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Herramientas moleculares empleadas en genotipificacion de Mycobacterium tuberculosis.

Molecular tools for Mycobacterium tuberculosis genotyping

Tuberculosis (TB) is an infecto-contagious disease caused by Mycobacterium tuberculosis complex that affects around a third of the world's population (1). M. tuberculosis genome is markedly homogeneous. The species of M. turberculosis, M. africanum, M. bovis, M. bovis BCG, M. caprae, M. pinnipedii, M. microti and M. canettii are genetically related. The presence of mutations in drug interaction places in genes rpoB (rifampicin), inhA, KatG, ahpC (izoniacid), rrs, rpsl (streptomycin), embB (ethambutol) and gyrA y gyrB (quinolones) results in resistance to these drugs producing a phenotypical change (2). This fact gave way to the development of methods capable of differentiating among this species isolates. Various techniques were used for the differential identification of M. tuberculosis, among them, those for determining unusual drug resistance, serotyping, multilocus enzyme electrophoresis (3), biochemical heterogeneity and phagus typing (4), being the latest one the standard method used until the late 1980's that presented however considerable disadvantages such as the low amount of identified phagotypes and its laboriousness.

The development of new molecular methods for M. tuberculosis genetic characterization has greatly contributed to the understanding of the transmission dynamics and pathogenesis of the disease (5,6). Today, the typing standard method for M. tuberculosis complex species is the restriction fragment length polymorphism (RFLP)-IS6110.

Molecular typing methods for M. tuberculosis complex are grouped in genomic methods for DNA studies such as RFLP IS6110 (7-10), polymorphic GC-rich sequence (PGRS) (11-13) analysis and pulsed-field gel electrophoresis (PFGE) on the one hand, and on the other hand, DNA-specific sequence amplification methods using polymerase chain reaction such as spoligotyping (spacer oligonucleotide typing) (14-18), ligation-mediated PCR (LM-PCR) (19,20), double repetitive element PCR (DRE-PCR) (21), fast ligation mediated PCR (FliP) (22), fluorescent amplified-fragment length polymorphism (FAFLP) (23,24), mycobacterial interspersed repetitive unit (MIRU) (25) analysis, amplification and sequencing of single nucleotide polymorphism (SNP), amplification of exact tandem repeats (ETR) and Queen's University Belfast (QUB) polymorphism of variable number tandem repeat (VNTR) (Table 1).


The characteristics, advantages and disadvantages of the different methods used for M. tuberculosis complex molecular typing always pose a problem at the time of choosing the most adequate. We undertook a review of literature by determining and analyzing the discrimination power for each method or combination of methods through the Hunter-Gaston discrimination index (HGDI) (26) as selection criterium at the time of implementing molecular methods in transmission studies because this is the first factor to consider at the initial stage of choosing a method (27).

Publications were searched in PubMed, Blackwell-synergy, ScienceDirect and EBSCOhost electronic data bases from March 2007 to August 2008 including the terms "Mycobacterium tuberculosis complex", "RFLP-IS6110", "spoligotyping", "MIRU", "VNTR", "Pulsed Field Gel Electrophoresis" "Direct repetition", "DRE-PCR", "Fluorescent amplifield fragment leng polymorphism", "FLip", "Genotyping AND Mycobacterium", "SNP", "LM-PCR", "HGDI AND Mycobacterium".

The selection criterium aimed at publications with titles related to M. tuberculosis complex genotyping and choosing those related to epidemiology and molecular characterization that included the Hunter-Gaston discrimination index value (HGDI) or data on the number of groupings and the number of isolates grouped in each of them to enable HGDI determination.


Our search exhibit as LM-PCR showed the highest HGID (0.9980), followed by FLiP (HGID 0.9945) and 15-loci MIRU (0.9620). RFLP-IS6110, the standard method used in M. tuberculosis molecular epidemiology, showed an average HGID of 0.9519. The combination of two or more methods showed higher HGDI values than those found when analyzing each method separately; these values were equal or close to one (28-54) (Table 2).


The different typing methods show discrimination powers expressed in a wide range of HGDI values that go from 0,6044 to 0,9985. A base HIGD value of >0,95 is required to differentiate among related organisms (26), and previous studies of each method should be undertaken in the development of M. tuberculosis complex molecular epidemiology research.

The analysis of HGDI average values for each method under study showed that spoligotyping has an HGDI average of 0.8630; we found values ranging from 0.6582 to 0.9817, the lowest of them resulting from the analysis of isolates obtained in Nigeria and the highest in the Netherlands, which shows the diversity of circulating strains in these places probably due to population migrations in each area.

