JPH1132793A - Diagnostic kit for urinary tract infectious disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diagnostic kit intended for more surely diagnosing upper and lower urinary tract infectious diseases without being based on the finding for bacteriuria, by assaying urine leukocytes through paying attention to neutrophils and macrophage. SOLUTION: This diagnostic kit is intended to assay urine leukocytes through paying attention to neutrophils and macrophage. By determining the concentration of myeloperoxidase(MPO) in urine, the presence of the urine leukocytes can be surely detected; when the MPO concentration is to be determined by an immunoassay technique, it is recommended to be e.g. an enzyme immunoassay, latex coagulation technique, immuno-nephelometry, immuno- chromatography, immuno-spectrometry.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a diagnostic kit for urinary tract infections, and more particularly, to the determination of the presence of leukocytes (neutrophils and macrophages) in urine by measuring the concentration of myeloperoxidase in urine to determine the presence of bacteria in urine. The present invention relates to a urinary tract infection diagnostic kit capable of diagnosing upper and lower urinary tract infections without proving its existence.

[0002]

BACKGROUND OF THE INVENTION Urinary tract infections are generally defined as colonization of microorganisms in urine and bacterial infection of urinary tract organs from the kidney to the urethra. Urinary tract infections are broadly classified into upper urinary tract infections including pyelonephritis and renal abscess, and lower urinary tract infections that have infected the bladder, urethra, prostate, epididymis and the like. As described above, urinary tract infection is nonspecific inflammation caused by general bacteria in the renal and urinary tracts.The findings of bacteriuria are the most important for diagnosis of urinary tract infection, and leukocyte urine associated with inflammation. The findings are said to be second. That is, since bacteria do not originally exist in normal urine, it can be said that the presence of bacteria in urine is abnormal. Bacteriuria is caused by bacterial infection of the urinary tract and is essential for the diagnosis of urinary tract infections. However, urine excreted by a usual method is contaminated by bacteria from the external genitals and the like, and contains a small amount of bacteria. So in general,
Bacterial urine is a case in which more bacteria are detected than can be found in normal urine. The standard of bacteriuria is to determine the number of bacteria in 1 ml of urine, and when the number of bacterium is 10 ⁵ / ml or more (10 ⁵ CFU / ml), it is defined as bacteriuria. Applicable when intermediate urine or catheter urine is used as a test material).

[0003] As a method for testing the number of bacteria in urine, a quantitative culture method in which the number of bacteria in 1 ml of urine is determined by a urine quantitative culture test, or a method in which the number of bacteria and bacterial clumps are directly calculated by observing urine sediment with a microscope. Urine sediment microscopy is used to estimate the number. Also,
Some tests measure urinary nitrite and indirectly estimate bacterial counts. Furthermore, in urinary tract infection, inflammation often increases the number of white blood cells in the urine, and thus is a useful finding in diagnosing urinary tract infection together with bacterial urine. In the detection of urinary leukocytes, usually, the presence or absence of leukocytes is examined by urinary sediment microscopy. Alternatively, there is a method in which urine itself is placed on a hemocytometer to perform microscopic examination and calculation. Further, as a chemical test method, there is a method of estimating the number of leukocytes from the strength of the reaction using the esterase activity of leukocytes. From the above situation, diagnostic criteria for urinary tract infections include:
Proof of bacteriuria is primary and the main diagnostic criteria is bacteriuria ≧ 10 ⁵ CFU / ml, and detection of urinary leukocytes is only used as a useful reference.

[0004]

As described above, calculation of urinary bacterial count is the most important diagnostic criterion for diagnosing urinary tract infections.
Most of the bacteria are contaminated by bacteria from the external genitalia, and urine is an environment suitable for bacterial growth.As the time from urine collection to testing increases, bacterial growth in urine increases. Is an indispensable phenomenon, and it is difficult to accurately determine the number of urinary bacteria. On the other hand, in leukocyte urine, which is useful information for diagnosing urinary tract infection, an increase in urinary leukocytes is not a manifestation of urinary tract infection as it is, but is only a nonspecific finding observed in many disease states. It is not. The present invention has been made in view of these problems, that is, since it is very difficult to accurately measure the number of bacteria in urine, the detection of urinary leukocytes alone can prevent urinary tract infection. It is an object of the present invention to examine in detail whether or not diagnosis is possible, and to provide a kit capable of more reliably diagnosing a urinary tract infection.

