Biomarkers for the diagnosis of respiratory tract infections

Abstract

The present invention relates to a method for diagnosing a respiratory tract infection in a subject, comprising determining the level of high mobility group protein B1 (HMGB1) in a sample from the subject, and / or determining the level of a histone protein in a sample from the subject, and / or determining the level of insulin-like growth factor binding protein, acid-labile subunit (IGFALS) in a sample from the subject, wherein if the level of IGFALS is below a predetermined threshold level and / or the level of the histone protein is above a predetermined threshold level, the subject is diagnosed with a bacterial respiratory tract infection, and / or if the level of HMGB1 is above a predetermined threshold level, the subject is diagnosed with a respiratory tract infection.

Description

Technical Field

[0001] The present invention relates to markers and methods for the diagnosis and differential diagnosis of respiratory tract infections (RTIs), particularly lower respiratory tract infections (LRTIs) such as pneumonia.

Background Art

[0002] A serious problem in clinical practice is the diagnosis of patients with disorders having overlapping symptoms or unspecified disease-related features. Furthermore, rapid patient management and accurate treatment initiation are desired, despite the increasing overload on clinical settings such as the emergency department (ED) or primary care.

[0003] The symptoms presented can vary from patient to patient depending on the severity of the disease and overlap with diseases or disorders caused by non-infectious diseases such as chronic obstructive pulmonary disease (COPD), acute coronary syndrome (ACS), asthma, heart failure, pulmonary embolism, tumors, particularly lung tumors, edema, idiopathic pulmonary syndrome (IPS) or atrial fibrillation. Thus, there are obvious problems in the triage of patients with symptoms of lower respiratory tract infections (LRTIs) such as shortness of breath, weakness, cough, fatigue, confusion, pleuritic chest pain or fever (Girish et al., Med Clin N Am 95 (2011) 1143-1161). Many underlying diseases can rapidly shift patients into a critical and life-threatening condition such as sepsis, leaving no room for time-consuming diagnostic procedures or evaluation systems that take hours or even days overall. This is a serious problem for medical staff, and there is a strong medical need for rapid and individualized treatment and medical decisions for such patients.

[0004] LRTIs such as pneumonia, acute bronchitis and bronchiolitis are caused by pathogenic infectious agents and require pathogen-specific antibacterial or antiparasitic treatment and protective isolation of the patient to avoid reinfection or further spread.

[0005] Among lower respiratory tract infections, pneumonia is a serious health problem and one of the leading causes of mortality and morbidity worldwide. It is caused by a wide range of bacteria, viruses, and, rarely, fungal pathogens or other parasites (Raeven et al., BMC Infectious Diseases (2016) 16:299; Li et al., Microbes and Infection (2020) 22(2):80-85). The financial burden is high, with hospitalization and patient length of stay being major cost factors.

[0006] The most common form of pneumonia is community-acquired pneumonia (CAP), which is a significant cause of death, primarily in children under 5 years of age, adults over 65 years of age, or patients with comorbidities. The incidence of CAP is expected to increase in the next decade due to the aging population and the subsequent increase in comorbidities. Therefore, the main risk factors for CAP may be major pathological conditions such as chronic disorders like asthma or COPD, chronic heart failure, immune system dysfunction, or the use of medications such as proton pump inhibitors, and respiratory stressors such as air pollution or smoking. Depending on the healthcare system and the patient's circumstances, mortality rates range from less than 1% to as high as 50% (Girish et al., loc.cit., Cillioniz et al., Int.J.Mol.Sci.(2016)17:2120, Savvateeva et al., BioMed Res Int(2019)1701276).

[0007] Pneumonia can be caused by a diversity of pathogens, which can lead to slightly different symptoms, and their spread can vary regionally and seasonally (Ho et al., Infect Dis Clin N Am(2019)33:1087-1103). The main factors for reducing patient mortality are rapid and effective pathogen-related treatment with appropriate antibiotics, and other adjunctive medical applications such as oxygenation or mechanical ventilation.

[0008] Disease-causing bacteria can be divided into typical and atypical groups.

[0009] The most common pathogens causing CAP are extracellular bacteria, including Streptococcus pneumoniae, Hemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA).

[0010] Atypical bacteria, the second most frequent class of CAP pathogens, amplify intracellularly in human cells and typically lack a typical bacterial cell wall. Representative examples of atypical bacteria include Legionella pneumophila, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, and Coxilla burnetii. The proportion of atypical pneumonia is often reported in 5–30% of cases, with 30% being mixed infections, and appears to be more common in patients admitted to the ICU (up to 20%). Patients infected with atypical bacteria often present with subacute symptoms such as a dry cough, low-grade fever, normal WBCs, and frequently associated extrapulmonary symptoms.

[0011] The prevalence of CAP infections caused by respiratory viruses varies from 2% to 30% of cases. Several studies have found that, in addition to pandemic or epidemic events, the frequency of viral-induced CAP is higher than previously thought, with a significant increase in cases. The most common viral particles are influenza virus, respiratory syncytial virus (RSV), coronavirus, rhinovirus, parainfluenza virus, human metapneumovirus, varicella, hantavirus, and adenovirus. One of the most problematic complications in viral infections is co-infection with other pathogens, which can frequently advance the patient's condition to a more serious state. The interaction between viral particles and bacteria is not fully understood, but it is likely that the interaction enhances bacterial pathogenicity, resulting in a worsening of clinical outcomes (Girish et al., loc.cit., Cillioniz et al., loc.cit., Savvateeva et al., loc.cit.), and further increases the burden on the immune system due to the increased load of pathogenic particles.

[0012] Identifying the underlying pathogen, or at least eliminating a specific pathogen, is key to accurate diagnosis, treatment decisions, and containing the spread of infection within a population.

[0013] An accurate diagnosis of pneumonia with an underlying pathogenic cause cannot be made based solely on the evaluation of a patient's symptoms and signs. Additional clinical tests or imaging can support the diagnosis of pneumonia, but they cannot distinguish between different pathogens. Imaging methods such as X-ray, ultrasound, or CT scans have drawbacks due to their limited availability, inter-observer variability in interpretation of results, and differences in the experience of radiologists or ultrasound technicians.

[0014] There is no standard serological test for all patients with RTIs, especially those with CAP, and certainly no rapid and simple clinical means to distinguish between intracellular and classical pathogens.

[0015] Direct microbiological identification of pathogens from sputum, bronchoalveolar lavage fluid, or blood is highly dependent on the quality of the obtained sample material and can take several hours to several days.

[0016] The current IDSA / ATS guidelines 2019 recommend sputum or blood cultures only for patients suspected of having resistant pathogens, and recommend empirical administration of antibiotics, but do not perform regular measurement of biomarkers or urinary antigen tests, nor follow-up radiographs (Metlay et al., Am J Respir Crit Care Med (2019) 200(7):e45-e67).

[0017] In the aforementioned cultures, the pathogen identification rate using CAP is less than 50%. One reason for this is that it is difficult to grow atypical bacteria in standard culture media because they are not easily identified due to their presence within cells and / or the absence of a typical cell wall.

[0018] Another method is identification using molecular biological techniques such as polymerase chain reaction (PCR), which is commonly used to identify infections caused by viruses. Here, it is important to know that the upper respiratory tract in healthy humans is often colonized with potentially pathogenic bacteria such as Pseudomonas aeruginosa. If the sample material is not taken from the lower respiratory tract, or contains colonized "harmless" microorganisms from the upper respiratory tract, the system tends to produce false positive results, which will result in unnecessary treatment (Savvateeva et al, loc. cit.). In contrast, the host response biomarkers detected by the approach described here are altered only when the patient's immune system responds to the infection, thus preventing false positives.

[0019] While current strategies using blood host biomarkers exist, the clinical use of single biomarkers is limited. Currently, there are no biomarker-based algorithms for establishing the pathogenesis of CAP. There is no known single biomarker that can reliably distinguish between different pathogens for making clinical decisions regarding treatment. For example, procalcitonin is a well-known biomarker for the diagnosis and severity of bacterial infections. Other published biomarkers, such as TRAIL (WO2016 / 059636 A1) or MxA (WO2014 / 137858 A1), have been used to detect viral causes. Nevertheless, the feasibility of known single biomarkers is limited to the identification or differentiation of viruses versus bacteria, particularly subdifferentiation between typical and atypical bacterial infections (Kruger et al., Respiratory Research (2009) 10:65).

[0020] Complex severity assessment scores such as the Pneumonia Severity Index (PSI), the CURB-65 criteria (a modified version of the British Thoracic Society assessment system), or SMART-COP are used to stratify patients according to their mortality risk and to guide medical care, but they are time-consuming and difficult to calculate.

[0021] In many cases, the causative pathogen remains unknown, so patient treatment often involves a time-consuming trial-and-error approach.

[0022] In current clinical practice, antibiotics are the first-line treatment for pneumonia. However, they are ineffective or not indicated for parasitic or viral infections; nevertheless, approximately 41% of all antibiotic use is related to respiratory conditions (Ho et al., loc. cit.).

[0023] Due to the need for therapeutic intervention within the first few hours of a patient's admission to the ED, or within a limited time slot in a typical physician's setting, treatment guidelines recommend empirical procedures (Thibodeau et al., American Family Physician (2004) 69(7):1699-1706).

[0024] This prescribing practice significantly increases antibiotic consumption and leads to overtreatment with a wide range of antibiotics, which can result in additional costs and drug side effects. Studies have shown that using antibiotics in non-infectious patients with respiratory infection-like symptoms, such as those with acute heart failure and dyspnea, increases the risk of side effects and poor outcomes (Girish et al., loc.cit., Maisel et al., Eur J Heart Fail. (2012) 14:278-286).

[0025] Another consequence is the emergence of multidrug-resistant bacteria, which significantly limits the effectiveness of new antibiotics.

