A Double Blind Randomized Clinical Trail: Comparing Lactobacillus plantarum 299v with Placebo in treating irritable bowel syndrome patients

Irritable bowel syndrome (IBS) is a gastrointestinal disease of intestinal mobility. IBS present with variable clinical symptoms making the treatment difficult. IBS is quiet prevalent around the globe with different frequency. Differences in frequency and gender is due to diet habit. It is less frequent where diary product and vegetable are frequently consumed as compared to those who consumed meat. Lactobacillus plantarum 299v (L. plantarum 299v) is the most widely studied strain in the IBS patients. It is resistant to the actions of intestinal acids and bile, colonizes the human colonic mucosa and is non-pathogenic in nature. The efficacy of Lactobacillus plantarum 299v L is different in different study. The present study was designed to find the efficacy of Lactobacillus plantarum 299v in comparison to placebo in randomized control trial.


Introduction
Irritable bowel syndrome (IBS) is a gastrointestinal disease characterized by altered intestinal motility, affecting about 11 % of the world's population [1]. The prevalence of IBS among different countries varies such as in Pakistan it is 13.34%, Iran 7.13 %, China 11.5%, United States of America 20.4%, United Kingdom 21.65% and in Japan 14.04 % [2]. This variation in the prevalence of this most common functional disorder is attributed to the wide clinical spectrum and the different criteria used for the diagnosis of this disorder.
IBS patients usually present with a complex of symptoms which is difficult to treat [3]. These symptoms harm the patient's value of life. The patients suffer from severe depression with a tendency of suicide. Treating patients usually involves surgeries and invasive measures [4,5] . The efficacy of the up-to-date treatment modalities for IBS is low mainly because of the heterogeneous nature of the disease pathophysiology includes hypersensitive viscera's, altered gut motility, distorted interactions of the brain with gut, intolerance to specific foods, altered intestinal permeability and inflammatory changes in the gut [6]. More recently the role of enteric infections and intestinal inflammation along with the implications of changing gut flora has been proposed in the pathogenesis of IBS [7]. Based on these facts it was suggested that the IBS patients may have bacterial overgrowth and the eradication or altering the gut flora may improve symptoms in these subsets of patients [8,9].

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To improve immunize the patients against environmental opportunistic, probiotics are used. These probiotics are usually live or attenuated bacteria or bacterial metabolites [10]. How probiotics improve the symptoms of IBS patients is still to be understood, but few concepts have been postulated with pieces of evidence. As enteric flora is thought to play some role in the disease pathogenesis [11], gut functions improve with changes in gut flora attributed to probiotics [7]. In postinfectious IBS the disease course is altered by the antibacterial and antiviral effects possessed by many probiotic organisms [12]. Anti-inflammatory actions of probiotics on the gut's mucosal surface may decrease the firing of enteric neurons, mediated by the immune system, resulting in lesser stimulus and response cycles between the gut's mucosa and brain [13]. The probiotics are considered to play a role in transforming short-chain fatty acids from the undigested carbohydrates, which in turn have their influence on intestinal handling of its contents by acting as nutrients for the enteric cells [14].
Lactobacillus plantarum 299v (L. plantarum 299v) is the most widely studied strain in IBS patients. It is resistant to the actions of intestinal acids and bile, colonizes the human colonic mucosa and is nonpathogenic [15]. While adhering to the colonic mucosal layer it reduces the inflammation by its antimicrobial activity against many potential pathogens importantly gram-negative bacteria which contains endotoxins [16]. The synthesis and secretion of the interleukin-10 from the macrophages and the T-cells of the inflamed colon are increased by the L.Plantarum which has got beneficial immunomodulatory activity [17]. The lowering of the colonic pH by small chain fatty acids helps in controlling the growth of the microbes in the gut, L.Plantarum increases the concentration of carboxylic, acetic and propionic acids in the feces [16].
The results from the previous international trails were very encouraging regarding the safety and efficacy of the probiotics use in IBS. The present study aimed to assess efficacy L. Plantarum 299v in the local population suffering from IBS with improved study design and larger sample size.

Study population
The patients were recruited through the outpatient clinic of the Gastroenterology and Hepatology 4 Department of the Lady Reading Hospital, Peshawar. All the adult patients of either gender fulfilling the Rome III criteria for IBS, willing to participate and committed for follow up throughout the study period were enrolled in the study. The patients having a history of major abdominal surgery, organic intestinal disease or chronic infectious diseases like HIV or tuberculosis were excluded from the study.
The pregnant and the female breastfeeding their babies or anyone with the current use of antibiotics were also not included in the study population.

Study Design
This is a double blind placebo-controlled study, conducted for six (6) months. Approval of the study was taken from the Institutional Research and Ethics Board reference number 31/IREB/PGMI/LRH.
After fulfilling the inclusion criteria for the study and signing the informed consent form the randomization was started by the lottery method and then patients were alternatively allotted to either of the two groups. All the patients received probiotics (L. Plantarum 299v) or placebo for four (4) weeks and had three follow up visits after the baseline investigation. The baseline and the first follow up after two weeks of the treatment were face to face. The second follow up, at the end of the treatment and the last one which was after four (4) weeks of finishing the treatment, were telephonic.

Study product:
The study drug and placebo contained 5x1010 cfu of L.Plantarum 299v and micro-crystalline cellulose powder respectively, both packed identically by the manufacturers (Genetex Pharma PVT Limited).
The study drug was labeled A and the placebo as B, that was disclosed at the end of the study.

