrated per os (p.o.) or intravenously (i.v.) according to ESPGHAN recommendations 11 and then realimentation was initiated. Additionally, children were randomly and double blindly assigned to receive, twice daily, either 1.2 × 1010 CFU of freeze-dried mixture of three L. rhamnosus strains (573L/1; 573L/2; 573L/3) (Lakcid L; Biomed, Lublin, Poland) or placebo (in appearance identical to the study product) – for five consecutive days. The preparations were kept refrigerated (2–8 °C) until use. Ten doses contained in glass vials were packed in identical, numbered envelopes for each child. Individual vials containing bacteria or placebo were rehydrated with 2 mL of 10% glucose, 30 min before the application. The solution was not diluted anymore before ingestion. Vials with placebo contained all auxiliary ingredients but without L. rhamnosus.

The hospitalized children were also examined for clinical parameters by the attending physician in the 6th and in the 24th hour after initiation of the study treatment and then on each day of their hospital stay. Out-patients were examined by the physician on their first visit and then on the 6th and on the 14th day. The parents of the children were instructed to fill-in patients’ diaries – containing data on the number of stools, number of vomits and oral rehydration solution (ORS).

Microbiology

The stools of patients enrolled to the study were checked on admission for the presence of rotaviruses and adenoviruses making use of latex reagents (Slidex Rota Kit 2; BioMerieux and Orion Diagnostica, Marcy L'Etoile, France and Espoo, Finland, respectively) as well as bacterial agents of acute gastroenteritis (Salmonella, Shigella and enteropathogenic strains of Escherichia coli) by using the standard methods.12 Incidence of Campylobacter and Yersinia in our epidemiological settings is negligible, so no attempt was made to isolate them. Colonization of the patients’ gut with the administered L. rhamnosus strains was proven by culture and molecular methods. The stool samples were taken from all enrolled patients on admission and again on the 5th and on the 14th day, then transported on swabs dipped into de Man, Rogosa, Sharpe (MRS) agar slabs (Oxoid, Basingstoke, UK) to specialized probiotic laboratory where the swabs were shaken in 1 mL of MRS broth (Oxoid), serially diluted in MRS broth and plated in a volume of 100 μL on MRS agar. The inoculated plates were incubated in anaerobic atmosphere (N 85%, H2 10%; CO2 5%) at 37 °C for 48 h.

All morphotypes of the grown colonies were subcultured on MRS agar and propagated as described above. The isolates were speciated using the polymerase chain reaction (PCR) method with primers designed for 16S–23S rRNA of L. rhamnosus.13 The identity of isolated L. rhamnosus strains was compared using pulsed-field gel electrophoresis (PFGE) technique.14 Briefly, DNA was isolated from bacteria present in agarose blocks by lysis with a lytic buffer [lysozyme 2.5 mg/mL (Sigma, St Louis, MO, USA) and mutanolysin, 20 U/mL (Sigma)], then incubated with proteinase K (1 mg/mL) (Roche, Basel, Switzerland) and cut with SfiI restriction nuclease (25 U/plug) (Roche) overnight at 37 °C. Electrophoresis was carried out with a CHEF DR II apparatus (Bio-Rad) in 1% certified agarose (Bio-Rad, Hercules, CA, USA) with 0.5×TBE buffer. The pulse time was 15–60 s, the current 5 V/cm, the temperature was 14 °C and the running time was 26 h. DNA fragments’ bands were stained with ethidium bromide (0.5 μg/mL) (Bio-Rad) and analysed using the Molecular Analyst Fingerprinting Software (Applied Maths, St-Martens-Latem, Belgium).

Objective

To determine whether L. rhamnosus strains (573L/1; 573L/2; 573L/3) (Lakcid L) would shorten acute diarrhoea of any aetiology and rotaviral diarrhoea in children. The hypothesis that was put forward was that Lakcid L can shorten duration of diarrhoea by at least 24 h.

Outcomes

The primary outcome measure of interest was the effect of the new probiotic preparation on the duration of diarrhoea measured in hours. The starting point for the intervention was the time of giving the first dose, whilst the end time was the time when no abnormal bowel movement was present for the last 12 h or the time of the second normal stool (whatever was first). Secondary outcome measures were: weight gain after rehydration, number of stools passed in the consecutive days of the illness, duration of parenteral rehydration, diarrhoea lasting for over 7 days, presence of adverse events and estimation of the extent of colonization of the gastrointestinal (GI) tract by the examined L. rhamnosus strains.

Sample size

Seventeen participants were required in each group for the study to detect a difference of at least 24 h (s.d. ± 24 h) in mean time to cessation of diarrhoea with a 5% level of significance and a power of 80% (unpaired Student's t test). We assumed that the study withdrawal rate would be at least 20%. Based on this, we estimated that for a minimum total sample size, 26 children needed to be enrolled in each group.

