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12 Recommendations for Standardized Operation of Blood Culture
一. Indication for blood culture
1. Fever (≥38 °C) or low temperature (≤36 °C).
3. Leukocytosis (>10 × 10 9 / L, especially "nuclear left shift" immature or banded leukocytosis.
4. Granulocyte reduction (mature polynuclear leukocytes <1 × 10 9 / L).
6. Skin and mucous membrane bleeding.
8. Multiple organ failure.
Blood culture should be taken when there are several signs mentioned above. In the evaluation of suspected neonatal sepsis, in addition to fever or low-grade fever, bacteria are rarely cultivated, and urine and cerebrospinal fluid culture should be supplemented. Children with Streptococcus pneumoniae and Haemophilus influenzae bacteremia (especially children under 2 years of age) are more common in outpatient clinics, often accompanied by significant fever (≥38.5 °C) and leukocytosis (≥20×109/L). Patients with senile bacteremia may not have fever or low fever, such as with physical discomfort, myalgia or stroke may be an important indication for infective endocarditis.
二. Skin disinfection procedure
lood culture To prevent skin parasite contamination, disinfectant (iodine or iodophor) can be used to strictly and carefully disinfect the skin to minimize skin contamination. Infective endocarditis, particularly infections of heart valve repair, may be caused by microorganisms that are parasitic on the skin (eg, Staphylococcus epidermidis or Corynebacterium). Therefore, the contamination of blood culture must be minimized during the collection process. Blood used for culture should not be drawn in a catheter of a vein or artery unless blood is not obtained by venipuncture or used to evaluate indicators associated with catheter infection. If catheter blood is drawn, non-catheter venous blood should also be punctured at other sites for blood culture.
Skin disinfection is strictly carried out as follows:
1. First, wipe the venipuncture site with 70% alcohol for more than 30 seconds.
2. Then use an iodine or iodophor cotton swab to disinfect the skin (1% - 2% iodine 30 seconds or 10% iodophor disinfection for 60 seconds), and sterilize from 1.5 to 2 cm in diameter from the puncture point.
3. Finally, the iodine is dehydrated with 70% alcohol.
After strict three-step disinfection, blood sampling can be performed by venipuncture. Note that patients who are allergic to iodine can only be disinfected with 70% alcohol for 60 seconds. After the alcohol is evaporated and dried, the blood is puncated.
三. Culture Bottle disinfection procedure
1. Sterilize the blood culture bottle with 70% alcohol or iodine solution (do not use iodine).
2. Alcohol takes 60 seconds.
3. Before the blood is injected into the blood culture bottle, the remaining alcohol on the surface of the rubber stopper is removed with sterile gauze and then injected into the blood.
Key points: The key to blood culture is to prevent skin parasites or environmental pollution. False positives caused by contaminated bacteria increase the amount of antibiotics used by patients, prolong hospitalization days, delay disease diagnosis and increase economic burden. However, under ideal disinfection conditions, there are still 3% to 5% of blood cultures mixed with contaminating bacteria, which are derived from the skin (S. epidermidis, Propionibacterium acnes, Fusobacterium, diphtheria) or from the environment. (Gram-positive Bacillus, Acinetobacter), these microorganisms sometimes have pathogenic effects. The same microorganism was grown in two different parts of the blood culture, and the same microorganism was grown in the different types of sterile parts, and the microorganisms grew rapidly (within 48 hours). In the above case, it should be considered a real infection. Blood culture results should be discussed frequently between clinical laboratory staff and doctors, and establishing a good communication relationship is beneficial to all aspects. For example, retrospective investigation of cases: 70 cases of coagulase-negative staphylococci isolated from blood in 1995~1999, 39.3% (24 cases) were contaminated by bacteria within 48 hours after admission, and were detected after 48 hours (9 cases) 100% is a contaminated bacteria, and the detection time of contaminated bacteria is significantly longer than that of pathogenic bacteria. It is suggested that the coagulase-negative staphylococci contamination rate in blood culture is high. Therefore, it is very important to strictly perform the skin disinfection procedure before blood collection.
四. Blood collection
The amount of blood drawn from each culture flask is the only important variable for obtaining microbes from blood cultures in patients with bacteremia or fungal bacteremia. When the blood volume of the culture is increased from 2 ml to 20 ml, the positive rate of blood culture is increased by 30% to 50%, because the amount of blood cultured is increased by 1 ml, and the positive rate is increased by 3% to 5%.
