Evaluation of the Impact of a Sequencing Assay for Detection of Drug Resistance on the Clinical Management of Tuberculosis.

BACKGROUND: In 2012, the California Department of Public Health began using pyrosequencing (PSQ) to detect mutations associated with resistance to isoniazid, rifampin, quinolones and injectable drugs in Mycobacterium tuberculosis complex. We evaluated the impact of the PSQ assay on the clinical management of tuberculosis (TB) in California. METHODS: TB surveillance and laboratory data for specimens submitted 1 August 2012 through 31 December 2016 were analyzed to determine time to effective treatment initiation. A survey of clinicians was used to assess how PSQ results influenced clinical decision making. RESULTS: Of 1957 specimens tested with PSQ, 52% were sediments and 46% were culture isolates, submitted a median of 8 and 35 days, respectively, after collection. Among 36 patients with multidrug-resistant (MDR) TB who had a sediment specimen submitted for PSQ, median time from specimen collection to MDR-TB treatment initiation was 12 days vs 51 days when PSQ was not used. Completed surveys were returned for 303 patients, 177 of whom reported a treatment change; 75 (42%) of clinicians reported PSQ as a reason for change. Twenty-one patients either had an MDR-TB risk factor and a smear-positive sputum specimen, but had PSQ performed on a culture isolate (9/36 [25%]); or did not have PSQ used for MDR-TB diagnosis (12/38 [32%]) and thus had an opportunity for earlier MDR-TB diagnosis with PSQ on sediment. CONCLUSIONS: Patients with MDR-TB initiated effective treatment 5 weeks earlier when PSQ was used compared to those without PSQ. Survey data suggest clinicians use PSQ to devise effective TB drug regimens. To maximize the benefit of PSQ, earlier submission of specimens should be prioritized.

Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries.

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex.

Cross-resistance in rifamycins has been observed in rifampin (RIF)-resistant Mycobacterium tuberculosis complex isolates; some rpoB mutations do not confer broad in vitro rifamycin resistance. We examined 164 isolates, of which 102 were RIF-resistant, for differential resistance between RIF and rifabutin (RFB). A total of 42 unique single mutations or combinations of mutations were detected. The number of unique mutations identified exceeded that reported in any previous study. RFB and RIF MICs up to 8 µg/mL by MGIT 960 were studied; the cut-off values for susceptibility to RIF and RFB were 1 µg/mL and 0.5 µg/mL, respectively. We identified 31 isolates resistant to RIF but susceptible to RFB with the mutations D516V, D516F, 518 deletion, S522L, H526A, H526C, H526G, H526L, and two dual mutations (S522L + K527R and H526S + K527R). Clinical investigations using RFB to treat multidrug-resistant tuberculosis cases harboring those mutations are recommended.

Molecular diagnosis of tuberculosis and drug resistance.

Molecular drug susceptibility testing (MDST) provides rapid diagnosis of tuberculosis (TB) and detection of drug resistance with commendable sensitivity and specificity. MDST reduces unnecessary isolation or treatment, when a negative result for TB is obtained. Because of the possibility of false detection of rifampin resistance by probe-based MDST, confirmation by sequencing is recommended, especially in regions where the prevalence of resistance is low. Revealing mutation identity by sequencing offers opportunities to study drug minimum inhibitory concentrations for each mutation. Such information enables prediction of resistance levels, and may be helpful in formulating optimal regimens, when treatment options are limited.