Appendix 1. Sputum specimen: collection, storage and shipment

1.1 Sputum collection techniques

Regardless the collection technique used, staff member present during sputum collection should wear a respirator to prevent bacilli inhalation.

1.1.1 Sputum obtained spontaneously

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

The first sample is collected on the spot, at the consultation, when the patient is identified as suspected TB case. If the patient has recently eaten, ask him/her to rinse his/her mouth with water in order to avoid the presence of food in the sample.

The second sample is collected the day after, in the early morning, right after the patient wakes up and before eating. The second sample may be collected at home then the patient brings it to the health facility.

Alternatively, two sputum specimens can be collected one hour apart (frontloaded microscopy).

Collection technique:
– The patient must be given a labelled sputum container (or a Falcon® tube, if the sample is to be shipped by air).
– Have the patient take a deep breath, hold for a few seconds, exhale, repeat two or three times, then cough: sputum is material brought up from the lungs after a productive cough. One or two minutes of chest clapping are of benefit.
– Collect at least 3 ml and close the container hermetically.

The quality of sample determines the reliability of the result. Always check that the sample contains solid or purulent material and not only saliva. Take a new sample if unsatisfactory.

If the sample is collected at home, make sure that the patient has understood the technique, including closing the container hermetically after collecting the sputum.

1.1.2 Sputum induction

Sputum induction is sometimes used in children when sputa cannot be spontaneously expectorated, and only in order to perform cultures or Xpert MTB/RIF.

Sputum induction must be performed under close medical supervision. The child should be observed for respiratory distress during, and for 15 minutes after, the procedure. Bronchospasm may occur. Salbutamol spray and oxygen must be ready at hand.

Equipment

– Gloves and respirator
– Suction catheter (6, 7, 8F)
– Sputum container
– 50 ml syringe, needle
– Mask and tubing for nebulizer
– Holding chamber with child’s mask (to be sterilized between each patient)
– Sterile hypertonic solution of 5% sodium chloride (to be kept refrigerated)
– Sterile solution of 0.9% sodium chloride (for the specimen)
– Salbutamol spray
– Oxygen

Procedure

The child should fast for at least 2 hours before the procedure.

– Prior to nebulization:
• Explain the procedure to the child and/or the person accompanying him/her (this person must wear a respirator).
• Place the child in a sitting position in the adult’s arms.
• Administer 2 puffs of salbutamol via a holding chamber, 10 minutes before nebulization.
• Prepare a sputum container.

– Nebulization:
• Fill the nebulizer with 5 ml of 5% hypertonic saline solution (sputum inducer).
• Place the nebulizer mask over the child’s mouth.
• Leave the child to inhale until the reservoir is empty.

– Nasopharyngeal suction:
• Do 1 to 2 minutes of clapping.
• Clean out the nasal cavity.
• During suction, the child is laid on his /her side, back to the operator, who is behind him/her.
• Fit a suction catheter to a 50 ml syringe. Lubricate the end of the catheter.
• Measure the distance from the tip of the nose to the angle of the jaw. Insert the suction catheter to that depth.
• When inserting and withdrawing the tube, pull on the plunger of the syringe to create suction.
• Once the syringe is filled with air and mucus, disconnect it from the suction catheter and purge the air (tip facing upward), so that only mucus is left in the syringe.
• To collect the mucus: draw 2 ml of 0.9% sodium chloride into the syringe to rinse, then empty contents into the sample container.

1.1.3 Gastric aspiration

Gastric aspiration is sometimes used in children when sputa cannot be spontaneously expectorated nor induced using hypertonic saline, and only in order to perform cultures or Xpert MTB/RIF.

Equipment

– Gloves and respirator
– Suction catheter (6, 7, 8F)
– Sputum container
– 50 ml syringe
– Sterile water

Procedure

– Prior to inserting the suction catheter:
• Explain the procedure to the child and/or the person accompanying him/her (this person must wear a respirator);
• 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.

– First suction to collect the gastric fluid and place it in the sputum container, then rinse the stomach with 30 ml of sterile water and suction again. Add the suctioned fluid to the first sample.

– Start culture within 4 hours of collecting the sample. If there will be more than four hours’ delay, neutralize with 100 mg of sodium bicarbonate.

1.2 Sputum specimen storage

When examinations are not performed on the site of collection:

Specimen for smear microscopy

Smears should be performed within three-four days of collection and in the meanwhile stored refrigerated (2 to 8°C) and protected from light.
Contamination does not affect microscopy but heat make specimen liquefy, with selection of mucopurulent part of the sample more difficult.

Specimen for culture in liquid medium

Keep the specimen refrigerated (2 to 8°C), protected from light. Do not use cethylpyrodinium chloride (CPC) as it is not compatible with MGIT.
The specimen should be processed as soon as possible.

Specimen for culture on Lowenstein-Jensen medium (LJ)

Specimens that can be cultured in less than 3 days after collection:
Keep refrigerated (2 to 8°C) and protected from light until transport OR immediately transport to the laboratory for processing.

Specimens that will be cultured more than 3 days after collection:
Use Falcon tubes and add 1% CPC to preserve the specimen for up to 2 weeks. Specimens with CPC should not be refrigerated, as the CPC will crystallize and be ineffective.
Samples with CPC can be inoculated on LJ. For inoculation on agar, they require prior neutralization by neutralizing buffer (Difco).
CPC can be used for specimens tested by Xpert MTB/RIF.

1.3 Sputum specimen shipment

To a local laboratory

– Without CPC transport medium: between 2 and 8°C and protected from light;
– With CPC transport medium: should not be refrigerated because at low temperatures the CPC will crystallize and ruin the sample. Specimens should be kept at room temperature, protected from heat and light.

By air to a reference laboratory for culture

Samples are collected and shipped in 50 ml Falcon® conical tubes with screw caps. The tubes are labelled UN 3373, corresponding to Category B infectious substances. If transport times are less than 12 hours, even specimens without CPC can be transported at room temperature.

Samples are triple-packaged, in accordance with IATA packing instruction 650:
1. Primary container holding the sputum sample: tube tightly closed and placed into a latex glove;
2. Secondary container intended to protect the primary container: leak-proof box with enough absorbent material to absorb the entire sample, should the primary container break;
3. Outer packaging intended to protect the secondary container, with UN 3373 labelling.

Information to be provided:
– Primary container: label with the patient’s name or identification number and the sample collection date and location;
– Outer package: indicate the name of the receiving laboratory, the complete address (name, street, postal code, locality, country), and telephone number.

All samples must be accompanied by the corresponding laboratory test request form (including clinical information).

Notes:
– Procedures for shipping bacterial strains obtained after culture are different, more complicated, and rarely feasible in practice. Cultures are classified as Category A infectious substances (UN 2814).
– For a detailed description of the shipment procedures, see MSF Medical catalogue, volume 4.