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Keywords:

  • Mycobacterium avium subspecies paratuberculosis;
  • etiology;
  • Crohn's disease

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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.

(Inflamm Bowel Dis 2007)

The histopathological and clinical similarities between human chronic granulomatous enteritis,1, 2 intestinal tuberculosis, and animal paratuberculosis3 has led to the suspicion that Mycobacterium avium subspecies paratuberculosis (MAP) has a role in the etiology of Crohn's disease (CD). The literature on the pathogenesis, pathophysiology, and clinical management of CD has evolved considerably over the last decade due to advances in molecular biology and genetics. Despite considerable medical debate, the etiology of CD remains an enigma. However, there is a consensus that CD results from a sustained immune response arising from an environmental stimulus in a susceptible host.4 MAP has been postulated to be a potential stimulus at least in a subset of patients.5

Unlike Mycobacterium tuberculosis, MAP is fastidious and slow-growing, with nutritional dependence for the iron chelating compound mycobactin.5 In vitro culture techniques have improved significantly in the last decade. Grant et al6 compared Mycobacterium Growth Indicator Tube (MGIT) and BACTEC culture media for the recovery of MAP from milk and found both media were able to detect 10 organisms per mL of milk. However, despite these improvements in culture media it remains a difficult organism to isolate from humans, for unclear reasons that may include mycobacterial dormancy or low bacillary burden. Similarly, serological assays are unlikely to provide the evidence required, as they are not reliable for the diagnosis of Johne's disease, a disease of animals caused by MAP.7

A number of studies have reported the more frequent detection of the insertion sequence IS900 in CD patients compared with controls using polymerase chain reaction (PCR) assays.5, 8 In contrast, other authors found no association between CD and the detection of IS900.9, 10 The 2 main methods that have been used include PCR and in situ hybridization (ISH) assays. Although other IS900-like elements have been described in environmental mycobacteria,11, 12 the entire IS900 gene is unique to MAP.13 The short fragments of the IS900 sequence targeted in various studies may not be unique, as evidence of partial cross-hybridization between M. avium strains has been reported in AIDS patients.14 Multiplex PCR simultaneously targeting several fragments of the IS900 gene has been developed to overcome this difficulty.15 In addition, crosshybridizing elements can be distinguished from IS900 by optimized sample processing and through the design of primer pairs.16

Various reviews have been conducted examining the MAP hypothesis that suggest that although there is insufficient evidence, a causal link in a subset of CD patients cannot be excluded.17 There has, however, been no formal systematic review and meta-analysis assessing all the studies using PCR and ISH assays. We present a systematic review of all published studies and, where appropriate, meta-analysis of studies using PCR and ISH to detect MAP in patients with CD and controls.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Search Strategy

Database searches of MEDLINE (1966 to January 2006), Embase (1980 to January 2006), Biosis (1993 to January 2006), Cochrane Library (2006 Issue 1), and Web of Science were conducted using keywords for the organism (Mycobacterium avium paratuberculosis, paratuberculosis, Mycobacterium paratuberculosis) and “Crohn's disease.” Other databases searched include Database of Abstracts and Reviews and Conference proceedings (World Congress of Gastroenterology, British Gastroenterology Society, United European Gastroenterology Week and American College of Gastroenterology). Bibliographies of published articles including previous reviews were checked for additional studies. The search was limited to studies conducted on human beings and no language restrictions were used. We contacted authors who have published studies relevant to PCR and ISH assays for the detection of MAP in CD patients and relevant material requested.

Inclusion Criteria and Data Extraction

All titles and abstracts from each of the searches were examined. The full text of each article was obtained and reviewed if the study compared the frequency of detection of MAP among CD patients with controls using PCR or ISH assays. A reviewer (I.A.) extracted data from each study. Any study without a control group was excluded.

Quality Assessment

The quality of each study was appraised using a form adapted from http://www.medepi.net/meta/. No studies were excluded on the basis of quality, but rather the criteria were used to check for evidence of heterogeneity in the analysis.

