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Chinese Standard: 'WST499-2017'
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WS/T 499-2017English479 Add to Cart Days<=4 Performance guideline for bacterial culture of lower respiratory tract infections Valid WS/T 499-2017
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Standard ID WS/T 499-2017 (WS/T499-2017)
Description (Translated English) Performance guideline for bacterial culture of lower respiratory tract infections
Sector / Industry Health Industry Standard (Recommended)
Classification of Chinese Standard C50
Classification of International Standard 11.020
Word Count Estimation 21,293
Date of Issue 2017-01-15
Date of Implementation 2017-07-01
Quoted Standard WS/T 503
Regulation (derived from) State-Health-Communication (2017) No. 1
Summary This standard specifies guidelines for routine bacterial culture of lower respiratory tract infections. This standard applies to medical institutions microbiology laboratory.

WS/T 499-2017
Performance guideline for bacterial culture of lower respiratory tract infections
ICS 11.020
C 50
People's Republic of China Health Industry Standard
Guidelines for bacterial culture of lower respiratory tract infections
2017-01-15 release
2017-07-01 Implementation
Published by the National Health and Family Planning Commission of the People's Republic of China
1 Scope
This standard specifies guidelines for routine bacterial culture of lower respiratory tract infections.
The wood standard applies to microbiological laboratories in medical institutions.
2 Normative references
The following documents are essential for the application of this document. For dated references, only the dated version applies to this document.
For undated references, the latest version (including all amendments) applies to this document.
WS/T 503 Code of Practice for Blood Culture in Clinical Microbiology Laboratory
3 terms and definitions
Column F terms and definitions apply to this document
Curschmann's spiral fibers
Kushman spiral fibers are spiral, which are mucus secreted by the small bronchus during chronic inflammation. Due to dyspnea and increased carbon dioxide tension in the lungs
High and condensed, and at the same time rolling and rolling due to intermittent blows of breathing gas. Caterpillars can be seen under the microscope after Gram staining
It is curled, blue-stained on the axis, and the edges are pale red.
Charcot-Leyden Crystal
Visible in the bronchi, rhombic or needle-like hexagonal colorless transparent crystals, which are sharp at both ends, vary in size, are highly refractive, and are composed of eosinophils.
After cell rupture, eosinophilic particles are fused with each other and can be stained with iodine. Heap of eosinophils can be found in slightly placed sputum. On the smoke
It is common in sputum from asthmatic patients with allergic bacteria or from patients with pulmonary infection with parasites.
Ciliated columnar epithelium cell
It is mainly distributed on the inner surface of the lower respiratory tract. On the free surface of columnar cells, there are cilia that can swing and are goblet cells that can secrete mucus.
The secreted mucus can stick and remove foreign matter such as dust and bacteria. With the rhythmic swing of the cilia, the mucus containing dust and bacteria can be eliminated.
To the throat.
Pulmonary macrophages
It is differentiated from monocytes and widely distributed in the interstitial lung. It is more around the tubes below the respiratory bronchioles and in the alveolar septum.
Alveolar giant thallium cells that swim into the alveolar cavity are called alveolar giant thallium cells. Lung macrophages are very active in phagocytosis, immunity and secretion.
Important defense functions. Inhaled dust particles, bacteria and other siblings enter the alveoli and interstitial lungs, and are mostly swallowed and cleared by macrophages, and some are removed from the lungs.
The cavities are coughed out by mucus flow and cilia movement in the respiratory tract.
4 Before analysis
4.1 Specimen collection
4.1.1 Sputum
Suitable for patients with pulmonary infection, especially in intensive care unit (ICU) and hospital-acquired community-acquired pneumonia (CAP), chronic obstructive pulmonary disease
Patients with acute exacerbation of the disease (eight ECOPDs), lung abscess (non-optimal specimens for sputum), patients with pulmonary infection caused by suspected bacterial pathogens (see appendix
A). Before sputum sputum, the patient rinses with sterile saline; instruct the patient to cough up deep sputum, and do not leave saliva and nasopharyngeal secretions.
4.1.2 Tracheal aspirate
Tracheal aspirate specimens can only be collected in patients with tracheal intubation (such as fever or infiltration). Sucking sputum from trachea,
Keep it in a sterile container for inspection.
Note. The trachea colonizes the bacteria 24 hours after intubation. If the tracheal aspirate is not tested when the lung infection is not indicated, the result may not be consistent with the disease.
4.1.3 Blood culture
When a patient with pulmonary infection is accompanied by fever (> 38 ° C) or hypothermia (<36 ° C), leukopenia or granulocytopenia, etc.
When indicated, blood culture is submitted for examination (for specific reference to the requirements for specimen collection and submission in WS/T 503).