Randomization

Computer generated, blocked randomization list was prepared by an independent subject, outside of the center carrying out the trial. We used a block size of four.

Allocation concealment

The randomization numbers were sequentially assigned to participants as enrolled.

Blinding

The code of randomization was broken at the end of the investigation.

Statistical methods

The differences between the two groups of children were evaluated with the unpaired t-test. Analysis of variance was used to compare multiple parameters and their frequency was compared with chi-squared test. The computer software package –statistica Pl. V.6.0. – was applied and P < 0.05 was regarded as significant. We report the analysis based on the allocated treatment, i.e. intention to treat – all of the participants in a trial for which outcome data were available were analysed according to the intervention to which they were assigned, whether or not they received it.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Participant flow diagram

Figure 1 is a flow diagram showing the subjects’ progression through the study.

image

Figure 1. Flow diagram of the subjects' progression through the study.

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Recruitment

The study was carried out between September 2003 and June 2004 at the Department of Pediatrics, St Hedwig of Silesia Hospital, Trzebnica, Poland.

Baseline data

The appropriate data on characteristics of the probiotic-treated and placebo groups are shown in Table 1. No differences between probiotic- and placebo-treated groups were found, except lower mean number of vomits in the probiotic-treated subgroup with rotaviral diarrhoea.

Table 1.  Characteristics of all patients with diarrhoea and those with rotaviral diarrhoea on admission
 All patientsPatients with rotaviral diarrhoea
Lakcid LPlaceboP/testLakcid LPlaceboP/test
Number of children4641 2217 
In-patients vs. out-patients31/1529/120.74/χ220/216/10.71/χ2
Age (months)24.6 ± 17.726.8 ± 20.80.59/t18.6 ± 13.318.2 ± 9.10.91/t
Sex (m/f)24/2221/200.93/χ212/108/90.64/χ2
Duration of diarrhoea before admission (hours)46.3 ± 27.141.8 ± 26.40.43/t50.6 ± 32.140.8 ± 26.90.31/t
Duration of breast feeding (months)7.7 ± 6.88.7 ± 6.90.53/t6 ± 5.87.9 ± 5.90.32/t
Mean number of stools8.5 ± 5.58.2 ± 4.10.77/t7.5 ± 5.28.7 ± 5.40.52/t
Mean number of vomitings3 ± 2.84.3 ± 4.10.08/t3.5 ± 2.26 ± 4.70.04/t
Fever (number of children)33260.41/χ221170.37/χ2
Dehydration (number of children)
 No12100.58/χ2310.33/χ2
 Mild2721 1610 
 Moderate710 36 

Numbers analysed

Altogether 93 patients were enrolled to the study. Data from six patients were judged not amenable to evaluation, because they were incomplete and/or not compliant with the protocol. Data from 87 patients (45 boys and 42 girls) were available for evaluation.

Outcomes

Aetiology of diarrhoea was identified in 53 of 87 (61%) children: 39 of 87 (45%) had rotavirus infection, five of 87 (6%) had adenovirus gastroenteritis and nine of 87 (10%) children showed a presence of bacterial agents of acute diarrhoea (Salmonella enteritidis, enteropathogenic E. coli serotypes O25, O26, O44 and O128).

Analyses based on the allocated treatment were performed. No differences in major clinical outcomes were found between the study groups: duration of diarrhoea in the group receiving probiotics was insignificantly shorter than the control one. However, duration of diarrhoea in children with rotavirus infection receiving probiotics (n = 22; 77.5 ± 35.4 h) was significantly shorter (P = 0.03) than the one of untreated patients (n = 17; 115 ± 66.9 h) (Table 2).

Table 2.  Characteristics of all patients and those with rotaviral diarrhoea after treatment
 All patientsPatients with rotaviral diarrhoea
No.Lakcid LNo.PlaceboPNo.Lakcid LNo.PlaceboP
  1. * RR 0.38; 95% CI: 0.11–1.26. ** RR 0.19; 95% CI: 0.03–1.15.

