Medical/SCIENTIFIC affairs GUIDELINE

Xpert MTB/RIF Alternate Specimen Testing Procedures

Disclaimer: This document, developed by Cepheid Medical/Scientific Affairs, is provided as a courtesy to
Cepheid customers to provide guidance for inquiries regarding the testing of alternate specimen types
with Xpert MTB/RIF. Cepheid does not endorse the testing of alternate specimen types (i.e., specimen
types not included in the product labeling) with FDA-cleared assays without proper validation, but
recognizes the medical need of such testing for some patients. The guidelines below reference published
studies and procedures that have been independently validated by the study authors. If you choose to
use Xpert MTB/RIF with alternate specimen types, it is your laboratorys responsibility to validate the
assay for each alternate specimen type in accordance with federal, state, and local laws.*

Background

A synopsis of the intended use of the Xpert MTB/RIF test is as follows (CE-IVD and US-IVD versions differ
slightly in wording):

The Xpert MTB/RIF test, performed on the GeneXpert System, is a qualitative, nested real-time polymerase
chain reaction (PCR) in vitro diagnostic test for the detection of Mycobacterium tuberculosis complex DNA in raw
sputum or concentrated sputum sediment prepared from induced or expectorated sputum. In specimens where
Mycobacterium tuberculosis complex (MTB-complex) is detected, the Xpert MTB/RIF test also detects rifampin
resistance-associated mutations of the rpoB gene.

The Xpert MTB/RIF test is intended for use with specimens from patients for whom there is clinical suspicion of
tuberculosis (TB) and who have received no anti-tuberculosis therapy, or less than three days of therapy. This
test is intended as an aid in the diagnosis of pulmonary tuberculosis when used in conjunction with clinical and
other laboratory findings.

The Xpert MTB/RIF test must always be used in conjunction with mycobacterial culture to address the risk
of false negative results and to recover organisms when MTB-complex is present for further characterization
and drug susceptibility testing. However, decisions regarding the removal of patients from Airborne Infection
Isolation (AII) need not wait for culture results. Spontaneously expectorated sputum should be representative
of secretions from the lower respiratory tract and should appear purulent (not saliva). Collection of specimens
should occur under healthcare personnel supervision and with active patient participation to facilitate collection
of quality specimens, which are critical for diagnosis of pulmonary TB.

The Xpert MTB/RIF test does not provide confirmation of rifampin susceptibility since mechanisms of rifampin
resistance other than those detected by this device may exist that may be associated with a lack of clinical
response to treatment.

*This guide is applicable to both the US-IVD and CE-IVD versions of the Xpert MTB/RIF test.

A better way.
Specimens that have both MTB-complex DNA and rifampin-resistance associated mutations of the rpoB gene
detected by the Xpert MTB/RIF test must have results confirmed by a reference laboratory. If the presence of
rifampin-resistance associated mutations of the rpoB gene is confirmed, specimens should also be tested for
the presence of genetic mutations associated with resistance to other drugs.

The Xpert MTB/RIF test should only be performed in laboratories that follow safety practices in accordance with
the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories and Guidelines for Safe Work Practices
in Human and Animal Medical Diagnostic Laboratories (Centers for Disease Control and Prevention. MMWR
2012;61[Suppl]:1-102. http://www.cdc.gov/mmwr/pdf/other/su6101.pdf) publications and applicable state or local
regulations or those recommended by the World Health Organization in the Tuberculosis laboratory biosafety
manual (versions in various languages available at http://www.who.int/tb/publications/2012/tb_biosafety/en/)
and the Xpert MTB/RIF implementation manual available at http://www.who.int/tb/publications/xpert_implem_
manual/en/.

Testing Guidelines

The table below, organized by published performance, summarizes the reported findings and refers readers to
the appropriate section(s) of the document. Additional meta-analyses of Xpert performance with extrapulmonary
sample types have been published by Maynard-Smith et al.1 and Denkinger et al.2

Quick Reference Guide

Sensitivity/ Guide
Specimen Type* Pros/Cons Specificity Section # References^

Pros: Easy sample handling. Gu et al.

Gu et al. 3
Bone and Joint 82%/100%
2.1 Held et al. 4
Aspirates Cons: Biopsy tissue or aspirates of pus must be available Held et al.
for testing. 95.6%/96.2%

Bronchial Aspirates
via fiberoptioc Pros: Samples can be obtained from patients who do not
produce sputum. 80-93%/
scope or Le Palud et al.5
87.7-98.6%
Bronchoalveolar Theron et al.6
In Mok et al., where
Lavages 2.2 Barnard et al.7
94% were smear-
(Note: Bronchial Mok et al.8
Cons: Invasive procedure; should be performed in medical negative samples,
washings are not
centers with trained personnel. 68%/98%
an appropriate
sample type.)

