New method to detect systemic MAP infections in Crohn's patients, V1
Research type
Research Study
Full title
Identification of Crohn’s disease cases with systemic Mycobacterium avium subsp. paratuberculosis (MAP) infections using the novel RAPIDvMAP assay
IRAS ID
339897
Contact name
Irene R Grant
Contact email
Sponsor organisation
Rapid-Myco Technologies Limited
Duration of Study in the UK
0 years, 11 months, 30 days
Research summary
Mycobacterium avium subsp. paratuberculosis (MAP) has long been linked with Crohn’s disease, although its precise role in the disease remains uncertain; largely due to the lack of diagnostics with sufficient speed, detection sensitivity and quantitative ability to detect the pathogen in human samples. A recently developed novel one-day phage-based method (RAPIDvMAP assay, Rapid-Myco Technologies Ltd) for detecting viable MAP in blood theoretically offers these test attributes, but feasibility of using it to detect viable MAP in human blood samples needs to be determined. During the feasibility study we propose to test buffy coat samples separated from the blood of Crohn’s patients (newly diagnosed and those with established disease) using the RAPIDvMAP assay in parallel with liquid and solid mycobacterial culture. Phage assay results will provide information on the prevalence of viable MAP in the blood of Crohn’s patients attending a NI hospital Inflammatory Bowel Disease clinic. Parallel culture results will permit an evaluation of the performance of the more rapid phage-based assay relative to culture ('gold standard'). Any MAP isolates obtained will be stored for future testing of susceptibility to antibiotics currently being used by some gastroenterologists in other parts of the world to treat MAP-infected Crohn’s patients. Ultimately, this study will: (1) provide new information on the prevalence of systemic MAP infection in a cohort of NI Crohn’s patients, and (2) provide information on the suitability of the novel phage-based RAPIDvMAP assay as a quick test for systemic MAP infection in humans.
Lay summary of study results: Identification of Crohn's disease cases with systemic Mycobacterium avium subsp. paratuberculosis (MAP) infections using a novel RAPIDvMAP assay (IRAS 339897)
Principal Investigator: Prof Irene Grant, Rapid-Myco Technologies Limited, Belfast
Co-Investigator: Mr Graham Morrison, Gastroenterologist, Royal Victoria Hospital Belfast
Sponsor: Rapid-Myco Technologies Limited, Belfast
Funding: Innovate UK (project 10106516) under the Northern Ireland Life and Health Sciences Launchpad Programme.
Start date: 1 May 2025 End date: 31 May 2026
This study was carried out to demonstrate the feasibility of using a new phage-based RAPIDvMAP assay that had been developed by Rapid-Myco Technologies Limited (a spin-out from Queen’s University Belfast) to detect systemic Mycobacterium avium subsp. paratuberculosis (MAP) infections in the blood of Northern Ireland Crohn’s disease (CD) patients (established cases and newly diagnosed cases) by separating and testing the buffy coat fraction. The new phage-based assay can detect viable MAP within 24 h of blood draw, whereas conventional mycobacterial culture takes 16 weeks or more to detect viable MAP. This was a non-intervention study.Crohn’s disease patient recruitment – Blood was obtained from a total of 205 CD patients (8 new and 197 existing, treated) attending Royal Victoria Hospital, Belfast, during a 7-month period (mid-May-early December 2025). Each gave written consent, completed a short questionnaire, and had a blood sample collected (into a 6 ml EDTA tube). We would wish to thank all CD patients who contributed to our study.
Viable MAP testing – The buffy coat fraction was separated from CD patient blood samples and subjected to the RAPIDvMAP assay and mycobacterial culture (Herrold’s egg yolk medium (HEYM) and Pozzato broth) within 24 h of blood collection. The equivalent of 2 ml of blood was tested per CD patient. Isolation of viable MAP was confirmed by testing any suspect positive broth cultures or colonies from HEYM slopes by IS900 qPCR and then subculturing onto QUB MAP medium to verify colonies obtained, i.e. viable MAP confirmed.
Results
Objective 1: to assess the analytical performance of the novel one-day phage-based assay (RAPIDvMAP) to detect viable MAP in human blood, by comparing with detection by MAP culture (solid and liquid).
MAP was successfully cultured from blood of only two CD patients, one of which also tested RAPIDvMAP positive. In contrast, the blood of 38 CD patients tested positive by the RAPIDvMAP assay overall. Agreement between the results of the two methods was ‘slight’; kappa statistic was 0.0321 (95% confidence interval -0.0.0496 to 0.1137, p-value 0.1249) Given the high number of extra MAP positives by the RAPIDvMAP assay (n=37), the two tests seem to be detecting viable MAP in different physiological states. Only a working metabolism is needed for phage amplification and burst whereas for replication and colony production a different physiological capacity is necessary. The calculated performance characteristics of the novel RAPIDvMAP assay relative to culture are: Diagnostic sensitivity (%) = 1/2 = 50.0%, Diagnostic specificity (%) = 166/203 = 81.8% and Diagnostic accuracy (%) = 167/205 = 81.5%
Objective 2: to ascertain the prevalence of viable MAP in NI Crohn’s patients by applying a novel, one-day phage-based assay (RAPIDvMAP) alongside mycobacterial culture to test buffy coat samples.
For the first time, information is available on the prevalence of systemic MAP infections in the blood of CD patients in Northern Ireland. The blood of 38 (18.5%) and 2 (1.0%) of the 205 CD patients tested positive for viable MAP by the new RAPIDvMAP assay and culture (solid or liquid, or both), respectively, and one CD patient (0.5%) tested positive for viable MAP by both tests. Therefore, the prevalence of viable MAP in the blood of NI CD patients detected by either or both tests was 19.0% (39/205).
Objective 3: to determine if a difference exists in the viable MAP detection rates between newly diagnosed Crohn’s patients and patients with established Crohn’s disease (that may have been treated with biologics or immunomodulators for variable time periods).
Unfortunately, there were insufficient numbers of new CD patient results recruited to permit a comparison of the two categories of CD patient.
Objective 4: to cryopreserve any MAP isolates obtained by PBMC culture so that they can be further characterised in the future.
As of 31 May 2026, after 6 months incubation at 37oC, two MAP isolates from patients Exist123 and New04 have been confirmed by sub-culture and stored away on cryobeads at -80oC for future DNA sequencing and antibiotic susceptibility testing.
Analysis of questionnaire results - Multivariate regression analysis was carried out on patient questionnaire responses. When viable MAP positivity by the RAPIDvMAP assay was the outcome considered, taking other medication (namely 5-ASAs or JAK inhibitor) was the only significant variable showing a negative association with MAP positivity (p=0.0161, coefficient -0.11822), with one other variable (Living in urban v rural location) approaching significance (p=0.0773, coefficient 0.427).How has this study helped patients and researchers?
This study has revealed that 19% of NI CD patients tested had evidence of systemic MAP infections and has demonstrated that the new phage-based RAPIDvMAP assay could be a useful test for CD patients to inform treatment decisions (anti-MAP antibiotic therapy is available). MAP cells circulating in the blood of CD patients, and particularly those treated with biologics or immunosuppressants, may not have been in a fully competent physiological state at point of testing, so the possibility that MAP recovered from human blood may benefit from a ‘resuscitation step’ before applying either RAPIDvMAP or culture merits further investigation.REC name
Wales REC 6
REC reference
24/WA/0143
Date of REC Opinion
20 Aug 2024
REC opinion
Favourable Opinion