Diagnosis of Pulmonary Tuberculosis and Detection of Resistance to Rifampin and Isoniazid through Direct Molecular Methods in Stool Samples

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Abstract

Background.

Tuberculosis (TB) is an infectious disease that is caused by Mycobacterium tuberculosis (M.tb). TB has high morbidity and mortality around the world.

Objective.

To evaluate molecular-based methods performed directly on stool samples for the diagnosis of pulmonary tuberculosis (PTB) and to determine the susceptibility to Rifampicin (RMP) and Isoniazid (INH).

Study design.

This is a descriptive study evaluating the performance of the PCR-based method for direct PTB diagnosis and to determine the susceptibility of RMP and INH using stool samples from PTB patients. The study was conducted between March 2011 and March 2014.

Methods.

Three stool samples and three sputum samples (n=300 stool and 300 sputum) were collected from 100 PTB patients (75 pretreatment and 25 follow up). Stool samples (n=60) were also collected from 20 healthy individuals to serve as controls. DNA was extracted from stool samples using Chelex®-100. PTB was diagnosed using a Genekam® kit. RMP and INH susceptibility testing was performed using the Genotype MTBDRplus® assay. The sputum Lowenstein Jensen (LJ) culture and agar proportion method (PM) of drug susceptibility testing for INH and RMP were considered to be the gold standard methods for comparing the results of the molecular methods.

Results.

The Genekam kit showed 100% sensitivity and 95.24% specificity for diagnosing new patients and showed 100% sensitivity and 80% specificity for follow-up patients. The Genotype MTBDR-plus assay showed 86.4% and 100% sensitivity and 98.1% and 97.8% specificity for determining INH and RMP sensitivity, respectively, in newly diagnosed patients and 85.7% and 94.4% sensitivity and specificity, respectively, for both INH and RMP in follow-up patients.

Conclusion.

Molecular-based methods are promising techniques for the diagnosis and susceptibility testing of PTB when the ease of sample collection and the speed of diagnosis are taken into consideration. However, they are not as useful for assessing follow-up patients.

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