In recent years, with the high incidence of malignant tumors, the prevalence of AIDS, the widespread use of chemotherapy drugs, broad-spectrum antibiotics, glucocorticoid and immunosuppressants, and the application of invasive diagnosis and treatment technologies such as artificial catheter and organ transplantation, the infection caused by fungi has increasingly become a huge challenge for clinical departments.
In order to improve the level of clinical diagnosis, it is very important to strengthen the ability of fungal detection. The common problems of fungal testing in clinical microbiology laboratory are summarized as follows for your reference.
1. Why should the culture be set at 35℃?
In order to prevent the influence of temperature fluctuation on the growth of bacteria or fungi, the temperature of the incubator is usually set to 35℃ in the laboratory. The optimum growth temperature of common clinical bacteria is 33~37℃ (except thermophilic bacteria and mesophilic bacteria, which account for a few). When it is set at 35℃, the culture will not be affected by the fluctuation of 2℃. If it is set at 37℃, it is obviously inappropriate. As for fungal culture, it depends on the source of the specimen.
Samples of dander, nail clippings, dandruff and hair suspected of superficial fungal infection were isolated and cultured at 26~28℃. Specimens from deep tissues of human body, such as sputum, pleural effusion, blood and deep abscess of bone marrow, are suggested to be cultured at 35℃ which is closer to human body temperature. Secondly, the most common clinical Candida albicans can form a typical pseudopodophyllum colony at 35℃, which is the same as the serum germination test at 35℃.
At the same time, Candida glabrata isolated at 35℃ has high enzyme activity. It is suspected that temperature-type biphasic fungal infection or a single fungus needs to grow at a higher temperature, and both temperatures need to be cultivated at the same time. Also pay attention to the humidity of the culture environment, which is generally required to be greater than 60%. If the humidity of the incubator is not enough, it is best to place a container with sterile water near the flat plate to ensure the humidity.
2. Does 2.CO2 have a great influence on the growth of fungi?
Fungi belong to heterotrophic microorganisms, which mainly obtain carbon source from organic matter, while autotrophic microorganisms only need to obtain carbon source from CO2, so fungi generally do not need to be cultured in carbon dioxide environment, and CO2 is not conducive to the growth and reproduction of fungi, but a certain concentration can stimulate spore formation.
3. Fungi were cultured twice in blood culture, but the child had no clinical manifestations, and no antifungal drugs were used in clinic. The child was in good condition. Is pollution a problem?
In this case, it is necessary to judge whether it is contaminated bacteria, investigate whether the blood collection process is standardized, whether the blood bottle transfer process meets the requirements, whether the pinhole is well sealed, whether the transfer box is contaminated and many other factors. The most important thing is the patient's clinical manifestations. If there are no symptoms of fungal infection, pollution or colonization should be considered (unlikely).
4. In the actual work of our primary hospital, it is almost impossible to carry out biochemical identification on fungal culture of vaginal secretions, and many people directly report Candida albicans. Is this appropriate?
This is inappropriate. At present, some clinical laboratories can't even tell yeast from mold. When candida is found in vaginal secretions, urine and feces under a microscope, it is reported as "mold discovery".
Mold refers to filamentous multicellular fungi; Yeast refers to unicellular fungi, and the two cannot be confused. Most of the yeasts infected in vagina are Candida albicans, but other yeasts are not excluded.
Candida crocus has natural resistance to fluconazole and 5- fluorocytosine. If you encounter similar strains with natural drug resistance, it will affect the follow-up treatment.
5. Is there a small amount of Candida in sputum samples that need to be reflected in the report? Still not reporting?
Specimen requirements and inspection procedures should be reflected, and clinicians will consider them comprehensively. Although the probability of pulmonary fungal infection caused by Candida albicans is very small, it is suggested that clinicians report and consider this fungus in combination with imaging results.
Late sputum specimens should be smeared directly. If the quality of the specimen is qualified, if true hyphae, false hyphae or a large number of spores and fungi grow in sputum culture, it should be reported to the clinician for comprehensive consideration. As for whether to do drug sensitivity, you can communicate with your doctor before deciding.