The amount of blood obtained by venipuncture is different for adults and children. It is difficult for children, especially newborns, to obtain a large amount of blood. For infants and children, 1ml~5ml of venous blood is generally used for blood culture. When the bacterial concentration is high enough, less than 1ml of blood is enough to detect bacteremia. The amount of specimen is greater than 1 ml, and the amount of bacteria is also increased. For infected children, there are more microorganisms per ml of blood than adults. For adult blood culture, the amount of specimens is less than 10 ml, and it is difficult to culture bacteria. The minimum amount of each bottle should be 10 ml of blood, 20 ml~30 ml is most suitable, and the ratio of blood and broth is generally recommended to be 1:5 to 1:10. Almost all modern blood culture systems have a blood volume of more than 10 ml. Although 30 ml of venous blood can increase the amount of bacteria, it is not practical, which can easily lead to hospital-acquired anemia.
五. Number of blood cultures and blood collection time
The number of blood cultures and the time of blood collection are related to the pathophysiology of bacteremia. A single venous blood injection into multiple culture flasks should be considered as a single blood culture. Studies have confirmed that the injection of the right amount of blood into 2 to 3 bottles of blood culture bottles is sufficient to detect all bacteremia and fungal bacteremia. For intermittent bacteremia, the blood used for culture should be collected before the estimated chill or temperature peak is reached, because the bacteria flow into the blood is usually separated from the onset of chills by 1 hour. In the case of fever, the blood may be free of bacteria. In fact, blood culture is usually After chills or fever. Since the bacteria will quickly clear from the blood, blood culture should be taken as soon as possible after chills or fever. It is for this reason that we generally do not recommend venous blood for culture at any time. The research data show that blood collection at any time does not increase the detection rate of microorganisms. It has in fact been demonstrated that the results of microbial cultures at the same time or at any time within 24 hours are similar. At any time, blood collection should be carried out before the use of antibiotics. It is recommended to collect 2 to 3 bottles at the same time, and each bottle of 20ml~30ml blood samples for initial evaluation, which is more realistic. Treatment with antibiotics prior to sampling may result in negative blood culture results and delayed growth of microorganisms is more common.
Studies have confirmed that in patients with streptococcal endocarditis, patients received antibiotic treatment within the first two weeks of blood culture, the positive rate of blood culture decreased from 97% to 91%. Due to good blood culture techniques, the positive rate of blood culture in patients with infective endocarditis should be above 95%.
Recommendations for collecting blood cultures for patients with special systemic and local infections:
1. Patients suspected of acute primary bacteremia, fungal bacteremia, meningitis, osteomyelitis, arthritis or pneumonia: 2 or 3 blood culture flasks should be taken immediately for rapid blood culture.
2. Fever of unknown source, such as recessive abscess, typhoid fever and wave heat: fever begins to collect 2 or 3 blood cultures. After 24h to 36h, it is estimated that more than 2 blood cultures are collected immediately before the temperature rise (usually in the afternoon).
3. Suspected bacteremia or fungal bacteremia, blood culture results continue to be negative, blood culture methods should be changed in order to obtain rare or harsh microorganisms.
4. Infective endocarditis, 3 blood cultures were collected from patients with acute endocarditis within 1 h (within 2 h). If all the results were negative after 24 h, 3 blood culture flasks were collected. Patients who received antibiotics within two weeks prior to admission were enrolled in blood culture for three consecutive days, 2 times a day.
Key points: Most clinicians have insufficient blood collection for adult patients. The number of blood cultures is not enough. The timing of blood collection is not suitable. Usually, when the patient's body temperature is high, a bottle of blood culture is collected within 24 hours, which reduces the positive rate of blood culture. In line with the basic procedures for blood collection, the laboratory should do a lot of publicity and communication work.
六. Transportation standard
Blood collection bottles or collection tubes should be sent to the clinical microbiology laboratory immediately after blood collection. The short-term built-in blood culture bottle and collection tube does not affect the detection of bacteria at room temperature. Do not refrigerate. If the blood culture bottle has to be placed for a period of time before being sent to laboratory culture or automated instrument testing, it should be placed in a 35°C~37°C incubator. in. However, blood-containing collection tubes should not be placed in the incubator. If bacteria grow, some gas will be released and the collection tube may be ruptured or leaking. Immediately after receiving the blood culture flask, the laboratory visually observed the growth of microorganisms.
