Methods of diagnosing infectious disease pathogens and their drug sensitivity

Patent metadata

Field	Value
Publication number	US-2019119728-A1
Application number	US-201816035240-A
Country	US
Kind code	A1
Filing date	Jul 13, 2018
Priority date	Feb 24, 2010
Publication date	Apr 25, 2019
Grant date	—

Abstract

The specification relates generally to methods of detecting, diagnosing, and/or identifying pathogens, e.g., infectious disease pathogens and determining their drug sensitivity and appropriate methods of treatment. This invention also relates generally to methods of monitoring pathogen infection in individual subjects as well as larger populations of subjects.

First claim

1 - 27 . (canceled)

28 . A method of identifying a pathogen in a test sample, the method comprising:
providing a test sample suspected of comprising a pathogen, wherein the test sample is a crude sample; exposing the test sample to a plurality of nucleic acid probes designed to bind specifically to one or more target nucleic acid sequences that uniquely identifies a pathogen, wherein the exposure occurs for a time and under conditions in which binding between the probe and the target nucleic acid can occur wherein the exposure occurs for less than one hour; and determining a level of target nucleic acid that indicates the pathogen in the test sample.

29 . The method of claim 28 , wherein the target nucleic acid is a target RNA.

30 . The method of claim 28 , wherein the test sample is obtained from a subject, optionally wherein the subject is human.

31 . The method of claim 28 , further comprising treating the test sample under conditions that release nucleic acid from cells of the test sample.

32 . The method of claim 28 , wherein the one or more probes that bind specifically to a target nucleic acid are designed to minimize non-specific hybridization.

33 . The method of claim 28 , wherein the pathogen is an infectious disease pathogen, optionally an infectious disease pathogen selected from the group consisting of Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus mitis, Candida albicans, Acinetobacter baumanii, Proteus mirabilis, Haemophilus influenza, Mycobacterium avium/paratuberculosis, Mycobacterium kansasii, Serratia proteamaculans, Plasmodium falciparum, Stenotrophomonas maltophilia, Enterobacter species, Stenotrophomonas maltophilia, HIV, HSV-1 and HSV-2, optionally wherein the pathogen is a drug-resistant strain of infectious disease pathogen.

34 . The method of claim 28 , wherein the probes are designed to bind to two or more different target nucleic acid sequences.

35 . The method of claim 28 , wherein the target nucleic acid is highly conserved across all strains of the identified pathogen.

36 . The method of claim 28 , wherein the cells are lysed mechanically, optionally wherein the cells are lysed via sonication, French press, electroporation or a microfluidic device comprising fabricated structures.

37 . The method of claim 28 , wherein the method comprises use of a microfluidic device or a microarray.

38 . The method of claim 28 , wherein two or more probes are used that bind specifically to a target nucleic acid that uniquely identifies a pathogen.

39 . The method of claim 28 , wherein the target nucleic acid is a target mRNA encoded by a gene of Table 2.

40 . The method of claim 28 , wherein the plurality of nucleic acid probes comprises a selection of probes selected from SEQ ID NOs: 1-227.

41 . The method of claim 28 , wherein the method further comprises exposing the test sample to a reporter nucleic acid probe that is conjugated to a fluorescent tag, optionally wherein the tag is a bar code.

42 . The method of claim 28 , wherein one or more of the nucleic acid probes is conjugated to a label, optionally wherein the label is selected from the group consisting of a fluorophore, biotin, digoxygenin, and a radioactive isotope.

43 . The method of claim 28 , wherein the pathogen is a bacterium, fungus, virus, or parasite.

