MXPA00004711A - System for analyzing images produced by bacterial reactions - Google Patents

Abstract

The system is based on an apparatus (1) forming a closed chamber (2) inside which is arranged a superior and centred digital camera (15) oriented towards a removable support (17) either a tray of wells wherein are situated the liquid cultivating means or a plate on which is placed a solid cultivating means;the support (17) is arranged between lower lighting means (18) and upper and lateral lighting means (19) and are completed with light diffusers (20 and 21). The apparatus is provided externally with operation indicators, switches and taps and connections which are intended to the proper operation and control via a central computer. The system captures and interprets the images produced by the reactions which take place in the referred liquid or solid cultivation means, thereby converting said bacterial reactions into values, as well as analyzing the growth of micro-organisms.

Description

SYSTEM ANALYZER OF IMAGES PRODUCED BY BACTERIAL REACTIONS E »DESCRIPTION 5 OBJECT OF THE INVENTION The invention relates to a system capable of converting images of bacterial reactions into numerical values, as well as analyzing the growth of microorganisms by means of the capture of the image of a panel or board. It is the object of the invention to carry out a capture and interpretation of the images produced by reactions that occur, either in liquid culture media "contained within the wells of panels, or on the surface of a solid culture medium contained in containers commonly used in the laboratory, called plates.

BACKGROUND OF THE INVENTION In liquid culture media, supports consisting of the so-called panels are used, which are usually polystyrene and rectangular in shape, which include wells intended to contain a variety of media.

I culture my cryptography, biochemical substrates, and / or antimicrobial agents, all of them in liquid form, in which metabolic reactions take place whose interpretation my cr io g 1 of a microorganism and / or the determination of its ability to be neutralized by one or several antibiotics. On the other hand, the crops are also made in another type of supports called plates and they are generally polystyrene containers, of variable form, which contain a single solid culture medium on which the bacterial reaction takes place. Well, in the case of the panels, in the laboratories they are inspected visually the different reactions produced in the different wells and depending on their shape and color, the probability of the result is determined through the timely consultation of tables of general use published for that purpose. 20 Currently, automatic reading systems are used that, by measuring the optical density of the wells, assign a value that is interpreted as positive or negative through a computer program. Subsequently, another computer module determines the probability of the result by combining the positivity of the partial reactions. In the case of plates, t r a di ci ona lme n t e f i * the approach to the result is more rudimentary, since that the number of parameters to be considered is smaller, based essentially on the quantity, size and color of the bacterial colonies. The advantage of the automatic systems described, which measure optical densities, on the traditional method of visual inspection, is that the user's subjectivity in the interpretation of reactions is eliminated, since each reaction is translated into a numerical value. However, these automatic systems 15 suffer from a serious problem and inconvenience, and that is that they can not interpret the morphological characteristics of the bacteria, nor their growth patterns, so they escape their method of obtaining results from certain elements of the bacteria. 20 importance, as the form of bacterial growth, which has been the basis for obtaining results my cr obiológico s during the last decades.

DESCRIPTION OF THE INVENTION The system proposed is designed to solve the aforementioned problems, permitting the conversion of reactions into numerical values that are subsequently used by a diagnostic software to determine the quantity of microorganisms present indicative of infection, either for the identification of the microorganism present in the sample, or to determine the pattern of ntimicrobial susceptibility of the causative organism. The reactions identified by the system of the invention are the following: Size, color, shape and quantity of the bacterial colonies. - Development of color in wells with biochemical substrates, indicative of the type of bacterial metabolism. - Development in the form of button or turbidity in wells with antibiotics, indicative of the capacity of the microorganism to develop in the presence of certain concentrations of antimicrobial agents.

- Circle around a disc with antibiotic, called "inhibition halo". For this, the system is based on an apparatus j of an eminently prismatic-rectangular configuration, whose interior determines a closed chamber with different shapes and dimensions, provided that they allow a suitable focal distance between the lens of a digital camera and the panel or plate. object of analysis. The digital camera is mounted internally on a support arranged upper and lower, while in a zone close to the lower part of the interior of the camera there is a support associated with a feeder for one way to deposit in it the corresponding panels or plates, or what is the same the objects to analyze in each case, with the particularity that the supports are interchangeable and can be of different types depending on the panels or plates to be processed. In each case, the support in question, located inside the chamber as previously stated, has the function of allowing the passage of light, from the lower part of the object that is processed or analyzed, and that of centered of the same one under the digital camera.