WHO Xpert MTB/

Pros: Other tissues, especially smear-positive, may be RIF Implementation
Lymph nodes 83.1%/97.8% 1.2
tested in the same manner. Manual9
Denkinger et al. 2

Pros: NAAT more sensitive than smear. WHO Xpert MTB/

Cerebrospinal Fluid RIF Implementation
80.5%/99.8% 1.1
(CSF) Cons: Higher volume of sample yields better results. Manual9
Concentrated sample has higher sensitivity. Denkinger et al. 2

Pros: Sample type readily available for patients who cannot

Moussa et al.10
produce sputum.
80.5-85.7%/ Kokuto et al.11
Stool Cons: Different processing methods yield varying results. 2.3
94-100% Hillemann et al.12
Centrifugation required. Overall number of samples tested Banada et al.13
is low.

Xpert MTB/RIF Alternate Specimen Testing Procedures | page 2

 Sensitivity/ Guide
Specimen Type* Pros/Cons Specificity Section # References^

Pros: Does not require patient to produce sputum. Pang et al.14

48.6-87.5%/
Gastric Aspirates 2.4 Singh et al.15
Cons: Gastric aspirate invasive procedure and can be 89.6-100%
Hillemann et al.12
difficult in children.

Pros: Does not require patient to produce sputum 19-44.4%/

Peter et al.16
Urine 98-100% in 2.5
Cons: Better sensitivity with centrifugation. Better Lawn et al.17
HIV+ patients
sensitivity in HIV+ patients with CD4<200.

Pros: If positive, will allow faster treatment than culture.

15-46.4%/ Meldau et al.18
Pleural fluid N/A
Cons: Sensitivity so low that this sample type is not 98-99.1% Porcel et al.19
recommended.

*By reported performance

^Not all references are used in protocol summaries

The following sections provide synopses of recommendations from the World Health Organization (WHO) and
peer-reviewed publications. Section 1 summarizes recommendations from WHO, focusing on testing CSF and
lymph node specimens. Section 2 summarizes recommendations from various peer-reviewed publications,
focusing on bone and joint, fiberoptically obtained respiratory secretions or tissues, stool, gastric aspirates,
and urine specimens. References on pleural fluid testing are provided in the table above, but routine testing of
that sample type is not recommended due to the very low sensitivity reported for the Xpert MTB/RIF test.

Note: All manipulations of non-sputum specimens containing viable organisms should be performed in a
biological safety cabinet.

Section 1. WHO Protocols for Testing Cerebrospinal Fluid (CSF) and Lymph Nodes

Protocols from WHO:

Document 1 (Policy): Xpert MTB/RIF test for the diagnosis of pulmonary and extrapulmonary TB in adults
and children. This is a comprehensive document summarizing multiple studies using the Xpert MTB/RIF test.
http://www.who.int/tb/publications/xpert-mtb-rif-assay-diagnosis-policy-update/en/

Document 2 (Implementation; laboratory protocols): Xpert MTB/RIF implementation manual. This document
contains standard operating procedures for processing lymph nodes and other tissues, and CSF for testing
with Xpert MTB/RIF. http://www.who.int/tb/publications/xpert_implem_manual/en/

This WHO manual states that the Xpert MTB/RIF test can be used to process CSF specimens and
homogenized tissue specimens (i.e., biopsies of lymph nodes or other tissues), or decontaminated specimens
(if not collected in a sterile manner), if culture is performed concurrently to enhance the sensitivity of detecting
true positives. Whenever possible, specimens should be transported and stored at 28 C prior to processing.
The maximum time for storage and processing is seven days.

Xpert MTB/RIF Alternate Specimen Testing Procedures | page 3

1.1 Protocol for CSF

The optimal method for processing CSF using Xpert MTB/RIF depends on the volume of specimen
available for testing. Blood-stained and xanthochromic CSF specimens may cause false-negative results
from Xpert MTB/RIF due to inhibition of PCR.