[0005]

As a result of the research described below, the present inventor measured urinary leukocytes (neutrophils and macrophages) to obtain urinary tract infection without proof of bacterial urine. Found that the disease can be diagnosed. And
The presence of leukocytes in the urine indicates that myeloperoxidase (MP
O) It can be reliably detected by measuring the concentration. In addition, it has been found that when the urinary MPO concentration is within a predetermined range, it can be diagnosed as asymptomatic bacteriuria, and when it is higher than that, it can be diagnosed as symptomatic bacteriuria. As mentioned above,
The diagnostic kit of the present invention diagnoses urinary tract infection by measuring the presence of urinary leukocytes at the MPO concentration. The method for measuring the MPO concentration is appropriate, but in the case of the enzyme immunoassay, an anti-human myeloperoxidase antibody, an enzyme-labeled anti-human myeloperoxidase antibody, and a diagnostic kit comprising the enzyme measurement reagent are used. good. Similarly, when the latex agglutination method is used, a diagnostic kit including anti-human myeloperoxidase antibody-sensitized latex particles may be used. When the immunoturbidimetry is used, a diagnostic kit including an anti-human myeloperoxidase antibody reaction solution may be used. When the immunochromatography method is used, a diagnostic kit including an anti-human myeloperoxidase antibody and a colloidal gold particle-sensitized anti-human myeloperoxidase antibody may be used. When the immunoluminescence method is used, a diagnostic kit including an anti-human myeloperoxidase antibody sensitized with a luminescent substance such as a luminol derivative may be used.

Hereinafter, the process leading to the present invention will be described in detail. Whether screening for urinary tract infections, which are bacterial infections, and understanding the pathological condition can be achieved only by detecting leukocyte urine can be determined by clarifying the actual state of nonspecific leukocyte urine and the mechanism of leukocyte transmigration into urine I can judge. Non-specific leukocyte urine is observed, in part, in inflammation of urinary tract organ tissues other than infectious diseases and in some systemic inflammatory diseases such as collagen disease, and in others,
It is known that it exists as unexplained pyuria expressed as asymptomatic leukocyte urine in school urinalysis for elementary and junior high school students. Thus, using a urine sample from kindergarten, elementary, junior high, and high school students, a group of specimens exhibiting asymptomatic leukocyte urine was extracted, and the formation mechanism of leukocyte urine indicated by these groups was clarified. In extracting the specimen, the following conditions should be satisfied in order to eliminate urinary tract infection cases as much as possible. First, general urinalysis tests (protein and occult blood) and nitrite tests are all negative, and urothelial cells are produced by bacterial infection as part of a nonspecific infection defense system. In order to exclude urinary bleeding cases, α ₂ M, which is a sensitive marker for retrorenal bleeding, was measured, and it was assumed that all of them satisfied the condition that they were below the reference value. Next, using these extracted samples, the distribution of urinary myeloperoxidase (MPO) value was determined for each sex and age (Table 1). Although similar results were shown, the female group showed significantly higher MPO levels throughout the age group than the male group, despite unexpectedly eliminating urinary tract infection cases. High MPO value (leukocyte urine) was observed (FIG. 1A). The white blood cell count (average MPO value of 4.3 ng / ml, white blood cell count of 3.9 / μl) present in the urine of this boy group was compared with the resident macrophages deployed in the urinary tract as part of the body defense mechanism. It can be regarded as the resident number of leukocytes mainly composed of neutrophils (monitoring role of foreign substance invasion).

