Method for accurately detecting cryptococcus neoformans

Abstract

The invention relates to the technical field of fungal etiology detection, and discloses a method for accurately detecting cryptococcus neoformans, which comprises the following steps: S1, selecting a sample to be detected; s2, direct smear microscopic examination; s3, culturing; and S4, detecting. One of cerebrospinal fluid, blood and sputum of a patient is adopted as a sample to be detected, and timely detection or proper preservation is needed after collection. In the step S2, the sample is mixed with a proper amount of Indian ink, the sample is observed under a microscope, the capsule of the cryptococcus neoformans is found, and the cryptococcus neoformans can present obvious capsule and a transparent halo in a black background and can be easily recognized. And S3, inoculating the sample on a proper culture medium, and observing whether cryptococcus neoformans grow or not. The method adopted by the invention is rapid, simple and convenient, not only has higher sensitivity and specificity, but also can improve the accuracy and reliability of cryptococcus neoformans detection.

Description

Technical Field

[0001] The invention relates to the technical field of fungal pathogen detection, and in particular to a method for accurately detecting Cryptococcus neoformans. Background Art

[0002] Cryptococcus is a pathogen that can cause cryptococcosis. The medium is usually dry pigeon droppings, and there are few reports of human-to-human transmission. There is no specific medicine yet, and symptomatic treatment is generally used. Cryptococcosis is a pulmonary or disseminated infectious disease caused by Cryptococcus, which mainly causes pneumonia and meningitis, and also causes skin, bone or internal organ infections. Clinically, the diagnosis is made by combining clinical manifestations and microscopic examination results, and then confirmed by fungal culture or tissue staining. Clinical treatment drugs include amphotericin B, triazole antifungal drugs and flucytosine. Cryptococcus neoformans belongs to the genus Cryptococcus. Cryptococcus neoformans is a yeast-type fungus with a thick capsule composed of a layer of polysaccharides. It is not colored by general staining methods and is difficult to detect, so it is called cryptococcus. Cryptococcus neoformans is widely distributed in nature and is most common in pigeon droppings. When people have low immunity, they are infected by inhaling air contaminated by pigeon droppings, mainly causing subacute or chronic infections of the lungs and brain. The lungs are the primary lesions, which can spread to the brain infection, causing chronic inflammation and abscesses. Cryptococcus neoformans and its variants are pathogenic and mainly invade the central nervous system. Cryptococcus neoformans is an environmental saprophyte that lives widely in soil and pigeon droppings. Normal people are often in an environment contaminated by Cryptococcus neoformans, but very few people develop the disease. When the body's resistance is reduced, it is easy to invade the human body and cause disease. Due to the use of tumors and chemotherapy drugs, the prevalence of AIDS, and the use of immunosuppressive drugs after transplantation, the incidence of Cryptococcus neoformans is getting higher and higher. It has become one of the common complications of AIDS patients abroad and an important cause of death in patients. The incidence of Cryptococcus neoformans in my country is also increasing year by year. Cryptococcus neoformans presents round yeast-like cells whether in tissues or under artificial culture conditions, with a thick colloid capsule on the periphery, called thick capsule. The diameter of the bacteria is 4-20um, the capsule is 3-5um wide, and there are one or more reflective particles in the bacteria, which are nuclear structures. Some bacteria can be seen budding. Clinical and environmental isolated strains generally grow in the yeast phase of the asexual period. In 1976, Kwon-Chung discovered the sexual phase of the new variant, and hyphae formation can be seen during the sexual phase. The culture induction experiment is the main method for observing the sexual phase of Cryptococcus. Non-pathogenic cryptococci have no capsule. Cryptococcus neoformans can grow on Sabouraud medium and blood agar medium at both 25°C and 37°C, while non-pathogenic cryptococci cannot grow at 37°C. Yeast-type colonies are formed after several days of culture, with a sticky surface that is milky white at first and then turns orange-yellow. This bacterium can decompose urea and can be distinguished from Candida. Since cryptococcal infection is easily misdiagnosed based solely on clinical manifestations, etiological testing is the main basis for diagnosis and treatment, resulting in low accuracy and reliability of the data.

[0003] To this end, we proposed a method for accurate detection of Cryptococcus neoformans to solve the problem. Summary of the invention

[0004] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides a method for accurately detecting Cryptococcus neoformans, which solves the problems in the above-mentioned background technology.

[0005] (II) Technical solution To achieve the above object, the present invention provides the following technical solution: a method for accurately detecting Cryptococcus neoformans, comprising the following steps: S1: Select the sample to be tested; S2: direct smear microscopy; S3: Cultivation; S4: Detection.

[0006] Preferably, the sample to be tested can be one of the patient's cerebrospinal fluid, blood, and sputum. After collection, it needs to be tested in time or properly stored.

[0007] Preferably, the method of step S2 is to mix the sample with an appropriate amount of India ink, observe the sample under a microscope, and look for the capsule of Cryptococcus neoformans. Cryptococcus neoformans will show a clear capsule and a transparent halo against a black background, which can be easily identified.

[0008] Preferably, the method of step S3 is to inoculate the sample on a suitable culture medium to observe whether Cryptococcus neoformans grows.

