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Publications

 
 
 

Publications on Mycobacterium Paratuberculosis and Crohn’s Disease
by Dr. Shafran et al.

Bacteria and Crohn's Disease
Ira Shafran1, M.D., Jonathan W. Decker, R.N., B.S.N.1, 2
Department of Molecular Biology and Microbiology and Center for Diagnostic and Drug Development, University of Central Florida, Orlando, Florida1; and University of Florida College of Nursing, Gainesville, Florida2.
Ameriacn Journal of Gastroenterology (accepted)

Mycobacterium avium subsp. Paratuberculosis as one cause of Crohn's Disease
W. Chamberlain, D.Y. Graham, K. Hulten, H.M.T. El-Zimaity, M.R. Schwartz, S. Naser, I. Shafran, F.A.K. El-Zaatari, Aliment Pharmacol Ther 15:000-000 (2001).
Full text (.pdf)

Use of Short Term Culture for Identification of Mycobacterium avium subsp. Paratuberculosis in Tissue from Crohn’s Disease Patients.
D. Schwartz, I. Shafran, C. Romero, N. Naser, C. Piromalli, J. Biggerstaff, W. Chamberlain and S.A. Naser, Clinical Microbiology and Infection. 6:1-6 (2000).

Isolation of Mycobacterium avium subsp. Paratuberculosis from Breast Milk of Crohn’s Disease Patients.
S. Naser, I. Shafran, D. Schwartz.
American Journal of Gastroenterology. 95(4) 1094-1095 (2000).

Fistula Healing in MAP Positive Crohn’s Disease Patients Following Rifabutin and Macrolide Antibiotic Treatment.
Shafran, L. Kugler, J. Sandoval
(Submitted to DDW: Gastroenterology).

A Comparative Analysis of Anti-Saccharomyces Antibodies (ASCA) and p35 and p36 Recombinant Clones for the Diagnosis of Crohn's Disease (CD)
I. Shafran, C. Piromalli, S. Naser,
Gastroenterology. 118(4): A697 (2000).

Cost Analysis of Antibiotic Treatment in Crohn’s Disease Patients Serologically Positive for Mycobacterium avium ss. paratuberculosis.
I. Shafran, C. Piromalli and J. Davies
Gastroenterology 118(4): A5071 (2000).

Mycobacterium Identification in Stool Cultures of IBD Patients Serologically Positive for Mycobacterium avium ss. paratuberculosis.
I.Shafran M.D, C.Piromalli B.S
Gastroenterology 118(4): A6207 (2000).

Identification of Mycobacterium avium ss. paratuberculosis in Crohn's Tissue.
I.Shafran, C. Piromalli, C.Romero, D.Schwartz and S.Naser
Gastroenterology 118(4): A1767 (2000).

Rifabutin and Macrolide Antibiotic Treatment in Crohn’s Patients Identified Serologically Positive for Mycobacterium avium ss. paratuberculosis.
I. Shafran, C. Piromalli, S. Naser
Gastroenterology 118(4): A4182 (2000).

Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.
Naser SA, Hulten K, Shafran I, Graham DY, El-Zaatari FA
Vet Microbiol 2000 Dec 20;77(3-4):497-504

Comparison of Serolgic Markers for the Diagnosis of Crohn’s Disease.
I.Shafran M.D., C.Piromalli, F.El-Zaatari Ph.D
Am J of Gastroenterol. 94, 2761 (1999).

Serum sickness-like illness in Crohn’s Disease Patients Treated With Antibiotics.
I.Shafran M.D.and C.Piromalli
Am J of Gastroenterol. 94, 2737 (1999).

Treatment of Crohn’s Disease with Rifabutin and Macrolide Antibiotics.
I.Shafran, C.Piromalli
Am J of Gastroenterol 94, 2761 (1999).

Mycobacterium Avium, SS Paratuberculosis, in Crohn’s Disease is an Affirmative.
D. Schwartz, D. Campbell, C. Romero, I. Shafran, S. Naser
presented at the American Society of Microbiology Meeting, 99th meeting, Chicago, May of 1999, poster session.

In Vitro Evaluation of Anti-Tuberculous Drugs Against M. Avium, Subspecies Paratuberculosis, for Treatment of Crohn’s’ Disease.
D. Campbell, D. Schwartz, C. Romero, I. Shafran, S. Naser
presented at the American Society of Microbiology 99th Meeting, May of 1999, poster session.

Endoscopic Healing of Crohn’s Disease After Antibiotic Treatment.
I. Shafran, C. Piromalli, S. Naser
Gastroenterology, A-929 (1999).

Humeral Immune Response of Crohn’s Patients for Mycobacterium Avium, Subspecies Paratuberculosis.
I. Shafran, W. Fenster, C. Piromalli, C. Romero, D. Schwartz, D. Campbell, F. El-Zaatari
Gastroenterology, A-929 (1999).

In Vitro Evaluation of Anti-Tuberculous Drugs Against Mycobacterium Avium Subspecies Paratuberculosis for Treatment of Crohn’s Disease.
S.A. Naser, W. Fenster, C. Romero, D. Schwartz, C. Piromalli, I. Shafran
Gastroenterology, A-783 (1999).

Rapid Culturing and PCR Detection of Mycobacterium Avium, SS. Paratuberculosis, from Crohn’s Disease Tissue.
S. Naser, W. Fenster, C. Romero, D. Schwartz, C. Campbell, C. Piromalli, I. Shafran
Gastroenterology, A-782 (1999).

Mycobacterium avium subsp. Paratuberculosis in Crohn’s Disease Is Serologically Positive
S. Naser, I. Shafran
Clinical and Diagnostic Laboratory Immunology, 6: 282 (1999).

Specific Seroreactivity of Crohn’s Disease Patients Against P 35 and P 36 Antigens of M. Avium Subspecies Paratuberculosis, S.A. Naser, K. Hulten, I. Shafran, D.Y. Graham, F. El-Zaatari, presented at the 1999 6th International Colloquium on Paratuberculosis in Melbourne, Australia.

