Diagnostic Method and System for Diagnosis

Abstract

The method for the diagnosis of a microbial infection in an organism makes use thereof that said microbial infection is at least partially present as elementary bodies in cellular material of the organism. The method comprises the steps of (1) providing a sample of cellular material from the organism; (2) processing the sample to obtain a test composition, that is enriched in elementary bodies, in so far as the sample contains any elementary bodies; and (3) Subjecting a volume of the test composition comprising at most a predetermined maximum of elementary bodies to a MALDI mass spectrometry method to identify presence of the microbial infection. The sample processing more particularly involves cell lysis and separation of elementary bodies from the lysed cell material. Subsequently, the elementary bodies (and any further material attached thereto) are contacted with a matrix material, that facilitates the MALDI mass spectrometry.

Claims

1.-24. (canceled)

25. A method for the diagnosis of a microbial infection in an organism, wherein said microbial infection is of a type that is present in one form suitable for multiplication known as a reticulate body and another form suitable for infection of cells known as elementary body, wherein said microbial infection is at least partially present as elementary bodies in cellular material of the organism, which method comprises the steps of: providing a sample of cellular material from the organism; processing the sample comprising the steps of lysing the cellular material so as to maintain the integrity of the elementary bodies, and separating the elementary bodies from the lysed cellular material into an enriched sample, and adding a composition of a matrix material thereto, therewith generating a test composition, that is enriched in elementary bodies, in so far as the sample contains any elementary bodies; and subjecting a volume of the test composition comprising at least one and at most a predetermined maximum of elementary bodies, in so far as the sample contains any elementary bodies, to a MALDI mass spectrometry method to identify presence of the microbial infection.

26. The method as claimed in claim 25, wherein the test composition that is enriched in elementary bodies comprises at least 50% by weight of elementary bodies, based on total solid matter in the test composition.

27. The method as claimed in claim 25, wherein the method further comprises the step of generating droplets of the test composition, which droplets are the volumes sequentially subjected to the MALDI mass spectrometry method.

28. The method as claimed in claim 27, wherein the step of subjecting a volume of the test composition to a MALDI mass spectrometry method comprises, subsequent to dispensing of said droplets, the steps of: crystallizing the matrix material onto solid matter, more particularly the elementary bodies, wherein a volatile solvent of the composition of the matrix material is evaporated, therewith generating an aerosol beam of elementary bodies coated with matrix material; irradiating said coated elementary bodies with a laser, resulting in ionization of proteins; and performing a time-of-flight mass spectrometry measurement on said ionized proteins.

29. The method as claimed in claim 25, wherein the predetermined maximum of elementary bodies per volume is 10.

30. The method as claimed in claim 29, wherein each volume contains at most 2 elementary bodies.

31. The method as claimed in claim 27, wherein the generated droplets are subjected to a selection step, which comprises registration of an optical image of a droplet, and selection of droplets based on analysis of the optical image.

32. The method as claimed in claim 25, wherein the lysing is carried out by means of sonication.

33. The method as claimed in claim 25, wherein the separation involves a centrifuge treatment.

34. The method as claimed in claim 33, wherein the centrifuge treatment provides a supernatant comprising the elementary bodies, separated from cell debris.

35. The method as claimed in claim 25, further comprising a step of concentrating a composition of the separated elementary bodies.

36. The method as claimed in claim 25, wherein the microbial infection is generated by at least one intracellular bacterial species.

37. The method as claimed in claim 36, wherein the bacterial species belongs to the Chlamydia genus.

38. The method as claimed in claim 25, wherein the microbial infection is asymptomatic.

39. The method as claimed in claim 25, wherein said MALDI mass spectrometry method comprises: processing the volume of the test composition, preferably the droplet, to crystallize the matrix material thereof onto the at least one elementary body contained in the volume, therewith obtaining an analyte; ionizing at least part of the analyte; separating the ionized components using a time-of-flight detector to obtain a spectrum; and comparing the spectrum with at least one reference for identification of the microbial infection.