[0007]

[Table 1]

[0008] That is, the urinary leukocyte resident count indicates that approximately 600 to 1 million leukocytes are excreted in the urine per day in a healthy person. This is considered to correspond to the conventional knowledge that about one or two pieces are observed in several fields or one field of view. On the other hand, leukocyte urine observed in the girls under the age of 10 corresponds to asymptomatic leukocyteuria or non-specific leukocyteuria pointed out by Murakami et al. (Matsumi Murakami, et al .: Pediatrics, 35: 1601-1607, 1994.). Given that, this group was calculated as the average of
2 S.D. (14.5 ng / ml: equivalent to 13 white blood cells / μl)
Average value of the following group (normal group) and the group of more than 10-year-old girl + 2 S.D. (14.6 to 167.8 ng / ml: leukocyte count 1)
(Corresponding to 4 to 152 cells / μl), and further divided into three groups, a high value group showing a higher MPO value (FIG. 2),
The mechanism of leukocyte urine formation in the medium-dose group was examined. It has been pointed out that nonspecific leukocyteuria is caused by inflammation of urinary tract organ tissues other than infectious diseases or by systemic inflammatory diseases. The urinary levels of various acute phase reaction proteins were measured and compared. It was thought that the increase in the medium concentration was reflected as an increase in urinary excretion, but there was no significant difference in the urinary acute phase reaction phase protein concentration between the two groups (Fig. 3). Was unlikely to be based on non-communicable inflammatory disease. Since urinary tract infections include infectious diseases of adjacent organs such as prostate and epididymitis, non-infectious target diseases are considered to be few. Therefore, leukocyte urine seen in the medium-value group corresponds to so-called asymptomatic leukocyte urine, and elucidation of the cause was the next task.

[0009] The specimens used in this study exclude urinary tract infection cases as much as possible at the sampling stage, and are considered to be free of significant bacteriuria. Taking into account the fact that leukocyteuria is unlikely due to inflammatory diseases, leukocyteuria, which is seen in the median group, is caused by a small amount of bacteria invading the urinary tract, i.e., asymptomatic bacteriuria. It was thought that the possibility was high. This asymptomatic bacteriuria is said to be due to ascending infection of resident bacteria present in the urethra and vulva.In men, however, the urethra is long and has two sphincter parts, which is anatomical. It is known that ascending infections do not occur easily, while women with short urethra are more likely to occur and are more common in younger people. This is evident from the results in which the urinary MPO level in the boy group was low and constant throughout the entire age group (Fig. 1A). When calculating the proportion of subjects who show more than the number, only 3.2% was recognized on average in each age in the boy group, but 58.5 of the first grade elementary school students in the female group.
% Is highest in the younger age group, and this phenomenon
It was strongly suggested from the fact that the same pattern (FIG. 1B) as the transition of the average value of the O value was shown.

[0010] Generally, when a pathogenic microorganism invades, an acute inflammatory reaction centering on neutrophil infiltration occurs first, and thereafter, monocytes and macrophages migrate and accumulate locally, and activated macrophages become inactive. It is known to phagocytose pathogenic microorganisms, kill them, and produce various cytokines. In addition, macrophages present antigens to infiltrating lymphocytes, promote the production of cytokines from the lymphocytes, induce a specific immune response, and finally eliminate pathogens. However, in the case of urinary tract infection, bacterial-derived factors (LPS, which is an outer membrane component of gram-negative bacteria, pneumolysin, which is a gram-positive bacterium, lipoteichoic acid or staphylococcus aureus, which is a cell wall component, have entered the urinary tract. Toxic shock sy
macrophages and neutrophils are activated by ndrome toxin-1), resulting in TNF-α, IL-1β, IL-8
Such cytokines are released. In the early stage of infection,
Eliminating pathogens by phagocytosis and bactericidal action of neutrophils recruited to the infected area by L-8 plays a major role, but the resistance of the invading bacteria is strong or the number is large, It has been known that, when exclusion cannot be eliminated, the so-called immune system is triggered by lymphocytes.

