Inactive IBD Research Articles

Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease

Objective: Our purpose was to study the molecular basis of infliximab (IFX) effect on colon mucosa in a colitis model and to identify new biomarkers of mucosal healing.Methods: Healthy rats and rats which were subjected to experimental colitis induced by dextran sulfate sodium, with or without IFX treatment (in the short- and long-term), were studied along with forty-seven IBD patients. Colon mucosal integrity by periodic acid Schiff (PAS) staining, intestinal damage by immunohistochemistry (proliferating cell nuclear antigen, β-catenin, E-cadherin, phosphotyrosine, p-p38, allograft inflammatory factor-1 (AIF-1) and colonic mucosal apoptosis by TUNEL staining were evaluated in rats while serum and colon AIF-1 levels were determined in IBD patients.Results: In rats with colitis, IFX reestablished the epithelial barrier integrity, recovered mucus production and decreased colon inflammation, as verified by reduced serum and colon AIF-1 levels; colon and serum AIF-1 levels were also lower in inactive IBD patients compare to active ones. P38 activation after IFX treatment tended to induce differentiation/proliferation of epithelial cells along the colonic crypt-villous axis.Conclusions: These findings support AIF-1 as a new biomarker of mucosal healing in experimental colitis and suggest that p38 activation is involved in the mucosal healing intracellular mechanism induced by IFX treatment.

Chemotherapy Tolerance and Oncologic Outcomes in Patients With Colorectal Cancer With and Without Inflammatory Bowel Disease

BackgroundInflammatory bowel disease (IBD), comprising Crohn disease and ulcerative colitis, is a risk factor for colorectal cancer (CRC). Chemotherapy toxicity may exacerbate IBD symptoms and vice versa, but data are limited. We evaluated chemotherapy tolerance and oncologic outcomes in patients with CRC with and without IBD. Patients and MethodsMedical records of patients with CRC with and without IBD treated between 2008 and 2013 were reviewed. Where possible, patients were matched by age, sex, stage, and diagnosis year. Chemotherapy tolerance and survival outcomes were compared between patients with IBD and without IBD. ResultsA total of 158 subjects with CRC were included: 80 patients had IBD and 78 matched control patients did not have IBD. Between cases and controls, there were no significant differences in demographic data, stage of CRC, and cancer treatments, with equivalent numbers of patients receiving surgery, radiation, and chemotherapy. Patients with IBD experienced more CRC treatment alterations than those without IBD (74% vs. 44%, P = .03), largely due to a higher frequency of treatment delays among patients with IBD. Differences in stage-specific 5-year overall survival (OS) and recurrence-free survival (RFS) in patients with and without IBD were not significant, except for stage IV patients with IBD who had significantly shorter OS than those without IBD. Patients with histologically active IBD did not require more chemotherapy alterations than patients with inactive IBD. ConclusionIn this series, patients with CRC with IBD experienced more treatment alterations (mostly delays) than those without IBD. Patients with stage IV CRC with IBD had shorter survival than patients without IBD.

Mo1811 Clinical Remission Induced by Exclusive Enteral Nutrition (EEN) in Pediatric Crohn's Disease Is Associated With Microbiome Metabolic Changes Toward Increased Xenobiotic Biodegradation and Metabolism

