Patent Application
20200049706
The present invention relates to the field of diagnosis and more particularly to determining the presence or absence of a bacterial infection in an individual by detecting proteins produced by said individual in response to a bacterial infection. It also relates to determining the severity of and/or monitoring a bacterial infection in said individual.
An infection occurs when invading pathogenic microorganisms gain a foothold, succeed in growing, and thereby disturb the normal physiological properties of the tissue [Peterson J W, 1996]. To minimize injuries to the patient, the microbe responsible for the infection must be identified, its susceptibility to antibiotics determined, and effective therapy initiated immediately [Tunkel A R, p. 1267, 2004]. The discovery and implementation of antibiotics is tremendously valuable in targeting specific pathogens, effectively eliminating them, and saving lives and resources [Infectious Diseases Society of America (IDSA), S397, 2011]. However, sufficient guidelines and proper diagnostic instruments are not always available to communities to support proper antibiotics administration. Insufficient compliance regarding intake and unregulated administration has, in part, resulted in development of resistant bacterial strains [Pechere J C, p. 245, 2007]. Among the factors that promote the global dissemination of antimicrobial resistance is the increasing international travel and extensive intercontinental commerce. The current circumstances in developing regions of the world also contribute to the spread of antimicrobial resistance—something that is undoubtedly related to population density, household purchasing power, drug availability, over-the-counter availability of antibiotics, hygiene and sanitation standards, and organized disease control and prevention [Bonelli R R, p. 24, 2014]. Antibiotic resistance rates in Gram-negative bacteria are rapidly increasing, especially with regard to resistance to third and fourth generation cephalosporin and mainly due to the production of extended-spectrum β-lactamases (ESBLs) (Falagas, p. 345-354, 2009). Infections caused by such enzyme-producing organisms are associated with a higher morbidity and mortality and greater fiscal burden (Richi H, 625170, 2012.). The prevalence of ESBLs varies worldwide, with reports from North America, South America, Europe, Africa, and Asia. Data from the global surveillance database shows the rate of ESBL production was highest among the Klebsiella pneumoniae isolates collected in Latin America, followed by Asia/Pacific Rim, Europe, and North America (44.0%, 22.4%, 13.3%, and 7.5%, resp.) (Reinert, p. 1018-1029, 2007). The incidence of ESBL infections per 10,000 discharges increased for both healthcare-associated infections, 1.9 per year (95% CI 1-2.8), and for community-acquired infections, 0.85 per year (95% CI 0.3-1.4) as reported in a recent study from USA (Kassakian, p. 9, 2014). Pseudomonas aeruginosa is an important pathogen frequently implicated in healthcare-associated infections, particularly in critically ill or immunocompromised patients (Hirsch, p. 441-451, 2010). Broad spectrum antimicrobial resistance in isolates significantly limits effective therapeutic options (Oliver, p. 44-59, 2015). ESBL-producing Salmonella isolates resistant to several antibiotic agents, especially 3rd generation cephalosporin, are increasingly reported (Uma, p. 201-204, 2010). In recent years, studies have suggested that international travel and/or gastroenteritis during travel is a risk factor for infection with multiple resistant ESBL producing enterobacteriaceae (Ostholm-Balkhed, p. 2144-53, 2013).
Urinary tract infection (UTI) remains to be the most common infection diagnosed in outpatients as well as in hospitalized patients. The studies have revealed a global high frequency of antimicrobial resistance and ESBL production in Enterobacteriaceae isolated from urine samples in patients (Cohen-Nahum p. 41-6, 2010). The rapid evolution and spread of antimicrobial-resistant bacteria in parallel with insufficient development of new active drugs seriously affect future anti-infective therapy of bacterial infections due to Gram-negative rods in the most common diseases; infectious gastroenteritis and urinary tract infection. Experts in the field estimate that by the next decade the world will have witnessed the wide dissemination of untreatable infections, both within and beyond hospitals (Laxminarayan R, p. 1057-98, 2013). Upper respiratory tract infections are a major reason for the prescription of antibiotics, even though many of these infections are due to viruses, where antibiotics are neither effective nor necessary.
