METHOD TO DETECT BACTERIAL ACTIVITY IN A BIOLOGICAL SAMPLE AND CORRESPONDING DETECTION UNIT

Abstract

Method and corresponding detection unit to detect bacterial activity in a biological sample, in particular, but not only, blood samples contained in a test tube with a stopper.

Claims

1. Method to detect bacterial activity in a biological sample, comprising: introducing the biological sample into a sealed and sterilized test tube; defining a headspace for the accumulation of gas inside said test tube and above the biological sample; taking a volatile sample from said headspace; and analyzing the content of the inorganic gaseous substances, such as in particular CO.sub.2, H.sub.2 and/or O.sub.2 present in said volatile sample by means of a micro gas chromatograph with a flow suitable to detect the presence of inorganic substances generated by the bacterial metabolism in said biological sample in a range comprised between 1 ppm and 10 ppm, said detection of the inorganic substances being carried out in a continuous flow at various sampling times to obtain a growth curve relating to the inorganic substances measured both with increasing CO.sub.2 and decreasing O.sub.2.

2. Method as in claim 1, and further comprising taking a volatile sample from the headspace and at the same time measuring the total variation in pressure in the test tube by means of a sensor.

3. Method as in claim 2, and further comprising: taking a volatile sample from said headspace by introducing a needle into the test tube through a stopper; and re-introducing the quantity of volatile sample taken from the test tube into the test tube using a second needle connected to an introduction member in order to allow the re-circulation of the gaseous volume present in the headspace.

4. Method as in claim 1, including carrying out, by means of said micro gas chromatograph, a dynamic measurement of the gaseous substances present in said headspace, in one or more determinate time intervals, in order to obtain a growth dynamic of the individual inorganic gaseous substances present in said headspace.

5. Method as in claim 3, and further including, after every taking of the volatile sample, re-introducing the volatile sample in said headspace, and performing a new measuring in sequence, in order to detect if the organic component inside it increases or is constant over time.

6. Method as in claim 1, and further introducing a culture medium or broth together with the biological sample inside the test tube.

7. Method as in claim 1, and exclusively introducing the native biological sample inside the test tube.

8. Method as in claim 1, and further introducing adjuvant substances into said test tube, suitable to speed up the bacterial replication.

9. Method as in claim 8, wherein said adjuvant substances are hydrocarbons, such as methane, ethane, propane or similar or comparable substances.

10. Method as in claim 8, and further providing lysant substances able to increase the presence and/or the detection capacity of the bacteria in said biological sample, said lysant substances being able to increase the detection of bacteria inside the red blood cells.

11. Method as in claim 1, and further introducing a magnetic element to stir the biological sample.

12. Method as in claim 1, and further introducing said biological sample into a test tube under vacuum, to take the biological sample directly from a patient.

13. Method as in claim 1, wherein sequestrant substances of possible antibiotic substances are introduced inside a biological sample or a bacterial culture.

14. Method as in claim 1, and further detecting the pressure present in the test tube.

15-17. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] These and other characteristics of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:

[0048] FIG. 1 is a schematic representation of a detection method according to one embodiment.

[0049] To facilitate comprehension, the same reference numbers have been used, where possible, to identify identical common elements in the drawings. It is understood that elements and characteristics of one embodiment can conveniently be incorporated into other embodiments without further clarifications.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

[0050] FIG. 1 is used to describe a method 10 to detect bacterial activity, that is, to detect the existence of live and replicating bacteria, by detecting the presence and quantity of CO.sub.2, H.sub.2 and/or O.sub.2 in a hermetically sealed test tube with a biological sample.

[0051] For example, biological sample 14 can comprise, but not only, blood, urine, saliva, mucus, tears or other suitable sample.

[0052] In one embodiment, the biological sample 14 is inserted in a test tube or vial 12, suitably sealed and sterilized.

