Appendix 1. Xpert assays

Update: October 2022

 

1.1 Specimen processing

Staff members present during specimen preparation should wear a respirator (FFP2 or N95) to prevent the inhalation of bacilli. 

A biosafety cabinet (Appendix 6) should be used to protect staff from aerosols when the specimen is to be centrifuged or cut/ground. 

1.1.1 Sputum specimens

See Xpert MTB/RIF package insert:

https://www.cepheid.com/Package%20Insert%20Files/Xpert-MTB-RIF-ENGLISH-Package-Insert-301-1404-Rev-G.pdf

 

See Xpert MTB/XDR package insert:

https://www.cepheid.com/Package%20Insert%20Files/Xpert%20MTB-XDR%20ENGLISH%20Package%20Insert%20302-3514%20Rev%20C.pdf

1.1.2 Lymph node and other tissue specimens

Xpert assay performed on a biopsy (adapted from WHO) [1] Citation 1. World Health Organization. Xpert MTB/RIF implementation manual: technical and operational ‘how-to’; practical considerations. Geneva; 2014. https://apps.who.int/iris/bitstream/handle/10665/112469/9789241506700;jsessionid=44788E3067C3F9DBF836E8BB6BB0F253?sequence=1

 

• Cut the tissue specimen in small pieces in a sterile mortar (or grinder).
• Add 2 ml of sterile phosphate buffer saline (PBS).
• Grind solution of tissue and PBS to obtain a homogeneous mixture.
• Transfer 0.7 ml of mixture into a centrifuge tube using a transfer pipette. Avoid transferring clumps that are not well homogenized.
• Add 1.4 ml of Xpert Sample Reagent (XSR) using a transfer pipette.
• Shake vigorously 10 to 20 times or vortex for at least 10 seconds.
• Keep at room temperature for 10 minutes.
• Shake vigorously 10 to 20 times or vortex for at least 10 seconds.
• Keep at room temperature for 5 minutes.
• Transfer 2 ml of the mixture to the Xpert cartridge using a transfer pipette.
• Load the cartridge into the Xpert instrument as per the manufacturer’s instructions.

 

Xpert assay performed on a lymph node specimen obtained by fine needle aspiration (FNA)

 

• Flush the needle and syringe into a sterile container containing 1 ml of sterile 0.9% sodium chloride or sterile PBS.
• Transfer 0.7 ml of mixture into a centrifuge tube using a transfer pipette.
• Add 1.4 ml of XSR using a transfer pipette.

For the next steps, continue as above.

 

For lymph node fine needle aspiration technique, see Appendix 7.

1.1.3 Cerebrospinal fluid specimens

Adapted from WHO [1] Citation 1. World Health Organization. Xpert MTB/RIF implementation manual: technical and operational ‘how-to’; practical considerations. Geneva; 2014. https://apps.who.int/iris/bitstream/handle/10665/112469/9789241506700;jsessionid=44788E3067C3F9DBF836E8BB6BB0F253?sequence=1

 

The processing method for cerebrospinal fluid (CSF) depends on the volume available for testing.

 

Volume of CSF	Procedure
0.1 to 1 ml	Add XSR to the CSF to obtain a final volume of 2 ml. Transfer 2 ml of the mixture into the Xpert cartridge. Load the cartridge into the Xpert instrument as per the manufacturer’s instructions.
1 to 5 ml	Add an equal volume of XSR to the CSF. Add 2 ml of the mixture directly into the Xpert cartridge. Load the cartridge into the Xpert instrument as per the manufacturer’s instructions.
> 5 ml	Centrifuge the CSF at 3,000g for 15 minutes. Pour the supernatant and add XSR to the sediment to obtain a final volume of 2 ml. Transfer 2 ml of the mixture into the Xpert cartridge. Load the cartridge into the Xpert instrument as per the manufacturer’s instructions.

 

Note: a volume of CSF less than 0.1 ml is insufficient for testing.

1.1.4 Stool specimens [2] Citation 2. de Haas P, Yenew B, et al. The Simple One-Step (SOS) Stool Processing Method for Use with the Xpert MTB/RIF Assay for a Child-Friendly Diagnosis of Tuberculosis Closer to the Point of Care. J Clin Microbiol. 2021 Jul 19;59(8).
https://doi.org/10.1128/JCM.00406-21

Stool specimens can be used within 3 hours if kept at room temperature.

• Add 0.8 to 1 g of stool into the 8 ml XSR bottle.
• Shake vigorously for 30  seconds.
• Keep at room temperature for 10 minutes.
• Shake vigorously for 30  seconds.
• Sediment at room temperature for 10 minutes.
• Without disturbing the sediment, transfer 2  ml of the supernatant into the Xpert cartridge.
• Load the cartridge into the Xpert instrument as per the manufacturer’s instructions.