[0026] The increasing antibiotic resistance of S. pneumoniae, the most common cause of CAP, to several antibiotics including cephalosporins, macrolides, and fluoroquinolones, could become a global problem. Within the last 20 years, 20% to 30% of pneumococcal infection cases worldwide have been resistant to three or more classes of antibiotics.

[0027] Based on the appearance within cells and the absence of a cell wall, the treatment of atypical pneumonia pathogens requires different types of antibiotics compared to the bacterial pathogens of typical pneumonia. Antibiotics for atypical bacteria must be able to penetrate human cells and must not be directed against the types of cell walls of typical bacteria. Thus, standard beta-lactams are not effective, and other antibacterial agents such as erythromycin and sometimes tetracycline have been conventionally used for atypical infections. Macrolide antibiotics are more tolerant than erythromycin. Doxycycline has fewer gastrointestinal side effects and is a cheaper alternative. Fluoroquinolones have excellent bioavailability that allows once-daily dosing (Thibodeau et al., loc.cit.).

[0028] Effective patient management helps reduce costs, but rapid and appropriate decisions are required regarding the clinical setting (ambulatory patients, hospitalization in a hospital or intensive care unit (ICU)) and the choice of appropriate treatment.

[0029] One solution to the current problem is the use of advanced technologies with rapid diagnosis that enables treatment targeting the causative agent (Ho et al., loc.cit.).

[0030] One of the most important clinical needs is the early detection of the most causative pathogen groups, the distinction between bacterial and viral pathogens in RTI, and also the early and rapid sub-differentiation of atypical and typical bacteria for effective patient management and accurate treatment, and the avoidance of the currently used empirical "trial and error" method is emphasized.

[0031] Therefore, the rapid and reliable detection of host biomarkers, particularly blood biomarkers, is a solution for the identification and differential diagnosis of pathogens causing infection-based disorders and / or the exclusion of non-infectious disorders with overlapping or similar symptoms. Rapid tests or automated assays are easy to handle and can support patient triage decisions and initial accurate and individualized treatment.

[0032] The clear medical advantages of these procedures are the reduction of the generally common antibiotic consumption, or at least the avoidance of its unnecessary application, which will also reduce the generation of further antibiotic resistance. Another advantage is the possibility of making rapid medical decisions and initiating intervention with appropriate pharmaceuticals. Another group of clinically relevant pathogens are viruses that tend to cause pneumonia. In crisis situations such as epidemics or pandemics, such as the previously unknown pathogenic variants like the coronavirus-related severe acute respiratory syndrome-CoV (SARS-CoV) in 2002, Middle East respiratory syndrome-CoV (MERS-CoV) in 2012 or SARS-CoV-2 / COVID-19 (M. Ashour et al. Pathogens 2020, 9(3)), there are a large number of patients in the healthcare system who are potentially at risk of being infected with the new pathogen, but there may also be patients suffering from non-infection-related serious disorders with similar symptoms and co-infections by bacteria. In that case, the rapid triage of patients is the most important means to determine the next clinical steps and to provide as much appropriate medical support as possible to reduce the risk of complications such as dyspnea, ARDS, sepsis, (septic) shock, etc. or the mortality of the patients.

Summary of the Invention

[0033] The present invention is a method for diagnosing a respiratory tract infection in a subject, comprising determining the level of high-mobility group protein B1 (HMGB1) in a sample from the subject, and / or determining the level of a histone protein preferably selected from histone H4, histone H2A, histone H2B, histone H3 and histone H1 in a sample from the subject, and / or determining the level of insulin-like growth factor binding protein, acid-labile subunit (IGFALS) in a sample from the subject, where if the level of HMGB1 exceeds a predetermined threshold level, the subject is diagnosed with a respiratory tract infection, and / or The present invention relates to a method for diagnosing a subject with a bacterial respiratory infection if the level of IGFALS is below a predetermined threshold level and / or the level of histone proteins is above a predetermined threshold.

[0034] In other words, in one aspect, the present invention is a method for diagnosing a respiratory tract infection in a subject, In the sample from the subject, (i) Level of high-mobility protein B1 (HMGB1), (ii) the level of histone proteins, and / or (iii) Determining the level of insulin-like growth factor binding protein, acid-unstable subunit (IGFALS), If the HMGB1 level exceeds a predetermined threshold level, the subject is diagnosed with a respiratory tract infection and / or The present invention relates to a method for diagnosing a subject with a bacterial respiratory infection if the level of IGFALS is below a predetermined threshold level and / or the level of histone proteins is above a predetermined threshold.

[0035] In one aspect, the present invention provides a method for diagnosing a respiratory tract infection (RTI) in a subject suspected of having a respiratory tract infection (RTI), This includes determining the level of high-mobility protein B1 (HMGB1) in a sample from the subject, This invention relates to a method for diagnosing a patient with a respiratory tract infection when the level of HMGB1 exceeds a predetermined threshold level.

[0036] In another aspect, the present invention provides a method for differential diagnosis of airway diseases in a subject, comprising determining the level of high-mobility group protein B1 (HMGB1) in a sample from the subject, This invention relates to a method for diagnosing a patient with a respiratory tract infection (RTI) when HMGB1 levels exceed a predetermined threshold level.

[0037] In this context, a subject may have one or more symptoms of a lower respiratory tract infection (LRTI), for example, one subject may exhibit one or more symptoms selected from shortness of breath, weakness, fever, sputum formation, cough, fatigue, wheezing, chest discomfort or pain, rapid breathing, dyspnea, congestion, common cold, and sore throat. The subject may also have a cough, as well as one or more symptoms selected from sputum formation, shortness of breath, wheezing, and chest discomfort or pain. This method is particularly useful for distinguishing subjects with overlapping symptoms between an RTI and other lower respiratory tract diseases ("RTI mimics").

[0038] In the context of the present invention, an RTI may be, for example, a lower respiratory tract infection (LRTI) selected from the group of acute bronchitis, pneumonia, and bronchiolitis. An LRTI may be an atypical bacterial infection, particularly a bacterial or viral infection such as atypical bacterial pneumonia. The method of the present invention is particularly useful for distinguishing between subjects with atypical bacterial LRTIs, such as atypical bacterial pneumonia, and healthy subjects or subjects with symptoms similar to those of an LRTI, such as COPD patients; see below.

[0039] In this specification, viral infections may be selected from the group consisting of, for example, influenza A, influenza B, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus infection 2019 (COVID-19).

[0040] In the context of the present invention, the levels of one or more further markers selected from the group consisting of procalcitonin (PCT), proadrenomedjulin (proADM) or fragments thereof, histone proteins, serum amyloid A1 (SAA1), fetuin-A (FetA), insulin-like growth factor-binding protein, acid-unstable subunits (IGFALS), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), and CXC-motif chemokine 10 (CXCL10) may be determined in a sample from the subject to improve the diagnosis. In particular, the level of MR-proADM may be determined in a sample from the subject, and in particular, the level of MR-proADM may indicate the severity of the infection.

[0041] In addition to HMGB1, the level of one or more histone proteins selected from histone H2B, histone H4, histone H2A, histone H3, and histone H1 may be determined in the sample from the subject, preferably the level of H4. If the level of at least one histone protein, preferably H4, exceeds a predetermined threshold level, the subject may be diagnosed with atypical bacterial pneumonia. On the other hand, if the level of at least one histone protein, preferably H4, exceeds a first predetermined threshold level but is below a second predetermined threshold level, the subject may be diagnosed with typical bacterial pneumonia.

[0042] In addition, the FetA level can be determined in the sample from the subject. If the FetA level is below a predetermined threshold level, the subject may be diagnosed with bacterial LRTI. On the other hand, if the FetA level exceeds a predetermined threshold level, the subject may be diagnosed with viral LRTI.

[0043] Furthermore, in addition to HMGB1, the level of IGFALS can be determined in the sample from the subject. If the level of IGFALS is below a predetermined threshold level, the subject may be diagnosed as a bacterial LRTI. On the other hand, if the level of IGFALS exceeds a predetermined threshold level, the subject may be diagnosed as a viral LRTI.

[0044] Furthermore, the level of CXCL10 can be determined in the sample from the subject. If the level of CXCL10 exceeds a predetermined threshold level, the subject may be diagnosed with viral LRTI.

[0045] Furthermore, the TRAIL level can be determined from the sample taken from the subject. If the TRAIL level exceeds a predetermined threshold level, the subject may be diagnosed with viral LRTI.

[0046] In a further embodiment, the present invention relates to a method for diagnosing a respiratory tract infection in a subject, comprising determining the level of a histone protein, preferably selected from histone H4, histone H2A, histone H2B, histone H3 and histone H1, in a sample from the subject, and / or This includes determining the levels of insulin-like growth factor-binding protein, acid-unstable subunit (IGFALS) in the sample from the subject, This invention relates to a method for diagnosing a subject with a bacterial respiratory infection if the level of IGFALS is below a predetermined threshold level and / or the level of histone proteins is above a predetermined threshold. In particular, the histone protein may be H4.

[0047] In this specification, a typical subject is one suspected of having a bacterial respiratory infection. Such subjects may have one or more symptoms of a lower respiratory tract infection (LRTI), particularly pneumonia. Such subjects may exhibit one or more symptoms selected from shortness of breath, weakness, fever, sputum formation, cough, fatigue, wheezing, chest discomfort or pain, rapid breathing, dyspnea, congestion, rhinitis, and sore throat. In particular, such subjects may have a cough, as well as one or more symptoms selected from sputum formation, shortness of breath, wheezing, and chest discomfort or pain.

[0048] In this context, the levels of one or more additional markers selected from the group consisting of procalcitonin (PCT), proadrenomedjulin (proADM) or its fragments, histone proteins, high-mobility group protein B1 (HMGB1), serum amyloid A1 (SAA1), fetuin-A (FetA), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), and CXC motif chemokine 10 (CXCL10) may be determined in a sample from the subject. For example, the level of MR-proADM may be determined in a sample from the subject, and in particular, the level of MR-proADM indicates the severity of the infection.