Efforts in order to decrease the antibiotic consumption might have an impact to control the world-wide problem of emerging multiple resistant microorganisms (Nielsen, p. 28-42, 2015). Therefore an effective approach to controlling the problem is directed antibiotic therapy [Gaieski D F, p. 1045, 2010], which requires developing methods to diagnose an infection and identify the microorganism prior to initiating therapy. Laboratory culturing methods and highly technological PCR-based assays that are employed to identify pathogenic microorganisms plus their susceptibility to specific antimicrobial agents are of immense value [Kerremans J J, p. 428, 2008]. But, to fully determine the need for antibiotic therapy, distinguishing harmless colonization of pathogens from active infection is crucial. Herein lays a certain level of complexity that needs unraveling because the host environment houses different constellations of pathogenic microorganisms that, together in symbiosis, can produce synergistic survival mechanisms called biofilms [Steart P S, p. 135, 2001]. In polymicrobial environments, slow-growing bacteria constitute infections that are difficult to detect due to rapidly-growing microorganisms whose rate of growth obscures the true result in culture. Using even the latest PCR methods, the multiple outcomes confuse practitioners [Wolcott R, p 107, 2013; Melendez J H, p. 1762, 2010]. Therefore, tools to assess the inflammatory reaction and response of tissue to the presence of corrosive pathogens are urgently needed. Direct examination of the body fluids such as stool for faecal leukocytes has been used for decades as an indicator of intestinal inflammation [Harris J C, 1972]. The methodology is simple enough that someone with microscopy skills and a very basic laboratory can perform the test reliably. The finding of >10 leukocytes per HPF is considered positive for infection. Markers with short half-lives provide a recent picture of organ status including recovery. Acute phase reactants, C-reactive protein and procalcitonin have been studied widely and are acknowledged by physicians as tools for indicating and monitoring acute injury, including those sustained by infection [Pepys M B, p. 1805, 2003; Xiao Z, p. 1093, 2015; Lam S W, 2015]. However, these markers are analyzed systemically and cannot locate the focus of the infection, especially during hazardous sub-acute and silent infections.
The extracellular matrix (ECM) is composed of molecules produced by the cells in organ tissues that provide structural and biochemical support to the surrounding cells [Theocharis A D, 2015]. By studying the physiological aspects of the ECM in healthy persons, alterations found in its counterparts and products might reflect disease. Heparan sulfate proteoglycan (HSPG) is an important component of the ECM that, inter alia, is involved in the activation and elimination of cytokines and growth factors produced by various cells during injury [Nadanaka S, p. 7, 2008]. One such well-studied growth factor is hepatocyte growth factor (HGF), which is known as a multifunctional regenerative factor that is delivered endocrinally (systemic) as well as paracrinally (local) to injured epithelial cells [Molnarfi N, p. 293, 2015; Nakamura T, p. 188, 2011]. Production of biologically-active HGF increased during acute bacterial infections [Nayeri F, p. 13, 2005; Nayeri F, p. 2092, 2000]. The binding profile to HSPG in body fluids could reflect the pathophysiological status of injured organs, identify an acute inflammation due to microbial invasion, and differentiate acute from chronic inflammation [Lönn J, p. 33, 2013].
Proteins can be detected based on their specific interaction with a corresponding antibody. However, this measurement system relies on specialized resources, limiting its usefulness in non-equipped centers or as a self-test. Methachromasy is a characteristic color change exhibited by certain aniline dyes upon binding to chromotropic substances [Bergeron J A, 1958]. This phenomenon has been widely used in histology. Methylene blue (0-Toluidine) is an excellent metachromatic dye that changes from blue to pink upon binding to high-molecular-weight polysaccharides, such as sulfated glycan [Pham N A, 2007]. The pink dye will then quickly turn back to blue following addition of a proportional amount of a protein with high affinity to sulfated glycan (inverted methachromacy).
We have previously in several studies shown that amounts and biological activity of Hepatocyte Growth Factor (HGF) could be used for diagnosis of acute inflammation in body fluids. However, there still remains a need to further develop and sensitize the diagnostic methods for bacterial infections available today. There is also a need for faster, more reliable measures for monitoring therapy, such as antibiotic therapy, of bacterial infections.
The above problems and drawbacks have now been overcome, or at least mitigated, by a device and an in vitro method provided herein. Hence herein, we present a test (or a kit) comprising a device according to the present disclosure to diagnose a bacterial infection by the determination of acute phase proteins in an individual e.g. making it possible to monitor an ongoing antibiotic therapy administered in less than 36 hours. The test may be performed via analysis of biological samples comprising body fluids without any preparations, in less than 5 minutes and less than 0.4 GBP cost per test.
