A METHOD FOR DIFFERENTIATION OF URINARY TRACT INFECTIONS

Description

Technical Field

[0001] The invention concerns a method of discriminating an urinary tract infections to the extent of differentiating between a urinary tract infection (UTI) and the urinary tract infection able to cause urosepsis. In particular, the invention is directed to a method to analyse an urinary sample to discriminate E.coli bacteria able to cause UTI from E. coli bacteria which cause urosepsis.Background Art

[0002] Sepsis is one of the causes of infections and deaths at hospitals. If sepsis results from a urinary tract infection (UTI), it is referred to as urosepsis. It is estimated that approximately one fourth of the general number of sepsis cases is caused by urinary tract infections, which represents ~12% of these infections. Early administration of proper medication (antibiotic) targeted at the specific causative factor (bacteria) increases the patient's chances to survive. Any delay of one hour or more in launching the therapy may increase the mortality rate from 20% to 33%. The most frequent etiological factors of urosepsis are: Gram-negative bacteria of the Enterobacteriaceae family (Escherichia, Proteus, Enterobacter, Klebsiella spp.), as well as non-fermenting bacteria (Pseudomonas spp.) and Gram-positive bacteria, less frequently by fungi. Majority of urosepsis cases is caused by the bacteria of the Escherichia coli intestinal bacteria.

[0003] The method commonly used today to differentiate generalized inflammatory response from sepsis consists in assaying the CRP protein level in combination with assaying the level of procalcitonin (PCT). The Escherichia coli bacteria which are the most frequent cause of urosepsis are detected by absorption microscopy using Fourier transform and static analysis.

[0004] Known in the prior art are methods of detecting and identifying bacteria.

[0005] Disclosed in patent application P.395007 is a method of assaying bacteria colonies cultured on solid media and an optical system to carry out the method.

[0006] According to the disclosure, the assay of the cultured bacteria colonies is conducted on solid media, where a collimated beam of light from a coherent light source is filtered using an amplitude filter, polarized, and expanded, whereupon a beam of light is formed using a diaphragm and a lens to the convergent-spherical form and the bacteria colony placed on a transparent medium is lit. The transmitted and deflected beam of light is recorded and the two-dimensional distribution of intensity of the light deflected on the bacteria colony is analyzed.

[0007] Disclosed in patent US9970063B2 is a PCR-based test kit and nucleic acid amplification process for identification of sub-species of infectious bacteria, such as uropathogenic E. coli causing cystitis, pyelonephritis and sepsis, for the purposes of predicting antibiotic resistance of the bacteria. The clonal E. coli strains are differentiated based on SNP (single nucleotide polymorphism) identification.

[0008] Disclosed in patent EP 3 110 963 B1 is a method of analysing a sample including a microorganism of interest, such as a bacterium responsible for causing a urinary tract infection ("UTI"). UV-Vis spectroscopy and other spectroscopic techniques (such as FT-IR and Raman) is used to obtain a fast and accurate diagnosis of the susceptibility / resistance of bacteria responsible for causing UTIs. to antibiotics. using a simple and inexpensive screening tool.

[0009] In order to diagnose urinary tract infections and implement an appropriate treatment as soon as possible, it is necessary to provide a swift and cost-effective method of diagnosing infections of the type, in particular a method which will enable differentiation between uropathogenic E. coli strains.

[0010] It has been found in the research works conducted by the Inventors that it is possible to differentiate uropathogenic E. coli strains using optical methods, where analysis is conducted on a liquid medium.Summary of Invention

[0011] The invention, defined by appended independent claim 1, concerns the provision of a method to analyse an urinary sample to discriminate E. coli bacteria able to cause urinary tract infection (UTI) from

[0012] E. coli bacteria which cause urosepsis using an optical system which employs measurements of absorbance of waves of the visible spectrum. The specific changes in the absorbance spectrum which occurred between the patient groups and control groups made it possible to develop an algorithm which differentiates between these two populations based on the recorded spectrum.

