Sputum - Sample Collection, Transport, Processing
Sputum sample collection
The patient is provided with a clean, dry, wide-necked, leak-proof container and requested to produce a sputum specimen.
It is collected for diagnosis in cases of respiratory tract infections such as bronchitis, pneumonia, bronchiolitis, pleural infection, or tuberculosis.
Expectorated sputum
Expectorated sputum sample is the primary means of determining the cause of bacterial pneumonia.
LRT secretions will be contaminated with URT secretions, especially saliva unless an invasive technique is used. So, sputum sample is considered the least clinically relevant specimen received in microbiology labs.
Food should not have been ingested 1-2 hours before expectoration, mouth should be rinsed with NS or water just before expectorate. The deep-coughed specimen should be expelled into a sterile container with nominal saliva contamination.
The specimen should be transported to the lab immediately. Even a moderate amount of time at room temperature can result in the loss of viable pathogens and the recovery of pathogens from specimens.
Induced (high diagnostic yield)
Sputum if unable to produce by patients may be obtained by postural drainage of thoracic percussion which stimulates the production of an acceptable sputum samples.
The specimen is collected after patients clean their oral cavities. Alternatively, the aerosol-induced specimen can be collected for the isolation of mycobacterial and fungal agents.
Aerosol-induced specimens are collected by allowing patients to breathe aerosolized droplets, using an ultrasonic nebulizer, containing 0.85-10% NaCl or until a strong cough reflex is initiated. Although these specimens obtained by this method contain material directly from alveolar spaces, it resembles saliva.
Gastric aspirates
The gastric aspirate is collected from patients who cannot produce sputum sample (particularly small children) and is used exclusively for the isolation of acid-fast bacilli.
Before the patient wakes up in the morning, a nasogastric tube is inserted into the stomach, and the contents are withdrawn. It is assumed that acid-fast bacteria from the respiratory tract will be swallowed during the night and will be present in the stomach. Some bacteria such as Mycobacteria are resistant to acidity which allows them to remain viable for a short duration of time.
The gastric aspirate specimen must be delivered to the lab immediately so that acidity can be neutralized. If immediate delivery is not possible, the specimen can first be neutralized and then transported to the lab.
Endotracheal or Trancheostomy suction specimens
Patients with tracheostomies are unable to produce sputum sample in a normal fashion and hence LRT secretions can be collected using a Lulens trap. The tracheotomy aspirates/suction should be treated as sputum.
Bronchoscopy
Bronchoscopy specimens include BAL, BW, BB, and transbronchial biopsies. Diagnosis of pneumonia in HIV-infected, immunocompromised patients, require mostly invasive forms of specimen collection.
The lung tissue collected via fiberoptic bronchoscopy can be sent for transbronchial biopsy for evaluation of lung cancer and other lung diseases.
Transbronchial biopsy is important but the procedure is associated with significant complications such as bleeding. The specimen should be transported in sterile 0.85% saline.
Bronchial washings/aspirates are collected by washing a small amount of sterile physiologic saline inserted into the bronchial tree and withdrawing the fluid. The specimens will be contaminated with URT flora (Viridans Streptococci and Neisseria spp). Lavages are very suitable for detecting pneumocystis cysts and fungal elements.
BAL is also a safe and practical method for diagnosing opportunistic pulmonary infections in immunocompromised patients. Non-bronchoscopic “mini BAL” using a Metras catheter has been introduced; typically 20 ml or less saline is installed.
Once received, the contents of the bronchial brush may be suspended in 1ml of broth solution with vigorous vortexing and incubated into culture media using a 0.01ml calibrating inoculating loop.
Colony counts greater than or equal to 106/ml in the original specimen have been considered to be arrested with infection.
Transtracheal aspirates
Percutaneous transtracheal aspirates (TTAs) are obtained by inserting a small plastic catheter into the trachea via a needle previously inserted through the skin and cricothyroid membrane.
This invasive procedure is uncomfortable for patients and is not suitable for all patients (cannot be used in patients with a bleeding tendency or with poor oxygenation)
The procedure reduces the likelihood that a specimen will be contaminated by URT flora and diluted by added fluids. It is a rarely used technique. Actinomycetes and those associated with aspiration pneumonia can be isolated from TTA specimens.
Other invasive procedures
In cases of pleural empyema, thoracentesis is used to obtain specimens. Blood culture should always be performed in patients with pneumonia.
The most invasive procedure for obtaining respiratory tract specimens is the open lung biopsy. It is helpful in diagnosing severe viral infections such as Herpes simplex pneumonia, for rapid diagnosis of Pneumocystis pneumonia, and other life-threatening pneumonia which are hard to diagnose.
Processing of sputum
The processing of sputum includes:
A smear (of sputum sample) is prepared on a slide for Ziehl-Neelson’s staining using any caseous particles and the most purulent materials. The slide is then allowed to dry in a safe place
The slide is fixed by using 1-2 drops of 70% ethanol
The slide is covered with filtered carbon fuschin stain
The stain is heated until the vapor just begins to rise
Allow the heated stain to remain on the slide for 5 mins
Wash well with clean water
Cover the smear with 3% v/v acid alcohol for 5 mins and wash well with clean water
Cover the smear with malachite green for 1-2 mins then wash with clean water
Blot dry the back of the slide and place it in the rack
Observe under oil immersion at 100x (pleural fluid, peritoneal fluid, and other exudates are collected in containers with citrate to prevent coagulation.)
* specimens should be transported asap. In case of delay, specimens are refrigerated at 4°C but not more than overnight.
Transport medium: 1% cetyl Dyridenium Chloride (CPC) + 2% NaCl or Tri Sodium Phosphate