Xpert MTB/RIF Ultra testing from tongue swabs – Diluted SR method
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
Abstract
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.
Attachments
Steps
Preparation of diluted 66% SR:PB
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
Method A: For testing of smaller batches of samples
Label a sterile conical tube with the date of the diluted SR:PB preparation.
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.
| A | B | C |
|---|---|---|
| Number of samples to be tested | Volume of SR (mL) | Volume of PB (mL) |
| 1-3 | 2 | 1 |
| 4-7 | 4 | 2 |
| 8-11 | 6 | 3 |
| 12-16 | 8 | 4 |
| 17-20 | 10 | 5 |
| 21-25 | 12 | 6 |
Vortex (maximum setting) to mix for 5 to 10 seconds.
Use the diluted SR:PB buffer to test tongue swabs collected on the same day the buffer is prepared.
Method B: For testing of larger batches of samples (up to 16 samples) or standardized daily preparation of diluted SR buffer
Label a sterile conical tube with the date of the diluted SR:PB preparation.
Add the entire contents of Cepheid SR buffer (8 mL) from 1 bottle to the labeled conical tube.
Add 4 mL of sterile PB to the same conical tube.
Vortex (maximum setting) to mix for 5 to 10 seconds.
Use the diluted SR:PB buffer to test tongue swabs collected on the same day the buffer is prepared.
Xpert MTB/RIF Ultra Processing
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.
Vortex to mix for 5 to 10 seconds.
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).
Label the Xpert MTB/RIF Ultra cartridge with the appropriate sample identifier.
Prefill the cartridge with 1.5 mL of PB (undiluted).
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.
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.
Proceed with the instructions for “preparing the Cartridge” and testing that are found in the Xpert MTB/RIF Ultra manufacturer’s package insert.
