SE466623B - SET AND TEST KIT FOR DIAGNOSTICATION OF AN ACTIVE INFECTION - Google Patents

Description

466 625 10 15 20 25 30 35 2 characterized epitopes on a microorganism of interest make it possible to detect the microorganism either directly from a patient sample or after prior amplification by culture.

DNA hybridization technology has recently become available for the detection of a number of infectious diseases. This technology can be applied both to direct samples and to cultured microorganisms.

A common feature of the above techniques is that they do not differentiate between active infection and infection which is superior. In addition, they are often labor-intensive and involve many work steps, and when culture methods are used, the time between sampling and an established diagnosis for certain microorganisms (viruses, parasites, certain bacteria) is very long.

The need for a rapid and sensitive diagnosis of an active infection caused by a microorganism is particularly important when dealing with, for example, patients who have been given immunosuppressive treatment and who have undiagnosed fever. A fever can be caused by, among other things, different types of microorganisms that require totally different treatment strategies, which can be more or less toxic to the patient.

Recently, W. van der Bij, et al. an article entitled 'Rapid Immunodiagnosis of Active Cytomegalovirus Infection by Monoclonal Antibody Staining of Blood Leucocytes' (Journal of Medical Virology 25: 1979-188 (1988)). Similar to the method of the present invention, detection of infected lymphocytes is used in a light microscope. However, the assay steps differ significantly from those used in the present invention, mainly due to the fact that enzyme-labeled anti-antibodies, which require staining for detection, are used. The described analysis can, if necessary, be performed in 3 hours. 10 15 20 25 30 35 466 625 3 would be useful for the purpose of determining the grounds for effective monitoring and treatment of an infection.

Description of the Invention The present invention provides a method of in vitro diagnosing an active infection caused by a microorganism, which method is quick and easy to perform without the need for any sophisticated equipment. The method is also sensitive and allows the detection of an approaching infection at an early stage and provides a tool that makes it possible to follow the patient from the beginning to the end of a specific microbial infection, as it reflects an active infection. In addition, the effectiveness of the selected treatment can be monitored using the method of the invention. The time between sampling and response is comparatively short, in one embodiment of the invention approximately 2 hours, and in another embodiment of the invention approximately 1/2 hour.

In one aspect of the invention there is provided a method of diagnosing in vitro an active infection caused by a microorganism, in which the presence of said microorganism, and / or phagocytosed parts thereof, exposed to white blood cells is determined in a sample of white blood cells separated from whole blood, wherein said sample of white blood cells in a liquid medium is incubated with either a) an excess amount of antibodies directed against said microorganism, followed by incubating the mixture with an excess amount of antibodies coated on a support having a particle size detectable, in a light microscope detectable in b) an excess amount of antibodies directed against said microorganism and coated on a carrier with a particle size detectable in a light microscope, followed by placement of a drop of the incubated mixture resulting from a) or b) on a microscope slide and determining, by means of a light microscope, the presence of said microorganism, and / or phagocytosed parts thereof, exposed on white blood cells such as so-called rosettes formed by said carrier particles attached to infected white blood cells, which indicates an active infection caused by said microorganism.

Separation of a white blood cell sample from whole blood can be performed by any method known in the art, for example by gradient centrifugation using a mixture of sodium metrizoate and Ficoll according to the method of Bøyum, A. ( Scand J. Clin.

Lab. Invest. 21, Suppl. 97 (1968)).

The term "fluid medium" as used in the present specification and claims refers to any suitable fluid medium which does not lyse the white blood cells.

Examples of such media are physiological saline or a culture medium, such as RPMI 1640 medium.

Antibodies directed against a microorganism of interest can either be selected from commercially available antibodies or prepared by standard procedures. (See, for example, Fazekas de St Groth, Scheidegger D., Production of monoclonal antibodies: Strategy and tactics. J.

Immunol. Meth. 35: 1, 1980). Both monoclonal and polyclonal antibodies can be used in the method.

The carrier particles used in the method of the invention should have a particle size detectable in a light microscope, and in a preferred embodiment of the invention the carrier particles are microspheres with an average diameter of 3-5 μm. The carrier material should be such that antibodies can be coated on the particles. There are several commercially available carrier particles, and in one embodiment of the invention latex particles are used. In a preferred embodiment of the invention, the incubations in the method of the invention are performed at about 4 ° C. In a preferred embodiment of the method according to the alternative of the invention a), washing of the first incubated mixture is performed with, for example, phosphate buffered saline, followed by separation of a mixture of free and antibody-bound white blood cells. The separated mixture of white blood cells is then diluted with a liquid medium before incubation with antibodies coated on a support.

