such as Chlamydia trachomatis. For this reason, methods that allow
sequencing directly from suspected M. tuberculosis positive samples,
forgoing the culturing step, are particularly attractive.
Treatment for TB requires an extended course (6–24 months)
of a combination of antibiotics; however, the development of
MDR-TB (multidrug resistant TB, defined as resistance to the
two front line drugs, isoniazid and rifampicin), XDR-TB (extensively drug-resistant TB, MDR-TB plus resistance to a quinolone,
and kanamycin, capreomycin, or amikacin), and most recently
TDR-TB (totally drug-resistant TB) has severe ramifications on
treatment. Traditional identification of M. tuberculosis typically
involves sputum smear microscopy, followed by culturing (three
to six weeks) and drug susceptibility testing (an additional three to
six weeks). Rapid determination of drug sensitivity profiles is critical
for providing specific and effective treatment; recent advances in
detecting front-line resistance based on SNP identification in rpoB
and inhA have improved this. One such test is the Cepheid GeneXpert XpertMTB/RIF assay, which has been endorsed by the
WHO for use in TB endemic countries [4]. The Xpert MTB/RIF
assay is a cartridge-based system that can identify M. tuberculosis
and rifampicin (RIF) resistance from sputum samples in 90 min,
using a molecular beacon approach to recognize a M. tuberculosisspecific rpoB sequence and mutations in the RRDR (rifampicin
resistance-determining region) associated with RIF resistance
[5]. However, RIF resistance in M. tuberculosis has been found to
be not just due to RRDR rpoB mutations but related to a number of
mutations, some in seemingly unassociated genes, plus the level of
resistance exhibited is strain dependent, and these cannot be
detected by methods targeting just SNPs in rpoB [6].
Currently, resistance testing for second-line TB drugs is limited
and typically is based on culture growth in the presence of antibiotics at a concentration greater than the MIC. A recent publication described the use of MODS (microscopic observation drug
susceptibility) assay for the determination of second-line drug susceptibility [7], and molecular assays have also been described, such
as the MTBDRs1 assay [8]. Whole-genome sequencing (WGS)
provides the ultimate information in terms of genome structure,
and utilizing this technology in a clinical diagnostic setting is now
being realized for the first time due to the advent of smaller scale
sequencers, such as Illumina’s MiSeq, which allows for a smaller
number of samples to be run in a single lane in a cost-effective
manner. Using WGS for identification of M. tuberculosis and resistance allows for all SNPs associated with resistance to be identified,
along with strain type in a single assay, in a more timely fashion than
currently achieved by culture based techniques. This would permit
a personalized medicine approach in the prescribing of antimycobacterials, reducing the wastage of ineffective drugs, and improving
the clinical outcome for patients. WGS of M. tuberculosis has been
460
Amanda Claire Brown
sequencing directly from suspected M. tuberculosis positive samples,
forgoing the culturing step, are particularly attractive.
Treatment for TB requires an extended course (6–24 months)
of a combination of antibiotics; however, the development of
MDR-TB (multidrug resistant TB, defined as resistance to the
two front line drugs, isoniazid and rifampicin), XDR-TB (extensively drug-resistant TB, MDR-TB plus resistance to a quinolone,
and kanamycin, capreomycin, or amikacin), and most recently
TDR-TB (totally drug-resistant TB) has severe ramifications on
treatment. Traditional identification of M. tuberculosis typically
involves sputum smear microscopy, followed by culturing (three
to six weeks) and drug susceptibility testing (an additional three to
six weeks). Rapid determination of drug sensitivity profiles is critical
for providing specific and effective treatment; recent advances in
detecting front-line resistance based on SNP identification in rpoB
and inhA have improved this. One such test is the Cepheid GeneXpert XpertMTB/RIF assay, which has been endorsed by the
WHO for use in TB endemic countries [4]. The Xpert MTB/RIF
assay is a cartridge-based system that can identify M. tuberculosis
and rifampicin (RIF) resistance from sputum samples in 90 min,
using a molecular beacon approach to recognize a M. tuberculosisspecific rpoB sequence and mutations in the RRDR (rifampicin
resistance-determining region) associated with RIF resistance
[5]. However, RIF resistance in M. tuberculosis has been found to
be not just due to RRDR rpoB mutations but related to a number of
mutations, some in seemingly unassociated genes, plus the level of
resistance exhibited is strain dependent, and these cannot be
detected by methods targeting just SNPs in rpoB [6].
Currently, resistance testing for second-line TB drugs is limited
and typically is based on culture growth in the presence of antibiotics at a concentration greater than the MIC. A recent publication described the use of MODS (microscopic observation drug
susceptibility) assay for the determination of second-line drug susceptibility [7], and molecular assays have also been described, such
as the MTBDRs1 assay [8]. Whole-genome sequencing (WGS)
provides the ultimate information in terms of genome structure,
and utilizing this technology in a clinical diagnostic setting is now
being realized for the first time due to the advent of smaller scale
sequencers, such as Illumina’s MiSeq, which allows for a smaller
number of samples to be run in a single lane in a cost-effective
manner. Using WGS for identification of M. tuberculosis and resistance allows for all SNPs associated with resistance to be identified,
along with strain type in a single assay, in a more timely fashion than
currently achieved by culture based techniques. This would permit
a personalized medicine approach in the prescribing of antimycobacterials, reducing the wastage of ineffective drugs, and improving
the clinical outcome for patients. WGS of M. tuberculosis has been
460
Amanda Claire Brown