[0049] In addition, the level of HMGB1 may be further determined in relation to the levels of histone proteins or IGFALS in a blood sample from the subject. If the level of IGFALS is below a predetermined threshold level, the level of histone proteins is above a predetermined threshold, and the level of HMGB1 is above a predetermined threshold level, the subject is diagnosed with a bacterial respiratory infection.

[0050] In this specification, if the level of at least one histone protein, preferably H4, exceeds a predetermined threshold level, the subject is diagnosed with atypical bacterial pneumonia. On the other hand, if the level of at least one histone protein, preferably H4, exceeds a first predetermined threshold level but is below a second predetermined threshold level, the subject may be diagnosed with typical bacterial pneumonia.

[0051] In the context of the present invention, the sample is a body fluid sample, preferably a blood sample, saliva sample, nasal swab, sweat sample, urine sample, or bronchoalveolar lavage fluid (BAL), more preferably serum, plasma, or whole blood, most preferably plasma.

[0052] The present invention also relates to an antibiotic for use in treating a bacterial respiratory infection in a subject, wherein if the subject is determined to have a bacterial respiratory infection by a method according to the present invention, the subject is treated with the antibiotic. [Brief explanation of the drawing]

[0053] [Figure 1] This shows the concentration (ng / ml) of HMGBI in blood samples from healthy donors and patients with typical bacterial pneumonia, atypical bacterial pneumonia, and viral pneumonia, as measured by ELISA. [Figure 2] This shows the relative concentrations of histone H4 in blood samples from healthy donors and patients with typical bacterial pneumonia, atypical bacterial pneumonia, and viral pneumonia, as measured by MS. [Figure 3] The relative concentrations of IGFALS in blood samples from healthy donors and patients with bacterial respiratory infections (11 pneumonia cases, 7 non-pneumonia cases), viral respiratory infections (4 pneumonia cases, 26 non-pneumonia cases), and atypical bacterial pneumonia, as measured by ELISA, are shown. [Figure 4] The relative concentrations of fetuin-A in blood samples from healthy donors, as well as from patients with bacterial respiratory infections (11 pneumonias, 7 non-pneumonias), viral respiratory infections (4 pneumonias, 26 non-pneumonias), and atypical bacterial pneumonia, as measured by a magnetic bead-based multiplex immunoassay, are shown. [Modes for carrying out the invention]

[0054] The object of the present invention is to provide markers and methods for the diagnosis and differential diagnosis of respiratory tract infections (RTIs), particularly lower respiratory tract infections (LRTIs). In particular, acute forms of RTI / LRTI are diagnosed by the methods of the present invention. As outlined above herein, the present invention is based on the remarkable discovery that the following markers can be used for the differential diagnosis of respiratory tract infections, particularly lower respiratory tract infections: histone proteins such as high mobility group protein B1 (HMGB1), histone H4, histone H2A, histone H2B, histone H3 and histone H1, insulin-like growth factor-binding protein, acid-unstable subunits (IGFALS), and fetuin-A (FetA). As is evident from the accompanying examples and drawings, these markers can be useful in this diagnosis, in particular in distinguishing RTI from diseases with similar or similar symptoms, or in distinguishing bacterial from viral RTIs, particularly LRTIs, more particularly from pneumonia, or in distinguishing typical from atypical bacterial pneumonia.

[0055] In this specification, this method is preferably used for the diagnosis of LRTI. Therefore, in all aspects and embodiments (unless otherwise specified), RTI is preferably LRTI. Pneumonia is one of the most severe forms of LRTI. Therefore, in all aspects and embodiments of the present invention, the aim is particularly to diagnose pneumonia. In the context of the present invention, RTI / LRTI / pneumonia may be of different origins, such as those caused by bacterial or viral pathogens. In the case of bacterial pneumonia, there are typical and atypical forms of pneumonia caused by different bacteria; see the description below.

[0056] Respiratory tract infections (RTIs) are infectious diseases that involve the respiratory tract. RTIs can be further classified as upper respiratory tract infections (URTIs) or lower respiratory tract infections (LRTIs).

[0057] The upper respiratory tract is generally considered to be the airway above the glottis or vocal cords. This includes the nose, sinuses, pharynx, and larynx. Typical infections of the upper respiratory tract include tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, certain types of influenza, and the common cold. Symptoms of URTI may include cough, sore throat, runny nose, nasal congestion, headache, low-grade fever, facial tightness, and sneezing.

[0058] The lower respiratory tract consists of the trachea (larynx), bronchi, bronchioles, and lungs. Lower respiratory tract infections, such as pneumonia, are generally more severe than upper respiratory tract infections. LRTIs are a leading cause of death among all infectious diseases. The two most common LRTIs are bronchitis and pneumonia. Another LRTI is bronchiolitis. Influenza or coronavirus can affect both the upper and lower respiratory tracts. Symptoms of LRTIs commonly include shortness of breath, weakness, fever, cough and fatigue up to acute respiratory distress syndrome (ARDS), organ damage, and sepsis.

[0059] Bronchitis is inflammation of the bronchi (large and medium-sized airways) in the lungs that causes coughing. Symptoms include expectoration of sputum, wheezing, shortness of breath, and chest pain. Bronchitis can be acute or chronic. Acute bronchitis usually involves a cough that lasts about three weeks. In over 90% of cases, the cause is a viral infection. These viruses can spread through the air when people cough or come into direct contact with others. Typically, these viral infections are rhinovirus, parainfluenza, coronavirus, or influenza. A small number of cases are caused by bacterial infections such as Mycoplasma pneumoniae or Bordetella pertussis.

[0060] Pneumonia is an inflammatory condition of the lungs, primarily affecting the small air sacs known as alveoli, and can be community-acquired (CAP) or hospital-acquired (HAP) infections. Typically, symptoms include several combinations of cough with sputum or dry cough, chest pain, fever, and shortness of breath. Bacteria are the most common cause of community-acquired pneumonia (CAP), with Streptococcus pneumoniae isolated in nearly 50% of cases. Other commonly isolated bacteria include Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, and Gram-negative bacilli. Drug-resistant forms of many of the above infections, including drug-resistant Streptococcus pneumoniae (DRSP) and methicillin-resistant Staphylococcus aureus (MRSA), are becoming increasingly common.

[0061] In adults, viruses account for about one-third of pneumonia cases, and in children, they account for about 15%. Common causes of viral pneumonia are influenza viruses A and B, respiratory syncytial virus (RSV), and human parainfluenza virus. Further viruses that commonly cause pneumonia include adenoviruses, metapneumoviruses, hantaviruses, certain specific forms of coronaviruses, variants / variants, or zoonotic viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 / COVID-19). SARS coronavirus causes severe acute respiratory syndrome (SARS). MERS coronavirus causes Middle East respiratory syndrome (MERS). SARS-CoV-2 causes coronavirus infection 2019 (COVID-19).

[0062] Bronchiolitis is a blockage of small airways in the lungs caused by a viral infection, and it usually occurs only in children under two years of age. Symptoms may include fever, cough, runny nose, wheezing, and difficulty breathing.

[0063] In particular, HMGB1 is useful in diagnosing RTIs (bacterial and viral), especially LRTIs, and is useful in distinguishing RTIs, especially LRTIs, from healthy individuals or patients with similar or overlapping symptoms, i.e., patients with diseases that resemble RTIs and LRTIs.

[0064] Similarly, histone proteins (including histone H4, histone H2A, histone H2B, histone H3, and histone H1) are useful markers for diagnosing RTIs (bacterial and viral), particularly LRTIs, in subjects, and for distinguishing RTIs, especially LRTIs, from healthy individuals or patients with similar or overlapping symptoms, i.e., patients with diseases resembling RTIs and LRTIs. Histones are also useful in distinguishing atypical bacterial pneumonia from typical bacterial pneumonia.

[0065] In contrast, fetuin A (FetA) is particularly useful in distinguishing between bacterial and viral RTI / LRTI infections, while IGFALS is particularly useful in distinguishing between typical bacterial pneumonia and atypical bacterial pneumonia, and between viral infections and typical bacterial infections.

[0066] Therefore, in one aspect of the present invention, the aim is to distinguish between subjects having RTI / LRTI and subjects having diseases with similar or overlapping symptoms such as acute coronary syndrome (ACS), asthma, chronic obstructive pulmonary disease (COPD), heart failure, pulmonary embolism, lung tumors, other tumors, or atrial fibrillation.

[0067] Considering their individual advantages, the markers of the present invention can be combined (as a panel of at least two, three, or four markers) to obtain a more detailed differential diagnosis and / or to direct or monitor appropriate treatment. Furthermore, the above markers can be combined individually or as a panel with additional markers or other clinical parameters to further improve the diagnosis. Such additional markers include procalcitonin (PCT), proadrenomedjulin (proADM) and its fragments, e.g., MR-proADM, mature ADM, PAMP, serum amyloid A1 (SAA1), interferon-induced GTP-binding protein Mx1 (Mx1), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), CXC-motif chemokine 10 (CXCL10; also known as IP10), and C-reactive protein (CRP).

[0068] Proadrenomedulline (proADM) and its fragments, preferably MR-proADM, can be used as an additional marker for the severity of RTI / LRTI, in particular. Higher levels of proADM, especially MR-proADM, indicate a more severe disease.

[0069] The present invention relates to several aspects, which are discussed below.

[0070] In one embodiment, the present invention relates to a method for diagnosing respiratory tract infections (RTIs), particularly LRTIs, in a subject suspected of having a respiratory tract infection, This includes determining the level of high-mobility protein B1 (HMGB1) in a sample from the subject, This invention relates to a method for diagnosing a patient with a respiratory tract infection when the level of HMGB1 exceeds a predetermined threshold level.

[0071] In related embodiments, the present invention is a method for differential diagnosis of airway diseases in a subject, This includes determining the level of high-mobility protein B1 (HMGB1) in a sample from the subject, This relates to a method for diagnosing a patient with a respiratory tract infection, particularly a LRTI, when the level of HMGB1 exceeds a predetermined threshold level.