Kremer et al (55) studied a group of 90 M. tuberculosis complex strains and determined the discrimination power based on the amount of different patterns obtained by typing them using the methods under study (RFLP-IS6110, mixed-linker PCR (Lm-PCR), and arbitrarily primed PCR (aPPCR), RFLP-PGRS, DRE-PCR, spoligotyping, VNTR, RFLP y RFLP-IS1081). They reported that for epidemiological research the methods of choice should be RFLP-IS6110 and LM-PCR, as they show a higher number of distinct patterns (84 and 81 patterns, respectively) (23).

Clinical isolates characterization at the Instituto Nacional de Salud de Colombia resulted in 11% of them with less than four IS6110 copies (47), contrasting with the finding reported by Asgharzadeh 2006, who found 8.6% of isolates with less than six copies (43). Due to the low number of IS6110 copies, a clear disadvantage of this method, complementary methods have appeared such as MIRU, that combined with spoligotyping, provide a higher discrimination power than that one obtained by using a single method (56). MIRU is a recently developed molecular typing method that has the greatest acceptance at the moment, as it shows an adequate balance between variability, an essential feature to differentiate among non related isolates, and molecular marker stability (57) with intermediate sensitivity and specificity as compared with spoligotyping sensitivity and RFLP-IS6110 specificity (58). Like spoligotyping (59), this method has shown to be useful for typing M. bovis species that generally present few copies of IS6110 sequence, and it has a greater discrimination power as compared with spoligotyping (60). However, its standardization has taken several years in a process where main changes refer to the primers used to enhance amplified product specificity, the magnesium chloride concentration in PCR mixture, the annealing temperature and even the loci analyzed (61), including a 24 loci, that have shown the same or greater discrimination power than RFLP-IS6110. This method has been the only one enabling discrimination of isolates with no IS6110 insertion sequences, excelling, therefore, spoligotyping and PGRS (62).

It is important to note that some methods are seldom used in genotyping and there are few publications reporting results from their implementation: FAFLP and LM-PCR data are reported in only one publication complying with our inclusion criteria. Analysis of HGDI average values in methods included in more than one publication shows that 15-loci MIRU presents the highest discrimination power (0.9620), followed by RFLP-IS6110 (0.9519), 12-loci MIRU (0.9491) and spoligotyping (0.8630) (Table 2). The average HGDI for RFLP-IS6110, excluding the 0.6044 HGDI reported by Dahle 2005, is 0.9808, this being the highest discrimination power agreeing with results reported by Kremer 2005 who found the highest discrimination power in RFLP-IS6110 (HGDI: 0.997) followed by MIRU (HGDI 0.995). Spoligotyping was not included in the analysis done by Kremer 2005 (20).

Our review methodology enabled us to establish the low number of publications determining discrimination power of molecular methods used in M. tuberculosis complex characterization (28 publications from 1,352). This was a limitation in the development of the present study as it has been in other review (44).

Based on the data analyzed in our study we can conclude that M. tuberculosis complex isolates molecular characterization requires at least two molecular markers to discriminate non related isolates as reported by Barlow 2001, who with combination of RFLP-IS6110 and VNTR- ETR obtained a higher HGID (0.988) than the one reported for each method taken separately. Spoligotyping reaches a higher discrimination power (HGDI 0.97) in the characterization of M. tuberculosis complex members by increasing spacing sequences from 43 to 65 (63), as happens with RFLP-IS6110 that provides greater evidence of epidemiological relation between two isolates when combined with other methods.

Implementation of molecular methods for M. tuberculosis complex typing requires an analysis of their advantages and disadvantages as regards tuberculosis surveillance and control programs because their use allows for an extra 52% detection of epidemiological relation among patients (64) and contributes to better understanding TB transmission dynamics.

The results obtained by our review evidence that laboratories should determine the discrimination power for each molecular method to enable a better selection, implementation and combination according to the specific conditions of each laboratory and the particular features of the geographic region and to guarantee their reproducibility and discrimination power.

Acknowledgements: This study was supported by the Instituto Nacional de Salud (INS) and the Centro Colombiano de Investigacion en Tuberculosis (CCITB).


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Juan C. Rozo-Anaya [1] y Wellman Ribon [2]

[1] Grupo de Micobacterias, Instituto Nacional de Salud. Bogota. Centro Colombiano de Investigacion en Tuberculosis-CCITB. Universidad de Pamplona.

[2] Grupo de Micobacterias, Instituto Nacional de Salud. Bogota. Centro Colombiano de Investigacion en Tuberculosis-CCITB. Escuela de Bacteriologia, Universidad Industrial de Santander, Bucaramanga. Colombia.