1.1.5 Urine specimens [3] Citation 3. MSF South Africa Medical Unit. SOP: Urine GeneXpert MTB/Rif Assay.

Urine specimens can be used within 3 hours if kept at room temperature.

• Centrifuge 4 ml of urine at 3,000g for 5 minutes.
• Pour the supernatant and add 2 ml of XSR to the sediment.
• Shake vigorously.
• Transfer 2 ml of the mixture into the Xpert cartridge.
• Load the cartridge into the Xpert instrument as per the manufacturer’s instructions.

1.2 Diagnostic accuracy of Xpert in specimens other than sputum

Specimens	Performances of Xpert MTB/RIF compared to culture [4] Citation 4. World Health Organization. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis - rapid diagnostics for tuberculosis detection. 2021 update. Geneva; 2021. https://www.who.int/publications/i/item/9789240029415
Lymph node biopsy or aspirate	Biopsy: sensitivity: 82%; specificity: 79% Aspirate: sensitivity: 89%; specificity: 86%
CSF	Sensitivity: 70%; specificity: 97%
Pleural fluid	Sensitivity: 50%; specificity: 99%
Pericardial fluid	Sensitivity: 67.6%; specificity: 99.4%
Nasopharyngeal aspirate (children with presumed PTB)	Sensitivity: 46%; specificity: 100%
Gastric aspirate (children with presumed PTB)	Sensitivity: 73%; specificity: 98%
Stool (children with presumed PTB)	Compared to culture of respiratory specimens: No HIV infection: sensitivity: 61%; specificity: 98% HIV infection: sensitivity: 70%; specificity: 98%
Urine (presumed genitourinary TB)	Sensitivity: 85%; specificity: 97%
Urine (HIV-infected patients with presumed disseminated TB)	Sensitivity: 40%; specificity: 98% [5] Citation 5. World Health Organization. Rapid communication on updated guidance on the management of tuberculosis in children and adolescents. Geneva: World Health Organization; 2021. https://www.who.int/publications/i/item/9789240033450
Synovial fluid	Sensitivity: 97%; specificity: 94%
Peritoneal fluid	Sensitivity: 59%; specificity: 97%
Blood (HIV-infected patients with presumed disseminated TB)	Child: sensitivity: 7%; specificity: 99% [6] Citation 6. Pohl C, Rutaihwa LK, Haraka F, Nsubuga M, Aloi F, Ntinginya NE, Mapamba D, Heinrich N, Hoelscher M, Marais BJ, Jugheli L, Reither K. Limited value of whole blood Xpert(®) MTB/RIF for diagnosing tuberculosis in children. J Infect. 2016 Oct;73(4):326-35. https://doi.org/10.1016/j.jinf.2016.04.041 Adult: sensitivity: 56%; specificity: 94%

 

Note: the performances of Xpert MTB/XDR in non-sputum specimens are considered similar to those of Xpert MTB/RIF as the tests are based on similar technologies [4] Citation 4. World Health Organization. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis - rapid diagnostics for tuberculosis detection. 2021 update. Geneva; 2021.
https://www.who.int/publications/i/item/9789240029415 .

1.3 Logistic requirements

All Xpert assays are performed with the same instrument.

The 10-colour module can read all Xpert cartridges.

The 6-colour module can read Xpert MTB/RIF and Xpert MTB/RIF Ultra cartridges.

1.3.1 Power supply

The instrument requires a constant and stable power supply.

If power cuts are short (less than 10 minutes), use a 1500VA "on line" UPS.

If power cuts are long, the system must be able to sustain a full cycle (approx. 45 minutes). Use a battery charger, a stationary battery, and a voltage stabilizer.

1.3.2 Storage and operating temperatures

Storage of cartridges and reagents: between 2 and 28 °C for 12 months from date of manufacture.

 

Operating temperature for the Xpert instrument: between 15 and 30 °C. According to climate conditions, air conditioning may be required.

1.3.3 Calibration

The Xpert modules require annual calibration performed by an authorized service provider or carried out by swapping out the modules. A detailed contract with the supplier should guarantee regular maintenance, calibration, repair, and replacement as and when needed.

1.3.4 Required space

The dimensions of the Xpert IV instrument (4 modules enabling the processing of 4 specimens at the same time) are:

Width: 29.8 cm; height 35.6 cm; depth 31.1 cm; weight: 12 kg.

 

The instrument is designed for indoor use only. Provide at least 5 cm of clearance on each side to ensure adequate ventilation. Do not place the instrument close to the vents of other instruments or air-handling units.

 

The dimensions of the kits containing cartridges and reagents are:

Xpert MTB/RIF kit 50 tests: 31 cm x 28 cm x 20 cm

Xpert MTB/XDR kit 10 tests: 24 cm x 16 cm x 7 cm

1.3.5 Waste disposal

Same procedure as for sputum containers.

Xpert assays generate large volumes of waste.