COMPOSITION FOR DIAGNOSING INFECTIOUS DIEASES OR INFECTIOUS COMPLICATIONS BY USING TRYPTOPHANYL-TRNA SYNTHETASE AND METHOD FOR DETECTING DIAGNOSTIC MARKER

Abstract

The present invention relates to a composition for diagnosing infectious diseases by using a tryptophanyl-tRNA synthase (WRS) and a method for detecting a diagnostic marker and, more specifically, to: a composition for diagnosing infectious diseases, containing a preparation measuring the WRS protein or mRNA expression level; a diagnostic kit; a method for detecting the WRS for providing information required for the diagnosis of infectious diseases, and a method for determining the infectious disease mortality risk by using the WRS. According to the present invention, the WRS is increased only in infection-induced infectious diseases, differentiating non-infectious diseases therefrom, and is rapidly increased in the early stage of infection. In addition, the level of the WRS is closely correlated with the severity and prognosis of diseases or complications induced by infection. Therefore, the WRS can be used as a marker for more rapid and accurate diagnosis, in comparison to a conventional marker for infectious diseases or complications thereof.

Claims

1. A method for diagnosing and treating an infectious disease in a subject in need thereof, the method comprising: measuring an expression level of tryptophanyl-tRNA synthetase in a sample collected from a subject suspected of suffering from an infectious disease; comparing the level of tryptophanyl-tRNA synthetase in the subject with the level of a healthy subject, wherein a higher expression level of tryptophanyl-tRNA synthetase in the sample of the subject than that of the healthy subject indicates that the subject suffers from the infectious disease; and administering an appropriate treatment to the subject for the infectious disease if the expression level of tryptophanyl-tRNA synthetase in the sample of the subject is higher than that of the healthy subject.

2. The method of claim 1, wherein the infectious disease is caused by an infection by one or more selected from the group consisting of viruses, bacteria and fungi.

3. The method of claim 2, wherein the bacteria are gram-negative bacteria or gram-positive bacteria.

4. The method of claim 1, wherein the measuring is conducted with an agent comprising an antibody that specifically binds to the tryptophanyl-tRNA synthetase protein.

5. The method of claim 4, wherein the tryptophanyl-tRNA synthetase protein comprises the amino acid sequence of SEQ ID NO: 1.

6. The method of claim 1, wherein the measuring is conducted with an agent comprising a probe or a primer set which specifically binds to tryptophanyl-tRNA synthetase mRNA.

7. The method of claim 6, wherein the tryptophanyl-tRNA synthetase mRNA comprises the nucleotide sequence of SEQ ID NO: 2.

8. The method composition of claim 6, wherein the primer set is represented by SEQ ID NO: 3 and SEQ ID NO: 4.

9. The method of claim 1, wherein the infectious disease is selected from the group consisting of salmonellosis, food poisoning, typhoid, paratyphoid, pneumonia, pulmonary tuberculosis, tuberculosis, sepsis, septic shock, urinary tract infection, cystitis, pyelonephritis, urethritis, prostatitis, upper respiratory tract infection, and otitis media.

10. (canceled)

11. The method of claim 1, wherein the method comprises comparing the level of tryptophanyl-tRNA synthetase in the subject with the level of a healthy subject, and determining that the subject having an increased expression level of tryptophanyl-tRNA synthetase compared to the healthy subject has been infected with the infectious disease.

12. The method of claim 1, wherein the sample is selected from the group consisting of blood, plasma, serum, saliva, nasal mucus, sputum, capsular fluid, amniotic fluid, ascites, cervical or vaginal discharge, urine and cerebrospinal fluid.

13. The method of claim 1, the step of measuring the expression level of the tryptophanyl-tRNA synthetase measures the protein or mRNA expression level of tryptophanyl-tRNA synthetase.

14. (canceled)

15. A method for determining the mortality risk of a subject afflicted with an infectious disease, the method comprising: (a) providing a sample of a subject; (b) measuring the expression level of tryptophanyl-tRNA synthetase in the sample; and (c) determining that the mortality risk of the subject increases proportionally to an increase in the level of tryptophanyl-tRNA synthetase.

16-18. (canceled)

19. The method of claim 1, wherein the appropriate treatment to the subject for the infectious disease comprises a treatment with at least one agent selected from the group consisting of an antibiotic agent, an antiviral agent and an antifungal agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0124] FIG. 1 shows the change of WRS with time after infection measured by ELISA in peritoneal lavage fluid of mice injected intraperitoneally with 10.sup.8, 10.sup.7 or 10.sup.8 CFU of S. typhimurium (ST). The abscissa represents the time at which the abdominal exudate was obtained after ST infection (Time after ST inoculation (hr.))