[0072] In this context, HMGB1 is a particularly useful marker for distinguishing patients with atypical LRTIs, especially atypical bacterial pneumonia, from other nonbacterial-related illnesses that resemble pneumonia or from healthy subjects.

[0073] In another aspect, the present invention relates to a method for diagnosing respiratory tract infections, particularly LRTIs, in a subject, In a sample from the subject, preferably, determine the level of at least one histone protein selected from histone H4, histone H2A, histone H2B, histone H3, and histone H1, and / or This includes determining the levels of insulin-like growth factor-binding protein, acid-unstable subunit (IGFALS) in the sample from the subject, The present invention relates to a method for diagnosing a subject with a bacterial respiratory infection if the level of IGFALS is below a predetermined threshold level and / or the level of histone proteins is above a predetermined threshold.

[0074] In related embodiments, the present invention relates to a method for differential diagnosis of bacterial pneumonia in a subject, This involves determining the level of at least one histone protein, preferably selected from histone H4, histone H2A, histone H2B, and histone H3, in a sample from the subject. If the level of the histone protein is below a predetermined threshold level, the subject is diagnosed with typical bacterial pneumonia, and This invention relates to a method for diagnosing atypical bacterial pneumonia in a subject if the level of the relevant histone protein exceeds a predetermined threshold level.

[0075] In another related aspect, the present invention relates to a method for differential diagnosis of respiratory tract infections in a subject, This includes determining the level of IGFALS in a sample from the subject, If the IGFALS level is below a predetermined threshold level, the subject is diagnosed with a bacterial respiratory infection, and This invention relates to a method for diagnosing a subject with viral LRTI if the level of IGFALS exceeds a predetermined threshold level.

[0076] Such differential diagnoses of viral versus bacterial RTI / LRTI / pneumonia can be improved by a combination of IGFALS and one or more markers selected from TRAIL, Mx1, FeA, CRP, and CXCL10.

[0077] The combination of histone proteins and IGFALS is particularly useful in the differential diagnosis of typical bacterial LRTIs, especially typical bacterial pneumonia.

[0078] In a similar embodiment, the present invention relates to a method for diagnosing respiratory tract infections, particularly LRTIs, in a subject. In a sample from the subject, preferably, the level of at least one histone protein selected from histone H4, histone H2A, histone H2B, and histone H3, and / or This includes determining the level of fetuin-A (FetA) in a sample from the subject, The present invention relates to a method for diagnosing a subject with a bacterial respiratory infection if the level of FetA is below a predetermined threshold level and / or the level of histone proteins is above a predetermined threshold.

[0079] The combination of histone proteins and FeA is particularly useful in the differential diagnosis of LRTIs (bacterial vs. viral), especially bacterial pneumonia (typical and atypical).

[0080] In related embodiments, the present invention relates to a method for differential diagnosis of bacterial respiratory tract infections in a subject, This includes determining the level of fetuin A in a sample from the subject, This invention relates to a method for diagnosing a subject with a bacterial respiratory infection if the level of fetuin A is below a predetermined threshold level.

[0081] The predetermined threshold may be based, for example, on the respective marker levels in samples from one or more individuals of a control group, e.g., healthy subjects. Depending on the differential diagnosis of interest, the control group may be patients with a specific form of RTI (e.g., bacterial / viral infection, atypical / typical pneumonia, etc.). Most preferably, the predetermined threshold is determined as a cutoff from the level of the control group, based on the desired specificity / sensitivity of the assay.

[0082] The present invention also relates to a method for treating bacterial RTIs, particularly bacterial LRTIs, and more particularly bacterial pneumonia, in a subject with antibiotics, wherein the subject has been diagnosed with bacterial RTI or bacterial LRTI or bacterial pneumonia by the method of the present invention. Accordingly, the present invention relates to antibiotics for use in such a method for treating bacterial RTIs, particularly bacterial LRTIs, and more particularly bacterial pneumonia. This also includes mixed bacterial / viral infections.

[0083] Different antibiotics are commonly prescribed for typical and atypical RTI / LRTI / pneumonia. Typical bacterial RTI / LRTI / pneumonia is generally treated with amoxicillin, erythromycin, cefuroxime, flucloxacillin, doxycycline, second-generation cephalosporins such as cefaclor, ciprofloxacin, or rifampicin. Atypical bacterial RTI / LRTI / pneumonia is generally treated with macrolide antibiotics such as azithromycin and clarithromycin, fluoroquinolones such as ciprofloxacin and levofloxacin, or tetracycline antibiotics such as doxycycline and tetracycline.

[0084] The most common pathogenic bacteria of typical pneumonia include Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. Therefore, antibiotics targeting these bacteria are preferred for the treatment of typical pneumonia.

[0085] The most common pathogenic bacteria of atypical pneumonia are those such as Chlamydophila pneumoniae, Chlamydophila psittaci, Coxiella burnetii, Francisella tularensis, Legionella pneumophila, and Mycoplasma pneumoniae (atypical bacteria exhibiting characteristics such as the absence of an intracellular survival cycle or cell wall). Therefore, antibiotics targeted at bacteria with an intracellular replication cycle or lacking a cell wall are preferred for the treatment of atypical pneumonia.

[0086] Therefore, the present invention relates to an antibiotic for the treatment of bacterial pneumonia in a subject, The antibiotic in question is selected from the group consisting of amoxicillin, erythromycin, cefuroxime, flucloxacillin, doxycycline, and second-generation cephalosporins, such as cefaclor, ciprofloxacin, and rifampicin. The present invention relates to an antibiotic to which a subject is treated if, by the method according to the present invention, the subject is determined to have typical bacterial pneumonia.

[0087] Similarly, the present invention relates to an antibiotic for the treatment of bacterial pneumonia in a subject, The antibiotic is selected from the group consisting of macrolide antibiotics, such as azithromycin and clarithromycin; fluoroquinolones, such as ciprofloxacin and levofloxacin; and tetracycline antibiotics, such as doxycycline and tetracycline. The present invention relates to an antibiotic to which a subject is treated if, by a method according to the present invention, the subject is determined to have atypical bacterial pneumonia.

[0088] In all embodiments of the present invention, the term “sample” refers to a biological sample obtained from a subject. “Sample” as used herein may also refer to a sample of body fluid or tissue obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. Preferably, as used herein, a sample is a sample of body fluid, such as blood, serum, plasma, urine, saliva, sputum, tears, sweat, nasal secretions, and bronchoalveolar lavage fluid (BAL). In particular, a sample is blood, plasma, serum, or urine. A sample may be treated (pre-treated) by fractionation or purification procedures, such as separation of whole blood into serum or plasma components. Such pre-treatment may also include, but is not limited to, dilution, filtration, centrifugation, concentration, sedimentation, precipitation, or dialysis. Pre-treatment may also include the addition of chemical or biochemical substances to a solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, and chelating agents. Preferably, a sample is a blood sample, more preferably a serum sample or plasma sample.

[0089] In the context of this invention, "plasma" refers to the substantially cell-free supernatant of blood containing an anticoagulant, obtained after centrifugation. Exemplary anticoagulants include calcium ion-binding compounds such as EDTA or citrate, and thrombin inhibitors such as heparin or hirudin. Cell-free plasma can be obtained by centrifugating anticoagulated blood (e.g., citrate-treated, EDTA- or heparin-treated blood) at, for example, 2000-3000 g for at least 15 minutes.

[0090] In the context of this invention, "serum" refers to the liquid fraction of whole blood collected after blood coagulation. When coagulated blood (blood clot) is centrifuged, serum can be obtained as the supernatant.

[0091] As used herein, “urine” is the bodily fluid product secreted by the kidneys through a process called urination (or micturition) and excreted through the urethra.

[0092] In those aspects and embodiments of the present invention in which more than one marker is determined in the sample of interest, at least two markers are typically, but not necessarily, determined in the same sample.

[0093] As used herein, “histone” or “histone protein” refers to standard histones such as H1, H2A, H2B, H3, or H4, as well as histone variants such as H3.3, H2A.Z, or fragments thereof. Histones form octameric particles in which DNA is encapsulated to assemble chromatin structures (Luger, Nature. 1997 Sep 18; 389(6648): 251-60). For example, histone proteins H2A, H2B, H3, and H4 (two of each) form an octamer, which is encapsulated by 165 base pairs of DNA to form a nucleosome, the basic subunit of chromatin. Thus, although H4 is determined in the examples of the present invention, other histone proteins can also be determined. Accordingly, in one embodiment, at least one histone in this specification may be selected from the group consisting of H1, H2B, H4, H2A, and H3. Therefore, the histone levels to be determined in the methods and kits of this embodiment of the present invention are, in particular, the levels of histones H1, H2B, H4, H2A, and / or H3. In another embodiment, at least one histone in this specification may be selected from the group consisting of H1, H2A, H2B, H3, and H4. Therefore, the histone levels to be determined in the methods and kits of this embodiment of the present invention are, in particular, the levels of histones H1, H2A, H2B, H3, and / or H4. The structures of histones and the sequences of histone proteins are known to those skilled in the art (Porto & Stein, Front Immunol. (2016) 7:311).

[0094] Exemplary histone sequences are shown in SEQ ID NOs: 4-8. Exemplary amino acid sequences of histone H4 are shown in SEQ ID NOs: 4. Exemplary amino acid sequences of histone H2A are shown in SEQ ID NOs: 5. Exemplary amino acid sequences of histone H3 are shown in SEQ ID NOs: 6. Exemplary amino acid sequences of histone H2B are shown in SEQ ID NOs: 7. Exemplary amino acid sequences of histone H1 are shown in SEQ ID NOs: 8. In particular, at least one histone is selected from the group consisting of H2B, H4, H2A, H1, and H3. More specifically, at least one histone is selected from the group consisting of H2B, H4, and H2A. More specifically, at least one histone is H2B and H4. More specifically, at least one histone is H2B or H4.

