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.
