METHOD FOR LIQUEFYING A RESPIRATORY SAMPLE AND FOR THE SUBSEQUENT DETECTION OF RESPIRATORY INFECTIONS IN SAID SAMPLE

Abstract

The present invention relates to a method for liquefying respiratory samples, such as sputum samples. Samples of this type are characterized in that they can be highly viscous or semisolid, which means that to detect pathogenic microorganisms in them, they require prior treatment in order to make them more liquid and homogeneous. The liquefaction method proposed in the present innovation enables pathogenic microorganisms that cause respiratory infections to be subsequently detected.

Claims

1. A method for liquefying a respiratory sample, characterized in that it consists of adding an aqueous solution of hydrogen peroxide to said sample.

2. The method, according to claim 1, wherein the aqueous solution has a concentration of hydrogen peroxide between 0.01 M and 1 M.

3. The method, according to claim 2, wherein the aqueous solution has a concentration of hydrogen peroxide of 0.3 M.

4. The method, according to any of the preceding claims 1 to 3, wherein the amount of the aqueous solution of hydrogen peroxide added to said sample is between 10 and 20 microliters per milligram of sample.

5. The method, according to any of the preceding claims 1 to 4, wherein the respiratory sample is a sputum sample, a sample of bronchial aspirate, bronchoalveolar lavage, nasopharyngeal swab or bronchial brushing.

6. A method for detecting respiratory infections caused by pathogenic microorganisms in a respiratory sample, characterized by comprising: a) a first step for liquefying the respiratory sample according to the method defined in any of claims 1-5, b) a second step comprising the detection of the pathogenic microorganism in the liquefied respiratory sample obtained in the first step.

7. The method, according to claim 6, wherein the pathogenic microorganisms are viruses, bacteria or fungi.

8. The method, according to claim 7, wherein the pathogenic microorganisms are bacteria selected from the list that comprises: Pseudomonas aeruginosa, Streptococcus pneumoniae, enterobacteria, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Haemophilus influenzae or Legionella pneumophila.

9. The method, according to claim 7, wherein the pathogenic microorganisms are viruses selected from the list that comprises coronavirus, influenza virus, rotavirus, cytomegalovirus and respiratory syncytial virus.

10. The method, according to claim 7, wherein the pathogenic microorganisms are fungi selected from the list that comprises: Histoplasma capsulatum, Paracoccidioides brasiliensis, Blastomyces dermatitidis, Coccidioides immitis, Sporothrix schenckii, Candida spp., Aspergillus fumigatus, Torulopsis glabrata, Aspergillus spp., Pseudallescheria boydii, Cryptococcus neoformans, and Zygomycota.

11. The method, according to any of the preceding claims 1 to 9, wherein the detection of the pathogenic microorganism in the second step is performed with a biosensor.

12. A kit for detecting respiratory infections caused by pathogenic microorganisms in a respiratory sample, characterized in that it comprises or consists of an aqueous solution of hydrogen peroxide and a biosensor configured to detect the pathogenic microorganism under study.

13. The kit, according to claim 12, wherein the aqueous solution has a concentration of hydrogen peroxide between 0.01 M and 1 M.

14. Use of the kit described in any of claims 12 or 13 for detecting respiratory infections in a respiratory sample.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0035] FIG. 1 shows a schematic representation of the method for liquefying a respiratory sample such as sputum A hydrogen peroxide solution (ii) is added to the semi-solid sputum sample (i) so that the catalase present in the sample generates oxygen bubbles (iii) that disperse the sample. After 1 minute the sample has been liquefied (iv) and can be applied to a biosensor (v) for pathogen detection.

[0036] FIG. 2 shows immunodetection of Pseudomonas aeruginosa after disaggregating sputum from a patient suffering an infection by Pseudomonas aeruginosa (red circle) or not suffering an infection (black square) with the proposed method. The colorimetric signal S increases when the concentration of peroxide increases because when more bubbles are generated more bacteria are released from the sputum infected by Pseudomonas aeruginosa. Error bars are the standard deviation of 3 experiments.

[0037] FIG. 3 shows the increase in luminosity measured as the absolute value of the variation of pixel intensity from a sputum sample according to the experiment performed in example 3 to which an inhibitor of the enzyme catalase has been added.

EXAMPLES

[0038] Next, the invention will be illustrated through experiments performed by the inventors, which shows the effectivity of the methods of the invention.

Example 1: Liquefaction of a Sputum Sample

[0039] Firstly, solutions of hydrogen peroxide in PBS with a concentration of 0.01 M, 0.03 M, 0. 1 M, 0.3 M and 1 M were prepared.

[0040] 200 microliters of each solution were added to 10 mg of sputum originating from a patient with a confirmed respiratory infection by Pseudomonas aeruginosa.