[0095] As used herein, the terms “proadrenomedjuline” or “proADM” refer to proadrenomedjuline or a fragment thereof, in particular MR-proADM. “Determining the level of proADM,” etc., is understood to refer to determining proADM or a fragment thereof. A fragment can have any length, e.g., at least about 5, 10, 20, 30, 40, 50, or 100 amino acids, as long as it allows for the clear determination of the level of proADM. In a particularly preferred embodiment of the present invention, “determining the level of proADM” refers to determining the level of proadrenomedjuline in the middle region (MR-proADM). MR-proADM is a fragment of proADM. The peptide adrenomedjuline (ADM) was discovered as a blood pressure-lowering peptide containing 52 amino acids, and it was isolated from human phenochromocytometry (Kitamura et al., 1993). Adrenomedjuline (ADM) is encoded as a precursor peptide containing 185 amino acids ("preproadrenomedjuline" or "preproADM"; SEQ ID NO: 9). An exemplary amino acid sequence of preproADM is shown in SEQ ID NO: 9. ADM contains positions 95-146 of the preproADM amino acid sequence and is their splice product. "Proadrenomedjuline" ("proADM") refers to preproADM (amino acids 1-21) without a signal sequence, i.e., amino acid residues 22-285 of preproADM. "Mid-region proadrenomedjuline" ("MR-proADM") refers to amino acids 42-95 of preproADM. An exemplary amino acid sequence of MR-proADM is shown in SEQ ID NO: 10. It is also assumed herein that peptides of preproADM or MR-proADM and fragments thereof may be used in the methods described herein. For example, the peptide or fragment may contain amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre-proADM (mature adrenomedjuline). The C-terminal fragment of proADM (amino acids 153-185 of pre-proADM) is called adrenotensin.A fragment of the proADM peptide or a fragment of MR-proADM may contain, for example, at least about 5, 10, 20, 30, or more amino acids. Therefore, the proADM fragment can be selected from the group consisting of, for example, MR-proADM, PAMP, adrenotensin, and mature adrenomedulline, and preferably, as specified herein, the fragment is MR-proADM.

[0096] High-mobility protein B1 (HMGB1) is among the most important chromatin proteins, similar to histone proteins. HMGB1 (Uniprot ID P09429; https: / / www.uniprot.org / uniprot / P09429) is encoded by the HMGB1 gene (NCBI gene number 3146). Exemplary amino acid sequences of human HMGB1 isoforms are shown in SEQ ID NO: 11. HMGB1 is known to be upregulated in certain RTIs (Zhou et al., Microbiol.Immunol.(2011)55:279-288, Patel et al., mBio 9(2):e00246-18).

[0097] Fetuin A (FetA), also known as alpha-2-HS-glycoprotein (AHSG) (Uniprot ID P02765; https: / / www.uniprot.org / uniprot / P02765), is encoded in humans by the AHSG gene (NCBI gene number 197). An exemplary amino acid sequence of human fetuin A is shown in SEQ ID NO: 12.

[0098] The insulin-like growth factor-binding protein, acid-unstable subunit (IGFALS) (Uniprot ID P35858; https: / / www.uniprot.org / uniprot / P35858) is encoded in humans by the IGFALS gene (NCBI gene number 3483). An exemplary amino acid sequence of human IGFALS is shown in SEQ ID NO: 13.

[0099] CXC-motif chemokine 10 (CXCL10), also known as interferon-gamma-inducible protein 10 (IP-10 or IP10) (Uniprot ID P02778; https: / / www.uniprot.org / uniprot / P02778), is an 8.7 kDa protein encoded in humans by the CXCL10 gene (NCBI gene number 3627). CXCL10 has been shown to increase in viral infections (van der Does et al., J Infect. (2016) 72(6):761-763, WO2016 / 092554).

[0100] Interferon-induced GTP-binding protein Mx1 (Uniprot ID P20591; https: / / www.uniprot.org / uniprot / P20591), also known as MX dynamin-like GTPase 1, is a protein encoded in humans by the MX1 gene (NCBI gene number 4599). Mx1 has been proposed as a marker for viral infections (WO2013 / 117746, WO2014 / 137858).

[0101] Serum amyloid A1 (SAA1) (Uniprot ID: P0DJI8; https: / / www.uniprot.org / uniprot / P0DJI8) is a protein encoded in humans by the SAA1 gene (NCBI gene number 6288). SAA1 has been proposed as a marker for tuberculosis (not LRTI in the context of this invention, and M. tuberculosis is not a bacterium that causes atypical pneumonia in the context of this invention) in pneumonia, healthy subjects, and COPD patients (Jiang et al., PLoS One (2017) 12(3):e0173304).

[0102] TNF-associated apoptosis-inducing ligand (TRAIL), also known as CD253 and TNFSF10 (Uniprot ID: P50591; https: / / www.uniprot.org / uniprot / P50591), is a protein encoded in humans by the TNFSF10 gene (NCBI gene number 8743). TRAIL has been shown to increase in viral infections (van der Does et al., J Infect. (2016) 72(6):761-763, WO2016 / 092554, WO2018 / 011796, WO2013 / 117746, US10209260, WO2018 / 011795, US20190041388).

[0103] Procalcitonin (PCT) is a peptide precursor of the hormone calcitonin and has 116 amino acids. PCT is involved in the diagnosis of respiratory and pulmonary infections or inflammatory diseases associated with heart failure, and the marker procalcitonin or a portion thereof is determined in the patient to be tested (WO2008 / 040328 A2). Methods for its detection (or fragment thereof) are also described in WO2000 / 022439 A2 and WO2008 / 104321 A1. PCT has been described as a marker in the differential diagnosis of pneumonia (Self et al., Clinical Infectious Diseases (2017) 65(2):183-90, Neeser et al., Clin Chem Lab Med. (2019) 57(10):1638-1646). PCT assays, such as the B·R·A·H·M·S·PCT high-sensitivity KRYPTOR assay (BRAHMS GmbH, Hennigsdorf, Germany), are commercially available.

[0104] Where used herein, “subject” (or “patient”) may be a vertebrate. In the context of the present invention, the term “subject” includes both humans and animals, particularly mammals and other living organisms. Accordingly, the methods provided herein are applicable to both human and animal subjects. Thus, the aforementioned subjects may be animals such as mice, rats, hamsters, rabbits, guinea pigs, ferrets, cats, dogs, chickens, sheep, cattle, horses, camels, or primates. Preferably, the subject is a mammal. Most preferably, the subject is a human.

[0105] The levels of a marker or marker panel, for example, at least one histone, IGFALS, HMGB1, MR-proADM, Mx1, PCT, TRAIL, CXCL10, and / or fetuin A, can be determined by any assay that reliably determines the concentration of the marker. In particular, mass spectrometry (MS) and / or immunoassays can be used as illustrated in the accompanying examples. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of macromolecules / polypeptides in a solution through the use of antibodies or antibody-conjugated fragments or immunoglobulins.

[0106] As used herein, the term “antibody” refers to a molecule containing an immunoglobulin molecule and an immunoglobulin (Ig) molecule with an immunoactive portion, i.e., an antigen-binding site that specifically binds to an antigen (responds to an immune reaction). According to the present invention, the antibody may be a monoclonal antibody or a polyclonal antibody. In particular, an antibody that specifically binds to a marker of interest is used. An antibody is considered specific if its affinity for the marker of interest is at least 50 times higher, preferably 100 times higher, and most preferably at least 1000 times higher, than its affinity for other molecules in the sample containing the molecule of interest. How to develop and select an antibody having a given specificity is well known in the art. In the context of the present invention, a monoclonal antibody is preferred. Furthermore, the antibody or its antigen-binding fragment is used in the method of the present invention that specifically binds to a marker of interest.

[0107] Alternatively, in place of antibodies, other capture molecules or molecular scaffolds that specifically and / or selectively recognize target sequences, epitopes, and the structural conformations of target proteins may also be included in the scope of the present invention. In this specification, the terms “capture molecule” or “molecular scaffold” include molecules that can be used to bind to a target molecule or molecule of interest from a sample, i.e., an analyte (i.e., a marker). Therefore, the capture molecule must be appropriately shaped spatially and with respect to surface features such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and / or acceptors, and must specifically bind to the target molecule or molecule of interest. Thus, binding may be mediated, for example, by ions, van der Waals forces, π-π, sigma-π, hydrophobic or hydrogen bonding interactions, or a combination of two or more of the aforementioned interactions or covalent interactions between the capture molecule or molecular scaffold and the target molecule or molecule of interest. In the context of the present invention, the capture molecule or molecular scaffold may be selected from the group consisting of, for example, nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, peptides, and glycoproteins. Capture molecules or molecular scaffolds include, for example, aptamers, DARpin (designed ankyrin repeat protein), and affimers.

[0108] Exemplary immunoassays may include luminescence immunoassays (LIA), radioimmunoassays (RIA), chemiluminescence and fluorescence immunoassays, enzyme immunoassays (EIA), enzyme-conjugated immunoassays (ELISA), luminescence-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid test formats, and rare-earth cryptotate assays. Furthermore, assays suitable for point-of-care testing and rapid test formats such as immunochromatography strip tests can be used.

[0109] In certain embodiments of an immunoassay utilizing two antibodies for a target marker, one antibody can be labeled, while the other antibody can be bound to a solid phase or selectively bound to a solid phase. In a particularly preferred embodiment of the assay, one of the antibodies is labeled, while the other is either bound to a solid phase or selectively bound to a solid phase. The first and second antibodies may be dispersed in a liquid reaction mixture, and a first labeling component, which is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, binds to the first antibody, and a second labeling component of the labeling system binds to the second antibody, thereby generating a measurable signal that allows detection of the resulting sandwich complex in the measurement solution after both antibodies to be detected have bound to the marker. The labeling system may include rare earth cryptotes or chelates in combination with particularly cyanine-type fluorescent or chemiluminescent dyes.