The testing using said device might be performed by the patient, relatives or the health workers throughout the world and the results can rule out bacterial infection or therapy failure with a 98% negative predictive value. The test is designed to be environmentally harmless, low weight, stable to temperature or mechanical strains and has over 2 years shelf-life. The range between positive (sky blue) and negative result (horizon red) is detectable by naked eye (normal vision) in daylight and has the capacity to give quantitative values using spectrophotometry or RGB Reader (Red Green Blue Reader). Using the test might result in huge decrease in social and individual costs and an objective antibiotic treatment strategy. Daily monitoring can detect a therapy failure due to antibiotic resistance within few days and therefore the high technical resources to identify antibiotic resistant genes can be used in the few but reasonable cases.
Accordingly, there is provided herein a device for quantitatively determining the presence or absence of one or more acute phase protein(s), such as Hepatocyte Growth Factor (HGF), in a biological sample from an individual, wherein a presence of one or more acute phase protein(s) in said biological sample is indicative of an acute bacterial infection in said individual, said device comprising
a) at least one surface on which a composition comprising a proteoaminoglycan and toluidine blue has been immobilized,
b) optionally a solid support onto which said at least one surface is attached,
wherein said at least one surface comprises a material suitable for immobilization of said composition comprising proteoaminoglycan and toluidine blue.
More particularly, the proteoaminoglycan may be dextran sulphate.
The ratio of the amount of proteoaminoglycan to the amount of toluidine blue in said composition is sufficient to detect the presence or absence of one or more acute phase protein(s) in said biological sample. Examples thereof are presented herein by specific predetermined ratios of a proteoaminoglycan, such as dextran sulphate to toluidine blue on a surface as examples of parameters sufficient to detect the presence of one or more acute phase protein(s) in a biological sample.
The different combinations of surfaces presented herein are particularly useful for detecting acute phase proteins in different biological fluids due to the composition of the acute phase proteins present in the various biological fluids. This may be due to the different elimination patterns of the acute phase proteins in the respective biological fluids. Therefore, different combinations of surfaces are presented herein.
The surface of a device according to the present disclosure can also be described as an “interacting” surface as it is capable of interacting with and binding a composition comprising toluidine blue and proteoaminoglycan.
A quantitative determination is possible by studying the colour of the at least one surface of the device after the biological sample has been added thereto. The stronger the blue colour, the more acute phase proteins are present in the biological sample. If the colour of all surfaces remains red or red-purple (horizon red), this means that there are no acute phase proteins in the sample.
Again, of course the “absence” of acute phase proteins in the biological sample shall not be construed as an absolute non-presence of acute phase proteins in said sample but rather at such a low level that there is essentially no acute phase proteins, or severe bacterial infection, present in said sample or said individual.
There is also provided herein, a kit of parts, said kit of parts comprising a device as disclosed herein, said kit further comprising a colour detecting means for quantitative determination of acute phase protein(s) detected by said device, and optionally instructions for use. Said kit of parts may comprise one, two or three (interacting) surfaces present on strip or said surface may be a tissue or a suitable material.
There is furthermore provided by the present disclosure, an in vitro method for quantitatively determining the presence or absence of one or more acute phase protein(s) in a biological sample from an individual, said presence of one or more acute phase protein(s) being indicative of an acute bacterial infection in said individual, said method comprising the steps of:
a) adding said biological sample to a device as provided herein,
b) incubating said biological sample on the surface of said device in step a), and thereafter
c) comparing the colour of the surface with a reference obtained with known amounts of acute-phase proteins to determine the presence or absence of one or more acute phase protein(s) in said biological sample.
The method is quantitative and fast and can instantly detect an increase or a decrease in a bacterial infection in an individual which makes it possible to continuously monitor an ongoing therapy, such as an antibiotic therapy.
In addition, there is provided herein a method for determining a suitable material for a surface as defined for a device herein, said method comprising the steps of:
a) soaking a material in a liquid solution comprising a composition comprising a proteoaminoglycan, such as dextran sulphate, and toluidine blue in any one of the ratios as defined herein,
b) determining the colour of the material after soaking in the solution of step a),
wherein if the material presents the colour of toluidine blue after soaking, said material is suitable for a surface as defined herein.
This introduces a simple method in order to choose which material is most appropriate to be used to immobilize a solution composed of proper amounts of proteoaminoglycan, e.g. dextran sulphate and Toluidine blue (interacting surface) in order to choose the most affordable one, and which material is most proper to be used as the supporting material for an (interacting) surface, in order to choose the most stable test configuration.
Various other objects and advantages of the present invention will become apparent from the drawings and the following description of the invention.
