Candida drug tolerance screening method

Abstract

The invention particularly discloses a method for screening drug tolerance of candida, and relates to the technical field of biological medicines. The invention provides a method for screening drug tolerance of Candida, which comprises the following steps: observing morphological characteristics of a Candida strain in contact with a drug, treating clinically separated Candida by using caspofungin, and observing morphological change of the strain under a microscope. The candida resistant to the echinocandin drugs can be quickly and effectively screened out. The invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for screening Candida drug resistance. Background Art

[0002] Candidemia is a serious invasive fungal infection, especially candidemia caused by tropical Candida, which has a high drug resistance, making clinical treatment more difficult. In recent years, the resistance of tropical Candida isolated in China to fluconazole has increased significantly, from 5.7% to 31.8%. In particular, in hematopoietic stem cell transplant patients in some hospitals, the resistance of tropical Candida isolated to azole drugs has even exceeded 80%. Therefore, echinocandins have become the first-line drug of choice for the treatment of candidemia. However, although the in vitro drug sensitivity test report provided by the clinical microbiology laboratory shows that tropical Candida is sensitive to echinocandins, in clinical applications, the commonly used echinocandins such as caspofungin are not ideal for the treatment of some patients, and the prognosis is poor.

[0003] In clinical microbiology laboratories, the microbroth dilution method is commonly used for drug sensitivity tests of Candida isolated from patients with Candida infections. This method is based on the determination of the minimum inhibitory concentration (MIC value) of Candida to different antifungal drugs to determine whether Candida is "sensitive" or "resistant" to these drugs. Based on this, clinicians will decide on the medication regimen for patients based on the drug sensitivity test report provided by the laboratory. However, more and more clinical cases have shown that even if the drug sensitivity test report shows that Candida is "sensitive" to caspofungin (a commonly used echinocandin drug), the actual effect of some patients with candidemia after treatment with caspofungin is not ideal, manifested as persistent candidemia and high mortality. This situation reveals the limitations of existing drug sensitivity test methods, that is, strains reported as sensitive by the laboratory may actually be resistant to echinocandin drugs, which may lead to treatment failure during monotherapy. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of this, one of the main purposes of the present invention is to provide a screening method for Candida drug resistance, the screening method comprising observing the morphological characteristics of Candida strains after contact with drugs. By using caspofungin to treat clinically isolated Candida, and observing the morphological changes of the strains under a microscope, Candida resistant to echinocandin drugs can be quickly and effectively screened out.

[0006] (II) Technical solution

[0007] In order to achieve the above object, the present invention provides a method for screening Candida drug resistance, which includes observing the morphological characteristics of Candida strains after contact with drugs.

[0008] In one embodiment, the screening method involves screening for non-diagnostic purposes only.

[0009] In one embodiment, the morphological characteristics include one or a combination of cell morphology, size, and colony morphology.

[0010] In one embodiment, the morphological characteristic is cell morphology.

[0011] In one embodiment, the cell morphology is a multicellular aggregate morphology.

[0012] In one embodiment, the screening method further comprises performing a drug sensitivity test before observing the Candida strains for exposure to the drug.

[0013] In one embodiment, the drug sensitivity assay comprises broth microdilution.

[0014] In one embodiment, the drug sensitivity test results show sensitivity.

[0015] In one embodiment, the drug sensitivity test results show drug resistance.

[0016] In one embodiment, the Candida species is freshly isolated.

[0017] In one embodiment, the Candida species is frozen resuscitated.

[0018] In one embodiment, the Candida includes one or a combination of Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida portugallis, Candida kefir, Candida rugosa, Candida dubliniensis, Candida asteroids, Candida pseudotropicalis, and Candida auris.

[0019] In one embodiment, the Candida species include Candida tropicalis and Candida auris.

[0020] In one embodiment, the drug is an antifungal drug.

[0021] In one embodiment, the antifungal drug comprises one or a combination of polyenes, azoles, echinocandins, allylamines, and pyrimidines.

[0022] In one embodiment, the antifungal drug is an echinocandin.

[0023] In one embodiment, the echinocandins include caspofungin and micafungin.

[0024] In one embodiment, the echinocandin is caspofungin.

[0025] In one embodiment, the Candida is derived from one or a combination of whole blood, plasma, serum, red blood cell fraction, saliva, cerebrospinal fluid, semen, feces, urine, nasal swab, wound swab, skin swab, rectal swab, bile, lymph sputum, lavage fluid.