esis of IBD, both Crohn's disease (CD) and ulcerative colitis (UC), is not well known. Most studies of human gut microbiota rely on the non-invasive collection of stool samples. However, the analysis of the fecal microbiota may not reflect the role of the mucosa-associated microbes which live in close proximity to the intestinal epithelium and that are in contact with the cells of the innate immune system directly involved in the inflammatory response. The aim of this study was to investigate the genotypes of Bacteroidetes microbiota from colon biopsies of IBD patients and to determine their relationship with the endoscopic activity of the disease. Methods: A prospective case-control study was designed. Colon biopsies from consecutive IBD patients and healthy controls (HC) (healthy subjects undergoing colonoscopy for colon cancer screening and having a healthy colon mucosa) were included. Inactive UC was defined as a Mayo endoscopic subscore of 0. Inactive CD was defined as a SES-CD score ≤ 2. Microbiota was characterized by using a restriction fragment length polymorphism (RFLP) analysis on PCR products targeting the 16SrRNA genes of Bacteroidetes digested with HinfI, PciI, DpnII and AciI. Results are shown as percentages. Results: 29 consecutive IBD patients (22 UC and 7 CD) and 15 HC were included. 8 patients presented with inactive UC (iUC) and 14 with active UC (aUC). All 7 CD patients presented with endoscopic activity (aCD). A total of 7 genotypes of Bacteroidetes called N1 and C1-C6 (of which N1 genotype is probably a strain of Bacteroides dorei and C1, and maybe C2, strains of B. vulgatus) were detected in all the biopsy samples analyzed. The number of genotypes present in biopsies from IBD patients was higher than that in HC, in whom only the N1 (66%) and C1 (34%) genotypes appear. While the presence of N1 genotypes was relatively constant in patients with active and inactive IBD, the percentage of C1 genotype in patients with aUC and aCD were very low (<5%) compared to controls. The C4 genotype never appeared in control samples and it was present in only 2% of iUC biopsies, whereas it was present in 15% and 16% of patients with aUC and aCD respectively. The C2, C5 y C6 genotypes appeared sporadically in biopsies of IBD with percentages of 1% in iUC (C2), 3% in aUC and 1% in aCD (C5), and 1% in iUC and aCD (C6), but never in HC. Conclusions: Bacteroidetes genotypes in colon biopsies differ between IBD patients and HC. These genotypes, and especially the C4 genotype, are highly specific for active IBD and further studies on their role on IBD pathogenesis are required.

The relationship between coping, health competence and patient participation among patients with inactive inflammatory bowel disease

BackgroundCoping is an integral part of adjustment for patients with Inflammatory Bowel Disease but has not been well described in the literature. This study explored the relationship between coping, perceived health competence, patient preference for involvement in their treatment, depression and quality of life, particularly among patients with inactive disease (in remission). MethodsSubjects (n=70) with active and inactive IBD completed questionnaires, including the Inflammatory Bowel Disease Quality of Life Questionnaire, Beck Depression Inventory, Perceived Health Competence Scale and the Coping Inventory for Stressful Situations. The Harvey Bradshaw Index measured disease activity. ResultsPatients with inactive IBD demonstrated significantly more interest in participating in their treatment (p<.05), more perceived health competence (p=.001), less depressive symptoms (p<.001), more task oriented coping (p=.02), and better quality of life than those with active disease. Only Task Oriented Coping was significantly negatively associated with the number of flares among inactive patients (p<.001). Patient preference for participation in treatment was inversely associated with Avoidance (p=.005), Distraction (p=.008), and Social Diversion (p=.008) coping among inactive patients. ConclusionAmong patients in remission, those who expressed a greater interest in treatment participation were also less likely to practice maladaptive coping. Our data demonstrate that a more active coping style may be associated with improved health outcome. Compared to patients with active disease, patients in remission are more likely to employ task oriented coping, demonstrate a higher interest in treatment participation, report greater perceived control of their health, and exhibit less depression symptoms. Our findings may increase awareness of the importance of identifying coping strategies for IBD patients, including those in remission.

Dynamics of vitamin D in patients with mild or inactive inflammatory bowel disease and their families

Background25(OH) vitamin D levels may be low in patients with moderately or severely active inflammatory bowel diseases (IBD: Crohn’s disease and Idiopathic Ulcerative Colitis) but this is less clear in patients with mild or inactive IBD. Furthermore there is limited information of any family influence on 25(OH) vitamin D levels in IBD. As a possible risk factor we hypothesize that vitamin D levels may also be low in families of IBD patients.ObjectivesTo evaluate 25[OH] vitamin D levels in patients with IBD in remission or with mild activity. A second objective is to evaluate whether there are relationships within IBD family units of 25[OH] vitamin D and what are the influences associated with these levels.MethodsParticipants underwent medical history, physical examination and a 114 item diet questionnaire. Serum 25[OH] vitamin D was measured, using a radioimmunoassay kit, (replete ≥ 75, insufficient 50–74, deficient < 25–50, or severely deficient < 25 nmol/L). Associations between 25[OH] vitamin D and twenty variables were evaluated using univariate regression. Multivariable analysis was also applied and intrafamilial dynamics were assessed.Results55 patients and 48 controls with their respective families participated (N206). 25[OH] vitamin D levels between patients and controls were similar (71.2 ± 32.8 vs. 68.3 ±26.2 nmol/L). Vitamin D supplements significantly increased intake but correlation with serum 25[OH] vitamin D was significant only during non sunny months among patients. Within family units, patients’ families had mean replete levels (82.3 ± 34.2 nmol/L) and a modest correlation emerged during sunny months between patients and family (r2 =0.209 p = 0.032). These relationships were less robust and non significant in controls and their families.ConclusionsIn patients with mild or inactive IBD 25[OH] vitamin D levels are less than ideal but are similar to controls. Taken together collectively, the results of this study suggest that patient family dynamics may be different in IBD units from that in control family units. However contrary to the hypothesis, intra familial vitamin D dynamics do not pose additional risks for development of IBD.