[0013] In greater detail, the invention, defined by appended independent claim 1, concerns a method to analyse an urinary sample 3 to discriminate E. coli bacteria able to cause urinary tract infection (UTI) from E. coli bacteria which cause urosepsis using a system consisting of a light source 1 which emits radiation having a central wavelength within the range of 200 - 780nm, a cuvette 2 into which an urine sample 3 to be tested is inserted or a container 2 with the urine sample 3 to be tested into which a measuring probe 6 is dipped, a detector 4, where the detector 4 is placed at a distance from the light source 1, and an analyzer 5 programmed to classify the urine sample to UTI / urosepsis based on the reading of the differentiating factor R, the method according to the invention, wherein the method progresses in the following stages: a) reference urine samples from patient groups of different age groups and sexes, with infections confirmed on genetically verified clinical strains representing confirmed UTI caused by E.coli and patients with confirmed Urosepsis caused by E.coli are provided; b) light absorbance measurements at the wavelength of 600 nm are performed for the reference urine samples and a differentiating factor R characteristic for UTI and Urosepsis infections is determined for each patient group so as to achieve 95% likelihood that it would indicate the correct UTI / Urosepsis group; c) from a patient's sterile urine container a specific volume of the patient's urine sample 3 is collected into the cuvette 2 or the measuring probe 6 is dipped in the urine, whereupon urine absorbance is measured in the radiation of the central wavelength of 200-780 nm emitted by the light source 1, wherein before measuring the absorbance, the urine sample is centrifuged to separate the supernatant from the sediment at 4 000 x g for 20 min at the temperature of 25°C, and wherein the preliminary selection of the urine samples is conducted using measurement of sample absorbance at the central wavelength of 600 + / - 20 nm, thus determining the normalizing parameter N, where referred for further analysis is the urine sample for which the parameter N is higher than 0.2; d) the light absorbance is measured using the detector 4, e) stages c) and d) are carried out for the entire range of 200-780 nm, thus obtaining the dependence of absorbance A on wavelength λ and the absorbance measurement results are transmitted to the analyzer 5, wherein the analyzer 5 determines the differentiating factor R by using the absorbance measurement results from the entire range of the 200-780 nm and calculating the surface area P from the following formula: P = ∫ 600 750 A λ dλ then calculating the differentiating factor R from the formula R P / N < 0 , − Log P / N P / N > 0 , Log P / N and, based on the differentiating factor R, the sample is classified to UTI / urosepsis by the analyzer 5.

[0014] Preferably, in the method according to the invention the differentiating factor R above 2 indicates that the sample was collected from a patient of the UTI group, factor R below -2 indicates that the sample was collected from a patient of the Urosepsis group, and factor R of <-2, 2> indicates an uncertain result.

[0015] Preferably, in the method according to the invention the differentiating factor R is determined for a reference liquid culture on genetically verified clinical strains or for urine samples from patients with confirmed infections, namely with confirmed UTI caused by E. coli and with Urosepsis caused by E. coli, respectively.

[0016] Compared to the solution disclosed in application P.395007, which presented a method of assaying bacteria colonies cultured on solid media and an optical system for carrying out the method and enabling differentiation between the assayed groups using measurements within the wavelength of 670 nm, the solution according to the invention, defined by claim 1, does not require culturing a colony on a solid base, and in addition used in the measuring system according to the present disclosure are measurements of sample absorbance instead of reflected light measurements, as is the case of the cited disclosure. Moreover, in the solution according to the present disclosure additional absorbance measurements are taken before and after the sample is centrifuged, which is vital for the correct interpretation of the results and differentiation between the groups. In addition, in the method according to the invention collected is a urine sample instead of blood sample, as in P.395007, hence the method according to the invention is less invasive and enables taking the measurements without the need for the presence of diagnostic laboratory personnel, the latter required to take a blood sample. Moreover, in accordance with the method according to the invention, defined by claim 1, absorbance measurements may be taken using both a regular spectrometer, and a solution based on measurement probe used to measure absorbance in in liquid samples, hence the measuring system in the method according to the invention is simplified, and sample analysis is less time consuming and less costly than that in the said disclosure.

