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.

In the Cartridge Based Nucleic Acid Amplification Test (CBNAAT) machine, Sample Processing Control (SPC) monitors sample processing.

Once the cartridge has been loaded into CBNAAT module, system control checks:

• The instrument and software readiness
• Conditions for sample processing
• The integrity of PCR reagents and PCR efficiency.

Instrument system check control verifies the:

• Optics
• Mechanical components
• Temperature of the module
• Physical integrity of each cartridge
• Favourable PCR reaction conditions.

If any aspect of system check control fails, an ERROR is reported.

The CBNAAT System automatically performs internal quality control for each sample. 

During each test, the system uses the following inbuilt controls:

System Check Control (SCC)

• Checks integrity of the instrument, cartridges and PCR reagents.

Sample Processing Control (SPC)

• Ensures that a sample is correctly processed.
• Included in the cartridge and is processed with the sample. The DNA is detected by a PCR assay.

Probe Check Control (PCC)

The External Quality Assessment (EQA) for Fluorescent Microscopy (FM) follows the same basic principles as for sputum smear microscopy using Ziehl-Neelsen (ZN) staining:

1. On-site Evaluation (OSE)
2. Panel Testing
3. Random Blinded Re-checking

Differences between EQA for FM and sputum smear stained by ZN method:

The Laboratory Technician (LT) must safely discard contaminated, biohazard waste generated by tuberculosis (TB) laboratories. This waste must be discarded along with the overall waste of the health facility in which TB services are provided.

There are 2 types of waste generated from DMC laboratory settings:

1. Sputum containers with specimen and wooden sticks
2. Stained slides

Disposal of Sputum Cups with Left-over Specimen, Lids and Wooden Sticks

One of the methods for internal monitoring of the quality of the microscopy process is the use of Quality Control (QC) slides.

QC is a systematic internal monitoring of working practices, technical procedures, equipment and materials, including quality of stains.

Quality Control Positive (QCP) and Quality Control Negative (QCN) slides are used for sputum smear microscopy quality monitoring.

Process for quality control using QCP and QCN is as follows: