HK1148580A - Device and method for microbiological analysis of biological samples - Google Patents

Description

Apparatus and method for microbiological analysis of biological samples

Technical Field

The present invention relates to a method and apparatus for analysing biological samples, and more particularly to microbiological analysis for testing biological samples for the presence of bacteriologically significant concentrations of microorganisms, such as in particular but not exclusively body fluid samples such as urine and blood.

Background

In order to test for the presence of pathogens, which are generally microorganisms that may have a detrimental effect on the health of humans or animals, it is well known to carry out microbiological analyses on biological samples, in particular body fluids. Such assays are typically performed on urine, blood, feces, and buffers. Generally, testing samples for the presence of pathogens within them is not sufficient and it is also necessary to classify them, i.e. to test which type of microorganism is involved, in order to determine the danger to health and to give the required treatment.

Traditional microbiological analysis methods for urine samples are based on so-called inoculation, which can be used to distribute the sample to be analyzed on a culture medium and hold it for a number of hours (typically 12 hours or more) in order to check whether a population of microorganisms is growing on the medium. If so, the microorganisms are examined to verify their properties.

When more samples have to be analyzed, the inoculation process will last for a very long time, requiring some preparation by the operator performing the process. Handling a large number of samples leads to biological risks and the risk that the samples may be confused with each other, thereby giving the patient wrong analysis results.

In "A head space chromatographic approach for the monitoring of the microbial cell activity and the cell viability evaluation" of F.Gardini et al, Journal of Microbiological Methods 29(1997)103-114, a method for detecting microbial activity within a sample to be analyzed based on gas chromatography is described. This gas chromatography is used to determine the carbon dioxide (CO) in the atmosphere in which the sample is located₂) Concentration, wherein such atmosphere is due to metabolism of microorganisms in the sample. This method requires complicated and expensive equipment and long analysis time.

US-A-4,971,900 describes A method and apparatus for detecting bioactivators in samples with different properties, such as urine. The method is based on the analysis of the carbon dioxide content in the atmosphere above a sample, which is placed on a culture medium. This analysis lasts for hours and is used to identify any pathogens by the trend of carbon dioxide over time. This analytical method requires a very long time and is not particularly reliable since it detects the microorganisms according to a calibrated trace of the time curve of carbon dioxide development. In particular, various problems arise when different types of pathogenic bacteria are present in the sample, which may develop at different times from each other.

US-A-6,709,857 describes A system for optically detecting the concentration of A gas in A vial containing A sample to be analysed. The gas concentration can be detected by means of photothermal spectroscopy.

US-A-5,155,019 describes A method of determining the presence and concentration of carbon dioxide in the atmosphere above A sample cultured within A container by detecting the presence of biological activity in the sample by infrared analysis of the sample sealed within the container. In this case, too, a particularly long time is required for the analysis, and complicated equipment is required.

US-A-5,217,876 describes A method for detecting the presence of microorganisms in A sample in A container. The method is based on the idea of optically detecting in a container in which the sample is grown a change in the colour of the indicator medium, which change is due to the presence of microbial activity within the sample and the development of carbon dioxide. As in the case mentioned before, also in this case, a long analysis time is required and the determination of the presence of pathogenic bacteria in the sample is not particularly reliable, since it is based on the trend of carbon dioxide development over time.

A similar process is described in US-A-5,094,955.

US-A-5,482,842 describes another method of detecting microorganisms in A body fluid, in particular A blood sample. The analysis is performed by an infrared light source and an infrared detector. Also in this case, the presence of carbon dioxide generated due to the presence of pathogenic microorganisms is detected. The infrared absorption coefficient of carbon dioxide is different from that of the atmosphere (in the absence of pathogenic bacteria) which is typically above the level of the sample in the vial.

US-A-5,856,175 describes A device for detecting pathogenic bacteriA in A sample of bodily fluid, which device is similar to that described in US-A-5,094,955 and US-A-5,217,876.

US-A-5,814,474 describes an apparatus for direct detection of microorganisms in culture flasks. The described device is used for analyzing urine, saliva or blood samples. Basically, the method described herein is based on the analysis of the atmosphere contained in a bottle into which a culture sample is inserted. The gas in the bottle is passed through a gas sensor to detect its composition, and then the presence of microorganisms in the sample is determined based on the results of the gas analysis. This analysis method is extremely complex, requiring very expensive sensors and long analysis times.

Disclosure of Invention

According to one aspect, the invention provides a method that simplifies and speeds up the operation of analysis of body fluids, particularly but not exclusively urine. According to some embodiments, the method according to the invention makes it possible to distinguish between a positive and a negative sample among a plurality of samples, i.e. to distinguish between the presence of at least one pathogenic bacterium in the sample, which must be identified in a second, more accurate analysis stage from a sample in which it is determined that no pathogenic bacterium is present, and therefore to perform further analyses without any use.