[0125] FIG. 2 shows ELISA test results for measuring the manner of WRS secretion following viral infection.

[0126] FIG. 2 A shows the time course of WRS levels present in the culture medium of peripheral blood mononuclear cells (PBMC) infected with RSV (MOI=2) or PRS virus (MOI=2).

[0127] FIG. 2 B shows ELISA results of measuring the level of the WRS present in the serum of a healthy (HC, n=20) and viral pneumonia (n=5) patient. The statistical significance uses the Mann Whitney test, and the p value denoted by * is 0.04.

[0128] FIG. 3 A shows ELISA results of measuring the level of the WRS present in the serum of healthy control (H.C), severe sepsis, and septic shock patients.

[0129] FIGS. 3 B and C show ELISA results of measuring the levels of GRS or KRS present in the serum of healthy controls (HC) and sepsis patients, respectively (Statistical significance in the FIG. 3A was determined by Dunn's comparison test after Kruskal wallis test. FIGS. 3 B and C were determined by the two-tailed Mann-Whitney test. *** indicates p<0.001, * indicates p<0.05, n.s indicates statistically not significant.).

[0130] FIG. 4 A shows ELISA results of measuring the level of the WRS present in the serum of healthy controls (H.C.) and fungi infected sepsis patients.

[0131] FIGS. 4 B and C show ELISA results of measuring the levels of GRS or KRS present in the serum of healthy controls (HC) and sepsis patients, respectively (statistical significance was determined by the two-tailed Mann-Whitney test. *** indicates p<0.001, * indicates p<0.05, ns indicates statistically not significant).

[0132] FIG. 5 A shows ELISA results of measuring the levels of the WRS present in the serum of patients with gram-positive bacterial infection and patients with fungal infection.

[0133] FIG. 5 B shows ELISA results of measuring the levels of the WRS present in the serum of single infected and multiple infected patients (statistical significance was determined by Dunn's comparison test after Kruskal wallis test. *** indicates p<0.001, * indicates p<0.05, ns indicates statistically not significant).

[0134] FIG. 6 is a graph showing the results of the level of the WRS in patients with a systemic inflammatory response syndrome (SIRS, FIG. 6 A), asthma (ASA, FIG. 6 B) such as sterile chronic inflammatory diseases, rheumatoid arthritis (RA, FIG. 6 C) and Sjogren's syndrome (SS, FIG. 6 C) compared to a healthy controls (HC).

[0135] FIG. 7 shows the ROC curve of the WRS.

[0136] FIG. 8 shows a graph showing the correlation between the WRS and the SOFA score of the CRP. r is a Pearson correlation coefficient, and p is a probability value.

[0137] FIG. 9 shows the WRS levels of patients who survived and died after 28 days of sepsis diagnosis. Mann Whitney test is used for statistical significance, and the p value indicated *** is 0.0007.

[0138] FIG. 10 confirmed the levels of the WRS and procalcitonin in serum by Spearman's association assay.

MODE FOR CARRYING OUT INVENTION

[0139] Hereinafter, the present invention will be described in detail.

[0140] However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Clinical Experiment

[0141] The experimental method was conducted under permission of the institutional review board in Seoul National University (Permit No. 1502-001-010). Serum samples from healthy controls were collected at the Seoul National University Health Center. Serum samples from 99 sepsis patients were obtained through severe sepsis or septic shock intensive care unit. The patients who provided the serum used in the experiment were those who were admitted to the intensive care unit (ICU) of the universityaffiliated hospital in Seoul. Only the patients who had confirmed the bacterial infection participated in the experiment. The diagnosis of severe sepsis or septic shock was made according to the ACCP/SCCM consensus conference (1992). The experiment was conducted with the informed consent of all patients participating in the experiment according to the policy of the review committee. The experiment was also approved by the Review Committee of Asan Medical Center in Seoul. Between January 2014 and July 2015, a total of 35 patients admitted to the Seoul Severance Hospital allergy-asthma clinic participated in the experiment. The experiment included 26 healthy controls and 35 stable asthmatics diagnosed with asthma based on the results of symptoms and pulmonary function tests (more than 12% increase in forced expiratory volume for 1 second (FEV1) after the used of bronchodilator) by an allergist. The stable asthma was defined as asthmatic patients who maintained the usual dose of the drug without increasing the drug administration for the past one month. Clinical trials were approved by Severance Hospital and Yonsei University Health System (Permission No. 4-2013-0397). The serum was collected from 42 patients with primary Sjogren's syndrome (pSS), 35 patients with rheumatoid arthritis (RA), and 20 healthy controls. Primary Sjogren's syndrome was diagnosed according to American-European Consensus Group criteria for pSS or 2012 American College of Rheumatology criteria. Rheumatoid arthritis was diagnosed according to the 1987 revised criteria for classification of rheumatoid arthritis or 2010 rheumatoid arthritis classification criteria. According to the principle of the Helsinki Declaration, all patients and healthy controls participating in the experiment received informed consent from the experiment. The experiment was approved by the Seoul St. Mary's Hospital Review Committee (KC13ONMI0646).