Inside it also includes a lighting system consisting of four fluorescent tubes, two of them installed on the sides, above the object to be processed, and the other two installed below the object to be processed, so that depending on the type of image The upper fluorescent, lower or both fluorescent lights will be activated, which may be the au- tomation, through the software that manages the different functions, or manual through the operation of a switch. Except in the corresponding front panel of the camera, a series of readout indicators and switches are included for the control of their functions, as well as the corresponding mechanisms of the feeder for the internal control of the panel or plate to process. The rear panel of the camera includes externally the corresponding connections to a central computer, as well as a TDM 436 card, and the actual power supply, both for the digital camera and for the lighting system, without prejudice to the fact that system needs to be installed or other connections can be modified or modified.

Also noteworthy is the fact that in this back panel an access t-apa for cleaning in erna, maintenance, replacement of components, calibration, etc. is included. The system also includes means that make it possible to identify the type of panel, as well as the identification of external information that is used to complement the idenfication tests included in the panel, as well as to determine the position of each well in the panel and calculate the area of the same that is to be used for reading. According to the described characteristics, the system must first perform the corresponding calibration of the camera, showing it a histogram on the screen of a personal control computer and reading values, so that once the indicator has been activated Once the calibration reading has been completed, a panel that has been inserted into the internal support of the feeder will be read, dividing the upper surface of the panel into three information areas, one corresponding to the identification area of the panel type , one corresponding to the external information area and another corresponding to the area riiii J. ü b Once activated in the control software xa corresponding reading option, ^ e closes the feeder and the reading instruction of the image is given, so that the information generated by the system is interpreted by the software that assigns to the analyzed bacterium a classification taxonomic ion and a coding as resistant or not to a certain antibiotic or group of antibiotics.

DESCRIPTION OF THE DRAWINGS To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical realization thereof, is accompanied as an integral part of said description, or set of drawings where the following has been shown as an illustrative and non-limiting character: Figure 1.- Shows an external representation and front of the apparatus or camera that forms part of the system object of the invention.

Figure 2 shows an external view, but in this case later, of the same apparatus or camera represented in the previous figure. Figure 3 shows a front view in section through a vertical plane of the apparatus or camera represented in the two previous figures. Figure A. - It shows a side view, also in section by a vertical plane, of the same apparatus or camera of the previous figure. i0 Figure 5.- Shows a general and superior view of a panel where the three identification sections corresponding to: type of panel; external information, and position of each well. Figure 6 shows a perspective view of an embodiment of a support for a well panel. Figure 7 shows another view also in top perspective of a plate holder. Figure 8 shows a detail of the way in which the selection of the reading area is carried out according to the position of each well in the corresponding panel. < * ? < *, 'Figure 9.- Shows a plan view of a plate in which printed discs are included < l ~ a selection of tibiates. The fi xure 10.- Finally shows the rafica corresponding to the calibration histogram.

