Drug Resistance in Mycobacterium Tuberculosis: An Investigation of Drug Transporter P-glycoprotein and an Evaluation of Improved Diagnosis of Drug Resistant Tuberculosis in Cameroonian Pulmonary Tuberculosis Patients

Abstract

Tuberculosis (TB) is a major health problem, especially in Africa and Southeast Asia. The disease, caused by Mycobacterium tuberculosis, (Mtb) is curable with a multidrug regimen of Rifampicin (RIF), Isoniazid (INH), Ethambutol and Pyrazinamide. However, treatment is not always successful, as ~ 2 million patients fail treatment each year, in part, because the bacterium has become resistant to these drugs. Thus, efficient diagnosis of drug-resistant Mtb is extremely important. This study sought to determine if a new molecular assay, the Genotype MTBDRplus, could accurately detect drugresistant Mtb in Cameroon. When compared to the conventional drug susceptibility testing assay, the molecular assay identified 98% (48/49) RIF-resistant isolates, 92% (55/60) INH-resistant isolates, and 94% (46/49) of Mtb resistant to both drugs in Cameroonian TB patients. Further evaluation of the molecular assay with an additional 50 Mtb isolates, identified 6% (16/275) of isolates with questionable RIF-resistant results. To determine if these 16 isolates were truly RIF-resistant, the Rifampicin Resistance-determining Region of the rpoB gene were sequenced. Mutations were found known as ‘disputed’ RIF mutations, i.e., mutations that are not always associated with resistance to RIF. Thus, sequencing results confirm that when DNA from Mtb isolates do not hybridize with the wild-type rpoB probe, it is wise to assume the Mtb isolate is RIF-resistant and treat accordingly. The Genotype MTBDRplus assay can be adopted by the government of Cameroon to diagnose drug-resistant TB. In addition to mycobacterial mutations, host factors that cause sub-therapeutic plasma drug concentration could lead to treatment failure. One of such host factors could be variation in the liver efflux pump, P-glycoprotein (p-gp). Therefore, the relative amount of p-gp V protein in 87 human liver samples was measured. P-gp was found to be present from birth reaching 90 % of adult levels by age 5. Since low variability (2.9 + 0.32 fold) in pgp occurred among individuals, it is unlikely variability in p-gp protein accounts for wide variation in RIF plasma levels. The data are important because expression p-gp in children provides a plausible explanation as to why, when given the same dose/kg of RIF as adults, children clear the drug faster.