The following detailed description will be more fully understood in view of the figures, in which:
Recognizing the clinical importance and differences between recommended therapies, differential diagnosis between inflammatory disorders in the body has been the subject of several investigations. One major clinical problem is determining whether infection or other inflammatory disorders cause the disease. There are several markers that typically are used by physician to establish the right diagnosis such as microscopic analysis and culture of body fluids, white blood cell count, C-reactive protein, plasma procalcitonin and lactate. However, there are still no golden standards to be used. Problems in establishing correct diagnosis occurs daily while treating inflammatory disorders in bowel, ulcers, joint diseases, CNS disorders, peritoneal, pleural and pericardial effusions, among others. The amounts of routine markers such as CRP and WBC might be high in several disorders and cultures are not always positive in spite of an infection.
The innate immune system, also known as the non-specific immune system is an important subsystem of the overall immune system that comprises the cells and mechanisms that defend the host from infection. Neutrophils are the primary white blood cells that respond to a bacterial infection, so the most common cause of neutrophilia is a bacterial infection, especially pyogenic infections. On the other hand, acute viral infections cause lymphocytosis.
Local acute phase proteins and regenerative factors are produced by neutrophils but not lymphocytes. Therefore detection of acute phase proteins in body fluids indicates a bacterial infection. The most important proteins produced during bacterial infection show high affinity to heparan sulphate proteoglycan (HSPG) in the extracellular matrix, as well as sulphated glycan such as dextran sulphate.
We have used this knowledge to produce a test comprising a device described herein resembling the affinity to extracellular matrix of acute phase proteins produced by neutrophils during bacterial infection. Hence, the present disclosure is related to a rapid method and device to visualize this phenomenon. We have established and herein describe a method based on inverted methachromacy. Addition of a proteoaminoglycan, e.g. dextran sulphate, to Toluidine blue causes change of blue colour to red. Addition of proteins with high affinity to proteoaminoglycan, dextran sulphate, (fast binding with minimal amounts) causes the red colour to change back to blue proportional to
Thus the test results are highly correlated to presence of neutrophils in body fluids during bacterial infection detected by light microscopy and widely used since decades to detect infection world-wide. The test is intended to assess the presence of acute phase proteins produced by neutrophils e.g. in body fluids during infections.
The diagnostic value of detection of acute phase protein in body fluids using methachromacy to detect the protein in body fluid has been presented before (WO2010151222). However, these findings have now been further developed. Accordingly, there is presented herein:
There is presented herein a suitable proportion of proteoaminoglycan, e.g. dextran sulphate, and Toluidine blue in a solution that can detect different amounts of acute phase protein such as HGF. Examples thereof are shown further in the below (Table 1). Notably in this regard, methylene blue may also be used in a device or method herein as a substitute for Toluidine blue, even though it is preferred to use Toluidine blue in the context of the present disclosure. It is also envisaged that other substitutes for Toluidine blue may be used in the present context if such substitutes are evident to the skilled person.
Accordingly, it was shown herein to be particularly useful to use the approach of inverted metachromasy for detecting the presence of acute phase proteins in a biological sample of an individual. By adding a liquid solution comprising a composition comprising a proteoaminoglycan, such as dextran sulphate, and toluidine blue to a surface of a device, the proteoaminoglycan binding to toluidine blue is immobilized on the surface before the biological sample is added thereto. By adding the biological sample after the addition of toluidine blue to a proteoaminoglycan, detection of acute phase proteins was shown to be possible in a more rapid and efficient manner than previously. Now, detection of acute phase proteins can be performed already about 1 minute after the biological sample has been added to the surface comprising a proteoaminoglycan and toluidine blue. It was also shown particularly efficient to immobilize a proteoaminoglycan and toluidine blue (initially present in an aqueous solution added to said surface) on a surface before the biological sample is added thereto.
An example of liquid solutions comprising compositions comprising a proteoaminoglycan and toluidine blue is described in Table 1. The different solutions used in diagnosis of infection in different body fluids in shown in Table 2. The proteoaminoglycan provided in the experiments is dextran sulphate. Hence, ratios in about the below amounts are very useful in a method or device herein as further explained in the below.
27 g/500 ml
Based on the knowledge that HGF is one of the proteins produced at the acute phase of bacterial infection and captured by Dextran sulphate, we have shown that the results from the (index) strip test were significantly correlated with the results from the antibodies-based ELISA test for HGF (n=47; Pearson's Correlation, R=0.71; P<0.0001) (Table 3).
The material used in preparation of the detecting/interacting surfaces and supporting material (solid support):
In order to immobilize the solutions containing dextran sulphate and Toluidine blue, different materials may be used. As a suggestion, Relia Flow 800 Ahlstrom is a good material that does not react to low or high pH or to high amounts of hemoglobin. However, in order to decrease the costs and during epidemics it is possible to test the materials and choose the one at the lowest price that still experiences the appropriate characteristics.
