Crohn’s disease (CD) together with ulcerative colitis belongs to the group of disorders known as inflammatory bowel diseases (IBD). The etiology of Crohn’s disease still remains not fully explained, although the changes in the composition and distribution of intestinal microbiome seem to play a crucial role in the development and persistence of inflammation in the gastrointestinal tract (Kostic et al. 2014; Scarpellini et al. 2015). It is still under debate if the changes in the microbiome in IBD are a cause or a consequence of inflammation.
Pediatric-onset CD is on the increase worldwide (Benchimol et al. 2017; Ng et al. 2017). Many young patients present with an extensive and aggressive course of the disease, which is a real therapeutic challenge. Treatment of CD is a complex, multistage process and depends on the type and clinical activity of the illness. ECCO/ESPGHAN guidelines recommend the usage of exclusive enteral nutrition (EEN) as a first-line therapy to induce remission in pediatric patients with mild to moderate CD (Ruemmele et al. 2014). EEN used as induction therapy should last from six to eight weeks and is usually based on standard, liquid, polymeric formulas. During this period, any other types of food are withdrawn and patients receive liquid diet orally or through a nasogastric tube, which covers full caloric and nutritional demand adjusted to the patient’s requirements. Maintenance of remission can be achieved with thiopurines or methotrexate. This therapeutic approach is called conventional therapy.
The biologic therapy with anti-tumor necrosis factor alpha (anti-TNFα) is recommended for treatment of patients with moderate to severe CD who didn’t respond to conventional treatment (with thiopurines or methotrexate). Infliximab (IFX) is the biologic agent used most often as a first-line treatment in the pediatric population. Induction doses of 5 mg/kg are given intravenously in 0, 2 and 6 weeks mode, followed by maintenance infusions every eight weeks (Hyams et al. 2007).
The majority of IBD therapies are focused on controlling inflammation, but the question of how or whether the clinical status of patients is related to the microbiome remains unanswered. The human gastrointestinal tract contains a wide range of archaea, prokaryota, eukaryota and viruses.
As mentioned above, studies are available in the literature that concern changes in the bacterial flora of the human gastrointestinal tract in the course of IBD; however, there are few articles describing the study of anti-
Patients aged 2 to 18 years diagnosed with CD according to the revised Porto criteria (Levine et al. 2014) were enrolled into two study groups.
The study protocol was approved by Jagiellonian University Ethics Committee – the decision no. 122.6120.68.2015. The informed consent was signed by patients’ parents or legal guardians and by patients themselves if above 16 years of age.
Group 1 consisted of newly diagnosed children, who received EEN for the induction of remission. In this group, we collected two stool samples: the first one (N1) before any therapeutic intervention and the second (N2) 2 to 4 weeks after completing EEN. In a group 2, there were CD patients who failed to respond or stopped responding to conventional maintenance treatment (with thiopurines or methotrexate) and therefore were qualified for biologic therapy. Stool samples were collected prior to the first dose of IFX (Remsima®, Celltrion Healthcare, Incheon, Korea) (B1) and then 4 weeks after the 3rd induction dose (B2).
The exclusion criteria comprised the following: 1) age of patient below two years old or above 18 years of age; 2) treatment with antibiotics (including antimycotic antibiotics) and probiotics during the period of 3 months before collecting the stool sample; 3) confirmed infections of the gastrointestinal tract; 4) any active neoplastic diseases (particularly of the gastrointestinal tract); 5) confirmed immunodeficiency.
The control group consisted of healthy children who didn’t meet the exclusion criteria. In this group, we collected one stool sample.
The stool samples were delivered to the Chair of Microbiology of the Jagiellonian University Medical College in deep-freeze conditions (–70°C).
A RT-PCR standard curve by plotting the threshold cycle (Cq) versus the number of
A total of 61 patients were enrolled in this study. Table I contains the baseline patient characteristics. The control group consisted of eight girls and nine boys, aged on average 140.76 months (± 34.58). Both therapeutic interventions resulted in a statistically significant decrease in disease activity assessed according to PCDAI. In group 2, the mean PCDAI was 47.5 points (ranged from 5 to 60 points) before induction therapy and decreased to a mean of 9.04 (ranged from 0 to 20) points (
Baseline patient characteristics.
Characteristics | Biologic therapy – IFX (n = 13) | EEN (n = 48) | Control group (n = 17) |
---|---|---|---|
Male:Female, n (%) | 7 (54%):6 (46%) | 29 (60%):19 (40%) | 9 (53%):8 (47%) |
Age at diagnosis, months; mean (± SD) | 137 (± 48.15) | 160.27 (± 37.11) | N/A |
Age at initial treatment, months; mean (± SD) | 157.15 (± 45.16) | 160.27 (± 37.11) | N/A |
Weight, kg; mean (± SD) | 41.97 (± 16.3) | 40.93 (± 14.05) | 42.8 (± 17.2) |
Height, cm; mean (± SD) | 149.95 (± 20.31) | 155.3 (± 19.1) | 148.7 (± 18.8) |
BMI, kg/m2; mean (± SD) | 17.89 (± 3.62) | 16.4 (± 2.92) | 18.3 (± 3.8) |
PCDAI-1; mean (± SD) | 47.5 (± 16.43) | 32.03 (± 15.01) | N/A |
PCDAI-2; mean (± SD) | 9.04 (± 6.5) | 5.93 (± 11.36) | N/A |
EEN – exclusive enteral nutrition; N/A – not applicable; PCDAI (Pediatric Crohn’s Disease Activity Index):
1 – prior to therapeutic intervention, 2 – after therapeutic intervention
The DNA sequences isolated from all 139 fecal samples were analyzed using qPCR. The presence of
Quantitative assessment of fungi of the genus
a – significant differences between children with CD and the control group; b – significant differences between children with CD before and after biologic treatment.
The numbers of
The total distribution of
We didn’t find any correlation between the numbers of
The increasing occurrence of Crohn’s disease and the decreasing age of patients stimulate researchers to find out the causes behind this illness (Benchimol et al. 2017; Ng et al. 2017). Although, until now, it has not been possible to associate a particular microorganism with CD etiology, microbial participation is still considered crucial, besides genetic and immunological disorders, for induction or intensification of inflammation in the gastrointestinal tract (Gosiewski et al. 2012; Wright et al. 2015). However, there is still insufficient knowledge concerning the role of fungi in the course of CD, as well as the impact of the treatment on gastrointestinal colonization with fungi of the genus
Research by Sokol et al. (2017) demonstrated a significant increase in the
Zwolinska-Wcislo et al. (2009) demonstrated, in an animal model, similar observations as regards the effectiveness of treatment of patients with inflammatory bowel diseases using antifungal agents and a significant clinical improvement following treatment with fluconazole.
Our research demonstrated that the IFX therapy translated into a statistically significant reduction in the number of fungi of the genus
A large number of
This study provides additional information to the multifactorial nature of CD and may contribute to the modification of therapeutic approach.