In this way, in a subsequent sample analysis phase, which may be performed by a seeding process or other known processes, only some of the initially considered samples are processed, without the need for further processing of the samples determined to be negative.

According to one embodiment, the method according to the invention comprises the steps of:

a) introducing a biological sample to be analyzed into a vessel containing a culture medium;

b) incubating the containers at a first time interval;

c) after the first time interval, analyzing an atmosphere in the container;

d) determining whether the biological sample comprises a pathology-related bacterial load or does not comprise a pathology-related bacterial load based on the amount of carbon dioxide detected in the atmosphere.

The invention is based on the idea, in general, of: the amount of carbon dioxide detected in the atmosphere within the sample is used as a parameter for distinguishing between the determination of a positive sample and the determination of a negative sample and is not used for determining the type of pathogenic bacteria that may be present in the sample as in the conventional method. Identification of the type of pathogen present in the positive sample can be carried out in a more accurate subsequent analysis stage, for example a vaccination process or other known systems, as described in the patent literature mentioned in the introductory part of the present description. However, the best methods currently able to identify in a reliable manner the type of microorganism present in a sample are those based on vaccination, which have the drawbacks described above.

In one embodiment of the present invention, two thresholds can be provided against which the amount of carbon dioxide produced after a given period of time in each individual culture sample container is compared. In some embodiments, a sample having a carbon dioxide content greater than a first limit value after a predetermined incubation time interval is classified as positive for determination, whereas a sample having a carbon dioxide content less than a second limit value over the same incubation period is classified as negative for determination. The intermediate sample is considered to be indeterminate and can be analyzed in detail to test for the presence and type of microorganisms for greater reliability of the analysis.

Vice versa, according to a modified embodiment of the invention, the undefined sample is not subjected to a detailed analysis such as a vaccination treatment, and if necessary is subjected to a second incubation interval to renew the atmosphere present in the single sample container. This renewal of the atmosphere can eliminate the presence of carbon dioxide and increase the oxygen, so that the development of the metabolism of the microorganisms present in the sample (if any) can take place. After a second incubation time interval, the uncertain sample is again examined for the carbon dioxide content of the container atmosphere. This content is then compared to a threshold value that distinguishes between positive samples (where the positive sample is determined to have a greater carbon dioxide content than the threshold value) and negative samples, where the negative sample is determined to have a lower carbon dioxide content than the threshold value.

By this second operating method, it is possible to further reduce the samples that must be subsequently subjected to the inoculation treatment, since the samples that have been determined as inconclusive by the first analysis stage are further divided into positive-determining samples and negative-determining samples. No inoculation or other analytical treatment is necessary for determining negative samples to determine the type of pathogenic bacteria contained therein.

Possible features of further advantageous embodiments and methods according to the invention are indicated in the appended claims and are described in more detail below with reference to one embodiment.

According to a different aspect, the invention relates to a device for microbiological analysis of a body fluid, such as urine or the like, comprising:

-an incubation area of a container containing said sample;

-an analyzer for analyzing the internal atmosphere of the container; and

-a sorting system for sorting the containers according to the carbon dioxide content detected by the analyzer.

Generally, in some embodiments, the device has an incubation area in which samples contained within a single container are incubated for a suitable period of time, such as about one hour. The samples are then analyzed by an analyzer and classified, i.e., divided into positive and negative samples. In a modified embodiment, the classification system classifies the first incubated sample into a positive-determined sample, a negative-determined sample, and an indeterminate sample. The device has a second incubation area for the indeterminate sample, which remains in the second incubation area for a second incubation time interval, if necessary after renewal of the atmosphere in the indeterminate sample container for the reasons described above. The second incubation stage may be performed in an incubation area where the first incubation is performed on a single sample. The device may be suitably controlled by the microprocessor such that it stores in memory information about the location of the containers with the sample having performed the first incubation period and the sample having performed the second incubation period, thereby avoiding errors in performing the first and second incubation periods on different samples contained within the analysis device or instrument.

Further advantageous features and embodiments of the device according to the invention are indicated in the appended dependent claims and are described in more detail below with reference to a non-limiting embodiment.

According to another aspect, another object of the invention is to provide a cuvette for use in said analysis method and use of the device according to the invention. More particularly, according to one embodiment, the test tube of the present invention is a vacuum test tube containing a culture medium suitable for the growth of microorganisms that may be present in the particular biological sample for which the test tube is intended, and a magnetic stirring element placed inside the test tube.

