WO2020035028A1

WO2020035028A1 - Detection of infection

Info

Publication number: WO2020035028A1
Application number: PCT/CN2019/100808
Authority: WO
Inventors: 文格佩尔; 孙立宁
Original assignee: 苏州芬瑞医疗诊断科技有限公司
Priority date: 2018-08-16
Filing date: 2019-08-15
Publication date: 2020-02-20

Abstract

The present invention relates to a device and method for determining whether the infection in a subject is caused by bacteria or viruses, more particularly, comprising: measuring amount of two or more biomarkers in a whole blood sample obtained from the subject, and measuring whether the amount of each biomarker changes compared with a normal control. At least one biomarker measured is derived from pre-activated whole blood HNL and the at least one biomarker is not HNL.

Description

Detection of infection

Technical field

The present invention relates to a device that can be used to determine whether an infection in a subject is caused by a bacterium or a virus. More specifically, the invention relates to such a device, an array for implementing the method, and a method. The present invention provides a fast and simple way to analyze whole blood samples at a location physically close to a potentially infected individual.

background

Distinguishing the bacterial and viral causes of respiratory infections is a major clinical challenge that often leads to unnecessary antibiotic prescriptions. As a diagnostic aid, white blood cell counts and C-reactive protein (CRP) plasma levels have been widely used for many years. However, despite their speed, none of these tests meet sufficiently accurate requirements, which has led to the need for microbial testing in many cases.

Previous reports (Xu SY, Pauksen K, Venge P. "Serum measures of human neurotrophil Lipocalin (HNL) discriminate between acute infections and viral infections". Scand J Clin Lab Invest 1995; 55 (2): 125-31; Yu ZZ , Jing H, Hongtao P, Furong J, Yuting J, Xu et al. "Distinction between bacterial and viral infections by serum" measurement of human neutrophil Lipocalin (HNL), and the impact act of "antibody body selection"; 432 : 82-6; Bjorkqvist M, Kallman J, Fjaertoft G, Xu S, Venge P, Schollin J. "Human Neutrophil Lipocalin: normal levels and use use as a marker for intrusive infection, innocent the newborn"; Acta2004Paedia 4): 534-9; Venge, P, Hakansson, LD, Garwicz, D, Peterson, C, Xu, Pauksen, K. "Human Human Neutrophil Lipocalin" in MLP-activated whole blood as well as "Diagnostic" means "Dangerous" Immunol M ethods 2015 May 20; and Venge P, Douhan-Hakansson L, Garwicz D, Peterson C, Xu S, Pauksen K. "Human Social Neutrophil Lia Pocalin as a Superior Superior Diagnostic Means, Too Distinguish, Between Acute, Bacterial, and Immunity 22 (9): 1025-32) has shown that human neutrophil apolipoprotein (HNL) may fulfill this unmet clinical need because serum HNL is released in whole blood after activation with tripeptide fMLP Both HNL and HNL have better diagnostic performance than modern biomarkers and new biomarkers, such as procalcitonin and CD64 expression on neutrophils.

US 9,476,880 (Venge, Per) describes a method for detecting acute kidney injury using HNL (also known as neutrophil gelatinase-associated apolipoprotein (NGAL)) in an individual, in which a bodily fluid sample The measurement device that detects the marker is contacted to allow the NGAL protein in the sample to be complexed with the NGAL antibody, and the amount of the complex formed between the NGAL protein from the sample and the NGAL antibody in the measurement device is measured using the detectable label. . The NGAL antibodies in the device have the ability to bind to more than two NGAL protein epitopes, and the amount of complex formed represents the level of acute kidney injury.

However, many other biomarkers have recently been proposed to distinguish acute infections caused by bacteria or viruses. Some of these markers, azurecidin (heparin-binding protein, HBP) and calprotectin, are derived from circulating neutrophils like HNL.

Other markers, such as TRAIL (TNF-related apoptosis-inducing ligand), IP-10 (interferon gamma-induced protein 10 kDa) (see Oved K, Cohen A, Boico O, Navon R, Friedman T, Etshtein L et al. "A novel host-proteome signature for distinguishing between acute bacterial and viral infections". PLoS One 2015; 10 (3): e0120012) and TK1 (thymidine kinase 1) (7) (see Gronowitz JS,

CFR, Diderholm H, Hagberg H, Petterson U. "Application of an in vitro assay for serum thymidine kinase: Results on viral disease and malignancies in humans". Int J Cancer 1984; 33: 5-12) has multiple cell sources, But in this regard it has been proposed as a useful biomarker.

WO2018 / 060999 (Memed Diagnostics) relates to the use of TRAIL, and more specifically describes a method comprising: (a) measuring the level of TRAIL protein in a patient's blood sample; (b) measuring at least one disease in a patient Levels of relevant parameters; and (c) providing a risk assessment based on a combination of said levels of TRAIL protein and levels of said disease-related parameters.

The inventors have directly compared the effectiveness of a number of potentially useful biomarkers in distinguishing the bacterial and viral causes of acute infections, and have also combined these biomarkers to find the most powerful algorithm for this distinction.

Summary of invention

It can be seen from the experimental part and the drawings that direct comparison of 10 candidate biomarkers shows a huge difference in AuROC, of which B-HNL is an excellent biomarker (0.91, 95% CI 0.86-0.95), and 9 other organisms The AuROC of the markers ranged from 0.63-0.79.

The combination of B-HNL with IP-10 and / or TRAIL further improves diagnostic performance, with AuROC reaching 0.94-0.97. The clinical manifestations of the combination of CRP with IP-10 and / or TRAIL varied between 0.85 and 0.87, and were significantly lower than the combination with B-HNL (p = 0.003-p = 0.03).