Received 27th July 2009/Sent for Modification 1th April 2010/Accepted 27th June 2010
Table 1. Several M. tuberculosis typing techniques

Metodos         Advantages                  Disadvantages

RFLP-IS6110     The number of copies and    Large amounts of DNA
                their positions in the      (1-2 ug). Technical
                genome may vary from        skills, it is slow and
                isolate to isolate (7)      has little discrimination
                providing identical         power in isolates with
                profiles in isolates        less than six IS6110
                from patients involved      copies(9),
                in recent transmission      reproducibility problems
                chains, and different       in isolates presenting
                genetic patterns In         large amounts of IS6110
                isolates from patients      copies (10)
                not associated by
                infection (8).

PGRS            Higher discrimination       Requiring large amounts
                power in isolates with      of high quality DNA
                six or less IS6110          (11.12) and lower
                copies                      discrimination power in
                                            isolates with multiple
                                            copies (13).

PFGE            Higher discrimination       Requiring large amounts
                power                       of high quality DNA and
                                            efforts to establish a
                                            standardized protocol
                                            have resulted in

PCR-based       Typing isolates with        It shows a 63 %
typing          less than six IS6110        sensitivity and 49%
methods         copies and it has shown     specificity compared
Spoligotyping   a 98 % identification       with the standard RFLP-
                specificity and 96 %        IS6110 method In
                sensitivity in clinical     molecular epidemiology
                samples (14,16).            studies (17.18)

LM-PCR          Requiring smaller DNA
                amounts than the
                RFLP-IS6110 (19) that
                has shown to be highly
                reproducible and to have
                great discrimination
                power (20).

DRE-PCR         Discrimination power is     This method has shown to
                high (21),                  be difficult to
                                            reproduce (58 %) due to
                                            the weak intensity of
                                            the lanes obtained.

FLiP            The main advantage is       low discrimination power
                that it is a quick          in isolates with less
                method based on the         than six IS6110 copies
                IS6110 sequence from 1      (22)
                ng DNA

FAFLP           Especially useful for       Its disadvantages
                differentiating isolates    Include the need of
                with less than five         isolates for culture.
                IS6110 copies (23).         DNA digestion and
                                            initial investment
                                            required to implement
                                            the method (24).

MIRU            Results in a code system    Show a 65 % specificity
                helpful in inter-lab        and a 35 % sensitivity
                comparisons. It has         as compared with the
                shown to be useful in       RFLP-IS6110 in molecular
                the Identification of       epidemiology studies.
                polyclonal infections
                (25) as its
                discrimination power is
                similar to that of the
                RFLP IS6110 and it is
                more specific than
                spoligotyping when the
                number of IS6110 copies
                is less than six

SNP             It has advantages such      It has shown similar
                as allowing multiple        results to those
                polymorphisms analysis      obtained with RFLP-
                in a short time,            IS6110 and
                rendering results in a      spoligotyping.
                binary format, and easy
                storage and inter-lab

Table 2. Comparison of several M. tuberculosis typing techniques

Author           Publication    Country           Number      Number
                 year                               of       of typed
                                                  samples     samples

Skuce (28)       2002           UK                  100         100

Kassama (29)     2006           UK                  44          44

Reisig (22)      2005           Germany             131         131

Burger (301      1998           Brazil              78          78

Banu (31)        2004           Bangladesh          46          44

Evans (32)       2004           UK                  70          70

Godreuil (33)    2007           Burquina Faso       120         120

Chin (34)        2005           Taiwan              502         502

Maes (35)        2003           Venezuela           41          41

Maes (35)        2006           Venezuela           41          41

Maes (35)        2006           Venezuela           41          41

Maes (35)        2008           Venezuela           41          41
Maes (35)        2006           Venezuela           41          41

Sun (36)         2004           Netherlands         68          68

Chin (34)        2006           Taiwan              502         502

Maes (35)        2006           Venezuela           41          41

Sun (36)         2004           Netherlands         68          68

Sun (36)         2004           Netherlands         68          68

Sun (36)         2004           Netherlands         68          68

Haas (37)        1997           Sierra Leona        138         135

Skuce (28)       2002           UK                  100         100

Skuce (28)       2002           UK                  100         100

Darte (38)       2005           Norway              14          14

Durmaz (39)      2003           Turkey              320         320

Garcia (40)      2002           Spain               147         147
Geri (15)        2005           Italy               64          64

