# US 12,007,392 B2 · Method and system for detection of mycobacteria in human samples

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- Number: US 12,007,392 B2
- Document ID: US12007392B2
- Status: Granted
- Kind: B2
- Application number: 16/585,391
- Filed: 2019-09-27
- Published: 2020-04-02
- Granted: 2024-06-11
- Earliest claimed: 2018-09-27
- Assignees: Temple University of the Commonwealth System of Higher Education
- Applicants: Temple University—Of The Commonwealth System of Higher Education; Queen's University Belfast; St. George's, University of London; University of Central Florida
- Inventors: Irene Grant; Antonio Foddai; John Todd Kuenstner; Raghava Potula; Tim Bull; Saleh Naser; Ira Shafran
- Web page: https://patentgrep.com/patent/US12007392B2

## CPC

- C12Q 1/06: Quantitative determination
- G01N 33/5695: {Mycobacteria}
- C12Q 1/689: for bacteria
- G01N 2333/35: from Mycobacteriaceae (F)
- G01N 2800/065: Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
- G01N 2800/56: Staging of a disease; Further complications associated with the disease
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention provides a semi-quantitative phage-based assay for Mycobacterial infection, in which the phage assay plaque count correlates to the number of viable Mycobacterial organisms in the subject sample, and methods of use thereof for diagnosing, treating or monitoring Mycobacterial infection.

## Claims

### Claim 1

A method of treating Crohn's Disease in a subject in need thereof comprising the steps of:
a) obtaining a human blood or peripheral blood mononuclear cell (PBMC) sample from a subject diagnosed with Crohn's Disease;
b) lysing cells of the sample to release any viable target mycobacterial cell;
c) admixing a broad host range mycobacteriophage with the sample under conditions suitable to allow the bacteriophage to infect any viable target mycobacterial cell present in the sample;
d) removing any bacteriophage not infecting a viable target mycobacterial cell;
e) admixing at least a portion of the admixture of step c) following the removal of step d) with bacteria capable of supporting replication of the bacteriophage;
f) plating at least a portion of the admixture of step e) on a substrate to support growth of the bacteria;
g) quantifying the number of bacteriophage plaques that form;
h) discriminating between infected and asymptomatic subjects, comprising identifying the subject as infected when the number of bacteriophage plaques is statistically significantly greater than a reference number of bacteriophage plaques, or as asymptomatic when the number of bacteriophage plaques is not statistically significantly greater than a reference number of bacteriophage plaques, wherein the reference number of bacteriophage plaques is a reference number of plaques in asymptomatic human hosts; and
i) administering a pharmaceutical composition for inhibiting or disrupting microorganism growth or treating a microbial infection to a subject identified as infected.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cells are lysed by osmotic shock.

### Claim 3 (depends on 1)

The method of claim 1, wherein the sample is from a human suspected of being infected with the target mycobacterial cell.

### Claim 4 (depends on 3)

The method of claim 3, wherein the target mycobacterial cell is selected from the group consisting of Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium complex (MAC), Mycobacterium avium hominissuis (MAH), Mycobacterium tuberculosis (MTB), Mycobacterium leprae, Mycobacterium vaccae, Mycobacterium celatum, Mycobacterium kansasii, Mycobacterium gordonae, Mycobacterium porcinum, Mycobacterium cheloni, Mycobacterium flavescens, Mycobacterium bovis, Mycobacterium sylvaticum and Mycobacterium bovis Bacillus Calmette Guerin (BCG).

### Claim 5 (depends on 1)

The method of claim 1 wherein the bacteriophage is D29 or TM4.

### Claim 6 (depends on 1)

The method of claim 1 further comprising the step of analyzing DNA from a lysed target mycobacterial cell obtained from a bacteriophage plaque to identify a signature DNA sequence that occurs in the target mycobacterial cell.

### Claim 7 (depends on 6)

The method of claim 6 wherein the DNA is analyzed by a method selected from the group consisting of whole genome sequencing (WGS) and a PCR based DNA amplification system.

### Claim 8 (depends on 6)

The method of claim 6 wherein the DNA is analyzed by PCR using primers that anneal specifically to a signature DNA sequence that occurs in the target mycobacterial cell.

### Claim 9 (depends on 8)

The method of claim 8, wherein the signature DNA sequence is selected from the group consisting of IS900, IS1245, f57 and IS6110.

### Claim 10 (depends on 1)

The method of claim 1, further comprising a step of identifying latent paratuberculosis in asymptomatic humans.