[0053] The test tube 12 is sealed by a stopper 16 which comprises a membrane, for example rubber, self-sealing and which allows needles to pass for example.

[0054] The biological sample 14 is analyzed by introducing inside the test tube 12 a minimum amount of biological sample 14, for example less than 5 ml.

[0055] This aspect advantageously allows to apply the present method even when small quantities of biological sample 14 are available, for example in the case of analyses for newborns.

[0056] According to one aspect of the present invention, a free volume or headspace 18 is left inside the test tube 12, defined between the stopper 16 and the level of the biological sample 14, that is, above the biological sample 14.

[0057] The accumulation of gas or volatile substances is allowed in the headspace 18, that is, inorganic gaseous substances produced by the bacteria (bacterial catabolism) during their growth inside the test tube 12.

[0058] Here and hereafter we will refer to volatile substances understood as inorganic gaseous substances, such as CO.sub.2, H.sub.2 and/or O.sub.2, which due to how they are defined in the present disclosure do not comprise organic substances.

[0059] In one embodiment, a volumetrically fixed quantity of gas is sent to a detection unit 22, described hereafter.

[0060] Therefore, at least one step is provided of taking a volatile sample from the headspace 18, present inside the test tube 12.

[0061] In another embodiment, the detection unit 22 is a gas chromatograph or micro gas chromatograph 22 to perform the GC analysis.

[0062] The micro gas chromatograph 22, which is suitably configured to be miniaturized and reduce to a minimum the transfer bulk, is also equipped with a device to perforate the stopper 16.

[0063] In one embodiment, taking the volatile sample from inside the headspace 18 provides to use a needle device 20 which perforates the stopper 16 of the test tube 12 and sucks up a desired quantity of gaseous mass above the biological sample 14 inside which there is the gas generated by the bacteria.

[0064] The volatile sample is subsequently analyzed by the micro gas chromatograph 22 to detect the presence and/or quantity of CO.sub.2, H.sub.2 and/or O.sub.2.

[0065] In particular, the micro gas chromatograph 22 provides a control and command device 28.

[0066] According to possible embodiments, the micro gas chromatograph 22 is configured to identify inorganic substances, such as for example CO.sub.2, H.sub.2 and/or O.sub.2, correlated to the presence of bacteria in the biological sample, in a range comprised between 1 ppm and 10 ppm.

[0067] The control and command device 28 is able to process a detection pattern of CO.sub.2 30, that is, of the gaseous catabolic substances produced and present in the headspace 18.

[0068] Furthermore, as an alternative or in addition to measuring the CO.sub.2, the control and command device 28 is able to process a detection pattern O.sub.2 32.

[0069] The possibility of measuring simultaneously the temporal variation of two or more inorganic gaseous substances such as CO.sub.2 and/or O.sub.2 allows to correlate the respective patterns and to obtain a precise, reliable and complete result.

[0070] Moreover, the measurement is carried out by re-introducing the gas into the headspace after every measurement, so that it is possible to carry out measurements in sequence and obtain the temporal evolution of each substance and hence of the correlated bacterial activity.

[0071] In particular, a removal and subsequent re-introduction procedure can provide that, in a measuring time 0, the whole gas component inside the test tube is removed. A second reading of the same test tube at time T1 would give a vacuum, alias a negative pressure, due to having aspirated the whole substance already before suction.

[0072] Therefore, re-introduction of the substance aspirated into the same test tube allows on the second, third or fourth reading at times T0, T1, T2, T3 . . . a measurement assessment for the components object of the detection of the bacteria.

[0073] In the temporal and subdivided measurements at times T0, T1, T2, T3 . . . it is therefore possible to detect whether the inorganic component increases or is stable over time. In one embodiment, the needle device 20 comprises a needle 24 to take a volatile sample from the headspace 18.

[0074] The taking of the volatile sample is made possible by the positive pressure in the test tube 12 which allows the volatile sample to enter inside the micro gas chromatograph 22.

