Rapid molecular detection of tuberculosis and rifampin resistance

Abstract

Background: Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect.

Methods: We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test.

Results: Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay.

Conclusions: The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Figure 1. Enrollment and Outcomes

Patients were enrolled at centers that have diverse populations with a high prevalence of tuberculosis. In Lima, Peru, patients with suspected tuberculosis were enrolled at 30 primary care clinics with a high rate of tuberculosis case notification, a rate of coinfection with the human immunodeficiency virus (HIV) of less than 3%, and a low rate of multidrug resistance. In Cape Town and Durban, South Africa, patients were enrolled at primary care tuberculosis clinics located within informal settlements with a high incidence of tuberculosis and an estimated rate of HIV coinfection of 70% and a rate of multidrug resistance of 4%. In Mumbai, India, patients with complicated tuberculosis and a rate of multidrug resistance as high as 50% were enrolled at a tertiary care center. In Baku, Azerbaijan, prisoners were enrolled on arrival at a tuberculosis screening and treatment facility, which reports a high rate of multidrug resistance (25%) among patients with tuberculosis and a rate of HIV coinfection of approximately 6%. LJ denotes Löwenstein–Jensen, MGIT mycobacteria growth indicator tube, and NALC–NaOH N-acetyl-l-cysteine and sodium hydroxide.

Figure 2. Assay Procedure for the MTB/RIF Test

Two volumes of sample treatment reagent are added to each volume of sputum. The mixture is shaken, incubated at room temperature for 15 minutes, and shaken again. Next, a sample of 2 to 3 ml is transferred to the test cartridge, which is then loaded into the instrument. All subsequent steps occur automatically. The user is provided with a printable test result, such as “MTB detected; RIF resistance not detected.” PCR denotes polymerase chain reaction.