Search

CN-122029289-A - Detection, differentiation and identification of candida yeasts, microbiological methods and tools therefor

CN122029289ACN 122029289 ACN122029289 ACN 122029289ACN-122029289-A

Abstract

The present invention relates to microbiological methods and culture media specific for the non-denaturing detection, differentiation and/or identification of candida species, in particular candida otorula, candida albicans, candida glabrata, candida tropicalis, candida parapsilosis and candida krusei. The invention more particularly relates to a method for identifying candida yeasts optionally present in a biological sample, comprising the steps of culturing the yeasts to be identified on or in a medium comprising-a nutrient component capable of allowing the development and growth of candida yeasts, and-an alpha-glucosidase inducer and optionally an alpha-glucosidase repressor, -an acid phosphatase inducer and phosphatase repressor, -an N-acetyl-beta-glucosidase inducer, and-detecting any alpha-glucosidase, acid phosphatase and/or N-acetyl-beta-glucosidase activity expressed by the yeasts, and-identifying the species to which the yeasts belong based on the alpha-glucosidase, acid phosphatase and/or N-acetyl-beta-glucosidase activity actually expressed by the yeasts.

Inventors

  • G. Shang Xige
  • GHIRARDI SANDRINE
  • S. ORENGA
  • ROCHE JEAN-MARC

Assignees

  • 生物梅里埃公司

Dates

Publication Date
20260512
Application Date
20241002
Priority Date
20231003

Claims (16)

  1. 1. A solid microbial culture medium for detecting, differentiating and/or identifying candida yeasts that may be present in a biological sample to be tested, said culture medium comprising: -a nutritional component capable of allowing the development and growth of candida yeasts, and -A differentiating component capable of allowing visual marking of yeast colonies growing on said medium, and Characterized in that the differentiating component comprises: an alpha-glucosidase inducer and a chromogenic and/or fluorogenic substrate for the alpha-glucosidase which releases a chromophore and/or fluorophore under the action of the alpha-glucosidase, emits a first detectable optical signal, An acid phosphatase inducer, a phosphatase repressor and chromogenic and/or fluorogenic substrates for the phosphatase which release chromophores and/or fluorophores under the action of the phosphatase, emit a second detectable optical signal which is different from the first detectable optical signal, -A chromogenic and/or fluorogenic substrate for the N-acetyl- β -glucosaminidase inducer and the N-acetyl- β -glucosaminidase, which releases a chromophore and/or fluorophore under the action of the N-acetyl- β -glucosaminidase, emitting a third detectable optical signal, which is different from the first and the second detectable optical signal.
  2. 2. The medium of claim 1, comprising a sugar selected from maltose and raffinose as an alpha-glucosidase inducer at a concentration between 0.05 g/L and 0.5 g/L.
  3. 3. The medium of claim 1 or 2, comprising a fermentable sugar metabolizable by yeast as an acid phosphatase inducer and a pH buffer adapted to maintain the medium at an acidic pH.
  4. 4. A culture medium according to claim 3, comprising a pH buffer which maintains the pH between 5.5 and 6.5.
  5. 5. The medium of claim 3 or 4, comprising glucose as fermentable sugar in a concentration of between 5 g/L and 20 g/L.
  6. 6. The medium according to any one of claims 1 to 5, comprising inorganic phosphate, preferably potassium phosphate, as phosphatase repressor in a concentration between 0.5 g/L and 3 g/L, preferably 2 g/L.
  7. 7. The culture medium according to any one of claims 1 to 6, comprising hexosamine as inducer of N-acetyl- β -glucosaminidase, preferably N-acetyl- β -glucosaminide.
  8. 8. The culture medium of any one of claims 1 to 7, wherein the alpha-glucosidase substrate is a chromogenic substrate with an indoxyl (1H-indol-3-yl) core conjugated to a cyclic amine.
  9. 9. The culture medium of any one of claims 1 to 8, comprising an alpha-glucosidase repressor selected from galactose and xylose at a concentration between 5 g/L and 30 g/L.
  10. 10. The culture medium of any one of claims 1 to 9, wherein the nutritional component is free of yeast extract.
  11. 11. A method for identifying candida yeasts that may be present in a biological sample, comprising the steps of: -culturing the yeast to be identified on or in a medium comprising: a nutritional component capable of allowing the development and growth of candida yeasts, An alpha-glucosidase inducer, Acid phosphatase inducer and phosphatase repressor, and -N-acetyl-beta-glucosaminidase inducer, -Detecting any alpha-glucosidase, acid phosphatase and/or N-acetyl-beta-glucosaminidase activity expressed by said yeast, and -Identifying the species to which the yeast belongs based on the α -glucosidase, acid phosphatase and/or N-acetyl- β -glucosaminidase activity actually expressed by the yeast.
  12. 12. The assay of claim 11, wherein the α -glucosidase, acid phosphatase and N-acetyl- β -aminoglucosidase activities are detected using chromogenic and/or fluorogenic substrates.
  13. 13. The identification method according to claim 11 or 12, wherein the α -glucosidase, acid phosphatase and/or N-acetyl- β -aminoglucosidase activity is detected using a solid microbial culture medium as defined in any of claims 1 to 10.
  14. 14. A method for detecting and/or identifying candida otorula that may be present in a biological sample, comprising the steps of: -culturing any yeast to be detected and/or identified on or in a medium comprising: a nutritional component capable of allowing the development and growth of candida yeasts, An alpha-glucosidase inducer, Acid phosphatase inducer and phosphatase repressor, and -N-acetyl-beta-glucosaminidase inducer, -Detecting any alpha-glucosidase, acid phosphatase and/or N-acetyl-beta-glucosaminidase activity expressed by said yeast, and -Identifying a yeast expressing alpha-glucosidase activity and not acid phosphatase activity nor N-acetyl-beta-glucosaminidase activity as candida otorula.
  15. 15. The detection and/or identification method of claim 14, wherein the α -glucosidase, acid phosphatase and N-acetyl- β -aminoglucosidase activities are detected using chromogenic and/or fluorogenic substrates.
  16. 16. The detection and/or identification method according to claim 14 or 15, wherein the α -glucosidase, acid phosphatase and N-acetyl- β -aminoglucosidase activities are detected using a solid microbial culture medium as defined in any of claims 1 to 10.