The use of automated continuous blood culture systems is a bit of a concern. Specimen shipments are sometimes delayed, which can lead to delayed microbial detection (although microbial growth is not affected), and few people pay attention to this. Although the automated continuous detection system has the principle of allowing the growth of microorganisms to be detected in the upper bottle, the culture bottle should be minimized during transportation.
七. Acceptance Criteria
After receiving blood culture in the laboratory, you should follow these steps:
1. Check the culture bottles to make sure they are safely placed.
2. Visually check the growth of microorganisms, pay attention to whether there is flocculent sediment on the blood layer, uniform or subsurface turbidity, hemolysis, solidification of liquid medium, surface film, gas generation, blood layer surface or white particles in deep layer. Etc. If there is such a situation, it indicates that there is microbial growth.
3. Check the label on the bottle to see if the information is complete and consistent with the patient data on the application form.
4. Guarantee to get the right amount of blood.
5. Check if the blood exceeds the required baseline.
6. Place in the incubator or on the machine for testing.
Since the detection of bacteremia and fungal bacteremia is very important for the diagnosis of clinical infectiousness, laboratory staff should carefully receive blood samples. For children and adults, regardless of the amount of blood drawn, they should be cultured and reported in the report. The blood volume is indicated, which may delay the detection of bacteremia or fungal bacteremia.
八. Non-standard culture treatment method
Blood samples sent to the laboratory should be handled carefully to reduce specimen errors and specimen contamination. Specimens are improperly handled and incorrectly collected, and the bacteria carried by the hospital and laboratory staff may cause specimen contamination. Therefore, it is recommended that the following irregular blood cultures should be handled in a timely manner.
1. The blood culture bottle or culture tube is unlabeled or mislabeled, the culture bottle or the culture tube is leaky, cracked or obviously contaminated. After the blood sample is collected, it is placed for more than 12 hours, and the specimen is collected with an inappropriate culture bottle or culture tube.
Treatment: Immediately contact the clinician and report: “Specific reasons for non-standard specimens”.
2. Collect specimens with a failed culture flask or culture tube.
Treatment: Contact the doctor and report: “Use the expired culture flask to collect blood samples, please collect the specimens and send them for inspection within the validity period.
3. The amount of blood samples sent is insufficient or only one bottle of blood is given.
Treatment: Contact the doctor and report: "The amount of blood culture specimens delivered is insufficient. Please replenish a sufficient amount of blood culture flasks."
4. Number of recommended flasks or types not sent
Treatment: Contact the doctor, report: If "only the oxygen bottle is not in compliance with the regulations, the basic procedures for blood culture are recommended to send two kinds of culture bottles: aerobic bottles and anaerobic bottles."
Key points: Each clinician should be responsible for the patient. After the laboratory staff finds an irregular blood sample, the clinician should be notified in time to repeat the blood culture for remediation.
九. Safety procedures to prevent infection
1. Bacterial room staff should wear latex gloves during all blood culture treatments.
2. Blood culture bottles or culture tubes that do not meet safety standards are rejected.
3. Open or puncture the culture flask or add a membrane protection device in the biosafety cabinet (eg, place the flask on the acrylate membrane or wear a mask).
4. A culture bottle with signs of microbial growth was visually observed in a biosafety cabinet.
5. The aerosol produced by the operation may contain high concentrations of pathogenic bacteria or fungi, using laboratory methods and techniques to take correct self-protection measures.
6. Plugs and tapes used in culture media may contain high concentrations of pathogens and should be protected from contact.
7. Properly handle contaminants associated with blood culture.
8. Immediately report blood culture results and antibiotic susceptibility test results.
十. Treatment of positive results
Traditional manual blood culture should be inspected at least once a day, and at least one aerobic seed culture should be carried out on culture bottles that have not grown for 48h~72h. Visually inspect the culture bottle and note the following points for microbial growth:
1. The mixture of blood and broth appeared cloudy.
2. There are flocculent deposits on the blood layer, and some streptococci have small "cotton-like" growth on the surface of the deposited red blood cells.
3. There is turbid growth in the broth.
4. The surface of the broth has a film growth.
5. Hemolysis occurs.
6. Producing gas, many fermenting bacteria can produce gas, Clostridium will produce a large amount of gas, and obvious hemolysis occurs.