Any material that after soaking in the solution keeps the purple red colour of solution, when dried, might be used for detecting surface. As an example Geotextile is a cheap and appropriate material.
For the support of the strip test, we use a material that does not bind to dextran sulfate in order to increase the stability of the test. We can test materials by soaking in a solution containing a proteoaminoglycan, dextran sulphate and Toluidine blue. The material is identified as appropriate for the test if it takes the blue colour. The natural products such as wood, paper, wool, cotton or silk do not bind to dextran sulphate and can be used as supporting (i.e. as a solid support) in the device herein (strip test).
The Inverted Methachromacy:
Methylene blue (O-Toluidine) is an excellent metachromatic dye that changes from blue to pink upon binding to high-molecular-weight polysaccharides, such as sulfated glycan [Pham N A, 2007]. The pink dye will then quickly turn back to blue following addition of a proportional amount of a protein with high affinity to sulfated glycan (inverted methachromacy).
This approach has now been used to develop a new strip test (device) to assess the presence of proteoaminoglycan (dextran sulphate)-binding proteins in sputum. Other biological samples may also be used in the context of the present disclosure.
Without wishing to be bound by theory, a theoretical support for the presently studied approach to detect presence of acute phase protein in body fluids during infection is suggested to be as follows. (1) Glucosaminoglycan (GAG) polymer chain with negative charges attaches to glucosaminoglycan binding material. (2) Positive toluidine blue (TBO) molecules attach to the polymer chain of the GAG, concentrate, and form stacks, and the color changes from blue to red. (3) Sample containing acute phase protein is added, and the protein binds to GAG. (4) TBO molecule stacks are destroyed, becoming free dissolved TBO molecules, and the color changes from red to blue. Hence, when the blue colour is detected, this means that there is a presence of acute phase proteins in said biological sample that is tested.
Accordingly, there is provided herein a device for quantitatively determining the presence or absence of one or more acute phase protein(s), such as Hepatocyte Growth Factor (HGF), in a biological sample from an individual, a presence of one or more acute phase protein(s) in said biological sample being indicative of an acute bacterial infection in said individual, said device comprising
a) at least one surface on which a composition comprising a proteoaminoglycan and toluidine blue has been immobilized,
b) optionally a solid support onto which said at least one surface is attached,
wherein said at least one surface comprises a material suitable for immobilization of said composition comprising proteoaminoglycan and toluidine blue.
The ratio of the amount of proteoaminoglycan to the amount of toluidine blue in said composition is sufficient to detect the presence or absence of one or more acute phase protein(s) in said biological sample. Examples thereof are presented herein.
Said biological sample usually comprises a biological fluid such as e.g. sputum, urine or the like or as further described herein.
Said device may comprise at least two surfaces, such as three surfaces, on which a composition comprising a proteoaminoglycan and toluidine blue has been immobilized, wherein the ratio of the amount of a proteoaminoglycan to the amount of toluidine blue in the composition on said first surface is different than the ratio of the amount of proteoaminoglycan to the amount of toluidine blue in the composition on said second surface and/or optionally further different from said third surface.
There is further provided herein a device comprising at least one surface, wherein the ratio of the amount of a proteoaminoglycan to the amount of toluidine blue in said composition present on said at least one surface is:
More particularly, said ratio for surface 1 may be about 100:1, for surface 2 may be about 138:1, for surface 3 may be about 165:1, for surface 4 may be about 180:1, for surface 5 may be about 200:1 and/or said ratio for surface 6 may be about 226:1.
There is provided a device herein comprising any combination of one or more these surfaces(s).
A device provided herein may comprise one or more of the above mentioned surfaces (ref. no. 2 in
It should be noted that the above mentioned ratios may also vary slightly. The ratios between the amounts of proteoaminoglycan, such as dextran sulphate, and toluidine blue are shown herein to present a particularly useful composition for detecting the presence of acute phase proteins in a biological sample.
The amounts of proteoaminoglycan and toluidine blue are amounts in weight of proteoaminoglycan and toluidine blue, of dry material that has been added to an aqueous liquid. As previously mentioned herein, firstly, toluidine blue may be added to MQ water and secondly, proteoaminoglycan, such as dextran sulphate, may be added to MQ, and thereafter, these two aqueous solutions are mixed.
Furthermore, there is provided herein a device, wherein said device comprises two or more, such as two, three, or four of the surfaces: surface 1, surface 2, surface 3, surface 4, surface 5, and surface 6. Such more than one surface(s) are usually presented on a test strip.
