Method and devices for treating biological samples

Patent metadata

Field	Value
Publication number	US-2017241878-A1
Application number	US-201515521595-A
Country	US
Kind code	A1
Filing date	Oct 23, 2015
Priority date	Oct 24, 2014
Publication date	Aug 24, 2017
Grant date	—

Abstract

A method of treating a biological sample, preferably a sample of blood or bodily fluids likely to contain one or more species of interest, and including a step of decomplexification by acoustophoresis (as well as associated systems, devices, substrates and connection devices).

First claim

1 . A method for treating a sample from a blood culture, or a sample of sterile body fluid which may contain one or more microorganism(s) of interest, comprising a decomplexification step, the decomplexification step comprising the following steps:
carrying out a step of enrichment of the sample by incubation, introducing all or part of this sample into a first inlet orifice of an acoustophoresis device without dilution prior to the introduction, introducing a buffer into a second inlet orifice of the acoustophoresis device, the inlet orifices being fluidically connected to at least two outlet orifices by at least one separation channel, the buffer and the sample being introduced at respective flow rates capable of generating a laminar flow in the separation channel, carrying out a step of separation of the biological sample by acoustophoresis so as to promote the concentration of the non-specific particles present in the sample in at least one of the outlet orifices of the acoustophoresis device, following this decomplexification step, carrying out a step of identification of the microorganism(s) present in the sample, optionally and following this step of identification of the microorganism(s), carrying out a step of determination of the antibiogram profile of the microorganism(s) that has (have) been identified.

2 . The treatment method as claimed in claim 1 , comprising a second decomplexification step comprising carrying out a second step of separation of the concentrated sample by acoustophoresis so as to promote a greater recovery rate of the microorganism(s) still present in the concentrated sample in at least one of the outlet orifices of the acoustophoresis device.

3 . The treatment method as claimed in claim 1 , comprising a second decomplexification step comprising carrying out a second step of separation of the decomplexified sample by acoustophoresis so as to promote a greater rejection rate of the non-specific elements present in the decomplexified sample in at least one of the outlet orifices of the acoustophoresis device.

4 . The treatment method as claimed in claim 2 , wherein the second separation step is carried out with a single acoustophoresis device comprising at least two successive separation channels.

5 . The treatment method as claimed in claim 1 , wherein the buffer used is of different nature and/or of different density relative to the biological sample.

6 . The treatment method as claimed in claim 1 , wherein the step of identification of the microorganism(s) following the obtaining of an enriched and decomplexified sample is carried out by means of a mass spectrometry method.

7 . The treatment method as claimed in claim 1 , comprising an antibiogram test of the microorganism(s) following the obtaining of an enriched and decomplexified sample by early imaging of the inhibitory zones obtained on culture medium using an antibiotic disk or an antibiotic strip, or by means of a biochemical test.

8 . The treatment method as claimed in claim 1 , comprising
measuring the optical density of the enriched and decomplexified sample during or following a step of separation by acoustophoresis so as to obtain a sample having a defined optical density, it being possible for the measurement to be carried out in the outlet orifice.

9 . The treatment method as claimed in claim 1 , comprising a subsequent step of incubation of the decomplexified and enriched sample, it being possible for the incubation to be carried out directly in the outlet orifice or collection tube.

10 . The treatment method as claimed in claim 9 , comprising a step of shaking the decomplexified and enriched sample during the incubation.

11 . The treatment method as claimed in claim 9 , comprising measuring the optical density of the enriched and decomplexified sample during or following the incubation step, so as to stop or prolong the incubation step or to adjust the sample by dilution when a defined optical density threshold is measured, it being possible for the measurement to be carried out in the outlet orifice.

12 . The treatment method as claimed in claim 6 , comprising a step of analysis of susceptibility of the microorganism(s) present in the sample to one or more antibiotic(s) by early imaging of the inhibitory zones obtained on culture medium using an antibiotic disk or an antibiotic strip, or by means of a biochemical test, the antibiotic(s) being determined by the result of the step of identification of the one or more species of microorganism(s) that is (are) present in the concentrated sample.

13 . The treatment method as claimed in claim 1 , all or part of the enriched and decomplexified sample being transferred into the wells of a microplate or into various wells in fluidic connection with at least one of the outlet orifices containing all or part of the sample, it being possible for each of the wells to contain an antibiotic which is different and/or an antibiotic in different concentrations.

14 . The treatment method as claimed in claim 13 , comprising carrying out a step of incubation of the enriched and decomplexified sample directly in the microplate.

15 . The treatment method as claimed in claim 13 , comprising carrying out a step of adjustment of the optical density of the enriched and decomplexified sample directly in the microplate.

16 . The treatment method as claimed in claim 13 , each of the wells being, after an incubation time greater than or equal to lh in the presence of the antibiotic(s), successively suctioned so as to be analyzed by flow cytometry.

17 . The treatment method as claimed in claim 16 , comprising the detection by flow cytometry analysis of a shift in the fluorescence signal between microorganisms which are sensitive, intermediate or resistant to the antibiotics tested with the enriched and decomplexified sample.

18 . The treatment method as claimed in claim 1 , comprising an additional step of mechanical or enzymatic lysis of the decomplexified sample, followed by a step of extraction of the nucleic acids and of analysis of said nucleic acids by PCR, qPCR or by sequencing.

19 . The treatment method as claimed in claim 1 , comprising a step of capture of the species present in the concentrated sample using magnetic particles followed by separation by magnetophoresis.

20 . The treatment method as claimed in claim 2 , wherein the first separation step is carried out using a first buffer, and the second separation step is carried out with a second buffer, distinct from the first buffer.

21 . The treatment method as claimed in claim 20 , wherein the first buffer is isotonic and comprises Percoll at a volume ratio of less than or equal to 10%.

Assignees

• Biomerieux Sa

Inventors

• Broyer Patrick
• Patel Pradip
• Pamme Nicole
• Bisceglia Emilie

Classifications

• G01N1/44

  Sample treatment involving radiation, e.g. heat · CPC title

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title

• B01L2300/0829

  Multi-well plates; Microtitration plates · CPC title

• G01N27/447

  using electrophoresis · CPC title

• B01L3/502776

  specially adapted for focusing or laminating flows · CPC title