Clinical ExperimentComparison with Procalcitonin

[0142] Based on the review committee's policy of each institution, informed consent was obtained on all patients who participated in the experiment including 120 healthy persons, 18 SIRS patients (due to causes other than infectious diseases), 166 sepsis patients and 160 septic shock patients. The systemic inflammatory response syndrome (SIRS), septicemia, and septic shock which were obtained from patients admitted to the ICU of Seoul National University Hospital were diagnosed according to the ACCP/SCCM consensus conference (1992).

[0143] Levels of procalcitonin (RayBiotech, USA Cat No: ELH-PROCALC) and WRS (CUSABIO, China, Cat No: CSB-E11789h) in serum were measured using ELISA kits.

[0144] Cell Culture

[0145] Human peripheral blood mononuclear cells (PBMC) were isolated using Cell Preparation Tube (CPTTM, Becton Dickinson) containing sodium citrate.

[0146] Bacterial Strain and Infection

[0147] Salmonella typhimurium (ATCC 14028) was obtained from the Center for Microbial Conservation, Seoul, Korea. Bacteria were routinely cultured using nutrient broth of BD bioscience. Bacteria were incubated overnight before infection and were obtained at a density of 110.sup.8 CFU. Bacterial density was estimated using absorbance and calibration curve at 600 nm. For PBMC or mouse infection experiments, the bacteria were washed with PBS and redispersed in serum-free medium or PBS.

[0148] Enzyme-Linked Immunosorbent Assay, ELISA

[0149] For quantitative analysis of the WRS and KRS of mouse and human, the amount of protein secreted in culture medium or serum was measured by ELISA kit. The WRS ELISA kit was purchased from Cusabio (Wuhan, China, catalog number CSB-E11789h) and KRS was purchased from USCN (Wuhan, China, catalog number SED002 Hu).

[0150] Statistics

[0151] Statistical significance was determined when the provability value (p-value) was less than 0.05. All statistical calculations were performed using Graphpad prism 5.0 (GraphPad Software).

Example 1

Increased WRS Levels Due to Bacterial or Viral Infection

[0152] <1-1> WRS Secretion by Salmonella Infection

[0153] Salmonella typimurium was injected into mouse abdominal cavity to confirm the amount of WRS present in the peritoneal exudate.

[0154] Salmonella of 10.sup.6, 10.sup.7 or 10.sup.8 CFU was intraperitoneally injected into female C57B116 at 9-10 weeks old, and peritoneal exudate was obtained from 5 to 11 mice every hour up to four hours after the bacterial infection to measure the amount of WRS present in the peritoneal exudate by ELISA (FIG. 1). WRS secreted by peritoneal exudate was secreted from the very early stage after salmonella infection and increased according to salmonella concentration. The amount of WRS was almost reached at 1 hour after infection.

[0155] <1-2> Secretion of WRS Due to Viral Infection

[0156] In addition to bacteria, we examined the secretion of WRS by viral infection by using serum of human peripheral blood mononuclear cells and viral pneumonia.

[0157] When human peripheral blood mononuclear cells (PBMC) are infected with respiratory syncytial virus (RSV) and PR8 virus such as influenza virus, the WRS was significantly increased in the cell culture medium from 30 minutes after infection, and it was confirmed that the level of secreted WRS was maintained by 4 hours after infection in which the experiment was conducted (A in FIG. 2). In addition, it was confirmed that the amount of WRS was significantly increased in the serum of patients with viral pneumonia compared with the healthy controls (H.C) (B in FIG. 2)

[0158] These results show that the level of WRS is increased by viral infection.