Statistical Analysis

Risk difference was estimated from the selected studies and pooled using meta-analysis. If significant heterogeneity (P < 0.20) between study results was evident using a chi-square test, a random effects model was used to calculate the summary risk difference. Meta-regression analysis was used to investigate the effect of heterogeneity due to study characteristics including measures of quality. This allowed us to assess the extent to which covariates affect the random effects summary measure estimate. Factors likely to result in differences in the ability of assays to detect MAP were agreed on a priori such as the inclusion of children, study year, reported inclusion of patients on immunosuppressive therapy, use of nested PCR, mechanical disruption and laser dissection, blinding, use of positive and negative controls, and the presence of granulomas in tissue samples. Publication bias was assessed based on funnel plots and a P-value of less than 0.20 using Begg's test.18 Statistical terms used in the article are explained in the box. All analyses were conducted using the Stata software (v. 9; StatA Corp., Texas).

Risk difference: the main outcome measure used in this study, is defined here as the difference between the risk in the Crohn's disease group (proportion of positive results) and the risk in the control group (proportion of positive results). The risk difference describes the actual difference in the probability of a positive result observed among Crohn's disease patients and controls.

Funnel plot: a method used to assess publication bias. It is a simple scatterplot of the study effect, risk difference in this case, estimated from individual studies against some measure of each study's sample size (standard error of the risk difference in this meta-analysis). In the absence of bias the plot should resemble a symmetrical funnel.

Statistical heterogeneity: excessive variation between studies beyond what would be expected by chance alone. The difference is usually due to clinical and/or methodological diversity among the studies.

Random effects model: assumes that the effects being estimated in the different studies are not identical, but follow some distribution. The center of this symmetric distribution describes the average of the effects, while its width describes the degree of heterogeneity. This type of model is usually used in the presence of heterogeneity.

Ethical Considerations

This study is a systematic review of the literature with no patient contact and does not raise any ethical issue of note.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The study selection procedure is outlined in Figure 1. Fifty-eight full text studies were reviewed, 47 of which were included in the analysis. Two studies reported the assessment of both a PCR and an ISH assay.19, 20 Therefore, a total of 49 studies were analyzed. The median study population size was 51 individuals, with a range of 5 to 200.

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Figure 1. Flow chart outlining the study selection process.

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Study Characteristics

Table 1 summarizes the characteristics of the included studies. The assessment of study quality was limited by the amount of information reported in published articles; however, additional data were obtained from authors (10/47) where possible. The studies are reported from laboratories in 15 countries. Forty-three used PCR, while 6 studies reported analysis of in situ or DNA hybridization. In 2 studies the only available comparison group were patients with ulcerative colitis (UC).21, 22

Table 1. Characteristics of Studies and Risk Difference for the Detection of Mycobacterium paratuberculosis in Crohn's Disease Patients Compared to Non-Inflammatory Bowel Disease Controls.
Study, Year (References)Assay Type (Target Sequence)Inclusion of ChildrenMechanical DisruptionGranulomaControlsImmunosuppressionBlindingCases (Positives/Total)Controls (Positives/Total)Risk Difference (95% Confidence Interval)Location
  1. CSLM, confocal scanning laser microdissection; RT, reverse transcriptase; OFG, orofacial granulomatosis.

