Journal of Applied Microbiology 2003, 94, 781–791
A REVIEW Molecular methods for Mycobacterium tuberculosis strain typing: a users guide E. Kanduma1, T.D. McHugh2 and S.H. Gillespie2 1
Clinical Laboratory, Kilimanjaro Christian Medical College, PO Box 3010, Moshi, Tanzania, and Department of Medical Microbiology University College London, London, UK
2
2002/6: received 30 July 2002, revised 17 January 2003 and accepted 30 January 2003
1. 2. 3. 4.
Summary, 781 Introduction, 781 Non-DNA typing methods, 782 Genotyping, 782 4.1 Restriction fragment length polymorphism, 782 4.2 Pulsed field gel electrophoresis, 782 4.3 RFLP with hybridization, 782 4.3.1 Methods based on repetitive elements, 783 4.3.2 IS6110, 783 4.3.3 IS1081, direct repeat and major polymorphic tandem repeat, 783 4.3.4 Polymorphic GC-rich repetitive sequence, 783 4.3.5 Other methods using repetitive elements, 784
1. SUMMARY There are now a wide range of techniques available to type Mycobacterium tuberculosis, the problem is to chose the correct technique. For large scale epidemiological studies the portability and standardization of IS6110 restriction fragment length polymorphism (RFLP) means that this remains the gold standard technique. In the next few years the internationally standard mycobacterial interspersed repetitive unit (MIRU) may come to challenge this primacy. Low copy number stains remain a problem and these can by typed by either polymorphic Guanine cytosine-rich repetitive sequence (PGRS) or MIRU-variable numbers of tandem repeat (VNTR). To confirm whether strains are part of a true cluster PGRS remains the method of choice. For local outbreaks and investigations of laboratory cross contamination where speed is of greatest importance suspect strains should be initially investigated using a PCR-based Correspondence to: S.H. Gillespie, Department of Medical Microbiology Royal Free Campus, University College London, Rowland Hill Street, London NW3 2PF, UK (e-mail:
[email protected]).
ª 2003 The Society for Applied Microbiology
4.4 Amplification-based methods, 784 4.4.1 IS6110-based methods, 785 4.4.2 16S- and 23S rRNA-based methods, 785 4.4.3 DR region-based methods, 786 4.4.4 Spoligotyping, 786 4.4.5 Minisatellite-based methods, 786 5. Application of typing methodology, 787 5.1 Large-scale national and international studies, 787 5.2 Local outbreak investigation, 787 5.3 Detecting laboratory cross contamination, 787 5.4 The contribution of molecular methods to the epidemiology of tuberculosis, 788 6. References, 788
method The superior reproducibility and discrimination of MIRU-VNTR means that these methods should be favoured. If matches are found, then further confirmation of identity can be achieved using IS6110 RFLP or PGRS if the strains prove to have a low IS6110 copy number. 2. INTRODUCTION The World Health Organization (WHO) estimates that if the effectiveness of tuberculosis (TB) control does not improve substantially, the number of TB cases will pass the 200 million mark in early 2001 and by 2020 nearly 1 billion people will be newly infected (data not shown), because of a combination of demographic factors, population movements, the expanding HIV epidemic and increasing drug resistance (Kochi 1994). Unlike many other diseases affecting the developing world, TB can be controlled and treated. Better case finding and treatment would considerably reduce the risk of transmission (Rodrigues and Smith 1990). Strain identification can be used as an additional tool in epidemiological investigations in order to gain a better understanding of factors that influence TB transmission, for
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identification of risk factors of TB transmission in a community and for evaluation of regional control programmes permitting a rational design of more adequate control measures (Maguire et al. 2002). Strain identification of Mycobacterium tuberculosis can help to address important epidemiological questions such as the origin of an infection in a patient’s household or community, and the spread and early detection of organisms with acquired antibiotic resistance (Edlin et al. 1992; Alland et al. 1994). Global transmission of M. tuberculosis can be studied by use of standardized molecular fingerprinting techniques that can be used for comparison of strains between laboratories, regions, countries and continents. With the advent of molecular techniques, TB investigators have new and powerful tools to further understand the transmission and phylogenetic properties of M. tuberculosis. Molecular techniques have been used to discriminate exogenous versus endogenous disease (Moro et al. 1998; Chaves et al. 1999) to investigate outbreaks (Moro et al. 1998; Edlin et al. 1992; Kenyon et al. 1997; Sahm and Tenover 1997) and cases of laboratory cross contamination (Small et al. 1993; Bauer et al. 1997; Carricajo et al. 1999). They have also been used to study transmission within a defined geographical setting (Yang et al. 1994; Gillespie et al. 1995; Hermans et al. 1995; Van Soolingen et al. 1995; Samper et al. 1998). Molecular typing can demonstrate the occurrence of exogenous superinfection in immunocompetent and immunocompromised patients; Godfrey-Faussett and Stoker (1992) reported that pairs of isolates from patients in Malawi with apparent relapse were infected with different strains. That exogenous re-infection after curative treatment has been demonstrated with these techniques (Das et al. 1993; Small et al. 1993; Van Rie et al. 1999). Wenger et al. (1995) have shown that molecular fingerprinting can be useful in informing the control measures necessary to break the chain of transmission of multi-drug resistance-TB (MDRTB). This paper will review these new molecular epidemiological tools and outline the way in which they can be applied to answer epidemiological, clinical and biological questions.
different susceptibility to antibiotics (Collins et al. 1982), but because of the limited number of possible patterns, this method is only useful for tracing spread of strains with unusual characteristics (data not shown). Serological methods have been used for diagnosis but do not differentiate between infections with different strains of M. tuberculosis (Grange and Laszlo 1990) and although some biochemical differences were observed between different isolates (Hoffner et al. 1993), reproducibility and limited strain variation is a problem.
