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Chapter 26 : Molecular Detection and Characterization of Mycobacterium tuberculosis
Category: Clinical Microbiology; Bacterial Pathogenesis
This chapter focuses on nucleic acid amplification based (NAA) assays that detect Mycobacterium tuberculosis directly in clinical specimens; specifics regarding the laboratory diagnosis of infections caused by the nontuberculous mycobacteria are not included. The chapter talks about on NAA assays that detect M. tuberculosis directly in clinical specimens. The salient features of commercially available NAA assays are first briefly described, followed by an overview of in-house-developed assays. Second, similar to commercially available NAA assays, evaluation of in-house NAA assays has used primarily respiratory specimens, but testing has also been performed on nonrespiratory specimens such as pleural fluids, gastric aspirates, formalin-fixed and paraffin-embedded tissues, fresh tissues, cerebrospinal fluids (CSF), and other sterile body fluids. Third, in-house assays have also been developed to detect both M. tuberculosis complex and drug resistance. Finally, other miscellaneous aspects of NAA assays including their use in monitoring response to therapy, cost and cost-effectiveness, and newer approaches and modifications are addressed. Identifications obtained by use of the specific deletion profiles correlated 100% with the original identifications for all M. tuberculosis complex members except M. africanum; further characterization resulted in profiles specific for all members. Subsequent studies using genomic deletions revealed that by testing an isolate for signature deletions, members of the M. tuberculosis complex can be identified.
Key Concept Ranking
- Restriction Fragment Length Polymorphism
Examples of GenoType MTDBRplus strips (Hain Lifescience, Nehren, Germany). Lanes: 1, M. tuberculosis, susceptible to INH and rifampin (RIF); 2, M. tuberculosis, INH monoresistant (katG S315T1 mutation); 3, MDR M. tuberculosis, rpoB S531L mutation and katG S315T2 mutation; 4, MDR M. tuberculosis rpoB S531L mutation and katG S315T1 and inhA C15T mutations; 5, M. tuberculosis, RIF monoresistant (mutation in rpoB 530-533 region); 6, MDR TB, rpoB D516V and katG S315T1 mutations; 7, MDR M. tuberculosis, rpoB S531L, and katG S315T2 mutations; 8, MDR TB, rpoB, D516V, katG S315T1 mutation, and inhA mutation at –15/–16; 9, uninterpretable result, no M. tuberculosis complex band; 10, negative control. (Reprinted from the American Journal of Respiratory and Critical Care Medicine [ 7 ] with permission of the publisher.)
Detection of amplified target DNA using the LAMP method can be visually achieved through the byproduct of amplification, magnesium pyrophosphate. (A) White turbidity can be observed because of the production of large amounts of amplified product. (B) Amplified product can also be detected by fluorescence that is clearly distinguished by the naked eye under UV light. Calcein, a chaotropic agent that is fluorescent unless combined with manganese ion, is added to the reaction mix; manganese binds to pyrophosphate produced during amplification, thereby releasing the calcein, resulting in fluorescence. (Reprinted with permission of Eiken Chemical Co. Ltd.)
Primers included in initial versions of the LAMP method. Shown are two sets of specially designed inner and outer primers; inner primers are called the forward inner primer (FIP) and the backward inner primer (BIP), and each contains two distinct sequences corresponding to the sense and antisense sequences of the target DNA, one for priming in the first stage and the other for self-priming in later stages ( 99 ). The sequences inside both ends of the target region for amplification are designated F2c and B2, respectively. Two inner sequences that are 40 nucleotides in length from the ends of F2c and B2 are designated F1c and B1, and two sequences outside the ends of F2c and B2 are designated F3c and B3. In the initial steps of the LAMP reaction, all four primers are used, but later during the cycling reaction only the inner primers are used for strand displacement DNA synthesis. (Reprinted with permission of Eiken Chemical Co. Ltd.)
Representative IS6110-based RFLP image. Isolates represented by lanes 3, 5, 6, 9, and 10 have the same pattern and were epidemiologically linked. Lane S shows the CDC molecular weight standard. (From U.S. Department of Health, Education, and Welfare, Public Health Service, CDC, Atlanta, GA.)
Two examples of spoligotype results showing the original banding patterns as well as the steps involved in converting the banding pattern results to the final octal code designation. The original banding pattern is converted to a series of 1 ’s and 0’s (1 means the band is present, and 0 means it is absent) that is 43 digits long (i.e., binary code). The binary code is further simplified by converting to a 15-digit octal code designation in a two-step process. The binary code is divided into 14 sets of three digits plus one additional digit (spacer 43). Then, each three-digit binary set is converted to its octal equivalent, with the final additional digit remaining as 1 or 0. The octal designation is the form of the result that is reported by the genotyping laboratories to tuberculosis programs. (From U.S. Department of Health and Human Services, CDC, Atlanta, GA.)
Examples of MIRU results. MIRU results are reported as a 12-digit designation, with each digit representing the number of repeats detected at the respective 12 MIRU loci. For loci with more than nine repeats, letters are used (e.g., “a” for 10 repeats, “b” for 11, etc.). (From U.S. Department of Health and Human Services, Public Health Service, CDC, Atlanta, GA.)
Commercially available molecular assays for the direct detection of drug-resistant MTB complex and/or MTB complex in clinical specimens a
Results of recent meta-analyses and systematic reviews regarding the accuracy of NAA assays for direct detection of M. tuberculosis complex in clinical specimens
Results of recent meta-analyses and systematic reviews regarding the accuracy of line probe assays for direct detection of M. tuberculosis complex