40. The method as claimed in claim 25, wherein the organism is a mammal.

41. A system for diagnosis of a microbial infection of a type that is present in one form suitable for multiplication known as a reticulate body and another form suitable for infection of cells known as elementary body, wherein said microbial infection is at least partially present as elementary bodies in cellular material of the organism, said system for diagnosis comprising in combination: means for lysis of a sample of cellular material configured to maintain the integrity of the elementary bodies; separation means for separating of elementary bodies from other material obtained by said means of cell lysis; mixing means for mixing a composition of a matrix material with the thus separated elementary bodies to obtain a test composition comprising said elementary bodies; a dispensing unit for dispensing volumes of said test composition each comprising a predetermined maximum of elementary bodies, wherein the dispensing unit is a droplet generator for generating droplets of said test composition; a MALDI TOF mass spectrometer apparatus configured for generating a mass spectrum of said test composition and comprising a flight path unit comprising: a drying chamber, wherein solvent of the composition of the matrix material is evaporated to crystallize the matrix material onto solid matter, more particularly the elementary bodies, to generate an aerosol beam of coated elementary bodies, which drying chamber is arranged such that droplets dispensed from the droplet generator enter the drying chamber and dry during flight through the drying chamber; an ionization chamber wherein said coated elementary bodies are irradiated with a laser to result in ionization of proteins and said ionized material is accelerated and passes a charged grid; and a time-of-flight tube, wherein individual ions of the ionized material are separated; wherein the MALDI TOF mass spectrometer apparatus furthermore comprises a detector for detecting the separated ions; and a processor for generating a mean spectrum based on a plurality of mass spectrum of individual volumes and for comparing the mean spectrum with a reference, typically from a database.

42. The system as claimed in claim 41, further comprising optical image analysis means comprising an optical image recorder and processing means for analysis a recorded optical image.

43. The system as claimed in claim 42, further comprising droplet removal means, which are driven on the basis of said analysis by said processing means.

44. The system as claimed in claim 41, wherein the lysis means comprises sonication means.

Description

BRIEF INTRODUCTION OF THE FIGURES

[0042] These and other aspects of the invention will be further elucidated with reference to the figures, which are purely diagrammatical and not drawn to scale, wherein:

[0043] FIG. 1 shows a schematic representation of an apparatus for MALDI mass spectrometry with a preferred pre-treatment for a liquid test composition, and

[0044] FIG. 2 shows a schematic representation of the particle flow path and mass spectrometer within the apparatus of FIG. 1.

[0045] FIG. 3A-B diagrammatically indicates process steps according to one embodiment of the method of the invention, wherein FIG. 3A indicates the generation and selection of droplets and FIG. 3B indicates MALDI TOF mass spectrometry.

[0046] FIG. 4 shows a flow chart for the processing of the sample according to one embodiment of the method of the invention;

[0047] FIG. 5 shows a mass spectrum obtained in a preliminary experiment according to the invention

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

[0048] FIG. 1 shows a schematic representation of a first embodiment of an apparatus for MALDI mass spectrometry. FIG. 2 shows in more detail the portion 200 of the apparatus, hereinafter also referred to as a flight path unit 200. MALDI mass spectrometry is particularly suitable for identification of biological material. One preferred type of biological material is micro-organisms such as bacteria, fungi and viruses. Other types of biological material that can be identified with MALDI include for instance blood cells, peptides.

[0049] The apparatus comprises a sample receiver 10, conduits 11, a first mixing unit 12, a second mixing unit 14, and a flight path unit 200. The flight path unit comprises a drying chamber 15, an ionization chamber 191 and a time-of-flight tube 194. A droplet is ejected by any droplet ejector 16, such as for instance based on a piezoelectric resonator. The droplet follows a droplet beam 24 that extends from the drying chamber 15 into the time-of-flight tube 194. Upon drying the droplet beam 24 is actually converted into a particle beam 192. Upon ionization by radiation from a pulse laser 18, the particle beam 192 is converted into a ion beam 195. The mass spectrometernot shownmeasures the ions of the ion beam 195 and creates spectra on the basis thereof According to one embodiment of the invention, use is made of a sensor 20, 22 for determining a morphology parameter so as to select particles that are ionized by a laser pulse of the pulse laser 18. This is particularly done so as to ionize only those particles that may lead to useful spectrum information.