PCR assays           
Rosenberg 199140IS900Not statedNot statedNot statedNot statedNot statedNot stated(0/21)(0/6)0.00 (−0.20–0.20)Oxford, UK
Sanderson 199241IS900YesNoYesYesNot statedNo(26/40)(5/40)0.53 (0.35–0.70)London, UK
Moss 199242IS900Not statedNoYesYesNot statedNot stated(6/18)(1/6)0.17 (−0.20–0.54)London, UK
Wall 199343IS900Not statedNoNot stated.YesNot statedNo(6/28)(0/13)0.21 (0.03–0.39)Surrey, UK
Cellier 199344IS900NoYesNot statedNot statedYesNo(0/45)(0/18)0.00 (−0.08–0.08)Paris, France
Dell Isola 199425IS900NoYesNot statedYesYesYes(13/18)(9/30)0.42 (0.16–0.69)Paris, France
Lisby 199445NestedYesNoYesYesYesNo(11/24)(3/28)0.35 (0.12–0.58)Copenhagen, Denmark
Fidler 199446IS900NoNoYesYesNot statedYes(4/31)(0/20)0.13 (−0.01–0.27)Surrey, UK
Rowbotham 199536IS900YesNoNot statedYesYesNot stated(0/68)(1/26)−0.04 (−0.13–0.05)Leeds, UK
Murray 199537IS900NoNoNot statedYesNot statedYes(2/9)(0/15)0.22 (−0.06–0.50)Palmerston N, NZ
Suenaga 199547IS900NoNoYesNot statedNot statedNot stated(10/10)(14/16)0.13 (−0.08–0.33)Kochi, Japan
Erasmus 199548IS900Not statedNot statedYesYesNot statedNot stated(10/26)(4/34)0.27 (0.06–0.48)Tygerberg, SA
Kreuzpainter 19952316s rDNAYesNoNot statedNot statedYesNo(0/4)(0/1)0.00 (−0.66–0.66)Hamburg, Germany
Mishina 199649RT IS900NoNoNot statedYesYesNot stated(8/8)(0/2)1.00 (0.55–1.45)New York, US
Dumonceau 199650Nested IS900NoNoNot statedYesNot statedNot stated(0/36)(0/23)0.00 (−0.07–0.07)Brussels, Belgium
Frank 19969Nested IS900NoNoYesYesNot statedNo(0/23)(0/11)0.00 (−0.13–0.13)Michigan, US
Gan 199751IS900Not statedNoYesYesNot statedNot stated(17/36)(3/20)0.32 (0.10–0.55)Chengdu, China
Al Shamali 199752IS900NoNoVarious typesYesNot statedNot stated(0/10)(0/21)0.00 (−0.14–0.14)Kuwait, Kuwait
Huatian 199753IS900Not statedNoYesYesNot statedNo(17/36)(3/20)0.32 (0.10–0.55)Chengdu, China
Riggio 199754Nested IS900Not statedNoOral tissue–yesYesNot statedNot stated(0/7)(0/12)0.00 (−0.20–0.20)Glasgow, UK
Clarkston 199855IS900NoNoYesYesNot statedYes(1/21)(0/11)0.05 (−0.11–0.20)Kansas City, US
Kallinowski 199856IS900NoNoNot statedYesNot statedNot stated(0/21)(0/24)0.00 (−0.08–0.08)Heidelberg, Germany
Del Prete 199857IS900YesNot statedStool samplesYesNot statedYes(15/32)(1/41)0.44 (0.27–0.62)Bari, Italy
Chiba 199858Nested IS900YesNot statedYesYesNot statedNot stated(0/30)(0/3)0.00 (−0.33–0.33)Akita, Japan
Cellier 199824Nested IS900NoYesYesYesYesNo(2/47)(2/20)−0.06 (−0.20–0.09)Paris, France
Kanazawa 199910IS900NoNoNot statedYesNot statedNot stated(0/13)(0/13)0.00 (−0.14–0.14)Hirosaki, Japan
Collins 200059Nested IS900NoNoNot statedYesYesYes(15/79)(3/48)0.13 (0.02–0.24)Wisconsin, US
Schwartz 2000IS900Not statedNoNot statedYesNot statedNot stated(10/27)(1/15)0.30 (0.08–0.53)US
Gibson 200060IS900Not statedNoOFGYesNot statedNot stated(0/24)(0/12)0.00 (−0.12–0.12)Glasgow, UK
Hulten 2001b19IS900NoNoYesYesNot statedYes(9/36)(0/22)0.25 (0.10–0.40)Houston, US
Ryan 200238Nested, CSLM IS900Not statedNoYesYesNot statedNot stated(6/15)(0/12)0.40 (0.14–0.66)Cork, Ireland
Fujita 200231IS900NoNot statedYesNot statedNot statedNot stated(0/16)(0/18)0.00 (−0.11–0.11)Tokyo, Japan
Ellingson 200361IS900Not statedNot statedYesYesNot statedNot stated(0/35)(0/21)0.00 (−0.07–0.07)Iowa, US
Bernstein 200362Nested IS900NoNoYesYesNot statedYes(0/24)(6/28)−0.21 (−0.38–−0.05)Manitoba, Canada
Bull 200316Nested IS900YesYesYesYesYesYes(34/37)(9/34)0.65 (0.48–0.83)London, UK
Naser 20048Nested, IS900NoNot statedBlood samplesYesYesYes(13/28)(3/15)0.26 (−0.01–0.54)Orlando, US
Sechi 2004b20IS900Not statedNot statedNot statedYesNot statedNot stated(8/82)(0/20)0.10 (0.01–0.19)Sassari, Italy
Sechi 200527IS900Not statedNot statedNot statedYesNot statedNot stated(25/37)(7/34)0.47 (0.27–0.67)Sassari, Italy
Sechi 200528IS900YesYesNot statedYesYesNot stated(25/30)(3/29)0.73 (0.56–0.90)Sassari, Italy
Romero 200563Nested CSLM IS900NoNoNot statedYesYesNo(10/12)(1/6)0.67 (0.30–1.03)Orlando, US
Autschbach 200529Nested IS900NoYesYesYesYesYes52/100)(5/100)0.47 (0.36–0.58)Heidelberg, Germany
Pooled risk difference         0.20 (0.12–0.28) 
Hybridization           
Butcher 198864pMB21 & pMB22Not statedNoNot statedYesNot statedNo(0/17)(0/4)0.00 (−0.27–0.27)London, UK
Yoshimura 198865DNA DNANot statedNoNot statedNot statedNot statedNot stated(10/19)(1/6)0.36 (−0.01–0.73)Houston, US
McFadden 199266DNA IS900 pMB22Not statedNoNot statedNot statedNot statedNot stated(2/82)(0/26)0.02 (−0.04–0.09)Surrey, UK
Hulten 200167insitu IS900NoNoYesYesNot statedYes(7/37)(0/22)0.43 (0.26–0.60)Houston, US
Sechi 200172In situ IS900 p89 & p92Not statedNot statedYesYesNot statedNot stated(27/33)(0/20)0.82 (0.67–0.97)Sassari, Italy
Sechi 200420In situ IS900 p89 & p92Not statedNot statedNot statedYesNot statedNot stated(73/82)(0/20)0.89 (0.80–0.98)Sassari, Italy
Pooled risk difference         0.43 (0.01–0.84) 