3. NON-DNA TYPING METHODS
4.3 RFLP with hybridization
Before molecular techniques were available, the most used method of differentiation of strains of the TB complex and of M. tuberculosis strains was phage typing (Bates and Fitzhugh 1967). This method is cumbersome and lacked sensitivity because of the limited number of mycobacterium phage types available. However, the technique proved useful in typing M. tuberculosis strains from outbreaks (Snider et al. 1984) and laboratory cross examination (Jones 1988). Members of the M. tuberculosis-complex have been differentiated through evaluation of biochemical features and their
DNA polymorphism can also be demonstrated through hybridization of digested nucleic acids with genomic DNA or cloned fragments. Total DNA can be used as the probe but the use of the complete genome as a probe usually results in considerable background and affects the interpretation of the results. Some study groups have used cloned repetitive DNA from M. tuberculosis as probes (Eisenach et al. 1986, 1988) and one of them appeared to differentiate all strains of M. tuberculosis analyzed (Zainuddin and Dale 1989).
4. GENOTYPING 4.1 Restriction fragment length polymorphism Differentiation of strains of M. tuberculosis complex using nucleic acid-based technology is based on strain specific differences and frequencies of certain DNA sequences in chromosomal DNA. This is usually demonstrated by digestion of the genomic DNA with specific restriction enzymes and analysis of the generated patterns after separation of the DNA fragments on agarose gel: restriction fragment length polymorphism (RFLP) (Collins and Lisle 1984; Patel et al. 1996). This kind of analysis is technically possible and no hybridization step with defined probes is needed. However, interpretation of the results is difficult because the large number of fragments generates a complex pattern and only a small number of different RFLP types are observed. 4.2 Pulsed field gel electrophoresis Pulsed field gel electrophoresis (PFGE) has been designed to simplify RFLP. The method uses a less frequently cutting enzyme that generates high molecular weight fragments and allows separation of these fragments under special conditions in PFGE. The main limitation of the technique is that the small polymorphism characteristic for different strains will not always produce sufficient discrimination (Varnerot et al. 1992; Zhang et al. 1992).
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 781–791
MOLECULAR METHODS FOR MTB STRAIN TYPING
4.3.1 Methods based on repetitive elements. Repetitive elements and insertion sequences are frequently used as target sequences for differentiation between mycobacterial strains. Five repetitive DNA elements are useful in strain differentiation of M. tuberculosis complex (Dale 1995; Poulet and Cole 1995). For use of repetitive sequences in epidemiological studies, polymorphism in different strains must be present. Examples of repetitive elements are shown in Table 1. 4.3.2 IS6110. IS6110 is the element most widely used as a probe for RFLP. It is an insertion sequence belonging to the enterobacterial IS3 family (McAdam et al. 1990). This sequence hybridized with a plasmid isolated from M. fortuitum (Zainuddin and Dale 1989) and, depending on the organism in which it was characterized, is called IS6110 or IS986 in M. tuberculosis (as the description of IS6110 was published first and it is the preferred name in M. tuberculosis) or IS987 in M. bovis-BCG (Eisenach et al. 1990; Hermans et al. 1990b; Thierry et al. 1990). IS6110 is a 1361 bp long sequence that was detected in members of the M. tuberculosis complex and differences of only a few nucleotides have been detected between the sequenced copies. The number of IS6110 copies present in the genome is species- and strain-dependent. Most strains of M. tuberculosis carry between eight to 15 copies in different positions of the genome although single copy strains are common (Fig. 1). This sequence is characterized by presence of inverted repeats (direct repeat) separated by a transposase gene. IS6110 typing is the most widely used method for molecular epidemiological studies because of the high degree of discrimination obtained with this element. Table 1 Repetitive DNA sequences in Mycobacterium tuberculosis Repeated sequence IS6110 (IS986, IS987)
IS1081
DR cluster
MPTR PGRS
Host range
Copy number
Polymorphism
M. tuberculosis
0–20
High
M. africanum M. bovis M. bovis-BCG M. tuberculosis M. africanum M. bovis M. bovis-BCG M. tuberculosis M. africanum M. bovis M. bovis-BCG Tuberculosis complex Tuberculosis complex
0–20 1–20 1–2 5–6 5–6 5–6 5–6 1 1 1 1 ±80 26–30
High High None Low Low Low Low High High High None Low High
Modified from Poulet and Cole 1995.
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The procedure has been standardized (Van Embden et al. 1993) so that results generated in different laboratories can be compared permitting national and international studies of disease transmission to be carried out (Kremer et al. 1999). The major disadvantages are that this method requires a live culture, high quality DNA, and the procedure takes up to 5 days to complete. In some communities low copy number strains (