The Use of Combined Antigens for Specific Diagnosis of Crohn’ Disease.
I. Shafran, W. Fenster, S. Ishak, F. El-Zaatari, S. Naser
presented at the American Society of Microbiology, 98th General Meeting, in Atlanta, Georgia, May of 1998.


Bacteria and Crohn's Disease
Ira Shafran1, M.D., Jonathan W. Decker, R.N., B.S.N.1, 2
Department of Molecular Biology and Microbiology and Center for Diagnostic and Drug Development, University of Central Florida, Orlando, Florida1; and University of Florida College of Nursing, Gainesville, Florida2.

Bacteria and Crohn's Disease
TO THE EDITOR: We wish to comment on the interesting work by Hulten, et. al. (1) in a recent issue of your journal regarding the detection of Mycobacterium avium ss. paratuberculosis (MAP) in the tissue of patients with Crohn's disease (CD).  Their work underscores a need for further investigation to pursue a microbial basis for CD using molecular diagnostic techniques.  Searching for the root cause of the disease will inevitably translate into better and safer treatment options for our patients.

The role of MAP in CD has been debated for the past several decades.   Our work has shown a significant serologic response of CD patients to recombinant cloned antigens as compared with non-IBD controls (2).  We have also documented the organism in cultures from surgically acquired tissue using special media confirmed by PCR (3).  Our open clinical trial has shown the combination of Rifabutin and Clarithromycin effective in achieving remission in 80% of treated patients to date with elimination of corticosteroids, significant reduction of CDAI and improvement of quality of life.  Correlating with clinical response is endoscopic healing and return to normal histology in the majority (75%) of patients in remission at one year.  The benefit of probiotics in the induction and maintenance of remission is being evaluated in our open trial.  Probiotic studies suggest the mechanism of action to be the up-regulation of anti-inflammatory cytokines (IL-10) and reduction of the most widely known proinflammatory cytokine (tumor necrosis factor [TNF]-a) (4-7).  The recent report by our group of fistula closure in serologically positive patients adds further support to a bacterial etiology and the role of antibiotics in the treatment of this subset of patients.

The recent discovery of the Nod-2 gene on chromosome 16 suggests the important role of the monocyte in the detection of cell wall lipopolysaccharides in a variety of bacteria.  This gene, which is an important determinant of innate immunity, is defective in as many as 15% of patients with CD (8, 9).  The unique cell wall features of Mycobacterial species and their interaction with receptors on the human intestinal epithelial cell fit well with a possible role of this organism in the pathogenesis of CD.

Since 1998, Infliximab, a monoclonal chimeric antibody to TNF-a, has been used extensively in the treatment of CD and has proven beneficial to chronic steroid dependent CD patients.  Several groups have published the benefit in fistula closure (10, 11).  The preliminary results of the ACCENT I trial showed the benefit of every eight-week administration of Infliximab with maintenance of remission and improved quality of life in over 550 patients with CD (12, 13).  Safety concerns regarding complications of the immunosuppressive effects of Infliximab need greater attention.  Documented miliary tuberculosis (14), aspergillosis, and other opportunistic infections, and recent reports of neoplasms (15) and disseminated infection in patients treated with Infliximab for CD and Rheumatoid arthritis raise concerns for our patients.

Our group is studying the effect of immunotherapy in patients serologically positive for MAP.  Our preliminary results show the elimination of antibody markers after treatment with Infliximab in patients known previously positive.  The loss of humoral antibody, after Infliximab infusion, and the known protective role of TNF in the recruitment of macrophages, may in fact be harmful to those with underlying bacterial infection.  The identification of these patients and the eradication of infection prior to using immunotherapy may prove safer.  We are currently studying serology and its role in selecting the appropriate and most effective treatment in this group of patients.

Clearly, important bacterial antigens play an important role in CD and its complication of fistula and abscess formation.  Whether these are primary or secondary invaders should not reduce our need to address these factors.  Elimination of harmful bacteria antigens and repopulation with beneficial ones (probiotics) appears logical and safe when compared to existing approved therapies which do not treat the root cause of this disease.  Further large, controlled antibiotic trials using serologic and genetic markers are desperately needed.

Ira Shafran, M.D.
Jonathan W. Decker, R.N., B.S.N.

REFERENCES
1. Hulten K, El-Zimaity HM, Karttunen TJ, et al. Detection of Mycobacterium avium subspecies paratuberculosis in Crohn's diseased tissues by in situ hybridization. Am J Gastroenterol 2001;96:1529-35.
2. Naser SA, Hulten K, Shafran I, et al. Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis. Vet Microbiol 2000;77:497-504.
3. Schwartz D, Shafran II, Romero C, et al. Use of short-term culture for identification of Mycobacterium avium subsp. paratuberculosis in tissue from Crohn's disease patients. Clin Microbiol Infect 2000;6:303-307.
4. Bengmark S. Colonic food: pre- and probiotics. Am J Gastroenterol 2000;95:S5-7.
5. Bengmark S. Ecological control of the gastrointestinal tract. The role of probiotic flora. Gut 1998;42:2-7.
6. Gorbach SL. Probiotics and gastrointestinal health. Am J Gastroenterol 2000;95:S2-4.
7. Borruel N, Casellas F, Antolin M, et al. Abstract #1442: Increased mucosal TNF-alpha production in Crohn's disease can be modulated locally by probiotics, In Digestive Diseases Week, Atlanta, GA, Gastroenterology, 2001.
8. Hugot J, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001;411:509-603.
9. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001;411:603-606.
10. Present DH, Rutgeerts P, Targan S, et al. Infliximab for the treatment of fistulas in patients with Crohn's disease. N Engl J Med 1999;340:1398-405.
11. Targan SR, Hanauer SB, van Deventer SJ, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med 1997;337:1029-35.
12. Hanauer SB. Avoiding the short-term fix: Long-term treatment solutions in inflammatory bowel disease, In Digestive Diseases Week, Atlanta, GA, 2001.
13. University.of.Chicago. (2001, May 21). Study Shows Infliximab Can Maintain Long-Term Remissions In Crohn's Disease, [internet press release]. PRNewswire. Available: http://www.gastrotherapy.com/reuters/articles/20010102publ001.html.
14. Woodman R. (2001, 01/02). [internet article]. Reuters Health. Available: http://www.gastrotherapy.com/reuters/articles/20010102publ001.html.
15. Sachmechian A, Vasiliauskas EA, Abreu MT, et al. Abstract #3144: Malignancy following Remicade therapy: Incidence and characteristics, In Digestive Disease Week, Atlanta, GA, Gastroenterology, 2001.
 


