Herramientas moleculares empleadas en genotipificacion de Mycobacterium tuberculosis.
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  y Wellman Ribon 
 Grupo de Micobacterias, Instituto Nacional de Salud. Bogota. Centro Colombiano de Investigacion en Tuberculosis-CCITB. Universidad de Pamplona. Colombia.email@example.com
 Grupo de Micobacterias, Instituto Nacional de Salud. Bogota. Centro Colombiano de Investigacion en Tuberculosis-CCITB. Escuela de Bacteriologia, Universidad Industrial de Santander, Bucaramanga. Colombia. firstname.lastname@example.org
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 considerable modifications. 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 comparison. 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 (43 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 +spoligotyping Maes (35) MIRU 15 loci 20 11 10 +poligotyping Maes (35) MIRU 24 loci 21 11 10 Maes (35) MIRU 24 loci 7. 11 10 +poligotyping 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 ABC-RFLP- IS6110 Sun (36) ABC-RFLP- 67 66 1 IS6110- Spoligotyping Sun (36) ABC- 62 59 3 Spoligotyping Haas (37) Mixed-linker 71 59 12 PCR Skuce (28) QUB-VNTR ND ND ND Skuce (28) QUB-VNTR- ND ND ND ETR 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 (43 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 Spoligotyping Author Total HGDI Average number of HGDI grouped strains 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 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 (43 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|
|Date:||Jun 1, 2010|
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