If there is more than 5 mL of CSF available for testing:

Transfer the CSF specimen to a conical centrifuge tube and concentrate the specimen at 3000 x g for
15 minutes.
Carefully pour off the supernatant through a funnel into a discard can containing 5% phenol or other
mycobacterial disinfectant. Make certain that you leave approximately 0.5 mL of the supernatant in the tube
to ensure that the pellet remains intact.

Note: Concentrated CSF should be decanted in a biological safety cabinet.

Resuspend the pellet to a final volume of 2 mL by adding Xpert MTB/RIF sample reagent. Mix the pellet
suspension by vortexing to ensure that none of the suspension remains on the sides or bottom of the
tube. After seven to eight minutes of incubation at room temperature, vortex the sample a second time as
indicated in Xpert MTB/RIF product insert. Incubate for an additional seven to eight minutes (15 minutes total
incubation) at room temperature.
Label an Xpert/MTB/RIF cartridge with the specimens identification number.
Using a fresh transfer pipette, transfer 2 mL of the resuspended CSF sample to the Xpert MTB/RIF
cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturers instructions.

If there is 15 mL of CSF available:

Add an equal volume of Sample Reagent to the CSF. Mix the specimen and the Sample Reagent by
vortexing as described above. After seven to eight minutes at room temperature, vortex the sample a
second time. Incubate for an additional seven to eight minutes (15 minutes total incubation) at
room temperature.
Add 2 mL of the sample mixture directly to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturers instructions.

If there is 0.11 mL of CSF available

Add enough Sample Reagent to the CSF specimen to achieve a final volume of 2 mL. Mix the specimen and
the Sample Reagent by vortexing as described above. After seven to eight minutes at room temperature,
vortex the sample a second time. Incubate for an additional seven to eight minutes (15 minutes total
incubation) at room temperature.
Add the 2 mL of sample mixture directly to the Xpert MTB/RIF cartridge.
Load the cartridge into the GeneXpert instrument following the manufacturers instructions.

If there is less than 0.1 mL of CSF available

This is an insufficient sample size for testing using Xpert MTB/RIF.

Xpert MTB/RIF Alternate Specimen Testing Procedures | page 4

1.2 Protocol for lymph nodes and other tissues (sterile collection)

Note: All manipulations should be performed in a biosafety cabinet.

Cut the tissue specimen into small pieces in a sterile mortar, homogenizer, or tissue grinder using sterile
forceps and scissors.
Add approximately 2 mL of sterile phosphate buffered saline (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.
Place approximately 0.7 mL of the homogenized tissue in a sterile, conical screw-capped tube using a
transfer pipette.

Note: Avoid transferring any clumps of tissue that have not been properly homogenized.

Use a transfer pipette to double the volume of the specimen with Xpert MTB/RIF Sample Reagent (i.e., add
1.4 mL of Sample Reagent to 0.7 mL of homogenized tissue).
Shake the tube vigorously 10 to 20 times or vortex for at least 10 seconds.
Incubate the Sample Reagent for 10 minutes at room temperature, and then shake the specimen again for
another 1020 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 manufacturers instructions.

Note: For samples not collected in a sterile manner, the WHO manual suggests a NaOH decontamination/
concentration protocol similar to that used for sputum. See section 3.3.2. of the WHO document.

Section 2. Peer-reviewed protocols for bone and joint specimens, fiberoptically obtained
bronchial aspirates or bronchoalveolar lavages, stool, gastric aspirates, and urine

2.1 Bone and Joint Specimens

Protocol from Gu et al3:

Obtain 1 mL of purulent specimen and mix with 2 mL of Sample Reagent.
Vortex the sample for at least 10 seconds, then incubate at room temperature for 10 minutes. Vortex for
another 10 seconds and incubate at room temperature for five minutes.
Add 2 mL of the mixture to the Xpert MTB/RIF cartridge and load the cartridge onto the instrument following
the manufacturers instructions.

2.2 Fiberoptically obtained bronchial aspirates or bronchoalveolar lavages

Protocol from Le Palud et al5:

Decontaminate the specimen with N-acetyl-cysteine- 2% NaOH and concentrate by centrifugation at
3,000 x g for 20 minutes. Discard the supernatant except for approximately 0.5 mL of the supernatant, and
resuspend the pellet in 0.5 mL of fluid.

Xpert MTB/RIF Alternate Specimen Testing Procedures | page 5

 Add Sample Reagent in a 2:1 ratio to the resuspended pellet.
Mix the specimen and the Sample Reagent by vortexing. After seven to eight minutes at room temperature,
perform a second vortexing step. Incubate for an additional seven to eight minutes (15 minutes total
incubation) at room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument following the manufacturers instructions.

