Acute rotavirus gastroenteritis is a significant pediatric infectious disease problem in developing and industrialized countries. Treatment is largely symptomatic and involves fluid and electrolyte replacement and maintenance of nutrition.1, 2 During diarrhea there is a decrease of protective commensal microflora including lactobacilli and bifidobacteria,3, 4 followed by overgrowth of urease-producing pathogenic bacteria.5 Exogenously administered lactobacilli may reverse such development and attenuate the clinical course of diarrhea. Clinical studies have shown that treatment with Lactobacillus GG (in which GG denotes Goldin and Gorbach) reduces the duration of acute rotavirus diarrhea.6-9 It is likely that several bacteria, including other strains of lactobacilli and bifidobacteria, have a beneficial effect in acute diarrhea.7, 10-13
Lactobacillus reuteri is a relatively newly recognized species of Lactobacillus, which occurs in the gastrointestinal tract of humans and animals.14, 15L. reuteri produces a broad spectrum antimicrobial carbohydrate metabolite called reuterin.14-16 In poultry L. reuteri provides effective protection against Salmonella challenge17, 18 and has shown therapeutic effect in a rat colitis model.19, 20
We have previously demonstrated colonization with L. reuteri and shortening of acute diarrhea in children.21 The purposes of the present study were to confirm that observation in rotavirus-induced diarrhea in children and to investigate dose dependency of the effect of L. reuteri.
Study design and population. This was a randomized, double blind study carried out in two pediatric infectious disease wards at the Tampere University Hospital, Tampere, and at the Aurora Hospital, Helsinki, Finland, during the rotavirus epidemic season from January 22 to July 15, 1996. Children 6 to 36 months of age with acute diarrhea evident for <7 days, who had had three or more watery stools during the preceding 24 h, were enrolled at admission. Parents or legal guardians of eligible children were contacted by the investigator or a designee and informed consent was obtained.
On admission the children were weighed and examined clinically. The degree of dehydration was estimated and treatment with a hypotonic oral rehydration solution (ORS) was prescribed. Only orally rehydrated children were included in the study. Oral rehydration was followed by realimentation with normal food for age, after oral rehydration was completed (usually 6 to 8 h).
The recruited patients were randomized to receive either a preparation of L. reuteri, in two dosages, or a matching placebo once a day. The first dose of the assigned preparation was given immediately after informed consent had been obtained. The study preparation was administered for the maximum of 5 days, even if hospitalization was extended beyond that.
The clinical outcome measures included presence or absence of diarrhea on each day after the onset of treatment, duration of diarrhea as counted to the last watery stool, frequency of watery stools and frequency of vomiting episodes per day and weight gain during convalescence at the end of episode; diarrhea was defined as the presence of watery stools in any 24-h period, and conversely the end of episode was defined as the absence of watery stools for a 24-h period. If diarrhea or vomiting continued after the day of discharge, the parents recorded these on a diary card kept for 1 week.
Study preparations.L. reuteri and placebo formulations were prepared, quality controlled and assured by a Good Manufacturing Practice-certified facility (BioGaia Biologics AB, Göteborg, Sweden). The large dosage of L. reuteri contained 1010 to 1011 colony-forming units (cfu) and the small dosage contained 107 cfu of freeze-dried viable bacteria in a 0.5-g aliquot with lactose as a carrier. The placebo consisted of the same amount of carrier alone. Each dose was packed in a capsule, and five capsules containing the same preparation were packaged in an individual flask assigned to a single patient.
All treatment preparations were kept at −20°C until the day of use. The content of a capsule containing L. reuteri or placebo was reconstituted in 20 to 50 ml of any fluid including infant formula or breast milk and mixed well. Infant formula was recommended as a diluent for the first dosing, except for breast-fed infants. Hot food was cooled before mixing with the treatment preparation. The final L. reuteri concentration was ∼5 × 108 to 2.5 × 109 cfu/ml in the large dosage and 2.5 × 106 to 5 × 106 cfu/ml in the small dosage. No other fermented milk products were allowed during the treatment.
Laboratory processing. Stool specimens for analysis of rotavirus antigen were collected from each subject as soon as possible after admission. Rotavirus antigen was sought using a commercial enzyme immunoassay (IDEIA® Rotavirus; DAKO Diagnostics Ltd., Ely, UK).