[0041] When adding the solution, the generation of bubbles was observed. The size and the number of bubbles increased with the concentration of peroxide in the solution. This was compared with the addition of water or PBS (without peroxide) which did not produce any bubbles, and therefore, did not liquefy the sputum sample. When the concentration of peroxide is 0.3 M, the sample liquefies in less than 1 minute.

[0042] The liquefied and homogenized samples obtained with this example were directly used to detect P. aeruginosa in them, as explained in Example 2.

Example 2: Detection of P. Aeruginosa in the Liquified Sputum Samples Obtained in Example 1

[0043] The sputum samples that were previously liquefied as indicated in example 1 originated from a patient infected by Pseudomonas aeruginosa and from another patient with no infection.

[0044] Pseudomonas aeruginosa was detected in the liquefied sputum samples with paper immunosensors using gold nanoparticles as colorimetric probes. The method for manufacturing paper biosensors has been previously disclosed in several publications: Russell SM et al. ACS Sensors. 2017; 2:848-853; Russell SM et al. ACS Sensors. 2018;3(9):1712-1718; Alba-Patiño A. et al. Sensors Actuators, B Chem. 2018;273:951-954; Alba-Patiño A et al. ACS Sensors. 2020;5:147-153 and Alba-Patiño A. et al. Nanoscale Adv. 2020;2:253-1260. The method for manufacturing the biosensor used in this particular example has been previously disclosed in detail in the publication Alba-Patiño A. et al. ACS Sensors. 2020, 5:147-153.

[0045] To detect Pseudomonas aeruginosa, 10 .Math.l of sputum (10 mg) liquefied with 200 .Math.L of H.sub.2O.sub.2 at different concentrations between 0 and 0.3 M in PBS were deposited on the receiving surface of a 2×8 cm paper strip and let dry at room temperature (between 15 and 30° C.) for 15 minutes. Subsequently, 1 mL of PBS supplemented with bovine serum albumin (5 mg/mL) was added to avoid the formation of non-specific interactions between nanoparticles and the paper substrate. Next, the paper was folded so that the reservoir containing nanoparticles decorated with an anti-Pseudomonas aeruginosa antibody comes in contact with the area containing the bacteria, pressing both surfaces with the aid of a clamp. After 5 minutes, excess nanoparticles were eliminated by washing the paper strip with PBS supplemented with Tween 20 (0.1 %) and the colorimetric signal S generated by the nanoparticles on the receiving surface of the immunosensor, dependent on the presence of Pseudomonas aeruginosa in the applied sample, was measured with densitometry. To this end, the paper tests were scanned with a Brother MFC -1910 printer and the pixel intensity was calculated with the Histogram function of the Imaged software. The signal S is taken as the absolute value of the number obtained after subtracting 255 from the pixel intensity value. The number 255 corresponds to the pixel intensity of pure white.

[0046] The results obtained are shown in FIG. 2. In this figure, the sample with an infection by Pseudomonas aeruginosa that was treated only with PBS without hydrogen peroxide (0 M) yields a very low signal indistinguishable from the signal obtained with the sample without infection by Pseudomonas aeruginosa. As the concentration of hydrogen peroxide increases, the signal from the sample with infection by Pseudomonas aeruginosa increases because more antigens are released. The signal generated by the samples without Pseudomonas aeruginosa does not vary when the concentration of hydrogen peroxide increases, which demonstrates that the increase in signal observed in samples containing Pseudomonas aeruginosa is produced by the bacteria and not by the treatment with hydrogen peroxide. The highest signal was obtained with samples infected by Pseudomonas aeruginosa when the liquefaction step was performed with 0.3 M hydrogen peroxide.

Example 3: Treatment of a Sputum Sample With Hydrogen Peroxide and a Catalase Inhibitor

[0047] 10 mg of sputum were weighed out and 100 microliters of solutions containing PBS (phosphate buffer saline) with increasing concentrations of sodium azide (0, 0.01, 0.1 and 1 M), which is an inhibitor of the enzyme catalase, were added. The samples were incubated for 2 hours at room temperature. After pre-treatment, the azide was aspirated with a micropipette without touching the sputum samples. Subsequently 200 microliters of PBS with 0.3 M hydrogen peroxide were added for 1 minute. Photographs were taken before and after the treatment. In the sample without azide (0 M), the sputum was totally dissolved and the highest increase in luminosity was recorded after calculating the pixel intensity with image processing software such as Imaged or Adobe Photoshop by selecting the area of interest and using the Histogram function. As the concentration of azide increased, the number of generated bubbles decreased and the sputum dissolved to a lesser extent. Therefore, the increase in luminosity decreased.

[0048] The results obtained in this experiment have demonstrated that the catalase activity in the samples is crucial for the generation of bubbles, and therefore, to dissolve or liquefy sputum, since when adding an inhibitor of this enzyme no dissolution or liquefaction of the sample is achieved.