Duration of diarrhoea (hours) 83.6 ± 55.6 96 ± 71.50.36 77.5 ± 35.4 115 ± 66.90.03
Duration of diarrhoea according to age (hours)
 <12 months12116 ± 82.78158 ± 90.10.30891 ± 40.74193 ± 28.40.001
 <24 months3088.7 ± 64.627108 ± 72.80.291779.9 ± 39.615122 ± 66.60.03
 <36 months3386.3 ± 62.133110 ± 71.50.161978.7 ± 37.516121 ± 64.60.02
Duration of diarrhoea in relation to the start of intervention
 <72 h3688.8 ± 5937102 ± 72.80.401585.3 ± 30.415125 ± 64.60.04
 >72 h1064.8 ± 37.8442.5 ± 17.50.29760.7 ± 41.9240.5 ± 26.20.55
 Weight gain on 1st day of intervention (gram)2660 ± 17025120 ± 2800.331640 ± 1601590 ± 3200.56
 Duration of parenteral rehydration (hours) 16 ± 19.3 24.3 ± 29.10.08 14.9 ± 13.7 37.7 ± 32.90.006
 Diarrhoea lasting over 7 days (number of patients) 3 70.13 test χ2* 1 10.13 test χ2**

The duration of diarrhoea in different age groups was also analysed. Although there were no general differences found amongst probiotic-treated and -untreated children, patients below 12 months of age with rotavirus infection treated with probiotics (n = 8; 91 ± 40.7) had significant reduction of the duration of diarrhoea (P = 0.001) in comparison with controls (n = 4; 193 ± 28.3). In rotavirus infected children, the duration of diarrhoea before starting the treatment correlated to its duration after treatment was begun. In fact, when the intervention started before the 72nd hour of diarrhoea, treated children had diarrhoea for a significantly shorter time than untreated ones (85.3 ± 30.4 h vs. 156 ± 124.2 h; P = 0.04). Treatment begun after 72 h of diarrhoea had no effect on the duration of diarrhoea. The mean number of watery stools in consecutive intervention days for all patients and for patients with rotaviral diarrhoea are shown in Figures 2 and 3, respectively.

image

Figure 2. Mean number of watery stools in consecutive intervention days – all patients. (bsl00036) Lakcid L; (bsl00000) placebo; ( inline image) standard deviation.

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image

Figure 3. Mean number of watery stools in consecutive intervention days – patients with rotaviral diarrhea. (bsl00036) Lakcid L; (bsl00000) placebo; ( inline image) standard deviation.

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No differences were found for other clinical outcomes considering either all patients or only those with rotavirus. Three diarrhoea cases lasting more than 7 days were observed amongst treated children, whilst seven such cases were found in the control group. This difference was also not statistically significant [P =0.13; chi-squared test, RR: 0.38; 95% CI: 0.11–1.26]. Positive effect of the probiotic treatment of the rotavirus infected children also influenced the duration of parenteral rehydration (14.9 ± 13.7 h vs. 37.6 ± 32.9 h; P =0.006).

The applied probiotic strains were able to colonize the gut of the treated children; amongst 46 children of the group receiving probiotics who completed the study, presence of the strains used was detected in stool samples of 37 of 46 (80%) children at day 5 and in 19 of 46 (41%) children at day 14 after initiating the treatment.

Adverse events

No adverse events of the probiotic treatment were noted during the study.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In our study, using the same approaches and criteria as in most of the preceding clinical trials on Lactobacillus therapy of ID, we have been able to confirm that L. rhamnosus given in a dose of 1.2 × 1010 CFU is able to shorten the duration of acute diarrhoea of rotaviral aetiology in children, especially in infants younger than 12 months of life. Both the general number of the studied children as well as the number of patients with rotaviral diarrhoea were comparable with the corresponding numbers of children reported in most of the other studies8, 15, with the exception of the largest study that enrolled 287 patients.5 These results confirm data contained in the meta-analysis of Van Niel and others1 that higher Lactobacillus doses, i.e. those exceeding 10 × 109 CFU, are more effective. As other authors5, 16, we have not been able to find a significant effect of Lactobacillus treatment in children without defined aetiology of diarrhoea. In a large proportion of these children, a norovirus infection has been confirmed when this study was already completed (not shown in this study). It is therefore possible that, in diarrhoea characterized by very short duration and of other than a rotaviral aetiology, the Lactobacillus effect cannot be observed making the use of standard methodology.

It is of interest that a high proportion of the treated patients was colonized. Colonization was in fact confirmed for all of the applied strains of L. rhamnosus and lasted for up to 2 weeks after stopping the treatment. This is, to our knowledge, the first report in which colonization with a probiotic strain was demonstrated during a clinical trial, making use of molecular approaches. The other studies on treatment and colonization with L. rhamnosus GG were based on the unique morphology of colonies of this Lactobacillus strain17 whilst the study of De Champs et al.18, in which molecular methods were also used, was carried out on experimental animals and human volunteers.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dr Piotr Kochan, M.D., for language consultation and technical adaptation of the manuscript. Tomasz Gosiewski, M.Sc., for help with figures. Prof. Hanna Szajewska, M.D., Ph.D., for reviewing this manuscript and helpful suggestions. The financial support of the Wellcome Travel Award (M.S.) is gratefully acknowledged.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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    Tynkkynen S, Satokari R, Saarela M, Mattila-Sandholm T, Saxelin M. Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains. Appl Environ Microbiol 1999; 65: 390814.
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