Re-assessments and compiling results
Two endpoint assessments were noted and interpreted. Daily frequency of abdominal pain was considered as a primary endpoint. The secondary endpoints were improvement in the severity of abdominal pain, the severity of the bloating and feeling of partial rectal emptying. Both the primary and the secondary endpoints were gauged on the visual analog scale from zero (0) to ten (10), 0 being normal and 10 being very severe. In each visit, the patients were examined thoroughly and all the parameters were noted along with the medication compliance. The patients were advised to avoid any change in their dietary habits and intake of medicine regularly in the study period.

Statistical Analysis
The data was collected and SPSS version 19 I.L Chicago was used to analyze the data. One way ANOVA was used to analyze the data.

Results
One hundred and ninety patients were assessed for eligibility 46 among them were excluded from the study and twenty-four declined to participate in the study (Figure 1). One hundred and twenty participants meet the criteria to participate in the study with sixty participants in each group. In the follow up period five participants in L.Plantarum group and seven in the placebo group discontinued the study. The mean age of participants in the L.Plantarum group was 37.53 ± 9.02 and the placebo group was 34.40 ±11.23 (p-value = 0.652). The male to female ratio in the L.Plantarum group was 34: 21 and it was 29:24 in the placebo group (p-value = 0.412). In both the groups the majority of the patients were having mixed-type IBS with no statistically significant difference were observed among the groups. Table 1.
Our primary end-point was the frequency of abdominal pain per day. Although mean frequency was decreased from baseline at the end of therapy, there was no statistically significant difference in the two groups (2.7 vs 3.4, p-value = 0.744). There was no difference in the frequency of abdominal pain in the two groups in any follow up assessment (Figure 2).
In the endpoint assessment for the abdominal pain between the two groups no statistically significant difference was observed. The mean scores for the severity of bloating were not significantly improved among the groups from the baseline in the entire study period. A complaint of feeling incomplete rectal emptying was improved from baseline to secondary endpoint with no statistically significant difference between L.Plantarum and the placebo group ( 3.6 vs 4.1, p-value = 0.211).

Discussion
In this study, we included all subtypes of IBS fulfilling the Rome III criteria. Although most trials have been done in IBS-D regarding probiotics microbiological studies indicate that changes in microbiota occur in all IBS subtypes. Therefore, all patients meeting Rome III criteria were included in the study.
In the present study, most of the patients were male as compared to other trials were approximately 6 2/3 of the population was female. The reason for male predominant complain of IBS as compared to females unlikely reported in European countries; that IBS is less prevalent in females as compared to males in Asian countries [18]. In our study, there was no significant difference in treatment and placebo group in relieving IBS symptoms. This is in contrast to a recent Indian study, a population that shows the same demographics as Pakistan [6,19]. However, this may be due to different eating behavior in India where a high percentage were pure vegetarians and half the population was consuming yogurt daily. This might have confounded the results as in that study relief in abdominal pain was more significant in the vegetarian group as compared to non-vegetarians. Furthermore, yogurt is also rich in probiotics other than L. Bacillus which may be responsible for better results. This is in contrast to our population which is not strictly vegetarian. The result of the study is suggestive of partial interaction of the luminal content and the strain with an enhancement of the other probiotic strain.
In a study done by Nobaek S et al administration of L. plantarum with known probiotic properties decreased pain and flatulence in patients with IBS [5]. The study is a little biased because the intake of the fermented products was 59% in L. Plantarum group as compared to the placebo group (i.e 73%), which may be the source of Probiotic strain other than L bacillus. Moreover, it is ambiguous in that medications for IBS like fiber supplementation and antispasmodics were controlled or not. The information about bloating among L.Plantarum group and placebo control group in 12-month follow up assessment is also not reported [5,20].
Treatment of colonic fermentation in untreated IBS patients by L bacillus found no significant difference between the control and L. Plantarum group. They assessed symptoms daily by validated composite score and 24-hour indirect calorimetry for fermentation. Breath hydrogen was determined three hours after 20ml lactulose intake. There was no significant difference between the median symptom score (8 vs 8.5), median maximum rates of gas production (0.92 ml/min vs 0.55ml/min) and the median hydrogen production (208.2 ml/24 hr vs 189.7 ml/24hr in L .bacillus group compared to the placebo group [19,21]. The largest RCT in this regard was done by Stevenson C Et al in Cape Town South Africa which 7 included 81 patients. These patients were treated with either L. plantarum 299 v at a dosage of 5 x 109 cfu per capsule for 12 weeks or placebo. No significant difference in abdominal pain relief was observed among the L.Plantarum and placebo group and control group. Similarly, no significant difference in QoL-IBS scores between the groups were observed [19,22]. However significant improvement in abdominal pain scores during the study was observed with an average of 251.55 to 197.90 (P < 0.0001) indicating a large placebo effect. They responded well to both probiotic therapy and placebo which is also the case in our study. Our study was similar in design to this study with an even larger number of patients thus validating the results of the aforementioned study. There is high degree variability in clinical outcomes in many IBS clinical trials. The cause of such a placebo effect could be due to complex pathophysiology of IBS with many mutually interacting features.. Furthermore, patients with IBS are usually anxious to be treated and may respond to any alteration in therapy, even if that alteration is a placebo.

Conclusion
This randomized trial failed to show any significant improvement in the IBS symptoms by the use of L.
Plantarum as compared to the placebo.

Declarations
Ethics approval and consent to participate: The study was conducted according to Helsinki deceleration and approved by the Institutional Research and Ethics Board of Lady Reading Hospital Peshawar (Ref.No. 31/IREB/PGMI/LRH). Written informed consent was obtained from all study subjects who agreed to participate.

Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests.

Funding
This is a self-supported study. No funding was obtained for this study.