[0026] In one embodiment, the Candida species is derived from one or a combination of whole blood, plasma, and serum.

[0027] (III) Beneficial effects

[0028] The present invention provides a screening method for Candida drug resistance, which includes observing the morphological characteristics of Candida strains after contact with drugs. Compared with the prior art, it has the following beneficial effects:

[0029] 1. By using caspofungin to treat clinically isolated Candida albicans and observing the morphological changes of the strains under a microscope, Candida albicans resistant to echinocandin drugs can be quickly and effectively screened out.

[0030] 2. The effectiveness of the screening method was verified by using Candida albicans strains that were confirmed to be resistant to caspofungin through bactericidal tests and randomly selected Candida albicans strains that were verified to be non-resistant, with an accuracy rate of 100%.

[0031] (IV) Terms and Definitions

[0032] As used herein, the term "drug tolerance" refers to a phenomenon in which the fungus's sensitivity to a drug decreases, resulting in a weakened inhibitory or bactericidal effect of the drug on the fungus, and this phenomenon cannot be detected by conventional drug sensitivity tests conducted in clinical microbiology laboratories. For example, a fungus that can be effectively inhibited from growing by a low concentration of a drug, after developing tolerance, requires a higher concentration of the drug to produce the same inhibitory effect, and can even survive and reproduce at very high concentrations, and there is no difference in the MIC value of the antifungal drug.

[0033] As used herein, "multicellular aggregation morphology" refers to the multicellular aggregation morphology that resembles a bunch of grapes when the daughter cells of the blastospores cannot separate from the mother cells due to cell division defects during the proliferation of Candida. The daughter cells that are continuously generated are close to each other in physical space and aggregate together. These cells can be of the same size or a combination of cells of different sizes. This aggregation is not a simple accumulation of cells, but is achieved through the continuous generation of daughter cells during cell division and proliferation, and then aggregation, and usually presents certain structural and functional characteristics.

[0034] As used herein, "multiple clones of tropical Candida" refers to two or more cell populations generated by asexual proliferation from different ancestral cells (tropical Candida cells). The tropical Candida cells within each clone are relatively consistent genetically, but there may be differences in genetic composition, phenotype (such as morphology, growth rate, drug resistance, etc.) between different clones. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 It is the resistance of Candida tropicalis to echinocandin drugs. Figure 1 A is the tolerance index of Candida tropicalis to echinocandin drugs. Figure 1 B is the survival rate of Candida tropicalis under the action of echinocandin drugs.

[0037] Figure 2 is the therapeutic effect of caspofungin on resistant Candida tropicalis infection, Figure 2 A is an analysis of the survival rate of patients with candidemia caused by resistant Candida tropicalis treated with caspofungin. Figure 2 B is the mortality analysis of G. mellonella larvae resistant to Candida tropicalis infection after treatment with caspofungin.

[0038] Figure 3 Caspofungin-resistant Candida tropicalis exhibits multicellular aggregates. Figure 3 A is the morphology of Candida tropicalis after treatment with caspofungin. Figure 3 B is the morphology of Candida tropicalis after co-culture with macrophages and treatment with caspofungin. Figure 3 C is the in vivo morphology of Candida tropicalis strains after infection with G. mellonella larvae and treatment with caspofungin.

[0039] Figure 4 This is a transmission electron micrograph of the multicellular aggregate morphology of Candida tropicalis.

[0040] Figure 5 This is the gene expression profile of Candida tropicalis.

[0041] Figure 6 This is the cell morphology of Candida tropicalis after treatment with caspofungin.

[0042] Figure 7 The cell morphology and survival rate of Candida auris after treatment with caspofungin. DETAILED DESCRIPTION

[0043] After an in-depth analysis of tropical Candida, it was found that about 16% of tropical Candida were resistant to echinocandin drugs. This discovery reveals an important problem: although the detection methods used in clinical laboratories may judge these tropical Candida as "sensitive" to echinocandin drugs, they may not be sensitive in fact, which is mainly due to the limitations of the detection methods. This was further confirmed by in vitro bactericidal experiments, which found that the survival rate of these 16% tropical Candida under drug treatment was significantly higher than that of non-tolerant strains. In addition, the 30-day mortality rate of patients with bloodstream infection with tolerant strains exceeded 65%, while this rate was only 15% in patients in the non-tolerant group. Similarly, in the infection experiment of greater wax moth larvae, the present invention found that caspofungin had a poorer therapeutic effect on tolerant strain infection than non-tolerant strains. In view of this, the present invention provides a screening method for Candida drug resistance, the screening method comprising observing the morphological characteristics of Candida strains after contact with drugs, treating clinically isolated Candida with caspofungin, and observing the morphological changes of the strains under a microscope, so as to quickly and effectively screen out Candida resistant to echinocandin drugs.