Stool samples for L. reuteri studies were collected from 40 patients (26 in Tampere and 14 in Helsinki) at baseline before study product administration, on Day 2 after study product administration and at discharge from the hospital. Stool samples were prepared as follows. Two grams of homogenized stool were diluted in 0.1% peptone water for a final ratio of 1:5, mixed well, divided into 5 aliquots of 1.6 ml each, quick frozen and stored at −70°C. The samples were shipped on dry ice to BioGaia. All cultures were coded.
Stool samples for the determination of fecal enzymes were collected from 32 patients; of the patients 10, 11 and 11 received the large and small dosages of L. reuteri and the placebo, respectively. Samples for enzyme activity determination were prepared as follows. Two grams of undiluted homogenized stool were placed in a 25-ml container, frozen and stored at −70°C as soon as possible after collection. The enzyme activities (urease, beta-glucuronidase and beta-glucosidase) were determined in the laboratory of the Department of Clinical Nutrition, University of Kuopio, as previously described.3, 22
Blood samples were collected on the day of admission or 1 day after admission and again 4 weeks later for determination of serum rotavirus antibodies from a subset of patients admitted to the Tampere University Hospital (from 7, 8 and 6 patients receiving the large and small dosages of L. reuteri and the placebo, respectively). Rotavirus IgA and IgG class antibodies were determined by enzyme-linked immunosorbent assay.23
Statistical methods. Statistical analysis was performed using BMDP Version 1990 (SUN/UNIX) and SPSS for Windows Version 6.0 programs. Analysis of variance (ANOVA), analysis of variance and covariance, chi square tests and a nonparametric test (Kruskal-Wallis one-way ANOVA) were applied.
Ethical review. The study protocol was approved by the Ethical Review Committee of Tampere University Hospital and that of the Health Care Center, City of Helsinki.
Of 97 patients enrolled from January 22, 1996, to July 15, 1996, 86 (89%) were positive and 11 (11%) were negative for rotavirus. Of the 86 rotavirus-positive patients 66 (77%) received oral rehydration; 18 (23%) received exclusively or mainly intravenous fluids for rehydration and were excluded. The rotavirus-positive patients treated with ORS were included in the analysis.
The clinical characteristics of the three treatment groups are presented in Table 1. The mean (±SD) age of all the patients was 16.4 (7.6) months, the mean (±SD) duration of diarrhea until treatment was 3.4 (1.4) days and the mean degree of dehydration (±SD) was 3.8 (0.9) percent. The duration of diarrhea before admission in the three treatment groups was uneven, being longer in the group receiving a small dosage of L. reuteri than in the other two groups (Table 1). Table 1 also describes the clinical characteristics of the patients during hospitalization and at discharge. There were no differences between the groups in weight gain, consumption of ORS or electrolyte and acid-base balance.
L. reuteri had a significant effect on the duration of watery diarrhea (F = 4.59; P = 0.01, ANOVA and covariance with duration of diarrhea before treatment as a covariate). There was not a significant difference in hospital stay between the groups.
The effect of L. reuteri on the number of patients with diarrhea was best seen in the second 24-h period of treatment (Fig. 1). On the first day of treatment watery diarrhea persisted in 100% of the placebo, 100% of the small dosage L. reuteri and 81% of the large dosage L. reuteri recipients (P = 0.01 placebo vs. large dosage of L. reuteri, chi square test). On the second day of treatment watery diarrhea persisted in 80% of the placebo, 70% of the small dosage and 48% of the large dosage L. reuteri recipients (P = 0.04 placebo vs. large dosage of L. reuteri; chi square test). The frequency of watery diarrhea per 24-h period was significantly reduced on the second day of treatment in the L. reuteri recipients as compared with placebo recipients. The mean (±SD) frequencies of watery diarrhea were 3.8 (2.8), 2.0 (2.1) and 1.8 (2.7) in the placebo, small dosage and large dosage L. reuteri groups, respectively (F = 3.95, P = 0.02, analysis of variance, Bonferroni test).
Figure 2 presents the Lactobacillus counts in stool samples. Both large and small dosages of L. reuteri resulted in colonization, the mean concentrations of L. reuteri being higher in the large dosage recipients. Total lactobacilli were also increased concomitantly. In the placebo-treated children the total counts were low both on admission and at discharge.