In addition, there is also provided a device comprising surface 1 and surface 2. A device comprising one or more of surface 1 and surface 2 may be used for detecting recombinant HGF. In addition, there is also provided a device comprising at least one, such as two or three of: surface 3, surface 4 and/or surface 5. A device comprising surface 3, surface 4 and/or surface 5 is particularly useful for determining the presence of acute phase proteins in biological samples comprising sputum, broncho-alveolar lavage, a nasal secretion, cerebrospinal fluid, urine or renal secrete. A device comprising surface 3 and/or surface 4 is useful for determining the presence of acute phase proteins in a biological sample comprising joint fluid. This may be used in determining the presence of bacterial osteoarthritis. A device comprising surface 3 is particularly useful for determining the presence of acute phase proteins in saliva and/or periodontal secretions. This is also illustrated in Table 2.
Furthermore, there is also provided a device comprising surface 5 and surface 6. A device comprising surface 5 and/or surface 6 is particularly useful for determining the presence of acute phase proteins in biological samples comprising ulcer secretion.
In a device according to the present disclosure, said proteoaminoglycan may be selected from the group consisting of HSPG (Heparan Sulphate Proteoglycan), dextran sulphate and heparin sulphate. More particularly, said proteoaminoglycan may be dextran sulphate.
There is also provided herein, a kit of parts, said kit of parts comprising a device as disclosed herein, said kit further comprising a colour detecting means for quantitative determination of acute phase protein(s) detected by said device, and optionally instructions for use. Said colour detection means may use spectrophotometry. In addition, said colour detecting means may be any means, device or the like, that is suitable for determining the colour of the at least one surface used in a device according to the present disclosure. It can be a device capable of automatically determining the colour, or it can be performed by using the mere eye and a reference chart with different colours (i.e. a colour chart) indicating the amount of acute phase proteins present in the sample for use in comparing with the results of the test.
The device according to the present disclosure can also be presented in a detector of choice for interpreting the results of the method as presented herein. For example, a hand-held detector for convenient use, such as in the form of a pen (pen-shaped detector) for rapid indication of a bacterial infection in a sample is envisaged herein, said detector providing for positioning of the device within the detector. The detector may have a replaceable single-use tip containing a transparent version of the detection reagent. Introduction of sample into the tip, may e.g. be passive (the sample enters the reactive area in the tip when in contact with the sample solution) or active (the detector includes a device for actively drawing sample solution into the tip). The detector may have integrated optics and electronics enabling a sensitive detection of the color change in the reaction area of the tip. The detector may also include a display indicating the result and optionally an ON/OFF button. The design/geometrical position of the light guides and the light detector will most likely be dependent of the chosen design of the tip. Accordingly, there is also provided herein a device, wherein said device is present in a hand-held device for detection of an acute bacterial infection.
There is furthermore provided by the present disclosure, an in vitro method for quantitatively determining the presence or absence of one or more acute phase protein(s) in a biological sample from an individual, said presence of one or more acute phase protein(s) being indicative of an acute bacterial infection in said individual, said method comprising the steps of:
The in vitro method may be performed in a manner wherein step c) is performed directly after step b), or at least within about 2 minutes, such as about 1 minute. This timeframe illustrates the efficacy and rapid nature of the method and device presented herein.
Hence, said method allows diagnosing a bacterial infection in an individual, and comprises contacting a body fluid sample from the individual with a means, e.g. a colorimetric device to determine relative levels of acute phase proteins by neutrophil's production stimulated by bacterial infection in the affected organ, and correlating the determined levels to an acute infection. This provides a method for rapid determination of amounts of acute phase proteins in the body fluids (biological samples), such as, for example, urine, cerebrospinal fluid, exhaled breath condensate, broncho-alveolar lavage, nasal secretion, renal secrete, periodontal secretions, sputum, semen, saliva, joint fluid and ulcer secretion. As mentioned herein, knowledge of the concentration of acute phase protein facilitates diagnosis and monitoring of disease.
The acute phase protein determined in said in vitro method may be Hepatocyte Growth Factor (HGF), a derivative thereof or a protein related thereto. Said acute phase protein may also be calprotectin.
Said method employs an inverted methachromic method for visualization of the binding between toluidine blue and proteoaminoglycan. A positive immunological reaction is indicative of the presence of acute phase protein, e.g. HGF (both active and inactive forms) in the sample. A positive immunological reaction provides a blue surface (toluidine blue has been released from the proteoaminoglycan).