[0050] The first mixing unit 12 comprises a first mixer 120, a container 122 for solvent and/or antisolvent, such as water, and a detector 124. Rather than one container 122, two separate containers may be present. Sample material that is for instance obtained from a patient, is diluted with the solvent and/or antisolvent in the first mixer 120. Detector 124 is suitably an optical detector configured to detect light scattered from individual micro-organisms when the micro-organisms flow through a measurement beam. From a count of micro-organisms that are detected on average per unit of time interval, the density may be determined. Such detector 124 is known per se and is for instance a cytometer or flow cytometer. Particle detector 124 is shown coupled to a control input of first mixer 120. The control mechanism is arranged to increase the amount of solvent and/or antisolvent, until the measured density has dropped to or below a predefined density. Preferably both are added in a predefined ratio. A liquid circulation circuit may be used to circulate the composition until the desired density has been achieved. The second mixing unit 14 comprises a second mixer 14 and a matrix material reservoir 142. Matrix material reservoir 142 is coupled to the second mixer 140. The second mixer 14 is configured to mix the matrix material into the test composition obtained from the first mixing unit 12.

[0051] The droplet generator 16 may be provided with means for evaluation whether a droplet contains a single microorganism or any other number of microorganisms. Such a detecting means may be arranged to view the suspension in a channel prior to ejection by a nozzle. The generator 16 may further be provided with means for directing an ejected droplet to a first position or to a second position depending on information obtained from the detecting means. The first position is then a target position, i.e. a flow path towards the position where a laser source may eject radiation on the particle so as to ionize it. The second position is a waste position. The directing means are configured for deflection of the droplet or a motorized stage configured for directing the nozzle. Such an apparatus is known per se from EP2577254B1, and is included herein by reference.

[0052] FIG. 3A shows one embodiment of a droplet generator 16 and the chamber 15 in more detail. In this figure, the flow path of the droplet beam 24 through the chamber 15 may have a vertical orientation. In this embodiment, the detecting means 161 are arranged to optically sense the droplets upon ejection from the nozzle of the droplet generator 16. The sensor 161 is coupled to a selection means 162, preferably an electromagnetic shutter, to remove those droplets that do not contain any microorganism.

[0053] Due to the small droplet size, it has been found that the droplets quickly, i.e. in the first few centimeters of the flow path, arrive at a constant velocity. This velocity is a balance of gravity and aerodynamic resistance. Due to the drying, the droplet beam 24 is converted into particle beam 192. The chamber 15 is provided with temperature controlled walls so as to keep the temperature in the chamber 15 constant. In one embodiment a temperature of 22-30 C. is chosen. The chamber 15 is further provided with an inlet 165 for gas generating a homogeneously distributed sheath flow. The gas comprises for instance air or nitrogen and is controlled with respect to the concentration of water vapor and optionally any solvent. Suitably, the water vapor concentration is controlled, for instance such that the relative humidity is 30% or more. During flight through the drying chamber 15, the matrix material in a liquid drop crystallizes on the analyte, typically a microorganism, while the drop dries in flight, resulting in a dried particle, which is also referred to as the test sample. Typically, the drop is launched with a diameter in the range of 30-60 m. The dried particle has an aerodynamic diameter of less than 3.0 m in a first embodiment, wherein the test sample contains a single bacteria. The sheath flow transports the droplets towards the inlet of the aerosol time-of-flight mass spectrometer.

