In the present study, incorporation of probiotics in the treatment of acute watery diarrhea in children emerged as effective, safe and was associated with a shorter duration of diarrhea, leading to a faster discharge from the hospital. These findings support the institution of probiotic approaches as a routine component of the management of acute watery diarrhea in Bangladesh, while closely monitoring opportunities to further improve on such intervention while attempting to minimize the use of antibiotics in this context.
Before we discuss the potential implications of our study, some specific comments on the ancillary findings are worthy of mention. All groups were comparable in their baseline characteristics. Common clinical features of diarrhea were loose watery stool, nausea/vomiting, fever, blood in stool. Laboratory findings were overall comparable to those of Salazar-Lindo et al. in 2004 in Peru, except for bicarbonate levels which were higher in the present study, suggesting less severe dehydration status [15]. The most prevalent enteropathogens detected at the time of enrollment differed considerably from those of the trials conducted in India and Finland. In the Finish trial, Rotavirus accounted for more than 80% of diarrheal cases [16], while in the study from India, Rotavirus (34.55%), E. coli (19.95%), no growth of pathogens (23.7%), Vibrio cholera (6.95%), and Shigella (2%) were reported [17]. Thus, the greater diversity of etiologic agents and frequent co-pathogen associations as identified in the prese may reflect more accurately the findings occurring in LMICs.
In the current study, no treatment failures occurred, similar to the trial in Finland, in which all infants recovered within 5 days, and no treatment failure was reported [17]. We used L. acidophilus, L. bulgaricus, and Bifidobacterium bifidum as probiotic preparation in this trial, while Dubey et al. (2008) used strains of L. Casei, L. bulgaricus, L. plantarum, S. thermophiles for the treatment of Rotavirus associated diarrhea in children [18]. Narayanappa et al. (2008) showed that Bifilac (a combination of several probiotics) was safe and effective in patients with acute viral diarrhea [19]. Two different studies that were conducted with the aim to evaluate the efficacy of L. rhamnosus GG strain in acute watery diarrhoea in children showed inconsistent effects [20]. Furthermore, no beneficial effects of Lactobacillus acidophilus were observed in children suffering from acute diarrhea [21]. Probable explanations for the inconsistency of the findings across the multiple studies may include: (i) the fact that probiotic preparations and doses were not standardized in the Indian context; (ii) data generated in Western countries cannot be extrapolated to Indian or LMIC settings; (iii) the poor nutritional status of Indian or LMIC children may alter the responses to the probiotic interventions; (iv) different food habits may also affect the response to therapy; (v) the presence of a wide variety of both helpful and harmful intestinal microflora that may interfere with the efficacy of the treatment [17]. Accordingly, the Indian trial showed that about 12% of patients had unresolved diarrhea and an additional 20% were classified as treatment failure mostly due to severe diarrhea [15]. Findings of two more studies conducted also in India found similar results. In a randomized controlled clinical trial of L. sporogenes as probiotic in clinical practice on acute watery diarrhea in children, no treatment failures or adverse effects and complications were reported [17]. However, the rate of treatment failure reported in a study conducted in Peru where L. Casei strain GG was used in the treatment of infants with acute watery diarrhea was 21.1% with LGG vs 18.0% with placebo [15].
We should also remark that the duration of diarrhea was significantly different among the three groups, indicative of a major beneficial effect of the probiotic intervention, particularly when in isolation and without the concurrent treatment with antibiotics. The duration of diarrhea in our study was comparable to that reported in two previous trials [15, 17]. Overall conclusions of a meta-analysis of 63 studies of probiotics involving more than 8,000 participants, mostly children, suggests that probiotics shorten the duration of diarrhea by ~24 hours with no evidence of adverse effects [17, 22].