① It is recommended to rinse your mouth with 5% soda water before leaving the sample;
② First sputum in the morning;
③ Whether the results of sending 3d images are consistent.
It is best to communicate with clinicians, or to train doctors and nurses in specimen collection methods. Before reporting, pay attention to the quality information of the specimen and the possibility of pollution combined with the results of sputum smear microscopy.
6. Excuse me: A few days ago, I accidentally found an extracted pus specimen, which was caught by blood culture. After the positive report, the smear showed fungal spores. After the transfer, the smear was round, and the ink staining was positive, but there was no envelope. After the transfer, Kemajia has no color, and the white is a bit yellow. This shows that it is almost smooth and drug sensitive. The questions are: ① Is this result credible? Does it matter whether ink is ordinary juice? ③ How to improve?
Ink staining is a negative staining test for the capsule of Cryptococcus neoformans, especially Cryptococcus neoformans, and can not be used as a differential test for pure culture Cryptococcus.
(1) Do 10%KOH wet biopsy on previous pus samples to find fungal spores and true and false hyphae.
(2) The culture results should be credible, and it is best to communicate with clinicians to see if patients have symptoms.
(3) I am unfamiliar with a domestic brand, and it is difficult to judge the identification results and drug sensitivity results.
(4) According to the national operating rules, biochemical tubes for identifying candida were prepared, and the coincidence rate of fungal identification controlled by the former Ministry of Health was still very good. You can consider making your own biochemical tube and using quality control bacteria for verification.
(5) Use each batch number to verify the quality control bacteria of a domestic identification tube. If the results are consistent, there should be no problem.
(6) It is best to participate in the microbial quality control training of the former Ministry of Health, which will help you improve your professional level and verify the quality of the reagents you use, whether bacteria or yeast.
(7) As long as the ink thickness is uniform, the finer the particles, the better. Cao Sugong ink is recommended, and Indian ink can be purchased from reagent company.
7. After Aspergillus was first detected in common clinical sputum specimens, how did this process proceed?
(1) Filamentous fungal colonies were cultured from sputum samples for the first time, provided that the sputum samples were fresh or could not be sent for inspection immediately, and should be kept in a refrigerator at 4℃.
The process is sputum specimen acceptance → microscopic examination → culture → identification.
After the sputum specimen is qualified, first do 10%KOH wet film microscopy, and look for fungal hyphae under low magnification, whether they are transparent or dark, and whether they have 45-degree branches.
If the sputum specimen is qualified and hyphae are found, call the clinic and tell the doctor that filamentous fungi are growing, which is suspected to be XXXX. It is easy to report directly to the clinic, but it is difficult to plant Shack agar plate or Chaz agar plate and then identify the progress.
(2) If the specimen is unqualified or qualified, but no hyphae are found in the specimen, communicate with the clinician by telephone to inform that there is fungus growth, so that the doctor can consider it comprehensively. The appraisal was carried out normally and the clinic reported it. This remark does not rule out the possibility of pollution.
8. Why did the yeast assimilation test use 28℃ instead of 35C? What is the difference between assimilation test and fermentation test? Why does the textbook say that anyone who can ferment a certain sugar must assimilate it, but assimilating a certain sugar may not necessarily ferment it?
The optimum growth temperature of most yeasts in vitro is 28-30℃, so this temperature is selected for in vitro experiments. Transformation and fermentation refer to aerobic respiration and anaerobic glycolysis of fungi respectively. The former decomposes sugar into carbon dioxide and water, while the latter decomposes sugar into carbon dioxide and alcohol.
Its metabolism is firstly aerobic respiration to get energy, and then anaerobic glycolysis, which is determined by related enzymes of bacteria and fungi. Most yeasts are obligate aerobic bacteria, so the experimental design is mainly based on sugar assimilation experiment. For example, Cryptococcus can only breathe aerobically, so it can only assimilate glucose but not ferment, while Saccharomyces cerevisiae can ferment grain into wine, which can ferment and assimilate glucose.
9. How to choose the culture medium, temperature, reporting time and method in view of the fact that the current fungal culture is not standardized? Please describe the specific process of the laboratory.