Drawings

The invention will be better understood by reference to the following description and to the attached drawings, which illustrate non-limiting embodiments of the invention. More specifically, in the figure:

figure 1 shows a plan view of an apparatus according to the invention;

FIG. 2 shows a section according to II-II in FIG. 1;

FIG. 3 shows a cross section according to III-III in FIG. 1;

FIGS. 4A and 4B show details of the analyzer and cannula or needle system for removing atmosphere from a single sample container;

FIGS. 5A to 5E show the order of processing positive samples;

FIGS. 6A to 6F show the sequence of treatment of an indeterminate sample;

FIG. 7 shows a longitudinal cross-section of a container with magnetic stirring elements and an external agitator; and

fig. 8 shows a flow chart of an analysis method according to the invention.

Detailed Description

With reference to fig. 1-3, the device according to the invention, designated as a whole by reference numeral 1, comprises a loading zone 3 for loading holders R of containers P, wherein the holders of individual containers P contain the biological sample to be analyzed and are inserted into the loading zone and processed by means of a first conveyor belt 5 according to the direction of arrow f 5.

In the illustrated embodiment the container is a vacuum tube with a sealed cap, but it will be appreciated that different types of containers may be used, sealed, if desired, to detect carbon dioxide (if any) that has accumulated inside the container due to the metabolism of microorganisms contained in the sample and that have grown due to the medium contained in the tube in which the sample is disposed.

An incubation area 7 is arranged in the vicinity of the loading area 3, in which a second conveyor belt 9 is arranged for moving the holders R of test tubes P according to the arrow f 9.

Reference numeral 11 generally designates a resting zone of the test tube P containing the indeterminate sample, preferably contained in the rack R, which must be subjected to the second incubation. Between the incubation area 7 and the rest area 11 there is arranged an analyzer, indicated as a whole with reference numeral 13, a sorter, indicated as a whole with reference numeral 15. The analyzer analyzes the atmosphere contained in the test tube in sequence for each test tube P in the rack R from the incubation area 7. Depending on the result of the analysis performed by the analyzer 13, the sorter 15 sorts the test tubes, subdividing them into: test tubes containing a positive sample, i.e. which must be analyzed in order to determine the pathogenic microorganisms contained in the sample; tubes containing negative samples, i.e. no further analysis thereof, is required as they do not contain significant pathogenic bacteria; and test tubes containing indeterminate samples, which are conveyed to the rest area 11 for the second incubation phase.

In particular in the loading zone 3, a single rack R is provided containing test tubes P, containing the samples to be analyzed, which can be inserted into the rack through holes closed by a door 17 (fig. 2). The conveyor belt 5 transfers the single rack R stepwise from the insertion position of the loading area 3 to the transfer unit 21, which transfers the single rack R of test tubes P from the loading area 3 to the incubation area 7. In some embodiments, the transfer unit 21 comprises a continuous flexible element 23 driven around pulleys 25, 27, at least one of which is driven by a motor. One or more pushers 29 are fixed to the flexible element 23, which can push the single rack R containing the test tubes P in the direction of the arrow f21, in a direction perpendicular to the direction of feed f5 of the conveyor belt 5, to process it. The conveying unit can also have a different structure, and can for example comprise a screw with which a cursor with a pusher engages. Rotation of the screw in one direction and the opposite direction can advance and return the cursor and associated pusher.

Whichever configuration has a transfer unit 21 which can be used to move a single rack R containing test tubes P of the sample to be analyzed from a loading zone 3, which is kept at a low temperature in order to inhibit or slow down the metabolism of the microorganisms (if any) present in the sample, to an incubation zone 7, which is preferably kept at a controlled temperature, for example about 37 ℃.

In the incubation area 7, the single rack R with the test tubes P is moved stepwise by the conveyor belt 9 from a position where the single rack is inserted from the transfer unit 21 into the incubation area 7 to the analysis and sorting area where the analyzer 13 and the sorter 15 are arranged.

In the analysis sorting zone there is provided a second transport unit 31, similar to the transport unit 21, for example comprising a flexible element 33 driven around pulleys 35, 37. One or more thrusters 39 are constrained to the flexible element 33 for thrusting the single support R towards the sorter in a step-by-step controlled movement. The transfer unit 31 is controlled by a programmable electronic control unit, not shown, in such a way as to feed each rack R step by step according to the arrow f31, away from the conveyor belt 9 and to pass the single test tube P contained in the rack R through the analyzer 13, respectively. In this way, each test tube is analyzed by aspirating the sample of atmosphere contained in the test tube and determining the carbon dioxide content developed in the test tube due to the metabolic action of the pathogenic microorganisms (if any) contained in the sample cultured in the test tube P during the incubation in the incubation zone 7.

With respect to the incubation time used in conventional analysis systems, which incubation has a suitable duration and lasts, for example, for about 1 hour, the conveyor belt 9 is programmed to move at a speed such that the incubation time is substantially equal to the time required for a single rack to move from the position where the transfer unit 21 is positioned to the position where the transfer unit 31 is positioned.