[0009] Preferably, the culture medium adopts Sabouraud medium. Culture on Sabouraud medium has certain requirements for temperature and humidity. Specifically, the culture medium can grow at a temperature between 25°C and 37°C, but around 30°C is most suitable. The humidity needs to keep the culture medium and the surrounding environment in a relatively humid state. Usually, the humidity is more suitable at around 80%-90%, so as to prevent the culture medium from drying out and cracking due to rapid evaporation of water, which affects the growth of the bacteria.

[0010] Preferably, the detection in step S4 includes antigen detection and molecular biology detection.

[0011] Preferably, antigen detection is specifically performed by mixing a patient sample with a sensitized latex reagent to observe whether agglutination occurs. If Cryptococcus neoformans antigen is present in the specimen, an agglutination reaction will occur, thereby determining a positive result.

[0012] Preferably, the molecular biological detection is specifically as follows: nucleic acid is extracted from the sample, primers are designed according to the specific gene sequence of Cryptococcus neoformans, PCR amplification is performed using the primers to replicate the target gene fragment in large quantities, and the presence of the target gene is quantitatively analyzed by detecting the change in the fluorescence signal.

[0013] (III) Beneficial effects Compared with the prior art, the present invention provides a method for accurately detecting Cryptococcus neoformans, which has the following beneficial effects: The method adopted by the present invention is rapid and simple, not only has high sensitivity and specificity, but also can improve the accuracy and reliability of Cryptococcus neoformans detection. DETAILED DESCRIPTION

[0014] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0015] A method for accurately detecting Cryptococcus neoformans comprises the following steps: S1: Select the sample to be tested. Here, the sample to be tested can be one of the patient's cerebrospinal fluid, blood, and sputum. After collection, it needs to be tested in time or properly stored; S2: Direct smear microscopy: Mix the sample with an appropriate amount of India ink and observe the sample under a microscope to look for the capsule of Cryptococcus neoformans. Cryptococcus neoformans will show a clear capsule and a translucent halo against a black background, which can be easily identified. S3: Cultivation: Inoculate the sample on a suitable culture medium to observe whether Cryptococcus neoformans grows. Here, the culture medium is Sabouraud culture medium. Cultivation on Sabouraud culture medium has certain requirements for temperature and humidity. Specifically, the temperature can generally grow between 25°C and 37°C, but around 30°C is the most suitable. The humidity needs to keep the culture medium and the surrounding environment in a relatively humid state. Usually, the humidity is more suitable at around 80%-90%, so as to prevent the water in the culture medium from evaporating too quickly and causing cracking, which affects the growth of bacteria. S4: Detection: including antigen detection and molecular biology detection; Among them, antigen detection specifically involves mixing the patient sample with a sensitized latex reagent to observe whether agglutination occurs. If there is Cryptococcus neoformans antigen in the specimen, an agglutination reaction will occur, thus judging it as a positive result; Molecular biology testing specifically involves extracting nucleic acid from samples, designing primers based on the specific gene sequence of Cryptococcus neoformans, using the primers to perform PCR amplification to replicate the target gene fragment in large quantities, and quantitatively analyzing the presence of the target gene by detecting changes in the fluorescent signal.

[0016] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for accurately detecting Cryptococcus neoformans, characterized in that: The following steps are involved: S1: Select the sample to be tested; S2: direct smear microscopy; S3: Cultivation; S4: Detection.

2. A method for accurately detecting Cryptococcus neoformans according to claim 1, characterized in that: The sample to be tested can be one of the patient's cerebrospinal fluid, blood, or sputum. After collection, it needs to be tested in time or properly preserved.

3. A method for accurately detecting Cryptococcus neoformans according to claim 1, characterized in that: The S2 step is to mix the sample with an appropriate amount of India ink, observe the sample under a microscope, and look for the capsule of Cryptococcus neoformans. Cryptococcus neoformans will show a clear capsule and appear as a translucent halo against a black background, which can be easily identified.

4. The method for accurately detecting Cryptococcus neoformans according to claim 1, characterized in that: The method of step S3 is to inoculate the sample on a suitable culture medium to observe whether Cryptococcus neoformans grows.

5. A method for accurately detecting Cryptococcus neoformans according to claim 4, characterized in that: The culture medium adopts Sabouraud medium. Culture on Sabouraud medium has certain requirements for temperature and humidity. Specifically, it can grow at a temperature between 25℃-37℃, but around 30℃ is most suitable. The humidity needs to keep the culture medium and the surrounding environment in a relatively humid state. Usually, the humidity is more suitable at around 80%-90%, so as to prevent the culture medium from evaporating too quickly and causing cracking, which affects the growth of bacteria.

6. The method for accurately detecting Cryptococcus neoformans according to claim 1, characterized in that: The detection in step S4 includes antigen detection and molecular biology detection.

7. A method for accurately detecting Cryptococcus neoformans according to claim 6, characterized in that: Specifically, the antigen test involves mixing the patient sample with a sensitized latex reagent to observe whether agglutination occurs. If Cryptococcus neoformans antigen is present in the specimen, an agglutination reaction will occur, resulting in a positive result.

8. The method for accurately detecting Cryptococcus neoformans according to claim 6, characterized in that: Molecular biology testing specifically involves extracting nucleic acid from samples, designing primers based on the specific gene sequence of Cryptococcus neoformans, using the primers to perform PCR amplification to replicate the target gene fragment in large quantities, and quantitatively analyzing the presence of the target gene by detecting changes in the fluorescent signal.