Electrochemical detection of bacterial and/or fungal infections

• US 10,106,847 B1
• Filed: 11/30/2017
• Issued: 10/23/2018
• Est. Priority Date: 08/24/2017
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Clinical Micro Sensors Inc. (Roche Holding AG)
• Inventors: Brown, Bradley Adam, Martinez, Milena Iacobelli, Freeman-Cook, Lisa Lynn, Harvey, John Jay, Shaw, Christine J., Al-Khouri, Anna Maria
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US15/828,074
• Time in Patent Office: 327 Days
• Field of Search: None
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 27/48 using polarography, i.e. measuring changes in current under a slowly-varying voltage
• CPC Class Codes: G16H 10/40 for data related to laboratory analysis, e.g. patient specimen analysis
• CPC Class Codes: G16H 15/00 ICT specially adapted for medical reports, e.g. generation or transmission thereof
• CPC Class Codes: G16H 80/00 ICT specially adapted for facilitating communication between medical practitioners or patients, e.g. for collaborative diagnosis, therapy or health monitoring
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The present disclosure relates to methods and devices for amplifying a plurality of targets in a single PCR run while distinguishing between clinically relevant amplification and amplification from other sources such as from background contamination. The methods and devices further enable discrimination between gram-positive, gram-negative and fungal infections as wells as identify antimicrobial resistance genes. When applying the methods and devices of the invention, the species or genus of an infection(s), and genus of a fungal co-infection(s) or category of bacterial (gram-positive or negative) co-infection(s) are identified. Species identification of co-infections can also be achieved. Further, when applying the methods and devices of the invention, organisms which are likely to be contaminating organisms from a blood draw are identified.

Claims

1. An in vitro method for detecting a viable gram-positive microorganism comprising:

a) subjecting a sample in blood culture medium, i) the sample comprising or suspected of comprising, the viable pram-positive microorganism and ii) the blood culture medium comprising nucleic acid from a non-viable gram-positive microorganism, wherein the non-viable cram-positive microorganism is the same pram-positive microorganism as the viable gram-positive microorganism, and is present in the sample at a lower concentration than the viable gram-positive microorganism, to a single detuned multiplex end-point polymerase chain reaction (PCR) to produce amplicons, the PCR comprising about 30 to about 35 cycles;

b) contacting amplicons from step a with a plurality of signal probes and a plurality of capture probes, one of the signal probes and one of the capture probes is specific for the amplicons to form a hybridization complex; and

c) electrochemically detecting an amount of hybridization complex above a threshold thereby detecting the viable gram-positive microorganism, and not detecting the nucleic acid from the non-viable gram-positive microorganism present in the blood culture medium.

2. The method of claim 1, further comprising, prior to step a, contacting the sample with a compound which hydrolyzes nucleic acids and extracting the nucleic acids.

3. The method of claim 1, wherein if four or more microorganisms are detected by the method, the method is repeated.

4. The method of claim 1, further comprising generating an epidemiology report on a clinical instrument.

5. The method of claim 1, further comprising reporting the detected viable gram-positive microorganism to a hospital'"'"'s laboratory information system (LIS).

6. The method of claim 1, wherein the viable gram-positive microorganism is a Bacillus cereus, a Micrococcus, a Bacillus subtilis, a Staphylococcus, a Staphylococcus aureus, a Propionibacterium acnes, a Staphylococcus epidermidis, a Staphylococcus lugdunensis, a Enterococcus faecalis, a Streptococcus, a Enterococcus faecium, a Streptococcus agalactiae, a Lactobacillus, a Listeria, a Streptococcus pneumoniae, a Listeria monocytogenes, or a Streptococcus pyogenes.

7. The method of claim 1, wherein the viable gram-positive microorganism is a S. anginosus, a Enterococcus spp., or a Corynebacterium and the single detuned multiplex end-point PCR comprises primers at a primer concentration of about 250 nM.

8. The method of claim 1, wherein the single multiplex PCR comprises shuttling, by electrowetting manipulation, the sample across thermal zones comprising a denature heater and an anneal/extension heater.

9. The method of claim 1, wherein a Streptococcus spp., a P. acnes and a Streptococcus pneumoniae gram-positive microorganism are amplified together in a multiplex pool.