[0017] According to the present disclosure two methods of preparing samples for testing were proposed, namely directly from patient's urine sample and by growing a culture on liquid medium inoculated with patient's urine. The method according to the invention, defined by claim 1, can be used to perform a swift and reliable discrimination of urinary tract infection from an infection able to cause urosepsis, thanks to which it is possible to take up proper treatment early.

[0018] The advantages of the proposed solution consist primarily in the swiftness of taking the measurement, mobility of the measurement-taking method (no medical personnel required to collect the sample), and the low cost of the test. The solution according to the invention can be used in medical laboratories and medical and veterinary clinics. Contrary to the solutions known in the prior art, where the differentiating method consists in testing the radiation dispersed on a solid medium, the solution according to the invention is based on measuring changes in the intensity of light in the visible spectrum, once it has passed through the liquid medium (urine, liquid bacteria culture, or any other liquid) without the need to grow a culture on a solid medium, which requires at least 24h of sample incubation and qualified personnel able to identify bacteria based on the colony phenotype.

[0019] As mentioned above, the solution according to the invention eliminates the need to collect a sample, i.e. patient's blood, by an invasive method. Currently, in order to classify a patient to the UTI or Urosepsis group, diagnostic laboratories grow cultures from blood and urine on a solid medium and differentiation is only possible afterwards. In accordance with the invention it is possible to classify a patient in a respective group solely based on the observations of changes in urine sample absorbance.

[0020] The solution according to the invention can be used in medical laboratories and medical and veterinary clinics.Brief Description of Drawings

[0021] Embodiments of the solution according to the invention is shown on drawing figures, where [Fig.1] shows a diagram of the arrangement of the measuring system to carry out the method according to an embodiment of the invention in the variant with a cuvette. The references used in the figure have the following meanings: 1 - light source, 2 - plastic cuvette, 3 - sample, 4 - optical radiation detector and 5 - data analyser. [Fig.2] shows a diagram of the arrangement of the measuring system to carry out the method according to an embodiment of the invention in the variant with a measuring probe. The references used in the figure have the following meanings: 1 - light source, 2 - container, 3 - sample, 4 - optical radiation detector, 5 - data analyser, 6-measuring probe. [Fig.3] shows the distribution of factor R calculated for cultures of clinical E. coli strains, as detailed in the description of embodiments of the invention. Description of Embodiments

[0022] The present invention is presented in more detail in embodiments which do not limit its scope. The scope of the present invention is solely limited and defined by the appended claims.Examples

[0023] In order to differentiate E. coli bacteria able to cause UTI from E. coli bacteria which cause urosepsis it is necessary to perform: (i) reference measurements which enable determination of the R parameter depending on the culture medium used or the regional changes in absorbance resulting from different diets of the patients if the measurements are conducted on patients' urine samples; (ii) - proper measurements which enable classifying patients in the UTI or Urosepsis group.Sample preparationA. Patient's urine

[0024] A sample of the patient's urine was collected in a sterile container. Then, 2 mL of the urine was collected and transferred to a measurement cuvette.

[0025] The sample was placed in a measuring device where the source of radiation was a diode emitting a wave of the central wavelength of at least 600 + / - 20 nm.

[0026] Then, the measurement was taken and the absorbance read for the wavelength of 600 nm, thus obtaining the normalising parameter N. Parameter N > 0.2 indicates that the biological material can be used for further analyses.

[0027] Subsequently, 3 mL of urine was collected again from the sterile container with the patient's urine and centrifuged to separate the supernatant from the sediment (4 000 x g, 20 min, 25°C).

[0028] The supernatant was the sample subject to absorbance measurements.B. Liquid culture

[0029] With the view of performing inoculation, a solution of the liquid medium was prepared as follows.