Forty-two PCR studies used the IS900 target sequence, while 1 used 16s rRNA.23 Four hybridization assays used the insertion sequence IS900 as the target. The methods used for DNA extraction also differed. Table 1 outlines the variation in quality between studies. Several measures that may enhance detection of MAP in humans, such as mechanical disruption of tissue and the use of nested PCR, were not used in many studies. Six studies had an additional step of mechanical disruption.16, 24–27 Eleven (25.6%) PCR studies and 1 (16.7%) hybridization study reported blinding of the investigator as to the source of tissues or samples. All studies describe measures to reduce the probability of cross-contamination; hence, we could not investigate this as a source of heterogeneity.

All Studies

A meta-analysis of all 49 studies (including 2 studies with both PCR and hybridization tests) revealed evidence of significant heterogeneity. The pooled estimate of risk difference was 0.23 (95% confidence interval [CI], 0.14–0.32) using a random effects model. The studies were divided into the 2 main test groups (PCR and hybridization assays).

Sensitivity analysis was carried out by excluding studies with no evidence of MAP from any CD patient or control. Thirty-two studies detected the organism in at least 1 person. Among these studies a risk difference of 0.33 (95% CI, 0.22–0.45) was estimated. Figure 2 shows the forest plot of the risk difference from the reviewed studies.

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Figure 2. Forest plot for risk difference in the detection of Mycobacterium paratuberculosis in CD patients compared with non-IBD controls using PCR and DNA hybridization assays. Each solid box represents the risk difference estimate from a study with error bars indicating 95% confidence intervals. The dotted vertical line is the random effects summary estimate of risk difference and 95% confidence intervals for overall effect is represented by the diamond box.

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PCR Studies

Thirteen studies showed a risk difference of zero. Three studies showed a negative risk difference suggesting greater detection of MAP in control specimens, although only 1 was statistically significant. The full range of risk difference estimates varied from 0.21 to 0.73.

There was statistically significant evidence of heterogeneity (chi-square = 468.99, P < 0.001). The summary measure of risk difference from a random effects model was 0.20 (95% CI, 0.12–0.28) and remained almost identical after excluding the study by Kreuzpaintner et al,23 which did not use IS900 (0.20; 95% CI, 0.12–0.28). Similarly, exclusion of any single study did not significantly alter the summary measure. When the analysis was limited to studies investigating tissue samples only (i.e., excluding blood), a risk difference of 0.23 (95% CI, 0.10–0.29) was observed.

Hybridization Studies

There was significant evidence of heterogeneity (chi-square = 301.29, P < 0.001); therefore, a random effects model was used. The summary measure of risk difference was 0.43 (95% CI, 0.01–0.84).

Detection of MAP in CD Compared with UC

Thirty-eight studies had data comparing samples from CD and UC patients. The pooled risk difference was 0.19 (95% CI, 0.10–0.28).