Tu1161 Adherence to Methotrexate (MTX) in Inflammatory Bowel Disease (IBD) Patients: An Observational Multicenter Study

of calcium homeostasis and inflammatory parameters in subjects with either active or inactive IBD. In the linear regression model, calprotectin was the strongest predictor of the disease activity, as expected [beta coefficient 0.75 (P< 0.001) in men and 0.42 (P< 0.05) in women, respectively], when adjusted for age and TNF-alpha inhibitor treatment. Ionized calcium and serum PTH did not demonstrate any significant associations with disease activity in the same regression model. Conclusions: IBD patients have an altered calcium homeostasis and ionized calcium levels are significantly lower in the active disease state. That may indicate that IBD patients with frequent relapses may suffer from underestimated calcium losses and could be at higher risk of osteoporosis.

263 Characterization of the Tissue-Associated Microbiome in Active and Inactive IBD

disease behavior and location between these 8 patients and the others. Anti-glycan and anti-Candida antibodies status were stable during active versus quiescent disease and anti-glycan antibodies levels did not change significantly. Conclusions: CD patients are frequently colonized by C. albicans and are more heavily colonized in active than in quiescent phase of the disease while the antiglycan antibodies status is not influenced by disease activity. Colonization by C. albicans may participate in triggering intestinal inflammation in CD.

P698 Characterization of the tissue-associated microbiome in active and inactive IBD

P698 Characterization of the tissue-associated microbiome in active and inactive IBD

PDCD4/miR-21 dysregulation in inflammatory bowel disease-associated carcinogenesis

Inflammatory bowel diseases (IBDs; both ulcerative colitis [UC] and Crohn's colitis [CC]) are well-established predisposing pathological conditions for colorectal cancer (CRC) development. In IBDs, both the endoscopy and the histology assessment of CRC precursors (i.e., dysplasia, also defined as intraepithelial neoplasia) are associated with low interobserver consistency, and no reliable dysplasia-specific biomarker is available. The programmed cell death 4 (PDCD4) tumor suppressor gene is involved in sporadic colorectal oncogenesis, but scanty information is available on its involvement in IBD-associated colorectal oncogenesis. One hundred twenty tissue samples representative of active and inactive IBD and of flat dysplasia were obtained from 30 cases of UC and 30 of CC who undergone colectomy. Twenty additional biopsy samples obtained from patients with irritable bowel syndrome acted as normal controls. PDCD4 expression was assessed by immunohistochemistry; the expression of miR-21 (a major PDCD4 regulator) was investigated by quantitative real-time PCR and in situ hybridization in different series of a hundred samples. Tissue specimens from both controls and inactive IBD consistently featured strong PDCD4 nuclear immunostain; conversely, lower PDCD4 nuclear expression was featured by both active IBD and IBD-associated dysplastic lesions. Significant PDCD4 down-regulation distinguished IBD-associated dysplasia (p < 0.001) versus active IBD. In both active IBD and dysplasia, PDCD4 down-regulation was significantly associated with miR-21 up-regulation. PDCD4 nuclear down-regulation (which parallels miR-21 up-regulation) is involved in the molecular pathway of IBD-associated carcinogenesis. PDCD4 nuclear expression may be usefully applied as ancillary maker in the histological assessment of IBD-associated dysplastic lesions.