Also as shown in particular in fig. 4, in this embodiment the analyzer doubles, comprising a first sensor 41A and a second sensor 41B for determining with sufficient accuracy the carbon dioxide content in a single test tube P. With this dual arrangement, the analysis speed of the apparatus can be doubled. Each sensor 41A, 41B may be manufactured by non-dispersive infrared (NDIR) technology, for example as described in US-A-6,255,653, the contents of which are incorporated herein by reference. Each sensor is connected to a piercing needle 45, one of which is visible in fig. 4, by a respective flexible tube 43A, 43B. The two needles 45 are carried by a slide 47 which can slide vertically along the guide column 49 according to a movement f47 exerted by an actuator not shown. The raising and lowering movement of the needle 45, integral with the slide 47, serves to make the two needles 45 penetrate the test tube P, each time the test tube is located below the slide 47. The lowering of the needle 45 is controlled in such a way that the needle remains in the area of the single test tube P where there is a gaseous atmosphere, indicated by G in figure 4, and without touching the biological sample C collected in the lowermost part of the test tube P, such as urine, blood or other body fluid sample. The descending movement of the needle 45 perforates the cap T of the test tube P, so that a portion of the gas above the sample C flows through the perforated needle 45 and the flexible tubes 43A, 43B towards the sensors 41A, 41B of the analyzer 13. Fig. 4A and 4B show the penetration movement of the piercing needle 45 through the cap T of the test tube P to position the piercing needle (fig. 4B) in the gas aspiration position. The gas in the single test tube P flows towards the sensors 41A, 41B through the respective tubes 43A, 43B under the effect of an overpressure generated inside the test tube due to the accumulation of carbon dioxide developed by the metabolism of the microorganisms (if any) present in the sample C.

The sensors 41A, 41B are able to detect the amount of carbon dioxide present in a single tube under analysis with sufficient accuracy for the purpose described below. There is no need for high precision and long-term or repeated measurements, and instead the trend in carbon dioxide concentration is used as a primary parameter in determining the type of microorganism present in the sample, as in conventional systems. In contrast, according to the invention, it is important that the presence of carbon dioxide is taken as an indicator of the presence of microbial metabolism in the sample, if desired, and that the nature of the microorganisms can be determined in a subsequent stage of quantitative analysis on positive samples.

The sorter 15 performs operations on the single test tube P contained in the rack R, according to the amount of carbon dioxide detected by the single sensor 41A, 41B, as will be described below with particular reference to fig. 5 and 6.

The sorter 15 can pick up the single test tubes P from the racks R fed in steps by the transfer unit 31 in order to sort them in the resting areas 11, or sort them in the underlying trays 51 (fig. 2) where the positive samples are accumulated, or can also retain the negative samples in the racks R, which are then taken by the operator and emptied of the test tubes P, or simply discharged from the analysis device for subsequent processing by the operator.

More particularly, in the embodiment shown, the sorter 15 comprises a slide 53 guided on a substantially vertical guide 55 having a movement according to the double arrow f53 (see in particular fig. 3). Cursor 59 is movable along a guide 57, along slide 53, which carries a gripper 61 with opening and closing elements controlled by an actuator 63 carried by cursor 59. The cursor 59 has a movement according to the double arrow f59 (fig. 3) along the longitudinal length of the slider 53. Thanks to these two movements f59 and f53, the gripper 61 can grip a single test tube P from a rack R which is pushed in a stepwise manner by the transfer unit 31 so as to be expelled through the well 65 into the space 51 below or inserted into one or the other of the two racks R in the rest area 11.

It should be understood that the number of supports R in the rest area 11 may be different from that shown. For example, it is possible to provide only one support R, or more than two supports R, in which case it is obviously necessary to expand the stroke of the gripper 61 and its cursor 59 in the direction f59 in a suitable manner so that all the supports R arranged in parallel in the rest area 11 can be reached.

FIGS. 5A-5E show a test tube P for containing a positive sample (i.e., the bacterial load present in the sample that requires further analysis-such as by inoculation-to detect the type of microorganism present) through a well 65⁺The movement of the gripper 61 discharged into the space 51 below. In fig. 5A, test tube P is facing under the gripper⁺The open gripper is lowered and the test tube can be carried in this position by following the movement f31 of the holders actuated by the transfer unit 31. In fig. 5B, the gripper is lowered and then closed to grasp the test tube P⁺. In FIG. 5C, the test tube P is removed by lifting it off the holder R⁺To lift the test tube so that it is moved above the well 65 by following the movement f59 (fig. 51D), where the gripper 61 is opened to allow the test tube P⁺Into the well (fig. 5E). Test tube P⁺The collection zone is reached through the well, where the operator can collect all the test tubes that must be analyzed according to known methods, in order to detect the type of microorganism present in the samples contained in these test tubes.

