Systems and methods for detecting infectious diseases

Patent metadata

Field	Value
Publication number	US-9460268-B2
Application number	US-201414479245-A
Country	US
Kind code	B2
Filing date	Sep 5, 2014
Priority date	Sep 6, 2013
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

Systems, methods, and devices for detecting infections in a clinical sample are provided. Small-volume clinical samples obtained at a point-of-service (POS) location and may be tested at the POS location for multiple markers for multiple diseases, including upper and lower respiratory diseases. Samples may be tested for cytokines, or for inflammation indicators. Dilution of samples, or levels of detection, may be determined by the condition or past history of a subject. Test results may be obtained within a short amount of time after sample placement in a testing device, or within a short amount of time after being obtained from the subject. A prescription for treatment of a detected disorder may be provided, and may be filled, at the POS location. A bill may be automatically generated for the testing, or for the prescription, may be automatically sent to an insurance provider, and payment may be automatically obtained.

First claim

What is claimed is:

1. A method of determining the stage of an infection in a subject suffering from an infection from a pathogen associated with an infectious disease, comprising:
I) Testing, within the housing of a sample analysis device, at least one sample, or an aliquot or aliquots thereof, obtained from said subject
1) for the presence of a target nucleic acid indicative of the pathogen using isothermal nucleic acid amplification methods comprising non-cycling nucleic acid amplification methods, wherein said target nucleic acid comprises a double-stranded nucleic acid having a first strand and a second strand; wherein said double-stranded nucleic acid is directly isolated from said pathogen or wherein nucleic acid is isolated from said pathogen and generated into double-stranded nucleic acid using DNA polymerase and/or reverse transcriptase; and
2) for the presence of an antibody indicative of the pathogen,
Wherein said non-cycling nucleic acid amplification methods are initiated with a strand-displacement polymerase and performed at a temperature of about 56° C. without thermal cycling, and comprise:
i) contacting at least a portion of said sample with primers consisting of pairs of primers, said pairs consisting of a first primer having a first nucleic acid sequence, and a second primer having a second nucleic acid sequence, wherein said first nucleic acid sequence is different than said second nucleic acid sequence, wherein: said first primer comprises a first tail region and a first template-binding region, wherein said first template-binding region is complementary to at least a first portion of said target nucleic acid, and wherein said first tail region of said first primer comprises
a) a 5′ terminal nucleotide of the primer,
b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide, and
c) a middle section comprising one or more nucleotides between the 5′ terminal nucleotide and the innermost nucleotide;
said second primer comprises a second tail region and a second template-binding region, wherein said second template-binding region is complementary to at least a second portion of said target nucleic acid, and wherein said second tail region of said second primer comprises
A) a 5′ terminal nucleotide of the primer,
B) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide, and
C) a middle section comprising one or more nucleotides between the 5′ terminal nucleotide and the innermost nucleotide; wherein at least a portion of the first tail region of the first primer is complementary to at least a portion of the second tail region of the second primer;
ii) generating an extension product of the first primer by treating the target nucleic acid with said strand displacement polymerase and treating the first primer under conditions such that the template-binding region of the first primer anneals to said first strand of the target nucleic acid,
iii) generating an extension product of the second primer by treating the extension product of the first primer with said strand displacement polymerase and treating the second primer under conditions such that the template-binding region of the second primer anneals to the extension product of the first primer,
iv) repeating steps ii) and iii) to provide further extension products comprising partially double-stranded nucleic acids comprising at least one tail-region sequence of said first primer tail and at least one tail region sequence of said second primer tail, wherein at least a portion of said first primer tail region sequence is hybridized to a complementary portion of said second primer tail region sequence,
v) generating a concatemer strand, wherein said concatemer strand is generated as a result of the annealing of at least two hybridized tail sequences from at least two partially double-stranded nucleic acid strands to generate cross-over structures, and
vi) detecting the amplification of the target nucleic acid, and

II) Determining whether the amounts of a) nucleic acids indicative of the pathogen and b) antibodies indicative of the pathogen indicate that the infection is a recent infection, wherein
1) detection of greater than 100 copies per microliter (c/μl) of the nucleic acids indicative of the pathogen and a failure to detect antibodies indicative of the pathogen indicate that the infection is a recent infection, and
2) detection of antibodies to the pathogen and either no detection of pathogen-related nucleic acids or detection of less than 100 c/μl of nucleic acids indicative of the pathogen in the at least one sample indicates that the infection is not a recent infection, wherein a recent infection is an infection which began less than about a week prior to said testing of the sample.

2. The method of claim 1 ,
further comprising extending said cross-over structures by a polymerase effective to form extension products of both of said nucleic acid strands.

3. The method of claim 1 , further comprising inserting a cartridge into said sample analysis device having a housing, wherein said cartridge comprises at least one sample, the reagents necessary for the detection of nucleic acids in said at least one sample, and at least one reagent for the detection of antibodies in said at least one sample.

4. The method of claim 1 , comprising testing said at least one sample for a marker for inflammation.

5. The method of claim 4 , wherein the marker for inflammation is selected from a prostaglandin, tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12), interferon gamma (IF-γ), bradykinin, a complement system molecule, a blood-clotting factor, C-reactive protein, erythrocyte sedimentation rate (ESR), white blood cell count, and a morphological change in a blood or other cell.

6. The method of claim 4 , wherein the marker for inflammation is a cytokine selected from a lymphokine, a chemokine, an interleukin, and an interferon.

7. The method of claim 1 , wherein said testing comprises testing to determine whether the subject suffers from a bacterial infection, a viral infection, a yeast infection, a mycoplasma infection, a fungal infection, other infection, or combination thereof.

8. The method of claim 1 , wherein said testing differentiates between a viral pathogen and a bacterial pathogen, and is effective to determine whether the subject suffers from a recent or non-recent bacterial infection or a recent or non-recent viral infection.

9. The method of claim 1 , further comprising providing suitable treatment of said infection.

10. The method of claim 9 , wherein providing suitable treatment of said infection comprises prescribing an antibiotic when the testing determines that the subject suffers from a bacterial infection.

11. The method of claim 9 , wherein providing suitable treatment of said infection comprises prescribing an anti-mycoplasmal drug when the testing determines that the subject suffers from a mycoplasmal infection.

12. The method of claim 9 , wherein providing suitable treatment of said infection comprises prescribing an anti-viral drug when the testing determines that the subject suffers from a viral infection.

13. The method of claim 9 , wherein providing suitable treatment of said infection comprises avoiding the prescription of an antibiotic, and providing the prescription of an anti-viral drug, when the testing determines that the subject suffers from a viral infection.

14. The method of claim 9 , wherein providing suitable treatment of said infection comprises prescribing an an

Assignees

• Theranos Inc

Inventors

• Holmes Elizabeth A
• Belhocine Kamila
• Lee Josephine
• Patel Pranav
• Richardson Aaron
• Tabakman Scott

Classifications

• G16H50/20

  for computer-aided diagnosis, e.g. based on medical expert systems · CPC title

• G01N33/56994

  Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• G16H10/40 Primary

  for data related to laboratory analysis, e.g. patient specimen analysis · CPC title