[0110] In certain embodiments, the method is carried out as a heterologous sandwich immunoassay, in which one antibody is immobilized on a solid phase of discretionary choice, such as the wall of a coated test tube (e.g., a polystyrene test tube; coated tube; CT) or on a microtiter plate made of polystyrene, or on particles such as magnetic particles, thereby having a group of antibodies that help detect the formed sandwich structure, which allows the other antibody to resemble or selectively bind to a detectable label. Temporary delay or subsequent immobilization using a suitable solid phase is also possible.

[0111] The method according to the present invention can be further embodied as a homogeneous method in which the antibody to be detected / a sandwich complex formed by multiple antibodies and a marker remains suspended in the liquid phase. In this case, when two antibodies are used, it is preferable that both antibodies are labeled in part of the detection system so that a signal is generated or induced when both antibodies are combined into a single sandwich. Such techniques should be embodied in particular as fluorescence-enhanced or fluorescence-quenched detection methods. Particularly preferred embodiments relate to the use of detection reagents used in pairs, such as those described in US 4 882 733 A, EP-B1 0 180 492, or EP-B1 0 539 477, and the prior art cited therein. Thus, it becomes possible to detect only the reaction product containing both labeled components directly in a single immunocomplex in the reaction mixture. For example, such techniques are provided as the trademark TRACE® (time-resolved amplification cryptotate release) or KRYPTOR®, implementing the teachings of the applications cited above. Therefore, in a particularly preferred embodiment, a diagnostic device is used to carry out the method provided herein.

[0112] Furthermore, the immunoassay method of the present invention may preferably utilize a first antibody and / or a second antibody that are specific to the epitope of the detected marker, or their antigen-binding fragments or derivatives.

[0113] In addition, host markers or infectious pathogens (see, e.g., US9,074,236) can be determined by mass spectrometry-based methods, such as those that determine the relative or absolute quantification of the marker of interest. Other detection methods, such as MS techniques or molecular-based methods, can be combined with immunological tests.

[0114] Relative quantification (rSRM) can be achieved by the following: 1. The presence of increased or decreased target protein is determined by comparing the SRM (Selective Reaction Monitoring) signature peak area from a given target fragment peptide detected in the sample with the same SRM signature peak area of ​​the target fragment peptide in at least two, three, four, or more biological samples. 2. The increase or decrease in the presence of the target protein is determined by comparing the SRM signature peak area from a given target peptide detected in the sample with the SRM signature peak area from a fragment peptide of another protein in another sample from a different, separate biological source. The comparison of SRM signature peak areas between two samples for peptide fragments is normalized, for example, to the amount of protein analyzed in each sample. 3. To normalize the levels of biomarkers to the levels of other proteins that do not change their expression levels under various cellular conditions, the increase or decrease in the presence of the target protein is determined by comparing the SRM signature peak area for a given target peptide with the SRM signature peak area from other fragment peptides derived from different proteins within the same biological sample. 4. These assays can be applied to both unmodified fragment peptides and modified fragment peptides of target proteins. Modifications include phosphorylation and / or glycosylation, acetylation, methylation (mono, di, tri), citrullination, and ubiquitination. The relative levels of modified peptides are determined in the same way as the relative amounts of unmodified peptides.

[0115] The absolute quantification of a given peptide may be achieved by the following: 1. The SRM / MRM signature peak area for a given fragment peptide from a target protein in individual biological samples is compared to the SRM / MRM signature peak area of ​​an internal fragment peptide standard spiked into the protein lysate from the biological sample. The internal standard may be a labeled synthetic version or a labeled recombinant protein of the fragment peptide from the target protein being studied. This standard is spiked into the sample in a known amount before (essential for recombinant proteins) or after digestion, and the SRM / MRM signature peak areas for both the internal fragment peptide standard and the native fragment peptide in the biological sample are determined separately, after which a comparison of both peak areas can be made. This can be applied to unmodified and modified fragment peptides, where modification is phosphorylation and / or glycosylation, acetylation, methylation (e.g., mono, di, or trimethylation), citrullination, or ubiquitination, where the absolute level of the modified peptide can be determined in the same way as the absolute level of the unmodified peptide. 2. Peptides can also be quantified using an external calibration curve. The normal curve approach uses a fixed amount of heavy peptide as an internal standard, and various amounts of light synthetic peptide spiked into the sample. A standard curve must be constructed to account for matrix effects using a representative matrix similar to that of the test sample. Furthermore, the inverse curve method avoids the problem of endogenous analytes in the matrix, where a fixed amount of light peptide spikes over the endogenous analyte to create an internal standard, and various amounts of heavy peptide spikes to create a set of concentration standards. The test sample to be compared to either the normal or inverse curve is spiked with the same amount of standard peptide as the internal standard spiked in the matrix used to construct the calibration curve.

[0116] Further diagnostic methods can be used to improve patient management, clinical decision-making, or infection monitoring by identifying pathogenic strains, key mutations, and antibiotics. These aforementioned methods may include molecular-based technologies such as (real-time) polymerase chain reaction (RT-PCT) or next-generation sequencing (NGS), mass spectrometry (MS), and culture-based applications. These further interventions can be tested simultaneously or at different points in time.

[0117] All diagnostic methods described herein can be performed from one or additional samples from the patient, such as in a series of measurements.

[0118] The term "real-time PCR" is intended to refer to any amplification technique that allows the progress of an ongoing amplification reaction to be monitored as it occurs (i.e., in real time). Therefore, data is collected during the logarithmic phase of the PCR reaction, rather than at an endpoint, as in conventional PCR. Measuring the reaction kinetics in the early stages of PCR offers a clear advantage over conventional PCR detection. In real-time PCR, the reaction is characterized not by the amount of target accumulated after a certain number of cycles, but by the point in time during cycling when amplification of the target is first detected. The higher the starting copy number of the nucleic acid target, the earlier a significant increase in fluorescence is observed. Conventional PCR methods can also be applied, using separation methods such as agarose gels to detect PCR amplification at the final stage or endpoint of the PCR reaction. In the case of qRT-PCR, quantification is performed in real time during the reaction, so PCR post-processing of unknown DNA samples is not required. Furthermore, the increase in the reporter fluorescence signal is directly proportional to the number of amplicons generated. Because this method is designed to use similar experimental conditions, PCR amplification for each multiplex can be performed using the same thermal cycling profile, which allows all nucleic acid targets to be amplified simultaneously in a single instrument (e.g., a thermocycler).

[0119] Nucleic acid amplification is often performed by PCR or RT-PCR, but other methods also exist. Non-exclusive examples of such methods include quantitative polymerase chain reaction (Q-PCR), digital droplet PCR (ddPCR), ligase chain reaction (LCR), transcription-mediated amplification (TMA), autologous persistent sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), helicase-dependent isothermal DNA amplification (tHDA), branched DNA (bDNA), cycling probe technology (CPT), solid-phase amplification (SPA), and rolling circle amplification. RCA amplification techniques include real-time RCA, solid-phase RCA, RCA combined with molecular padlock probes (MPP / RCA), aptamer-based RCA (aptamer-RCA), anchored SDA, pre-elongation amplification (PEP), degenerate oligonucleotide-primed PCR (DOP-PCR), sequence-independent single-primer amplification (SISPA), linker-adapter PCR, nuclease-dependent signal amplification (NDSA), branched amplification (RAM), multiple substitution amplification (MDA), real-time RAM, and whole-genome amplification (WGA) (Westin, L. et al.). al.,2000,Nat.Biotechnol.18:199-204, Notomi,T.et al.,2000,Nucleic Acids Res.28:e63,Vincent,M.et al.,2004,EMBO reports 5:795-800,Piepenburg,O.et al.,2006,PLoS Biology 4:E204, Yi, J. et al., 2006, Nucleic Acids Res. 34: e81, ​​Zhang, D. et al., 2006, Clin. Chim. Acta 363:61-70; McCarthy, ELet al., 2007, Biosens. al.,2007,Anal.Chem.79:7492-7500, Coskun,S.and Alsmadi,O.,2007,Prenat.Diagn.27:297-302,Biagini,P.et al.,2007,J.Gen.Virol.This includes 88:2629-2701, Gill, P. et al., 2007, Diagn. Microbiol. Infect. Dis. 59:243-249, and Lasken, R. and Egholm, M., 2003, Trends Biotech. 21:531-535.

[0120] The scope of the present invention is not limited to specific detection techniques, and it should be understood herein that different techniques such as immunoassays, PCR, and MS can be combined within the context of the present invention.

[0121] The sensitivity and specificity of the diagnostic or prognostic method as described in the present invention are determined by more than just the quality of the test analysis, but also by the definition of what constitutes a particular outcome, e.g., an abnormal (pathological) or normal (healthy) outcome. The distribution of marker levels for subjects with and without a particular condition (e.g., RTI / LRTI, typical / atypical pneumonia, bacterial / viral, healthy / pathological) may overlap. Under such conditions, the test will not absolutely distinguish between subjects with and without a particular condition with 100% accuracy. In other words, a balance must be found between a mixture of false-negative and false-positive results. Those skilled in the art will recognize that the health status of the subject itself, or at least one further manufacturer and / or parameter of the subject, can aid in the interpretation of the data, and that this further information enables a more reliable diagnosis in the overlapping area.