Mycobacterium avium subsp. paratuberculosis as one cause of Crohn's disease.

Chamberlin W, Graham DY, Hulten K, El-Zimaity HM, Schwartz MR, Naser S, Shafran I I, El-Zaatari FA

A number of theories regarding the aetiology of Crohn's disease have been proposed. Diet, infections, other unidentified environmental factors and immune disregulation, all working under the influence of a genetic predisposition, have been viewed with suspicion. Many now believe that Crohn's disease is a syndrome caused by several aetiologies. The two leading theories are the infectious and autoimmune theories. The leading infectious candidate is Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of Johne's disease, an inflammatory bowel disease in a variety of mammals including cattle, sheep, deer, bison, monkeys and chimpanzees. The evidence to support M. paratuberculosis infection as a cause of Crohn's disease is mounting rapidly. Technical advances have allowed the identification and/or isolation of M. paratuberculosis from a significantly higher proportion of Crohn's disease tissues than from controls. These methodologies include: (i) improved culture techniques; (ii) development of M. paratuberculosis-specific polymerase chain reaction assays; (iii) development of a novel in situ hybridization method; (iv) efficacy of macrolide and anti-mycobacterial drug therapies; and (v) discovery of Crohn's disease-specific seroreactivity against two specific M. paratuberculosis recombinant antigens. The causal role for M. paratuberculosis in Crohn's disease and correlation of infection with specific stratification(s) of the disorder need to be investigated. The data implicating Crohn's as an autoimmune disorder may be viewed in a manner that supports the mycobacterial theory. The mycobacterial theory and the autoimmune theory are complementary; the first deals with the aetiology of the disorder, the second deals with its pathogenesis. Combined therapies directed against a mycobacterial aetiology and inflammation may be the optimal treatment of the disease.

Full text (.pdf)
 
 

 Abstracts Submitted to Gastroenterology

(Digestive Disease Week 2001)

Fistula Healing in MAP Positive Crohn's Disease Patients Following Rifabutin and Macrolide Antibiotic Treatment.

Ira Shafran, Lynn Kugler, Javier Sandoval

Background: Prior studies have shown the beneficial role of Rifabutin and Macrolide Antibiotic Therapy (RMAT) in the treatment of intractable, fistulizing, Crohn's disease. Methods: We identified a subset of 10 MAP positive patients (3 males and 7 females) ages 41-20 presenting acute lower GI Crohn's with fistulization. These patients failed to respond to prior corticosteroid and anti-inflammatory therapy and presented a mean Crohn's Disease Activity Index (CDAI) score of 269 (250 – 300). The distribution of fistula was scrotal 1 (10%), scrotal-anal 1 (10%), vaginal-anal 1 (10%), perianal 5 (50%), ileocecal 1(10%) and colonic 1 (10%). These patients were treated with RMAT consisting of 250 mgm 1 po bid Clarithromycin and 150 mgm 1 po bid Rifabutin. Antibiotic therapy was complemented with 200 mgm po bid of probiotic containing equal amounts of Lactobacillus acidophilus and Lactobacillus rhamnosus. Results: Patients treated with RMAT had complete closure of fistuli in all cases within an average time of 32 weeks (8 – 60) of treatment and presenting a mean of 126.5 points in their final CDAI score. All patients reached a state of remission classically defined by a CDAI score below 150 points with a minimum differential of 100 points from initial to final score. Subsequent follow up showed a recurrence of fistuli within an average time of 8 months (2-13) in 4 (40%) patients who chose to discontinue RMAT. Conclusion: This subset of MAP positive patients may represent a particularly responsive group of Crohn's patients whose disease is remarkably responsive to antimycobacterial therapy but who require effective alternative therapies for the maintenance of remission in their Crohn's disease. Double blind, placebo controlled trials are required to further examine the effectiveness of antibiotics in the management of fistulizing Cronh's disease.
 
 

Abstracts Submitted to Gastroenterology

(Digestive Disease Week 2000)
 
 

A Comparative Analysis of Anti-Saccharomyces Antibodies (ASCA) and p35 and p36 Recombinant Clones for the Diagnosis of Crohn's Disease (CD)

I. Shafran, C. Piromalli, S. Naser

BACKGROUND: Numerous studies have confirmed the utility of ASCA as a serological marker for CD. Recent investigations have identified the utility of another potential CD diagnostic serum marker, p35 and p36 recombinant clones specific for Mycobacterium avium ss. paratuberculosis. Our lab is currently investigating the utility of these serologic markers for the diagnosis of CD. AIM: To evaluate the accuracy of ASCA and both p35 and p36 serologic markers for the diagnosis of CD. METHODS: Serum samples were obtained from 62 patients with confirmed and documented CD. The sera samples were sent to Prometheus Laboratories to determine the IgA and IgG ASCA titers with the Prometheus IBD Diagnostic System. The serologic response to p35 and p36 were analyzed by Western blotting at the University of Central Florida. RESULTS: The ASCA titer levels for IgA and/or IgG were positive for 61% (38/62) of CD patients. The p35 and/or p36 recombinant clones were positive for 85% (53/62) of CD patients. The 24 CD patients not detected by ASCA were identified as positive by the p35 and/or p36 recombinant clones in 92% (22/24) of those CD patients missed by ASCA. The 9 CD patients not detected by p35 and/or p36 were identified by the ASCA serologic markers in 78%(7/9) of those CD patients missed by p35 and p36. CONCLUSION: This investigation further establishes the utility of p35 and p36 recombinant clones for the diagnosis of CD. These results also reveal added evidence to demonstrate the complimentary role that ASCA may have with p35 and p36 for increasing the sensitivity and specificity of these serologic tests. Larger studies are needed to investigate the combined use of these serologic markers for the diagnosis of CD and as predictive markers to facilitate the development of treatment strategies.
 