The mechanism of the urinary immune system is to induce antigen-specific secretory IgA under the mucosal immune mechanism. Specifically, the immune system is formed in the epithelial cell layer of urogenital-associated lymphoid tissue. An antigen that recognizes the invading pathogen is taken up by existing epithelial cells called M cells that specialize in antigen uptake, and is transported to a group of cells having an antigen presenting function, such as macrophages and dendritic cells, and the antigen information is transferred to the MHC class. Present to other immunocompetent cells together with II molecule. These immunocompetent cells enter the thoracic duct via the intestinal vein lymph node, reach the lamina propria of the urogenital tract along the blood circulation system, and undergo Th2-type cytokines (IL-5, IL-
6, IL-10) to produce plasma cells and produce and secrete antigen-specific IgA. From this point of view, in order to verify whether leukocyte urine in the younger age group of women is due to invasion of minute amounts of bacteria into the urinary tract by ascending infection, the normal group, the median group, the high group, For the infectious disease group, urinary PGE2, IL-8, IL-6 and SIgA were measured and compared. As a result, the characteristic of the median group presenting asymptomatic leukocyte urine is that there is no recruitment of macrophages (PGE2
Value is not increased), and no inflammatory symptoms are observed (IL-6
There is no increase in the urinary excretion of urine of the acute reaction phase protein induced by IL-6 induced by IL-6), and, naturally, a significant increase in the secretion of SIgA, which indicates activation of the immune system, was also observed. Although it shows characteristics close to the normal group, such as no
It was clarified that only the MPO value (neutrophils) increased in an IL-8 concentration-dependent manner (FIG. 4).

Regarding the mechanism of formation of leukocyte urine and IL-8 (Takayusu, Tadashi Kawai: Clinical Pathology, 43:
329-334, 1995.) have demonstrated that IL-8 is involved in neutrophil migration in urine of urinary tract infection patients. That is, by invasion of the pathogenic bacteria into the urinary tract, IL-8 is produced and released from vascular endothelial cells, fibroblasts in the interstitium, macrophages, smooth muscle cells, and neutrophils present in the urinary tract, and peripheral blood is released. Neutrophils are chemotactic factors (IL-8)
The mechanism of migration to the infectious lesion according to the concentration gradient of is assumed. In addition, as a new neutrophil migration factor, FMLP (formyl-methionyl-leucyl-phenylala) produced by bacteria is used.
With the discovery of nin), bacterial infection and leukocyte urine formation are considered to be closely related. The relationship between the MPO value and the IL-8 value was also close as shown in FIG. At this time, it has also been revealed that neutrophils migrating into urine have the ability to produce active oxygen.

When these factors are taken into consideration, the medium value group is in a state of so-called asymptomatic bacteriuria in which a small amount of bacteria has invaded the urinary tract. It is a state in which neutrophils are recruited by activation of a nonspecific defense mechanism, and asymptomatic bacteriuria and asymptomatic leukocyteuria are considered to have a corresponding cause-effect relationship. In addition, even in adults, although there is an increase in the incidence of inflammatory diseases other than bacterial infections compared to the school urinalysis group, the urinary MPO value in the boy group is similar to the school urinalysis group, and Significant differences in urinary total protein concentration as a measure of urinary tract bleeding and urinary urinastin concentration reflecting systemic inflammatory disease were observed between the urinary MPO level of less than 14.5 ng / ml and the above groups. Therefore, it is considered that the mechanism of urinary leukocyte appearance in adults is mainly the case of asymptomatic bacteriuria. Regarding the relationship between invasion of pathogens into the urinary tract and activation of the immune system, a significant increase in SIgA, which indicates activation of the immune system, was seen from the high value group, but a marked increase was from the urinary tract infection group, It is from this stage that an intermittent rise in IL-6, which is considered to reflect the local inflammatory response, is observed (FIG. 4). This IL-6 originally acts on the plasma cellization of activated B cells in the lamina propria of the urinary tract and is involved in the induction of antigen-specific IgA production. It is thought that it was mixed in.

As is conventionally known, 95% or more of the leukocyte cell types appearing in urine are neutrophils, but there is hardly any detailed description of the reason. In our results, neutrophils were the main cell type in the urinary tract urinary tract infections, and the macrophage increase rate was only 1.7 times that of the normal group (neutrophil increase rate). Is about 190 times). Regarding this mechanism, the mechanism of defense of the urogenital system is based on the mucosal immune system, and macrophages and lymphocytes accumulate and act in the mucosa-associated lymphoid tissue of the lamina propria, do not appear in the urinary tract, and It is thought that the mechanism that depends on only neutrophils to eliminate pathogens that invaded the road was considered. In infectious chronic inflammation, an increase in monocytes and lymphocytes in the urine is thought to be caused by inflammation that damages the lamina propria and cells accumulated in the lamina propria enter the urine. Conceivable.