[0044] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0046] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0047] The experimental strains used in this study were derived from the Microbiology Laboratory of the Department of Clinical Laboratory, the First Affiliated Hospital of University of Science and Technology of China, covering a total of 50 strains of tropical Candida isolated from patients with candidemia from January 2017 to May 2023, as well as the control strain AT CC750, the known echinocandin-resistant strain SHR001 and 9 strains of Candida auris.

[0048] The clinical information data of patients were obtained from the hospital electronic medical record system. This retrospective observational study has been reviewed and approved by the hospital ethics committee with approval number 2023-RE-346.

[0049] The experimental drugs used in the present invention, including azole drugs, caspofungin and micafungin, were purchased from Selleck Company in the United States.

[0050] Example 1

[0051] Screening of Candida tropicalis for resistance to echinocandins:

[0052] 1. Microbroth dilution method for sensitivity test of tropical Candida albicans to echinocandins: 50 frozen tropical Candida albicans were revived and the drug sensitivity test was performed using the US CLSI reference method - microbroth dilution method. The experimental results showed that all strains were sensitive to echinocandins, which was consistent with the results reported by the clinical laboratory.

[0053] 2. Screening of tropical Candida albicans resistant to echinocandin drugs: The above strains were cultured overnight in YPD medium. After the culture was completed, the medium was removed by centrifugation and washing steps, and then the strains were resuspended in fresh YPD medium to 2×10 5 The strains were divided into two groups, one group was added with 1 μg / mL of caspofungin, and the other group was added with 1 μg / mL of micafungin, and then cultured at 37°C for 24 hours. After the culture was completed, the culture was appropriately diluted and spread on YPD solid medium. After another 24 hours of culture at 37°C, the colonies were counted. Figure 1 As shown in A, 8 strains (SLT6, SLT9, SLT13, SLT21, SLT22, SLT34, SLT38, SLT44) of tropical Candida albicans resistant to echinocandin drugs were screened out. (The tolerance index is calculated by calculating the ratio of the number of colonies formed by each strain under the action of the drug to the initial inoculation amount. If the tolerance index exceeds 0.5, it indicates that the strain is resistant to echinocandin drugs).

[0054] 3. Survival rate experiment of tropical Candida albicans under the action of echinocandin drugs: In order to further verify the tolerance of resistant strains to echinocandin drugs, caspofungin was used to conduct bactericidal survival experiments at different time points and doses. Figure 1As shown in B, the resistance of these strains (SLT6, SLT9, SLT13, SLT21, SLT22, SLT34, SLT38, SLT44) to echinocandin drugs in vitro was confirmed, and the survival rates of these strains and the known echinocandin-resistant strain SHR001 under the action of drugs were significantly higher than those of non-resistant strains (SLT2, SLT14) and the control strain ATCC750.

[0055] Example 2

[0056] Caspofungin for the treatment of resistant Candida tropicalis infections:

[0057] 1. Analysis of the survival rate of patients with candidemia caused by tolerant Candida tropicalis infection after treatment with caspofungin: A detailed analysis was conducted on 20 subjects with complete clinical data. These subjects all experienced candidemia caused by a single pathogen and received at least one week of caspofungin as initial treatment, and had no signs of concurrent infection with other pathogens. Based on the tolerance of tropical Candida isolated from the patients to echinocandin drugs, we divided the subjects into two groups: "tolerant" and "non-tolerant". Survival data were analyzed using the Kaplan-Meier method, and the statistical significance was evaluated by the log-rank (Mantel-Cox) test. The analysis results show that Figure 2 As shown in A, the 30-day survival rate in the tolerant group was only 33%, while this proportion was as high as 85% in the non-tolerant group.

