Annex 2Standard Operating Procedure (SOP) for processing extrapulmonary specimens (CSF, lymph nodes and other tissues) for Xpert MTB/RIF assay
1. Scope
This standard operating procedures (or SOP) describes methods for processing specimens of cerebrospinal fluid (CSF), lymph nodes and tissues for testing using the Xpert MTB/RIF assay and for purposes of culturing Mycobacterium tuberculosis culture on solid media or liquid media.
2. Definitions and abbreviations
- BSC
biological safety cabinet
- CSF
cerebrospinal fluid
- ID
patient's specimen identification, usually laboratory number
- LJ
Löwenstein–Jensen
- NALC
N-acetyl-L-cysteine
- NaOH
sodium hydroxide
- PBS
phosphate buffer 0.067mol/ litre, pH 6.8
- RCF
relative centrifugal force
3. Procedure
3.1. Principle
WHO has issued recommendations about using of Xpert MTB/RIF to diagnose extrapulmonary TB and to detect rifampicin resistance:
Xpert MTB/RIF should be used in preference to conventional microscopy and culture as the initial diagnostic test for CSF specimens from patients suspected of having TB meningitis (strong recommendation given the urgency of rapid diagnosis, very low-quality evidence).
Xpert MTB/RIF may be used as a replacement test for usual practice (including conventional microscopy culture, and histopathology) for testing specific nonrespiratory specimens (lymph nodes and other tissues) from patients suspected of having extrapulmonary TB (conditional recommendation, very low-quality evidence).
In order to reach a quick diagnosis using CSF specimens, Xpert MTB/RIF should be preferentially used instead of culture if the specimen volume is low or if additional specimens cannot be obtained. If a sufficient volume of material is available, concentration methods should be used to increase yield.
Individuals suspected of having extrapulmonary TB but who have had with a single negative result from Xpert MTB/RIF should undergo further diagnostic testing; the processing of their tissue specimens (lymph nodes and other tissues) for Xpert MTB/RIF should include a decontamination step to enable specimens to be cultured concurrently.
Pleural fluid is a suboptimal specimen for the bacterial confirmation of pleural TB using any method. A pleural biopsy provides the preferred specimen.
These recommendations do not apply to specimens of stool, urine or blood, given the lack of data on the utility of Xpert MTB/RIF on these specimens.
3.2. General considerations
Important points about specimen processing procedures
All specimens should be processed as soon as possible, to obtain optimal culture recovery of M. tuberculosis. Longer transportation times of specimens should not affect the use of Xpert MTB/RIF.
Ensure that the Xpert MTB/RIF cartridge and any culture media to be inoculated are labelled correctly and clearly.
Tissues must be processed within a biological safety cabinet, given the risk of producing aerosols while grinding and homogenizing samples.
CSF samples are paucibacillary and can be processed using the same precautions as those used for sputum EXCEPT when they are concentrated by centrifugation.
It is important to use Safe Working Practices to avoid contamination by bacteria other than tubercle bacilli and especially to avoid cross-contamination with tubercle bacilli from other specimens.
When a sufficient volume of sample is available, culture should be performed concurrently with Xpert MTBR/RIF testing.
Exposure time to decontamination reagents must be strictly controlled for samples requiring decontamination.
Decontaminate samples for culture using either 4% NaOH or NaOH-NALC depending on usual practice. The example below uses 4% NaOH.
3.3. Specimen processing
The Xpert MTB/RIF assay can be used directly on CSF specimens and homogenized extrapulmonary specimens (from biopsies of lymph nodes or other tissues) or on decontaminated specimens if culture is performed concurrently.
Whenever possible, specimens should be transported and stored at 2–8 °C prior to processing (the maximum time for storage and processing is 7 days).
3.3.1. Lymph nodes and other tissues (for Xpert MTB/RIF only)
Using sterile pair of forceps and scissors, cut the tissue specimen into small pieces in a sterile mortar (or homogenizer or tissue grinder).
Add approximately 2 ml of sterile phosphate buffer (PBS).
Grind the solution of tissue and PBS using a mortar and pestle (or homogenizer or tissue grinder) until a homogeneous suspension has been obtained.
Use a transfer pipette to transfer approximately 0.7 ml of the homogenized tissue specimen to a sterile, conical screw-capped tube.
NOTE: Avoid transferring any clumps of tissue that have not been properly homogenized.
Use a transfer pipette to add a double volume of the Xpert MTB/RIF Sample Reagent (1.4 ml) to 0.7 ml of homogenized tissue.
Vigorously shake the tube 10 to 20 times or vortex for at least 10 seconds.
Incubate for 10 minutes at room temperature, and then shake the specimen vigorously again for another 10–20 times or vortex for at least 10 seconds.
Incubate the specimen at room temperature for an additional 5 minutes.
Using a fresh transfer pipette, transfer 2 ml of the processed sample to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturer's instructions.