Therefore, the present invention has shown that the diagnostic efficacy of whole blood-activated HNL is superior to any other known independent biomarker in the difference between acute infections caused by bacteria or viruses.

Adding IP-10 and / or TRAIL to the diagnostic algorithm further improves the diagnostic performance of (whole blood activated) B-HNL, where bacterial and viral infections are almost completely distinguished.

Therefore, the rapid and accurate HNL analysis provided by the present invention that reflects the bacterial challenge of the body, together with one or two biomarkers that reflect viral infections, should be our ideal combination of diagnostic markers against antibiotic abuse.

More specifically, the present invention relates to a device for determining whether an infection in a subject is caused by a bacterium or a virus, which includes a module for absorbing test blood samples and reagent storage, wherein the absorption module includes A first reagent for testing a blood sample, and a second reagent for measuring an amount of a HNL biomarker in the pre-activated test blood sample;

Wherein a module for absorbing a test blood sample is a syringe or a needle, which is arranged to collect whole blood from a subject and apply the blood to a substrate; and a module for signal detection and result display;

Wherein the module displays a signal indicating whether the amount of the measured HNL biomarker has changed compared to a normal control.

The invention also relates to an array for determining whether an infection in a subject is caused by a bacterium or a virus, said array comprising antibodies or two or more biomarkers in a sample obtained from the subject. An antigen-binding fragment, wherein at least one of the biomarkers is HNL obtained from a whole blood sample after being preactivated with an N-formylated peptide, and at least one of the biomarkers is not HNL.

Further, the present invention relates to a method for determining whether an infection in a subject is caused by a bacterium or a virus, which comprises measuring two or more biomarkers in a whole blood sample obtained from the subject. And measure whether the amount of each biomarker is changed compared to the normal control, at least one of the biomarkers is HNL obtained from a whole blood sample after pre-activating it with an N-formylated peptide, and At least one biomarker is not HNL.

Finally, the present invention relates to a method of treating a bacterial infection in a subject suspected of having a bacterial or viral infection, the method comprising: (a) measuring the HNL biomarker in a whole blood sample obtained from the subject (B) if the measured amount is changed compared to a normal control, the subject is diagnosed with a bacterial infection; (c) a subject diagnosed with a bacterial infection in step (b) is selected for treatment; and (d) administering an appropriate antibacterial therapy to said subject, in which the whole blood sample has been pre-activated with an N-formylated peptide.

definition

The term "human neutrophil apolipoprotein" (HNL) as used herein for biomarkers sometimes means neutrophil gelatinase-associated apolipoprotein (NGAL), apolipoprotein-2 (LCN2), or carcinogen Gene 24p3.

The term "B-HNL" as used herein refers to HNL from whole blood.

The term "P-HNL" as used herein refers to HNL from plasma.

As used herein, the term "activation" of HNL sometimes means "stimulation" in a sense and relates to its signal transduction properties.

Brief description of the drawings

Figures 1a-j show the concentration of 10 illustrative and tested biomarkers in healthy people compared to patients with bacterial or viral infections. Box plots indicate median and quartile ranges.

Figure 2 provides a comparison of individual biomarkers to distinguish between bacterial and viral infections. Results are given in AuROC and 95% CI.

Figure 3 provides a comparison of single and combined biomarkers to distinguish between bacterial and viral infections. Results are given in AuROC and 95% CI.

The table in FIG. 4 shows the results when the pre-activated whole blood-derived biomarker HNL was combined with other biomarkers TRAIL and IP-10.

Detailed description of the invention

A first aspect of the invention is a device for determining whether an infection in a subject is caused by a bacterium or a virus, comprising a module for absorbing test blood samples and reagent storage, wherein the absorption module includes a means for pre-activating A first reagent for testing a blood sample, and a second reagent for measuring an amount of a HNL biomarker in the pre-activated test blood sample;

In the device according to the invention, the signal may provide a numerical reading of the percentage above the baseline, which is representative of the presence of a bacterial infection in the test sample.

The first reagent may include an N-formylated peptide, such as N-formylmethionyl-leucine-phenylalanine (fMLP); and the second reagent may include at least one biomarker -Specific capture antibodies or antigen-binding fragments thereof.

A second aspect of the invention is for determining whether an infection in a subject is caused by a bacterium or a virus, the array comprising binding to two or more biomarkers in a sample obtained from the subject. Of the antibody or antigen-binding fragment, wherein at least one biomarker is HNL obtained from a whole blood sample after pre-activating it with an N-formylated peptide, and at least one biomarker is not HNL. As discussed above, the N-formylated peptide may be N-formylmethionyl-leucine-phenylalanine (fMLP).

The biomarker according to the invention and the use for its measurement will be described below in the context of a method for determining the cause of infection and a method of treatment or diagnosis. All details discussed below apply equally to the uses and concepts of the first aspect of the invention.

Therefore, a third aspect of the present invention is a method for determining whether an infection in a subject is caused by a bacterium or a virus, which comprises measuring two or more organisms in a whole blood sample obtained from the subject. The amount of markers, and whether the amount of each biomarker is changed compared to normal controls, at least one of which is obtained from a whole blood sample after pre-activating it with an N-formylated peptide HNL, and at least one biomarker is not HNL.

Herein, the terms "bacteria" and "viruses" should be understood to refer to one or more species, respectively.

As the skilled person will understand, there are many possible ways to activate the signal transduction of the biomarker HNL, and various pathways have been discussed in the literature. An illustrative reagent suitable for this purpose is N-formylmethionyl-leucine-phenylalanine (fMLP), which is readily available from commercial sources.