[0058] 2. Analysis of mortality of G. mellonella larvae resistant to Candida tropicalis infection after treatment with caspofungin: G. mellonella larvae with a length of 2.5 to 3.0 cm and a weight of 0.25 to 0.3 g were selected and subjected to a rigorous health assessment. Subsequently, the larvae were randomly assigned to multiple experimental groups, each containing 20 larvae. During the infection experiment, 1×10 6 To determine the concentration of CFU, 10 μL of a standardized yeast suspension of tolerant strains (SLT9, SLT13, and SLT34) and the non-tolerant strain ATCC750 was injected into the left hind leg of each larva. As a control group, larvae were injected with an equal volume of 0.1% dimethyl sulfoxide in a solvent. During the antifungal treatment phase, larvae were injected with caspofungin (0.5 μg per larva) into the right hind leg 1 hour after infection. The treated larvae were placed in an environment of 37°C and survival was assessed daily during the 14-day observation period. The experimental results are shown in Figure 2. Figure 2As shown in B, caspofungin significantly reduced the mortality of larvae infected with the non-tolerant strain ATCC750. However, for larvae infected with tolerant strains (SLT9 and SLT34), caspofungin treatment did not significantly improve their mortality. Although caspofungin treatment led to a significant reduction in the mortality of larvae infected with the tolerant strain SLT13, the degree of reduction was much lower than that of larvae infected with ATCC750 after caspofungin treatment (ATCC750: 75% vs SLT13: 20%).

[0059] Example 3

[0060] Caspofungin-resistant Candida tropicalis exhibited multicellular aggregates associated with cell division defects both in vitro and in vivo:

[0061] 1. Strain treatment and observation: The tropical Candida albicans (SLT9, SLT34, ATCC750 and SHR001) in the logarithmic growth phase were adjusted to a concentration of 0.2 OD, then treated with 1 μg / mL caspofungin and cultured at 37°C with shaking. After 6 hours of culture, it was found that the tropical Candida albicans SLT9 and SLT34 began to form multicellular aggregations associated with abnormal cell division. After 18 hours of culture, this phenomenon became more significant ( Figure 3 A). The strain morphology of the control group (ATCC750) showed swelling and cell wall defects, which was in sharp contrast to the multicellular aggregate morphology of the tolerant group. SHR001 is a resistant strain and is not affected by the drug.

[0062] 2. Macrophage co-culture experiment: The strains (SLT9, SLT34, ATCC750 and SHR001) were co-cultured with macrophages at an MOI of 10, and treated with different concentrations of caspofungin, respectively, and incubated at 37°C for 18 hours. Figure 3 As shown in B, in this culture system, tolerant Candida tropicalis still formed a multicellular aggregate morphology, and as the concentration of caspofungin increased, the multicellular aggregate morphology became more obvious.

[0063] 3. Experiment on the treatment of wax moth larvae infection: The wax moth larvae in Example 2 were subjected to histopathological examination, and the tissue sections were stained with HE and calcofluor white. The formation of multicellular aggregates of tolerant Candida tropicalis was also observed in vivo, further confirming the reliability of the in vitro experimental results. The scale mark (bar) in the figure is 20 μm.

[0064] 4. Transmission electron microscopy observation: Through transmission electron microscopy, it was further observed that the multicellular aggregation morphology of tropical Candida tolerant was caused by defects in intercellular separation (see Figure 4 ), the bar in the figure is 500nm.

[0065] Example 4

[0066] Gene expression analysis of caspofungin-resistant Candida tropicalis:

[0067] C. tropicalis (SLT9, SLT22, SLT34 and ATCC 750) in the logarithmic growth phase were adjusted to a concentration of 0.2 OD and treated with 1 μg / mL caspofungin or an equal amount of PBS. The treated strains were cultured at 37°C with shaking for 1 hour. Subsequently, total RNA was extracted, and RT-qPCR technology was used to detect the mRNA expression levels of specific genes. After caspofungin treatment, we found that the expression levels of genes CH S2, MNN22, MNN45 and CDA2 related to cell wall synthesis in tolerant C. tropicalis were significantly upregulated, as shown in Figure 2. Figure 5 This indicates that under drug pressure, the strain improves tolerance by enhancing the synthesis of cell walls. At the same time, the expression levels of genes CHS1, ACE2, and IQG1 related to cell division were significantly downregulated, which may affect the normal division process of cells, thus forming a multicellular aggregate morphology under the action of drugs.

