The emergence of multi-and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.DNA fingerprinting of Mycobacterium tuberculosis isolates is useful for determining the extent of recent transmission in a community and the potential risk factors for recent transmission, for identifying previously unsuspected transmission, for monitoring the transmission of drug-resistant strains, and for confirming laboratory cross contamination (reviewed in reference 19). Although a large number of DNA-fingerprinting methods for typing M. tuberculosis isolates have been developed in recent years, all have significant drawbacks, and only a few have been adopted for widespread use. In the most widely used method, IS6110 restriction fragment length polymorphism (RFLP) (18), both the number of copies and the location of the IS6110 insertion element generate variation in the RFLP pattern. The molecular clock of IS6110 RFLP pattern variation is slow enough to be useful for outbreak investigations and yet fast enough for this to be the most discriminatory of the typing techniques (8). Unfortunately, it is technically demanding and requires culturing of the organism to obtain the necessary quantity of DNA. In contrast, spacer oligonucleotide typing (spoligotyping) (12), a PCR-based technique which detects the presence or absence of 43 spacers in the direct-repeat locus, is easy to perform and requires only a limited number of organisms. Spoligotyping, however, is less useful than IS6110 RFLP in discriminating between strains and provides more conserved genetic information, allowing the grouping of isolates into families, such as the Beijing and Haarlem genotype families (8). Spoligotyping has been useful both as a prescreening method to reduce the number of isolates to be typed by IS6110 RFLP (3) and as a secondary typing method for isolates with a low copy number for IS6110, whose RFLP patterns are often more stable than ...

The prevalence of extrapulmonary disease in the young, US-born Hispanic population suggests recent transmission of M. bovis, possibly related to foodborne exposure. Because of its significantly different epidemiologic profile, compared with that of M. tuberculosis TB, we recommend routine surveillance of M. bovis TB. Routine surveillance and an improved understanding of M. bovis TB transmission dynamics would help direct the development of additional control measures.

Genotyping ofOur results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.

Phylogenetic lineage of M. tuberculosis is associated with the site of tuberculosis disease.

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for highthroughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.Spacer oligonucleotide typing (spoligotyping) is a commonly used, amplification-based method for genotyping Mycobacterium tuberculosis isolates (3). It is based on the polymorphisms found in the direct repeat (DR) locus that is present in all M. tuberculosis complex isolates. The DR locus contains multiple 36-bp DRs separated by 30-to 40-bp unique spacer sequences (5). The traditional spoligotyping method detects the presence or absence of 43 different spacer sequences by hybridizing labeled amplicons of the DR locus to oligonucleotide probes for each of the spacers arrayed on a membrane (a reverse line blot hybridization). Because the assay is inexpensive, quick, and robust, it is often used as a first-line genotyping method.Although the assay is quite functional in its original form, a new technology is available as an alternative to reverse line blot hybridizations. The Luminex (Austin, Tex.) system can rapidly detect multiple DNA sequences. The system uses microspheres (beads) that contain two fluorochromes; a microsphere is given a distinct spectral address by varying the ratio of the concentration of the fluorochromes. Oligonucleotide capture probes are covalently attached to 1 of 100 available microsphere sets. During analysis, individual microspheres are interrogated by two lasers. The first laser excites the fluorochromes within the microsphere and allows identification of the microsphere set. The second laser excites a reporter fluorochrome bound to the hybridized PCR product and allows quantification of the PCR product. A sample can be analyzed in less than 15 seconds. Here, we report the successful transfer of the spoligotyping assay to the Luminex platform.The 43 spacer oligonucleotide probes used in this study were the same as those used in the traditional spoligotyping assay (3). The probes were synthesized with a 5Ј-terminal amino group with a six-carbon spacer to allow covalent attachment to membranes or microspheres. Spoligotyping membranes were prepared as previously described (3). To couple the probes to the microspheres (Luminex Corp.), 200 pmol of oligonucleotide, 2.5 ϫ 10 6 microspheres, and 25 g of freshly purchased N-(3-dimethylaminopropyl)-NЈ-ethylcarbodiimide (Pierce Chemical, Rockford, Ill.) were combined in 25 l of 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 4.5 (Sigma, St. Louis, Mo.); the reaction mixtures were incubated at room temperature in the dark for 30 min. The N-(3-dimethylaminopropyl)-NЈ-ethylcarbodiimide addition and subsequent incubation were repeated once. After coupling, the microspheres were washed with 0.5 ml of 0.02% Tween 20 followed by 0.5 ml of 0.1% sodium dodecyl sulfate. The prepared microsphere...

Molecular characterization of Mycobacterium tuberculosis complex isolates (genotyping) can be used by public health programs to more readily identify tuberculosis (TB) transmission. The Centers for Disease Control and Prevention's National Tuberculosis Genotyping Service has offered M. tuberculosis genotyping for every culture-confirmed case in the United States since 2004. The TB Genotyping Information Management System (TB GIMS), launched in March 2010, is a secure online database containing genotype results linked with case characteristics from the national TB registry for state and local TB programs to access, manage and analyze these data. As of September 2011, TB GIMS contains genotype results for 89% of all culture-positive TB cases for 2010. Over 400 users can generate local and national reports and maps using TB GIMS. Automated alerts on geospatially concentrated cases with matching genotypes that may represent outbreaks are also generated by TB GIMS. TB genotyping results are available to enhance national TB surveillance and apply genotyping results to conduct TB control activities in the United States.

These tools may enable direction of resources to populations with high transmission rates.