Human Para’s inaugural MAP testing study is nearing completion. A total of 201 participants donated a blood sample at locations in Orlando, Philadelphia and New York City between May and September 2018. The final samples were drawn on September 10, 2018.
Since this was a blinded study comprised of both IBD patients and control subjects, each sample was assigned a number and sent to 6 participating researchers. The laboratories of Dr. Saleh Naser, Dr. Tim Bull and Dr. Irene Grant received buffy coats (the white blood cell layer) which were extracted at the Temple University laboratory. They tested all positive culture samples for two PCR markers: IS900 and f57. More detailed methodology follows:
Methodology
Dr. Saleh Naser: Peripheral blood leukocytes (PBL) were harvested from the Buffy coats of each specimen and were inoculated into BACTEC MGIT ParaTB medium with supplements but without antibiotics and incubated for 6 months at 37 C. After incubation, the culture was centrifuged, DNA extracted from the pellet, and nested IS900 PCR was performed. Subcultures were done on all positive MGIT cultures to attempt recovery of MAP in pure culture.
Dr. Tim Bull: The Buffy coats were prepared for culture using the proprietary TiKa culture method. The specimens were then incubated tightly sealed and static at 37°C for 2 days, after which they were inverted to mix and transferred into a MGIT tube containing MGIT-PANTA antibiotics, Mycobactin J (Final 2µg/ml), TiKa supplement A, M1, M2 and M3. Samples were incubated and read over 42 days using the automated MGIT320 system. Samples were tested for IS900. All positive samples were aliquoted onto TiKa solid media to obtain colonies suitable for long term collection/storage and further typing analysis.
Dr. Irene Grant: The phage amplification assay was adapted to test the human blood samples for the presence of viable MAP. Upon receipt of samples, peripheral blood mononuclear cells (PBMCs) were centrifuged and resuspended in 1 ml Middlebrook 7H9 broth supplemented with 10 % OADC (both Difco) and 2 mM CaCl2 (Sigma), the osmotic shock of which resulted in lysis of the PBMCs and release of internalized MAP cells, making them available for detection by the phage amplification assay or culture. The phage assay then proceeded and mycobacteriophages were added to each sample to infect any MAP cells present. Samples were then incubated at 37 C. After 2 hours, the extraneous seed phages were inactivated by addition of a chemical virucide followed by neutralisation, and the sample was returned to the incubator until a total of 3 hours and 30 minutes had elapsed from addition of the phages. The sample was then plated with Mycobacterium smegmatis sensor cells and 5 ml molten Middlebrook 7H9 agar in Petri dishes. The samples were incubated overnight at 37 C and examined next day for evidence of zones of clearing (‘plaques’), the presence of which would indicate viable mycobacteria in the sample.
In order to confirm the identity of the Mycobacterium species that had given rise to the plaques in a sample, up to 10 plaques per sample were collected from positive agar plates. DNA was extracted from the plaques and an IS900 PCR carried out to confirm the presence or absence of MAP DNA. Isolation of MAP by culture in Pozzato medium was also attempted. Cultures were incubated at 37 C for up to 16 weeks and optical density was measured every two weeks. Whenever an increase in optical density was observed, an aliquot of the culture will be subjected to Ziehl-Neelsen (acid-fast) staining. If acid-fast cells are observed, a MAP-specific PCR will be performed. If this PCR is positive, attempts will be made to obtain colonies of MAP from the sample.
Dr. Raghava Potula: The plasma sample for each patient was assayed using IDEXX Mycobacterium paratuberculosis antibody test kit for detection of antibody to MAP in bovine serum, plasma and milk. This kit was adapted for human use as described previously by Brenstein et al (Journal of Clinical Microbiology, Mar. 2004, p. 1129–1135). Human plasma controls optical density (OD) values were used to calculate sample/positive (S/P) ratios and interpret the assay.
Dr. Horacio Bach: Plasma samples were tested for antibodies to antigens, including PtpA, PknG and CL1, using previously published methods. ELISA (enzyme-linked immunosorbent assay) was also performed. All the experiments were performed in triplicate.
Dr. Peilin Zhang: Antibody testing was performed on all samples. An aliquot of plasma/serum was diluted 1:50, and the diluted samples added into 96-well plate previously coated with specific recombinant antigens from various microbes (S. aureus, MAP, and E. coli). The diluted samples were incubated with the recombinant antigens in the presence of 5% BSA for 1 hour at the room temperature, and the plate was washed with PBST for 5 minutes for three times. The secondary anti-human IgG conjugated with horseradish peroxidase (1:4000 in 5% BSA) was added to the 96-well plate for 1 hour at the room temperature, and the plate was washed with PBST for 5 minutes for three times. The TMB reagent was added and incubated for 5 minutes at room temperature. The color development was stopped by 2N sulfuric acid and the data was obtained at a reading of OD450 using a plate reader.