(1) medium: Shaikh's glucose agar, potato glucose agar, brain and heart extract agar and other media suitable for fungus growth. Clinically, it is suspected that yeast and yeast-like bacteria are infected. If conditions permit, the laboratory can inoculate chromogenic medium in parallel. The spiral tube slant culture method is recommended, and the specimens are inoculated in the middle and lower part of the slant of the culture medium after treatment. For most aerobic bacteria, it is recommended to inoculate two tubes in parallel.
(2) Culture conditions and reporting time: Deep fungi should be cultured at 28 65438 0℃ for 7 ~ 65438±04d. If biphasic fungal infection is suspected, it should be cultured at 65438 0℃ at 28℃ and 35℃ at the same time.
If it is suspected that rare fungi are infected with slow-growing fungi or clinical antifungal drugs have been used, the observation time should be extended for at least 4 weeks.
For example, cerebrospinal fluid should be cultured at 28℃ and 35℃ for at least one week;
Phlegm/urine/feces can generally be 35℃ for 48 hours. If no filamentous fungi grow, the culture time will be prolonged.
Tissue specimens are usually stored at 30℃ for 2~4 weeks.
10. Can Komajia chromogenic medium be used as the primary medium of fungi?
As one of the primary media, it is suggested to inoculate sabouraud medium or yeast nitrogen medium (YPDA). SDA and YPDA are usually the standard media for yeast-like bacteria.
1 1. Is it meaningful to cultivate fungi with throat swab?
It is of little significance to cultivate fungi with throat swab, unless the patient has clinical manifestations, there will be colonization.
12, I would like to ask how many spores and pseudohyphae are reported in sputum smear because the colonization rate of candida is so high.
Personally, I think it is still necessary to report to the clinician truthfully and inform the clinician whether the specimen quality is qualified. As for infection or not, clinicians will comprehensively consider the imaging data and the clinical manifestations of patients.
13. The patient has eczema-like papules on his hands, which is suspected of fungal infection. How can I take samples?
For the collection of skin lesions, it is necessary to disinfect the affected area with 70% alcohol first, then scrape the dander at the edge of the skin lesions with a sterilized stainless steel scraper or scalpel and put it into a sterile container for inspection.
14, can I use disposable gloves for SDA tablets?
Disposable gloves, whether PV or latex, will produce a relatively low oxygen environment, while fungi are aerobic bacteria, which will affect the growth of fungi. This operation is not recommended.
You can seal the flat plate with breathable adhesive tape (for fixing needles for intravenous infusion), and be careful not to seal it too tightly.
15, the ink used is not a uniform black background under the microscope, but some small black particles. Is this poor ink quality? Also, what's the difference between a biosafety cabinet, a clean workbench cabinet and a fume hood?
Ink itself is formed by adding some tiny particles into the solvent, and the quality of ink lies in the size and uniformity of the particles.
In general, when ink is used to detect Cryptococcus, it should be observed that it is only used as the background, the capsule is unstained and transparent, and the background is black.
The difference between biosafety cabinet and clean workbench lies in the direction of wind blowing:
The wind direction of the biosafety cabinet is vertically downward first, then enters the cabinet, and is discharged after being filtered and circulated by HEPA, thus forming a relatively negative pressure environment in the cabinet;
The wind direction of the clean workbench is downward and then outward to ensure the cleanness and dust-free in the cabinet. It is usually used to configure the flat plate and operate some simple molecular cloning related experiments.
The fume hood exhausts the air in the cabinet to the outside of the laboratory through a fan, which is usually used to prepare volatile chemical reagents and exhaust harmful gases to the outside.
16. What is the clinical significance of seeing aspergillus head in sputum smear? Is it possible to be contaminated by Mucor? Still a fixed value? Penicillium was detected in sputum, is it meaningless?
Aspergillus infection should be considered if aspergillus head is found in sputum samples by direct microscopic examination. Culture can determine what kind of Aspergillus it is.