 

Cost Analysis of Antibiotic Treatment in Crohn's Disease Patients Serologically Positive for Mycobacterium avium ss. paratuberculosis

I. Shafran, C. Piromalli and J. Davies

Previous studies have shown the benefits of Rifabutin and Macrolide Antibiotic Therapy (RMAT) in the treatment of Crohn's disease. The average annual cost for Crohn's disease patients has recently been estimated at $12,417, with surgical patients having an average annual cost approaching $37,135. Utilizing our database of serologically Mycobacterium avium ss. paratuberculosis positive Crohn's patients who were treated with antibiotics, we have shown a 60% response rate to combined antibiotics after three months of treatment. A decision analysis model was utilized to compare current accepted therapy, both medical and surgical, with alternative therapy utilizing antibiotics. Through the analysis of our data with the combined cost of medical care and medications, the average cost per patient on RMAT for one year was $6,806, achieving a 60% complete durable response. These findings show a significant cost savings with RMAT as compared to the current annual cost of care utilizing conventional therapy. Further cost analysis studies comparing RMAT with other treatments will be helpful in defining the costs and benefits of this therapy.
 
 

Mycobacterium Identification in Stool Cultures of IBD Patients Serologically Positive for Mycobacterium avium ss. paratuberculosis

I.Shafran M.D.and C.Piromalli B.S.; Florida Hospital, Orlando, FL.

Background: The role of Mycobacterium in the etiology of Inflammatory Bowel Disease has been the subject of much debate. Previous studies have identified that over 70% of Crohn's patients are serologically positive to both p35 and p36 recombinant clones specific for Mycobacterium avium ss. paratuberculosis (MAP). Purpose: To culture and identify mycobacteria in stool of patients serologically positive for p35 and/or p36 recombinant clones. Methods: Stool specimens were obtained during colonoscopy procedures from 15 Inflammatory Bowel Disease (IBD) patients who were identified as being serologically positive to the recombinant clones p35 and/or p36 [12 patients with Crohn's Disease (CD), 1 patient with Ulcerative colitis (UC) and 2 patients with Nonspecific colitis (NC)]. 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. Results: Of our total number of stool cultures, 7/15 (47%) were identified as positive for mycobacterial growth and 8/15 (53%) were negative for mycobacterial growth. Please refer to the table below. Conclusion: The variety of mycobaterial organisms found in these stool cultures demonstrate the presence of mycobateria in the digestive tract of IBD patients. Further studies must be done to explore whether these enteric organisms play a possible role in the pathogenesis of IBD. Larger studies must also be done to determine the utility of mycobaterial identification of stool cultures to facilitate in designing treatment strategies for IBD patients.

Mycobacterium Identification of Stool Cultures

Mycobacterium Identification CD  UC NC 
avium complex
gondonae
scotochromogen
Total
 
 

Identification of Mycobacterium avium ss. paratuberculosis in Crohn's Tissue

I.Shafran, C. Piromalli, C.Romero, D.Schwartz and S.Naser

Background: Mycobacterium ss. paratuberculosis (MAP) is known to cause chronic granulomatous enteritis in ruminants and primates (Johne s Disease). Many groups have implicated the possible role of the spheroplastic form of MAP in Crohn's Disease (CD) but the isolation and identification of this bacteria have been difficult due to the uncertain location and rigid growth requirements of this organism. Purpose: To investigate the role of MAP in CD using short-term mycobacterial culture media. Methods: 27 CD tissue specimens (7 surgical resected tissue and 20 biopsies) and 36 controls (3 surgical resected tissue and 33 biopsies) 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. Results: Microbial growth was found in both culture media, although only MGIT cultures contained MAP. MAP bacterial isolates were confirmed to be acid fast bacilli, mycobactin dependent and positive for the presence of IS900 and hybridization. Presence of MAP in MGIT cultures were detected in 12 weeks of incubation for resected tissue and 40 weeks for biopsies. See table for results. Conclusion: MAP was much more readily found in CD specimens as compared to control specimens. Additionally, MAP was found more readily in resected tissue and required short-term incubation in contrast to endoscopic biopsies. This may indicate that MAP resides submucosally. Our findings suggest that surgical resected tissue cultured in MGIT media is favorable for the rapid cultivation of MAP versus endoscopically acquired biopsies.

MAP Culture Results From CD and Control Tissue

  Total #  Positive #  Negative # 
CD Resected Tissue  6 (86%)  1 (14%) 
CD Biopsies  20  4 (20%)  16 (80%) 
Control Resected Tissue  0 (0%)  3 (100%) 
Control Biopsies  33  2 (6%)  31 (94%) 
Total  63  12 (19%)  51 (81%) 
Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.

Naser SA, Hulten K, Shafran I, Graham DY, El-Zaatari FA
Vet Microbiol 2000 Dec 20;77(3-4):497-504

Crohn's disease (CD) is a chronic inflammatory bowel disease that is similar to Johne's disease in ruminants. Recent data have strengthened the association of M. avium subsp. paratuberculosis (M. paratuberculosis) with CD. To provide more evidence of an etiological association, antibody reactivities from CD patients were tested by immunoblotting against recombinant antigens that were identified previously from our M. paratuberculosis genomic library. Two clones (designated pMptb#40 (3.2-kb insert) and #48 (1.4-kb insert) expressing a 35K (p35)- and 36K(p36)-antigens showed specific reactivities with serum samples from CD patients.Serum samples from 75% of 53 CD patients, 14% of 35 normal individuals and 10% of 10 ulcerative colitis patients reacted to p35 antigen. Reactivities were also observed with serum samples from 89% of 89 CD patients, 14% of 50 normal controls and 15% of 29 ulcerative colitis patients reacted with p36 antigen. When the reactivity results from p35 and p36 were combined, the background from the controls was eliminated, i.e. only the CD patients reacted to both p35 and p36. The positive predictive value was 98% with specificity of 98% and the negative predictive value was 76% with sensitivity of 74% (39 positive out of 53). A statistical significance (p<0.0001) was observed when the results from CD serum samples reacting with either or both antigens were compared to the controls. The reactivity of anti-M. paratuberculosis (specifically against p35 and p36 antigens) antibodies in a significant proportion of CD patients would suggest a causal role for the organism in CD.
 