Activation of REG family proteins in colitis

Aims. To do a genome-wide gene expression study of active and inactive ulcerative colitis and Crohn's disease (inflammatory bowel disease – IBD) and examine the most differentially expressed genes. As the study showed an extreme upregulation of all regenerating islet-derived genes (REG proteins) in active IBD, we further studied the expression of REGs on protein level in active and inactive IBD, as well as in non-IBD (pseudomembranous) colitis. Methods. Microarray analysis was done on a total of 100 pinch biopsy samples from healthy controls and patients with Crohn's disease or ulcerative colitis. Tissue samples from IBD and pseudomembranous colitis were examined with routine histology and immunohistochemical analysis for REGIα, REGIV, DEFA6, and serotonin. Results. REG mRNAs were up to 83 times overexpressed in diseased mucosa compared with mucosa from healthy individuals. REGIα and REGIV were overexpressed at immunohistochemistry and located to different mucosal cell types. REGIα was expressed in basal half of crypts, REGIV in mid and outer parts of crypts and in surface epithelium and seems to be stored in, and secreted from, goblets. Pseudomembranous colitis samples showed similar staining patterns, and some IBD samples stained REG positive without inflammation on routine histology. Conclusions. All REG family mRNAs are upregulated in IBD. REGIα and REGIV have different cellular localization, possibly reflecting different biological functions. REG protein expression also in pseudomembranous colitis shows that REG family proteins are regulated in inflammatory injury and repair, not specifically for IBD as previously thought.

A Prospective Population-Based Study of Symptoms and Life Stresses in Active and Inactive IBD

A Prospective Population-Based Study of Symptoms and Life Stresses in Active and Inactive IBD

W1295 Azathioprine (AZA) is Associated With Less Histological Inflammation of the Colon in Clinically Inactive IBD

W1295 Azathioprine (AZA) is Associated With Less Histological Inflammation of the Colon in Clinically Inactive IBD

Similarity of Mast Cell Density and Distribution in Terminal Ileum and Colon of IBD and IBS Patients and Normal Controls

Purpose: Background: In vitro and animal studies showed that Mast Cells (MC) are critical element of inflammatory cascade. However, the role of MC in IBD and IBS is still controversial. One possible reason for this controversy is lack of standard method to assess the size of gastrointestinal mucosal MC population in various region of the gastrointestinal tract. Hypoythesis: The number of MC in various part of GI tract is different among subjects with IBD, IBS in comparison to healthy controls. Aim: In this study we tried to evaluate the number of mucosal MC in subjects with IBD, IBS and compare the data with healthy controls. Methods: 24 subjects (8 Controls, 5 inactive IBD, 11 IBS) were studied. Total of 55 biopsy samples were taken during colonoscopy from sigmoid, ascending colon and ileum. Biopsies were stained using immunohistochemical techniques using monoclonal antibody against CD-117. A total of 9 random distinct fields, which were completely covered by transversely cut glands, were selected for MC counting and the mean value was considered as the number of MC per HPF. The counting was done by two independent observer. The number of MC was compared using ANOVA test. Results: There was on average 17 MC per HPF in the ileum, 16 in the ascending colon and 14 in the sigmoid colon. The number of MC in right colon was 20 in IBS-c while this number was 13 in healthy controls and 12 in subjects with IBS-d. However, this difference did not reach statistical significance (p=0.06). The number of MC in other part of the colon and ileum was not significantly different among the subjects group. The inter-observer variability was also not different among the groups. Conclusion: There was no significant difference in the number of MC in various region of lower GI tract in subjects with IBD, IBS compared to healthy counterparts. This may suggest that MC quantity may not be a determining factor in evaluating the role of MC in these disorders. Further studies that assess the function of MC may be more appropriate method of investigating the role of MC in various region of the GI tract in subjects with IBD or IBS.

Altered status of antioxidant vitamins and fatty acids in patients with inactive inflammatory bowel disease