Example 2

[0159] WRS-Specific Secretion in Infectious Inflammatory Disease

[0160] <2-1> Increase in WRS in Sepsis and Septic Shock Patients

[0161] The level of WRS in patients with septicemia and septic shock due to infection was compared with healthy controls.

[0162] The sepsis patients who participated in this experiment were patients with severe sepsis and septic shock who were admitted to the ICU of Asan Medical Center in Seoul. Bacteria and fungi detected in the patients are as described in Table 1.

[0163] The levels of WRS were significantly increased in the severe sepsis patients and septic shock patients compared to healthy controls (H.C) (FIG. 3A). The measured WRS values were increased by about 20 times in 2.630.62 ng/ml (n=37) patients with septic patients compared with 0.180.06 ng/ml (n=20) in the healthy controls. In addition, the WRS level in patients with septic shock was 6.350.90 ng/ml (n=63), which was about 50 times higher than that of the healthy controls. The WRS level in patients with septic shock is much higher than that in patients with severe sepsis, indicating that the level of WRS is closely related to the severity of sepsis.

[0164] In contrast to the significant increase in WRS levels in patients with sepsis compared with healthy controls, glycyl-tRNA synthetase (GRS) and lysyl-tRNA synthetase (KRS) such as aminoacyl tRNA synthetase (ARS) secreted into different types showed no significant changes (FIGS. 3B and 3C).

TABLE-US-00001 TABLE 1 Gram () bacteria N Gram (+) bacteria N Multi-infection N Fungi N Acinetobacter 1 Enterococcus 2 CRAB, E. coli 1 Candida 2 baumanii faecalis albican Acinetobacter 7 Enterococcus 1 CRAB, P. mirabilis 1 Candida 1 baumanii (CRAB) faecalis galabrata (VRE) Acinetobacter 2 Staphylococcus 14 K. pneumonia, 1 Bowel perfor- 1 baumanii (CSAB) aureus Citrobacter candida (MRSA) tropicalis Aeromona 3 Staphylococcus 3 K. pneumonia, E. coli 1 Aspergillus 3 hydrophila aureus (MSSA) Burkholderia 2 Staphylococcus 1 K. pneumonia, A. baumanii 1 cepacia epidermidis (CRAB) Enterobacter 1 Streptococcus 3 K. pneumonia, P. aeruginosa 1 aerogenes pneumoniae Enterobacter 1 Streptococcus 1 E. Coli, P. aerunginosa 1 cloacae agalactiae (CSPA) Escherichia coli 21 MRSA, A. baumanii 1 Haemophilus 1 MRSA, ESBL, 1 Influenza Serratia Klebsiella 13 Enterococcus, 1 pneumonia Bacteriodes, Streptococcus Morganella morganii 1 Fungus + MRSA 1 Proteus mirabilis 1 unknown 3 Stenotrophomonas 1 maltophilia Pseudomonas 1 aeruginosa (CRPA) Pseudomonas 4 fluorescens Vibrio vufnificus 2

[0165] Meanwhile, as shown in FIG. 4, the levels of WRS were increased (n=8, 4.521.83 ng/mL, Mann-Whitney test) in serum of patients with bacterial infections as well as fungi sepsis. The serum levels of GRS and KRS were not significantly changed.

[0166] Among the above sepsis patients, no significant difference was observed between Gram-negative bacterial infection patients (n=62, 5.980.93 ng/mL), Gram-positive bacterial infection patients (n=25, 2.870.72 ng/mL), and fungal infection patients (n=8, 4.521.83), and no significant differences were observed between patients with multiple pathogens (n=11, 3.531.22 ng/mL) and those with single pathogens (n=94, 5.010.67 ng/mL) (FIG. 5).

[0167] <2-2> WRS Level in Non-Infectious Inflammatory Disease

[0168] The levels of WAS in patients with non-infectious inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), asthma such as sterile chronic inflammatory diseases, rheumatoid arthritis, and Sjogren's syndrome were compared with healthy controls.

[0169] The levels of WAS present in the serum of these non-infectious inflammatory disease patients were not significantly different from healthy controls (FIG. 6). The WRS detected in the serum of systemic inflammatory response syndrome (FIG. 6A), asthmatic patients (FIG. 6B), rheumatoid arthritis patients (FIG. 60), and Sjogren's syndrome patients (FIG. 6C) was no statistically significant difference.

Example 3

[0170] Efficiency of WRS as a Diagnostic Marker of Infectious Inflammatory Diseases

[0171] <3-1> Performance of WRS as an Infectious Inflammatory Disease Marker

[0172] In order to confirm the performance of WRS as an infectious inflammatory disease marker, a receiver operating characteristic curve (ROC curve) was prepared.