 

Rifabutin and Macrolide Antibiotic Treatment in Crohn's Patients Identified Serologically Positive for Mycobacterium avium ss. paratuberculosis

I. Shafran, C. Piromalli, S. Naser

The use of antibiotics in Crohn's disease has recently been studied and our group has shown previously the efficacy and safety of RMAT in an open clinical trial. Forty-two Crohn's patients, who were serologically positive with the p35 and p36 serologic markers specific for Mycobacterium avium ss. paratuberculosis (MAP), were chosen for treatment with Rifabutin and Macrolide Antibiotic Therapy (RMAT). The RMAT medications included 250 mgm 1 po bid clarithromycin and 150 mgm 1 po bid rifabutin and 200 mgm po bid of a probiotic containing equal amounts of Lactobacillus acidophilus and Lactobacillus rhamnosus. These patients were followed serologically and clinically each month. The serial blood samples were stored to measure, at a later date, the quantitative antibody response to MAP. All of the patients were assessed to determine their overall response to treatment. The period of treatment ranged from 3 months to 17 months with the average treatment period being 12 months. 61.9% (26/42) of the patients reached a sustained state of clinical remission while being off all other Crohn's medications, such as sulfasalizine and corticosteroids. The majority of these patients had acute presentations of Crohn's disease when placed on RMAT. 5 patients also had documented endoscopic healing correlating with treatment response. 9.5% (4/42) of patients noticed significant improvements but were still using Crohn's medications with RMAT. 9.5% (4/42) were nonresponders noticing no marked improvement while on RMAT. 19.1% (8/42) of patients were unable to tolerate the RMAT medications and stopped therapy. These findings add further evidence to support the role of RMAT in the treatment of Crohn's disease in patients serologically positive for MAP. A large multicenter clinical trial is needed to further explore these findings.

RMAT CLINICAL TRIAL RESULTS

  RMAT Patients  Percent (%) 
Responders  26  61.9 
Partial Responders  9.5 
Nonresponders  9.5 
Intolerant to RMAT  19.1 
 
 

Abstracts published in The American Journal of Gastroenterology

(September 1999 Vol.94, Num.9)
 
 

COMPARISON OF SEROLOGIC MARKERS FOR THE DIAGNOSIS OF CROHN'S DISEASE

I.Shafran M.D., C.Piromalli, F.El-Zaatari Ph.D., S.Naser Ph.D.; Dept. Molecular Biology, UCF, Orlando,FL; Baylor College of Medicine, Houston,TX.

BACKGROUND: Considerable attention has been focused on the anti-saccharomyces cervicae antibody (ASCA) as a serological marker for Crohn's disease. Recent investigations have identified the utility of another potential Crohn's diagnostic serum marker, p35 and p36 recombinant clones specific for Mycobacterium avium ss. paratuberculosis.

AIM: To determine and compare the diagnostic accuracy of ASCA to both p35 and p36 serologic markers for the diagnosis of Crohn's disease.

METHODS: Serum samples were obtained from 27 patients with Crohn's disease. The sera samples were sent to Prometheus Laboratories to determine the IgA and IgG ASCA titers with the Prometheus IBD Diagnostic System. The serologic response to p35 and p36 were analyzed by Western blotting at the University of Central Florida.

RESULTS: The ASCA titer levels for IgA and/or IgG were positive for 63% (17/27) of the Crohn's patients. The p35 and/or p36 recombinant clones were positive for 85% (23/27) of the Crohn's patients. The 10 patients with negative ASCA titer levels for IgA and/or IgG each reacted positively with p35 and/or p36 recombinant clones. The 4 patients with negative reactivity to p35 and/or p36 each had positive ASCA titer levels for IgA and/or IgG.

CONCLUSION: This study reveals the complimentary role that ASCA may have with p35 and p36 for increasing the sensitivity and specificity of these serologic tests. Larger studies are needed to determine the enhanced accuracy for diagnosing Crohn's disease by combining the ASCA and p35 and p36 serologic markers.
 
 

SERUM SICKNESS-LIKE ILLNESS IN CROHN'S DISEASE PATIENTS TREATED WITH ANTIBIOTICS

I.Shafran M.D.and C.Piromalli; Florida Hospital, Orlando, FL.

Thirty-five patients with Crohn's disease are being treated with Rifabutin and Macrolide Antibiotic Therapy (RMAT) for a duration of six months to one year based upon their overall response to treatment. 37% (13/35) of these patients developed a serum sickness-like illness during the first four to six weeks of treatment. The patients experienced flu-like symptoms such as fever, chills, moderate to severe arthralgia, back pain, anorexia and fatigue. These symptoms generally lasted for a full week and dissipated over the following three weeks. With each patient, the majority of symptoms stopped within the first month of treatment. It was also found that these symptoms responded well to Cox-2 inhibitors (celecoxib - 200mgm po qd) with no adverse effects or worsening of colitis noted during treatment. These observations suggest that the Cox-2 inhibitors may help in controlling the initial side-effects of RMAT. It is also thought that this serum sickness may be a Jarisch-Herxheimer reaction in response to the antimicrobial therapy. Further studies will be needed to determine the exact cause and significance of these symptoms.
 
 

TREATMENT OF CROHNS DISEASE WITH RIFABUTIN AND MACROLIDE ANTIBIOTICS

I.Shafran M.D.and C.Piromalli; Florida Hospital, Orlando, FL.

