Detection of Mycobacterium avium subspecies paratuberculosis from patients with Crohn's disease using nucleic acid-based techniques: a systematic review and meta-analysis

This study is a systematic review and meta-analysis of studies using nucleic acid-based techniques to detect Mycobacterium avium paratuberculosis (MAP) in patients with Crohn's disease (CD) compared with controls. Database searches were conducted and risk difference estimates were calculated using meta-analysis. Fifty-eight studies were reviewed, 47 of which were included in the analysis. The pooled estimate of risk difference from all studies was 0.23 (95% confidence interval [CI], 0.14-0.32) using a random effects model. Similarly, MAP was detected more frequently from patients with CD compared with those with ulcerative colitis (risk difference 0.19, 95% CI, 0.10-0.28). Year of study, assay type, and inclusion of children explained some but not all of the observed heterogeneity. The data confirms the observation that MAP is detected more frequently among CD patients compared with controls. However, the pathogenic role of this bacterium in the gut remains uncertain. Our analysis demonstrates that there is an association between MAP and CD, across many sites, by many investigators, and controlling for a number of factors; however, this association remains controversial and inconclusive. Future studies should determine whether there is a pathogenic role.     

Mycobacterium avium subspecies paratuberculosis and its relationship with Crohn's disease

The hypothesis postulating that Mycobacterium avium paratuberculosis (MAP) is the cause of Crohn's disease (CD) has been circulating for many years. Advances in molecular techniques, such as polymerase chain reaction and culture methods, have enabled researchers to demonstrate that there is an association between MAP and CD. Recently, genome-wide association studies have identified novel susceptibility genes for CD, which are critical for generation of an adaptive immune response that is protective against intracellular pathogens,including M. tuberculosis infection. However,the role of MAP as a cause of CD suffered a setbackwith the report that administration of antimycobacterial therapy failed to lead to a sustained response in CD patients. Accordingly, this review sought neither to confirm nor refute this, but instead to survey recent literature on the role of MAP in CD.     

Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.     

Mycobacterium avium subspecies paratuberculosis, Crohn's disease and the Doomsday scenario

Johne's disease is chronic inflammation of the intestine caused by Mycobacterium avium subspecies paratuberculosis. Infection and disease are mainly in domestic livestock but can affect many species including primates. Johne's is a new disease which emerged at the turn of the 19^th and 20^th centuries and principally involved Europe and North America. It has since spread to former low incidence regions to become a global problem. Crohn's disease is a chronic inflammation of the intestine in humans which emerged in Europe and North America mid 20^th century and increased to become a major healthcare problem. It has now spread to former low incidence regions. Infected animals shed Mycobacterium avium subspecies paratuberculosis in milk and into the environment. Human populations are widely exposed. Outcomes maybe influenced by microbial phenotype. Exposure to extracellular forms of these pathogens may confer some natural protection; exposure to intracellular forms which have passaged through milk macrophages or environmental protists may pose a greater threat to humans particularly individuals with an inherited or acquired susceptibility. Hot spots of human disease such as in Winnipeg which sits on rock at the junction of two rivers may result from local exposure to high levels of waterborne pathogens brought down from farmland. When appropriate methods are used most people with Crohn's disease are found to be infected. There are no data which demonstrate that these pathogens are harmless to humans. An overwhelming balance of probability and Public health risk favours the conclusion that Mycobacterium avium subspecies paratuberculosis is also pathogenic for people. A two tier co-operative pathogenic mechanism is proposed in Crohn's disease. Intracellular infection with the primary pathogen widely distributed throughout the gut causes an immune dysregulation and a specific chronic enteric neuropathy with loss of mucosal integrity. Segments of gross inflammatory disease result from the perturbed neuroimmune response to penetration into the gut wall of secondary pathogens from the lumen. These include both normal gut organisms and educated members of the enteric microbiome such as more aggressive E. coli. More new diseases may arise from failure to apply a range of remedial measures to this longstanding zoonotic problem.     

Mycobacterium avium subspecies paratuberculosis causes Crohn's disease in some inflammatory bowel disease patients