2.3 Stool specimens

Protocol from Kokuto et al11:

Dilute approximately 2 mm3 of stool in 10 mL of sterile water, cap the tube tightly, and vortex vigorously to
produce a smooth suspension.
Allow the suspension to sit at room temperature for 15 minutes to sediment particulates.
Aspirate the entire supernatant into a conical centrifuge tube and centrifuge at 3000 x g for 20 minutes.
Remove the supernatant and resuspend the sediment in 1-3 mL of phosphate buffered saline, depending
on number of additional tests to be performed on the specimen.
Mix 1 mL of the resuspended sediment with 2 mL of Cepheid Sample Reagent. Mix the specimen and
the Sample Reagent by vortexing. After seven to eight minutes at room temperature, perform a second
vortexing step. Incubate for an additional seven to eight minutes (15 minutes total incubation) at
room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument following the manufacturers instructions.

2.4 Gastric Aspirates

Protocol from Pang et al14:

Neutralize 5 mL of gastric aspirate with an equal volume of sterile 1% sodium bicarbonate. Store frozen at
-20C if the sample is not to be tested immediately.
Thaw sample (if previously frozen), homogenize, and digest in 1.5% N-acetyl-L-cysteine-NaOH-Na citrate
(1.5% final concentration) and vortex for 30 seconds.
Incubate specimen for 15 minutes at room temperature, followed by neutralization with phosphate buffered
saline (PBS), then centrifuge at 4000 x g for 15 minutes.
Discard the supernatant and resuspend the sediment in 2 mL of PBS.
For testing on Xpert MTB/RIF, add 1.5 mL of Sample Reagent to 0.5 mL of the concentrated sediment.
Mix the specimen and the Sample Reagent by vortexing. After seven to eight minutes at room temperature,
vortex the sample a second time. Incubate for an additional seven to eight minutes (15 minutes total
incubation) at room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument following the manufacturers instructions.

Xpert MTB/RIF Alternate Specimen Testing Procedures | page 6

2.5 Urine Specimens

Protocol from Peter et al16:

Obtain 1 mL of unprocessed, undiluted urine. Add Sample Reagent in a 2:1 ratio to the urine and mix
vigorously or vortex. After seven to eight minutes at room temperature, vortex the sample a second time.
Incubate the sample for an additional seven to eight minutes (15 minutes total incubation) at
room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument.
Alternatively, centrifuge 2 to 20 mLs urine at 3000 x g for 15 minutes. Discard the supernatant except
for approximately 0.5 mL of the supernatant, and resuspend the urine specimen pellet in 1 mL of sterile
phosphate-buffered saline.
Add Sample Reagent in a 2:1 ratio to the resuspended urine. Mix the specimen and the Sample Reagent by
vortexing. After seven to eight minutes at room temperature, perform a second vortexing step. Incubate for
an additional seven to eight minutes (15 minutes total incubation) at room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument following the manufacturers instructions.

Protocol from Lawn et al17:

Obtain 2 mL of unprocessed, undiluted urine. Centrifuge the urine at 3000 x g for 15 minutes, remove the
supernatant, and resuspend the pellet in 0.75 mL of phosphate buffer.
Add Sample Reagent at a 2:1 ratio to urine pellet.
Mix the specimen and the Sample Reagent by vortexing. After seven to eight minutes at room temperature,
vortex the sample a second time. Incubate for an additional seven to eight minutes (15 minutes total
incubation) at room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument following the manufacturers instruction.
For frozen urines (30-40 mL volume), thaw and centrifuge the specimen at 3,000 x g for 15 minutes.
Remove the supernatant carefully making sure to retain the pellet; save ~1mL of supernatant.
Take the pellet, and resuspend it in 0.75 mL of the saved supernatant.
Add Sample Reagent at a 2:1 ratio to the resuspended urine.
Mix the specimen and the Sample Reagent by vortexing. After seven to eight minutes at room temperature,
vortex the sample a second time. Incubate for an additional seven to eight minutes (15 minutes total
incubation) at room temperature.
Add 2 mL of the Sample Reagent-treated specimen to the Xpert MTB/RIF cartridge and load the cartridge
onto the instrument following the manufacturers instructions.