[0030] Added to 1 litre of ultrapure water were: 6g / L of disodium hydrogen phosphate, 3g / L of potassium dihydrogen phosphate, 0,5g / L of sodium chloride, and 1g / L of ammonium chloride, and mixed until the components dissolved. The final pH of the solution was identified as 7.4 at 25°C.Then, the solution was autoclaved at 121°C for 15 min, following which added were: 2 mL of 1M magnesium sulphate solution, and 20 mL of 20% glycerol solution, where both solutions had been first subject to sterilisation by filtration through a filter with pores of the diameter of 0.2 µm.

[0031] Next, a sample of the patient's urine was collected in a sterile container and inoculated. To that aim, 2 mL of the sampled urine was collected and added to 8mL of sterile liquid medium. Then, culturing was performed for 24h at 37 °C shaking it at the speed of 70 rpm.

[0032] Once the culturing was completed, 2 mL of the culture was collected, the sample placed in the measuring device where the source of radiation was a diode emitting the wave of the central wavelength of at least 600 + / - 20 nm.

[0033] Then, the absorbance read for the wavelength of 600 nm, thus obtaining the normalising parameter N, where parameter N of > 0.2 indicates that the biological material can be used for further analyses.

[0034] Next, a sample of 3 mL of the culture was collected again and centrifuged to separate the supernatant from the sediment (4 000 x g, 20 min, 25 °C). The supernatant was the sample subject to the absorbance measurements.Measurement

[0035] The measurement system incorporates such devices as an optical radiation detector (e.g. optical power meter), and a source of radiation.The radiation source and the detector are arranged as appropriate so that having passed through the tested sample the light falls on the detector (the measurement is conducted by analysing the beam of unreflected light - opposite to invention P.395007 where analysed is the dispersed radiation), then the data from the detector are directed to the analyser which processes the obtained data. The diagram of the measuring system is presented on Fig.1.

[0036] The measurement was performed so that radiation of the central wavelength of 200-780nm was emitted from the source of light, i.e. a diode, the beam of light was directed onto a plastic cuvette 2 containing the tested sample 3, and having passed through cuvette 2 the beam of light fell on the detector 4 placed at a specific distance from the source of light 1, the absorbance of the sample for the given wavelength is then read in the detector 4, the reading data are recorded in the analyser which, based on the differentiating factor R obtained for the reference samples, classifies the samples as UTI / Urosepsis.

[0037] The absorbance was measured for the whole range of wavelength 200-780 nm obtaining the dependence of absorbance A on wavelength λ and the surface area P was calculated from the following formula: P = ∫ 600 750 A λ dλ

[0038] Finally, the differentiating factor R was calculated from the following formula: R = P / N < 0 , − Log P / N P / N > 0 , Log P / N Reference measurements

[0039] Reference measurements were performed in order to determine the differentiating factor R characteristic for UTI and Urosepsis infections. The measurement of sample absorbance was taken at the wavelength of 600 nm.

[0040] The tested urine samples came from 50 patients of different age groups and sexes, with infections confirmed on genetically verified clinical strains representing, as appropriate, confirmed UTI caused by E. coli and patients with confirmed Urosepsis caused by E. coli.

[0041] Liquid cultures were grown for all collected samples (according to the procedure described in pt. B) using the collected urine, and absorbance was measured for each of the obtained samples as described above. The results are presented on Fig.3.

[0042] Based on an analysis of the reference results the ranges of the R factor were determined for each patient group so as to achieve 95% likelihood that it would indicate the correct UTI / Urosepsis group.

[0043] It was established that whenever R > 2 one should assume that the patient represents the UTI patient group, whereas for R <-2 one should assume that the patient falls in the Urosepsis patient group. The R results in the range of <-2, 2> were assumed less diagnostic, hence defined as uncertain results.Proper measurement

[0044] Once the diagnostic range of parameter R had been determined for the reference samples, proper measurements were performed for the urine samples from patients suffering from urinary tract infection of unknown origin to discriminate between UTI / Urosepsis.

