Comparative evaluation of molecular based detection methods for drug resistant tuberculosis
Abstract
Tuberculosis (TB) is a globally problematic infectious disease.
According to the World Health Organization (WHO) multidrug resistant tuberculosis (MDR-TB) has recently increased by an annual
rate of N20%. In 2015; the WHO noted 580,000 MDR-TB cases of which
b65,000 were cured. This highlights the need for new and improved
methods for TB drug resistance profiling. The aim of this study is to
evaluate and compare three molecular based drug resistance profiling
kits to determine which is the most effective in targeting mutations in
the MTBC genome known to confer resistance to first- and second-line
anti-tuberculosis drugs. The three kits are: A commercially available
kit (HAIN Genotype MTBDR VER 2.0), a kit in the process of validation
(Kit A) and a kit of our own design (Kit B). A total of 100 blinded and
randomized Mycobacterium tuberculosis complex (MTBC) DNA isolates
were collected from the Centre for Tuberculosis (CTB) at the National
Institute for Communicable Diseases (NICD). Isolates will be subjected
to analysis with each kit. The kits will be evaluated in terms of cost,
turnaround time, ease of use and sensitivity and specificity using
whole genome sequencing data available on the isolates at the CFB as
reference. Five isolates have been analysed during pre-evaluations on
Kit A. The kit was able to identify 4 gene mutations i.e. rpoB, katG, embB
and rrs and did not present any false positive phylogenetic markers.
The kit however failed to detect a gyrA mutation in one isolate.
Mutations in the gyrB, pncA and inhA promotor region are not
detectable with Kit A. The sensitivity and specificity for the selective
gene mutations were 90% and 100% respectively. A further six isolates
with known gyrA mutations were evaluated using Kit A to determine if
the prior result was due to a low analytical sensitivity or suboptimal
reagent design. The sensitivity for identifying the gyrA mutations was
50% and reflected that the kit was not able to detect the Asp94His
mutated codon as confirmed by the supplier. In addition, the results
concurred with RIF and INH resistance on available phenotypic data.
Results of pre-evaluations on Kit A show high sensitivity and
specificity for selective gene mutations. It was determined though
that Kit A lacks in sensitivity for the gyrA mutations and will need
optimization of the current reagent design. The kit however has a low
coverage of the 11 different conferring mutations of interest. Kit A is
robust and takes less than two hours but requires repetitive pipetting,
which could lead to cross contamination and false positive results.
Further evaluations are in progress
URI
http://hdl.handle.net/10394/33326https://www.sciencedirect.com/science/article/pii/S1056871919303260
https://doi.org/10.1016/j.vascn.2019.106608