The present invention also encompasses the method described above, wherein biomarkers other than HNL are characterized by an independent ability to distinguish between bacterial and viral infections.

Illustrative biomarkers other than HNL that are suitable for combination with the HNL described herein are, for example, heparin-binding protein (HBP), such as azurol; a mammalian protein complex, such as calprotectin; from polymorphonuclear leukocytes Integrated membrane glycoproteins, such as PMN-CD64; pentraxines, such as C-reactive protein (CRP); chemokines, such as IFN-γ-induced protein 10 (IP-10, also known as CXCL10) Peptide precursors, such as procalcitonin (PCT); enzymes, such as thymidine kinase 1 (TK1), carcinoembryonic antigen-related cell adhesion molecules, such as carcinoembryonic antigen-related cell adhesion molecules 8 (CEACAM8 ); And apoptosis-related proteins, such as TNF-related apoptosis-inducing ligand (TRAIL).

Thus, such other biomarkers can be selected from the group consisting of: azurin, calprotectin, PMN-CD64, CRP, IP-10, PCT, TK1, CEACAM8, and TRAIL, all of which are well known and commercially available.

More specifically, such other biomarkers may be selected from the group consisting of azurin, calprotectin, PMN-CD64, CRP, IP-10, PCT, TK1, and TRAIL.

As can be seen from the experimental section below, other biomarkers IP-10 and TRAIL have been successfully used in the methods described herein.

In the method according to the present invention, the biomarker measurement may include binding to HNL or a capture antibody or antigen-binding fragment thereof specific to each biomarker, preferably binding of a monoclonal antibody. In some forms (such as arrays discussed below), biomarker measurements can be incorporated into further antibodies that are detectably labeled according to known techniques, such as radiolabels, enzyme labels (e.g. horseradish peroxidase, alkaline phosphate Enzymes), or fluorescent labels (eg, luciferin, Alexa, green fluorescent protein, rhodamine).

Therefore, for measurement purposes according to the present invention, antibodies can be conjugated to a solid support suitable for use in a diagnostic array according to known techniques, such as ELISA or biochip array technology.

Biomarker-specific antibodies and antigen-specific fragments thereof can be obtained from commercial sources including, for example, Thermofisher, Affymetrix or Diagnostics Development. Alternatively, the skilled artisan can routinely prepare antibodies for measurements described herein in a suitably equipped laboratory.

A fourth aspect of the invention is a method of treating a bacterial infection in a subject suspected of having a bacterial or viral infection, the method comprising: (a) measuring a HNL biomarker in a whole blood sample obtained from the subject The amount of the substance; and optionally also measuring the amount of at least one other biomarker that is not HNL; (b) if the measured amount is changed compared to a normal control, the subject is diagnosed with a bacterial infection; (c) Selecting a subject diagnosed with a bacterial infection in step (b) for treatment; and (d) administering an appropriate antibacterial therapy to said subject, in which N-formylated peptide has been used Pre-activated whole blood samples.

As will be understood by the skilled person, the methods described herein can also be used to monitor the effectiveness of the treatment given to a subject in need thereof. Therefore, the method according to the invention may be the fourth aspect of the invention as defined above, which further comprises the step of monitoring the effectiveness of the therapy by measuring the HNL biomarker in accordance with the invention together with at least one non-HNL biomarker (e).

Alternatively, the present invention is to monitor the effectiveness of antibacterial treatment using the HNL biomarkers described herein together with at least one other biomarker other than HNL, where a preliminary diagnosis of infection has been performed by any suitable method.

Accordingly, the present invention includes a combination of a HNL biomarker and at least one non-HNL biomarker for use in the diagnosis of a bacterial infection.

Optionally, the invention includes a combination of a HNL biomarker and at least one non-HNL biomarker for use in the diagnosis of a viral infection.

In the combination according to the invention, the HNL biomarker can be obtained from a whole blood sample.

Further, in the combination according to the present invention, N-formylated peptides, such as N-formylmethionyl-leucine-phenylalanine (fMLP), have been used to preactivate the sample.

Advantageously, the biomarker that is not HNL is selected from IP-10; TRAIL; and a combination of IP-10 and TRAIL.

All details discussed above regarding biomarkers and their measurements apply equally to this fourth aspect of the invention.

Finally, the invention also encompasses a diagnostic method performed in accordance with the teachings provided in this patent application and administering one or more of the above aspects.

Detailed drawings

In Figures 1a-j, the concentrations of 10 illustrative biomarkers in healthy people compared to patients with bacterial or viral infections are shown. Statistical differences between the results of patients with bacterial infections and healthy or virally infected patients were assessed by Mann-Whitney nonparametric tests and are shown in the figure. Box plots indicate median and quartile ranges.

More specifically, Figure 1a relates to the biomarker azurolin in plasma; Figure 1b relates to the biomarker HNL in whole blood; Figure 1c relates to the biomarker calprotectin in plasma; Figure 1d relates to the biomarker on PMN The marker PMN-CD64; Figure 1e relates to the biomarker CRP in plasma; Figure 1f relates to the biomarker IP-10 in serum; Figure 1g relates to the biomarker procalcitonin in serum; Figure 1h relates to the plasma The biomarker HNL; Figure 1i relates to the biomarker TK1 in serum; and Figure 1j relates to the biomarker TRAIL in serum.

Figure 2 is a comparison of a single biomarker to distinguish between bacterial and viral infections. Results are given in AuROC and 95% CI.

Figure 3 is a comparison of single and combined biomarkers distinguishing between bacterial and viral infections. Results are given in AuROC and 95% CI. Three vertical lines are given for comparing CRP only, AuROC for B-HNL only, or AuROC for 1.0.

