Report of Expert Consultations on Rapid Molecular Testing to Detect Drug-Resistant Tuberculosis in the United States

Appendix 1: Molecular Basis of Drug Resistance and Molecular DR tests

Drug resistance in Mycobacterium tuberculosis bacteria arises mainly through the acquisition of mutations in the chromosomal sequence that encode changes that 1) block the activity of a drug (mutations in rpoB prevent binding of rifampin to RNA polymerase and inhibition of transcription), 2) block activation of a prodrug (e.g., mutations in katG lead to loss of the ability of catalase to activate the prodrug isoniazid to its active form), or 3) produce an activity that binds or destroys the drug (e.g., mutations in inhA increase the amount of InhA protein which interferes with the activity of isoniazid by binding sufficient isoniazid to reduce its effective concentration in the bacterium to below an inhibitory level) (1,2). The mutations associated with resistance to many of the antituberculosis drugs have been identified, though much work remains to be done to identify the molecular basis of resistance for some of the drugs and to determine the predictive value of finding a particular mutation in a strain of M. tuberculosis (1,2). For example, approximately 95% of rifampin-resistant M. tuberculosis strains carry mutations within the rifampin-resistance determining region (RRDR), an 81-bp region encoding codons 507 through 533 of the rpoB gene.

Molecular genetic tests for detecting drug-resistance are, in general, just a variation of nucleic acid amplification (NAA) tests and can reliably provide information on the presence of mutations associated with drug resistance in 1 to 2 days. Typically, PCR is used to amplify a target sequence followed by a second assay to determine if the sequence contains a mutation associated with resistance. Methods that have been described for the latter include DNA sequencing, pyrosequencing, electrophoretic detection methods (e.g., single strand conformation polymorphism), methods for detecting mismatches in heteroduplexes (e.g., temperature gradient HPLC analysis or branch migration inhibition), and hybridization assays (e.g., molecular beacons, microarrays, membrane hybridization, or line-probe assays). Kits for detecting mutations associated with rifampin resistance that are commercially available in Europe and elsewhere include line-probe assays (INNO-LiPA® Rif.TB, Innogenetics and GenoType® MTBDR(plus), Hain LifeScience GmbH) and microarray assays (CombiChip Mycobacteria DR, GENE IN). Some also detect mutations associated with isoniazid resistance. In-house PCR-based tests using molecular beacons have also been used for diagnostic purposes in a few clinical laboratories.

For the hybridization assays, the region of the target gene associated with resistance is PCR amplified, and the labeled PCR products hybridized to oligonucleotide probes immobilized on a nitrocellulose strip or in a microarray. Mutations are detected by lack of binding to wild-type probes and/or by binding to probes specific for commonly occurring mutations. The performance of the line-probe assays relative to culture-based DS tests was evaluated in meta-analyses (3–5). For the INNO-LiPA Rif.TB assay, the pooled sensitivity was 0.97 (95%CI 0.95–0.98) and the pooled specificity was 0.99 (95%CI 0.98–1.00) for detecting rifampin resistance in M. tuberculosis isolates. Overall discriminatory ability of the assay was 99% and overall accuracy was 97%, with all studies yielding consistently high performances. In four studies, the INNO-LiPA Rif.TB showed 100% specificity and sensitivity ranging from 80% to 100% for detecting rifampin resistance directly from clinical specimens. For the MTBDR and MTBDR(plus) assays, the pooled sensitivity was 0.98 (95%CI 0.96–0.99) and the pooled specificity was 0.99 (95% CI 0.97–0.99) for detecting rifampin resistance in isolates or directly from clinical specimens. Overall discriminatory ability of the assay was 99% and overall accuracy 97%, with all studies yielding consistently high performances.

Molecular beacons are hybridization probes which emit fluorescence only when hybridized to their target. Molecular beacons can discriminate between targets differing by a single nucleotide. Because molecular beacons can use different fluorophores, real-time PCR assays can be designed in which different DNA fragments or mutations can be amplified and detected simultaneously in the same tube. For example, a single-well assay has been developed that uses five molecular beacons to detect mutations associated with rifampin resistance in M. tuberculosis bacteria and appears to perform similarly as the line-probe assays. In the California Microbial Diseases Laboratory, molecular beacons were designed to detect mutations in rpoB, katG, and inhA promoter region genes and directly applied to clinical specimens or to cultures. Comparison of molecular beacons results with results of culture-based drug-susceptibility testing showed 96% to 97% agreement in a series of approximately 1,000 clinical specimens and cultures (6, E. Desmond, personal communication).

Validation studies were conducted at the Wadsworth Center of an approach that combines PCR-amplification of the RRDR with rapid (< 2hrs) DNA sequencing (K. Musser, personal communication). A PyrosequencingTM protocol utilizing two primers was developed to sequence the 81-bp RRDR of the rpoB gene and obtain a clear and accurate pyrogram. The detection limit was determined and the pyrosequencing approach was evaluated in primary specimens positive for M. tuberculosis complex DNA by real-time PCR. Final results were compared with conventional susceptibility testing results and/or DNA sequencing. This test has a detection limit of <1 colony forming unit, 100% specificity, and 99% agreement in the 188 cultures and specimens tested. (7)

Molecular genetic tests for the other antituberculosis drugs are much less developed and studied than the tests for rifampin resistance. A meta-analysis of the performance of the Hain MTBDR(plus) assay for detecting isoniazid revealed a pooled sensitivity of 0.85 (95%CI 0.77– 0.90) which ranged from 57%–100% and a pooled specificity of 0.99 (95%CI 0.98–1.00) which was fairly consistent across studies. Validation studies conducted in the California Microbial Diseases Laboratory that used archived cultures revealed that the molecular beacon test displayed 82.7% sensitivity, 100% specificity, 100% positive predictive value, and 98.1% negative predictive value for detecting isoniazid resistance (6). Tests for the other key resistances, especially the XDR TB defining resistances, are in various stages of development from discovery of the mutations associated with resistance to development of prototype assays and laboratory-based evaluations.

References

1. Zhang Y and Telenti A. (2000) Genetics of drug resistance in Mycobacterium tuberculosis. In Hatfull GF and Jacobs WR Jr (Eds.). Molecular genetics of Mycobacteria (ASM Press, Washington, D.C) pp. 235–54.
2. Johnson R, Streicher EM, Louw GE, Warren RM, van Helden PD, Victor TC. Drug resistance in Mycobacterium tuberculosis. Curr Issues Mol Biol. 2006 Jul;8(2):97-111.
3. Morgan M, Kalantri S, Flores L, Pai M. A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. BMC Infect Dis. 2005; 5:62.
4. Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis. Eur Respir J. 2008;32:1165–74.
5. WHO Expert Group Report. Molecular line probe assays for rapid screening of patients at risk of multi-drug resistant tuberculosis (MDR-TB). Geneva: World Health Organization; 2008. http://www.who.int/tb/features_archive/expert_group_report_june08.pdf.
6. Lin S-YG, Lin S-Y, Probert W, Lo M , Desmond E. Rapid detection of isoniazid and rifampin resistance mutations in Mycobacterium tuberculosis complex from cultures or smear-positive sputa by use of molecular beacons. J Clin Microbiol 2004 42: 4204-08.
7. Halse TA, Edwards J, Driscoll JR, Escuyer VE, and Musser KA. A pyrosequencing approach to rapidly assess mutations in the rpoB gene associated with rifampin resistance in clinical specimens of Mycobacterium tuberculosis. 48th Annual ICAAC/IDSA Annual Meeting. Washington, DC. 2008.