Twenty-nine patients with Crohn's disease were selected for treatment with Rifabutin and Macrolide Antibiotic Therapy (RMAT) for a duration of six months to one year based upon their overall response to treatment. The RMAT medications included 250mgm 1 po bid clarithromycin, 150mgm 1 po bid rifabutin and 200mgm po qd of a probiotic containing equal amounts of Lactobacillus acidophilus and Lactobacillus rhamnosus. At three months on RMAT, all of the patients were assessed to determine their overall response to treatment. 28% (8/29) of the patients reached a state of clinical remission (defined by the CDAI criteria with a score <150) while being off all other medications. The majority of these patients had acute presentations of Crohn's disease when placed on RMAT. 31% (9/29) of patients were not in clinical remission but experienced significant improvements as they discontinued the use of all other Crohn's medications. 28% (8/29) of patients noticed some improvements on RMAT but were still using traditional medications, such as sulfasalazine and corticosteroids. 14% (4/29) were non-responders since they were unable to tolerate the RMAT medications and stopped therapy. These preliminary findings add further evidence to support the role of RMAT in the treatment of Crohn's disease. A large multi-center clinical trial is needed to further explore these findings.
 
 

Poster Abstracts at the 99th American Society of Microbiology General Meeting: Atlanta, Georgia May, 1999

In Vivo Evaluation of Anti-Tuberculosis Drugs For Treatment of Crohn's Disease Patients

I.Shafran, D.Campbell, C.Romero, D.Schwartz and S.Naser

Department of Molecular Biology and Microbiology/ Center for Discovery of Drugs and Diagnosis

University of Central Florida, Orlando

In a previous abstract, we reported that a regimen consisting of Rifampicin:  Clarithromycin at concentrations of 0.5:1.2 ug/ml or 1.0:0.75 ug/ml resulted in significant eradication of M.avium ss paratuberculosis (M para) in vitro where MIC99.9% was achieved. M para is the causative agent of Johne's Disease in ruminants and has been implicated in the pathogenisis of Crohn's disease (CD), a chronic inflammatory bwel disease in humans. A 65 year old patient diagnosed with CD who was found to be PCR positive for M para (using IS900 specific primers) with humoral immune response against recombinant antigens of M para, has demonstrated significant healing (>80%) of an ulcer seen in the ileum shown by endoscopy. This healing was observed afyer the patient underwent a combination of Clarithromycin 250mg. Twice a day and rifabutin 150 mg. Daily. He was endoscoped through his stoma and found to have a 4.0 cm aphthous ulcer. The remaining ileum was unremarkable to a depth of 120 cm. Histology showed typical features of CD. He became asymptomatic in 2 weeks and a follow-up endoscopy was done after completing 1 month of treatment. The 4 cm ulcer, noted 1 month before, had decreased to 1 cm with excellent reepithelialization from the edge of the ulcer inward. The remaining ileum to 120 cm was normal. The patient has remained symptom free and continues to be on antibiotics. The endoscopic observations reported further substantiate the possible role of M para in CD.
 
 

Mycobacterium avium ss paratuberculosis In Crohn's Disease Tissue Is an Affirmative!

D.Schwartz, C. Romero, D. Campbell, I. Shafran and S. Naser

Department of Molecular Biology and Microbiology/ Center for Discovery of Drugs and Diagnostics

University of Central Florida, Orlando, Florida

The insertion sequence IS900 of Mycobacterium avium ss paratuberculosis (M para) has been used as a fingerprint-type marker for identification of M para or M para-like microorganisms in clinical specimens from patients with Crohn's disease (CD), a chronic inflammatory bowel disease. However, amplification of IS900 in these specimens does not confirm the virulent form of the bacterium or the status of disease. Culturing M para from CD tissue has been reported but with limited success. This, in part, is significantly related to the type of culture media and the integrity of the specimen used. Amplification of the IS900 followed by DNA hybridization from cultured tissue, provide accurate diagnosis of CD. In this study, 59 tissue specimens from 59 patients (20 CD consisting of 7 resected tissue and 13 biopsies and 39 control consisting of 1 resected tissue and 38 biopsies from Ulcerative colitis and noninflammatory bowel disease patients) were homogenized, decontaminated and inoculated into BACTEC bottles and MGIT media (radioactive and non-radioactive methods, respectively). Monthly aliquots of incubated cultures were examined microscopically and by PCR/hybridization using IS900-based primers for the presence of M para. Acid fast staining and immunoscreeening using rabbit poly clonal anti-M para antibodies by confocal laser microscopy revealed the presence of M para in 6 CD specimens. The positive specimens consisted of 6 resected tissue and 1 biopsy. This was confirmed with PCR and hybrodization of the 39 control specimens, none were positive with any of the assays. Our data also suggest that M para may be isolated from tissue specimens, especially those resected (if present) within weeks in the MGIT media.
 
 


In Vitro Evaluation of Anti-Tuberculosis Drugs Against Mycobacterium avium ss paratuberculosis For Treatment of Crohn's Disease

D.Campbell, C.Romero,D.Schwartz, I.Shafran and S.Naser

Department of Molecular Biology and Microbiology/Center for Discovery of Drugs and Diagnostics

University of Central Florida

Mycobacterium avium ss paratuberculosis (M para) is the causative agent of Johne's Disease in ruminants and has been implicated in the pathogenesis of Crohn's disease (CD), a chronic inflammatory bowel disease in humans. Like members of the M avium complex, M para is resistant to the most anti-tuberculosis (anti-TB) drugs. In this study, seven anti-TB drugs (rifampicin;RIF, streptomycin; SM, kanamycin; KM, clarithromycin; CLR, isoniazid; INH, pyrazinamide; PZA and ethambutol; EMB) have been tested against M para using the Bactec system. The MIC50 and MIC99.9%, for the drugs (where 50% and 99.9%, respectively, of the M para cells were inhibited or killed) were determined with KM, CLR, and EMB produced significant inhibition activity against M para. To minimize induction of drug resistance strains of M para during patient treatment, regimens of these drugs have been tested. Our data suggest that a regimen consisting of RIF:CLR at concentrations of 0.5:1.2 ug/ml or 1.0:0.75 ug/ml resulted in significant eradication of M para in vitro where MIC99.9%, was achieved. The RIF:CLR combination produced similar results when used against two clinical strains of M para that have been isolated from resected tissue of CD patients. RIF:CLR is recommended for in vivo eradication of M para because the spheroplast form of M para has been suggested as the virulent form of the bacterium in CD. These data also provide guidelines and possible protocols for the first-line drugs to be used in the treatment of CD.