Experimental part

This embodiment is provided for illustrative purposes only and should not be construed as limiting the scope of the invention as defined by the appended claims. All references provided below and throughout this application are incorporated herein by reference.

General method

Patients were recruited from infectious disease departments and primary care units (n = 581). Clinical diagnosis of bacterial or viral infections is confirmed by objective microbiological / serological tests.

Measured in whole blood after neutrophil activator N-formylmethionyl-leucine-phenylalanine fMLP (B-HNL) or pre-activation in plasma (P-HNL) HNL. Commercially measure azurenicin (also known as cationic antibacterial protein 37 (CAP37) or heparin-binding protein (HBP)), calprotectin, PMN-CD64, CRP (C-reactive protein) in plasma / serum, IP-10 (interferon gamma-inducing protein 10 kDa), PCT (procalcitonin), TK1 (thymidine kinase 1), TRAIL (TNF-related apoptosis-inducing ligand). The area under the ROC (Recipient Operating Characteristics) curve (AuROC) was used to assess the clinical performance of the biomarkers alone or in combination.

method

The study group included a total of 725 participants. Patients with signs and symptoms of acute infection were 253 men (age 52.7 ± 20.0 years ± SD) and 328 women (age 46.4 ± 19.3 years). The mean age of 144 healthy controls was 43.6 ± 12.8 years, and consisted of 57 men (age 41.3 years ± 12.7 years) and 87 women (age 45.0 years ± 12.8 years). As mentioned earlier, a total of 581 patients with signs and symptoms of acute infection were recruited (Xu, SY, Pauksen, K, Venge, P. Serum, measurements of human neurotrophil, Lipocalin (HNL) discriminate between acute bacterial and viral infections. Scand J Clin Lab Invest 1995; 55 (2): 125-31; Yu Z, Jing H, Hongtao P, Furong J, Yuting J, Xu et al. Distinction between bacterial and viral infection measures by human neutrophic measures of human neutrophil (HNL) and of antibody selection. J Immunol Methods 2016 May; 432: 82-6.).

The inclusion criteria were fever> 38 ° C, and signs and symptoms of acute infection. In addition to clinical results, the judges also knew about CRP, white blood cell counts, X-ray results, and microbiological tests. Based on this information, it is determined whether the patient has a bacterium or a virus as the pathogen causing the infection. Exclusion criteria are known chronic viral infections, such as human immunodeficiency virus (HIV) infection or hepatitis C. In addition, children under 18 years of age and patients who were unable to give informed consent were excluded from this study. Patients were admitted to the Department of Infectious Diseases of the Uppsala University Hospital (Infectious Disease Department of the University of Uppsala) or the primary health care unit of Uppsala. Blood is drawn before starting antibiotic treatment. The study was approved by the Uppsala Ethics Committee.

The etiology of acute infection was confirmed in 288 patients (49.6% of all patients). Of these patients, 185 had bacterial infections, 54 were viral infections, 26 were mycoplasma infections, and 23 were bacterial infections secondary to influenza.

Biomarker assay

As previously mentioned, HNL was measured in whole blood after activation with fMLP, or in EDTA-plasma (Venge, P, Hakansson, LD, Garwicz, D, Peterson, C, Xu, S, Pauksen, K. Human, Neutrophil, Lipocalin, and MLP-activated whole blood, as well as diagnostics, discrimination, and bacterial and viral infections. J Immunol Methods 2015 (May 20).

As described, the expression of CD64 on polymorphonuclear leukocytes was assessed by flow cytometry. CRP in plasma is measured by the routine clinical chemistry department of university hospitals. Measurement of azurenicin (HBP) (Hycult Biotech) in EDTA-plasma. Serum calprotectin (Gentian Diagnostics), IP-10 (Invitrogen), procalcitonin (Thermo-Fisher), TK1 (Arocell Diagnostics, Uppsala, Sweden), and TRAIL (Affymetrix) were measured. All biomarkers are run according to the manufacturer's instructions. For all assays, the inaccuracies of duplicate samples were between 4-10% CV.

statistics

As shown, the data are expressed as median or quartile range or full range. Group comparisons were performed by a non-parametric Mann-whitney test for independent groups. To assess the clinical performance of the biomarker assay, a receiver operating characteristic (ROC) analysis was performed and the area under the c-statistical analysis curve was compared. The diagnostic performance of the combination of biomarkers is expressed as the area under the ROC curve (AuROC) and calculated by logistic regression analysis.

For statistical calculations, use the MedCalc statistical software version 18.2.1 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org; 2018).

result

It can be seen from Figs. 1a-1j that the distribution of biomarker concentrations in blood, serum, or plasma of patients whose etiology of acute infection has been confirmed compared with the results of the health reference. From these results, it can be seen that the biomarkers are elevated in bacterial infections compared to the results of the healthy reference. However, one exception is the result of TRAIL, as TRAIL results in bacterial infections are significantly lower than the healthy reference. Compared to viral infections, most biomarkers are elevated in bacterial infections. However, IP-10, TK1, and TRAIL results were significantly higher in viral infections compared to bacterial infections.

In the next step, the diagnostic power of a single biomarker in distinguishing the bacterial and viral causes of acute infection is assessed by the ROC curve. As can be seen from Figure 2, all biomarkers have significantly elevated AuROC. However, AuROC varies between 0.63 and 0.91. TK1, HBP and PCT are the three biomarkers with the lowest AuROC. Three neutrophil biomarkers: calprotectin, CD64 and P-HNL have an intermediate AuROC between 0.70-0.72, while TRAIL and IP-10 have a significantly higher AuROC of 0.79. CRP with AuROC of 0.81 is shown for comparison, keeping in mind that the results of this marker are known to the judges. The highest AuROC was found in activated whole blood HNL (0.91, 95% CI 0.86-0.95). The AuROC of B-HNL was significantly larger than any other biomarker (p = 0.003-p <0.0001).

