On the prevalence of M. avium subspecies paratuberculosis DNA in the blood of healthy individuals and patients with inflammatory bowel disease

Abstract

Background: Mycobacteria, such as M. leprae and M. tuberculosis infect billions of humans. However, because of appropriate immune responses and antibiotic therapy, overt mycobacterial diseases occur far less frequently. M. avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, an affliction evocative of inflammatory bowel disease (IBD). Several agents used to treat IBD (5-ASA, methotrexate, azathioprine and its metabolite 6-MP) have recently been shown to be antiMAP antibiotics. We herein evaluate the prevalence of MAP DNA in healthy individuals and compare them with IBD patients on antiMAP antibiotics.

Methods: We studied 100 healthy individuals (90 blood donors) and 246 patients with IBD. IS900 MAP DNA was identified using a nested primer PCR in the buffy coat of blood. Positive signal was confirmed as MAP by DNA sequence analysis. PCR positive results frequencies were compared according to medications used. Significance was accepted at p<0.05.

Results: 47% (47/100) healthy controls and 16% (40/246) IBD patients were IS900 positive (p<0.0001). MAP DNA was identified in 17% of 143 patients receiving mesalamine and 6% of 16 receiving sulfasalazine. None of the IBD patients receiving methotrexate (n = 9), 6-MP (n = 3), ciprofloxacin (n = 5) or Tacrolimus (n = 3) had MAP DNA detectable in their blood.

Discussion: We found a disquietingly large percentage of healthy individuals have MAP DNA in their blood, the significance of which remains to be determined. Counter-intuitively, the incidence of MAP DNA was significantly lower in patients with IBD. Agents with the most potent in vitro antiMAP activity were associated with clearance of blood MAP DNA. We posit that the use antiMAP antibiotics was responsible for the decreased prevalence of MAP DNA in patients with IBD.

Figure 1. Nested PCR detection of MAP DNA from peripheral blood samples.

Shown are representative samples of IBD patients and Controls. M = molecular weight marker, lane A = negative control of first round of PCR, lane B = negative control of second round of PCR, +  =  DNA from MAP strain ATCC 19698.

Figure 2. Shown is a bar graph of the % of 100 control subjects who were MAP DNA positive (first column) and all 246 patients who had IBD (second column).

We next stratify IBD patients by the class of medication that they were taking. “SAD” = salicylic acid derivatives. “Anti-metabolites” were 6-MP and its precursor azathioprine, methotrexate and the “immuno-suppressive” Tacrolimus®. The conventionally accepted antibiotics used in this study were ciprofloxacin and metronidazole. The % MAP DNA positive are shown on the ordinate. The total number of 270 is greater than the number of IBD subjects (246) because some individuals were getting multiple medications and some (37) were receiving no medications at all. All IBD patients, whether combined or sub-stratified are significantly different from the non-IBD controls.

Figure 3. Shown is a post hoc analysis of the 159 IBD patients who were receiving salicylic acid derivatives.

The control group comprises all IBD patients who were NOT receiving the agent identified. Mesalamine® is a proprietary name for 5-ASA. Sulfasalazine is a conjugate of 5-ASA and the antibiotic sulfapyridine. Although only 16 patients were taking sulfasalazine, the incidence of MAP DNA is significantly less than in the IBD group as a whole.

Figure 4. Shown is a post hoc analysis of the 62 IBD patients who were receiving “anti-metabolites”, agents recently shown to be potent antiMAP antibiotics.

The control group comprises all IBD patients who were NOT receiving the agent identified. The majority were receiving the precursor of 6-MP, azathioprine. No MAP DNA is found when 6-MP, methotrexate and Tacrolimus are used.

Figure 5. Shown is a post hoc analysis of the 16 IBD patients who were receiving conventional antibiotics, ciprofloxacin and metronidazole.

There was no MAP DNA detected in the small number of patients taking ciprofloxacin.

Figure 6. Shown are the data for the individual steroids used; prednisone, budesonide and triamcinolone.

There are no significant differences noted.

Figure 7. Shown is a comparison of the percentage of IBD patients who reported, at the time of phlebotomy to have “Active” (# = 40) or “Inactive disease” (# = 200).

For the remaining 6 patients “Disease Activity” data were not provided on their questionnaire. In this post hoc analysis, with group sizes that are not comparable, there is no difference in the presence or absence of MAP DNA among the groups. Our questionnaire did NOT obtain information on medications that had been used PRIOR to the day of phlebotomy.