There is provided herein an efficient proportion (ratio) between a proteoaminoglycan (dextran sulphate) and Toluidine blue that can detect different amounts of acute phase protein, such as HGF or calprotectin. This is illustrated by the respective surfaces presented herein. Hence, it is shown herein that the efficient proportion of proteoaminoglycan and Toluidine blue results in a solution that by immobilization on proper filter can diagnose grade of bacterial infection.
There is also provided an in vitro method, wherein said device comprises surface 1 and surface 2 as defined herein. Said device used in said in vitro method may also comprise at least one, such as one, two or three of: surface 3, surface 4 and surface 5 as defined herein. When said surfaces are used in said device, said biological sample added to said device particularly comprises sputum, broncho-alveolar lavage, a nasal secretion, cerebrospinal fluid, urine or renal secrete. There is also provided an in vitro method, wherein said device comprises surface 5 and surface 6, as defined herein and wherein said biological sample particularly comprises ulcer secretion.
Thus, herein it is shown that certain proportions (ratios) of amounts of proteoaminoglycan in relation to toluidine blue are particularly useful for determining the presence or absence of acute phase proteins in biological samples, and even more, certain ratios are particularly suitable for biological samples of a particular type (e.g. sputum or urine sample). This is illustrated in Table 2. For example, for sputum and for a self-test at home, it is useful with a device comprising surface 4. For professional use, said device still comprises surface 4, but it also comprises surface 3 and surface 5 for even more sensitive results. The optimization of these characteristics presents an even more efficient way of determining the amounts of acute phase proteins in a biological sample, and thereby of following infection progression and/or (antibiotic) therapy.
Accordingly, the present disclosure in broader terms relates to diagnosis of bacterial respiratory infection; community acquired or nosocomial, from viral or chronic respiratory disorder by analysis of sputum, broncho-alveolar lavage or nasal secretion as further defined herein. An appropriate (interacting) surface for these particular biological samples is presented herein (surface 3, surface 4, and/or surface 5).
There is also provided a use of a device and a method presented herein in the diagnosis of proximal urinary tract infection in need of wide-range and long antibiotic therapy from distal urinary tract infection or no infection, by analysis of urine or renal secretion as further defined herein. An appropriate (interacting) surface for these particular biological samples is presented herein (surface 3, surface 4, and/or surface 5).
There is also provided a use of a device and a method in the diagnosis of bacterial meningitis; community-acquired or nosocomial, from other causes of pleocytosis, by analysis of cerebrospinal fluid as further defined herein. An appropriate (interacting) surface for these particular biological samples is presented herein (surface 3 and/or surface 4).
There is also provided a use of a device and a method in the diagnosis of acute bacterial ulcer infection by analysis of ulcer secretion as further defined herein. An appropriate (interacting) surface for this particular biological sample is presented herein (surface 5 and/or surface 6).
In addition, there is also provided a use of a device and a method in the diagnosis of bacterial osteoarthritis by analysis of joint fluid. An appropriate (interacting) surface for this particular biological sample is presented herein (surface 3 and/or surface 4).
There is also provided the use of a device and a method for determining of periodontal status by analysis of saliva or periodontal secretions. An appropriate (interacting) surface for this particular biological sample is presented herein (surface 3 or surface 4).
There is also provided an in vitro method as defined herein, wherein determining the presence or absence of one or more acute phase protein(s) as explained herein is used for monitoring a therapy, such as antibiotic therapy in an individual. In such a method, the concentration of acute phase proteins may be monitored and quantitatively rapidly determined and with regular intervals and it may efficiently be determined e.g. if the wrong therapy is being used for a certain individual, or how efficient the therapy is. This e.g. avoids unnecessary use of antibiotics, when the antibiotics used may not be appropriate for the individual/particular infection.
It is also shown herein that an efficient proportion of proteoaminoglycan and Toluidine blue presents a solution that by immobilization on proper filter can diagnose grade of bacterial infection and/or monitor an antibiotic therapy. It is shown herein the value of a test based on a described method to rule out cases of non-bacterial infection and thereby reduce antibiotic consumption.
In a device according to the present disclosure, a material for the at least one surface for detecting acute phase proteins needs to be selected, based on properties that it will be suitable for the purpose. This is further described herein.
Accordingly, in addition, there is provided herein a method for determining a suitable material for a surface as defined for a device herein, said method comprising the steps of:
a) soaking a material in a liquid solution comprising a composition comprising a proteoaminoglycan, such as dextran sulphate, and toluidine blue in any one of the ratios as defined herein,
b) determining the colour of the material after soaking in the solution of step a),
wherein if the material substantially presents the colour of toluidine blue bound to proteoaminoglycan after soaking, said material is suitable for a surface as defined herein.