To investigate the diagnostic performance of biomarker combinations, a logistic regression analysis including all biomarkers was performed. As entered in the B-HNL (p <0.0001), IP-10 (p = 0.019) and TRAIL (p = 0.005) models, the distinction between bacterial and viral infections is significantly and independently increased, with AuROC being 0.96 ( 95% CI 0.92-0.99) (Table 1). By excluding whole blood assays, ie, CD64 expression on B-HNL and neutrophils, and calculating only plasma / serum biomarkers other than CRP, an AuROC of 0.87 (95% CI 0.82-0.91) was obtained. In this model, calprotectin (p = 0.01), TK1 (p = 0.009) and IP-10 (p = 0.001) independently contribute. When CRP was added, AuROC increased to 0.91 (95% CI 0.86-0.94). Figure 3 shows a comparison of AuROC with some biomarker combinations with the greatest potential. It can be seen that adding IP-10 and / or TRAIL to the CRP increased the AuROC from 0.81 (0.76-0.86, 95% CI) to 0.87 (95% CI 0.83-0.91), p = ns. However, whether it is the addition of IP-10 or TRAIL, AuROC is similar. Adding CRP to HNL slightly affects AuROC of HNL alone (0.91, 0.87-0.95 vs. 0.92, 0.88-0.96 95% CI), while adding IP-10 and TRAIL to HNL significantly increases AuROC (0.98, 0.97-0.99 95% CI) ), P = 0.01. The latter combination of AuROC is the same as AuROC in the model that contains all 10 biomarkers. However, when B-HNL was removed from the model, ie, only plasma / serum biomarkers were included, AuROC was significantly reduced (0.98, 0.97-0.99 vs. 0.90, 0.86-0.94, 95% CI), p = 0.003.

discuss

In our previous reports, we showed that in distinguishing the causes of bacteria or viruses from acute infections, HNL in the serum (Yu Z, Jing H, Hongtao P, Furong J, Yuting J, Xu et al. Distinction between bacterial and viral infections by measurement of human neutrophil Lipocalin (HNL) and the impact of anti body selection. J Immunol Methods 2016 2016 May; 432: 82-6; Xu SY, Pauksen K, Venge P. Serum measurement measures of human neutrophil NLlipocaliin Acute bacterial and viral infections. Scand J Clin Lab Invest 1995; 55 (2): 125-31; and Bjorkqvist M, Kallman J, Fjaertoft G, Xu S, Venge P, Schollin J. Human Human Neutrophil Lipocalin: Normally strong levels a marker for invasive infection in the new born. Acta Paediatr 2004 April; 93 (4): 534-9.), or HNL (Venge P, Hakansson LD, Garwicz D, Peterson C, Xu S, measured after activating whole blood) Pauksen, K. Human, Neutrophil, Lipocalin, MLP-activated, whole blood as a differentiating means of difference between distinguishing and bacterial and viral infections. J Immunol Methods 2015 May 20. 20. Venge P, Douhan-Hakansson L, Garwicz D, Peterson C, Xu s, Pauksen, K.Humana, and vocabulary. Distinguish between Acute Bacterial and Viral Infections. Clin Vaccine Immunol 2015 (September; 22 (9): 1025-32) is better than any other inhibited biomarker, such as procalcitonin in plasma or CD64 expression on neutrophils . These data make HNL a very attractive candidate for future acute infection management to avoid antibiotic abuse (Dupuy AM, Philippart F, Pean Y, Lasocki S, Charles PE, Chalumeau Met et al. Role of biomarkers therapy: an expert panel review: I-currently available biomarkers for clinical applications. Ann Intensive Care 2013; 3 (1): 22; Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, SupraraditN Antibiotic resistance-the need for global solutions. Lancet Infect Dis. 2013 (December; 13 (12): 1057-98). However, the most widely used biomarker in the management of acute infection is CRP, and CRP is indeed a valuable tool in the management of patients with acute infection (Hopstaken RM, Muris Jw, Knottnerus JA, Kester AD, Rinkens PE, Dinant GJ .Contributions of symptoms, signs, erythrocytesedimentation rate, and C-reactive protein protein diagnostics of pneumonia, a lower-level initiative, reduction, infection. BrJ Gen Practitioner 2003; May (53): 358-64.). However, it is also well known that CRP will respond to almost any process involving inflammation in the body, which makes CRP very specific. In our Bio-X study, CRP is known to judges and is used to classify patients into those that may have a bacterial or viral cause of infection. In this study, we evaluated an even larger group of potential candidate biomarkers to distinguish bacterial or viral causes of acute infection. The goal is to achieve the best possible identification of the cause of infection. It is therefore of great interest that the combination of our whole blood assay in which activated blood neutrophils release their specific protein HNL with several other biomarkers in serum makes this distinction almost absolute as we achieve The area under the ROC curve is close to 100%. This is true whether we evaluate our entire cohort of confirmed infections or limit the assessment to those patients presenting with respiratory disease (data not shown). This level of differentiation has been achieved only in our previous studies on the determination of serum HNL (references above). However, as mentioned earlier, serum measurements place quite stringent requirements on the end user, as the results depend on the coagulation length and ambient temperature. The biomarkers that appear to complement B-HNL most strongly are IP-10 (interferon gamma-induced protein 10 kDa) and TRAIL (TNF-related apoptosis-inducing ligand), and in fact, these biomarkers have recently been shown Is an attractive discriminator in these diagnoses, and especially when combined with CRP (Oved, K, Cohen, Boico, O, Navon, R, Friedman, T, Etshtein, L, et al. A novell host-proteome signature for distinguishinguishing bacterial and viral infections. PLoS One 2015; 10 (3): e0120012.). As shown in this study, both IP-10 and TRAIL have diagnostic potential as separate biomarkers and are superior to any other biomarker except B-HNL. Therefore, we evaluated the combination of these biomarkers along with many other candidate biomarkers. Disappointingly, we cannot repeat the results of the recently shown studies, because the combination of the three biomarkers CRP, IP-10 and TRAIL did not perform significantly better than any of the three markers alone. It was not until we combined the results of the last two biomarkers with those of B-HNL that we discovered their true potential. B-HNL is a reflection of human bacterial infections, and IP-10 and TRAIL also respond to viral infections. In fact, during bacterial infections, the concentration of TRAIL was even lower than that seen in healthy non-infected controls. Therefore, the addition of specific bacterial biomarkers to two biomarkers with a viral profile looks very successful.