Description

Detection, differentiation and identification of candida yeasts, microbiological methods and tools therefor Technical Field The present invention relates to the detection, differentiation and identification of candida yeasts by analytical microbiological methods and tools. More precisely, the invention relates to microbiological methods and culture media for the non-denaturing detection and identification of microorganisms, in particular candida yeasts, which may be present in a biological sample to be analyzed. Following detection/identification using such microbiological methods and media, microorganisms that remain active may optionally be recovered/collected, amplified and/or stored for further analysis (denatured or non-denatured). Background Candida yeasts (or "candida species") are ubiquitous microorganisms, widely distributed in our environment. These yeasts also exist in animals and humans, and remain in symbiotic relationship with them, and colonize the skin, oral cavity, auditory canal, respiratory tract, digestive tract, genital tract, etc. In high risk individuals (particularly immunocompromised patients, diabetic patients, patients receiving broad spectrum antibiotics and corticosteroids, renal failure patients, chronic stressors, infants, elderly, pregnant women, etc.), candida species may exhibit highly pathogenic opportunistic organisms. Its massive and uncontrolled proliferation leads to an infection called candida disease, which can be particularly severe or even fatal. These infections are described as "superficial" when they affect only mucous membranes and/or skin, and are often benign. In the worst case, the infection may reach deep organs (called "visceral candida") and/or spread throughout the body (called "invasive candida" or "candida"). In health, candida is often identified as a cause of nosocomial infections, often involving contamination of medical equipment and tools (catheters, etc.) used in invasive procedures. They are believed to be one of the main causes of hospital acquired sepsis, 40% of cases being fatal within 30 days. Although nearly 400 candida species have been described so far, only a few cause human disease. In 2020, the U.S. center for disease control and prevention (Centers for Disease Control and Prevention, US) indicated that, worldwide, 39% of invasive infections associated with candida species were attributed to candida albicans (c.albicans), 28% to candida glabrata (c.glabra) (reformed according to the recently proposed nomenclature, also known as candida glabrata (Nakaseomyces glabrata) (Kidd et al, 2023, ;Open Forum Infectious Diseases (2023): "Fungal Nomenclature: Managing Change is the Name of the Game")) and 15% to candida parapsilosis). In france, the society of france fungi (French Society of Mycology, SFM) was published in 2015 (Clinical Microbiology Reference Guide, CANDIDIASIS, fifth 2015 edition), about 50% of cases of candida disease in france (i.e., infections associated with candida species) are attributed to candida albicans. The second are candida glabrata (10-20%), candida parapsilosis (10-20%), candida tropicalis (c. tropicalis) (10%) and candida krusei (c. krusei) (3%; candida krusei, also known as pichia kudriavzevii (Pichia kudriavzevii) according to the recently proposed nomenclature). Other candida species are less common and their occurrence is generally dependent on specific ecological conditions. In connection with the special case of candida otophylla (c. auris), this species appears in japan in 2009 and is hereinafter referred to as "killer fungus". In addition to its high risk, there are three reasons that make it currently of increasing concern and concern: Firstly, it can spread rapidly in medical institutions, easily between patients, medical staff and visitors. In a hospital environment (particularly on the surfaces of hospital equipment such as a treatment vehicle, an infusion pump, a thermometer, a sphygmomanometer and the like), candida otorula can survive for a plurality of weeks; Second, its multi-drug resistance to antifungal drugs (e.g., fluconazole, itraconazole, voriconazole, or amphotericin B), while widely varying among different geographical branches, significantly limits the available treatment options. Also, in terms of health, measures to disinfect the environment and medical devices using conventional quaternary ammonium compounds have proven to be relatively ineffective; Finally, there are few analytical and diagnostic tools currently available for their identification. Because of the prevalence and risk of the six yeast species described above (i.e., candida otophylla, candida albicans, candida glabrata, candida tropicalis, candida parapsilosis, and candida krusei), it is referred to herein as the "main candida species" (or "candida species of major interest"). Since they infect the same type of tissue and organs (mucosa, skin, digestive system, respiratory system, genitals, etc.) and cause very similar o