Mucor may be polluted or exist in the air. However, due to its high invasiveness, the report should be cautious, and the possibility of setting a fixed value is very small, but there are still many cases of pollution in operation. If the specimen is qualified, and there are 90-degree branches and thick, undivided hyphae in wet film microscopy, it should be considered, otherwise there is the possibility of pollution, and it is best to contact the clinician in time.
Penicillium is a conditionally pathogenic fungus. If hyphae are directly detected in sputum by microscope and culture is positive, infection should be considered.
17. In the initial stage of experience accumulation, how to get enough strains to practice various dyeing and reading films?
If possible, it is best to go to the fungus room of the relevant hospital for further wet microscopy and fungus identification. After transferring the preserved strains in batches, carefully study the changes of colony color, size, texture and microscopic examination, and make records. Or take part in the professional training class of medical fungi for systematic study.
18, how to preserve fungal strains?
The best methods are vacuum drying and low temperature freezing. This method has been adopted by international strain preservation institutions.
(1) freezing the growth slope directly at-10℃ is not suitable for the preservation of dermatophytes and many zygomycetes.
(2) Distilled water preservation method: the mycelium, spores or yeast cells of mature colonies grown on potato glucose medium are scraped off or washed with sterile distilled water, then sucked out with a straw and placed in a sterile small tube, sealed to prevent evaporation, and stored at room temperature.
(3) Another method is to directly add sterile mineral oil to the agar slope where the mold grows, which can be stored for at least half a year.
(4) adding 15%~30% glycerol to the basic liquid culture medium, picking fresh and mature colonies and storing them at -80℃.
19, is it a fungal infection? What kind of patients will we focus on?
Immunodeficiency patients, organ transplant patients, tumor patients, neutropenia patients and patients who use broad-spectrum antibiotics for a long time.
20. What should I do after the mold incubator (28C) is polluted? Contaminated bacteria grow quickly after the culture medium is put into the incubator, especially on the sealing strip on the door. What should I do?
Fumigating with formaldehyde after power failure, wiping with 5% phenol (carbolic acid) for 30 minutes, wiping with clean water, and using after ventilation for 24 hours.
2 1. What filamentous fungi may be cultivated by blood culture? What is pollution?
The filamentous fungi with positive blood culture mainly include histoplasma, Marneffei blue bacteria, Fusarium, Candida, Cryptococcus, Trichosporon and Sedosporium. Paecilomyces purpurea was cultured in human eyeball tissue (ground and put into blood culture bottle). Pump the abdominal water into the blood culture bottle to cultivate pythium brownii.
Literature reports that blastomyces dermatitidis, Aemon and coccidiosis can all be cultured from blood culture.
Aspergillus fumigatus was polluted, and it was reported that Aspergillus was unearthed from the peripheral blood of patients with endocarditis [I clin microbiol.1998 nov; 36( 1 1):3347-5 1]。
22. Respiratory patients 1 case, male, 6 1 year, diagnosed as bronchopneumonia, HI Ⅳ negative, cyanobacteria Marneffei 3+ cultured from qualified sputum. Can it be diagnosed? Is it necessary to re-examine?
Does he have a big liver and spleen? Is there any bone involvement? Skin involvement and? Or is it just confined to the lungs? Does the patient have any other underlying diseases?
It is suggested that the specimen be sent again, and you should be alert if the light light blue marneffei is repeatedly cultivated. Penicillium marneffei cultivated in sputum should be considered as pathogenic fungi.
The difference between cyanobacteria Marneffei and Aspergillus;
If in the direct clinical specimen (in vivo), cyanobacteria Marneffei is a yeast-like spore, and Aspergillus is a mycelium with branches at a 45-degree angle.
For example, blue bacteria Marneffei can be light yellow and yellow-green, with red back and red pigment penetrating the culture medium at 28℃ in vitro;
Aspergillus is white at first, and the surface can be particles of different colors to distinguish which one is Aspergillus.
Blue fungus Marneffei can see broom branches, and Aspergillus can see Aspergillus's head.
Source of the above content: Inspection and Illustration of Medicinal Fungi edited by Lu Hongzhou, Qian Xueqin and Xu Heping.
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