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Antimycobacterial susceptibility testing is valuable in the proper treatment of patients with tuberculosis. The treatment of tuberculosis is commonly through a multiple drug regimen that includes the antimycobacterial drug pyrazinamide. It is important that the antimycobacterial drugs prescribed show appropriate activity against Mycobacterium tuberculosis, i.e., susceptibility of the isolate to the drug.

Multidrug resistant Mycobacterium tuberculosis (MDR-TB) has recently become a serious public health problem.¹ Resistance to any of the primary drugs, including pyrazinamide, makes the disease more difficult and expensive to treat. The rapid detection of these resistant isolates is critical to effective patient management.

Two methods have been widely used for antimycobacterial susceptibility testing. The first method, known as the Method of Proportion,² uses Middlebrook and Cohn 7H10 Agar. It compares colony counts on drug-containing and drug-free media. The testing for pyrazinamide requires some modification from the general methods because the drug is active in vitro only at lower pH values.³ A modification to the method of proportion method was developed using a 7H10 agar medium at pH 5.5, with a drug concentration of 25-50 μg/mL.⁴ A limitation of the method is that at a pH of 5.5, many isolates of M. tuberculosis either fail to grow or grow poorly. Agar-based methods such as the agar proportion method have not proven to be satisfactory for PZA susceptibility testing because of the failure of many isolates to grow when the agar has been acidified for the PZA test.

The second method, known as the BACTEC 460TB radiometric susceptibility method,⁵ is based on the production of radioactive ¹⁴C-labeled carbon dioxide by the growing mycobacteria, manifested by a Growth Index increase in the system. A modification to the BACTEC 460TB susceptibility method was developed using a modified 7H12 radiometric medium, BACTEC PZA Test Medium, with a reduced pH of 6.0.⁶ At this pH, PZA activity against mycobacteria can be determined without inhibiting the growth of most M. tuberculosis isolates. The BACTEC 460TB PZA susceptibility test uses a pyrazinamide drug concentration of 100 μg/mL. Susceptibility testing in the BACTEC 460TB System has proven to be satisfactory and is presently considered the reference method for PZA susceptibility testing. The Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) recommends the BACTEC 460TB method for PZA susceptibility testing.²

Use of the BACTEC MGIT instrument in combination with the BACTEC MGIT 960 PZA kit is a non-radiometric method of determining antimycobacterial susceptibility to PZA. The BACTEC MGIT 960 PZA Kit has been developed to allow susceptibility testing at a pyrazinamide concentration of 100 μg/mL. This concentration correlates with the concentration used in the BACTEC 460TB System.

Upon receipt of a new shipment or lot number of BACTEC MGIT 960 PZA Kit vials or BACTEC MGIT 960 PZA Medium, it is recommended that the control organism shown below be tested. The control organism should be a pure culture and the culture should be prepared according to "INOCULUM PREPARATION" instructions.

The quality control (QC) AST Set should be prepared according to the "Inoculation Procedure for BACTEC MGIT 960 PZA Susceptiblity Test" instructions. Important considerations when preparing the QC AST Set are the proper reconstitution of the lyophilized drug, use of pure culture and the proper dilution of the QC organism for the Growth Control and PZA tubes. It is important to add drug only to the corresponding MGIT tube labeled "PZA."

The same control organism should be run as batch QC once each week when susceptibility testing is performed. Observation of the proper results, as shown below, within 4–20 days indicates that the BACTEC MGIT 960 PZA reagents are ready for use in testing patient isolates.

If the proper results are not observed, do not report patient results. Repeat QC and any patient isolates affected by the initial QC failure. If the repeat QC does not perform as expected, do not report patient results. Do not use the product until you have contacted Technical Services at (800) 638-8663 (United States Only).

During the external evaluation of the BACTEC MGIT 960 PZA Kit the average time to result for the control organism was seven days with a range of four to eleven days. The most common causes of QC failures during the external evaluation were overinoculated PZA Sets and contaminated QC cultures.

The BACTEC MGIT 960 PZA Medium tube contains 110 μL of fluorescent indicator and 7 mL of PZA broth. The indicator contains Tris 4,7 - diphenyl-1, 10 phenanthroline ruthenium chloride pentahydrate in a silicone rubber base. The tubes are capped with a polypropylene cap. The pH is adjusted to 5.9.

BACTEC MGIT 960 PZA Kit contains two lyophilized vials of pyrazinamide and six vials of PZA Supplement.

BACTEC MGIT 960 PZA Supplement contains 15 mL of enrichment

For in vitro Diagnostic Use.

Working with M. tuberculosis growth in culture requires Biosafety Level (BSL) 3 practices, containment equipment and facilities.

Read and follow directions contained in all appropriate package inserts including the BBL™ MGIT 7 mL Mycobacteria Growth Indicator Tube.

Prior to use, the user should examine the tubes and vials for evidence of contamination or damage. Discard any tubes or vials if they appear unsuitable. Dropped tubes should be examined carefully. If damage is seen, the tube should be discarded.

In the event of tube breakage: 1) Close the instrument drawers; 2) Turn off the instrument; 3) Vacate the area immediately; 4) Consult your facility/CDC guidelines. An inoculated leaking or broken tube may produce an aerosol of mycobacteria; appropriate handling should be observed.

Autoclave all inoculated MGIT tubes prior to disposal.