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Introduction

Genotyping methods

Recent transmission

Clinical and public health applications

References

INTRODUCTION

Tuberculosis (TB) control programs prevent transmission of Mycobacterium tuberculosis by identifying and treating active cases of TB disease and ensuring that contacts of infectious cases are also identified, screened, and treated for active disease or latent infection. Genotyping and molecular surveillance methods can identify clusters of cases that share genetically similar M. tuberculosis strains, which may be related by recent transmission. Public health tools to investigate TB transmission have advanced by combining technologies for DNA sequencing and phylogenetic analysis with clinical and epidemiological information. Outbreak investigations are a priority because they can establish epidemiological links among patients and direct prevention measures toward populations and settings with ongoing transmission. The chapter reviews the use of genotyping to investigate transmission as well as how genotyping methods have been applied to study transmission dynamics, lineage, recurrent TB disease, mixed infections, and laboratory contamination.

GENOTYPING METHODS

Genotyping methods for M. tuberculosis can be divided into two main categories: (1) conventional genotyping methods that examine variation affecting a small portion of the M. tuberculosis genome and (2) whole-genome sequencing (WGS), which examines most of the M. tuberculosis genome at the nucleotide level. The three most commonly used conventional genotyping methods are restriction fragment length polymorphism (RFLP), spacer oligonucleotide typing (spoligotyping), and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR). These methods are all based on repetitive DNA sequences found either interspersed throughout the bacterial

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genome (insertion sequences) or at specific loci. Changes in these repetitive DNA sequences serve as a proxy for genetic diversification that is occurring throughout the entire genome. However, as DNA sequencing technologies have advanced, WGS is replacing the use of conventional genotyping methods for public health purposes to provide direct analysis of phylogenetic relationships at the nucleotide level.

Conventional methods based on repetitive sequences

RFLP typing for M. tuberculosis is based on the 1.3 kilobase repetitive insertion sequence IS6110, which is unique to M. tuberculosis complex (MTBC).1 IS6110 varies in the number of copies from 0 to 25 per strain and also by insertion site in the genome.2 These variations produce highly diverse hybridization patterns that are used to discriminate between strains (Figure 5.1). In the 1990s, IS6110 was the gold standard for genotyping M. tuberculosis due to the high discriminatory power and the stability of the biomarker, which has a half-life of approximately 3.2 years (i.e., the time it takes for a hybridization pattern to change).3 Disadvantages of IS6110-RFLP are that the technique itself is time-consuming, technically demanding, lacks inter-laboratory reproducibility, and requires specialized software for the analysis of results, which are difficult to compare between different laboratories and over time. Furthermore, the discriminatory power is poor for strains without a copy of IS6110 and for strains with a low (<6) copy number, such as M. bovis.

Spoligotyping is based on the presence or absence of 43 unique DNA “spacer” sequences that are interspersed between 36 base pair sequences.4,5 The results consist of binary data and the number and position of absent spacer sequences can be compared between strains (Figure 5.2). Results are reproducible, easy to interpret,