Methods and systems for detecting a pathogen in a biological sample

• US 10,640,807 B2
• Filed: 12/27/2012
• Issued: 05/05/2020
• Est. Priority Date: 12/29/2011
• Status: Active Grant

• Current Assignee: S.D. Sight Diagnostics Ltd
• Original Assignee: S.D. Sight Diagnostics Ltd
• Inventors: Pollak, Joseph Joel, Levner, Daniel, Bilu, Yonatan, Yafin, Arnon, Yorav-Raphael, Noam, Greenfield, Yuval
• Primary Examiner(s): Lankford, Blaine
• Application Number:
  US14/369,251
• Publication Number:
  US 20150037806A1
• Time in Patent Office: 2,686 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• 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

Disclosed herein, inter alia, is method, kit and systems for detecting a pathogen infection in a bodily sample, the method comprising (i) staining said bodily sample with two or more dyes, comprising at least one dye predominantly staining DNA to thereby provide differential staining between DNA and at least one other cellular component being different from DNA; (ii) identifying at least a first stained area comprising the DNA, if exists in the sample, and at least one other stained area comprising the other cellular component; (iii) extracting structural features for the first stained area and the at least one other stained area, said structural features comprise at least one of (i) size of at least one of the first stained area and one other stained area and (ii) location of said first stained area and said at least one other stained area one with respect to the other; and (iv) determining the presence of a suspected pathogen in the bodily sample if a first stained area was identified and said structural features conform to structural features predetermined as characterizing the suspected pathogen.

Claims

1. A method for use with a blood sample, the method comprising:

staining the blood sample with at least Hoechst stain and Acridine Orange stain;

using a microscope, acquiring a plurality of images of the blood sample, at least a first one of the images being acquired while the blood sample is illuminated by a light source having an illumination wavelength in which the Hoechst stain is visible, and at least a second one of the images being acquired while the blood sample is illuminated by a light source having an illumination wavelength in which the Acridine Orange stain is visible; and

using at least one computer processor, detecting that a parasite is within a red blood cell within the blood sample, by;

identifying an entity as a candidate of a parasite disposed within a red blood cell, by;

identifying an object within the plurality of images as being a red blood cell candidate, based upon the object having characteristics of a red blood cell;

identifying that a first stained area, which is stained by the Hoechst stain, is disposed within the red blood cell candidate, by analyzing the first one of the images; and

identifying that a second stained area, which is stained by the Acridine Orange stain, is disposed within the red blood cell candidate, by analyzing the second one of the images; and

validating that the entity is a parasite disposed within a red blood cell, by determining that structural features of the first and second stained areas satisfy predetermined criteria associated with the parasite, the structural features selected from the group consisting of;

(i) sizes of the first stained area and the second stained area, and (ii) locations of the first stained area and the second stained area with respect to each other,

the validating comprising distinguishing the entity from other entities within the blood sample that are stained by at least one of the Hoechst stain and the Acridine Orange.

2. The method according to claim 1, wherein distinguishing the entity from other entities within the blood sample that are stained by at least one of the Hoechst stain and the Acridine Orange comprises distinguishing the entity from white blood cells within the blood sample that are stained by at least one of the Hoechst stain and the Acridine Orange.

3. The method according to claim 1, wherein distinguishing the entity from other entities within the blood sample that are stained by at least one of the Hoechst stain and the Acridine Orange comprises distinguishing the entity from platelets within the blood sample that are stained by at least one of the Hoechst stain and the Acridine Orange.

4. The method of claim 1, wherein staining the blood sample with the Hoechst stain and the Acridine Orange stain comprises staining the blood sample using the Hoechst stain and the Acridine Orange stain at a Hoechst:

Acridine Orange ratio in a range of between 50;

1 to 1;

1.

5. The method of claim 1, wherein detecting that the parasite is within the red blood cell comprises detecting that a Plasmodium parasite is within the red blood cell.

6. The method of claim 5, wherein detecting that the Plasmodium parasite is within the red blood cell comprises identifying that the size of the first stained area satisfies a predetermined relation with respect to the size of the other stained area.

7. The method of claim 5, wherein detecting that the Plasmodium parasite is within the red blood cell comprises determining that an area of the first stained area is in a range of 0.2 μ

m² to 20 μ

m².

8. The method of claim 5, wherein detecting that the Plasmodium parasite is within the red blood cell comprises determining that an area of the second stained area is in a range of 0.8 μ

m² to 65 μ

m².

9. The method of claim 5, wherein detecting that the Plasmodium parasite is within the red blood cell comprises determining that the first stained area occupies between 12% and 60% of the second stained area.

10. The method of claim 5, wherein detecting that the Plasmodium parasite is within the red blood cell comprises identifying one or more structural features selected from the group consisting of:

the first stained area and the second stained area having predetermined shapes;

the first stained area comprising variability in stain intensity; and

the first stained area being maintained within boundaries of the second stained area, during movement of the object.

11. The method of claim 5, wherein detecting that the Plasmodium parasite is within the red blood cell comprises identifying at least one set of structural features of the stained object selected from the group consisting of:

i. the first stained area abutting with boundaries of the second stained area;

ii. the first stained area residing within boundaries of the second stained area; and

iii. the size of at least one of the first stained area and of the second stained area being within a predetermined range.

12. The method of claim 5, further comprising detecting a count per unit volume of said Plasmodium parasite in the blood sample, and determining that the blood sample is infected with a Plasmodium infection based on the count per unit volume of the Plasmodium parasite.