Juan C. Rozo-Anaya¹ y Wellman Ribón²

¹ Grupo de Micobacterias, Instituto Nacional de Salud. Bogotá. Centro Colombiano de Investigación en Tuberculosis-CCITB. Universidad de Pamplona. Colombia.juancarlosrozoanaya@hotmail.com
² Grupo de Micobacterias, Instituto Nacional de Salud. Bogotá. Centro Colombiano de Investigación en Tuberculosis-CCITB. Escuela de Bacteriología, Universidad Industrial de Santander, Bucaramanga. Colombia. wellmanribon@yahoo.es

Received 27th July 2009/Sent for Modification 1th April 2010/Accepted 27th June 2010

ABSTRACT

Objective The present work studied molecular typing methods used for Mycobacterium tuberculosis characterization in order to learn about their advantages, disadvantages and discrimination power as regards the implementation of tuberculosis surveillance and control programs.
Methods To analyze the discrimination power of each method we studied articles that included Hunter-Gaston discrimination index (HGDI) values or data allowing their calculation.
Results The highest discrimination power was registered for LM-PCR followed by FLiP and 15-loci MIRU. The most frequently used methods showed an HGDI of 0.9491, 0.9519 and 0.8630 for 12-loci MIRU, RFLP-IS6110 and spoligotyping, respectively.
Conclusion M. tuberculosis isolates molecular characterization requires at least two molecular markers to discriminate non related isolates, as well as previous analysis to their implementation.

Key Words: Tuberculosis, Mycobacterium tuberculosis, molecular epidemiology, genotype, DNA, Polymorphism, Restriction Fragment Length (source: MeSH, NLM).

RESUMEN

Objetivo En el presente trabajo se estudiaron las metodologías de tipificación molecular empleadas para caracterizar Mycobacterium tuberculosis con el objetivo de conocer las ventajas, desventajas y poder discriminatorio para ser consideradas al momento de la implementación en los programas de vigilancia y control de la tuberculosis.
Métodos Para el análisis del poder discriminatorio de cada metodología se estudiaron los artículos que suministraban el valor del Hunter-Gaston discrimination index (HGDI) ó los datos que permitían su determinación.
Resultados Se documentó que el LM-PCR tiene una mayor capacidad discriminatoria seguida de FLiP y MIRU de 15 loci. Las metodologías más comúnmente empleadas mostraron un HGDI de 0.9491, 0.9519 y 0.8630 para MIRU de 12 loci, RFLP-IS6110 y spoligotyping respectivamente.
Conclusión La caracterización molecular de aislamientos de M. tuberculosis requiere mínimo el análisis de al menos dos marcadores moleculares para discriminar aislamientos no relacionados y la necesidad de realizar análisis previos a la implementación de estas metodologías.

Palabras Clave: Tuberculosis, Mycobacterium tuberculosis, epidemiologia molecular, genotipo, ADN, polimorfismo de longitud del fragmento de restricción (fuente: DeCS, BIREME).

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 GCrich 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), my-cobacterial 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, mixedlinker 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 Investigación en Tuberculosis (CCITB).

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