Appendix 3. Collection, storage, and shipment of respiratory specimens

Update: September 2023

 

3.1 Respiratory specimen collection  

Staff members (and attendants if necessary) present during sputum collection or collection of any other respiratory specimen should wear a respirator (FFP2 or N95) to prevent bacilli inhalation. 

 

When a patient cannot expectorate spontaneously, respiratory specimens can be obtained by sputum induction (in children and adults) or by nasopharyngeal or gastric aspiration (in children only). These procedures must be performed under close medical supervision and only if the specimen is collected for molecular tests, culture, or genome sequencing.  

They should be well explained to the patient and the person accompanying them beforehand.  

 

Specimens are collected in specific containers:  

• Sputum containers a Citation a. Sputum container: plastic, screw top lid, single use, non-sterile, without additive. for sputum obtained spontaneously or by induction
• Conical tubes b Citation b. Conical bottom tube, polypropylene, screw top lid, sterile, without additive. for specimens obtained by aspiration

Containers should be labelled with the patient’s name or identification number, as well as collection date, time and location. 

3.1.1 Required number of specimens  

Spontaneously obtained sputum: 2 specimens  

• Rapid molecular tests (RMTs): the 2 specimens are collected on the spot. The RMT is performed on the best quality specimen. The second specimen is used if the RMT needs to be repeated. If the patient provides only one specimen on the spot and the RMT needs to be repeated, a new specimen should be collected. 
• Microscopy: the first specimen is collected on the spot. The second specimen is preferably collected by the patient in the morning of the next day c Citation c. The concentration of bacilli is higher in the morning sputum, which improves the detection of AFBs. as soon as they wake up and before eating. The specimen is then brought to the laboratory by the patient. 

If not feasible, the 2 specimens are collected one hour apart on the spot. 

• Culture and phenotypic drug-susceptibility tests (pDST): the 2 specimens are collected on the spot. The 2 specimens are cultured. The second specimen is cultured as a precaution i.e. in case of contamination or negative culture of the first specimen.  
• Genome sequencing: the 2 specimens are collected on the spot. The second specimen is used if there is not enough DNA in the first specimen.   

 

Sputum induction: if possible, 2 specimens are collected during the same session. However, as this procedure is invasive, if only one specimen is collected, do not repeat the procedure to obtain a second specimen. 

 

Nasopharyngeal or gastric aspiration: only one specimen is collected.  

3.1.2 Quality of specimens 

The quality of the specimen determines the reliability of the result. A minimum volume of specimen is required to perform the tests. The specimen should contain mucoid or purulent material.  

Rejection criteria: saliva (watery fluid) or specimens containing food particles. 

3.1.3 Sputum obtained spontaneously 

When possible, specimens are collected outside in the open air and far away from other people. 

Equipment 

• Gloves 
• Labelled sputum container  

Procedure

• Ask the patient to rinse their mouth with water. 
• Give the patient the sputum container.  
• Do 1 to 2 minutes of chest clapping if needed. 
• Have the patient take a deep breath, hold for a few seconds, exhale, repeat 2 or 3 times, then cough: sputum is material brought up from the lungs with cough.  
• Collect at least 2 ml of sputum and close the container tightly. 
• If the specimen is collected at home, make sure that the patient has understood the procedure, including closing the container tightly after sputum collection. 
• Take a new specimen if unsatisfactory. 

3.1.4 Sputum induction 

Patients should be observed for respiratory distress (including SpO₂ monitoring) during the procedure and for 15 minutes after the procedure. Oxygen must be ready at hand (risk of bronchospasm). 

Equipment

• Gloves 
• Labelled sputum container 
• Mask and tubing for nebulizer 
• Holding chamber (spacer) with masks of different sizes (to be sterilised between each patient)  
• Sterile hypertonic solution of 3 to 6% sodium chloride (sputum inducer)
• Sterile solution of 0.9% sodium chloride (for the specimen) 
• Salbutamol metered dose inhaler 
• Pulse oximeter and oxygen 

Procedure

Patients should fast for at least 2 hours before the procedure to reduce the risk of vomiting and aspiration. 

• Seat the patient comfortably. For young children, sit upright in an adult’s arms. 
• Give the patient the sputum container.  
• Administer 200 micrograms (2 puffs) of salbutamol via a holding chamber, 10 minutes before nebulization. 
• Fill the nebulizer with 5 ml of 3 to 6% hypertonic saline solution. 
• Place the nebulizer mask over the patient’s mouth. 
• Leave the patient to inhale until the reservoir is empty. 
• Encourage the patient to cough and spit at any time if they feel to urge to do so. 
• Collect at least 2 ml of sputum and close the container tightly. 
• Terminate the procedure if unsuccessful after 15 minutes. 

3.1.5 Nasopharyngeal aspiration 

Equipment

• Gloves 
• Suction catheter (CH6 for children 1-11 months; CH8 for children 1-10 years)
• 50 ml syringe or equipment for electric suction 
• Sterile solution of 0.9% sodium chloride 
• Labelled collection container 

Procedure

Children should fast for at least 2 hours before the procedure to reduce the risk of vomiting and aspiration. 