When the sample in the test tube P that has to be picked up by the gripper 61 is negative, i.e., when the analyzer 41A or 41B does not detect a considerable amount of carbon dioxide content in the atmosphere taken out from the upper portion of the test tube P after the incubation for about 1 hour in the incubation area 7, the test tube remains in the rack R and then exceeds the position where the controller 15 is positioned without gripping by the gripper 61.

A sample whose carbon dioxide content has been detected in the atmosphere inside the test tube, which is greater than a minimum value below which the test tube is considered negative, but less than a maximum value above which the test tube is considered positive, can be picked up by means of a gripper 61 and processed according to the cycle schematically illustrated in figures 6A-6E. In FIG. 6A, the open gripper is ready to be lowered to P^？The tubes shown, which require further incubation. In FIG. 6B, the gripper has been lowered and closed on the test tube P^？The above. Gripper 61 then withdraws test tube P according to arrow f53^？And is conveyed towards a rack R in the rest area 11, which has a movement beyond the drainage well 65 as shown in figures 6C and 6D. When this position is reached, lowering the gripper 61 will not determine the test tube P^？Inserted in the rack R of the rest area 11 (fig. 6E), then the grippers are opened and raised to leave the test tubes in the rack, and then returned to the gripping position to grip new test tubes P contained in the rack R, which is fed in a stepwise manner by the transfer unit 31 (fig. 6F).

In this way contained in test tube P^？The single indeterminate sample of (a) accumulates in the rack R of the rest zone 11, the carbon dioxide content of said indeterminate sample being between two static values, i.e. a maximum value and a minimum value, for which further incubation is required.

In a modified embodiment, it is also possible to carry out a further analysis of all the indeterminate samples in order to detect the type of microorganism contained inside them, so that it is not necessary to provide the rest area 11, or to inactivate the samples and simply sort the tubes by subdividing them into positive and negative, so as to expel the indeterminate samples together with the positive samples directly above the well 65 according to the method described above. Instead of providing a drainage well, it is also possible to transfer the positive and indeterminate samples in the area 11, picked up manually from the area 11, for a vaccination or other treatment for detecting pathogenic microorganisms inside the sample, while the test tubes with determinate negative samples remain on the initial rack R from the first incubation area.

According to other embodiments, the negative sample may be placed in the drain well such that there are no tubes on the rack R from the incubation area. In another variant of embodiment, the positive samples are transferred into the resting zone 11, the positive samples are expelled from the wells into the zone below, without being sure that the samples can be held in the rack in order to insert them again into the loading zone.

In general, it is important to classify at least between positive and negative tubes, preferably between positive tubes, negative tubes and indeterminate tubes, which require the second incubation phase.

According to some embodiments, the incubation means are arranged in the rest area 11 so that it is possible to maintain indeterminate test tubes P directly in the rest area 11 in an incubation state at a controlled temperature, for example about 37 ℃⁺And treated here in the manner described above, in the rest area 11, for example a second analytical device is arranged, or a single rack is transferred from the rest area or second incubation area 11 to the area where the sensors 41A, 41B are active.

However, according to a preferred embodiment, the rack R, which has filled the rest area 11, is picked up by the operator and inserted again in the loading area 3, so that it is subjected to a new incubation cycle in the incubation area 7.

In order to contain the test tube P (P) in the resting zone 11^？) The indeterminate sample in question undergoes the microbial metabolism present here, and according to some embodiments, a device generally designated by the reference 71 can be arranged in the resting zone 11, which can inject oxygen or a gas containing oxygen anyway into the single test tube P, which is in the resting zone 11. This device 71 comprises, for example, a slide 73 vertically movable along a guide 74 and carrying a pair of perforating needles 75A, 75B able to be fed to a resting zoneThe test tube P in 11 is perforated and inside it is blown oxygen or ambient air, fed for example by a compressor connected to the needles 75A, 75B by flexible tubes. The carriages R can be fed in the rest area 11, for example by means of two transfer units 81A, 81B, so as to be able to be fed with indeterminate samples P^？Filled and a perforation is obtained through the device 71, said transfer units 81A, 81B having substantially the same structure as the transfer units 21, 31 and not being described in greater detail.

In this way, the single rack R is gradually filled with indeterminate test tubes P passing under the slide 53^？And carries each test tube P below the needles 75A, 75B, so that it receives oxygen capable of developing the metabolism of the microorganisms, thus pushing the rack R outside the rest area 11 in order to reintroduce it into the loading area 3.

The device has a user interface that can communicate with the central unit of the machine in which the racks R inserted in the loading area 3 have passed the first incubation phase and therefore contain indeterminate test tubes, and are then loaded therein with new test tubes that must perform the first incubation in the incubation area 7.