  RIF  ST  KM  CLR INH  PZA  EMB  RIF: EMB RIF: CLR
                   
MIC50 (ug/ml)  >1.0  >0.4  <1.3  <0.25  >20  >20  <0.5  <1.0: 0.2  <0.5: 0.3 
MIC99.9 (ug/ml)  >2.6  >3.0  <3.0  <1.25  >20  >20  <3.0  <1.0:1.0  <0.5:1.2 or 1.0:0.76 
Status  R
R = Resistant        S = Sensitive
 
 

Abstracts published in Gastroenterology

(April 1999 Vol.116, Num.4)

Endoscopic Healing of Crohn's After Antibiotic Treatment

I. Shafran, C. Piromalli, S. Naser

In this case report, a Crohn's patient was found to be PCR positive for Mycobacterium avium subspecies paratuberculosis (M para) with a positive serologic response to p35 and p36 antigens (recombinant clones expressing a 35K and 36K protein from M para genomic library), and has demonstrated significant healing (>80%) of an ulcer seen in the ileum at endoscopy. This healing was observed after the patient underwent a combination of rifabutin and macrolide therapy. This 65-y/o male had Crohn's disease (CD), diagnosed at age 30, that was confined to his colon and treated with corticosteroids for 17 yrs. A proctocolectomy and Brookes ileostomy were done in 1980. He remained symptom free and off all medications until April, 1998 when he developed colicky abdominal pain and weight loss. He was endoscoped through his stoma and found to have a 4.0 cm. aphthous ulcer. The remaining ileum was unremarkable to a depth of 120 cm. Histology showed typical features of CD. The patient consented to treatment with macrolide antibiotics 250 mg. twice a day and rifabutin 150 mg. daily. He became asymptomatic in 2 weeks and a follow-up endoscopy was done after completing 1 month of treatment. The 4 cm. ulcer, noted 1 month before, had decreased to 1 cm. with excellent re-epithelialization from the edge of the ulcer inward. The remaining ileum to 120 cm. was normal. The patient has remained symptom free and continues antibiotics. In conclusion, this is the first known case of CD treated with this combination of antibiotics with documented clinical and endoscopic healing. The endoscopic observations reported further substantiate the possible role of M para in the etiology of CD and the possible use of antibiotic treatment for CD. Currently, 14 CD patients are undergoing similar treatment to further validate the role of M para in CD and the efficacy of antibiotic treatment.

     
Humoral Immune Response of Crohn's Patients for Mycobacterium avium subspecies paratuberculosis

I. Shafran, W. Fenster, C. Piromalli, C. Romero, D. Schwartz, D. Campbell,

F. El-Zataari and S. Naser

This study analyzed the humoral immune response of Crohn's Disease (CD) patients compared with age-matched controls against two recombinant clones of Mycobacterium avium subspecies paratuberculosis (M para). The recombinant clones designated p35 and p36, expressing a 35K and 36K protein respectively, were identified from a previously constructed genomic library of M para and screened by immunoblot against rabbit hyperimmune anti-M para antibodies. A total of 110 human sera consisted of 63 CD patients and 47 controls (35 volunteers with no history or symptoms of GI tract disorder and 12 with Ulcerative Colitis [UC]) were analyzed by immunoblot. The donated sera were free of tuberculosis, leprosy and/or have not received the BCG (Bacillus Calmet Guerin) vaccine. The following table summarizes the serologic results. The data confirm reasonable significance of P<0.001 between CD and control samples. These findings add further evidence that M para may have a close association in the etiology of CD. The data also suggest that there is great potential for using these recombinant antigens in a serologic test for clinical diagnosis.
 
 

Antigen CD UC Control
P35 49 (78%)  1 (8%)  5 (14%) 
P36  57 (90%)  1 (8%)  4 (11%) 
Combined  48 (76%)  1 (8%)  0 (0%) 
Either  58 (92%)  1 (8%)  9 (25%) 
Total (110)  63  12  35 
 
 
 


In Vitro Evaluation of Anti-Tuberculosis Drugs Against Mycobacterium avium ss. paratuberculosis For Treatment of Crohn's Disease

I. Shafran, W. Fenster, C. Piromalli, C. Romero, D. Schwartz, D. Campbell, and S. Naser

Mycobacterium avium ss.paratuberculosis (M para) is the causative agent of Johne's Disease in ruminants and has been implicated in the pathogenesis of Crohn's disease (CD), a chronic inflammatory bowel disease in humans. Like members of the M avium complex, M para is resistant to anti-tuberculosis (anti-TB) drugs. In this study, seven anti-TB drugs (rifamipcin; RIF, streptomycin; SM, kanamycin; KM, clarithromycin; CLR, isoniazid; INH, pyrazinamide; PZA and ethambutol; EMB) have been tested against M para using the Bactec system. The table below summarizes the MIC50 and MIC99.9 for the drugs(where 50% and 99.9% respectively of the M para cells were inhibited or killed). To minimize drug resistance-induced strains, regimens of these drugs have been used for the treatment of mycobacterial infection. Our data show that a regimen consisting of RIF:EMB or RIF:CLR resulted in the significant eradication of M para in vitro. In fact, the RIF:CLR combination produced similar results when used against two clinical strains of M para that have been isolated from resected tissue of CD patients. RIF:CLR is recommended for in vivo eradication of M para because the spheroplast form of M para has been suggested as the virulent state of the bacterium in CD. These data also provide guidelines and possible protocols for the first-line drugs to be used in the treatment of CD.