• Do 1 to 2 minutes of clapping. 
• Clean out the nasal cavity with 0.9% sodium chloride. 
• Lie the child on their back or side. 
• Lubricate the end of the suction catheter. 
• Put 2 drops of 0.9% sodium chloride into each nostril. 
• Measure the distance from the tip of the nose to the angle of the jaw, which represents the depth to which the catheter should be inserted. Gently insert the suction catheter to this depth without applying suction. 
• Once the catheter is in the posterior nasopharynx, suction with the 50 ml syringe or the electric suction device d Citation d. If an electric suction device is used, the suction pressure should be 80-100 mmHg for children 1-11 months; 100-120 mmHg for children 1-10 years.  and slowly pull out the catheter whilst suctioning.  
• Collect 2 to 3 ml of respiratory secretions. If insufficient (< 2 ml), put 2 drops of 0.9% sodium chloride into each nostril, then suction on the other nostril. 
• Close the container tightly. 

3.1.6 Gastric aspiration 

Equipment

• Gloves 
• Nasogastric tube (CH6 for children 1-11 months; CH8 for children 1-10 years)
• 50 ml syringe 
• Sterile water 
• Labelled collection container 

Procedure

Children should fast for 4 to 8 hours before the procedure. In practice, the specimen is collected early in the morning in order to obtain the sputum swallowed during the night. 

• Place the child in a half-sitting or sitting position in the adult’s arms. 
• Insert a nasogastric tube and check that it is correctly placed. 
• Suction with a 50 ml syringe. 
• Collect 5 to 10 ml of gastric fluid. If insufficient (< 5 ml), rinse the stomach with 10 ml of sterile water and suction again.  
• Close the container tightly. 
• Start culture within 4 hours of collecting the specimen. If there will be more than 4 hours delay, neutralize with an equal volume of sodium bicarbonate. 

3.2 Specimen inactivation  

Two methods can be used for specimen inactivation:  

• Add 90 or 95% ethanol to the specimen to reach a final concentration of 70%. Let the solution stand overnight. Transfer 1 ml of the solution into a 2 ml tube. 
• Immerse the specimen for 20 minutes in a water bath at 80 °C. 

These procedures can be performed in a well-ventilated room or using a ventilated workstation (Appendix 6).  

 

Inactivated specimens can be used for Sanger sequencing and targeted sequencing.  

They cannot be used for cultures, phenotypic drug susceptibility tests or whole genome sequencing. 

3.3 Specimen storage 

If tests are not performed immediately or on the site of collection, specimens should be protected from light. The storage conditions vary according to the test required.  

Fresh specimens

• For RMTs: store at 2-8 °C. Process as soon as possible and within 7 days. Alternatively, if stored at 35 °C max, process within 3 days max.  
• For microscopy: store at 2-8 °C. Process as soon as possible and within 3-4 days. Specimens stored at room temperature liquefy. This makes it more difficult to select mucopurulent material. 
• For cultures: 
  • on liquid medium (MGIT): store at 2-8 °C. Culture as soon as possible and within 3-4 days. 
  • on solid medium (LJ) cultured ≤ 3 days after collection: store at 2-8 °C. Culture as soon as possible.  
  • on solid medium (LJ) cultured > 3 days after collection: use cetylpyridinium chloride (CPC) to preserve specimen (i.e. add an equal volume of CPC to the specimen), store at room temperature (20-30 °C). Culture as soon as possible and preferably within 7 days.  

Do not refrigerate specimens as CPC crystallises and becomes ineffective.  

Note: CPC is not compatible with MGIT. 

• For targeted genome sequencing: store at room temperature (however storage at 2-8 °C will not interfere with the results). Process as soon as possible and preferably within 4 days.  

Inactivated specimens

Store at room temperature.  

Note: inactivated specimens can be kept for several years.  

3.4 Specimen shipment 

Fresh specimens

For specimens shipped to reference laboratory by air or road transport company, use a triple packaging as per P650 instructions e Citation e. For more information, see: Guidance on regulations for the transport of infectious substances 2021-2022. Geneva: World Health Organization; 2021.
https://www.who.int/publications/i/item/9789240019720 : 

1. Primary receptacle (containing the specimen f Citation f. If the specimen was collected in a sputum container, it should be transferred to a conical tube for shipment. ): leak-proof conical sterile tube tightly closed, labelled with the patient’s name and identification number, collection date, time and location. 

2. Secondary packaging (to protect the primary container): tightly closed leak-proof plastic box or sachet. The secondary container contains sufficient absorbent material to wrap the primary receptacle and absorb all of the specimen in case of leakage or breakage. 

3. Third/outer packaging (to protect the secondary packaging): rigid cardboard box with UN 3373 pictograph Biological substance, Category B. 

The outer packaging should display the following information:  

• Name, full address and telephone number of the receiving laboratory, as well as name and telephone number of the person to whom the specimen is sent. 
• Name, full address and telephone number of the sender. 

Specimens stored at 2-8 °C should be shipped in cold chain using an isothermal triple packaging.  

Inactivated specimens

Specimens can be shipped at room temperature without triple-packaging and UN labelling.

If specimens are inactivated with ethanol, the volume of 70% ethanol should not exceed 1 ml per 2 ml tube and 100 ml per package (ethanol is considered a dangerous good).  

 

Any fresh and inactivated specimen should be accompanied by the corresponding laboratory request form (Appendix 34 and Appendix 35).