Data Analysis
We are pleased to report that all 6 locations have completed testing and results have been reported to our principal investigator, Dr. Todd Kuenstner. Since testing is now complete, the study was recently unblinded. The Temple research team is currently analyzing the data from the study and will perform statistical analyses to determine what this data tells us about mycobacteria in Crohn’s, other immune conditions and control patients. They will also compare the testing methods side by side to assess the benefits and drawbacks of each.
During the initial analysis, it became apparent that more information about BCG vaccination status would be helpful. In May, Dr. Kuenstner reached out to participants via email with two additional questions to supplement the intake form that they completed.
Stage 2 Whole Genome Sequencing Extension
A secondary portion of the study which may be critical to understanding the data is the ability to conduct Whole Genome Sequencing (WGS) on mycobacterial isolates obtained from patients. (NOTE: Genomic sequencing will NOT be performed on the participant’s DNA, but only on the mycobacteria which was isolated from the participant’s blood sample.)
WGS can rapidly sequence whole bacterial genomes. This technology will allow the researchers to definitively identify the isolated mycobacteria, or may provide DNA evidence of a new mycobacterial species. 32 samples from the labs of Dr. Bull, Dr. Naser and Dr. Grant have been identified as of particular interest and WGS is currently being performed on those samples in Dr. Bull’s lab.
In addition, 8 participants who were MAP positive by Dr. Grant’s phage assay have provided a second blood sample for retesting at Dr. Grant’s lab. If any of these redrawn samples are again MAP positive, and sufficient DNA can be obtained, we will then conduct WGS on the mycobacteria obtained at Dr. Bull’s lab.
A MAP positive culture at a single point in time allows for the possibility that MAP bacteremia in a subject is due to an acute infection that is subsequently cleared by the host. MAP bacteremia demonstrated in 2018, and then again in a redrawn sample in the same host a year later demonstrates that this infection is persistent and cannot be cleared by the host.
The correlation of Dr. Grant’s Pozzato cultures with the IS900 positive MAP/phage positive samples is good evidence that the MAP/phage assay identifies viable MAP organisms. However, the incontrovertible and highest level of proof that Dr. Grant’s phage assay in fact detects MAP (or other non-contaminating mycobacteria) is to sequence the DNA harvested from the MAP/phage assay plaques. This process is a direct proof, and move MAP research ahead by leaps and bounds.
When Will the Study Conclude and the Results be Published?
As mentioned above, the researchers are analyzing the raw data from the first phase of the study and have begun performing genomic sequencing on up to 40 samples. This process will take an additional 2-3 months, but the researchers and Human Para felt that the delay to conduct WGS justified extending the study. Scientific research is not always predictable. Weighing all of the alternatives, we decided to extend the study and delay the results in order to produce the best, most evidence-based piece of research possible, rather than rushing to judgment. Our goal has always been to serve our community of patients and doctors to the best of our ability.
An article setting out the Stage 1 study findings is being drafted while the Stage 2 WGS is conducted. Once Stage 2 is completed, these results will be added into the publication which will be submitted to a medical journal. Every journal has a team of scientists who review submitted articles. These editors decide whether to accept or reject the article for publication. Sometimes the editor will request clarification on a specific point, or ask that portion of the article to be rewritten for clarity or accuracy. This process of fine tuning the article can delay publication. Due to the uncertainty in this process, we can only estimate the publication date at this time.
With the information we have available now, we hope to have the results published in Spring 2020.
When Will I get my Test Results?
We understand and appreciate the dedication of the study participants, and their interest in obtaining their personal test results. We are diligently working to get these results to participants as soon as possible.
Releasing individual results prior to the article being submitted for publication (or published) would severely jeopardize the study’s credibility. Medical journals will only accept articles which contain unpublished data. If we released individual results early, the journal may consider this “publication” and may refuse to publish our study. Since this study is critical to the future of MAP science, a conservative approach on the decision to release individual results is wisest.
We know our participants are patiently waiting for their own results, and we thank you for your willingness to help MAP science and Human Para.
Thank You!
A huge THANKS to the researchers, participants and doctors who have contributed to this landmark study, particularly our Principal Investigator, Dr. J. Todd Kuenstner, who has worked tirelessly for the last 18 months to coordinate hundreds of details and data points. Special thanks goes out to Dr. and Anita Shafran who collected the majority of the patient samples a month prior to retirement. To the Joly family and the Denver, NC community for hosting a golf fund raiser which provided the resources necessary to get this study off the ground. And to the entire Human Para community for donating their time, talents and funds to make this endeavor possible. Together we are stronger!