Investigating Heterogeneity and Publication Bias

We used a regression analysis model to investigate potential sources of heterogeneity. Factors considered a priori to be potential sources of heterogeneity were chosen either because they relate to the ability to detect MAP or can lead to a bias in estimating the measure of association. Table 2 shows the results of the meta-regression model for all studies, PCR assays and hybridization tests. The year of study, type of assay, and inclusion of children in the study were associated with the outcome measure. The positive association suggests that more recent studies, those that include children and PCR studies, were more likely to report a positive result.

Table 2. Meta-regression of the Relationship of Risk Difference and Study Characteristics in 49 Studies Comparing the Detection of M. paratuberculosis in Crohn's Disease and Control Patients.
VariableYesNoMissingCoefficientP-value
  1. CSLM, confocal scanning laser microdissection; PCR, polymerase chain reaction.

Inclusion of children921190.220.045
PCR versus hybridization43600.240.040
Year   0.020.027
Granuloma26221−0.010.877
Blinding1210270.070.485
Immunosuppression13036−0.020.118
CSLM247 0.300.164
Mechanical disruption629140.210.056
Nested PCR1336 −0.500.598
Controls409 0.190.099

The funnel plots for publication bias showed evidence of asymmetry (Fig. 3) (Begg's test; z = 3.08, P = 0.002).

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Figure 3. Funnel plot for the assessment of publication bias in all studies. Each circle represents a study in the meta-analysis. The central line represents the random effects summary risk difference. The presence of studies outside the funnel-shaped lines diverging from the summary effect is suggestive of publication bias. This is supported by the statistically significant results of the Begg's test. RD, risk difference; SE, standard error.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The results suggest that there is sufficient evidence for the presence of MAP in the gut of patients with CD. A summary risk difference of 0.23 (95% CI, 0.14–0.32) was calculated, which remained consistent after sensitivity analysis excluding various sets of studies. Earlier studies have suggested that MAP may also be found among controls but recent investigations using appropriate extraction techniques and more sensitive assays have revealed a significantly higher level of detection by PCR assays in CD patients compared with controls.16, 20, 27, 28, 29

Interpreting the Findings

The pathogenic role of MAP in CD is uncertain. Our analysis reveals a high strength of association with a reasonable level of consistency among recently conducted studies. However, for any agent to be considered a cause of a disease, evidence that the infection precedes the pathology is essential. The studies in this review do not provide evidence of temporality in the association.

The strength of the association and consistent detection of MAP DNA does not prove causation, and may arise from prior ingestion of nonviable organisms in water or milk. However, the detection of MAP DNA in the blood of CD patients8 may suggest that a viable form of the organism is present in CD. This study by Naser et al did not report the detection of MAP in intestinal tissue, nor explore the possibility that the results are a secondary phenomenon due to increased gut permeability or the inability of macrophages in CD to kill MAP. The increased gut leakiness hypothesis is supported by the more frequent detection of other organisms in CD patients compared with controls30, 31 and suggest a lack of specificity of MAP detection. For instance, the risk difference for Bacteriodes and Escherichia coli from the Fujita et al31 study was 0.5 and 0.4, respectively. In contrast, the frequent detection of MAP in CD compared with UC argues against increased gut permeability as the sole explanation.

Subgroup analyses were undertaken since IS900-based ISH has been discredited as a nonspecific tool.32 Therefore, the results of PCR assays were considered more reliable. Similarly, the analysis was restricted to those studies investigating intestinal tissue. This did not alter the findings.

It has been suggested that most of the positive results have been reported by a limited number of laboratories.33 However, data from this review indicates that positive results have been observed by several laboratories in different parts of the world. Low assay sensitivity used by certain laboratories may contribute to this observation, whereby only groups that have developed sufficiently sensitive tests are successful. Conversely, many studies detect MAP DNA in the control patients. This may be because MAP is potentially common in the environment. Alternatively, this may suggest that it is not pathogenic to all exposed individuals.

Strengths and Weaknesses

A comprehensive search strategy was used in this review. However, despite this, significant evidence of publication bias was found, suggesting that not all studies were identified.

In addition to presenting summary measures alone, meta-regression analysis was used to explore heterogeneity. Factors included in the meta-regression model were agreed a priori. However, limited availability of data hampered the exploration of sources of heterogeneity, for example, the effect of factors such as contamination in the meta-regression analysis. Nevertheless, this review investigated important factors related to the variation in the results of published studies.

