TITLE: Xpert MTB/RIF Ultra testing from tongue swabs – Diluted SR method
AUTHORS: Charlotte Ahls, Anura David, Gayatri Shankar Chilambi, Adithya Cattamanchi, Margaretha de Vos, Kelsey Heard, Karen Heichman, Adam Penn-Nicholson, Lesley Scott, Amy E Steadman, Lindsey Turnbull, David Alland

[DESCRIPTION]
This standard operating procedure (SOP) outlines the methodology for processing dry dorsum tongue swabs with Xpert MTB/RIF Ultra (Ultra) (Cepheid, Sunnyvale, CA). The procedure is intended as a consensus protocol for clinical evidence generation to evaluate whether tongue swabs can be used as an additional sample type to diagnose tuberculosis (TB) using Ultra. Tongue swabs will be collected for Ultra testing at the clinical facility. Please refer to the tongue swab collection SOP. Several groups have shown that the use of Xpert Sample Reagent (SR) at a starting concentration of 100% can result in decreased sensitivity for the detection of Mycobacterium tuberculosis from tongue swabs. This protocol makes use of diluted SR in phosphate buffer (PB), with a starting concentration of SR of 66%. 

[BEFORE_START]
Upon receipt at the laboratory, keep the collected tongue swab at 2-8°C until ready for processing. Processing and testing should be done within 24 hours of collection. If processing cannot be done within 24 hours, please store the swab at -80°C for later testing.
    
For stored swabs that will be tested from frozen, please remove the swabs from the freezer and keep on ice (2-8°C) for at least ten minutes and until ready for testing. 

Please refer to the attached schematic workflow.

[GUIDELINES]
Personnel testing the tongue swabs must wear a fitted N95 mask to protect from TB aerosols, eye protection (safety glasses or face shield), and disposable gloves. These should be worn during the entire collection process and should only be removed after leaving the ward/ location where the sample was collected. Wash hands thoroughly after handling samples.

[STEPS]
SECTION: Preparation of diluted 66% SR:PB
1. Dilute SR in a 2:1 ratio with PB, daily in a biosafety cabinet.
Please use step 2 for testing of smaller batches of samples, and step 3 for testing of larger batches (up to 16 samples) or standardized daily preparation of diluted SR buffer
 

SECTION: Preparation of diluted 66% SR:PB
2. Method A: For testing of smaller batches of samples
SECTION: Preparation of diluted 66% SR:PB
2.1. Label a sterile conical tube with the date of the diluted SR:PB preparation.
SECTION: Preparation of diluted 66% SR:PB
2.2. Depending on the number of samples to be tested, add the volume of SR and PB to the
labeled conical tube as per the table below. The total volume allows for 10% overage. 
 
 
 
SECTION: Preparation of diluted 66% SR:PB
2.3. Vortex (maximum setting) to mix for 5 to 10 seconds.
SECTION: Preparation of diluted 66% SR:PB
2.4. Use the diluted SR:PB buffer to test tongue swabs collected on the same day the buffer is prepared.

SECTION: Preparation of diluted 66% SR:PB
3. Method B: For testing of larger batches of samples (up to 16 samples) or standardized daily preparation of diluted SR buffer
SECTION: Preparation of diluted 66% SR:PB
3.1. Label a sterile conical tube with the date of the diluted SR:PB preparation.
SECTION: Preparation of diluted 66% SR:PB
3.2. Add the entire contents of Cepheid SR buffer (8 mL) from 1 bottle to the labeled conical tube.
SECTION: Preparation of diluted 66% SR:PB
3.3. Add 4 mL of sterile PB to the same conical tube.
SECTION: Preparation of diluted 66% SR:PB
3.4. Vortex (maximum setting) to mix for 5 to 10 seconds.

SECTION: Preparation of diluted 66% SR:PB
3.5. Use the diluted SR:PB buffer to test tongue swabs collected on the same day the buffer is prepared.
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4. As per local safety requirements for sputum processing, add 700 µl of the prepared diluted SR:PB buffer to each swab in the tube and close the tube lid. 
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5. Vortex to mix for 5 to 10 seconds.
SECTION: Xpert MTB/RIF Ultra Processing
6. Incubate the swabs at room temperature for 15 minutes. Vortex again halfway through the incubation time for 5 to 10 seconds (i.e. at between 7-8 minutes of the incubation time). 
 

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7. Label the Xpert MTB/RIF Ultra cartridge with the appropriate sample identifier.
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8. Prefill the cartridge with 1.5 mL of PB (undiluted).
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9. Before loading the samples to the cartridge, briefly pulse vortex each sample. Over vortexing may generate bubbles that can adversely affect the test and cause difficulty for accurate pipetting.
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10. Carefully add 0.5 mL of the SR:PB-treated swab to the cartridge, dispensing slowly against the interior wall of the cartridge to not generate bubbles. 
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11. Proceed with the instructions for “preparing the Cartridge” and testing that are found in the Xpert MTB/RIF Ultra manufacturer’s package insert.