Crohn’s disease (CD) is a chronic inflammatory condition that plagues millions all over the world. This debilitating bowel disease can start in early childhood and continue into late adulthood. Signs and symptoms are usually many and multiple tests are often required for the diagnosis and confirmation of this disease. However, little is still understood about the cause(s) of CD. As a result, several theories have been proposed over the years. One theory in particular is that Mycobacterium avium subspecies paratuberculosis (MAP) is intimately linked to the etiology of CD. This fastidious bacterium also known to cause Johne’s disease in cattle has infected the intestines of animals for years. It is believed that due to the thick, waxy cell wall of MAP it is able to survive the process of pasteurization as well as chemical processes seen in irrigation purification systems. Subsequently meat, dairy products and water serve as key vehicles in the transmission of MAP infection to humans (from farm to fork) who have a genetic predisposition, thus leading to the development of CD. The challenges faced in culturing this bacterium from CD are many. Examples include its extreme slow growth, lack of cell wall, low abundance, and its mycobactin dependency. In this review article, data from 60 studies showing the detection and isolation of MAP by PCR and culture techniques have been reviewed. Although this review may not be 100% comprehensive of all studies, clearly the majority of the studies overwhelmingly and definitively support the role of MAP in at least 30%-50% of CD patients. It is very possible that lack of detection of MAP from some CD patients may be due to the absence of MAP role in these patients. The latter statement is conditional on utilization of methodology appropriate for detection of human MAP strains. Ultimately, stratification of CD and inflammatory bowel disease patients for the presence or absence of MAP is necessary for appropriate and effective treatment which may lead to a cure.     

Genotype profiles of Mycobacterium avium subspecies paratuberculosis recovered from suspected and Crohn's disease patients in India

Present study aimed to genotype Mycobacterium avium subspecies paratuberculosis (MAP) recovered from suspected and Crohn' s disease patients. A total of 32 MAP and DNA (directly from clinical samples) recovered from human origin were genotyped using IS 1311 PCR-REA. Isolates were cultured from stool, biopsies and blood clots of Crohn's disease patients, and stool samples of suspected (animal attendants, lab workers etc). Of the 32 MAP isolates belonging to 28 human beings, majority (84.3%) were genotyped as 'Bison type', while 21.7% were of 'cattle' and none was 'sheep' genotype. Study first time reports distribution of 'Cattle' and 'Bison type' 'genotypes in suspected and Crohn's patients on pilot scale in India. 'Bison type' genotype was predominant in the surveyed human population.     

Incidence of Mycobacterium avium subspecies paratuberculosis in a population-based cohort of patients with Crohn's disease and control subjects

OBJECTIVE:  To define the incidence of Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease (CD) and in control subjects.
METHODS:  Blood samples from 361 CD patients from a previously described population-based inflammatory bowel disease (IBD) cohort and 200 blood donor controls, of known NOD2 genotype, were screened by PCR for MAP-specific IS900 DNA. These results were correlated with NOD2 genotype.
RESULTS:  The PCR assay was capable of detecting 20 fg of purified MAP DNA, equivalent to roughly 100 MAP cells/mL of blood. MAP-specific IS900 DNA was detected in 33.8% of CD cases and 21.5% of controls (OR 1.86, 95% CI 1.247–2.785, P = 0.002). All study participants were genotyped for the NOD2 mutations 2104C>T (R702W), 2722G>C (G908R), and 3020insC (1007fs). Carriage of one or two NOD2 mutations was not associated with a significantly higher risk of CD (OR 0.75, 95% CI 0.465–1.207, P = 0.234). No significant association was seen in the CD cohort for carriage of one or two NOD2 mutations and MAP status (OR 0.883, 95% CI 0.494–1.579, P = 0.675).
CONCLUSIONS:  Screening peripheral blood using IS900 PCR indicated that MAP DNA could be detected in a significant proportion of CD cases from a large population-based cohort, and also, in control subjects. The over-representation of MAP DNA in CD suggests either a role or a probable role for MAP in the etiology of CD.     

Detection of Mycobacterium avium subspecies paratuberculosis in surgicalpathology blocks from patients with Crohn's disease in China

AIM To determine whether MAP can be detected in archival paraffin embedded full thickness samples ofintestinal tissue from patients in China with Crohn's disease (CD), ulcerative colitis (UC), and in controlsubjects (NIBD) having surgery for bowel cancer.METHODS Optimized procedures for the removal of paraffin, recovery of tissue and access to MAP DNA,followed by MAP-specific nested IS900 PCR. Confirmation of specific amplification by Southern blotting andDNA sequencing.RESULTS IS900 PCR positive tests identified MAP in 9 (69%) of 13 CD, 1 of 3 UC and 2 (14%) of 14NIBD in the presence of correctly reporting positive and negative sample and reagent control reactions. DNAsequence analysis of the 298bp IS900 PCR amplification product from MAP in 2 Chinese CD patientsdemonstrated 99% homology with the GenBank IS900 sequence accession number X16293.CONCLUSION Although larger numbers of Chinese samples need to be studied, these initial results areconsistent with an exposure of human populations in China to MAP, and an involvement of this pathogen inchronic inflammation of the intestine of the Crohn's disease type. The results are in agreement with similarpositive studies reported from China, from Western Europe and elsewhere.