[0122] In practice, receiver operating characteristic curves (ROC curves) are typically calculated by plotting a variable against its relative frequency values ​​in “normal” (e.g., obviously healthy individuals without prenatal defects or conditions) and “disease” populations (similarly, for two different conditions, such as viral and bacterial RTIs or typical and atypical pneumonia). For any given marker, the distribution of marker levels for subjects with and without the disease / condition is likely to overlap. Under such conditions, the test will never perfectly distinguish between normal and disease with 100% accuracy, and the area of ​​overlap may indicate the portion where the test cannot distinguish between normal and disease. A threshold is chosen, below which the test is considered “abnormal,” above which the test is considered “normal,” or below or above which the test indicates a specific condition. The area under the ROC curve (AUC) is a measure of the probability that the perceived measurement would allow for the correct identification of a condition. ROC curves can be used even when test results do not necessarily assign exact numbers. ROC curves can be constructed as long as the results can be ranked. For example, the results of tests on “disease” samples may be ranked according to degree (e.g., 1 = low, 2 = normal, and 3 = high). This ranking may correlate with the results of the “normal” population, and an ROC curve may be constructed. These methods are well known in the art (see, for example, Hanley et al. 1982. Radiology 143:29-36). Preferably, the threshold is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, still more preferably greater than about 0.85, and most preferably greater than about 0.9. In this context, the term “about” refers to + / - 5% of a given measurement.

[0123] The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Therefore, for a specific cutoff selected, the value of (1-specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will enable accurate identification of a disease or condition. Thus, the area under the ROC curve (AUC) can be used to determine the effectiveness of this test.

[0124] In other embodiments, positive likelihood ratios, negative likelihood ratios, odds ratios, or hazard ratios are used as measures of a test's ability to predict risk or diagnose a disorder or condition ("affected group"). For a positive likelihood ratio, a value of 1 indicates that a positive outcome is equally likely to occur among subjects in both the "affected" and "control" groups; a value greater than 1 indicates that a positive outcome is more likely in the affected group; and a value less than 1 indicates that a positive outcome is more likely in the control group. For a negative likelihood ratio, a value of 1 indicates that a negative outcome is equally likely to occur among subjects in both the "affected" and "control" groups; a value greater than 1 indicates that a negative outcome is more likely in the test group; and a value less than 1 indicates that a negative outcome is more likely in the control group.

[0125] In the case of odds ratios, a value of 1 indicates that a positive result is equally likely to occur in both the "affected" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the affected group; and a value less than 1 indicates that a positive result is more likely in the control group.

[0126] In the case of hazard ratios, a value of 1 indicates that the relative risk of an endpoint (e.g., death or a specific outcome) is equal in both the “affected” and “control” groups, a value greater than 1 indicates a higher risk in the affected group, and a value less than 1 indicates a higher risk in the control group. The “affected” and “control” groups used herein represent two groups with different conditions.

[0127] Those skilled in the art will understand that associating diagnostic or prognostic indicators with prognostic risk of diagnosis or future clinical outcomes is a statistical analysis. For example, a marker level lower than X may indicate that a patient is more likely to suffer an adverse outcome than a patient with a level of X or higher, if determined by the level of statistical significance. Furthermore, changes in marker concentrations from baseline levels may reflect a patient's prognosis, and the degree of change in marker levels may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining confidence intervals and / or p-values; see, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. The preferred confidence intervals for the present invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, while the preferred p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0128] As outlined above in this specification, in addition to the specific markers or marker panels detected in embodiments of the present invention, further parameters may be taken into consideration for specific diagnoses or differential diagnoses. As used herein, parameters are characteristics, features, or measurable factors that can help define a particular system. Parameters are important elements for health and physiological estimates such as disease / disorder / clinical condition risk. Furthermore, parameters are defined as characteristics that are objectively measured and evaluated as indicators of normal biological processes, pathogenesis, or pharmacological responses to therapeutic interventions.

[0129] Exemplary parameters can be selected from a group consisting of results from the body mass index, weight, age, sex, diagnostic scores, imaging methods such as X-rays, white blood cell count, body temperature, blood pressure, respiratory rate, heart rate, oxygen saturation, breath sounds, and smoking behavior.

[0130] The present invention further relates to a “kit” for the in vitro diagnosis of RTI / LRTI / pneumonia or the use of such a kit, wherein the determination of at least one marker selected from the group of HMGB1, histone proteins, IGFALS, and fetuin A is carried out in the subject under investigation, particularly in the method according to the present invention. The kit comprises a detection reagent containing a capture molecule such as an antibody, and optionally further reagents such as a buffer and / or a calibrator. The following markers and combinations of markers are preferred (i.e., the kit comprises a detection reagent for the following combinations or markers): -HMGB1; -HMGB1+MR-proADM; -HMGB1+FetA; -HMGB1+TRAIL, IP10, PCT and / or MX1; -HMGB1+PCT, MR-proADM, histone proteins (especially H4), SAA1, FeA, IGFALS, TRAIL and / or CXCL10; - Histone proteins (especially H4) + IGFALS; -IGFALS+HMGB1; - IGFALS+TRAIL, IP10, PCT and / or MX1; -IGFALS+MR-proADM; -IGFALS+PCT, MR-proADM, histone proteins (especially H4), SAA1, FetA, TRAIL and / or CXCL10; - Histone proteins (especially H4) + TRAIL, IP10, PCT and / or MX1; -Histone proteins (especially H4) + PCT, MR-proADM, SAA1, FetA, IGFALS, TRAIL and / or CXCL10.

[0131] Embodiments of the present invention In certain embodiments, the present invention relates to the following: 1. A method for diagnosing respiratory infections (RTIs) in subjects suspected of having respiratory infections (RTIs), This includes determining the level of high-mobility protein B1 (HMGB1) in a sample from the subject, A method by which a subject is diagnosed with a respiratory tract infection if the level of HMGB1 exceeds a predetermined threshold level.

[0132] 2. A method for differential diagnosis of airway diseases in a subject, This includes determining the level of high-mobility protein B1 (HMGB1) in a sample from the subject, A method by which a subject is diagnosed with a respiratory tract infection (RTI) if the level of HMGB1 exceeds a predetermined threshold level.

[0133] 3. The method according to embodiment 1 or 2, wherein the subject has one or more symptoms of a lower respiratory tract infection (LRTI).

[0134] 4. The method according to embodiment 3, wherein the subject exhibits one or more symptoms selected from shortness of breath, weakness, fever, sputum formation, cough, fatigue, asthma, chest discomfort or chest pain, rapid breathing, difficulty breathing, congestion, common cold, and sore throat.

[0135] 5. The method according to aspect 4, wherein the subject is suffering from a cough and one or more symptoms selected from sputum formation, shortness of breath, wheezing, and chest discomfort or chest pain.

[0136] 6. The method according to any one of the preceding embodiments, wherein the RTI is a lower respiratory tract infection (LRTI).

[0137] 7. The method according to embodiment 6, wherein the LRTI is selected from the group consisting of acute bronchitis, pneumonia, and bronchiolitis.

[0138] 8. The method according to embodiment 7, wherein the LRTI is a bacterial or viral infection.

[0139] 9. The method according to aspect 8, wherein the LRTI is an atypical bacterial infection, particularly atypical bacterial pneumonia.

[0140] 10. The method according to aspect 8, wherein the viral infection is selected from the group consisting of influenza viruses such as influenza A or influenza B, respiratory syncytial virus (RSV), coronaviruses, particularly severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or coronavirus infection 2019 (COVID-19), rhinovirus, parainfluenza virus, human metapneumovirus, varicella, hantavirus, and adenovirus.

[0141] 11. The method according to any one of the preceding embodiments, wherein the level of one or more further markers selected from the group consisting of procalcitonin (PCT), proadrenomedjulin (proADM) or fragments thereof, histone proteins, serum amyloid A1 (SAA1), fetuin-A (FetA), insulin-like growth factor-binding protein, acid-unstable subunits (IGFALS), tumor necrosis factor-associated apoptosis-inducing ligands (TRAIL), and CXC-motif chemokine 10 (CXCL10) is determined in a sample from the subject.

[0142] 12. The method according to embodiment 11, wherein the level of MR-proADM is determined in a sample from the subject, and preferably the level of MR-proADM indicates the severity of the infection.

[0143] 13. Furthermore, the level of one or more histone proteins selected from histone H2B, histone H4, histone H2A, histone H3, and histone H1 is determined in the sample from the subject, preferably the level of H4 is determined. If the level of at least one histone protein, preferably H4, exceeds a predetermined threshold level, the subject is diagnosed with atypical bacterial pneumonia and / or The method according to any one of the preceding embodiments, wherein the subject is diagnosed with typical bacterial pneumonia if the level of at least one histone protein, preferably H4, exceeds a first predetermined threshold level and is below a second predetermined threshold level.

[0144] 14. The method according to any one of the preceding embodiments, wherein the sample is a body fluid sample, preferably a blood sample, a saliva sample, a nasal swab, sweat, a urine sample, or a bronchoalveolar lavage fluid (BAL), more preferably serum, plasma, or whole blood, most preferably plasma.

[0145] 15. An antibiotic for use in the treatment of a bacterial respiratory infection in a subject, wherein the subject is treated with the antibiotic if it is determined that the subject has a bacterial respiratory infection by the method described in Embodiment 13.

[0146] 16. A method for diagnosing respiratory tract infections in subjects, In a sample from the subject, the level of a histone protein preferably selected from histone H4, histone H2A, histone H2B, histone H3, and histone H1, and / or This includes determining the levels of insulin-like growth factor-binding protein, acid-unstable subunit (IGFALS) in the sample from the subject, A method by which a subject is diagnosed with a bacterial respiratory infection if the level of IGFALS is below a predetermined threshold level and / or the level of histone proteins is above a predetermined threshold.

[0147] 17. The method according to embodiment 16, wherein the histone protein is H4.

[0148] 18. The method according to aspect 16 or 17, wherein the subject is suspected of having a bacterial respiratory infection.

[0149] 19. The method according to aspect 18, wherein the subject has one or more symptoms of a lower respiratory tract infection (LRTI), particularly pneumonia.

[0150] 20. The method according to aspect 19, wherein the subject exhibits one or more symptoms selected from shortness of breath, weakness, fever, sputum formation, cough, fatigue, asthma, chest discomfort or chest pain, tachypnea, dyspnea, congestion, common cold, and sore throat.

[0151] 21. The method according to aspect 20, wherein the subject is suffering from a cough and one or more symptoms selected from sputum formation, shortness of breath, wheezing, and chest discomfort or chest pain.

