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To guide disease control departments at all levels and other relevant institutions to carry out laboratory testing for pneumonia of novel coronavirus infection, this technical guide is developed.
I. Collection objects. Pneumonia suspected cases of novel coronavirus infection, patients with suspected aggregated cases, others who require diagnosis or differential diagnosis of novel coronavirus infection, or other environmental or biological materials that require further screening tests (such as traceability analysis).
Second, specimen collection requirements.
1. Technical personnel engaged in specimen collection for novel coronavirus testing should be trained in biosafety (training qualified) and have the appropriate experimental skills. Sampling personnel professional protective equipment (personal protective equipment, PPE) requirements: N95 masks, goggles, protective clothing, latex gloves, waterproof boot covers; if contact with patient blood, body fluids, secretions or excretions, wear double latex gloves.
2. Specimens of hospitalized cases are collected by the medical and nursing staff of the hospital under the guidance of professionals from the local CDC.
3. Specimens from close contacts are collected by the local CDC.
4. According to the needs of laboratory testing work, can be combined with the course of the disease multiple sampling.
Third, the type of specimen collection. Each case must collect acute respiratory specimens and acute blood specimens; in severe cases, priority is given to collecting lower respiratory specimens (such as bronchial or alveolar lavage fluid), which can be collected according to the clinical presentation and sampling interval.
Other study materials were collected according to design requirements.
Specimen types.
1. Upper respiratory tract specimens: including pharyngeal swabs, nasal swabs, nasopharyngeal extracts, and deep cough sputum.
2. Lower respiratory tract specimens: including airway extracts, bronchial lavage, alveolar lavage, and lung tissue biopsy specimens.
3. Blood specimens: Try to collect anticoagulated blood in the acute phase within 7 days after the onset of the disease. The volume of blood collected should be 5 ml, preferably fasting blood, and it is recommended to use vacuum blood tubes containing anticoagulants to collect blood.
4. Serum specimens: Try to collect double copies of serum from the acute and recovery phases. The first serum should be collected as soon as possible (preferably within 7 days after the onset of the disease), and the second serum should be collected in the third to fourth week after the onset of the disease. The amount of blood collected should be 5 ml, preferably on a fasting basis, and the use of a vacuum blood tube is recommended.
IV. Specimen collection methods.
1. Pharyngeal swab: Swab the tonsils and posterior pharyngeal wall bilaterally with two polypropylene fiber-tipped plastic rods, dip the swab head into a tube containing 3 ml of sampling solution, discard the tail, and screw the cap tightly.
2. Nasal swab: insert a polypropylene fiber-tipped plastic rod swab gently into the nasal tract at the nasal palate, stay for a moment and then slowly turn to withdraw. Take another polypropylene fiber-tipped plastic rod swab to collect the other nostril in the same way. The above two swabs are dipped into the same tube containing 3 ml of sampling solution, the end is discarded and the cap is screwed on tightly.
3. Nasopharyngeal extractions or respiratory extractions: Mucus is extracted from the nasopharynx or respiratory secretions from the trachea using a collector connected to a negative pressure pump. Insert the head of the collector into the nasopharynx or trachea, connect the negative pressure, rotate the head of the collector and withdraw it slowly to collect the extracted mucus, and rinse the collector once with 3ml of sampling solution (the collector can also be replaced by a pediatric catheter connected to a 50ml syringe).
4. Deep coughing sputum: After asking the patient to cough deeply, collect the coughing sputum in a 50 ml screw-top plastic tube containing 3 ml of sampling fluid.
5. Bronchial lavage fluid: Insert the collector head into the trachea (about 30cm deep) through the nostril or tracheal socket, inject 5ml of saline, connect the negative pressure, rotate the collector head and withdraw it slowly. Collect the extracted mucus and rinse the collector once with the sampling fluid (a pediatric catheter connected to a 50 ml syringe can also be used to collect instead).
6. Alveolar lavage fluid: After local anesthesia, insert the fiberoptic bronchoscope through the mouth or nose through the pharynx into the bronchus of the middle lobe of the right lung or the lingual segment of the left lung, fit its tip into the opening of the bronchial branches, and slowly add sterilized saline through the tracheal biopsy hole, 30~50 ml each time, total 100~250 ml, should not exceed 300 ml.
7. Blood specimens: It is recommended to collect 5 ml of blood specimens using vacuum blood tubes containing anticoagulant, leave at room temperature for 30 minutes, centrifuge at 1500-2000 rpm for 10 minutes, and collect plasma and blood cells in sterile screw-top plastic tubes, respectively.