In this way, thanks to the presence of the encoders associated with all the actuators for handling the racks passing through the different zones of the machine, the machine is able to know at any moment which rack contains the test tubes that have already undergone the first incubation and are currently in the second incubation phase, and which rack contains the test tubes that must undergo the first incubation, the analysis and, if necessary, the second incubation if the results of the test tubes are uncertain. Alternatively, instead of tracking individual test tubes by controlling the feed motion, a system for reading barcodes or other codes associated with the test tubes may be provided to identify each test tube at a primary location on its path through the machine.

While in the incubation zone 7 (or, in a modified embodiment, directly in the resting zone 11) the test tubes containing indeterminate samples (test tubes P) coming from the resting zone 11 are subjected to^？) During the second incubation period, the cells are analyzed by an analyzer13 a new analysis is performed on it. The carbon dioxide content detected in the second analysis is preferably compared with a single threshold value. Samples containing an amount of carbon dioxide greater than the threshold value are considered positive and therefore expelled into the well 65 by the sorter 15, while samples containing an amount of carbon dioxide less than the threshold value are considered negative and remain in the rack, being gradually expelled from the incubation area 7 by the action of the conveying unit 31.

The threshold for distinguishing after the second incubation may be equal to the minimum or maximum threshold for sorting and distinguishing the tubes in the first incubation stage.

The flow diagrams of fig. 8A, 8B schematically summarize the overall process. The flow chart shows how a single cuvette is filled with a sample to be analyzed and then inserted into the device. Subsequently, the tube is incubated for a period of time Δ T, and when the incubation is finished, the atmosphere in the tube is removed. The detected carbon dioxide amount is compared with a first threshold value S_maxAnd a second threshold value S_minA comparison is made. If the content of carbon dioxide is larger than the threshold value S_maxThe sample is considered positive and is discharged by the sorter 15 through the well 65 into the space 51 below. If the content of carbon dioxide is less than the threshold value S_minThe sample is considered negative and is held on the holder and then discharged from the machine. If neither of these conditions occurs, the carbon dioxide content is between S_maxAnd S_minIn between, oxygen is added to the test tube to allow metabolism, a second incubation is performed with a time interval, in this example of the same duration Δ T as the first incubation, although this is not absolutely necessary, and the two incubation phases may also be of different duration. When the second incubation is finished, the atmosphere is removed from the test tube and the carbon dioxide content is compared with a single threshold value, which in the example shown is S_maxBut it may also be S_minOr is with S_maxAnd S_minDifferent thresholds. If the sample produces more carbon dioxide than S_maxIt is considered to be positive, otherwise it is considered to be negative.

In order to optimize the incubation of the sample, according to some embodiments stirring elements are arranged in the incubation area 7, positioned in a suitable manner along the feeding path of the rack. To simplify the drawing, the agitators are not shown in fig. 1 to 6, but one of them is shown schematically in fig. 7 below a single test tube P. The agitator of fig. 7 is generally referred to by the reference numeral 100. It comprises an actuator 101, such as an electric motor, which can rotate a magnetic element 103, such as a magnetic rod inserted inside a disc keyed to the shaft of the motor 101. The magnetic element 103 can act as a magnetic bearing for the stirring element 107 contained in the test tube P and immersed in the sample C in the same sample test tube P. The magnetic coupling between element 103 and element 107 may cause element 107 to rotate due to the rotating action of shaft 101. The element 107 can have a suitable shape, for example with fins, to enable the sample C contained in the test tube P, which also contains the culture medium, to move upwards, so as to optimize its incubation inside the test tube P.

Figure 7 also schematically shows other possible features of the device according to the invention, constituted by a sensor generally indicated with 111 and suitable for detecting the level of the sample C in the test tube P. The sensor 111 may be a capacitive sensor or any other type of sensor. It may for example comprise a transmitting/receiving device for detecting the level of the sample C by transparency. The sensor 111 has a vertical movement parallel to the axis of the test tube P to detect the level of the sample C in the test tube. The sensor 111 may be arranged at any suitable position in the device 1, for example at a free area between the loading area 3 and the incubation area 7, as schematically indicated with 111 in fig. 1, so that the sample level in each individual test tube can be determined during the movement of the test tube accommodated in the rack R from the loading area 3 to the incubation area 7 performed using the transfer unit 1.

Determining the level of the sample C in each test tube P can avoid inadvertently submerging the tip of the needle or cannula 45 into the biological sample, as this may damage the sensors 41A, 41B. The central control unit of the machine can store the level of the sample C detected in each test tube P, allowing the slide 47 carrying the needles 45 to always perform a sufficient lowering movement to perforate the caps T of the test tubes P, but also so that these needles do not come into contact with the sample.

