Innovative approaches to tuberculosis diagnosis with emphasis on nucleic acid amplification tests in a resource constrained high burden tuberculosis setting

Abstract

The global control of tuberculosis (TB) is currently hindered by the low sensitivity of microscopy and the prolonged time-to-result of culture. Recent technical progress has improved both diagnostic accuracy and turnaround, namely, nucleic acid amplification tests (NAAT). The World Health Organization (WHO) has recently endorsed two NAATs, which South Africa has been in the forefront of adopting. Based on WHO recommendations, the Xpert MTB/RIF assay (Xpert) has replaced microscopy as the firstline test in the National Algorithm. With current research and development primarily focused on rapid molecular tests, innovative methods of deployment are essential. In the work reported here, a contribution is offered towards fulfilling this need. This study aimed to show non-inferior diagnostic efficiency for the molecular detection of Mycobacterium tuberculosis from clinical sputum specimens in a novel specimen transport medium PrimeStore® - Molecular Transport Medium (PS-MTM). Technical evaluations of the parameters offered by the transport medium when applied to M. tuberculosis were performed; its ability to inactivate the organism, stabilize its deoxyribonucleic acid (DNA) in specimen over time and show compatibility with silica and magnetic bead-based DNA extraction systems for downstream molecular detection. Additionally, a novel and innovative sputum collection method, where a swab from sputum specimen placed into PS-MTM for the molecular detection of M. tuberculosis, is described. This collection system was evaluated in a routine clinical laboratory against mycobacterial culture, the reference standard. Collection method performance was further validated on sputum from suspected TB patients, at healthcare facilities in rural South Africa to a centralized laboratory for testing. Complete inactivation of M. tuberculosis occurred by 30 minutes after exposure, with a 1:3 sputum to PS-MTM ratio. The specimen remained stable with no significant change over time by real-time polymerase chain reaction (PCR) detection (<5% on a mean starting value) for PS-MTM samples over 28 days at ambient temperature. PS-MTM showed compatibility with all extraction systems; however, the automated bead-based extraction systems displayed better performance, with an estimated 170 CFU/ml lower limit of detection. Of 256 sputum specimens evaluated using the novel collection system, 10.2% were culture positive (routine specimen) and 11.0% positive by real-time PCR (PS-MTM swab from routine specimen). Against culture, detection of M. tuberculosis from swabbed sputum in PS-MTM had a sensitivity of 77% (CI 95%: 56-91%) and specificity of 96% (CI 95%: 93- 98%). Specimens obtained from 141 patients were included for the validation analysis, a subset of a larger cohort study. Concordance between the collection system under evaluation was 82% (McNemar, p=0.55) and 84% (McNemar, p=0.05) for culture and Xpert assay, respectively. Our findings suggest that PS-MTM is capable of improving safety and is an ideal solution for collecting, transporting and stabilizing sputum at ambient temperatures for centralized molecular TB testing. This system provides opportunities for resource-limited settings to introduce or further scale-up molecular diagnostics. PS-MTM samples are capable of bringing forward a significant number of positives, in addition to culture and Xpert testing, that could be regarded as real due to the system’s lower limits of detection and not just false-positives. Application of this system provides quality samples allowing for better discrimination, which in turn could provide adequate management of low bacillary load patients prior to transmission of infection.