8. Serum specimen: Collect 5ml of blood specimen by vacuum negative pressure blood collection tube, leave it at room temperature for 30 minutes, centrifuge it at 1500-2000rpm for 10 minutes, and collect the serum in a sterile screw-top plastic tube.
9. Other materials: collected according to the design requirements specification.
V. Specimen packaging. Specimens are collected and packed in a biosafety cabinet in a biosafety level II laboratory.
1. All specimens should be placed in a sample collection tube of suitable size with a screw cap with a gasket inside and freezing resistance, tightened. The specimen number, type, name and date of sampling should be indicated on the outside of the container.
2. The specimens will be sealed in a plastic bag of suitable size, each bag contains a specimen.
Six, specimen preservation. Specimens used for virus isolation and nucleic acid detection should be tested as soon as possible, and specimens that can be tested within 24 hours can be stored at 4℃; specimens that cannot be tested within 24 hours should be stored at -70℃ or below (if no -70℃ storage conditions, then stored temporarily in a -20℃ refrigerator). Serum can be stored at 4℃ for 3 days, and below -20℃ for long term storage. Specimens should be stored separately in special storage or special cabinets. Repeated freezing and thawing of specimens should be avoided during transport.
VII. Specimen delivery. Specimens should be sent to the laboratory as soon as possible after collection. If specimens need to be transported over long distances, it is recommended to use dry ice and other refrigeration methods for preservation.
1. Send specimens: the first positive test results in each province (autonomous region, municipality directly under the Central Government), suspected aggregated cases and aggregated cases of specimens, sent to the Chinese Center for Disease Control and Prevention, Viral Disease Prevention and Control Institute for testing and review, and attach the sample delivery form (see Annex).
2. Transportation of pathogens and specimens
2.1 Domestic transportation
The transport packaging classification of novel coronavirus strains or other potentially infectious biological materials belongs to category A, corresponding to the UN number UN2814, and the packaging conforms to the PI602 classification packaging requirements of the ICAO document Doc9284 "Technical Rules for the Safe Transport of Dangerous Goods by Air"; environmental samples belong to category B, corresponding to the UN number UN3373, and the packaging conforms to the ICAO document Doc9284 "Dangerous Goods by Air". Doc9284 "Technical Rules for the Safe Transport of Dangerous Goods by Air" PI650 classification packaging requirements; transported by other means of transport can refer to the above standard packaging.
The transport of new coronavirus strains or other potentially infectious materials should be in accordance with the "Regulations on the Transport of Highly Pathogenic Microorganisms or Samples that can Infect Human Beings" (former Ministry of Health Decree No. 45) for the "Certificate of Carriage".
2.2 International transportation
If the novel coronavirus strains or samples are transported internationally, the packaging should be standardized, and the relevant procedures should be handled in accordance with the "Regulations on Health and Quarantine Management of Special Items on Entry and Exit", and meet the relevant national and international requirements.
How was this pathogenic identification carried out?
The organizing laboratory uses genome sequencing, nucleic acid detection, virus isolation and other methods to conduct pathogenic testing on the patient's alveolar lavage fluid, pharyngeal swab (Warrican virus sampling tube), blood and other samples.
What procedures are required to confirm the pathogen?
(1) Suspected pathogens must be found in all patients, and pathogenic nucleic acids must be detectable in the patient's clinical sample.
(2) The pathogen can be successfully isolated from the patient's clinical sample.
(3) The isolated pathogen can cause the same disease symptoms when it infects the host animal. A 4-fold increase in antibody titers to the pathogen in the patient's recovery serum may help identify the pathogen.
What is the role of the Varicom virus sampling tubes in this pathogen identification?
The Warrican Virus Sampling Tubes served as the "foundation" for this pathogenic study. The HURIKOM virus sampling tubes and swabs were used to sample the patient's throat for subsequent nucleic acid testing and virus isolation.
History of the Warrican Virus Sampler
Harecon virus sampling tubes can be used for the collection, preservation and transport of human nasopharyngeal virus samples (influenza, avian influenza, hand, foot and mouth, measles, rubella and other viral samples).
H1N1
H1N1 is sweeping the world, Hualicon virus sampling tubes help the pathogenesis of influenza A detection.
H7N9 Avian Influenza
The H7N9 avian influenza epidemic has found a number of cases in China. The H7N9 Avian Influenza pathogenic test is facilitated by Hualikon virus sampling tubes.