4.1.4 Bronchoscopy specimen (see Appendix B) Precautions for collecting bronchoscopy specimens
The precautions for collecting bronchoscopy specimens are as follows.
a) Bronchoscopy can collect specimens of infected sites;
b) In order to prevent contamination, inhalation anesthetic should be used, and do not suck the lavage fluid standard from the working chamber;
c) The specimen collection sequence is. BW, BAL, PSB, and biopsy specimens. Blood should be avoided. BAL
Infuse small saline into the small bronchus and alveolar lavage with fiber bronchoscope, in 40 mL ~ 80 mL of recovered lavage fluid
Contains approximately 1 mL of secretions from the bronchial tip and alveoli; discard the part that may be contaminated in the previous section, and collect the rest for immediate inspection. PSB
Insert the bronchoscope into the suspicious infection site of the bronchus of the subsegment and push out the brush in the double-layer cannula through the bronchoscope brush hole
Polyethylene glycol), brush the purulent secretion, brush the hair back into the double-layer cannula and exit the fiber bronchoscope after sampling, and cut the hair with sterile scissors
Brush, put it in a sterile container containing 1 mL of physiological saline (for aerobic culture only), and quickly submit for inspection.
4.2 Specimen shipping
Collect the specimens in a sterile leak-proof container, and paste the specimen information (barcode) to the microbiology laboratory within 2 hours (room temperature). If delayed
Submission will cause excessive growth of non-harsh oropharyngeal colonization bacteria, although the number of clinically significant pathogenic bacteria will be relatively reduced.
4.3 Specimens screened and rejected
The rejected samples were screened as follows.
a) Sputum bacterial culture standard collected repeatedly within 24 hours;
b) saliva;
c) Nasal rinses and secretions, nostril swabs;
d) throat specimens;
e) Bronchial brush culture standards collected without protective sleeves;
f) sputum anaerobic bacteria culture specimens;
g) Inducing sputum.
Note 1. Except for bronchial aspirate aspirate, secretion collected by PSB, double-layer cannula protection, bedside vaccination (anaerobic culture), biopsy specimen, pleural fluid or other
Except for uncontaminated specimens, other specimens are not suitable for anaerobic culture.
Note 2. Induced sputum specimens are only suitable for the detection of Pneumocystis carinii and Mycobacterium tuberculosis, and the detection of other pathogenic bacteria is poor.
5 Analyzing
5.1 Treatment of sputum and tracheal aspirate specimens
5.1.1 Basic requirements for specimen processing
The basic requirements for specimen processing are as follows.
a) Vaccination standards and smear operations should be carried out in an n-level biological safety cabinet;
b) All specimens should be processed as soon as possible, especially those from outpatient and emergency departments, newly admitted patients, ICU patients, and invasive methods (such as.
BAL and lung biopsy specimens) to ensure the activity of pathogenic bacteria and avoid causing repeated collection of specimens;
c) The laboratories using the all-A inoculation pretreatment system should process the specimens according to the manufacturer's instructions.
5.1.2 Inoculation specimen
Use sterile swabs to pick appropriate (sufficient to inoculate plates and smears) sputum or tracheal aspirate from purulent or bloodless sites, inoculate blood plates
Acrylic plates, MacConkey or Chinese blue plates (or other medium for target pathogens, such as fungi, etc.) were cultured immediately after partitioning and streaking.
5.1.3 Training
5.1.4 Specimens smear and Gram stain
After inoculating the plate, smear the slide with the swab that has just been inoculated. The smear should be thin and uniform (the print below can be seen clearly through the smear site)
Handwriting). Natural drying or t. after ten drying on a warm dryer, fixed with methanol (chemically pure, stored in brown glass bottles or plastic containers) or
The flame is fast fixed 3 times, gram stained, and read after drying.
Gram dyeing and decoloring time varies with the choice of different decoloring agents, specifically.
a) The decolorization time of 95% ethanol is 30 s;
b) Acetone-ethanol (volume ratio of 3. 7, brown bottle stored at room temperature, valid for 1 year) decolorization time 1 s ~ 5 s (even decolorization);
c) The decolorization time of acetone (reagent pure) is shorter, and the reagent to be decolorized is colorless.
Note. The laboratory stained with Gram stainer is carried out according to the manufacturer's operating instructions. Pay attention to the optimization of the conditions to achieve a satisfactory result of the smear staining. Conventional plate counting
Plate 1. Use a sampler to take 10 pL specimens (or use a 10 fxL inoculation loop) to inoculate blood plates and chocolate plates, respectively, with sterile L-shaped glass
The plate was coated with a rod, and the number of colonies after culture was equal to the number of colonies of the same form multiplied by the dilution factor (102). 10 colonies in the culture
To the threshold (103 CFU/mL).
