Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium Tuberculosis by Droplet Digital PCR.

Abstract

The drug resistance of Mycobacterium tuberculosis constitutes a major public health threat. Existing approaches make it challenging to detect low levels of drug-resistant TB, also known as heteroresistance (HR), in a population. The recently found droplet digital PCR (ddPCR) is a sensitive method for determining the precise amount of nucleic acid in a sample. We used ddPCR to test the Mycobacterium tuberculosis heteroresistance because it delivers more exact quantitative data without the need for a reference curve. A TaqMan-MGB probe mutation detection assay was developed in order to determine the mutant and wild-type sequences of the isoniazid resistance katG (315) gene. We produced heteroresistant MTB combinations, which were subsequently identified by ddPCR, qPCR, and MeltPro/INH. In addition, 21 clinical sputum samples with positive smears were used to validate each method's capacity to determine HR in sputum. We discovered that ddPCR can detect mutant sequences in as few as 0.01% of a combination. DeepMelt TB/INH, which is less sensitive in comparison, cannot detect HR with high resolution and requires a mutation rate of 50% to identify. qPCR likewise has a high resolution of 0.02%, but unlike ddPCR, it cannot determine the exact number of mutations. Our assay is applicable to sputum as well. ddPCR found a katG 315 substitution in two sputums with extremely low values of HR (0.26% and 0.14%). In 21 samples of clinical sputum, the HR prevalence of INH was 9.5%. This work demonstrates that a well-designed ddPCR HR detection test can detect low levels of HR with high accuracy and consistency and gives new information for the clinical diagnosis of drug resistance.