10. The method of claim 1, further comprising detecting a viable gram-negative microorganism, fungal microorganism or antimicrobial resistance gene.

11. The method of claim 1, wherein false positives from the non-viable microorganism is less than 5%.

12. The method of claim 1, wherein the method has a limit of detection of 1×

10⁵ CFU/mL, 1×

10⁴ CFU/mL, or 1×

10⁶ CFU/mL.

13. An in vitro method for detecting a viable gram-negative microorganism comprising:

a) subjecting a sample in blood culture medium, i) the sample comprising or suspected of comprising, the viable gram-negative microorganism and ii) the blood culture medium comprising nucleic acid from a non-viable gram-negative microorganism, the non-viable gram-negative microorganism is the same gram-negative microorganism as the viable gram-negative microorganism, and is present in the sample at a lower concentration than the viable gram-negative microorganism, to a single detuned multiplex end-point polymerase chain reaction (PCR) comprising about 30 cycles to produce amplicons;

b) contacting amplicons from step a with a plurality of signal probes and a plurality of capture probes, one of the signal probes and one of the capture probes is specific for the amplicons to form a hybridization complex; and

c) electrochemically detecting an amount of hybridization complex above a threshold thereby detecting the viable gram-negative microorganism, and not detecting the nucleic acid from the non-viable gram-negative microorganism present in the blood culture medium.

14. The method of claim 13, wherein the viable gram-negative microorganism is a Acinetobacter baumannii, a Klebsiella pneumoniae, a Bacteroides fragilis, a Morganella morganii, a Citrobacter, a Neisseria meningitides, a Cronobacter sakazakii, a Proteus, a Enterobacter cloacae complex, a Proteus mirabilis, a Enterobacter, a Pseudomonas aeruginosa, a Escherichia coli, a Salmonella, a Fusobacterium necrophorum, a Serratia, a Fusobacterium nucleatum, a Serratia marcescens, a Haemophilus influenza, a Stenotrophomonas maltophilia, or a Klebsiella oxytoca.

15. The method of claim 13, further comprising detecting a viable gram-positive microorganism, fungal microorganism or antimicrobial resistance gene.

16. An in vitro method for detecting a viable fungal microorganism comprising:

a) subjecting a sample in blood culture medium, i) the sample comprising or suspected of comprising, the viable fungal microorganism and ii) the blood culture medium comprising nucleic acid from a non-viable fungal microorganism, the non-viable fungal microorganism is the same fungal microorganism as the viable fungal microorganism, and is present in the sample at a lower concentration than the viable fungal microorganism, to a single detuned multiplex end-point polymerase chain reaction (PCR) to produce amplicons wherein the PCR comprises multiple pairs of primers and at least one pair of primers comprises mismatches compared to a gene of the viable fungal microorganism;

b) contacting amplicons from step a with a plurality of signal probes and a plurality of capture probes, one of the signal probes and one of the capture probes is specific for the amplicons to form a hybridization complex; and

c) electrochemically detecting an amount of hybridization complex above a threshold thereby detecting the viable fungal microorganism, and not detecting the nucleic acid from the non-viable fungal microorganism present in the blood culture medium.

17. The method of claim 16, wherein the viable fungal microorganism is a Candida albicans, a Candida lusitaniae, a Candida auris, a Candida dubliniensis, a Candida famata, a Cryptococcus gattii, a Candida glabrata, a Cryptococcus neoformans, a Candida guilliermondii, a Fusarium, a Candida kefyr, a Malassezia furfur, or a Candida krusei.

18. The method of claim 16, wherein the hybridization complex comprising a Candida parapsilosis or a Candida tropicalis fungal microorganism is detected in two detection zones.

19. The method of claim 16, wherein the viable fungal microorganism is a Rhodotorula or a Trichosporon and the primers for the Rhodotorula or the Trichosporon viable fungal microorganism contain mismatches compared to a wildtype gene for Rhodotorula or Trichosporon.

20. The method of claim 16, wherein the single detuned multiplex end-point PCR comprises primers consisting of the nucleic acid sequence of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, or SEQ ID NO;

9.