Plate 2. Although 1 set inoculation ring (calibrated), take 1 // L standard wood to inoculate blood plate and chocolate plate, and coat with sterile L rod
On the plate, the number of colonies after culture is equal to the number of colonies of the same form multiplied by the dilution factor (103 CFU/mL). Counting dilution plates
5.2.3 Inoculate and culture other media
After the blood plate and chocolate plate are diluted and plated, 100 BAL specimens or PSB specimens can be taken to inoculate MacConkey or Lao Guolan Pei
The substrate is underlined. The culture steps are the same as 5.1.3; the lung tissue specimens collected in an invasive manner are ground by adding an appropriate amount of broth or sterile physiological saline,
The grinding solution was inoculated into blood and chocolate plates, and the culture was extended to 4 days.
5.2.4 Gram-stained specimen processing
After completing the halo culture of the specimen, take a suitable halo BAL specimen for cell centrifugation and protect the brush specimens by smearing directly. After natural drying,
Methanol fixation or flame fast fixation 3 times, Gram stain.
5.2.5 The remaining BAL specimens can be used for other pathogen detection
After centrifuging the remaining BAL specimens, related diseases such as Mycobacterium, Legionella, Fungi, and Pneumocystis carinii and viruses can be performed as needed.
The original culture, smear, molecular, immune and other inspections.
5.3 Continuous culture and observation of slow-growing bacteria
Observe the plate after 18 h to 24 h of culture, in order to detect possible growth of filamentous fungi, slow-growing bacteria, harsh gram-negative bacilli such as Bo
Deuteromyces sp.), The plate was continuously cultured after inspection and observed for 72 h, and K. spp. May be found.
5.4 Isolate and identify important pathogens of the lower respiratory tract (see Appendix C)
5.4.1 Streptococcus P-hemolytic streptococcus
P-hemolytic streptococcus is an important pathogen in the respiratory tract. It is important to observe the presence of tritium-hemolytic colonies on blood plates.
Although the quality of 5% sheep blood is very critical, it is difficult to observe the transparent hemolysis of colonies. Specific steps are as follows.
a) Examine P-hemolytic colonies and identify cocci that are catalase-negative and chain or paired;
b) Identified as Streptococcus pyogenes. paste the bacitracin paper (0.04 U/piece) after dense coating, and culture overnight at 35 ° C. If there is a bacteriostatic ring, report it
Inform Streptococcus pyogenes, any number M should be reported. Streptococcus pyogenes pyrrolidone arylamine enzyme (PYR) test was positive;
c) Children's specimens are checked for narrow hemolytic rings, and group B streptococci (streptococcus agalactiae) are identified. If the CAMP test (golden grapes)
Cocci), any number M should be reported. Streptococcus pneumoniae
Identify Streptococcus pneumoniae and Streptococcus pneumoniae, check for hemolytic colonies that are similar in form to Streptococcus pneumoniae, and do the following tests.
5.4.2 Harmful Gram-negative Bacteria Haemophilus influenzae
Haemophilus influenzae is a coccus that produces K on chocolate plates. It does not grow on blood plates, but it can grow in satellites around staphylococci. Bordetella
The Bordetella which has important clinical significance grows on the blood plate, and both the catalase and urease are positive, and the colonies are visible to the naked eye after 48 h of culture. Other Gram-negative Bacteria
Unless predominantly grown or in large numbers, it is usually not necessary to identify other harsh gram-negative bacilli, such as Eikenella, as these are the above
The normal flora of the respiratory tract rarely causes respiratory diseases.
5.4.3 Gram-negative diplococci
Gram-negative diplococci with clinical significance, mainly Moraxella catarrhalis and Neisseria meningitidis, are screened according to the following rapid tests.
5.4.4 Gram-negative bacilli
Gram-negative bacilli that grow well on MacConkey or China Net Blue plates are screened according to the following rapid tests.
a) If only one kind of bacteria has been grown and reached a clinically significant number M, but no other pathogenic bacteria, through preliminary test screening, this bacteria
For Enterobacteriaceae bacteria, especially Klebsiella pneumoniae, identification and drug sensitivity tests are required;
b) For T hospitalized patients, regardless of the presence of other pathogenic bacteria, check for significant numbers of Pseudomonas aeruginosa, Acinetobacter baumannii, and onions
Burkholderia and Stenotrophomonas maltophilia, because these bacteria are typical multi-drug resistant bacteria, which can cause epidemic in hospitals;
c) If one or more other Gram-negative bacilli are grown, conduct preliminary tests and report (such as. indole, oxidase,
Smell and morphology on the plate, colony pigments and results of the Krebs test).
5.4.5 Staphylococcus
The treatment method for the cultivated Staphylococcus is as follows.