[0054] FIG. 3B illustrates the identification process based on the generated test samples in a particle beam. A laser pulse is fired at the dried particle from pulse laser 18. This results in ionization of material from the test sample. The ionized material is then accelerated in a ionization chamber 191, in which high voltages are present to accelerate the ionized material. The ionized elements passes a charged grid 216. As a consequence, individual ions of a ion beam 195 are separated in a drift region 194, that is free of an electric field. This drift region is also known as a time-of-flight tube. The separated ions are detected by a detector 220. The processor that is coupled thereto processes the obtained data to generate a spectrum or data set 230 (fingerprint) that can be compared with known data sets. Such known data sets are typically stored in a library.

EXAMPLE

[0055] A test was carried out with Chlamydia trachomatis species that had been grown in a cell line of HeLa cells. The latter is a cell type in an immortal cell line used in scientific research. The sample preparation is shown in FIG. 4. The starting composition contained A (the said species in the HeLa cells) in a medium M. The HeLa cells were lysed by sonication, using three sonication periods each of 20 seconds at 70 W, interrupted by intermittent periods of 2 seconds using vortexing. This resulted therein that the elementary bodies (EB) could be collected, while other cell material was destructed to cell debris (D). After the sonication there was thus EB+D in medium (M). The cell material was then transferred to a centrifuge, for separation of the cell debris (D) and the elementary bodies (EB). Three subsequent steps (C1, C2, C3) were carried out in this example, using increasing spinning rates, i.e. 500 g, 2500 g and 15,000 g. As a consequence, first the medium M was removed, then a portion of the cell debris D was removed, and then the remaining cell debris D was removed. This led to pellets of elementary bodies.(EB). In the removal of the cell debris, typically first the more rough cell debris is removed and thereafter the finer cell debris. The figure refers for sake of simplicity to 1/2D, as if both steps each remove 50%. That is merely schematically and not a hard requirement. It will be understood that the exact spinning rates can be amended Furthermore, while in one embodiment according to the invention, three steps are carried out, this number may be varied. The pellets were then resuspended into a buffer (BUF) for storage (for instance at 80 C.), or for direct use.

[0056] While the figure shows a specific embodiment for characterization of the method of the invention, it will be understood that when used for diagnostic purposes, the use of C. trachomatis and HeLa cells is typically replaced by the use of cells that could contain the C. trachomatis. In such diagnostic method, the cells may be mixed with a medium, before the destruction step. Following the destruction step, the elementary bodies EB will be separated from the cell debris and the medium. The use of a centrifuge is therein preferred, although the centrifuge may be replaced in part or entirely by other separation techniques, including filtering, membrane filtering and/or activated (para)magnetic beads.

[0057] A test composition was then generated by bringing the pellets of elementary bodies (EB) into contact with a matrix material. In this example, use was made of resuspension, with 2-mercapto-4,5-dimethylthiazole as the matrix material. Droplets were generated with a droplet generator. Each droplet has a volume in the range of 10-100 picoliter. An image sensor, more particularly a camera, was provided at a location so as to enable recording of the droplets at the exit of the droplet dispenser. The image sensor was coupled to a processor to check whether dot-shaped elements, typically darker than other parts of the droplet were present in the droplet. A droplet was selected when the droplet contained one dot-shaped element. If a droplet was not selected, it was taken out of a droplet beam by means of an electro-magnetic shutter. The droplets thereafter passed a drying section. This resulted in the generation of coated particles suitable for MALDI mass spectrometry.

[0058] MALDI mass spectrometry on the individual droplets was thereafter carried out. Mass spectra of individual droplets were generated. The spectra contained a sufficient signal were accumulated, such that signal-to-noise ratio became sufficiently large to distinguish 20-30 significant peaks within the spectrum. Herein, the base line, which corresponds with the spectral level caused by a signal part that varies from particle to particle, is subtracted from the signal. The intensity has been normalized with local variance of the height of the base line. A peak with an intensity of at least one is considered sufficiently significant. The spectrum shown in FIG. 5 was obtained.

[0059] In case that it is expected that other objects than only elementary bodies are present, a classification may be carried. Use can be made therein of the method specified in EP2836958, which is included herein by reference.