Other biomarkers tested as individual biomarkers have not shown any guarantee as a powerful diagnostic tool to distinguish bacterial and viral infections. As discussed earlier, although many publications advocate PCT as a tool to manage antibiotic treatment for such patients (Muller B, Becker KL. Procalcitonin: how to be a camera, and a mediator of swepsie. SwissMed Wkly 2001 2001 October 20; 131 (41-42): 595-602; Meili, M, Muller, B, Kulkarni, P, Schutz, P. Management, with patients inspiring, infections, primary care: procalcitonin, C-reactive, protein, orboth? Expert, Rev, Respir, Med, 2015, October, 2015 (5): 587-601; Drozdov D, Dusemund F, Muller B, Albrich WC. Efficacy and Safety of Procalcitonin-Guided Antibiotic Therapy In Lower Respiratory Tract Infections. Antibiotics (Basel) 2013; 2 (1): 1-10) However, PCT does not play a significant role in diagnosing the bacterial and viral causes of acute infections (Ip, M, Rainer, TH, Lee, N, Chan, C, Chau, SS, Leung, et al. Value of serum procalcitonin, neopterin, and C-reactive protein indifferentiating bacteria from viral etetologies Patients presenting with lower respiratory tract infections. Diagn Microbiol Infect 2007 2007 October; 59 (2): 131-6; Toikka P, Irjala K, Juven T, Virkki R, Mertsola J, Leinonen M Met et al. Serum Cprocalciton protein and interleukin-6 for distinguishing and bacterial and viral and pneumonia in children. Pediatr Infect Dis Dis J2000 2000 July; 19 (7): 598-602; Le BJ, Hausfater P, Chenevier-Gobeaux C, Blanc FX, Benjoar C M, Ficko al. Diagnostic accuracy of C-reactive protein and procedural citizenship-spectrum community-acquired community-acquisition consultation department department and having a systematic thoracic CT scan. (Crit, Care 2015; 19: 366). Maybe our patients are less severe than many of these studies, but our previous results on sepsis also show that the diagnosis of HNL is better than PCT (Martensson J, Bell M, Xu S, Bottai M, Ravn B, Venge Pet et al .Association of plasmaneutrophil gelatinase-associated Lipocalin (NGAL) with epsis and acute kidney disease function. Biomarkers 2013 June; 18 (4): 349-56.). Similarly, the protein HBP (azuricidin) secreted from the primary granules and secretory vesicles of neutrophils and proposed for the diagnosis and monitoring of sepsis (Linder, A, Christensson, Herwald, Bjorck, L, Akesson, P Heparin-binding protein: an early marker of circularity failures. Clin Infect Dis (2009 October 1; 49 (7): 1044-50) does not have the ability to distinguish between bacterial and viral infections. In either of our logistic regression analyses, HBP also did not show a significant discriminator for this distinction. Calprotectin is a well-known biomarker for inflammatory bowel disease, and measurement of this protein in stool is incorporated into management guidelines. However, in some diseases (including infections), calprotectin derived from isolated neutrophils has also been analyzed in serum / plasma with variable success (Jonsson N, Nilsen T, Gille-Johnson P, Bell, M, Martling, CR, Larsson, et al. Calprotectin, as well as biomarkers of bacterial infections, critically ill patients: an explorery assets. Crit CareCare Resusc 2017 (September; 19 (3): 205-13). Our results show the moderate but significant ability of calprotectin to distinguish between bacterial and viral acute infections. This ability is most apparent when we exclude CRP from calculations in our logistic regression analysis. As shown, calprotectin and CRP are highly correlated, which may explain these results.

Thymidine kinase (TK1) is derived from all dividing cells and is used to monitor cancer cell growth (Wang N, He Q, Skog S, Eriksson S, Tribukait B. Investigation on cell proliferation with a new antibody against thymidine kinase 1. Anal Cell Pathol 2001; 23 (1): 11-9), but it has also been shown to increase in viral infections in previous reports (Gronowitz JS,

CFR, Diderholm H, Hagberg H, Petterson U. Application of an in vitro assay for serum thymidine kinase: Results on viral disease and malignancies in humans. Int J Cancer 1984; 33: 5-12). As a single biomarker, TK1 does not have the ability to distinguish between bacterial and viral infections. However, in a logistic regression analysis that only analyzed plasma / serum biomarkers, TK1 contributed significantly to this distinction. Plasma HNL is shown in this report for comparison with B-HNL. Therefore, plasma HNL is significantly inferior to B-HNL. However, in the absence of B-HNL in logistic regression analysis, the contribution of plasma HNL was significant. In fact, our previous research on sepsis showed that plasma HNL is a better diagnostic biomarker than CRP and PCT in distinguishing SIRS (systemic inflammatory response syndrome) from sepsis (Martensson J , Bell M, Xu S, Bottai M, Ravn B, Venge P et al. Association of plasma neutrophil gelatinase-associated lipocalin (NGAL) with sepsis and acute kidney dysfunction. Biomarkers 2013 June; 18 (4): 349-56).