[0068] Example 5

[0069] Screening for Candida tropicalis resistant to echinocandins:

[0070] Pick multiple clones of tropical Candida on the culture plate, smear them in a clean test tube containing YPD liquid culture medium, and prepare a bacterial suspension with a turbidity of 1.0. Add 1 μg / mL of caspofungin to the bacterial suspension and incubate at 37°C for more than 6 hours. Take 1 ml of bacterial solution and centrifuge it, add 4% paraformaldehyde to fix it at room temperature for half an hour or use proteinase K digestion for half an hour to remove the interference of cell adhesion and aggregation. Take an appropriate amount of bacterial solution smear, add calcium fluorescent white dye solution to stain, and observe the morphological changes of the strain under a fluorescence microscope. When observing the results, the spores are irregularly enlarged and not significantly aggregated for non-tolerant strains; the spores are oval and normal spore morphology is a resistant strain; and the multicellular aggregation morphology with spore separation defects is a tolerant strain. It is worth noting that in principle, the aggregation morphology can be observed after 6 hours of incubation, but as the incubation time increases, the aggregation morphology of tolerant strains will become more obvious and easier to identify.

[0071] Example 6

[0072] Validation of screening methods:

[0073] The method was validated for the tropical Candida strains confirmed to be resistant to caspofungin by the bactericidal test in Example 1, and 12 randomly selected tropical Candida strains verified to be non-resistant, and the specific steps followed the operating procedures described in Example 5. The results are shown in Figure 6As shown in the figure, all tropical Candida albicans strains resistant to caspofungin (SLT6, SLT13, SLT21, SLT22, SLT38, SLT44) showed obvious multicellular aggregates. Meanwhile, those tropical Candida albicans strains identified as non-resistant (SLT2, SLT8, SLT14, SLT17, SLT19, SLT23, SLT25, SLT29, SLT33, SLT39, SLT42, SLT47) showed swollen cells without obvious aggregates ( Figure 6 ). Therefore, all 6 caspofungin-resistant strains of Candida tropicalis showed multicellular aggregate morphology, with an accuracy of 100%, while none of the 12 non-resistant strains showed multicellular aggregate morphology, with an accuracy of 100%.

[0074] Example 7

[0075] Validation of the screening method in Candida auris:

[0076] Nine strains of Candida auris frozen in the laboratory were revived and subjected to drug sensitivity tests using the microbroth dilution method. It was found that four strains of Candida auris (Ca1, Ca2, Ca3 and Ca4) were sensitive to caspofungin (MIC < 1.0 μg / mL). Referring to Examples 1 and 5, these four strains of Candida auris were screened for resistance to echinocandin drugs, and a caspofungin bactericidal experiment was performed to verify strain tolerance.

[0077] The results are as follows Figure 7 As shown, Candida auris Ca2 showed obvious aggregation morphology, while Candida auris Ca1, Ca3 and Ca4 showed no obvious aggregation morphology. According to the screening method, Ca2 was a tolerant strain, and the other strains were non-tolerant strains. At the same time, the bactericidal experiment confirmed that the survival rate of Ca2 under the action of caspofungin was nearly 10,000 times higher than that of Ca1, Ca3 and Ca4 strains, which confirmed that Ca2 was a tolerant strain, while Ca1, Ca3 and Ca4 strains were non-tolerant strains. Therefore, the screening accuracy of this method in Candida auris was 100%.

[0078] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening Candida drug resistance, characterized in that: The screening method includes observing the morphological characteristics of the Candida strain after exposure to the drug.

2. The screening method according to claim 1, characterized in that The morphological characteristics include one or a combination of cell morphology, size, and colony morphology.

3. The screening method according to claim 2, characterized in that The morphological feature is cell morphology.

4. The screening method according to claim 3, characterized in that: The cell morphology is a multi-cell aggregation morphology.

5. The screening method according to any one of claims 1 to 4, characterized in that: The Candida species include one or a combination of Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida portugallis, Candida kefir, Candida rugosa, Candida dubliniensis, Candida asteroides, Candida pseudotropicalis, and Candida auris.

6. The screening method according to claim 5, characterized in that: The Candida species include Candida tropicalis and Candida auris.

7. The screening method according to claim 1, characterized in that: The drug is an antifungal drug.

8. The screening method according to claim 7, characterized in that: The antifungal drug is an echinocandin.

9. The screening method according to claim 8, characterized in that The echinocandins include caspofungin and micafungin.

10. The screening method according to claim 9, characterized in that: The echinocandin is caspofungin.

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