Martinez (41)    2006           Spain               154         154

Sun (36)         2004           Netherlands         68          68

van der          2002           Netherlands         314         314
Zanden (42)

Asgharzadeh      2006           Iran                125         105

Barlow (44)      2001           Tanzania            93          90

Jou (45)         2005           Taiwan              155         155

Lari(46)         2005           Italy               248         248

Gomez (47)       2002           Colombia            53          53

Maes (35)        2008           Venezuela           41          41

Sun (36)         2004           Netherlands         68          68

Author           Method or          Number      Number       Number
                 combination          of          of           of
                 of methods        distinct     single     groupings
                                   patterns    isolates

Skuce (28)       ETR                  ND          ND           ND

Kassama (29)     FAFLP                32          24           8

Reisig (22)      FLiP                 94          61           33

Burger (301      LM-PCR               72          66           6

Banu (31)        MIRU 12 loci         32          25           7

Evans (32)       MIRU 12 loci         56          44           12

Godreuil (33)    MIRU 12 loci         71          51           20

Chin (34)        MIRU 12 loci         69          ND           ND

Maes (35)        MIRU 12 loci         15           7           8

Maes (35)        MIRU 12 loci         16           8           8

Maes (35)        MIRU 15 loci         20          11           10

Maes (35)        MIRU 24 loci         21          11           10

Maes (35)        MIRU 24 loci         7.          11           10

Sun (36)         MIRU 15 loci         61          58           3

Chin (34)        MIRU 15 loci         84          ND           ND

Maes (35)        MIRU 15 loci         18           9           9

Sun (36)         MIRU-ETR             67          66           1

Sun (36)         ABC-RFLP-            67          66           1

Sun (36)         ABC-                 62          59           3

Haas (37)        Mixed-linker         71          59           12

Skuce (28)       QUB-VNTR             ND          ND           ND

Skuce (28)       QUB-VNTR-            ND          ND           ND
Darte (38)       RFLP-IS6110           6           5           1

Durmaz (39)      RFLP-IS6110          206         155          51

Garcia (40)      RFLP-IS6110          94          69           25
Geri (15)        RFLP-IS6110          61          58           3

Martinez (41)    RFLP-IS6110          133         122          11

Sun (36)         RFLP-IS6110          47          43           4

van der          RFLP-IS6110          175         132          43
Zanden (42)

Asgharzadeh      RFLP-IS6110          81          70           11

Barlow (44)      RFLP-IS6110          61          55           6

Jou (45)         RFLP-IS6110          137         122          15

Lari(46)         RFLP-IS6110          201         166          35

Gomez (47)       RFLP-IS6110          51          49           2

Maes (35)        RFLP-IS6110          18           9           9

Sun (36)         RFLP-IS6110-         62          59           3

Author             Total       HGDI     Average
                 number of                HGDI

Skuce (28)           ND       0.8100     6.8100

Kassama (29)         20       0.9799     0.9799

Reisig (22)          70       0.9945     0.9945

Burger (301          12       0.9980     C.9980

Banu (31)            19       0.9715

Evans (32)           26       0.9930

Godreuil (33)        69       0.9600     0.9491

Chin (34)            ND        0.951

Maes (35)            34       0.8700

Maes (35)            31       0.9100
Maes (35)            30       0.9300     0.9300

Maes (35)            30       0.9500     0.9500

Maes (35)            30       0.9500     0.9500

Sun (36)             10       0.9940     0.9940

Chin (34)            ND       0.9720     0.9720

Maes (35)            32       0.920C     0.9200

Sun (36)             2        1.0000     1.0000

Sun (36)             2        1.0000     1.0000

Sun (36)             9        0.9950     0.9950

Haas (37)            76       0.9067     0.9067

Skuce (28)           ND       0.9100     0.9100

Skuce (28)           ND       0.9600     0.9600

Darte (38)           9        0.6044

Durmaz (39)         165       0.9879

Garcia (40)          78       0.9874
Geri (15)            6        0.9985

Martinez (41)        32       0.9971

Sun (36)             25       0.9310

van der             182       0.9845     0.9519
Zanden (42)

Asgharzadeh          35       0.9897

Barlow (44)          35       0.9693

Jou (45)             33       0.9982

Lari(46)             82       0.9979

Gomez (47)           4        0.9985

Maes (35)            32       0.9300

Sun (36)             9        0.9950     0.9950

ND: no data, case in which HGDI was obtained directly from
the publication under study
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Author:Rozo-Anaya, Juan C.; Ribon, Wellman
Publication:Revista de Salud Publica
Article Type:Report
Date:Jun 1, 2010
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