In our previous studies in China, it was shown that serum HNL is better than CRP (Yu Z, Jing H, Hong Tao P, Furong J, Yuting J, Xu et al. Distinction between bacterial and viral infections by serum measures measurement of human human neutrophil lipocalin ( HNL) and the impact of antibody selection. J Immunol Methods 2016 (May: 432: 82-6). In this study, CRP was not biased as it may be in our current Bio-X study. However, even though the clinical performance of serum-based HNL is very powerful, the disadvantages of serum measurement are obvious, as the pre-analysis step of preparing serum requires several hours, and the measurement of HNL requires additional time. Therefore, for emergencies, point-of-care applications are the most attractive. In this report, we used a 20-minute protocol for the activation of whole blood. However, our previous experiments on the kinetics of HNL release from blood neutrophils (Venge, P, Hakansson, LD, Garwicz, D, Peterson, C, Xu, S, Pauksen, K. Human, Neutrophil, Lipocalin, Inf, MLP-activated, Whole, Blood, As, Diagnostic, Meme The method can be shortened to 3 to 5 minutes, which should meet the requirements of fast and accurate decision-making whether to use antibiotics to treat infected patients clinically.

HNL is a fairly complex molecule with several sources and several names, namely NGAL (neutrophil gelatinase-associated apolipoprotein) or apolipoprotein 2 (Xu S, Venge P. Lipocalins as biochemical markers of disease Biochim Biophys Acta 2000 October 18; 1482 (1-2): 298-307). In blood, the main source is blood neutrophils, in which the cells HNL exist in a pre-formed state (Cai, L, Rubin, J, Han, W, Venge, P, Xu. The Origin of Multiple Multiple Molecular Forms, Urine, of HNL / NGAL .Clin, J, Soc, Nephrol, September 9. (25); XuS, CaiL, ZhaoL, Douhan-HakanssonL, Kristjansson G, PauksenKetetal.Tissue localization, and the establishment of the awareness of the greater awareness Phospholipase B-precursor (PLB-P). J Immunol Methods 2010 (February 28; 353 (1-2): 71-7.). However, HNL production may be induced in epithelial cells, such as in renal tubular cells of patients with acute kidney injury (AKI) (Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C et et al. Neutrophil gelatinase-associated Lipocalin (NGAL) as a biomarker for acute care after surgery.Lancet 2005 April 2; 365 (9466): 1231-8; Cai L, Borowiec, J, Xu, S, Han, W, Ass. urine levels of HNL / NGAL in patients undergoing cardiac surgery and the impact of anti-body configuration on their clinical performance. Clin Chim Acta 2009 2009; 403 (1-2): 121-5.). If measured in serum or plasma, this HNL production can potentially affect the diagnostic performance of HNL. HNL derived from neutrophils is largely released as a dimer, while HNL released from epithelial cells is a monomeric form. This means that for best diagnostic performance, the assay should be able to distinguish between these forms. As shown recently, when measuring in serum or plasma, this distinction can be achieved through the measurement configuration of certain antibody pairs (Yu Z, Jing H, Hongtao P, Furong J, Yuting J, Xu et al. Distinction between bacterial and viral infections by measures of human neurotrophil and lipocalin (HNL) and impact of anti body selection. J Immunol Methods 2016 2016; May: 432: 82-6.). In this report, we circumvent these potential confounders by directly measuring substances released from circulating neutrophils.

The limitation of our study is the accuracy of the diagnosis, which is to distinguish the bacterial or viral cause of respiratory infections. As we all know, this distinction is very difficult (Hopstaken RM, Muris JW, Knottnerus JA, Kester AD, Rinkens PE, Dinant GJ.Contributions of symptoms, signs, erythrocyte, sedimentation rate, and C-reactive protein protein to reduce the acute diagnosis. Lower Respiratory Infection. Br J Gen Pract 2003 May; 53 (490): 358-64.), but it is very important if you want to study any biomarkers for these purposes. In this report, we strive to make accurate diagnoses through microbiological and serological tests of various body fluids, but through more extensive testing, we may have reached a higher diagnostic accuracy of HNL >> 0.9. However, it seems very important in our research that the diagnostic efficacy of HNL is still an excellent biomarker in direct comparison with other potential candidate biomarkers, but by adding, for example, IP-10 and / or TRAIL biomarkers can further increase the diagnostic efficacy of HNL. The strength of our research is that biomarkers judge each other, and the limitations of diagnosis have the same effect on biomarkers. In recent studies, it was found that the algorithm of three biomarkers is better than HNL (Ashkenazi-HoffnungL, Oved K, Navon R, Friedman T, Boico O, Paz M. et al. A-host- protein signature is superior to other biomarkers for differentiating between bacterial and viral diseases in patients with respiratory infections and sources without a source: a prospective observational study.Eur J Clin Microbiol Infectl Disorder 2018. The reported results for plasma HNL are consistent with our own findings in this report. Unfortunately, however, the study failed to follow the instructions for serum preparation, which made the comparison of serum HNL as a discriminator severely ineffective.

