Appendix 3. Xpert MTB/RIF


Xpert MTB/RIF assay is based on hemi-nested real time PCR for simultaneous detection of M. tuberculosis (MTB) and rifampicin (RIF) resistance. The target is the rpoB gene, critical for detection of mutations associated to rifampicin resistance.

Xpert MTB/RIF automates all aspects of real time PCR analysis, with results available in 2 hours.

3.1 Sample processing

The test can be performed using fresh sputum samples or decontaminated samples prior culture inoculation.

Procedure with fresh sputum samples

– Ask the patient to rinse the mouth twice before collecting the sample.
– Collect a minimum of 1.5 ml good quality sputum.
– Follow the procedures outlined below:

Procedure for Xpert MTB/RIF*

Step 1



Add the reagent 2:1 (v/v) to the sample and shake 10-20 times.
Incubate at room temperature for 15 min.; during incubation repeat once shaking for 10- 20 times.

Step 2

With a pipette transfer the diluted sample into a cartridge.

Step 3

Insert the cartridge in the machine and start the test.

* Source: National Health Laboratory Services, South Africa.

Procedure for sediment samples

– Sediments can be prepared according to standard decontamination procedure (NALCNaOH method) and re-suspend with phosphate buffer.
– Ensure 0.5 ml is available for the test; add 1.5 ml of reagent for 0.5 ml of re-suspended sediment.
– Follow the procedure described in the above figure.

3.2 Interpretation of the results

Proper test performance is ensured by 2 internal controls:
– Sample processing control (SPC) ensures adequate processing and monitors presence of inhibition.
– Probe check control (PCC) verifies that the steps of the tests (rehydration, filling of the cartridge, etc.) take place correctly.

When the test is completed the display can show:
– “MTB detected” expressed by levels (the higher the level, the higher the amount of MTB detected in the sample) or “MTB not detected”;
– RIF results expressed as “detected”, “not detected” or “indeterminate” are available only if MTB is detected.

Other possible results:
– Invalid: MTB invalid and SPC failed due to one of several reasons, such as inhibition;
– Error: MTB no result, SPC no result, PCC failed; fail of system components;
– No result: e.g. tests stopped during processing.

3.3 Storage of samples and cartridges

Samples

– For a period ≤ 3 days: store at 35°C maximum. A cold chain is not required for up to 3 days after collection. During this period of time, overgrowth of normal flora does not have negative influence on the test. However, if a cold chain is available, samples should be stored at 2 to 8°C in order to help their preservation.

– For a period of 4 to 10 days: store refrigerated at 2 to 8°C. If samples require other testing (i.e. smear microscopy and/or culture), sample storing conditions adequate for microscopy and culture have to be followed. CPC does not interfere with Xpert MTB/RIF testing.

Cartridges

– To be stored at 2 to 28°C.
– The cartridge should be used within 30 minutes of opening the cartridge lid.
– Cartridges are stable for 7 days after opening the packaging.

3.4 Logistic requirements

Power supply

The device requires stable and uninterrupted power supply.
Each GeneXpert instrument will need a uninterruptible power supply (UPS).
The minimum requirement for the functioning of the GeneXpert instrument is to have a 800VA UPS.

Operating temperature

The operating temperature for GeneXpert instrument device is 15 to 30°C. According to climate conditions, the installation of air conditioning can be recommended to keep the area within the temperature ranges indicated by the manufacturer.

Calibration

The GeneXpert modules require annual calibration, which must be performed by an authorised service provider or carried out by swapping out the modules. A detailed commercial sales contract and customer support plan should be negotiated with the supplier, guaranteeing regular maintenance, calibration, repair and replacement (when needed).

Cartridges and reagents shelf-life

12 months from date of production.

Storage space

Each kit contains 10 cartridges and all reagents necessary to run 10 tests.
The dimensions of the kit are 27 x 20 x 17 cm and the weight is 800 g.

Lab space

The GeneXpert IV instrument (4 modules allowing the processing of 4 specimens at the same time) has the following dimensions:
29.8 cm wide, 35.6 cm high, 31.1 cm deep; weight: 12 kg.
It is designed for indoor use only.
Provide at least 5 cm of clearance on each side of the instrument to ensure adequate ventilation.
Do not place the instrument close to the vents of other instruments or air-handling units.

Safety

The personal protection requirements for microscopy should be adopted, including use of gloves and respirators.

Waste disposal

Same procedure as for sputum containers. To be noted is the large volume of additional waste generated by Xpert MTB/RIF compared to smear microscopy.

3.5 Predictive values for detection of rifampicin resistance with Xpert MTB/RIF

Positive predictive value (PPV) is defined as the proportion of subjects with a positive test result who are correctly diagnosed. A high PPV means that when the test yields a positive result, it is most likely correct. Negative predictive value (NPV) is defined as the proportion of subjects with a negative test result who are correctly diagnosed. A high NPV means that when the test yields a negative result, it is most likely correct. PPV and NPV are influenced by the prevalence of disease in the population being tested.

Predictive values according to the prevalence of rifampicin resistance

Source: World Health Organization. Rapid implementation of the Xpert MTB/RIF diagnostic test1.

Rifampicin resistance prevalence

PPV

NPV

True positive*

False negative*

False positive*

True negative*

1%

32.4%

99.9%

9.5

0.5

19.8

970.2

2%

49.2%

99.9%

19

1

19.6

960.4

3%

59.5%

99.8%

28.5

1.5

19.4

950.6

4%

66.4%

99.8%

38

2

19.2

940.8

5%

71.4%

99.7%

47.5

2.5

19

931

6%

75.2%

99.7%

57

3

18.8

921.2

7%

78.1%

99.6%

66.5

3.5

18.6

911.4

8%

80.5%

99.6%

76

4

18.4

901.6

9%

82.4%

99.5%

85.5

4.5

18.2

891.8

10%

84.1%

99.4%

95

5

18

882

11%

85.4%

99.4%

104.5

5.5

17.8

872.2

12%

86.6%

99.3%

114

6

17.6

862.4

13%

87.7%

99.2%

123.5

6.5

17.4

852.6

14%

88.5%

99.2%

133

7

17.2

842.8

15%

89.3%

99.1%

142.5

7.5

17

833

20%

92.2%

98.7%

190

10

16

784

25%

94.1%

98.3%

237.5

12.5

15

735

* Sensitivity (95%) and specificity (98%) for Xpert MTB/RIF rifampicin resistance, compared with reference method (culture).