Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for a standardized methodology

Unit Molecular Microbiology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Journal of Clinical Microbiology (Impact Factor: 3.99). 03/1993; 31(2):406-9.
Source: PubMed


DNA fingerprinting of Mycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We propose a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns. General use of this technique will permit comparison of results between different laboratories. Such comparisons will facilitate investigations into the international transmission of tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug resistance.

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Available from: Jan Embden Van, Oct 01, 2015
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    • "A study sample included 101 M. tuberculosis isolates obtained from different regions of the country and previously characterized by spoligotyping and other methods [15]. The DNA of the studied strains was extracted from 4 to 6 weeks Lö wenstein–Jensen medium culture using the recommended method [16]. Spoligotyping was performed as described previously [17]. "
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    ABSTRACT: Tuberculosis (TB) control remains an important public health priority for Bulgaria. The population structure of Mycobacterium tuberculosis is clonal and certain genetic families of this species (e.g., Latin-American-Mediterranean [LAM]) have attracted more attention due to their global dissemination and/or particular pathogenic properties, e.g., association with multidrug resistance (MDR). The aim of this study is to evaluate the prevalence of the M. tuberculosis LAM family in Bulgaria based on the use of different molecular markers. A total of 101 previously spoligotyped M. tuberculosis strains were studied by LAM-specific PCR assay to detect an insertion of IS6110 in the specific genome region. On the whole, clear-cut results were obtained for most strains; spoligotype-based family was reassigned in some of them. At the same time, double bands were amplified in some cases and warrant further validation studies of this method. The higher MDR rate among LAM versus other genotype isolates was observed (P = 0.04). In conclusion, these results suggest a low (<4%) prevalence rate of LAM in Bulgaria (that is similar to its Balkan neighbors) and highlight the importance of using robust markers for correct detection of the LAM family.
    International Journal of Mycobacteriology 05/2015; 4(3):191-195. DOI:10.1016/j.ijmyco.2015.04.003
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    • "The ability to distinguish isolates has shown that individuals can be re-infected with M. tuberculosis, and this poses clear challenges for vaccine development since even natural infection at best provides partial immunity. Furthermore, the advent of high resolution tests for genetic variation has revealed that individuals may simultaneously harbor infections with more than one distinct strain of M. tuberculosis (Warren et al., 1999; Sola et al., 2003; Kremer et al., 1999; van Embden et al., 1993; Imaeda, 1985). This phenomenon, which we will refer to as " mixed infection " , has been linked with poor treatment outcome when the co-infecting strains differ with respect to drug susceptibility (van Rie et al., 2005; Hingley-Wilson et al., 2013) and is predicted to influence the impact of population-level interventions against tuberculosis (Cohen et al., 2008; Rodrigues et al., 2007; Colijn et al., 2009; Sergeev et al., 2011; Mills et al., 2013). "
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    ABSTRACT: High resolution tests for genetic variation reveal that individuals may simultaneously host more than one distinct strain of M. tuberculosis. Previous studies find that this phenomenon, which we will refer to as "mixed infection", may affect the outcomes of treatment for infected individuals and may influence the impact of population-level interventions against tuberculosis. In areas where the incidence of TB is high, mixed infections have been found in nearly 20% of patients; these studies may underestimate the actual prevalence of mixed infection given that tests may not be sufficiently sensitive for detecting minority strains. Specific reasons for failing to detect mixed infections would include low initial numbers of minority strain cells in sputum, stochastic growth in culture and the physical division of initial samples into parts (typically only one of which is genotyped). In this paper, we develop a mathematical framework that models the study designs aimed to detect mixed infections. Using both a deterministic and a stochastic approach, we obtain posterior estimates of the prevalence of mixed infection. We find that the posterior estimate of the prevalence of mixed infection may be substantially higher than the fraction of cases in which it is detected. We characterize this bias in terms of the sensitivity of the genotyping method and the relative growth rates and initial population sizes of the different strains collected in sputum. Copyright © 2014. Published by Elsevier Ltd.
    Journal of Theoretical Biology 12/2014; 368. DOI:10.1016/j.jtbi.2014.12.009 · 2.12 Impact Factor
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    • "During the last decades, various molecular techniques of DNA fingerprinting have been used to discriminate between clinical strains of MTBC (reviewed in [12]). Restriction fragment length polymorphisms (RFLP) typing is based on differences in copy number and the differential genomic location of the insertion sequence (IS) 6110, and became the first gold standard method for genotyping MTBC [15]. This technique has been used successfully to define chains of ongoing TB transmission, discriminate relapse from re-infection, and to detect laboratory cross-contaminations [16] [17]. "
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    ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis complex (MTBC), comprises seven phylogenetically distinct lineages associated with different geographical regions. Here we review the latest findings on the nature and amount of genomic diversity within and between MTBC lineages. We then review recent evidence for the effect of this genomic diversity on mycobacterial phenotypes measured experimentally and in clinical settings. We conclude that overall, the most geographically widespread Lineage 2 (includes Beijing) and Lineage 4 (also known as Euro-American) are more virulent than other lineages that are more geographically restricted. This increased virulence is associated with delayed or reduced pro-inflammatory host immune responses, greater severity of disease, and enhanced transmission. Future work should focus on the interaction between MTBC and human genetic diversity, as well as on the environmental factors that modulate these interactions. (C) 2014 The Authors. Published by Elsevier Ltd.
    Seminars in Immunology 10/2014; 26(6). DOI:10.1016/j.smim.2014.09.012 · 5.17 Impact Factor
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