This review does not systematically identify and critique other studies that may be important in the understanding of the role of MAP in CD such as the use of antimycobacterial therapy. This is the subject of a Cochrane review34 that showed that antibiotics may have a role in maintaining remission. The results of the largest randomized controlled trial to date does not support the use of antibiotics in CD.35

Exploring Heterogeneity

The aim of a review should not be limited to producing a summary estimate of divergent results but also to explore the reasons for the observed differences. Heterogeneity is inevitable in a meta-analysis of observational studies. Furthermore, the studies were carried out over a long time period during which laboratory methods evolved. A significant positive association with year of study with more recent studies more likely to have positive results was identified. This is very likely a proxy measure of changes over time in the assay techniques. The sensitivity of assays improved over the last 2 decades due to a combination of better extraction methods and enhanced detection limits. A significant positive association was observed when studies including children were compared with those with an adult population. It has been suggested that if the initial MAP infection occurs in childhood, its detection in studies among children25 would be more likely. This implies that earlier in the course of the disease, MAP is present. However, others have found no difference in the detection of MAP based on age using PCR.36 Other factors considered a priori to be potential causes of heterogeneity were not statistically significant.

Due to the hypothesized paucibacillary nature of MAP infection in CD, it is possible that there are very low numbers of infecting organisms, below the detection limits of the assay. Limiting the studies in the meta-analysis to those that have detected MAP in at least 1 sample either from a patient with CD, UC, or a control did not alter the results.

The exclusion of the study with a different target sequence23 did not alter the results. A number of studies included immunosuppressed patients. The results of this review showed no association with reported use of immunosuppressive agents in the study population; however, the majority of studies did not explicitly look for an association. Authors reporting negatives results have argued that contamination is a major factor in the detection of MAP.24 Unfortunately, due to variation in techniques for reducing contamination and limitations of the data available in published articles, this factor was not assessed. The use of appropriate controls and the meticulous steps taken by most of the studies suggest that this is unlikely to be the explanation for the detection of MAP.

There are other factors that may be associated with differences in the ability of the studies to detect MAP that we did not investigate. These include limitations of sampling techniques, failure to process fresh tissue samples (fixing results in fragmentation of DNA), and the role of concomitant use of antimicrobial drugs. Tissues from operative procedures provide more quantity and reduce sampling errors, but it is unclear whether patients with early lesions are more likely to have MAP compared with those that have developed complications requiring surgery.37 Sampling error due either to low abundance of MAP in tissues or lower numbers in nongranulomatous tissue has been suggested as an explanation for studies failing to detect MAP. Laser capture microdissection techniques have been used to specifically isolate granulomas that were subsequently assayed, resulting in the detection of MAP more frequently in CD patients compared to controls.38 However, the authors of the same report detected the DNA of other bacteria more frequently in CD tissue compared to controls using the same technique.39 Furthermore, other studies24, 36 that include intestinal granulomas and lymph nodes failed to detect MAP, while some detected the organism in various sites including noninflamed tissue.41 This meta-analysis did not show that investigating granulomas is a significant source of heterogeneity. Considerable heterogeneity remained after controlling for the effect of all the factors investigated.

It is unlikely that similar studies using PCR assays are going to change the current state of evidence. 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. While the summary effect measures from this study show an association between CD and MAP, this association remains controversial and inconclusive. Future studies should determine whether there is a pathogenic role. Such studies should also assess the evidence of temporality and specificity of the association.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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    Cousins DV, Whittington R, Marsh I, et al. Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis. Mol Cell Probes. 1999; 13: 431442.
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    Englund S, Bolske G, Johansson KE. An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol Lett. 2002; 209: 267271.
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    Bartos M, Hlozek P, Svastova P, et al. Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards. J Microbiol Methods. 2006; 64: 333345.
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    Naser S, Shafran I, El Zaatari F. Mycobacterium avium subsp. paratuberculosis in Crohn's disease is serologically positive. Clin Diagn Lab Immunol. 1999; 6: 282.
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    Bull TJ, Hermon-Taylor J, Pavlik I, et al. Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing. Microbiology. 2000; 146(Pt 12): 3285.
  • 16
    Bull TJ, McMinn EJ, Sidi-Boumedine K, et al. Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease. J Clin Microbiol. 2003; 41: 29152923.
  • 17
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