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One of the common problems encountered while using the CBNAAT machine is the non-detection of the module.

Usually, there is a loss of communication between modules and software, leading to non-detection.

Origins:

• Ethernet connection between Personal Computer (PC) and CBNAAT is bad
• Power supply issue (main power or Universal Power Supply (UPS) fluctuations)
• Bad connection points between gateway board and modules
• Too high room temperature 

Solutions:

If the barcode scanner is not working, enter the cartridge barcode manually (Figure):

• Step 1: Click on “Create Test”
• Step 2: A dialogue box - Scan Cartridge Barcode will appear. Click on “Manual Entry”
• Step 3: Manually type the 2-line numbers of the cartridges

Figure: Process for Manual Entry of the Cartridge Barcode

The most common situations that need troubleshooting while using CBNAAT are:

• Hardware or instrument problems
• Failures without error codes 
• Failures with error codes 

Troubleshooting Approach

1. In case of an issue, a message will be displayed (often with an error code). 
2. Check if the error affects one particular module. 
3. Refer to the CBNAAT user manual and follow the recommended corrective action.

The Cartridge-based Nucleic Acid Amplification Test (CBNAAT) test has some limitations such as:

The test results of the Cartridge-based Nucleic Acid Amplification Test (CBNAAT) assay are displayed in the ‘View Results’ window, of the CBNAAT software.

For visualizing the results after the test is completed:

The assay needs to be repeated by using a new cartridge if one of the following test results occur:

INVALID: An INVALID result indicates that Sample Processing Control (SPC) failed. The sample was not properly processed, or Polymerase Chain Reaction (PCR) was inhibited.

ERROR: An ERROR indicates that Probe Check Control (PCC) failed, and the assay was aborted possibly due to the reaction chamber being filled improperly, a reagent probe integrity, syringe pressure issues, or failure of the CBNAAT module.

The following procedures are recommended when processing various body fluids with the Cartridge-based Nucleic Acid Amplification Test (CBNAAT):

Bronchoalveolar Lavage (BAL): 

Processing of BAL for CBNAAT assay is given here. However, it is important that each laboratory optimizes this protocol to minimize the error rate.

If the BAL volume is sufficient (approx. 5 ml), centrifuge and dissolve sediment into 1 ml sterile phosphate buffer/ saline, then add sample reagent in a 1:2 ratio.

The following Standard Operating Procedures (SOPs) are recommended when processing the following extrapulmonary samples:

SOPs for Lymph Node and Tissues Samples

The molecular beacons targeting the rpoB gene cover all the mutations found in more than 99.5% of Mycobacterium tuberculosis rifampicin-resistant strains.

The molecular beacon consists of:

• Loop: 18-30 base pair region of the molecular beacon, which is complementary to the target sequence
• Stem: 5-7 bp complementary sequence on both the ends of the loop
• 5’ Fluorophore: Fluorescent dye covalently linked at the 5' end
• 3’ Quencher: Nonfluorescent dye covalently linked at the 3' end

Probe Check Control (PCC) of Cartridge Based Nucleic Acid Amplification Test (CBNAAT) Technology tests the fluorescence readings of probes at different temperatures, before the start of thermal cycling.

PCC verifies:

• Re-hydration of beads
• Filling of the Polymerase Chain Reaction (PCR) tube
• Integrity of probes
• Stability of a dye or the reagents/ quencher.

The results are automatically compared to the pre-established factory settings in the software.