Objectives

Objectives

Treat a subset of Crohn’s patients with Rifabutin and Macrolide Antibiotic Therapy who have been identified as being serologically positive for MAP through the use of the p35 and p36 recombinant clones.

Monitor these patients over the course of their treatment 

Purpose

A) Evaluate the overall response of serologically MAP positive Crohn’s patients to RMAT

B) Provide insights into the use of p35 and p36 serologic markers for the stratification and identification of candidates for RMAT

Approach

Analyze the sera of confirmed CD patients for antibody to the antigens p35 and p36 recombinant clones through the University of Central Florida. Patients selected for treatment with RMAT were those CD patients testing positive for antibodies to the p35 and/or p36 MAP recombinant clones and demonstrating active, refractory CD. These patients were monitored for duration of at least six months to two years.

Methods

Patient Selection

MAP Diagnostic Techniques: Each patient provided a 2 ml aliquot of sera that was sent and analyzed at the University of Central Florida against two recombinant clones of MAP. The recombinant clones designated p35 and p36 were screened by immunoblot against rabbit hyperimmune anti-MAP antibodies. Inclusion criteria required each patient to be identified as positive for one or more of these recombinant clones.

Inclusion Criteria: Patients selected demonstrated active, refractory CD with disease involvement present in the duodenum, colon or small bowel.

Exclusion Criteria: Patients were excluded from the study if their age <18 years, if they were pregnant or breast feeding, exhibited hepatic or renal disease, or demonstrated intestinal stenosis with clear obstructive symptoms.

Procedures

Treatment regimen:

RMAT treatment included 150 mg bid Rifabutin and 250 mg bid Clarithromycin 

Patients were encouraged to take nutritional acidophilus supplements (200 mgm po bid of a probiotic containing equal amounts of Lactobacillus acidophilus and Lactobacillus rhamnosus) 

Patients remained on their standard Crohn’s medications and were gradually taken off these medications in conjunction with the patient’s demonstrated response to RMAT. 

Study design:

Written informed consent was obtained from all patients who were eligible for treatment. 

This study was an open clinical trial. 

Photographic documentation of CD was established in patients who necessitated a colonoscopy before treatment. 

These patients were reevaluated endoscopically at the end of treatment to document any evidence of healing. 

All patients were treated and followed for at least 6 months. Each patient was seen routinely at times determined by clinical need and assessed for general response to treatment. 

At each visit, a Comprehensive Metabolic Profile, Hemogram w/ Platelet & Differential was obtained to monitor adverse drug induced reactions such as neutropenia and hepatic abnormalities. 

Blood was also sera banked throughout the course of treatment for future quantitative analysis of Mycobacterial antibody titer levels. 

Patients who were initially endoscoped, stool specimens and biopsy specimens were collected for future MAP analysis. 

Patients completed CDAI and Quality of Life evaluations (including IBDQ). 

At the end of the observational period, patients were assessed and categorized into three groups: Responders, Partial Responders and Non-responders. 

Responder: Patient off all standard CD medications (Corticosteroids and Immunosuppresants) and noticing marked improvement in their CD.

Partial Responder: Patient using one or more standard CD medications (Corticosteroids and Immunosuppresants) but noticing marked improvement in their refractory CD.

Non-responder: Patient who remained on some or all of their standard CD medications (Corticosteroids and Immunosuppresants) and demonstrated no marked improvement in their CD.

Stool Culture Analysis:

Stool specimens were obtained during colonoscopy procedures. These stool specimens were cultured and analyzed at the Florida Hospital Department of Microbiology in Orlando, Florida. Cultures positive for mycobacterial growth were further identified through nucleic acid hybridization with an AccuProbe (Gen-Probe) test kit.

Identification and Culturing of MAP in Resected Tissue and Biopsy Specimens:

Biopsy and tissue specimens were collected from patients before RMAT. The specimens were sent directly to the University of Central Florida. They were processed and cultured in both 12B* Bactec bottles and Mycobacterial Growth Indicator Tubes (MGIT) with OADC enrichment, Mycobactin J and PANTA antibiotic mixture for up to one year. Bacterial detection and identification was done through Acid fast staining, mycobactin dependency, PCR analysis using two IS900-derived oligonucleotides and hybridization with an internal probe.