  RIF  ST  KM  CLR  INH  PZA  EMB RIF: EMB RIF: CLR
                   
MIC50 (ug/ml)  >1.0  >0.4  <1.3  <0.2  >1.0  >5.0  <0.5  <1.0: 0.2  <0.5: 0.2 
MIC99.9 (ug/ml)  >2.6  >3.0  <3.0  <0.5  >1.0  >5.0  <0.8  ND  <0.5:1.2 or 1.0:0.75 
Status 
 
 

R = Resistant

S= Sensitive
 
 

Rapid Culturing and PCR Detection of Mycobacterium avium ss paratuberculosis from Crohn's Disease Tissue

I. Shafran, W. Fenster, C. Piromalli, C. Romero, D. Schwartz, D. Campbell, and S. Naser

The insertion sequence IS900 of Mycobacterium avium ss paratuberculosis (M para) has been used as a fingerprint-type marker for the identification of M para or M para-like microorganisms in clinical specimens from patients with Crohn's disease (CD), a chronic inflammatory bowel disease. However, amplification of IS900 in these specimens does not confirm the virulent form of the bacterium. Culturing M para from CD tissue has been reported but with limited success. This, in part, is significantly related to the type of culture media used and the integrity of the specimen. Amplification of the IS900 followed by DNA hybridization from cultured tissue, provide accurate diagnosis of CD. In this study, 59 tissue specimens (20 CD consisting of 7 resected tissue and 13 biopsy and 39 controls consisting of biopsies from Ulcerative colitis and non-inflammatory bowel disease patients) were homogenized, decontaminated and inoculated into BACTEC bottles and MGIT media (radioactive and non-radioactive methods, respectively). Aliquots of culture were analyzed by PCR assays specific for M avium complex (MAC) and M para (IS900). This was followed with hybridization using specific DNA probes. Of the 20 CD specimens, 15(75%) were positive for MAC with 6 (30%) confirmed for the IS900 as M para. Of the 39 control specimens, none were positive with either assay. Our data confirm previous studies reporting the association of M para or M para-like microorganisms in CD. Our data also suggest that M para may be isolated from tissue specimens, especially those resected (if present) within 10 weeks in the MGIT media. Ultimately, this provides a rapid culturing protocol for the diagnosis of CD especially at its proliferative stage.
 
 
 

Mycobacterium avium subsp. paratuberculosis in Crohn's Disease Is Serologically positive 

S. Naser, I. Shafran 

Crohn's disease, similar to Johne's disease (a Mycobacterium avium subsp. paratuberculosis - caused inflammatory bowel disease in ruminants and primates), is an inflammatory bowel disease with suspected mycobacterial etiology (1). The disease emerged perceptibly in Western Europe and North America in the late 1940s and early 1950s. The incidence then increased progressively on both continents to a level which in some areas, such as northeast Scotland (11.6/100,000 per year), now approaches that of an epidemic (4). Granulomas and lymph node alteration in Crohn's disease patients resemble those of tuberculosis, leprosy, sarcoidosis, and bovine paratuberculosis (3). The latter has been suggested as a reservoir for epidemiologic transmission of the microorganism through contaminated dairy and meat products and possibly even in water (5). 

In this study, we analyzed the humoral immune responses of Crohn's disease patients compared with those of age-matched controls against two recombinant clones of M. avium subsp. paratuberculosis. The recombinant clones, designated p35 and p36 and expressing 35- and 36-kDa proteins, respectively, were identified from a previously constructed expression genomic
library of M. avium subsp. paratuberculosis that was screened by Western blotting against rabbit hyperimmune anti-M. avium subsp. paratuberculosis antibodies (2). A total of 110 human serum specimens, consisting of samples from 61 Crohn's disease patients and 47 controls (35 volunteers with no history or symptoms of gastrointestinal tract disorder and 12 individuals diagnosed with ulcerative colitis) were analyzed by Western blotting (5). The participants who donated the sera used in this study were free of tuberculosis, leprosy, and/or had not received the Mycobacterium bovis BCG vaccine. As shown in Table 1, of the 61 Crohn's disease-positive serum samples tested, 48 (79%) reacted with p35, 56 (92%) reacted with p36, 47 (77%) reacted with both antigens, and 57 (93%) reacted with either antigen. As expected, a small portion of Crohn's disease-positive serum specimens, four (7%), did not react with either antigen. Of the 35 serum samples from healthy controls, 5 (14%) reacted with p35, 4 (11%) reacted with p36, none (0%) reacted with both antigens, and 9 (26%) reacted with either antigen. Of the 12 ulcerative colitis-positive serum specimens, only 1 (8%) reacted with p35 or p36, either individually or combined. The only ulcerative colitis patient who reacted positively with these antigens is under further evaluation for possibility of misdiagnosis. Crohn's and ulcerative colitis diseases are inseparable at the early stage of infection. Many physicians refer to them as colitis diseases. 
 

Antigen CD UC Control
P35 49 (78%)  1 (8%)  5 (14%) 
P36  57 (90%)  1 (8%)  4 (11%) 
Combined  48 (76%)  1 (8%)  0 (0%) 
Either  58 (92%)  1 (8%)  9 (25%) 
Total (110) 63  12  35 
These data confirm that there is a statistically significant difference in reactivity between Crohn's disease-positive serum samples and serum samples from controls (P < 0.001). This ultimately adds more evidence to strengthen the proposed association of Mycobacteria with Crohn's disease. The data also suggest that there is great potential for using M. avium subsp. paratuberculosis recombinant antigens (p35 and p36) individually or combined in a serologic test for diagnosis and possibly in vaccine development. 

1. 
   Chiodini, R. J., H. J. Van Kruiningen, W. R. Thayer, and J. A. Coutu. 1986. Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease. J. Clin. Microbiol. 24:357-363.
 2. 
   El-Zaatari, F. A. K., S. A. Naser, L. Engstrand, et al. 1994. Identification and characterization of Mycobacterium paratuberculosis recombinant clones expressed in E. coli. Curr. Microbiol. 29:177-184.
 3. 
   Graham, D. Y., D. G. Markesich, D. C. Kalter, et al. 1992. Mycobacterial aetiology of sarcoidosis. Lancet 340:52-53. 
 4. 
   Kyle, J. 1992. Crohn's disease in the northeastern and northern Isles of Scotland: an epidemiological review. Gastroenterology 103:392-399.
 5. 
   Millar, D., J. Ford, J. Sanderson, S. Withey, M. Tizard, T. Doran, and J. Hermon-Taylor. 1996. IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales. Appl. Environ. Microbiol. 62:3446-3452.

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