[0173] The AUC of the WRS on the ROC curve of 100 patients with bacterial infected sepsis was 0.90 (p<0.0001), the cut off value was 0.28 ng mL, the sensitivity was 82% and the specificity was 80% (FIG. 7).

[0174] <3-2> Correlations Among Level of WRS, Infectious Inflammatory Disease Severity and Prognosis

[0175] To further confirm the performance of WRS as an infectious inflammatory disease marker, we examined the correlations among the level of WRS, the severity of sepsis and the prognosis of sepsis.

[0176] The sequential organ failure assessment score (SOFA score), which indicates the prognosis of sepsis patients, and the level of WRS or CRP detected in the patient's serum were shown as a graph, and the Pearson correlation coefficient is calculated (FIG. 8).

[0177] The Pearson correlation coefficient of WRS was 0.43, which was significantly higher than that of CRP of 0.15, and statistically significant correlation was shown. In other words, the level of WRS is more closely correlated with the SOFA score of patients with sepsis than the CRP, which is an existing inflammation index.

[0178] In addition, the survival and the level of WRS after 28 days of the diagnosis of sepsis were measured as other indicators of the prognosis of sepsis patients, and statistical significance was determined by the Whitney test (FIG. 9). There was a statistically significant difference in WRS levels among patients who died after 28 days of the diagnosis of sepsis (death, n=27, WRS 9.14+1.63 ng/ml, survival, n=72, WRS 3.36+0.49 ng/ml). Thus, the higher the WRS level was, the worse the survival prognosis of sepsis was.

Example 4

[0179] Comparison with Procalcitonin

[0180] <4-1> Comparison of Contents in Serum

[0181] The amount of WRS (CUSABIO, China, Cat No: CSB-E11789h) and procalcitonin (RayBiotech, USA Cat No: ELH-PROCALC) known as major inflammatory markers in serum of 120 healthy subjects, 18 SIRS patients (due to non-infectious cause), 166 sepsis patients and 160 septic shock patients was quantitatively determined by ELISA method according to the manufacturer's instructions.

[0182] As a result of Table 2, procalcitonin was distinguished from non-infectious systemic inflammatory symptom SIRS in comparison with healthy persons and a more amount of it was detected in sepsis or septic shock, which is a complication of infectious disease. However, WRS was found to be specific only for complications of infectious diseases.

TABLE-US-00002 TABLE 2 Comparison of WRS and Procalcitonin in healthy Controls and Patients Healthy controls SIRS Sepsis Septic shock P Classification (n = 120) (n = 18) (n = 166) (n = 160) value WRS (ng/mL) 0.06 0.02 0.20 0.12 1.29 0.20 6.78 0.79 0.000 Procalcitonin (ng/mL) 0.03 0.01 1.02 0.36 2.02 0.23 2.98 0.31 0.000

[0183] <4-2> Comparison of Content by Survival

[0184] Sepsis and septic shock patients were divided into survivor group and death group according to the survival after 28 days. The content of WRS and procalcitonin in patient serum of each group was quantitatively measured as in the Example <4-1> above.

[0185] As shown in the following Table 3, in the case of procalcitonin, there was no distinction between survivors and deaths, but WRS was classified as statistically meaningful, and it was confirmed that patients with the fear of death could be selected.

TABLE-US-00003 TABLE 3 Comparison of WRS and Procalcitonin in Survival and Death Patients Survivals Deaths P value WRS (ng/mL) 2.47 0.40 6.22 0.84 <0.05 Procalcitonin (ng/mL) 2.29 0.22 2.73 0.35 NS

[0186] <4-3> Correlation Analysis with Procalcitonin

[0187] In order to confirm the correlation between the conventional diagnosis of procalcitonin and WRS, Spearman's correlation analysis was performed using WRS and procalcitonin measurements for sepsis patients.

[0188] As shown in FIG. 10, it was confirmed that Spearman's rho value (r) was 0.127 and p value was 0.022 between WRS and procalcitonin.

INDUSTRIAL APPLICABILITY

[0189] As we have seen, the WRS is increased only in infection-induced infectious diseases, differentiating non-infectious diseases therefrom, and is rapidly increased in the early stage of infection. In addition, the level of the WRS is closely correlated with the severity and prognosis of diseases or complications induced by infection. Therefore, the WRS can be used as a marker for more rapid and accurate diagnosis, in comparison to a conventional marker for infectious diseases or complications thereof.