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High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF09766-AF059853 and AF060279-AF060367.]For patients infected with Mycobacteria, especially those coinfected with the human immunodeficiency virus type 1 and type 2 (HIV-1, HIV-2), the identity of the Mycobacterium species and the presence of mutations that confer both biologically and clinically important phenotypes are of critical importance. Both of these issues have implications for the appropriate care and treatment of the infected patient. For example, although M. avium complex (MAC) is the most common cause for both disseminated Mycobacterium disease and death in patients with AIDS in the developed world (∼25%-50% of adults and 10% of children with AIDS are infected [Inderlied et al. 1993], Mycobacterium tuberculosis infections are also found in these patient populations. Important public health and patient management decisions (e.g., the need for clinical isolation and the choice of the appropriate therapeutic regimen) depend on a timely and accurate identification of the infecting agent. (Moore et al. 1997).On the basis of the insights provided by previously characterized RIF resistant mutants in Escherichia coli (Ovchinnikov et al. 1983; Jin and Gross 7 Corresponding author. E-MAIL tom gingeras@affymetrix.com; FAX (408) 481-0422.

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Objectives: With the spread of multidrug-resistant tuberculosis (MDR-TB) there is increasing demand for new accurate and cost-effective tools for rapid drug susceptibility testing (DST), particularly for developing countries. The reference standard method used today for DST is very slow and cumbersome. Colorimetric assays using redox indicators have been proposed to be used in low-resource countries as rapid alternative culture methods for the detection of resistance especially to rifampicin and isoniazid. These methods appear as promising new tools but their accuracy has not been systematically evaluated. Methods:We did a meta-analysis to evaluate the accuracy of the colorimetric assays for the detection of rifampicin and isoniazid-resistant tuberculosis among clinical isolates. We searched Medline, PubMed (NCBI), Global health-CAB, EJS-E (EbscoHost), ISI Web, Web of Science and IFCC databases and contacted authors if additional information was needed.Results: Eighteen studies met our inclusion criteria for rifampicin resistance detection and 16 for isoniazid. We used a summary receiver operating characteristic (SROC) curve to perform meta-analysis and summarize diagnostic accuracy. For both drugs, all studies had a sensitivity and specificity that ranged between 89% and 100%.Conclusions: There is evidence that colorimetric methods are highly sensitive and specific for the rapid detection of MDR-TB. These new tools could offer affordable technologies for TB laboratories especially in places where resources are limited and where the prevalence of MDR-TB is important and make TB control efforts more effective. Additional studies are needed in high MDR prevalence countries and cost-effectiveness analysis to have more evidence on the utility of these methods. Future developments to detect resistance directly from smear-positive sputum specimens should be taken into consideration to speed up the process.Pooled sensitivity = 0.98 (0.97-0.99) χ 2 = 18.12; df = 17 (P = 0.3812) Inconsistency (I 2 ) = 6.2 % Yajko, 1995 (Alamar) 0.89 (0.52-1.00) Franzblau, 1998 (Alamar)

Mycobacterium africanum is thought to comprise a unique species within the Mycobacterium tuberculosis complex. M. africanum has traditionally been identified by phenotypic criteria, occupying an intermediate position between M. tuberculosis and M. bovis according to biochemical characteristics. Although M. africanum isolates present near-identical sequence homology to other species of the M. tuberculosis complex, several studies have uncovered large genomic regions variably deleted from certain M. africanum isolates. To further investigate the genomic characteristics of organisms characterized as M. africanum, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of M. tuberculosis deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that M. africanum subtype II isolates are situated among strains of "modern" M. tuberculosis. In contrast, other M. africanum isolates (subtype I) constitute two distinct evolutionary branches within the M. tuberculosis complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of "M. africanum-like" isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the M. africanum subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for M. tuberculosis complex members, challenging the traditional approach to M. tuberculosis complex speciation. Furthermore, the genomic results suggest a rational framework for defining M. africanum and provide tools to accurately assess its prevalence in clinical specimens.Species of the Mycobacterium tuberculosis complex traditionally consist of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium bovis. Genomically, species of the M. tuberculosis complex present near-identical sequence homology (15,16,40) but are most notably differentiated by large sequence polymorphisms (6,30,31,34). Variants of these original species have since been identified via characteristic phenotypic or genotypic attributes, challenging taxonomic classification within the complex (30). As a result, other members of the M. tuberculosis complex have emerged, including "Mycobacterium canettii" (35, 44), Mycobacterium caprae (1, 2), Mycobacterium pinnipedii (10, 12), and the dassie bacillus (11,30).In contrast to M. tuberculosis and M. bovis, for which phenotype-based speciation is reliant upon concordant results for a number of biochemical tests, M. africanum isolates can present the entire spectrum of biochemical properties employed to discriminate among the M. tuberculosis complex. As a result, "M. tuberculosis-like" and "M. bovis-like" forms of M. africanum have been reported, potentially confounding laboratory identification and complicating attempts to accurately determine the true prevalence of M. a...