We conclude that the diagnostic performance of whole blood-activated HNL is superior to any other known biomarker in the differentiation of acute infections caused by bacteria or viruses. However, the addition of IP-10 and / or TRAIL to the diagnostic algorithm further improves the diagnostic performance of B-HNL, where a bacterial infection and a viral infection are almost completely distinguished. A fast and accurate HNL analysis that reflects the body's bacterial challenges, along with one or two biomarkers that reflect viral infections, can be our ideal combination of diagnostic markers to fight antibiotic abuse.

Claims

Device for determining whether an infection in a subject is caused by a bacterium or a virus, comprising a module for absorbing test blood samples and reagent storage, wherein the absorption module includes a first for preactivating the test blood samples A reagent, and a second reagent for measuring the amount of the HNL biomarker in the pre-activated test blood sample;

Wherein a module for absorbing a test blood sample is a syringe or a needle, which is arranged to collect whole blood from a subject and apply the blood to a substrate; and a module for signal detection and result display;

Wherein the module displays a signal indicating whether the amount of the measured HNL biomarker has changed compared to a normal control.
The device according to claim 1, wherein said signal provides a numerical reading of a percentage above baseline, which is representative of the presence of a bacterial infection in the test sample.
A device according to claim 1 or 2, wherein said first reagent comprises an N-formylated peptide, such as N-formylmethionyl-leucine-phenylalanine (fMLP).
A device according to any one of the preceding claims, wherein the second reagent comprises a biomarker-specific capture antibody or an antigen-binding fragment thereof.
Device according to any one of the preceding claims, comprising at least one other reagent for measuring the amount of other biomarkers selected from the group consisting of azurenicin, calprotectin, PMN-CD64, CRP, IP -10, PCT, TK1, CEACAM8 and TRAIL.
An array for determining whether an infection in a subject is caused by a bacterium or a virus, the array comprising antibodies or antigens that bind to two or more biomarkers in a sample obtained from the subject A fragment in which at least one biomarker is HNL obtained from a whole blood sample after pre-activation with an N-formylated peptide, and at least one biomarker is not HNL.
The array according to claim 6, wherein said N-formylated peptide is N-formylmethionyl-leucine-phenylalanine (fMLP).
An array according to claim 6 or 7, comprising an antibody or antigen-binding fragment thereof that binds a biomarker other than NHL, said biomarker other than HNL being characterized by an independent ability to distinguish bacterial and viral infections.
The array according to claim 8, wherein said other biomarkers are selected from the group consisting of azurin, calprotectin, PMN-CD64, CRP, IP-10, PCT, TK1, CEACAM8, and TRAIL.
The array according to claim 9, wherein said non-HNL biomarker is IP-10 and / or TRAIL.
A method for determining whether an infection in a subject is caused by a bacterium or a virus, comprising measuring the amount of two or more biomarkers in a whole blood sample obtained from the subject, and measuring each Whether the amount of the biomarker is changed compared to the normal control, wherein at least one biomarker is HNL obtained from a whole blood sample after being pre-activated with an N-formylated peptide, and at least one biomarker Not HNL.
The method according to claim 11, wherein said N-formylated peptide is N-formylmethionyl-leucine-phenylalanine (fMLP).
A method according to claim 11 or 12, wherein said non-HNL biomarker is characterized by an independent ability to distinguish between bacterial and viral infections.
The method according to claim 13, wherein said non-HNL biomarker is selected from the group consisting of azurin, calprotectin, PMN-CD64, CRP, IP-10, PCT, TK1, CEACAM8, and TRAIL.
The method according to claim 14, wherein said non-HNL biomarker is IP-10 and / or TRAIL.
A method according to any one of claims 11-15, wherein said biomarker measurement comprises the binding of a capture antibody or antigen-binding fragment thereof specific for each biomarker, preferably the binding of a monoclonal antibody.
A method according to any one of claims 11-16, wherein said biomarker measurement incorporates a detectably labeled further antibody.
A method according to any one of claims 11-17, wherein the amount of biomarkers in said sample is measured using biochip array technology.
A method of treating a bacterial infection in a subject suspected of having a bacterial or viral infection, the method comprising: (a) measuring an HNL biomarker in a whole blood sample obtained from the subject and at least one other than HNL (B) if the measured amount is changed compared to a normal control, diagnose that the subject has a bacterial infection; (c) select a subject diagnosed as having a bacterial infection in step (b) For treatment; and (d) administering to said subject an appropriate antibacterial therapy in which a whole blood sample has been pre-activated with an N-formylated peptide.
A method of detecting the effectiveness of an antibacterial treatment, said method comprising measuring the amount of a HNL biomarker and at least one non-HNL biomarker in a whole blood sample obtained from a subject; wherein N-formylation has been used Peptide pre-activated whole blood samples.
The method according to claim 19 or 20, wherein the N-formylated peptide is N-formylmethionyl-leucine-phenylalanine (fMLP).
A method according to any one of claims 19 to 21, comprising binding an HNL biomarker and at least one antibody or antigen-binding fragment thereof that is not a HNL biomarker
The method according to any one of claims 19 to 22, wherein the non-HNL biomarker is selected from the group consisting of azurin, calprotectin, PMN-CD64, CRP, IP-10, PCT, TK1, CEACAM8 and TRAIL .
A combination of a HNL biomarker and at least one non-HNL biomarker, which is used in the diagnosis of a bacterial infection.
A combination of a HNL biomarker and at least one non-HNL biomarker, which is used in the diagnosis of a viral infection.
A combination according to claim 24 or 25, wherein the HNL biomarker is obtained from a whole blood sample.
The combination according to claim 26, wherein the sample has been pre-activated using an N-formylated peptide, such as N-formylmethionyl-leucine-phenylalanine (fMLP).
The combination according to any one of claims 24-27, wherein the biomarker other than HNL is IP10 and / or TRAIL.