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In Situidentification of
mycobacteria in Crohn’s disease patient tissue using confocal scanning laser
microscopy.
Effiacy & Tolerablity of Rifaximin, A
non-Absorbable, Gut-Selective, Oral Antibiotic in the Treatment of Crohn's
Disease: Results of an Open-Label Trail Open
clinical trial of Rifabutin and Clarithromycin therapy in Crohn’s Disease.
Mycobacterium
avium subsp. Paratuberculosis as one cause of Crohn's Disease Use of Short Term
Culture for Identification of Mycobacterium avium subsp. Paratuberculosis in
Tissue from Crohn’s Disease Patients. Isolation of
Mycobacterium avium subsp. Paratuberculosis from Breast Milk of Crohn’s
Disease Patients.
Fistula Healing in MAP
Positive Crohn’s Disease Patients Following Rifabutin and Macrolide Antibiotic
Treatment.
A Comparative Analysis of
Anti-Saccharomyces Antibodies (ASCA) and p35 and p36 Recombinant Clones for the
Diagnosis of Crohn's Disease (CD)
Cost Analysis of Antibiotic
Treatment in Crohn’s Disease Patients Serologically Positive for
Mycobacterium avium ss. paratuberculosis.
Mycobacterium Identification in
Stool Cultures of IBD Patients Serologically Positive for Mycobacterium avium
ss. paratuberculosis.
Identification
of Mycobacterium avium ss. paratuberculosis in Crohn's Tissue.
Rifabutin
and Macrolide Antibiotic Treatment in Crohn’s Patients Identified
Serologically Positive for Mycobacterium avium ss. paratuberculosis. Specific seroreactivity of Crohn's
disease patients against p35 and p36 antigens of M. avium subsp.
paratuberculosis.
Comparison of Serolgic
Markers for the Diagnosis of Crohn’s Disease.
Serum sickness-like
illness in Crohn’s Disease Patients Treated With Antibiotics.
Treatment of
Crohn’s Disease with Rifabutin and Macrolide Antibiotics.
Mycobacterium
Avium, SS Paratuberculosis, in Crohn’s Disease is an Affirmative.
In
Vitro Evaluation of Anti-Tuberculous Drugs Against M. Avium, Subspecies
Paratuberculosis, for Treatment of Crohn’s’ Disease.
Endoscopic
Healing of Crohn’s Disease After Antibiotic Treatment.
Humeral
Immune Response of Crohn’s Patients for Mycobacterium Avium, Subspecies
Paratuberculosis.
In
Vitro Evaluation of Anti-Tuberculous Drugs Against Mycobacterium Avium
Subspecies Paratuberculosis for Treatment of Crohn’s Disease.
Rapid
Culturing and PCR Detection of Mycobacterium Avium, SS. Paratuberculosis,
from Crohn’s Disease Tissue.
Mycobacterium avium subsp.
Paratuberculosis in Crohn’s Disease Is Serologically Positive 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.
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
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 Conrtol Tissue
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
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
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
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.
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.
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. 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.
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.
R = Resistant S= Sensitive
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
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.
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