[0045] The urine samples were prepared according to the procedure described above (pt. A or B)

[0046] The results of the measurements taken on the urine sampled from the patients are presented in Table 1. Table 1PatientNSample preparation procedureRUTIUrosepsisUncertain1>0.2A1.65+2>0.2A2.70+3>0.2A-+0.214>0.2A-+2.405>0.2B-+1.126>0.2B-+2.347>0.2B2.21+8>0.2B2.09+9>0.2B0.34+10>0.2B2.11+11>0.2A-+2.18

[0047] In order to validate the method, absorbance measurements were taken for ultrapure water, urine from a healthy patient, and culture medium M9, with the obtained results presented in Table 2. Table 2.SampleNRUTIUrosepsisUncertainH2O<0.2None+Healthy<0.2None+M9<0.2none+

Claims

1. A method to analyse an urinary sample (3) to discriminate E. coli bacteria able to cause urinary tract infection (UTI) from E. coli bacteria which cause urosepsis using a system consisting of a light source (1) which emits radiation having a central wavelength within the range of 200-780nm, a cuvette (2) into which an urine sample (3) to be tested is inserted or a container (2) with an urine sample (3) to be tested into which a measuring probe (6) is dipped, a detector (4), wherein the detector (4) is placed at a distance from the light source (1), and an analyzer (5) programmed to classify the urine sample (3), wherein the method progresses in the following stages: a) reference urine samples from patient groups of different age groups and sexes, with infections confirmed on genetically verified clinical strains representing confirmed UTI caused by E.coli and patients with confirmed Urosepsis caused by E.coli are provided; b) light absorbance measurements at the wavelength of 600 nm are performed for the reference urine samples and a differentiating factor R characteristic for UTI and Urosepsis infections is determined for each patient group so as to achieve 95% likelihood that it would indicate the correct UTI / Urosepsis group; c) from a patient's sterile urine container, a specific volume of the patient's urine is collected into the cuvette (2) as an urine sample (3) to be tested; alternatively, the measuring probe (6) is dipped in a specific volume of the patient's urine in the container (2) as sample (3) to be tested, whereupon urine absorbance is measured using the radiation having the central wavelength within the range of 200-780 nm emitted by the light source (1), wherein before measuring the absorbance, the urine sample (3) is centrifuged to separate the supernatant from the sediment at 4 000 x g for 20 min at the temperature of 25°C, and wherein the preliminary selection of the patient's urine is conducted by measuring the absorbance of the urine sample at the central wavelength of 600 + / - 20 nm, thus determining a normalising parameter N, where referred for further analysis is the urine sample (3) for which parameter N is greater than 0.2; d) the light absorbance is measured using the detector (4), e) stages c) and d) are carried out for the entire range of 200-780 nm, thus obtaining the dependence of absorbance A on wavelength λ, and the absorbance measurement results are transmitted to the analyzer (5), wherein the analyzer (5) determines the differentiating factor R by using the absorbance measurement results from the entire range of the 200-780 nm and calculating the surface area P from the formula ∫ 600 750 A λ dλ , then calculating the differentiating factor R from the R P / N < 0 , − Log P / N P / N > 0 , Log P / N , and based on the determined differentiating factor R, classifying the sample to UTI / Urosepsis.

2. A method according to Claim 1 wherein a differentiating factor R above 2 indicates that the sample was collected from a patient of the UTI group, and a differentiating factor R below -2 indicates that the sample was collected from a patient of the Urosepsis group, and factor R of <-2, 2> indicates an uncertain result.

3. A method according to Claim 2 wherein the differentiating factor R is determined for a reference liquid culture on genetically verified clinical strains or for urine samples from patients with confirmed infections, namely, with confirmed UTI caused by E. coli and patients with confirmed Urosepsis caused by E. coli, respectively.