3.3.2. Lymph nodes and other tissues (nonsterile collections for Xpert MTB/RIF and culture)
Using a sterile pair of forceps and scissors, cut the tissue sample into small pieces in a sterile mortar (or homogenizer or tissue grinder).
Add approximately 2 ml of sterile PBS.
Grind the solution of tissue and PBS with a mortar and pestle (or homogenizer or tissue grinder) until a homogeneous suspension has been obtained.
Use a sterile transfer pipette to add the suspension to a 50 ml conical tube.
Add an equal volume of 4% NaOH and tighten the screw-cap.
Vortex thoroughly to homogenize the suspension.
Let the tube stand for 15 minutes at room temperature.
Fill the tube to within 2 cm of the top (that is, to the 50 ml mark on the tube) with PBS.
Centrifuge at 3000 g for 15 minutes.
Carefully pour off the supernatant through a funnel into a discard can containing 5% phenol or other mycobacterial disinfectant.
Resuspend the deposit in approximately 1–2 ml PBS.
Use another sterile transfer pipette to inoculate deposit into liquid media and/or onto two slopes of egg-based medium labelled with the specimen's identification number.
Label an Xpert/MTB/RIF cartridge with the specimen's identification number.
Using a transfer pipette, transfer approximately 0.7 ml of the homogenized tissue specimen to a conical, screw-capped tube to be used for the Xpert MTB/RIF test.
NOTE: Avoid transferring any clumps of tissue that have not been properly homogenized.
Using another transfer pipette, add a double volume of the Xpert MTB/RIF Sample Reagent (1.4 ml) to 0.7 ml of homogenized tissue.
Vigorously shake 10–20 times or vortex for at least 10 seconds.
Incubate for 10 minutes at room temperature, and then shake the specimen vigorously again for another 10–20 times, or vortex for at least 10 seconds.
Incubate the specimen at room temperature for an additional 5 minutes.
Using a fresh transfer pipette, transfer 2ml of the processed specimen to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturer's instructions.
3.3.3. Lymph nodes and other tissues (sterile collection for Xpert MTB/RIF and culture)
Using a sterile pair of forceps and scissors, cut the tissue specimen into small pieces in a sterile mortar (or homogenizer or tissue grinder).
Add approximately 2 ml of sterile PBS.
Grind the solution of tissue and PBS with a mortar and pestle (or homogenizer or tissue grinder) until a homogeneous suspension has been obtained, and add PBS to adjust to a final volume of approximately 2 ml.
Using a sterile transfer pipette, transfer the suspension to a 50 ml conical tube.
Use another transfer pipette to inoculate suspension into liquid media and/or onto two slopes of egg-based medium labelled with the specimen's identification number.
Label an Xpert/MTB/RIF cartridge with the specimen's identification number.
Using a transfer pipette, transfer approximately 0.7 ml of the homogenized tissue specimen to a conical, screw-capped tube to be used for the Xpert MTB/RIF testing.
NOTE: Avoid transferring any clumps of tissue that have not been properly homogenized.
Using a transfer pipette, transfer a double volume of the Xpert MTB/RIF Sample Reagent (1.4 ml) to 0.7 ml of homogenized tissue.
Vigorously shake the tube 10–20 times or vortex for at least 10 seconds.
Incubate for 10 minutes at room temperature, and then shake the specimen vigorously again for another 10–20 times or vortex for at least 10 seconds.
Incubate the sample at room temperature for an additional 5 minutes.
Using a fresh transfer pipette, transfer 2 ml of the processed sample to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturer's instructions.
3.3.4. CSF
The preferred processing method for CSF in Xpert MTB/RIF depends on the volume of specimen available for testing.
NOTE: Blood-stained and xanthochromic CSF specimens may cause false-negative results from Xpert MTB/RIF.
If there is more than 5 ml of CSF
Transfer all of the specimen to a conical centrifuge tube, and concentrate the specimen at 3000 g for 15 minutes.
Carefully pour off the supernatant through a funnel into a discard can containing 5% phenol or other mycobacterial disinfectant.
NOTE: Concentrated CSF should be decanted within a biological safety cabinet
Resuspend the deposit to a final volume of 2 ml by adding the Xpert MTB/RIF sample reagent.
Label an Xpert/MTB/RIF cartridge with the specimen's identification number.
Using a fresh transfer pipette, transfer 2 ml of the concentrated CSF specimen to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturer's instructions.
If there is 1–5 ml of CSF
Add an equal volume of sample reagent to the CSF.
Add 2 ml of the sample mixture directly to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturer's instructions.
If there is 0.1–1ml of CSF
Resuspend the CSF to a final volume of 2 ml by adding the Xpert MTB/RIF sample reagent.
Add 2 ml of the sample mixture directly to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturer's instructions.
If there is less than 0.1 ml
This is an insufficient sample for testing using the Xpert MTB/RIF assay.
4. Related documents
Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF assay for the diagnosis of pulmonary and extrapulmonary TB and rifampicin resistance in adults and children. Policy update.