cTreating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization. Regional increases in the prevalence of tuberculosis (TB) with drug resistance and a broad distribution of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB (1, 2) may reverse recent gains in global TB control (1). Molecular detection of mutations associated with drug resistance has facilitated rapid detection of drug resistance in the Mycobacterium tuberculosis complex (MTBC) (3-7), and the use of such molecular tools has become increasingly important in TB control and TB patient management (5, 7-10). Recognizing the advantages and disadvantages of various molecular methods will help in the selection of optimal methods for improved prediction of drug resistance in MTBC, which is critical in advancing molecular diagnostic approaches for this defined purpose.In 2003, the Microbial Diseases Laboratory (MDL) at the California Department of Public Health developed a real-time, probe-based assay using molecular beacons for screening for MDR TB (11). Remarkable improvements in turnaround times for predicting resistance to isoniazid (INH) and rifampin (RIF) and significant impacts on the management of MDR TB (9) were realized. However, several incidents of detection of rpoB mutations not conferring RIF resistance resulted in erroneous reporting of false RIF resi...

fIn an effort to update and clarify policies on tuberculosis drug susceptibility testing (DST), the World Health Organization (WHO) commissioned a systematic review evaluating WHO-endorsed diagnostic tests. We report the results of this systematic review and meta-analysis of the diagnostic accuracy and reproducibility of phenotypic DST for first-line and second-line antituberculosis drugs. This review provides support for recommended critical concentrations for isoniazid and rifampin in commercial broth-based systems. Further studies are needed to evaluate critical concentrations for ethambutol and streptomycin that accurately detect susceptibility to these drugs. Evidence is limited on the performance of DST for pyrazinamide and second-line drugs.T he global epidemic of drug-resistant tuberculosis (DR-TB), particularly multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampin), is one of the most serious problems facing TB care and control efforts. In their most recent worldwide survey, the World Health Organization (WHO) documented the highest rates of MDR-TB ever reported (1). Effective management of drug-resistant TB relies on multiple components, including detection, treatment, prevention, surveillance, and continuous program evaluation (2). Expanding the capacity to diagnose cases of drug-resistant TB is a priority for global TB control, requiring clear policies on the use of diagnostic tests and strengthened laboratories in which testing can be safely and effectively carried out (3).Conventional phenotypic drug susceptibility testing (DST) using the proportion method (PM) on solid media has been well studied for isoniazid and rifampin, with a general consensus achieved regarding methodology, critical concentrations, and expected performance (4). However, the diagnostic accuracy and reproducibility of DST for other first-line and second-line drugs are inadequate (5). DST for second-line anti-TB drugs has not been standardized internationally, which is reflected in the wide variability of practices among supranational reference laboratories, underscoring the need for standardization of the methods and interpretive criteria for second-line DST (4, 6). Further complicating the lack of consensus is the increasing number of different DST methods that are available. In 2008, the WHO Stop TB Department published interim laboratory policy guidance for DST of second-line anti-TB drugs (4). Guideline recommendations for a specific DST method should ideally be based on the diagnostic test accuracy, reproducibility, ease of use, cost, and rapidity of result availability.In an attempt to address gaps in the evidence base, WHO initiated an update to the 2008 interim guidelines with an expanded scope to include DST for all first-line and second-line drugs. As part of the update, we conducted a systematic review to determine the diagnostic accuracy and reproducibility of WHO-endorsed phenotypic DST methods and commercial genotypic DST methods for first-line and second-line anti-TB drugs. Whi...

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 MDR TB cases harboring those mutations are recommended.