Pyrosequencing, a new tool in management of tuberculosis.
In the traditional management of tuberculosis, the gold standard test for detecting antibiotic resistance is the bactec mgit system. However it takes 3 to 6 weeks for the results to come and this delays proper att regimen for each patient.
To overcome this drawback there is a new genetic technology called as pyrosequencing assay. This can be performed on sputum, CSF and other body fluids. Principle is that it detects resistance to 6 att drugs which is INH, RCIN, ETB, Aminoglycosides, quinolones. The turn around time is fast and it gives results in 2 days time.
Principle:
During synthesis of complimentary DNA strand by the DNA polymerase, when nucleotides are added to the chain pyrophosphate is released. This emits light which is picked up by a sensor and a peak is generated. The height of the peak is proportional to dNTP. Pyogram then reveals the mutated sequence.
Pyrosequencing detects the following mutations suggesting drug resistance:
katG--INH
rpoB--- RCIN
embB---ETB
rrs--- aminoglycosides
gyrA---quinolones
Presence of these mutations implies that particular drug cannot be used.
Pyrosequencing results are 95% concordant with results from traditional bactec mgit system as per clinical studies and trials.
Clinical applications: now if we have a critical case of meningitis in icu or a suspected MDR TB of lungs its easy to perform this test and start appropriate regimen within 2 days time. No more delay.
Drawbacks: Expensive, not available at all labs, can detect resistance to only 6 drugs.
What if there is a new mutation affecting a particular group of drugs. It may get missed out since the system detects small regions of dna.
If used correctly pyrosequencing can be a game changer in management of tuberculosis.
Dr Hardik Thakker,
MD MED, ECFMG (USA).
August 2023
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