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EP-4737879-A1 - METHOD FOR LIQUEFYING A RESPIRATORY SAMPLE AND FOR THE SUBSEQUENT DETECTION OF RESPIRATORY INFECTIONS IN SAID SAMPLE

EP4737879A1EP 4737879 A1EP4737879 A1EP 4737879A1EP-4737879-A1

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.

Inventors

  • DE LA RICA QUESADA, ROBERTO
  • CLEMENTE XIMENIS, Antonio
  • OLIVER PALOMO, Antonio
  • ROJO MOLINERO, Estrella

Assignees

  • Fundació Institut D'Investigació Sanitària Illes Balears
  • Servei de Salut de Les Illes Balears - Ibsalut

Dates

Publication Date
20260506
Application Date
20210505

Claims (14)

  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

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. Therefore, the present invention belongs to the area of medicine, and in particular, relates to the detection of respiratory tract infections. BACKGROUND OF THE INVENTION Respiratory tract infections are the origin of a high percentage of cases of sepsis and they are particularly worrying in patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis. Among these infections, nosocomial infections such as those caused by the bacteria Pseudomonas aeruginosa must be detected as quickly as possible because they require an antibiotic treatment different from other pathogens due to their multidrug resistant character. Currently, this requires culturing the sputum of the patient, which may take several days. Therefore, even though bacteriological culture is the gold standard for bacteria detection, it is not suitable for guiding the first antibiotic regimen. In cases of sepsis or COPD exacerbations, administering inadequate antimicrobial therapy may result in grave consequences or even the patient's death. Moreover, it is not an adequate procedure for rapid detection at the point of care because it requires too much time and infrastructure. Methods for pathogen detection in sputum samples are also known which require the extraction of nucleic acids, but again, this requires an additional time-consuming method that it is difficult to perform outside a specialised laboratory (Kukhtin A. V. et al. Anal. Chem. 2020, 92, 5311-5318). In recent years, a new generation of biosensors has emerged as a rapid alternative to microbiological culture and nucleic acid extraction methods for detecting bacteria. In the context of detecting bacteria in sputum, such devices could reduce the time required to identify the pathogen, thus enabling the antibiotic treatment to be customised. However, the detection of bacteria and, in general, any pathogenic microorganism in sputum samples, or in another type of respiratory sample, is problematic because said samples are very viscous or semisolid and the pathogens may not be homogenously distributed within them. The main component of respiratory samples are mucins, glycoproteins that oligomerise to generate a cross-linked scaffold where pathogenic microorganisms are trapped. Furthermore, in the case of some bacteria such as Pseudomonas aeruginosa, they are usually in the form of biofilms. This makes it even harder to detect them because, although the total concentration of bacteria in the sample may be high, they could be hidden inside the biofilm, and therefore they are undetectable. Due to this, a method that simultaneously liquefies the sample and disperses bacterial biofilms whenever they are present, in a few seconds and with simple procedure at the point of care, is required in order to rapidly detect pathogens in respiratory samples. Nowadays, some methods are known for processing sputum samples for the subsequent detection of pathogenic microorganisms with a biosensor, which are detailed as follows. The use of thiolated compounds such as N-acetyl-cysteine (He, F. & Zhang, L. Anal. Sci. 18, 397-401 (2002); Kim, J. H. et al. Lab Chip 12, 1437-1440 (2012)) or a commercially available solution of dithiothreitol known as Sputasol (Massai, F. et al. Biosens. Bioelectron. 26, 3444-3449 (2011)) has been proposed for sputum liquefaction. These methods require incubation times of between 10 minutes and 1 hour and may require additional instruments such as a vortex mixer to disperse the sample or a thermostatic bath, which makes them harder to implement in field applications. Furthermore, these compounds are used for dispersing sputum, but they are not able to disperse the bacterial biofilm when said biofilm is present in the sample. Other methods based on the use of NaOH and surfactants suffer from the same issues (Inoue, S. et al. PLoS One 9, 1-7 (2014)). Finally, other methods have been proposed that use organic solvents such as chloroform (Buzid, A. et al. Sci. Rep. 6, 1-9 (2016)) or dichloromethane (Wen, K. Y. et al. ACS Synth. Biol. 6, 2293-2301 (2017)), which are flammable and can only be manipulated in a fume hood, making them inadequate for detecting pathogenic microorganisms at the point of care. Among the biosensors known for detecting microorganisms, one could cite, for example, those published in the following documents: Kumar N. et al. Analyst. 2018;143(2):359-373; Radhakrishnan R. et al. Biosensors. 2017;7(4):1-12; Cesewski E. et al. Biosensors and Bioelectronics. 2020, 159: 12214.