Data regarding the nutritional status, antioxidant compounds and plasma fatty acid (FA) composition in inactive IBD are conflicting. We compared plasma levels of antioxidants and FA of patients with inactive IBD with active IBD and controls. Plasma levels of vitamin C, vitamin E, carotenoids, saturated, monounsaturated and polyunsaturated FA, inflammatory markers and nutritional status were determined after an overnight fast in 132 patients with quiescent IBD (40.6+/-13.2 years, 87F/45M), 35 patients with active disease (37.9+/-12.1 years, 25F/10M) and 45 age- and BMI-matched healthy controls (38.1+/-10.5 years, 39F/6M). Results are expressed as mean+/-SD or median [25th percentile;75th percentile]. Body mass index (BMI) was normal in inactive (23.9+/-4.7 kg/m(2)), active IBD (22.7+/-4.2 kg/m(2)) and controls (22.3+/-1.9 kg/m(2)). Compared with controls patients with quiescent IBD showed significantly decreased plasma levels of carotenoids (1.85 [1.37;2.56] vs 1.39 [0.88;1.87] micromol/L) and vitamin C (62.3 [48.7;75.0] vs 51.0 [36.4;77.6] micromol/L), increased levels of saturated FA (3879 [3380;4420] vs 3410 [3142;3989] micromol/L) and monounsaturated FA (2578 [2258;3089] vs 2044 [1836;2434] micromol/L) and similar levels of vitamin E and polyunsaturated FA. Results in active disease were similar to inactive disease. This study shows that antioxidant status and FA profile in a larger population of IBD patients are disturbed independently from disease activity and despite normal overall nutritional status.

Initial Application of the ShapeLock™ Endoscopic Guide for Small Bowel Enteroscopy

Background: Currently small bowel enteroscopy (SBE) is limited by loss of vector forces and curling of scope within the stomach. Attempts to overcome these limitations have led to widespread application of capsule endoscopy (CE), development of double-balloon enteroscopy (DBE), and use of conventional overtubes. CE is limited by inability to undertake endotherapy. DBE requires a significant capital expenditure. Conventional overtubes prove difficult to get through the pylorus and have been associated with inadvertent strip mucosectomy and even procedural pancreatitis from damage to the papilla. A shape-locking guide, developed for difficult colonoscopy, (USGI Medical, San Clemente, CA) was adapted for SBE and utilized clinically in patients with small bowel disorders in whom no diagnosis/treatment was noted with capsule and/or conventional SBE. Materials and Methods: A 54 Fr. ShapeLock™ (SL) device, 80 or 100 cm was used investigationally in conjunction with an Olympus pediatric colonoscope (2 patients) or SB enteroscope (6 patients), respectively. Conscious sedation was achieved with Demerol/Fentanyl and Versed. After initial scope passage into the ligament of Trietz, the SL was passed under fluoroscopic control through the pylorus and locked in place within the C-loop to prevent scope looping. Subsequent scope and SL passage to definable pathology or the limits of the scope length were undertaken using push and pull maneuvers with and without unlocking and advancement of the SL. Results: The SL was placed beyond the pylorus in 5/8 patients aged 32-85 years. Two of the three remaining patients had surgically altered anatomy eliminating the pylorus. The enteroscope/pediatric colonoscope could be advanced into the terminal ileum in 1 patient, mid to distal jejunum in 5 patients, and proximal to mid-jejunum in another two. Bleeding sites were found in 4/6 patients (3 subsequently treated with heater probe), an anastomotic stenosis in 1, and inactive IBD in another. An SL related proximal esophageal abrasion was seen in 3 patients, a non-significant cricopharyngeal tear in 1, and minor pyloric abrasions in 2 patients. An asymptomatic Mallory-Weiss tear was seen in another patient. Conclusions: 1) A ShapeLock™ Endoscopic Guide has been developed that precludes scope looping in the stomach and appears to facilitate SB enteroscopy. 2) Further studies are required to define safety of the ShapeLock™ and its role relative to CE and DBE. Background: Currently small bowel enteroscopy (SBE) is limited by loss of vector forces and curling of scope within the stomach. Attempts to overcome these limitations have led to widespread application of capsule endoscopy (CE), development of double-balloon enteroscopy (DBE), and use of conventional overtubes. CE is limited by inability to undertake endotherapy. DBE requires a significant capital expenditure. Conventional overtubes prove difficult to get through the pylorus and have been associated with inadvertent strip mucosectomy and even procedural pancreatitis from damage to the papilla. A shape-locking guide, developed for difficult colonoscopy, (USGI Medical, San Clemente, CA) was adapted for SBE and utilized clinically in patients with small bowel disorders in whom no diagnosis/treatment was noted with capsule and/or conventional SBE. Materials and Methods: A 54 Fr. ShapeLock™ (SL) device, 80 or 100 cm was used investigationally in conjunction with an Olympus pediatric colonoscope (2 patients) or SB enteroscope (6 patients), respectively. Conscious sedation was achieved with Demerol/Fentanyl and Versed. After initial scope passage into the ligament of Trietz, the SL was passed under fluoroscopic control through the pylorus and locked in place within the C-loop to prevent scope looping. Subsequent scope and SL passage to definable pathology or the limits of the scope length were undertaken using push and pull maneuvers with and without unlocking and advancement of the SL. Results: The SL was placed beyond the pylorus in 5/8 patients aged 32-85 years. Two of the three remaining patients had surgically altered anatomy eliminating the pylorus. The enteroscope/pediatric colonoscope could be advanced into the terminal ileum in 1 patient, mid to distal jejunum in 5 patients, and proximal to mid-jejunum in another two. Bleeding sites were found in 4/6 patients (3 subsequently treated with heater probe), an anastomotic stenosis in 1, and inactive IBD in another. An SL related proximal esophageal abrasion was seen in 3 patients, a non-significant cricopharyngeal tear in 1, and minor pyloric abrasions in 2 patients. An asymptomatic Mallory-Weiss tear was seen in another patient. Conclusions: 1) A ShapeLock™ Endoscopic Guide has been developed that precludes scope looping in the stomach and appears to facilitate SB enteroscopy. 2) Further studies are required to define safety of the ShapeLock™ and its role relative to CE and DBE.