PREFERRED EMBODIMENT OF THE INVENTION To the aforementioned figures, it can be observed how the system of the invention is based on an apparatus with a general housing (1) of prismatic configuration-r e_c tangular, that internally efine a closed chamber (2) , with the particularity that in its front anel '3) it incorporates eminently a series of led indicators (4) that indicate the following functions: the indicator (4a) corresponds to the current input to the chamber (2); the indicator '4b) corresponds to the end of calibration. The three following indicators (4c) correspond to 25%, 50% and 1 5, respectively, of the reading made, while the indicator (4 d) corresponds to the end of reading. Likewise, in that front panel (3) the corresponding power supply (5) is included, and its function will be explained later, an aper ura button (6) for action not of that, a switch and contrast (7) and a switch * general (8), so that both the indicators (4) 5 and the elements (5 to 8) referred to constitute 1. means for the control of the system functions, as well as for the introduction of the panel or plate to be read. On the other hand, in the rear panel (9) of the apparatus (1) includes a cover '10) for access to internal components, as well as the vile power socket) for auxiliary lighting systems; the current take (12) for a digital camera that will be described below; a port or port of communications (13) with the central computer, as well as the connections (14) to a TD 436 module in the ue resides the calculation program of ?? version of color images of the image in rmeric values, corresponding those connections (14; to 20 the colors blue, green and red, indicating the signal corresponding to the percentage of those colors or colorimetric signals. the camera (2) includes the aforementioned digital camera (15) mounted on a support (16) located at the top, the camera (15) being arranged centrally and aligned with the n ° of a: support (17). ) on which the object to be analyzed will be placed, write me: "1, the corresponding plate or anel." The support (17) is exchangeable and may be variable in its configuration, according to the feeder (5) and The type of panels or plates that are to be supported In any case, that support 17 forms part of the feeding device itself (5) and constitutes the means that allows the introduction of the supports to be processed from outside. ecir, of the panels and pracas, since the action on the button opening (6) carries with it the displacement of a! i me n t a o o r r (5) and by i. ".) -ancho de s port (17), guindose m ore railes and being driven in its displacement - outward by any conventional system, from a simple thrust spring to use pneumatic or other mechanisms. Inside the camera (2), in addition to the digital camera (15) and support (17) referred to, are included the corresponding lighting means consisting of a pair of fluorescents (18) located below and a pair of fluorescents (1 ) located at an intermediate and laterally, complementing those fluorescents 18) and (19) with respective diffusers laugh light (20) and i '21), so that the former are below the object to be processed, ie, below of the -opor e (17), while the seconds are above, with the particularity that the latter -i co plemen an with a plate to protection. { !? '), onstituti a to a metallic screen and clinada to prevent that his radiations interfere with the ecc ra of digital camera - •) The fusadores of light.20) and '21) are preferably of white crystal and are provided for the homogeneous distribution of the light, in the first c.o or on the object to be processed, and in the second case through the transparent polystyrene wells of the panel < from 1 to p 1 to c a. Depending on the type of image to be processed, the upper fluorescents will be activated (19), or the inferoresores inferieres (13), or both simultaneously, being able to carry out the accionation automatically, through the sof software that manages the different functions, or manually by means of the i ctionami ent of the contrast switch (7) located on the front panel (3).

Figure 6 shows a support (171) for a pan of wells, soup rte (1 ') that is affected by the corresponding holes (22) for the wells of the panel, as well as with the recesses (23) and (24) for the illumination of the panel identification area and for the illumination of the external information area, also including that support (17 ', and some pivots (25) for fastening to the corresponding feeder. a support (17") aa plates, counting with the -. pivot s (25) of s feción ai aiimentador, as well as with the orifice '26) for the lower illumination of the plate above it, and some tabs (27 The device's operation is different depending on whether it is intended to analyze the image of a panel with its biochemical reactions and its antimicrobial susceptibility patterns that take place in different sections of the same panel, or the image of a plate with growth of colonies and / or halos of inhib In any case, prior to the execution of a reading session of the same type of product, panel or plate, the device must be calibrated, choosing for it the support > , 17) that is going to be used which, in turn is in function c? e 1 panel or plate to be processed. For one hundred, once the support (17) is arranged, which as previously stated can have the configuration shown in figure 6 and referenced r (17 '), or bi n the configuration r presented c- n figure 7 and refere with i 1"), or other configuration depending on the type of panel or plate, without introducing any panel or plate to allow free passage of light through the holes (22) or (26) of the support, the option "calibrate" from the control software, which resides on a personal computer that communicates with the device through the aforementioned communication port (13), then the camera shows on the personal computer screen a control hystogram, which corresponds to the graph shown in figure 10, and some reading values, while the indicator (4b) is activated, indicating that the calibration reading has ended, if the calibration is within the stable ranges this process is considered finished so, and otherwise the necessary adjustments are made in the camera (2). The hi-togram offered by the system according to Figure 10, should have the peaks of the ends as close as possible and the valley as close as possible to the axis of ab solutes. The way to perform the readings is as follows: When it comes to panels, which as already said '- < > Priority are polystyrene supports containing a number of wells in which biochemical substrates are included that are used to identify a bacterium and others in which concentrations are included, increasing antimicrobial agents that are used to determine the capacity of the bacteria. Said bacterium to be ombatida with the lowest possible concentration of antibiotic, is started by operating a reading algorithm that divides the surface (28) of the panel shown in Figure 5 in three corresponding to the references i 2 i, ( 30) and .31), so that reference (29) (co-sponde < i identification area of panel type, reference (30) to external information source, and reference (31) to the wells area.