7. There are white particles on the surface or deep layer of the blood layer.
8. The liquid medium is solidified.
If the blood culture is suspected to be positive, Gram staining should be carried out immediately, and 2 to 3 drops of the broth should be taken from the culture flask aseptically, naturally dried, fixed by heating and stained. The results of staining should be reported as soon as possible. For example, Gram-positive cocci are paired or piled or chained, and Gram-negative bacilli are small bacilli or cocci. Gram staining results in instructing physicians to choose antibiotic therapy for patient experience and as a reference for laboratory supplementation of bacterial identification, which is useful for identifying staphylococcus and streptococcus. If blood culture-positive Gram-staining does not detect bacteria, acridine orange staining should be performed for secondary microscopy, which is helpful for detecting Campylobacteremia and Brucellosis, and Gram staining rarely finds these bacteria. .
When Gram staining shows uniform bacterial morphology, the value of anaerobic culture is small. For all culture-positive, Gram-negative blood culture flasks, anaerobic and aerobic culture dishes should be seeded before testing again.
Gram staining is a pure bacterium that can be initially identified and used as a standard procedure for direct antibiotic susceptibility testing and is not commercially available under FDA approval. Commercial reagents that can be used for preliminary identification include: bile dissolution, DNase, latex agglutination typing experiments, and the like.
The antibiotic susceptibility test recommended by the American Committee for Clinical Laboratory Standards (NCCLS) does not have a direct sensitivity test for blood culture. However, many scholars have suggested that according to the preliminary analysis of Gram staining, it is possible to flexibly select antibiotics for direct antibiotic susceptibility testing.
The positive broth cultures in various blood culture methods were seeded and immediately incubated for further experiments. After 5h-7h of seeding, there were obvious colony growth, and the agar surface colonies were used for bacterial identification and antibiotic sensitivity. test. Use a blood culture system containing solid medium, such as: biphasic method, colony growth can directly carry out bacterial identification and standard antibiotic susceptibility test.
Although probe hybridization and DNA amplification techniques can now be used to identify bacteria in blood cultures and to detect antibiotic susceptibility, this technique is not practical and expensive, and the FDA does not recommend routine use.
Bacteria isolated from blood culture should be kept for at least a few weeks in case further research is needed. In order to keep the bacteria alive, facultative aerobic and facultative anaerobic bacteria should be transplanted into semi-solid media, such as kinetic medium. Strict anaerobic bacteria should be stored in broth without glucose or frozen in rabbit blood or skim milk medium. The bacterium can be inoculated onto the chocolate agar slant and sealed with mineral oil. For long-term storage, the bacteria can be suspended in 15% glycerol broth and frozen at -70 °C.
十一. Reporting procedure for positive results
Positive blood culture results are important and should be reported orally to the patient's attending physician immediately. The date and time of the report and the name of the recipient should be recorded on the patient's report. Important information should be provided to the clinician, including the morphology of the Gram stain, the number of positive blood cultures, and other identification data, such as Gram-positive cocci, suspected to be Staphylococcus. Before processing the report, review the recent development of the patient's specimens, which will help explain the source of the infected microorganism. A written initial report should be made immediately after the oral report. The results of the preliminary report should not be changed unless there is an error in the initial report or a new discovery can change the patient's treatment plan.
After 24h and 48h of culture, a preliminary report was issued on the results of the negative blood culture in the shortest time. Some laboratories make preliminary reports on the length of the culture, for example: "There is no bacterial growth after 3 days of blood culture."
十二. Fungal culture
A variety of methods can increase the detection rate of fungi in the blood, including the use of ventilated vacuum blood culture bottles, biphasic media, lysis-centrifugation techniques and special nutrient broth media (such as brain heart infusion broth). Lysis-centrifugation is an effective method for isolating fungi, especially for nutrient-demanding biphasic fungi. In fact, most aerobic blood culture flasks (usually incubated for 5 to 7 days) provide sufficient nutrients to support the growth of Candida albicans. However, for other Candida species, Saccharomyces cerevisiae, Cryptococcus neoformans, Histoplasma capsulatum and other biphasic fungi, the highest detection rate is obtained using a lysis-centrifugation technique. Mold inhibition agar, brain heart agar agar and chocolate agar are usually used in combination with lsolator tubes. For the isolation of M. sphaeroides, the surface of the medium may be covered with a layer of olive oil or other long-chain fatty acids. Negative fungal blood cultures should be discarded after incubation for 4 weeks at 22-30 °C.