A few studies have demonstrated the presence of significant associations of probiotic species with altered gut microbiota composition. In our trial, stool analysis of participants at the 1-month follow-up revealed that Bifidobacterium and Lactobacillus were detectable and predominant among the majority patients treated with probiotics, while they were much less likely to be detected in those not treated with probiotics. If we assume that the presence of such probiotic strains is indicative of intestinal health and also signifies potential prevention of future diarrheal episodes, then administration of probiotics during the acute diarrheal episode may have long-term benefits that will need to be quantified in future studies. In experimental settings in rodents, a recent metagenomic analysis of 8-week-old Swiss mice fed a high-fat diet showed that treatment with a probiotic mixture of Lactobacillus and Bifidobacterium (L. rhamnosus, L. acidophilus, and Bifidobacterium bifidum) significantly altered the composition of the gut microbiota [23]. Similar work on obese mice revealed that several Lactobacillus spp., Bifidobacterium spp., and other coliform bacteria increased in the gut microbiota in mice with a high-fat diet treated with various Lactobacillus probiotic strains (L. acidophilus IMV B-7279, L. casei IMV B7280, B. animalis VKL, and B. animalis VKB) [24]. Studies have demonstrated that Bifidobacterium and Lactobacillus can inhibit harmful bacteria, improves gastrointestinal barrier function and Bifidobacterium alters the function of dendritic cells to regulate the intestinal immune homeostasis to harmless antigens and bacteria or initiate protective measures against pathogens [25–28]. Such basic studies have been somewhat corroborated by clinical trials as well. Indeed, a clinical study demonstrated that patients who received L. plantarum DSM 9843 showed the presence of L. plantarum in rectal samples of patients, along with reduced amounts of enterococci in fecal specimens [29]. In another study, analyses of the fecal microbiota of patients treated with a probiotic mixture of L. acidophilus, L. plantarum, L. rhamnosus, Bifidobacterium breve, B. lactis, B. longum and Streptococcus thermophilus. and analyses of the fecal microbiota of these patients revealed that the similarity of the microbial composition was more similar in probiotics-treated patients than that of the placebo group.[30] Another study analyzed the fecal microbiota of 6-month-old infants treated with daily supplements of L. rhamnosus (LGG), and showed an abundance of LGG and an increased index of evenness in the fecal microbiota of these infants, suggesting ecological stability [31].
Many probiotic bacteria have been tested for their immunomodulatory properties, especially Lactobacillus sp. and Bifidobacterium sp. [32–35]. In our study, all children during follow-up on day 30 showed an increase of serum IgM, IgG, IgA antibodies with highest increase in serum concentration of IgG in patients treated with only probiotics. These findings may suggest that the elimination of pathogenic organisms and reestablishment of normal gut flora induce improvements in immune status. Indeed, a double blind, randomized controlled trial in healthy adults reported that oral administration of Bifidobacterium lactis Bl-04 and Lactobacillus Acidophilus La-14 changed the serum immunoglobulin concentrations compared with controls [36]. Shin et al. in a study in pigs also reported that administration of L. plantarum strain JDFM LP11 led to increased serum IgG was increased [37]. Oral introduction of Bifidobacterium bifidum was shown to enhance antibody response to ovalbumin and Bifidobacterium breve was shown to stimulate IgA response to cholera toxin in mice [38, 39]. An increased humoral immune response, including an increase in rotavirus specific antibody-secreting cells in the IgA class, was detected in children with acute rotavirus diarrhea who received L. rhamnosus GG during the acute phase of diarrhea [40]. The mean serum rotavirus IgA antibody concentration at the convalescent stage was also higher in those individuals receiving L. rhamnosus GG [41]. In another trial, oral administration of L. acidophilus LBKV3 strain as probiotic showed enhancement of IgG immunoglobulin levels, and regulation of gut microflora [42].
Limitations
To date, insufficient data justify the routine use of probiotics in diarrhea in Bangladesh. Although the sample size was small, this was the first clinical trial in Bangladesh, and provides initial support to expand these observations. However, we should also indicate that the present study has the following limitations: (a) It was conducted involving only a small population.; (b) It was conducted only in one tertiary care teaching hospital in Dhaka city; (c) It does not represent the whole pediatric population of Bangladesh; (d) it did not conduct an in-depth analysis of gut microbiome using metagenomic approaches. A continuation of this study involving a large number of patients (both children and adult patients) involving all age groups from all areas in Bangladesh (rural and urban areas) and addressing the current limitations will be required to provide more accurate and definitive recommendations.