In the first case, and through rail area (29) is identified, as said, the type of panel that is being used (there are different types according to the microorganism, a group of micro-isms to which it is intended, or samples of Onde ocedan). The area (29) is divided into rectangles, although they could have another geometric shape, preferably being printed on the upper left of the panel (28) (for example, if the first and third rectangles of the area are full), 29 ), indicates that it is a panel for Enterobacteriaceae. Obviously, information on the type of panel is essential for the inter- pretation of the behavior of the bacterium by certain antibiotics. Regarding the area (30), which is planned to be used for external information, it uses r i r ci to supplement the tests; of identification included in the panel, includes six squares, pu that could also have another geometric shape, in which the user marks with a point that covers, at least, 0 'of its surface, if the bacteria in question has been positive to a certain technique d laboratory (for example, a point covering the first table of the upper part, i dica that the microorganism has given cultivation, to the Ir, hours, to the wheel of oxi ci asa). In the area or area of wells (31), it determines the position d each no cilio in the panel and calculates the area of the same that is to be used for reading, generating four values for each well. As seen in figure 8, in which the reference (32) corresponds to the focus of the digital camera (15), the wells (33) .- on cylindrical with the lower part or bottom (33 '). It is also possible to verify that the choice of the area to be considered with respect to the digital chamber of the fixed digital chamber in the upper part of the apparatus is essential to detect possible bacterial growth. In that figure the references (34) correspond to the liquid culture medium, and the references (35) to the zone of detection of bacterial growth, which in each case is different according to the situation of the corresponding well (33) with respect to the vertical in which the focus (32) of the digital camera is located.Of the four values generated by the system when processing a panel, the first two are used to detect colorimetric reactions c used by the bacterial metabolism, and correspond to color code; to the saturation of -olo, while the erce and fourth values are used to detect the bacterial growth itself, corresponding to the growth area and to the amount of light that passes through each well. The values obtained for each well '33) are subsequently tabulated by the control software, in order to determine the microorganism causing the infection and the most suitable anti-iotics, at their lowest concentration, to beat it. In Figure 9, a plate 3 'can be served to it, which is: n circular support of: > oliestireno, udiendo e er different f irmas and sizes, - having a medium of solid culture on whose sup ficie se. extends a suspension of the presumed microorganism causing the infection. Once all the surface is inoculated with the bacterial suspension, a choice of antibiotics in the form of discs impregnated with them is deposited on it, so that the antibiotic contained in a disc dissolves in the medium of (Culti or inoculated Around this, the lowest concentration of antibiotics can be found in the futs furthest from the edge of the disc, in this region 9, the area referenced - • on (38) is the zone • reconstruction of perimeters by superposition of the "halos of inhibition" of two of the discs (37) The ability of an icroo aanismo to grow around an antibiotic disc, and to do so in less, or no measure, determines its qualification as sensitive c resistant to said antibiotic and, therefore, its usefulness as the agent of choice to fight the infection.The microorganism forms around each disk a circular area that is previously called a "halo of inhibition". It is subject to the measurement of the device. Well, once the plate '35 is inserted in the camera (2), and given the order of processing the image, a reading algorithm is put into operation that divides the surface into as many sections as disks deposited on the medium. Next, check that there is no superposition of haloes, which is the phenomenon that occurs when neighboring discs produce a halo of such diameter. that both overlap, so that if this overlap occurs, the system reconstructs the most probable halo for each one, which is the Previously referred. Next, the mean diameter f of each inhibition halo is calculated, starting from the m diameter say .ca of the diameter p. opiamea e said, or c or any other parameter that leads to the ermination of it. For the calculation of the median diameter three measurements are made in each ;,. il. ', although it could also be a number different. Once the measurements have been made, the system associates each measurement with an disk (37), depending on whether or not on the plate 36). - i i- the part- superior and following e .. sense of _? C. ujas to l watch. The - obtained values for disk, 7) are tabulated by the software of (control in order to determine the most appropriate anti-erotic drugs to combat the microorganism causing the infection. Obviously, in addition to the readings of anels and plates according to the description previously made, a colony count must be carried out which, obviously, consists of "the number of bacterial groups that grow on the surface of a solid dioxide, so that according to the year of the inception, that colony count is initiated by the technique consistent in" seeding "a bacterial suspension. On the surface of a medium contained in a plate, incubate it at 37 ° C and determine the number of clusters (colonies) that have appeared after a period of 13 to 14 hours of incubation .The system, with the means associated to it, will perform an evaluation the color of the base medium on which the suspension was inoculated, being -i "numerical content between 0 and 256. In this regard, a colony considered as the enormous amount of adjacent pixels that have a color difference with respect to the of the base medium ie 20%, in or less. Well, once the surface of a colony has been determined, its surface is extrapolated to all the areas where the color has a difference of 207, then calculating how many times the surface of a colony is contained in the total surface, to finally bring the tabulation of the obtained numerical value, operation that is carried out by means of the control software associated to the subject, in order to determine the quantity of cologias suggests the presence, or not, of a so in bear fv