) If Gram staining shows that the dominant piles of cocci are related to leukocytes, but there are no other significant pathogenic bacteria, only for clinical
Identification of Staphylococcus aureus
b) In the case of hospitalized patients, cefoxitin should be used to detect resistance to oxacillin, even in small quantities, in accordance with the principles of infection control;
c) Only when the coagulase-negative staphylococcus is almost purely cultured on the plate, the species level and/or drug sensitivity test need to be identified, which
He staphylococci are normal flora of the respiratory tract.
5.4.6 Enterococcus
The treatment of cultured Enterococcus is as follows.
Under normal circumstances, a small amount of Enterococcus need not be reported, when the growth is almost pure culture, identification and confirmation with preliminary biochemical tests;
Many Gram-positive cocci are normal respiratory flora, PYR-positive and even bile aescin and leucine peptidase (LAP) -positive.
5.4.7 Gram-positive bacteria
Only treat Gram-positive bacteria grown in the following cultures, as other Gram-positive bacteria usually do not cause pneumonia, as follows.
a) Screen and exclude any number of Nocardia and Rhodococcus (mucus-like colonies, urease positive) from A immunosuppressed patients
b) For large Gram-positive Bacillus, exclude Bacillus anthracis and Bacillus cereus
c) Limited identification of coryneform bacteria. Gram-positive rods are used when the following two conditions are present and the bacteria are growing in large numbers
Commercial kits for identification of bacteria, including.
1) When the strain's rapid urease test is positive (Corynebacterium pseudodiphtheria, Corynebacterium pseudotuberculosis urease positive);
2) Specimens are from intubated patients in the ICU ward.
5.4.8 Fungi
Treatment of growing filamentous fungi or yeast-like fungi is as follows.
a) Identification of filamentous fungi (except laboratory or environmentally contaminated molds, such as penicillium);
b) Isolate colonies of biphasic fungi (such as capsular cytoplasmic bacteria and cocci) or yeast-like fungi from flat roars that have been cultured for more than 48 h;
c) Inspection of stale cultures to exclude Cryptococcus neoformans without further identification of other yeast-like fungi
d) Candida belongs to the normal colonized flora of the oral cavity. Generally, for people with normal immunity, even from the lower respiratory tract specimens,
For Candida, no further identification is required unless there is evidence of histopathology; however, for patients with cancer (eg, leukemia), lungs,
Transplant patients or newborns should be further identified from yeast-like fungi and candida in A lower respiratory tract specimens.
5.4.9 Smears visible but not growing in culture
If bacteria are visible when smeared, but no bacterial growth in this form is seen after cultivation, it may be due to the use of antibacterial drugs; but it cannot be ruled out
There may be Legionella, Bordetella pertussis, and Mycobacterium. The laboratory should contact the clinic in time to expand the scope of pathogen inspection.
5.5 Quality control
5.5.1 Gram stain
The frequency and results of the quality control in the Gram dyeing room are as follows.
a) In-house quality control should be done for newly purchased or new batches of commercial Gram staining reagents, and the regular quality control frequency is once a week;
b) Gram-staining positive quality control strain. Staphylococcus aureus ATCC25923, deep purple coccus is qualified;
c) Gram-negative quality control strain. Escherichia coli ATCC25922, red bacteria is qualified.
5.5.2 Acid-resistant staining
The frequency and results of the quality control in the acid-proof dyeing room are as follows.
a) Indoor quality control should be carried out for newly purchased or new batch of commercial acid-proof staining reagents, and the regular quality control frequency is once a week;
b) Acid-fast staining positive control strain. Mycobacterium tuberculosis ATCC25177 (weak strain), or use fast-growing Mycobacterium as positive
Quality control strain, Mycobacterium red is qualified;
c) Acid-fast staining negative control strain. Escherichia coli ATCC25922, blue bacteria is qualified.
5.5.3 Medium Blood plate
The 5% sheep blood plate purchased or manufactured by A for each batch of indoor quality control strains and the results are. Chocolate tablet
Purchased or homemade chocolate tablets, each batch do the indoor quality control strains and the results are.
Haemophilus influenzae ATCC49247, full growth, typical moist, translucent colonies were qualified.
5.5.4 Reagents Quality control reagents for each batch and weekly
Reagents for each batch and weekly quality control include. Quality control reagents for each lot and test
The quality control reagents required for each batch and test include.
a) 3% hydrogen peroxide (enzyme test). The positive control strain is Staphylococcus aureus ATCC25923, which produces severe bubbles; the negative pair
The strain was Enterococcus faecalis ATCC29212, which did not produce air bubbles or slowly produced a small amount of air bubbles.
b) Rabbit plasma or latex agglutination reagent (agglutination factor test, slide coagulase test). The positive control strain is Staphylococcus aureus
ATCC25923, irregular agglutination, the negative control strain is Staphylococcus epidermidis ATCC12228, which does not agglutinate.
Related standard:   WS/T 493-2017  WS/T 494-2017
Related PDF sample:   GB/T 20468-2006
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