[0015] As a result of examining the actual state of non-specific leukocyte urine and the mechanism of its formation, it is considered that leukocyte transmigration into urine is based on bacterial infection from any viewpoint.
It was considered that measuring the number of leukocytes in urine was equivalent to knowing the number of bacteria in urine. This can be said, of course, from the fact that leukocytes (polymorphonuclear leukocytes) play a central role in the biological defense mechanism against bacteria and the like. Therefore, if a urine leukocyte detection method (detection of MPO, which is an intragranular protein of neutrophils and monocytes / macrophages) with excellent accuracy and quantification, is used,
It was suggested that urinary tract infections could be screened and pathological conditions could be determined without using bacteriuria as the first choice.

Regarding the concept of the pathological condition discrimination value (cut-off value), the resident number of leukocytes present in urine irrespective of gender (existing as a monitor of foreign substance intrusion: MPO value 14.5 ng / m)
l, an increase in urine leukocyte count greater than or equal to 13 white blood cell counts / μl) is considered to be the presence of bacterial infection and has an MPO value of 14.6 to
167.8 ng / ml (14-152 cells / μ in terms of the number of white blood cells)
It was considered appropriate to consider l) as asymptomatic bacteriuria and 167.9 ng / ml or more, in which urinary bleeding accompanying inflammation was frequently observed, as symptomatic bacteriuria (FIG. 6). Although this asymptomatic bacteriuria is in a state where the defense mechanism of the living body exceeds the virulence, it has the potential to develop symptomatic, and some observers say that it is often seen in the early stage of urinary tract infection. Therefore, it is possible to monitor the progress of infection by conducting re-examination, as a subject for follow-up observation. The present inventors have now discovered that a urinary tract infection can be diagnosed only by accurate detection of urine leukocyte count. As described above, leukocytes (neutrophils, macrophages)
Urine tract infection can be diagnosed by measuring myeloperoxidase which is an intragranular protein.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION

[Example 1] 1. Preparation of sample Urine diluted 2-fold with physiological saline containing 0.1% of Triton X-100 (Wako Pure Chemical Industries, Ltd.) was used as a sample. 2. Measurement Method The MPO measurement was performed by a sandwich EIA method using a 96-well microplate (Sumitomo Bakelite: MS-8596F). The rabbit polyclonal anti-human MPO antibody (DAKO) used for the solid phase was pH 8.4, 0.05mo.
The mixture was diluted to a concentration of 5 μg / ml with 1 / l Tris-HCl buffer, dispensed in 0.1 ml portions to each well of a microplate, allowed to react at 4 ° C. for 24 hours, and adsorbed. Blocking was performed at pH 8.0 containing 1% bovine serum albumin (BSA).
0.1 mol / l Tris-HCl buffered saline (BSA-T
BS) was dispensed into each well in an amount of 0.1 ml. The enzyme-labeled antibody used for the secondary reaction was prepared by labeling calf intestinal alkaline phosphatase (Boehringer Mannheim) with a rabbit polyclonal anti-MPO antibody by the periodate method.

3. Measurement operation (1) 0.05 ml of urine or standard solution was added to wells in which 0.1 ml of BSA-TBS was dispensed, and reacted at 37 ° C. for 1 hour. (2) The reaction solution in the well was removed, and the well was washed 5 times with deionized water, and 0.1 ml of the enzyme-labeled antibody adjusted to 2.5 μg / ml with BSA-TBS was added to each well, followed by reaction at 37 ° C. for 1 hour. Was. (3) The reaction solution in the well was removed, and the well was washed 5 times with deionized water. Then, 0.1 ml of an alkaline phosphatase substrate solution (containing 3 mg / ml sodium phenylphosphate and 1 mg / ml 4-aminoantipyrine) was added to each well. The mixture was reacted at 37 ° C. for 30 minutes. (4) 0.1 ml of a coloring solution (containing 5 mg / ml of sodium periodate) was added to the well, and a microplate colorimeter S was added.
The absorbance at 510/630 nm was measured using J-eia (Sanko Junyaku), and the MPO derived from human neutrophil was measured simultaneously.
The MPO value in urine was determined by doubling the concentration calculated from the calibration curve of (Attends Research).