Xpert MTB/RIF Alternate Specimen Testing Procedures | page 7

References (including links to full text articles where available):
1 Maynard-Smith L, et al. Diagnostic accuracy of the Xpert MTB/RIF assay for extrapulmonary and pulmonary tuberculosis when testing non-
respiratory samples: a systematic review. BMC Infect Dis 2015, 14: 709. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298952/

2 Denkinger CM, et al. Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis. The European
respiratory journal 2014, 44: 435-446. http://tbevidence.org/wp-content/uploads/2014/04/Denkinger-ERJ-2014-Xpert-EPTB.pdf

3 Gu Y, et al. Xpert MTB/RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis. Int J Infect Dis 2015, 36: 27-30.
http://www.ijidonline.com/article/S1201-97121500122-8/pdf

4 Held M, et al. GeneXpert polymerase chain reaction for spinal tuberculosis: an accurate and rapid diagnostic test. Bone Joint J 2014, 96-B: 1366-
1369. http://www.bjj.boneandjoint.org.uk/content/96-B/10/1366

5 Le Palud P, et al. Retrospective observational study of diagnostic accuracy of the XpertR MTB/RIF assay on fiberoptic bronchoscopy sampling
for early diagnosis of smear-negative or sputum-scarce patients with suspected tuberculosis. BMC Pulm Med 2014, 14: 137. http://bmcpulmmed.
biomedcentral.com/articles/10.1186/1471-2466-14-137

6 Theron G, et al. Accuracy and impact of Xpert MTB/RIF for the diagnosis of smear-negative or sputum-scarce tuberculosis using bronchoalveolar
lavage fluid. Thorax 2013, 68: 1043-1051.

7 Barnard DA, et al. The utility of Xpert MTB/RIF performed on bronchial washings obtained in patients with suspected pulmonary tuberculosis in a
high prevalence setting. BMC Pulm Med 2015, 15: 103. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573925/pdf/12890_2015_Article_86.pdf

8 Mok Y, et al. Do we need transbronchial lung biopsy if we have bronchoalveolar lavage XpertR MTB/RIF Int J Tuberc Lung Dis 2016, 20: 619-624.

9 WHO. Xpert MTB/RIF implementation manual: technical and operational how-to; practical considerations. ed. Geneva: World Health Organization
2014. http://www.who.int/tb/publications/xpert_implem_manual/en/

10 Moussa H, et al. GeneXpert for direct detection of Mycobacterium tuberculosis in stool specimens from children with presumptive pulmonary
tuberculosis. Ann Clin Lab Sci 2016, 46: 198-203.

11 Kokuto H, et al. Detection of Mycobacterium tuberculosis MTB in Fecal Specimens From Adults Diagnosed With Pulmonary Tuberculosis Using the
Xpert MTB/Rifampicin Test. Open Forum Infect Dis 2015, 2: ofv074. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462888/

12 Hillemann D, et al. Rapid molecular detection of extrapulmonary tuberculosis by the automated GeneXpert MTB/RIF system. J Clin Microbiol 2011,
49: 1202-1205. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122824/

13 Banada PP, et al. A novel sample processing method for rapid detection of tuberculosis in the stool of pediatric patients using the Xpert MTB/RIF
assay. PLoS One 2016, 11: e0151980. http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0151980

14 Pang Y, et al. Evaluation of the Xpert MTB/RIF assay in gastric lavage aspirates for diagnosis of smear-negative childhood pulmonary tuberculosis.
Pediatr Infect Dis J 2014, 33: 1047-1051.

15 Singh S, et al. Xpert MTB/RIF assay can be used on archived gastric aspirate and induced sputum samples for sensitive diagnosis of paediatric
tuberculosis. BMC Microbiol 2015, 15: 191. http://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-015-0528-z

16 Peter JG, et al. The diagnostic accuracy of urine-based Xpert MTB/RIF in HIV-infected hospitalized patients who are smear-negative or sputum
scarce. PLoS One 2012, 7: e39966. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039966

17 Lawn SD, et al. Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic
testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa. BMC Med 2015, 13: 192. http://bmcmedicine.biomedcentral.
com/articles/10.1186/s12916-015-0432-2

18 Meldau R, et al. Comparison of same day diagnostic tools including GeneXpert and unstimulated IFN-gamma for the evaluation of pleural
tuberculosis: a prospective cohort study. BMC Pulm Med 2014, 14: 58. http://bmcpulmmed.biomedcentral.com/articles/10.1186/1471-2466-14-58

19 Porcel JM, et al. XpertR MTB/RIF in pleural fluid for the diagnosis of tuberculosis. Int J Tuberc Lung Dis 2013, 17: 1217-1219.

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