This intermediate washing procedure, which removes unbound antibodies, facilitates the detection of rosettes on the microscope slide.

So-called rosettes (white blood cells infected in the present invention to which antibody-bound carrier particles are attached) are well known to those skilled in the art (see, e.g., Jondahl M., Holm G., Wigzell H., J. Exp. Med., 136; pp. 207, 1972).

The method of the invention can be applied to the in vitro diagnosis of an active infection caused by any specific microorganism, provided that antibodies directed against said microorganism are, or can be made, available. Particularly important microorganisms that cause active infections are Cytomegalovirus (CMV), Candida, Aspergillus, Human immunodeficiency virus (HIV), Hepatitis virus, Toxoplasma, Epstein-Barr virus (EBV), Pneumocytis carinii and Herpes virus. In a preferred embodiment of the invention, the microorganism is Cytomegalovirus, and in another preferred embodiment of the invention is the microorganism Candida.

When using the method of the invention, it is possible to calculate the percentage of infected white blood cells in the white blood cell sample by calibrating the white blood cell sample to a predetermined number of cells per ml of liquid medium, and then, after the last incubation, scan an appropriate number of cells for the number rosettes.

In a variant of the method according to the invention, the presence of at least two different microorganisms is determined simultaneously by means of at least two differently colored carrier particles. This is possible due to the fact that the color of the carrier particle is detectable in a light microscope. There are commercially available carrier particles with different colors, such as blue, red, brown and white.

In another aspect of the invention, a diagnostic assay kit is provided for determining an active infection caused by a microorganism. The test kit comprises in separate containers either the ingredients a) a calibrated amount of antibodies directed against a selected microorganism in a liquid medium, and a calibrated amount of carrier particles precoated with antibodies in a liquid medium, or the ingredient b) a calibrated amount of carrier particles pre-coated with antibodies directed against a selected microorganism, and possibly washing solutions, media for separating white blood cells from whole blood and reagents for testing the activity of the antibodies.

The test kit is adapted for use in the method according to the invention. In one embodiment of the assay kit, the calibrated suspensions are in lyophilized form for reconstitution with a calculated amount of a liquid medium. Optionally, the liquid medium is provided in a separate container in the test kit. The assay kit may comprise at least two different ingredients as defined in a) or b) of this aspect of the invention, each of said ingredients comprising carrier particles which are differently colored with respect to each other.

DIAGNOSTIC ANALYSIS PROCEDURE Separation of white blood cells from whole blood A sample of whole blood from a patient is mixed 1: 2 with NaCl, and 8 ml of the resulting blood suspension is layered on top of 3 ml Lymphoprep in a centrifuge tube (Lymphoprep is a trademark of Nycom, Norge Comcom , and contains sodium metrizoate 9.6% (w / v) and Ficoll 5.6% (w / v)). The mixture is centrifuged for 20 minutes at 466 626 7 at 2000 rpm. The layer between the phases, which contains white blood cells, is aspirated using a pasteur pipette and transferred to another centrifuge tube. The cells are washed 3 times with physiological NaCl for 6 minutes at 1500 rpm. The cells are counted in a Bürker chamber and diluted in an RPMI 1640 medium (manufacturer Gibco BRL) to a predetermined number of cells per ml of liquid medium, eg 10 x 10 6 cells / ml.

Assay 1 according to alternative a) according to the invention 1. 50 μl of a suspension comprising 10 x 106 white blood cells per ml of RPMI 1640 medium is placed in a test tube on ice. 2. 5 μl of a suspension of antibodies directed against the microorganism of interest was added to the test tube.

The test tube is gently shaken to mix the tube contents uniformly. 3. The test tube is incubated on ice for 30 minutes (ie at 0-4 ° C). The test tube is gently shaken from time to time during the incubation, to keep the antibodies suspended in the liquid medium. 4. The contents of the test tube are washed twice with 1 ml portions of phosphate buffered saline (PBS), pH 7.4, comprising 2.0% fetal calf serum, and centrifuged at 1500 rpm. 5. The resulting pellet is suspended in 0.9 ml of RPMI 1640 medium, and SO μl of a suspension comprising 4 x 108 microspheres coated with anti-antibodies per nl of PBS is added while operating in a cold room. The test tube is rotated and incubated for 30 minutes in a cold room (at 4 ° C). 7. The contents of the test tube are mixed using a pasteur pipette, to suspend the microspheres and cells to an even distribution. A drop of the suspended mixture is placed on a microscope slide, which is then covered with a coverslip, followed by visual inspection in an ordinary light microscope. 466 625 10 15 20 25 30 35 B 8. A suitable number of cells, eg 200 cells, is scanned for the number of rosettes by means of a suitable magnification lens, eg 40x magnification. Optionally, the percentage of infected white blood cells in the starting sample of white blood cells (using the number of rosettes) is calculated.