[Embodiment 2] 1. Preparation of sample Urine diluted 2-fold with physiological saline containing 0.1% of Triton X-100 (Wako Pure Chemical Industries, Ltd.) was used as a sample. 2. Measurement Method The MPO measurement was performed by a sandwich EIA method using a 96-well microplate (Sumitomo Bakelite: MS-8596F). Mouse monoclonal anti-human MPO antibody (DAKO) used for the solid phase was pH 8.4, 0.05mo
The mixture was diluted to a concentration of 5 μg / ml with 1 / l Tris-HCl buffer, dispensed in 0.1 ml portions to each well of a microplate, allowed to react at 4 ° C. for 24 hours, and adsorbed. Blocking was performed at pH 8.0 containing 1% bovine serum albumin (BSA).
0.1 mol / l Tris-HCl buffered saline (BSA-T
BS) was dispensed into each well in an amount of 0.1 ml. The enzyme-labeled antibody used for the secondary reaction was prepared by labeling calf intestinal alkaline phosphatase (Boehringer Mannheim) with a rabbit polyclonal anti-MPO antibody by the periodate method.

3. Measurement operation (1) 0.05 ml of urine or standard solution was added to wells in which 0.1 ml of BSA-TBS was dispensed, and reacted at 37 ° C. for 1 hour. (2) The reaction solution in the well was removed, and the well was washed 5 times with deionized water, and 0.1 ml of the enzyme-labeled antibody adjusted to 2.5 μg / ml with BSA-TBS was added to each well, followed by reaction at 37 ° C. for 1 hour. Was. (3) The reaction solution in the well was removed, and the well was washed 5 times with deionized water. Then, 0.1 ml of an alkaline phosphatase substrate solution (containing 3 mg / ml sodium phenylphosphate and 1 mg / ml 4-aminoantipyrine) was added to each well. The mixture was reacted at 37 ° C. for 30 minutes. (4) 0.1 ml of a coloring solution (containing 5 mg / ml of sodium periodate) was added to the well, and a microplate colorimeter S was added.
The absorbance at 510/630 nm was measured using J-eia (Sanko Junyaku), and the MPO derived from human neutrophil was measured simultaneously.
The MPO value in urine was determined by doubling the concentration calculated from the calibration curve of (Attends Research).

[0021]

As described above, according to the present invention, the urinary leukocyte count can be accurately determined by measuring the concentration of urinary myeloperoxidase, and the diagnosis of urinary tract infection can be performed without proof of bacterial urine. It is possible.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing changes in urinary MPO value and the ratio of the number of indigenous persons or more by sex and age.

FIG. 2 illustrates the distribution of MPO values by gender in a young age group.

FIG. 3 illustrates the comparison of the concentration of various trace proteins in urine.

FIG. 4 is a diagram illustrating comparison of various test values in a young age group and a urinary tract infection group.

FIG. 5 illustrates the correlation between urinary MPO value and IL-8 value.

FIG. 6 is a diagram illustrating setting of a cut-off value of urinary MPO.

Claims (4)

[Claims] 1. A diagnostic kit characterized in that urine leukocytes are measured by focusing on neutrophils and macrophages, and urinary tract infection is diagnosed without proof of bacterial urine. 2. Urinary leukocytes (neutrophils, macrophages)
2. The diagnostic kit according to claim 1, wherein the presence of is detected by measuring the concentration of myeloperoxidase. 3. When the urine myeloperoxidase concentration is within a predetermined range, it is diagnosed as asymptomatic bacteriuria, and when it is higher than this value, it is diagnosed as symptomatic bacteriuria. The diagnostic kit according to claim 2, wherein: 4. The method according to claim 1, wherein an immunological assay such as an enzyme immunoassay, a latex agglutination assay, an immunoturbidimetric assay, an immunochromatographic assay, or an immunoluminescent assay is used.
A diagnostic kit according to the item.