METHODS FOR DIAGNOSING INFECTIOUS DISEASES USING ADSORPTION MEDIA

• US 20160084835A1
• Filed: 10/16/2015
• Published: 03/24/2016
• Est. Priority Date: 11/08/2013
• Status: Active Grant

• Current Assignee: ExThera Medical Corporation
• Original Assignee: ExThera Medical Corporation
• Inventors: McCrea, Keith R., WARD, ROBERT S.
• Granted Patent: US 10,487,350 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01D 15/3828 Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/005 from viruses
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 2333/43556 from ticks
• CPC Class Codes: G01N 2333/44 from protozoa
• CPC Class Codes: G01N 2333/445 Plasmodium
• CPC Class Codes: G01N 2469/10 Detection of antigens from microorganism in sample from host
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56905 Protozoa
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides an in vitro method for concentrating infectious pathogens found in a biological sample obtained from an individual who is suspected of being infected with the pathogens. Provided herein is also an in vitro method for reducing or eliminating blood cells from a sample obtained from an individual suspected to being infected with an infectious pathogen. The present invention also provides a method for diagnosing malaria and a method for determining if an individual is infected with a pathogen. Provided herein is also a concentrator and a kit for use with the methods.

Claims

1. An in vitro method for concentrating infectious pathogens present in a biological sample obtained from a subject who is suspected of being infected with said pathogens, the method comprising:

(a) contacting the biological sample obtained from the subject with an adsorption media under conditions to form an adhering complex comprising the adsorption media and said pathogens;

(b) separating the adhering complex from components of the sample that are not included in the complex while maintaining the complex;

(c) washing the adhering complex with a wash buffer; and

(d) collecting pathogens of the adhering complex by applying an elution buffer to the complex, thereby concentrating the infectious pathogens in an eluent.

2. The method of claim 1, wherein the wash buffer is a saline solution.

3. The method of claim 1, wherein the elution buffer is a high ionic strength or hypertonic saline solution.

4. The method of claim 1, wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, feces, sputum, tears, saliva, bronchial lavage fluid, other bodily fluid, and combinations thereof.

5. The method of claim 1, wherein the adsorption media is a solid substrate of high surface area having at least one polysaccharide adsorbent on the surface thereof.

6. The method of claim 5, wherein the at least one polysaccharide adsorbent is attached to the surface of the solid substrate by end-point attachment.

7. The method of claim 5, wherein said solid substrate comprises a plurality of rigid polymer beads.

8. The method of claim 7, wherein said plurality of rigid polymer beads are rigid polyethylene beads.

9. The method of claim 6, wherein the at least one polysaccharide adsorbent is a member selected from the group consisting of heparin, heparan sulfate, mannose, dextran sulfate, hyaluronic acid, salicylic acid, chitosan, and a combination thereof.

10. The method of claim 9, wherein the mannose is D-mannose or a D-mannose polymer.

11. The method of claim 9, wherein the at least one polysaccharide adsorbent is heparin and mannose.

12. The method of claim 1, wherein said pathogens are selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Ebola virus (EBOV), filovirus, Flaviviridae, Streptococcus aureus, Escherichia coli, carbapenem-resistant enterobacteriaceae (CRE) bacteria, an ESBL-producing pathogen, vancomycin-resistant enterococci (VRE) bacteria, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella oxytoca, Enterococcus faecalis, Enterococcus faecium, Candida albicans, cytomegalovirus (CMV), Adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), and any combination thereof.

13. The method of claim 1, further comprising detecting the isolated infectious pathogens.

14. The method of claim 13, wherein detecting the isolated infectious pathogens comprises a colorimetric assay, an immunoassay, an enzyme-linked immunosorbent assay (ELISA), a PCR-based assay, a pathogen growth assay, or a combination thereof.

15. A concentrator for use according to the method of claim 1 comprising a barrel filled with adsorption media, a plunger and a needle.

16. A kit comprising the concentrator of claim 15, a wash buffer and an elution buffer.

17. An in vitro method for reducing blood cells from a biological sample obtained from a subject who is suspected of being infected with an infectious pathogen, the method comprising:

(a) contacting the biological sample obtained from the subject with an adsorption media under conditions to form an adhering complex comprising the adsorption media and a pathogen present in the sample; and

(b) separating the blood cells of the sample and the adhering complex while maintaining the adhering complex, thereby reducing blood cells of the sample.

18-31. -31. (canceled)

32. An in vitro method for diagnosing malaria in a subject who is suspected of being infected with Plasmodium, the method comprising:

(a) contacting a sample obtained from said subject with an adsorption media under conditions to form an adhering complex comprising the adsorption media and a cell present in the sample which is infected with Plasmodium;

(b) determining the presence of the adhering complex by detecting a physical change to the adsorption media; and

(c) predicting that the subject has malaria based on the physical change to the adsorption media compared to a reference adsorption media that has been contacted with a control sample.

33-44. -44. (canceled)

45. An in vitro method for determining that a subject is infected with an infectious pathogen, the method comprising:

(a) contacting a whole blood sample obtained from said subject with an adsorption media to form an adhering complex comprising the adsorption media and a pathogen present in the sample; and

(b) determining presence of the adhering complex by detecting a physical change to the adsorption media; and

(c) predicting that the subject is infected by the infectious pathogen based on the physical change to the adsorption media compared to a reference adsorption media that has been contacted with a control sample.

46-55. -55. (canceled)

56. A kit, the kit comprising:

a concentrator; and

packaging for before and after use.

57. (canceled)

58. An in vitro method for detecting an infectious pathogen present in a biological sample obtained from a subject who is suspected of being infected with said pathogen, the method comprising:

(a) contacting the biological sample obtained from the subject with an adsorption media under conditions to form an adhering complex comprising the adsorption media and said pathogen;

(b) separating the adhering complex from components of the biological sample that are not included in the complex while maintaining the complex; and

(c) collecting the pathogen of the adhering complex, wherein the pathogen or a component of the pathogen is collected after using an enzymatic digestion while the pathogen is resident on the adsorption media.

59-74. -74. (canceled)