Using the method and apparatus described above, it is possible to classify a large number of samples contained in the test tube P after a first incubation period (for example about 1 hour), into positive determinate samples, negative determinate samples and indeterminate samples, if any. For safety and simplicity, these indeterminate samples can be considered as positive or subjected to a second incubation cycle, whereby a second classification between positive and negative samples is performed according to the method outlined schematically in the flow chart of fig. 8A, 8B. Finally, whatever the method chosen, the first reliable results with respect to the determination of negative samples can be obtained from a large number of samples, over a period of up to two hours, significantly reducing the samples that must be analysed longer and more accurately, for example by inoculation, to detect which microorganisms present in the sample are classified as positive. Thus, only samples that can save significant costs and risks are inoculated or otherwise analyzed.

This allows considerable advantages to be obtained with respect to all the traditional analytical methods, in particular those described in the patent documents mentioned in the introductory part of the present description.

In order to automate the analysis performed by the above-described apparatus, the individual test tubes P may be marked with a single code during the production phase. Furthermore, each test tube has a label, identification or the like carrying a code, for example in the form of a bar code, which is connected in a doubly unique manner to the patient data with which the sample C contained in the test tube is associated. The device 1 has bar code reading means or the like which can read both the single code applied to the test tube during the production phase and the code associated with the patient to whom the sample contained in the test tube belongs. These two codes are matched by the central unit of the device 1 so that the code associated with the patient, applied for example by the identification around the cap of the test tube P, can be subsequently removed in order to carry out an analysis operation on the sample considered positive. These analyses-for example vaccination-actually require breaking the test tube and therefore risk damaging the barcode that can identify the patient and be applied to the cap. The matching between the test tube code and the patient code is performed by reading means associated with the device 1, avoiding the risk of losing the data of the patient to whom the sample belongs when breaking the test tube for inoculation.

Subsequent analysis operations may be performed automatically by storing the identification codes of the test tubes, which correspond one-to-one to the patient codes, and associating the analysis results with the test tube codes. When the analysis has been performed and the results obtained, it can be matched again with the patient data, so that the data can be recovered simply by means of the patient identification code and the test tube identification code associated with each other.

It is understood that the drawing only shows an example provided by way of a practical arrangement of the invention, which may vary in forms and arrangements without however departing from the scope of the concept underlying the invention. Any reference signs in the appended claims are provided merely as a convenience in reading the claims in light of the description and drawings, and do not in any way limit the scope of protection represented by the claims.

Claims (42)

1. A method of performing a biological assay on a biological sample, comprising the steps of:

a) introducing a biological sample to be analyzed into a vessel containing a culture medium;

b) incubating the containers at a first time interval;

c) after the first time interval, analyzing an atmosphere in the container;

d) determining whether the biological sample comprises a pathology-related bacterial load or does not comprise a pathology-related bacterial load based on the amount of carbon dioxide detected in the atmosphere.

2. The method of claim 1, wherein if a biological sample contains a pathologically relevant bacterial load, said biological sample is classified as positive and is further analyzed to determine the presence of microorganisms.

3. A method according to claim 1 or 2 wherein if a biological sample does not contain a pathologically relevant bacterial load, the biological sample is classified as negative and is not subjected to further analysis to determine the presence of microorganisms.

4. A method as claimed in claim 1, 2 or 3, comprising the steps of:

-comparing the atmosphere inside the container with a maximum and a minimum of carbon dioxide concentration after the first time interval;

-if the atmosphere contains an amount of carbon dioxide greater than the maximum value, classifying the sample as positive and analyzing it to detect the presence of microorganisms;

-if the atmosphere contains an amount of carbon dioxide less than the minimum value, classifying the sample as negative and not analyzing it for the presence of microorganisms;

-keeping the biological sample incubated for a second time interval if the atmosphere contains an amount of carbon dioxide between a maximum and a minimum.

5. The method of claim 4, wherein oxygen is injected into the container prior to the second incubation time interval.

6. The method according to claim 4 or 5, wherein at the end of the second incubation time interval the atmosphere in the container is analyzed again and the biological sample is classified as positive or negative depending on the carbon dioxide content detected in the atmosphere.

7. The method of claim 6, wherein the biological sample analyzed after the second incubation interval is classified as positive or negative based on whether the carbon dioxide content of the container atmosphere is greater than or less than a threshold value.

8. A method according to claim 6 or 7 wherein if the biological sample is classified as positive after the second incubation interval it is analysed to determine the presence of microorganisms and if the biological sample is classified as negative it is not analysed to determine the presence of microorganisms.

9. The method according to one or more of the preceding claims, wherein during said first incubation time interval the biological sample is kept in a thermostated state.

10. The method according to one or more of the preceding claims, wherein the biological sample is agitated during said first incubation time interval.

11. The method according to one or more of the preceding claims, wherein during said second incubation time interval the biological sample is kept in a thermostated state.

12. The method according to one or more of the preceding claims, wherein the biological sample is agitated during said second incubation time interval.