[0152] 22. The method according to any one of embodiments 16 to 21, wherein the level of one or more further markers selected from the group consisting of procalcitonin (PCT), proadrenomedjulin (proADM) or a fragment thereof, histone proteins, high mobility group protein B1 (HMGB1), serum amyloid A1 (SAA1), fetuin-A (FetA), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), and CXC motif chemokine 10 (CXCL10) is determined in a sample from the subject.

[0153] 23. The method according to embodiment 22, wherein the level of MR-proADM is determined in a sample from the subject, and preferably the level of MR-proADM indicates the severity of the infection.

[0154] 24. The method according to any one of embodiments 26 to 23, wherein the level of high-mobility group protein B1 (HMGB1) is determined in a blood sample from the subject, and if the level of IGFALS is below a predetermined threshold level, the level of histone proteins exceeds a predetermined threshold, and the level of HMGB1 also exceeds a predetermined threshold level, the subject is diagnosed with a bacterial respiratory infection.

[0155] 25. The method according to any one of embodiments 16 to 24, wherein a subject is diagnosed with atypical bacterial pneumonia if the level of at least one histone protein, preferably H4, exceeds a predetermined threshold level.

[0156] 26. The method according to any one of embodiments 16 to 25, wherein the subject is diagnosed with typical bacterial pneumonia if the level of at least one histone protein, preferably H4, is above a first predetermined threshold level and below a second predetermined threshold level.

[0157] 27. The method according to any one of embodiments 16 to 26, wherein the sample is a body fluid sample, preferably a blood sample, a saliva sample, a nasal swab, a sweat sample, a urine sample, or a bronchoalveolar lavage fluid (BAL), more preferably serum, plasma, or whole blood, most preferably plasma.

[0158] 28. An antibiotic for use in the treatment of a bacterial respiratory infection in a subject, wherein the subject is treated with the antibiotic if it is determined that the subject has a bacterial respiratory infection by the method of any one of embodiments 16 to 27.

[0159] array An exemplary sequence listing for the markers of the present invention is shown in the attached sequence protocol. The sequence protocol includes the following sequences: Sequence ID 1: Histone H4 peptide fragment detected by mass spectrometry. Sequence ID 2: Peptide fragment of fetuin A detected by mass spectrometry. Sequence ID 3: Peptide fragment of SAA1 detected by mass spectrometry. Sequence ID 4: Amino acid sequence of histone H4. Sequence ID 5: Amino acid sequence of histone H2A. Sequence ID 6: Amino acid sequence of histone H3. Sequence ID 7: Amino acid sequence of histone H2B. Sequence ID 8: Amino acid sequence of histone H1. Sequence ID 9: Amino acid sequence of pre-proADM. Sequence ID 10: Amino acid sequence of MR-proADM. Sequence ID 11: Amino acid sequence of human HMGB1. Sequence ID 12: Amino acid sequence of human fetuin A. Sequence ID 13: Amino acid sequence of human IGFALS. [Examples]

[0160] method Biomarker proteins were quantified in samples from different patient populations from different hospitals. Different biomarker levels were analyzed in patients with respiratory tract infections (RTIs), including viral RTIs, bacterial RTIs, typical bacterial pneumonia, and atypical bacterial pneumonia. Furthermore, biomarker levels were measured in samples from non-infected patients with RTI-like symptoms (RTI "mimics"), pneumonia-like symptoms (pneumonia mimics), and healthy patients.

[0161] Patient registration and sample collection were performed in the emergency department, and the samples were classified as either viral or bacterial infections according to subsequent procedures. In patients suspected of having a viral infection, the viral infection was confirmed by molecular testing. In patients suspected of having a bacterial infection, the bacterial infection was confirmed by a positive bacterial culture result or a positive bacterial antigen test. Specific pathogen biomolecules were detected in blood, lung specimens, or urine (Legionella antigen assay). Two patients with mixed bacterial and viral infections were excluded from evaluation.

[0162] Only samples from patients with confirmed viral or bacterial infections were included in further testing.

[0163] To compare different types of pneumonia, samples were collected from patients with confirmed typical and atypical pathogens. Cases of typical pneumonia were diagnosed according to the local pathway for pneumonia diagnosis. Only patients with pneumonia-specific symptoms and positive identification of typical pathogens in airway-derived samples were included as typical pneumonia patients. Atypical pneumonia was diagnosed in patients with pneumonia symptoms by molecular methods or detection of specific atypical antigens in urine.

[0164] The so-called RTI mimic group is characterized by samples from non-(bacterial) infected patients who have overlapping RTI symptoms such as dyspnea, cough, or chest pain. Samples were collected from patients diagnosed with, for example, heart failure, asthma, and COPD. The other so-called pneumonia mimic group is also characterized by samples from non-infected patients with ACS, asthma, COPD, heart failure, pulmonary embolism, lung tumors or other tumors, or atrial fibrillation. Patients with proven bacterial infections were excluded from the RTI or pneumonia mimic group. [Table 1]

[0165] biomarkers Determination by immunoassay: IGFALS The levels of the (insulin-like growth factor-binding protein complex acid-unstable subunit) were measured by enzyme-linked immunosorbent assay (ALS human ELISA) performed by BioVendor in the Czech Republic.

[0166] HMGB1 The values ​​for the high mobility group box 1) were determined by enzyme-linked immunosorbent assay (HMGB1 ELISA) performed by IBL International in Germany.

[0167] MR-proADM (Mid-region proadrenomedjuline) and PCT (procalcitonin) levels were measured using the KRYPTOR random access analyzer from Thermo Fisher Scientific, Germany, with ultra-high sensitivity assays BRAHMS MR-proADM and BRAHMS. PCT This was determined in plasma samples using [the specified method].

[0168] Fetuin A This was measured using a magnetic bead-based multiplex assay on the Luminex platform from R&D Systems, Inc., USA.

[0169] TRAIL (TNF-related apoptosis-inducing ligand) and CXCL10 (Interferon-gamma-induced protein 10kD) was also measured using a magnetic bead-based multiplex assay on the Luminex platform manufactured by R&D Systems.

[0170] Determination by mass spectrometry (MS) : Histone H4 (The detected peptide sequence, "VFLENVIR", SEQ ID NO: 1), Fetuin A (The detected peptide sequence, "FSVVYAK", SEQ ID NO: 2) and SAA1 (The detected peptide sequence, "EANYIGSDK", SEQ ID NO: 3) were determined in plasma samples by selected reaction monitoring or multiple reaction monitoring (SRM / MRM) assays.

[0171] The SRM assay was developed on a triple quadrupole mass spectrometer TSQ Quantiva combined with HPLC Ultimate 3000 (Thermo Fisher Scientific). Peptides were identified by co-eluting light and heavy labeled transitions in chromatographic separation. Pinpoint (Thermo Fisher Scientific) and Skyline (MacCoss Lab) software were used for time alignment, relative quantification of transitions and quantification of target proteins.

[0172] Statistical analysis Biomarker levels in immunoassays <LoD (limit of detection) were ascribed at half of LoD (about 10% of the measured values were below LoD). Patient samples were excluded from each comparison due to missing biomarker levels. The diagnostic accuracy when separating two diagnostic subgroups (e.g., healthy vs. diseased) was evaluated for all biomarkers with available data. No statistical outlier handling was performed in any analysis, so all observations received the same weight in the calculations. The area under the receiver operating characteristic curve (AUC) independent of cut-off functioned as the main measure of diagnostic accuracy. All analyses were performed in R 3.5.1 (R Core Team 2018).

[0173] For a single biomarker, the AUC was calculated based on the Wilcoxon statistic W, which belongs to the Wilcoxon rank-sum test that compares biomarker levels from two diagnostic subgroups for sample sizes n1 and n2, according to the formula AUC = W / (n1*n2).

[0174] For combinations of two and three biomarkers, AUC was calculated based on the C-statistic of a logistic regression model fitted by maximum likelihood estimation. The model included biomarkers as predictors and diagnostic subgroups as binary dependent variables. Before multivariate modeling, all biomarker levels were logarithmically transformed after substituting all zeros with 0.005. The regression model was fitted using the R package rms (Frank E Harrell Jr (2019)). rms: Regression modeling strategy. R package version 5.1-4

[0175] The ROC plot was created using the R package ROCR (Sing et al, Bioinformatics (2005) 21(20):3940-3941). The box plot was created using the R package ggplot2 (Wickham, ggplot2: Elegant Graphics for Data Analysis, Springer Verlag (2016)). [Table 2] [Table 3]

Claims

1. A method for detecting atypical bacterial pneumonia in a subject suffering from one or more symptoms selected from cough, sputum formation, shortness of breath, wheezing, chest discomfort or chest pain, and dyspnea, distinguishing between atypical bacterial pneumonia and respiratory tract infection (RTI) mimicry, Here, RTI mimic is a non-infectious or non-bacterial infectious disease that presents with one or more symptoms selected from cough, sputum formation, shortness of breath, wheezing, chest discomfort or chest pain, and difficulty breathing. The method includes determining the level of high-mobility protein B1 (HMGB1) in a blood sample from the subject, The method wherein the level of HMGB1 exceeding a predetermined threshold level indicates that the subject is distinguished from a subject having RTI mimicry and is an indicator of having atypical bacterial pneumonia.

2. The method according to claim 1, wherein the subject exhibits one or more symptoms selected from cough with sputum or dry cough, chest pain, fever and dyspnea.

3. The method according to claim 1 or 2, wherein the pneumonia is community-acquired pneumonia (CAP) or hospital-acquired pneumonia (HAP).

4. The method according to any one of claims 1 to 3, wherein the level of one or more further markers selected from the group consisting of procalcitonin (PCT), proadrenomedulin (proADM) or a fragment thereof, histone proteins, serum amyloid A1 (SAA1), fetuin-A (FetA), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), and C-X-C motif chemokine 10 (CXCL10) is determined in a blood sample from the subject.

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