[0075] In one embodiment, the volatile sample can be taken from the test tube 12 by means of aspiration using a suction member 29 integrated into the circuit of the needle device 20 and connected to the needle 24, to facilitate the measuring of the gaseous species inside the headspace 18, also for small concentrations of the gaseous species present and being examined, if the pressure inside the test tube 12 were equal to atmospheric or negative pressure.

[0076] In another embodiment, the needle device 20 comprises two needles 24. In particular, the second needle 24 is suitable to re-introduce the quantity of gas taken from the headspace 18 inside the test tube 12.

[0077] The second needle 24 can be connected to an introduction member 33 of the quantity of volatile sample inside the test tube 12.

[0078] The presence of two needles 24 allows to apply a recirculation of the gas inside the test tube 12, preventing waiting times, previously described in the state of the art, to release CO.sub.2 into the headspace 18.

[0079] Furthermore, the periodic measurement of CO.sub.2, O.sub.2 will be increased as the metabolism of the bacteria increases, also facilitating the possible periodic measurement of O.sub.2. The periodic measuring of the headspace, carried out at one or more defined time intervals, gives a dynamic measurement, that is, correlated to time, as a function of the quantity of bacteria present. The micro gas chromatograph 22 thus allows to detect growing quantities of gaseous substances to be detected and measured, so as to obtain a growth dynamic, possibly represented by a graph, of the gaseous substances detected with respect to time.

[0080] In one embodiment, the biological sample 14 can be introduced, inoculated, inside a test tube 12 where there is a culture broth.

[0081] The biological sample 14, together with the culture medium or broth forms a bacterial culture 26 after waiting for the incubation period.

[0082] In another embodiment, the method provides exclusively to introduce the native biological sample 14 inside the test tube 12 without eugonic broth.

[0083] In another embodiment, adjuvant substances can be introduced inside the test tube 12, which are able to accelerate the metabolic process of the bacterial species possibly present in the biological sample 14.

[0084] For example, by adjuvant substances we mean gaseous substances such as methane, ethane, propane or other gases, or liquid or solid substances.

[0085] In another embodiment, lysant substances can be introduced inside the biological sample 14 or the bacterial culture 26, so as to liberate the bacteria inside the red blood cells.

[0086] For example, by sequestrant substances we mean carbon or resins or other substances able to perform a sequestrant action on the antibiotic substances present in the sample following the start of an antibiotic therapy.

[0087] In another embodiment, not shown in the drawings, a magnetic element is introduced into the bottom of the test tube 12 to stir the bacterial culture 26.

[0088] The magnetic element interacts with a stirring device that causes it to rotate, and thus facilitates contact of the bacteria with the metabolic substances present in the culture medium or broth, increasing their growth and improving the mixing of the lysant substances in order to break the red blood cells inside which bacteria can exist.

[0089] The analysis of the gases, in particular CO.sub.2 and/or O.sub.2, present in the volatile sample, using a micro gas chromatograph 22, allows to obtain a result very quickly, for example from 5 to 40 seconds.

[0090] In one embodiment, the method can also be applied with particular types of vacuum test tubes 12.

[0091] In this way it is possible to take the biological sample 14 directly from a system to remove biological liquids, for example blood, directly from the patient.

[0092] According to a variant embodiment, if it became necessary, the method 10 can provide a step of detecting the pressure inside the test tube 12. In this way it is possible to detect an additional parameter to identify the class or species of bacteria present in the biological sample.

[0093] For example, it is possible to measure and detect the gaseous species present in the headspace 18, produced by the bacterial catabolism, and at the same time to measure the total variation in pressure in the test tube 12 by means of a miniaturized sensor.

[0094] It is clear that modifications and/or additions of parts may be made to the method 10 and detection unit 22 as described heretofore, without departing from the field and scope of the present invention. It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art shall certainly be able to achieve many other equivalent forms of method 10 and detection unit 22, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.