13. The method according to one or more of the preceding claims, wherein a plurality of biological samples are analyzed sequentially, the atmosphere in each biological sample container is analyzed after the first incubation time interval, the respective biological sample is classified into one of the following groups according to the carbon dioxide content detected in the atmosphere:

-a positive sample which is further analyzed to detect the presence of microorganisms in the biological sample;

-a negative sample which is not analysed to detect the presence of microorganisms;

-indeterminate sample, subjected to a second incubation for a second incubation time interval.

14. The method of claim 13, wherein after the second incubation, the indeterminate sample is subsequently classified into one of the following groups according to the carbon dioxide content detected in the atmosphere of the respective container:

-a positive sample which is further analyzed to detect the presence of microorganisms in the biological sample;

-a negative sample, on which said analysis is not performed.

15. An apparatus for performing microbiological analysis on a bodily fluid sample, comprising:

-an incubation area of a container containing said sample;

-an analyzer for analyzing the internal atmosphere of the container;

-a sorting system for sorting the containers according to the carbon dioxide content detected by the analyzer.

16. The apparatus of claim 15, comprising a laydown area for placing samples requiring a second incubation period.

17. The apparatus of claim 15 or 16, wherein the sorting system sorts the containers from the incubation area to subdivide the containers into:

-positive containers that must be analyzed to identify the microorganisms present in each sample;

-a negative container not having to perform an analysis to identify the microorganism;

-an indeterminate container for carrying out the second incubation.

18. The apparatus according to claims 16 and 17, wherein the sorting system conveys positive containers to a positive container pick zone and uncertain containers to the second laydown zone.

19. The apparatus of claim 15, 16, 17 or 18, wherein the sorting system comprises a conveying element for the containers.

20. The device of one or more of claims 15-19, wherein the analyzer comprises a non-dispersive infrared carbon dioxide sensor.

21. The apparatus according to one or more of claims 15 to 20, wherein said analyzer comprises an aspiration duct for aspirating the gas inside said container and terminating with a perforation needle, said perforation needle and said container having a relative penetration movement in order to perforate said container by means of said perforation needle.

22. The device according to one or more of claims 15 to 21, wherein said analyzer comprises two carbon dioxide sensors and two aspiration ducts for simultaneously aspirating gases from the inside of two containers and terminating with respective piercing needles, said piercing needles and said containers having a relative penetration movement of the piercing needles in said containers.

23. Device as claimed in one or more of the claims from 15 to 22, wherein at least the incubation zone is maintained at a controlled temperature.

24. Device as claimed in one or more of the claims from 15 to 23, wherein an agitation element is arranged in said incubation area in order to agitate the samples in said containers.

25. An apparatus as claimed in claim 24, wherein the agitating element is a magnetic agitator.

26. Device according to one or more of claims 15 to 25, comprising a loading zone for loading a sample to be analyzed.

27. The apparatus of claim 28, wherein the loading zone is cooled.

28. Apparatus according to claim 26 or 27, comprising a conveyor belt located in the loading zone for conveying the containers along the loading zone.

29. The apparatus of claim 28, wherein the conveyor in the loading zone is designed to receive and convey carriers of containers.

30. An apparatus according to claim 28 or 29, comprising a transfer unit for transferring containers from the loading zone to the incubation zone.

31. Device as claimed in one or more of the claims 15 to 30, wherein a conveyor belt is arranged in the incubation area for handling containers from an incubation start position to an incubation end position.

32. The apparatus according to claims 30 and 31, wherein said transfer unit is arranged near a cultivation start position.

33. The apparatus of claim 31 or 32, wherein a extractor is arranged near the incubation end position, said extractor taking the container out of the first incubation area and moving the container towards the analyzer.

34. An apparatus according to claim 31, 32 or 33, wherein the conveyor of the incubation area is arranged and designed to convey racks of containers.

35. Device as claimed in one or more of the claims 15 to 34, wherein a collection zone for collecting positive sample containers is arranged below the level at which the incubation zone is arranged.

36. The device of claim 35, comprising a well for gravity transfer of a positive sample of an incubation area to said collection area.

37. The apparatus as claimed in one or more of claims 15 to 36, comprising a conveyor belt at said laydown area for processing said containers along said laydown area.

38. The apparatus of claim 37, wherein the conveyor belt at the laydown area is designed to receive and convey racks of containers.

39. The apparatus of one or more of claims 15 to 38, comprising at least one sleeve for injecting a gas or a gas mixture comprising oxygen into the container.

40. Apparatus according to claim 21 or 22, comprising a measuring instrument for measuring the level of the biological sample contained in the container, said measuring instrument being associated with said suction duct to avoid said perforating needles reaching the level of the sample in the container.

41. A device as claimed in one or more of claims 15 to 40, comprising a second incubation zone, into which the sorter inserts the samples that have to undergo a second incubation.

42. A container, in particular a vacuum test tube for biological analysis, comprises a culture medium and a magnetic agitator inside the culture medium.