Assay according to alternative b) according to the invention 1. 50 μl of a suspension comprising 10 x 106 white blood cells per ml of RPMI 1640 medium is placed in a test tube on ice. 50 μl of a suspension comprising 4 x 108 microspheres with an average diameter of 3-S pm coated with antibodies directed against a microorganism of interest per ml of PBS is added, together with 0.9 ml of RPMI 1640 medium. 2. The test tube is rotated and incubated for 30 minutes in a cold room (at 4 ° C). 3. The contents of the test tube are mixed using a pasteur pipette to suspend the microspheres and cells to an even distribution. A drop of the suspended mixture is placed on a microscope slide, which is then covered with a coverslip, followed by visual inspection in a standard light microscope. 4. A suitable number of cells, eg 200 cells, are scanned for the number of rosettes by means of a suitable magnification lens, eg 40x magnification. Optionally, the percentage of infected white blood cells is calculated in the starting sample of white blood cells (using the number of rosettes).

EXAMPLE 1 The method according to alternative of the invention a) was used to determine the percentage of Cytomegalovirus-infected white blood cells in blood samples taken from nine patients who had received a transplant at Huddinge University Hospital, Sweden.

The microspheres that carried anti-antibodies were sheep anti-mouse IgG 1 (FC) coated on activated microspheres, Dynabeds, from Dynal AS, Norway. 10 15 20 25 30 35 466 623 9 Two different antibodies directed against Cytomegalovirus were used, namely MAS 127, monoclonal antibody against Cytomegalovirus with specificity for human Cytomegalovirus early antigen, a product from Sera-lab, UK, purchased from AB Kemila Preparat, Sollentuna, Sweden.

CH 16, monoclonal antibodies directed against Cytomegalovirus with specificity for DNA binding protein, purchased from Dr. Lenore Pereira, University of California, USA.

The results are given in Table I.

In Table I, patient l. Was a pancreatic transplant patient; patients 2nd, 3rd, 4th and 9th were kidney transplant patients; and patients 5., 6., 7. and 8. were bone marrow transplant patients. M denotes man and F denotes woman.

Serology had been performed by Stockholm County Council's microbiological central laboratory, Sweden, + indicates an increase in IgM or IgG titers; denotes unchanged IgM or IgG titers.

Culture in fibroblasts and virus detection had been performed by Stockholm County Council's microbiological central laboratory, Sweden, and B denotes blood, U denotes urine and Bal denotes pulmonary alveolar lavage. 466 625 10 TABLE I% infected white blood- Patient bodies: antibodies 5 (sex / age) Serology Culture MAS 127 CH 16 1. 14/32 + + (B, U) 5 5 2. F / 4? + - 13 3. M / 37 + + (B, U, Bal) 4 10 4. H / 1 + + (U) 4 ll 5. F / 2 - - 0 6. M / 21 - - O 7. M / S8 - + (U) 2 8. M / 32 + (B, U) 3 15 9. M / 24 + + (B, Bal) 9 8 From the data given in Table I, it can be deduced that there is a good correlation between serology + culture results and the results obtained when the method according to alternative a) is used: the difference is the time required from sampling to results, which is approximately 2 hours when the method according to the invention is used, and the fact that the method of the invention always reflects an active infection.

EXAMPLE 2 Determination of the percentage of Cytomegalovirus-infected white blood cells in blood samples taken from four patients who had undergone organ transplantation at Huddinge University Hospital, Sweden, was performed using both method alternative a) and method alternative b) according to the invention.

In alternative a), the microspheres carrying anti-antibodies were the same as those used in Example 1; and the antibodies directed against Cytomegalovirus were M 127 as in Example 1. In alternative b) the antibodies were MO 53, monoclonal antibodies directed against Cytomegalovirus matrix protein (64-68 kD) from BioInvent International , Lund, Sweden.

The antibodies were coated on red carboxylated microspheres (Polyscience) using the carbodiimide test kit from Polyscience, Inc., USA, according to the manufacturer's instructions.

The results are given in Table II.

TABLE II Patient% infected white blood cells; No. / age alternative a) alternative b) l./32 0% 0% 2./8 1% 1% 3./19 1% 1% 4./31 0% 0% The data given in Table II indicate that the same results are obtained with the two alternatives a) and b) of the method according to the invention.

EXAMPLE 3 The method according to alternative of the invention a) was used to determine the percentage of Candida-infected white blood cells in blood samples taken from eight patients who had received a transplant at Huddinge University Hospital, Sweden.