Hypertension prevalence and risk factors in adults in Istanbul

FGD was significantly higher (P 0.0001) in inactive IBD (81.9%; 78.8-85.0) than in Canadian householders (61.7%; 58.964.5). The prevalence of a functional bowel disorder (51.7%), irritable bowel syndrome (20.9%) and functional diarrhea (15.5%) in inactive IBD were significantly greater (P 0.01) than in Canadian householders at 41.6%, 12.1% and 8.5%, respectively. The prevalence of functional anorectal disorders at 53.7% and functional incontinence at 35.8% were also significantly higher (p 0.001) in inactive IBD compared with Canadian householders at 22.6% and 6.9%, respectively. Age and smoking were the strongest predictors of a FGD in inactive IBD while age and anxiety were the strongest predictors of FGD in inactive CD. CONCLUSION: FGDs, primarily bowel and anorectal, are significantly more prevalent in patients with inactive IBD compared to Canadian householders and may be easily confused with active IBD. (partially funded by IOIBD)

The prevalence and correlates of asthma in American Indian adults

FGD was significantly higher (P 0.0001) in inactive IBD (81.9%; 78.8-85.0) than in Canadian householders (61.7%; 58.964.5). The prevalence of a functional bowel disorder (51.7%), irritable bowel syndrome (20.9%) and functional diarrhea (15.5%) in inactive IBD were significantly greater (P 0.01) than in Canadian householders at 41.6%, 12.1% and 8.5%, respectively. The prevalence of functional anorectal disorders at 53.7% and functional incontinence at 35.8% were also significantly higher (p 0.001) in inactive IBD compared with Canadian householders at 22.6% and 6.9%, respectively. Age and smoking were the strongest predictors of a FGD in inactive IBD while age and anxiety were the strongest predictors of FGD in inactive CD. CONCLUSION: FGDs, primarily bowel and anorectal, are significantly more prevalent in patients with inactive IBD compared to Canadian householders and may be easily confused with active IBD. (partially funded by IOIBD)

Pregnancy and nursing in inflammatory bowel disease

The peak age of onset for IBD coincides with the peak age for conception and pregnancy. Women with inactive IBD who become pregnant do not have increased complications compared with age-matched controls. Most medications for IBD are safe in pregnancy. The greatest danger to a normal conception and pregnancy is active disease, not the medicine used to treat it. This article outlines fertility in IBD, the effect of IBD on pregnancy, the effect of pregnancy on IBD, and the medical therapy available.

Biodiversity in the fecal flora of patients with active and inactive IBD: Molecular assessment with temporal temperature gradient gel electrophoresis

Biodiversity in the fecal flora of patients with active and inactive IBD: Molecular assessment with temporal temperature gradient gel electrophoresis

Biodiversity in the fecal flora of patients with active and inactive IBD: Molecular assessment with temporal temperature gradient gel electrophoresis

Biodiversity in the fecal flora of patients with active and inactive IBD: Molecular assessment with temporal temperature gradient gel electrophoresis