The urease activities in stools were generally low on admission in all treatment groups. The mean urease activities increased in the placebo group but decreased in both L. reuteri groups; the decrease was greater in the small dosage group (Fig. 3). The differences of mean (±SD) activities between discharge and admission were −4.2 (19.8), 11.6 (11.1) and 0.5 (17.3) nmol/min/mg in the patients receiving the placebo, small dosage and large dosage L. reuteri, respectively (P = 0.058, Kruskal-Wallis one-way ANOVA test). If the differences for each patient group were divided into those greater than the median difference (−4.75 nmol/min/mg) and smaller or equal than the median difference, the ratios of patients in the two categories were 9:2, 2:9 and 5:5 in the placebo, small dosage and large dosage L. reuteri, respectively (P = 0.01, Kruskal-Wallis one-way ANOVA, median test). There were no differences between the groups in beta-glucuronidase and beta-glucosidase activities on admission or at discharge (data not shown).
Rotavirus antibody titers in convalescent sera were not different in the L. reuteri and placebo groups. The mean (±SD) serum rotavirus IgG antibody titers were 101 (11) enzyme immunoassay units (EIU) in the placebo group, 97 (13) EIU in the small dosage and 103 (11) EIU in the large dosage L. reuteri group. The mean (±SD) serum rotavirus IgA antibody titers were 104 (47), 44 (23) and 77 (32) EIU in the three groups, respectively.
Stool cultures indicated that L. reuteri established colonization in the gastrointestinal tracts of the patients with rotavirus diarrhea. This provides suggestive evidence that L. reuteri was specifically responsible for limiting duration of rotavirus diarrhea. The benefits of L. reuteri therapy were observed about 24 h after the initiation of treatment. While the presence of L. reuteri in the gastrointestinal tract was necessary for the observed beneficial effect, the mechanism is not known. L. reuteri might act by suppressing the growth of harmful bacteria, the overgrowth of which may prolong the course of rotavirus diarrhea. Administration of Lactobacillus GG has been shown to decrease activities of some bacterial enzymes in the stools, indicating suppression of growth of urease-producing pathogenic bacteria.22 In a study of acute diarrhea in children, some patients receiving Lactobacillus GG had lower concentrations of urease in stools than did control patients.3 We also studied urease activities in stools on admission and at discharge. There were more patients with decreased urease activity at discharge in the two groups receiving L. reuteri than in the placebo group, but the effect was not seen in all recipients of L. reuteri. Therefore the significance of this mechanism on the clinical reduction of diarrhea remains uncertain.
The responses of rotavirus IgG and IgA serum antibodies were similar in the L. reuteri and placebo recipients. In earlier studies with Lactobacillus GG in rotavirus gastroenteritis, Lactobacillus GG therapy was associated with enhancement of IgA and rotavirus-specific IgA-secreting cell responses.8, 24 These modalities of immune response were not investigated in the present study, and it was not possible to conclude on possible immunostimulating properties of L. reuteri on the basis of humoral antibody responses only.
Even if the mechanism of action remains unclear, the previously found beneficial clinical effect of Lactobacillus on acute rotavirus diarrhea in young children received further confirmation from the results of this study. It should be noted that in this hospital-based study the treatment started at a late stage of the disease. Further studies should be conducted to determine whether earlier administration of L. reuteri might result in greater benefit. In view of the rapid effect of L. reuteri in diarrhea, resembling one of a pharmacologic therapy, it would be of interest to study whether the effect can be obtained with inactivated bacteria.
L. reuteri should also be studied in combination with other probiotics. In Mexico studies have been conducted with a probiotic blend containing L. reuteri, Lactobacillus acidophilus and Bifidobacterium infantis added to a liquid nutritional beverage (PediaSure®). Preliminary results indicate that the probiotic mixture containing viable L. reuteri was effective in the prevention of diarrhea in Mexican children.25 The investigators suggested that Lactobacillus spp. can reduce the risk of diarrhea in young children when consumed as part of the daily diet.26 The prophylactic effect of L. reuteri alone should also be studied. At present L. reuteri is available in commercial acidophilic milk products in Sweden and Finland, but the clinical effectiveness of these specific products has not been established.
We conclude that administration of freeze dried viable L. reuteri once daily is a simple, safe and effective therapy of acute rotavirus diarrhea in children. Further studies are warranted to confirm the findings; at the same time a more practical formulation of L. reuteri should be developed.
We thank the nurses of Children's Ward 4 at Tampere University Hospital and Children's Ward 14 at Aurora Hospital, Helsinki, for their cooperation. These studies were financially supported by BioGaia Biologics AB.
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