Furthermore, examples of suitable materials for the surface are: e.g. geotextile or filters comprising polyester fibers.
The invention is now further described by the following examples. The examples are illustrative and should not be construed as limiting the scope of the invention.
Clinical Performance Study of Dexact-Resp (a Device of the Present Disclosure) on Left-Over Sputum in Diagnosis of Bacterial Pneumonia
Background:
In patients with clinical symptoms of respiratory infection, rapid identification of cases requiring antibiotic therapy is crucial to avoid development of multiple resistant bacteria. Identification of local acute-phase reactants can help assess the host's response to bacterial infection at the injury site. Here we developed an affordable, stable, feasible, and accurate diagnostic tool based on a locally produced protein with specific binding affinity to polysaccharides. We further evaluated the ability of the novel test strip to detect bacterial infection in cases of pneumonia.
Methods
A strip test (the so called index test) was developed based on inverted metachromacy. Leftover sputum samples (n=467) from patients with suspected pneumonia were blindly tested using the index test (Table 4). These results were compared to the ultimate outcomes determined based on independent clinical and laboratory assessments performed by the patient's physician.
Results
The index test showed a 98.9% sensitivity and 90.2% specificity for detecting the host reaction to high levels of pathogenic bacteria within 2 minutes (Table 5). The golden standard used was sputum culture and PCR. However, the sensitivity of standard tests such as sputum cultures, PCR, antigen detection and lung X-ray to detect bacterial pneumonia (judged and treated by physician in charge) was <70%.
Examples of devices useful for this test are presented in
Conclusions
The novel screening test based on inverted methachromacy has the potential to reduce mortality and morbidity in cases of pneumonia, to guide antibiotic prescription, and to conserve resources, which is especially vital in poorly equipped centers and rural areas (
Table 5: The test evaluation was performed between groups: Positive control: Invasive bacterial infection caused by Streptococcus pneumonia, Pseudomonas aeruginosa, Hemophylus influenza, Streptococci, Mycoplasma pneumonia, Staphylococcus aureus, Negative control: Infection was ruled-out. Sensitivity was 98.97, Specificity was 90.21, Positive predictive value was 83.48 and Negative Predictive value was 99.43.
The standard diagnostic procedures inclusive cultures, PCR, antigen detection and X-ray were performed on the patients, in which the left-over sputum was collected (n467) and used for evaluation of the strip test (Table 6). The ultimate diagnosis was set by the physician in charge after medical evaluation on basis of standard tests and clinical signs and symptoms of patient, without knowledge about the result from the strip test. The test performance for the standard diagnostic tests was 0.68 sensitivity, 0.85 specificity, 0.86 positive predictive value and 0.65 negative predictive value.
Clinical Performance Study of Dexact-Urine on Left-Over Urine in Diagnosis of Urinary Tract Infection
The urine strip test could differentiate between cases of severe proximal urinary tract infection from uncomplicated distal urinary tract infection and healthy (Table 7).
Table 7 shows the left-over samples included in the validation of Renal-ITIS test, divided in groups based on clinical diagnosis established by the medical teams at different departments at the region hospital in Linkoping, Sweden in May 2016. The control group was enrolled 2014-2016. Mann-Whitney U-test: Significant difference in Elisa concentration between Renal-ITIS positive and negative samples p<0.001.
Clinical Performance Study of Dexact-CSF on Patients with Nosocomial Bacterial Meningitis Included at the Department of Neurosurgery
Currently, the diagnosis of bacterial meningitis remains based on standard methods of direct microscopy, differential analyses of white blood cells, lactate, and protein, and cultures of blood and CSF [Baty V, 2000]. However, post-neurosurgical infections are difficult to distinguish from the effects of neurosurgical procedures [Baty V, 2008; Walti L N, 2013]. For example, when patients develop fever within days after surgery, a determination of cells and lactate in the CSF cannot provide reliable information. Moreover, due to prophylaxis treatments, the cultures are negative in a large group of patients, and the presence of skin flora, like Coagulase negative Staphylocococcus or Propionbacterium acnes, may indicate either infection or contamination.
Study on cerebrospinal fluid from cases that were admitted to the Department of Neurosurgery, University in Linkoping, Sweden has shown a 100% sensitivity and Negative Predictive value for the Strip test (Table 8).
| Number | Date | Country | Kind |
|---|---|---|---|
| 1651433-3 | Oct 2016 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/077916 | 10/31/2017 | WO | 00 |