Claims (6)

1. System for analyzing images produced by bacterial reactions, which is intended to convert images of microbiological reactions into numerical values that are then used by a diagnostic soft are to determine: the amount of microorganisms present indicative of infection; in the sample, and the pattern of antimicrobial susceptibility of the causative organism, and where the reactions that produce the images that have to be captured and interpreted by the system itself, occur in contained liquid culture media, inside the wells of panels, or on the surface of a solid culture medium known as a plate, is characterized in that it comprises an apparatus of preferably prismatic-rectangular configuration, determining in an interior of a closed and hermetic chamber, in which it is provided internally a digital camera mounted on a stand, or n removable support for the panel or plate in question, lighting means, supplemented with light diffusers, while the apparatus and in correspondence with its front panel, includes a series of luminous indicators, a feeder to which it is attached. associated with the corresponding removable support, a button • opening for feeder operation, a contrast switch and a general switch; having been anticipated that an access cover to the internal components is included in the rear panel of said apparatus, as well as sockets for current 10 of lighting, current to the camera and communications port with the central computer, respectively, also including on that rear panel connections to a TDM 436 control module.

2. System for analyzing e-s images produced by bacterial reactions, according to claim 1, characterized in that the support is mounted with a mating / uncoupling character to the feeder, guiding it in its movements 20 towards the inside and towards the outside, to allow the introduction of the corresponding well panel or plate.

3. Sis ema analyzer of produced images 25 for bacterial reactions, according to the previous claims, characterized in that the support for the well panels will have the appropriate configuration to the ~! e said panels, counting that with recesses in certain laterals, as well as with fastening pivots to the feeder and holes for the t-bolts of the corresponding panel.

4. System for analyzing images produced by bacterial reactions, according to claims 1 and 2, characterized in that the support for the plate has fastening pins to the limiter and a hole for the disc constituting the plate, as well as with tabs for the fastening of such a plate.

5. System for analyzing images produced by bacterial reactions, according to claims 1, 2 and 3, characterized in that three areas are determined on the upper surface of the panel, the first one for the identification of the panel type, the second for the identification of the information external, and the third for the identification of the well, the areas corresponding to the recesses of the support.

6. System for analyzing images produced by bacterial reactions, according to the above indications, because the support inside the camera of the apparatus is located above the fluorescent lighting means, and below the fluorescent lighting means, those located in the lower part, while the lighting means or fluorescent tubes are arranged laterally at an intermediate height, complemented both with the light diffusers, while the fluorescent tubes are they complement with metal plates q < = cut the beam of light that could reach the fall between the digital camera located superiormente and the panel located on the support.