44 . The method of claim 28 , wherein:
(a) the pathogen is Escherichia coli, optionally wherein the target nucleic acid is one or more of recA, carA, deoC, figF, htrL, uvrA, ybhK, uup, fabD, cysD, glnA, opgG, ftsQ, murC, putP, secA, uup, b1649, dinD, pyrB, wbbK, and hdeA mRNA; (b) the pathogen is Mycobacterium tuberculosis, optionally wherein the target nucleic acid is one or more of kasA, fadD32, accD6, efpA, Rv3675.1, bioD, hisI, era, Rv2296, Rv0813, rpsR, alkA, recA, ltpl, lhr, CHP, bcpB, gcvB, and groEL mRNA; (c) the pathogen is Pseudomonas aeruginosa, optionally wherein the target nucleic acid is one or more of PA_4175, mpl, proA, sltB1, nadD, dacC, and lipB mRNA; (d) the pathogen is Klebsiella pneumoniae, optionally wherein the target nucleic acid is one or more of lrp, ycbK, clpS, ihfB, and mraW mRNA; (e) the pathogen is Staphylococcus aureus, optionally wherein the target nucleic acid is one or more of mecA, proC, rpoB, fabD, ileS, ppnK, pyrB, rocD, and uvrC mRNA; (f) the pathogen is Enterococcus faecalis; (g) the pathogen is Streptococcus pyogenes; (h) the pathogen is Streptococcus pneumoniae; (i) the pathogen is Streptococcus agalactiae; (j) the pathogen is Streptococcus mitis; (k) the pathogen is Candida albicans; (l) the pathogen is Acinetobacter baumanii; (m) the pathogen is Proteus mirabilis; (n) the pathogen is Haemophilus influenza; (o) the pathogen is Mycobacterium avium/paratuberculosis; (p) the pathogen is Mycobacterium kansasii; (q) the pathogen is Serratia proteamaculans; (r) the pathogen is Plasmodium falciparum; (s) the pathogen is Stenotrophomonas maltophilia, optionally wherein the target nucleic acid is one or more of clpP, dnaK, purC, purF, sdhA, and secD mRNA; (t) the pathogen is HIV; (u) the pathogen is HSV-1; or (v) the pathogen is HSV-2.

45 . The method of claim 28 , wherein the method is used to monitor a pathogen infection.

46 . The method of claim 28 , wherein the method further comprises determining or selecting a treatment for the subject, and optionally administering the treatment to the subject.

47 . A method of identifying a pathogen in a test sample, the method comprising:
providing a test sample suspected of comprising a pathogen, wherein the test sample is a crude sample; exposing the test sample to one or more nucleic acid probes that are designed to bind specifically to target nucleic acids that identify one or more pathogens in the test sample, wherein the exposure occurs for a time and under conditions in which binding between the probe and the target nucleic acids can occur wherein the exposure occurs for less than one hour; and determining the target nucleic acids by imaging or counting reporter tags to indicate the pathogen in the test sample.

48 . The method of claim 47 , wherein the target nucleic acids are target RNAs.

49 . The method of claim 47 , wherein the reporter tag is a fluorescent tag.

50 . The method of claim 47 , wherein the plurality of nucleic acid probes comprises a selection of probes selected from SEQ ID NOs: 1-227.

51 . The method of claim 47 , further comprising treating the test sample under conditions that release nucleic acid from cells of the test sample.

52 . The method of claim 47 , wherein the probes are designed to bind to two or more different target nucleic acid sequences.

53 . The method of claim 47 , wherein one or more of the target nucleic acids is highly conserved across all strains of the identified pathogen.

54 . A kit comprising a plurality of nucleic acid probes for use in the method of claim 28 , and instructions for its use.

55 . A kit comprising a plurality of nucleic acid probes for use in the method of claim 47 , wherein the reporter probes have a fluorescent tag, and instructions for its use.

Assignees

• Broad Inst Inc
• Massachusetts Gen Hospital
• Brigham & Womens Hospital Inc

Inventors

• Gomez James
• Hung Deborah
• Barczak Amy

Classifications

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• C12Q1/6895

  for plants, fungi or algae · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title

• G01N2800/56

  Staging of a disease; Further complications associated with the disease · CPC title

• C12Q1/70

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title