The microspheres carrying anti-antibodies were the same as those used in Example 1.

The antibodies directed against Candida were MAB 806, a mouse anti-Candida albicans monoclonal antibody that has no cross-reactivity with other yeasts, Lactobacillus Streptococcus / Pediococcus, from Chemicon International, USA.

A 66 623 12 The results are given in Table III.

In Table III, the patients were 1st, 2nd, 4th, 5th, and 7th kidney transplant patients; the patient 3. was a liver transplant patient; and patients 6th and 8th were 5 renal plus pancreatic transplant patients. M denotes man and F denotes woman.

Serum samples from each of the patients had been tested by the Swedish Bacteriological Laboratory, Sweden, to determine the presence of the Candida glycoprotein antigen. The test was performed using Cand-Tece kit from RAMCO Laboratories Inc., USA. In Table III, + denotes the presence of said antigen (Ag).

TABLE III 15 Patient% infected (sex / age) Ag white blood cells 1. M / 58 + 3% 20 2. M / 37 + 9% 3. F / S4 + 11% 4. M / 54 + 11% 5. M / 8 + 16% 6. F / 29 + 7% 25 7. M / 51 + 13% 8. M / 44 + 1.5% From the data given in Table III it can be seen that 30 infected white blood cells, and thus an active Candide infection in the patient, could be detected using the sweetening alternative a) according to the invention.

Claims (11)

10 15 20 25 30 35 466 623 13 PATENT REQUIREMENTS A method of diagnosing in vitro an active infection caused by a microorganism, in which the presence of said microorganism, and / or phagocytosed parts thereof, exposed to white blood cells is determined in a sample of white blood cells separated from whole blood, characterized by wherein said sample of white blood cells in a liquid medium is incubated with either a) an excess amount of antibodies directed against said microorganism, followed by incubating the mixture with an excess amount of antibodies coated on a carrier having a particle size detectable, in a light microscope detectable in a b) an excess amount of antibodies directed against said microorganism and coated on a support having a particle size detectable in a light microscope, followed by placing a drop of the incubated mixture resulting from a) or b) on a microscope. slides and determination, by means of a light microscope, of the presence of said microorganism, and / or phagocytic parts thereof, exposed on white blood cells such as so-called rosettes formed by said carrier particles attached to infected white blood cells, which indicates an active infection caused by said microorganism. A method according to claim 1, wherein the following intermediate steps are performed between the two incubation steps in alternative a): washing the incubated mixture, separating a mixture of free and antibody-bound white blood cells and diluting the separated mixture of white blood cells with a liquid medium. 466 625 10 15 20 25 30 35 14 A method according to claim 1 or 2, wherein the microorganism causing an active infection is selected from the group consisting of Cytomegalovirus (CMV), Candida, Aspergillus, Human immunodeficiency virus (HIV), Hepatitis virus, Toxoplasma, Epstein-Barr. virus (EBV), Pneumocytis carinii and Herpes virus. The method of claim 3, wherein the microorganism is Cytomegalovirus. A method according to claim 3, wherein the microorganism is Candida. A method according to any one of the preceding claims, wherein the starting sample of white blood cells is calibrated to a predetermined number of cells / ml of liquid medium, and after the last incubation a suitable number of cells for the number of rosettes is scanned, followed by calculating the percentage of infected white blood cells in the starting sample of White blood cells. A method according to any one of the preceding claims, wherein the antibodies are monoclonal antibodies, the incubations are performed at about 4 ° C and the carrier particles are microspheres with an average diameter of 3-5 μm. A method according to any one of the preceding claims, wherein the presence of at least two different microorganisms is determined simultaneously by means of at least two differently colored carrier particles. 9. A diagnostic assay kit for determining an active infection caused by a microorganism, characterized in that it comprises in separate containers either the ingredients a) a calibrated amount of antibodies directed against a selected microorganism in a liquid medium, and a calibrated amount of carrier particles pre-coated with antibodies in a liquid medium, or the ingredient b) a calibrated amount of carrier particles pre-coated with antibodies directed against a selected microorganism, and optionally washing solutions, media for separating white blood cells from whole blood. and reagents for testing the activity of the antibodies. The diagnostic assay kit of claim 9, wherein the calibrated solutions are in lyophilized form for reconstitution with a calculated amount of a liquid medium, optionally provided in a separate container in the test kit. 11. ll. A diagnostic assay kit according to claim 9 or 10, wherein the test kit comprises at least two different ingredients as defined in a) or b), each of the ingredients comprising carrier particles which are differently colored with respect to each other.