e-ISSN 2477-9148

Genetic diversity of M. tuberculosis

Jiménez et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

Volumen 39. No. 1, Mayo 2018

Comparative study of the genetic diversity of Mycobacterium tuberculosis

Complex by Simplied Amplied Fragment Length Polymorphism

and Mycobacterial Interspersed Repetitive

Unit Variable Number Tandem Repeat Analysis

Estudio comparativo de la diversidad genética de Mycobacterium

tuberculosis complex mediante análisis de Polimorsmo

de Longitud de Fragmentos Amplicados y Número variable

de Repeticiones en Tándem

de Unidades Repetitivas Interespaciadas de micobacterias

Ana Patricia Jiménez Arias

1,2,3

*, María José Lahiguera

, Rafael Borrás

, Concepción

Gimeno Cardona

, Marcelo Grijalva Silva

2,5,

María José Vallejo López

, María del Remedio

Guna Serrano

Servicio de Microbiología, Hospital General Universitario de Valencia, España.

Laboratorio de Biotecnología Humana, Departamento de Ciencias de la Vida y la Agricultura, Universidad de las

Fuerzas Armadas ESPE, Sangolquí-Ecuador.

Departamento de Biotecnología, Universidad Politécnica de Valencia, España.

Servicio de Microbiología, Hospital Clínico Universitario de Valencia, España.

Centro de Nanociencia y Nanotecnología Universidad de las Fuerzas Armadas ESPE, Sangolquí-Ecuador.

* Corresponding author e-mail: apjimenez@espe.edu.ec

doi.org/10.26807/remcb.v39i1.568

Recibido 16-01-2018 ; Aceptado 24-04-2018

ABSTRACT.- Species within the Mycobacterium tuberculosis (MTBC) Complex are genetically monomorphic, hence the need for

genotyping methods for a comprehensive understanding of the disease’s epidemiology. The genetic diversity of a Spainish collec-

tion of sixty-three, GenoType MTBC® -conrmed Mycobacterium tuberculosis clinical isolates was assessed by simplied AFLP

and 15-loci MIRU-VNTR. AFLP results showed 7 patterns (P1-P7); Dice’s coecient was 71% for P1 vs P7 and 96% for P1 vs P2

and P2 vs P4. MIRU-VNTR showed 25 unique patterns and 14 clusters. Lineages found were as follows: Haarlem (23, 36.51%),

Cammeroon (2, 3.17%), LAM (12, 19.05%), West African (6, 9.52%) and EAI (1, 1.59%). Discrimination indexes were 0.61 for

AFLP and 0.92 for MIRU-VNTR. In conclusion, MIRU-VNTR is robust and reproducible for MTBC genotyping. Simplied AFLP

is a relatively easy-to-perform approach that might be useful for the screening of isolates or in low resource settings.

KEYWORDS: Mycobacterium tuberculosis, MIRU-VNTR, AFLP, clonal.

RESUMEN.- Las especies dentro del complejo Mycobacterium tuberculosis (MTBC) son genéticamente monomórcas, por lo

tanto, existe una gran necesidad de métodos conables de genotipicación para la comprensión de la epidemiología de esta enfer-

medad. Esta investigación evalúa la diversidad genética de una colección española de sesenta y tres aislados mediante el uso de

polimorsmo de longitud de fragmentos amplicados (AFLP) y número variable de repeticiones en tándem de unidades repetitivas

interespaciadas (15-loci MIRU-VNTR). Los resultados obtenidos mostraron 7 patrones de AFLP (P1-P7) cuyos coecientes de

Dice fueron: 71% para P1 vs P7 y 96% para P1 vs P2, y P2 vs P4. MIRU-VNTR demostró 25 patrones únicos y 14 clusters. Los

linajes encontrados fueron: Haarlem (23, 36.51%), Cammeroon (2, 3.17%), LAM (12, 19.05%), West African (6, 9.52%) y EAI (1,

1.59%). Los índices de discriminación para AFLP fueron de 0.61 y 0.92 para MIRU-VNTR. En conclusión, este estudio demostró

que MIRU-VNTR es robusto y reproducible para genotipicar MTBC. Adicionalmente, AFLP simplicado es relativamente sen-

cillo de realizar y puede ser útil en el análisis de aislados con recursos limitados.

PALABRAS CLAVES: Mycobacterium tuberculosis, MIRU-VNTR, AFLP, clonal.

Artículo científico

REMCB 39 (1): 63-71, 2018

INTRODUCTION

In its 2017 Global Tuberculosis Report, the World Heal-

th Organization (WHO) reported an estimated 10.4 mi-

llion tuberculosis (TB) cases worldwide with 1.3 mi-

llion deaths from the disease plus 374000 deaths among

HIV-positive people, in 2016. In Spain, the incidence of

TB+VIH per 100 000 population is 8.7–12, in 2016. Al-

though the number of TB deaths fell by 22% between

2000 and 2015, TB remained one of the top ten causes of

death worldwide in 2017 (WHO 2017).

The Mycobacterium tuberculosis complex is composed

of the closely related organisms M. tuberculosis, M. afri-

canum, M. bovis, and M. bovis BCG, M. caprae, M. mi-

croti, M. canettii, and M. pinnipedii (Brosch et al. 2002).

Rapid and reliable identication of the members of the

M. tuberculosis complex is critical in guiding public

health and primary care decision-making. This is becau-

se each organism exhibits a dierent epidemiology, host

spectrum, geographic range, pathogenicity, and drug

susceptibility prole (Van Soolingen et al. 1997).

Mycobacterium species related to the Mycobacterium tu-

berculosis complex (MTBC) are considered genetically

monomorphic bacterial pathogens, due to the fact that

they present, on average, a single nucleotide variation

per 200bp (Achtman 2008). Consequently, understan-

ding the this disease’s epidemiology by genotyping re-

quires dierent technical approaches.

Given the evolution of DNA-based assays in recent de-

cades, many genotyping methods have been used eecti-

vely in taxonomic and identication studies of a range of

bacterial genera, including M. tuberculosis. DNA nger-

printing of M. tuberculosis isolates is a helpful technique

for the study of recent transmission episodes in a popu-

lation, as well as for identifying potential risk factors.

Fingerprinting has also made it possible to characterize

earlier unsuspected transmission paths, to screen for the

transmission of drug-resistant strains, and to conrm la-

boratory cross contamination (Cowan et al. 2002).

For routine laboratory work, dierent automated me-

thods such as the GenoType MTBC assay (Hain Lifes-

cience GMbH, Nehren) have been implemented for spe-

cies-level identication of M. tuberculosis complex. The

assay is a multiplex PCR-based, solid-phase reverse hy-

bridization that relies on the detection of single nucleoti-

de polymorphisms of the gyrB gene, and on the presence

or absence of RD1 (Richter et al. 2003; Richter et al.

2004). The GenoType MTBC assay provides a rapid and

accurate method for the detection of various members of

the M. tuberculosis complex when used on grown-cul-

tures (Richter et al. 2003; Richter et al. 2004; Gómez et

al. 2007; Neonakis et al. 2007; Somoskovi et al. 2008).

Several alternative PCR-based methods have been deve-

loped and are currently widely used as molecular tools

for the characterization of Mycobacterium tuberculosis

isolates, mostly for TB epidemiology studies (Lam-

bregts-Van Weezenbeek et al. 2003; Cattamanchi et al.

2006; Clark et al. 2006; Iñigo et al. 2007; Allix-Béguec

et al. 2008). Restriction fragment length polymorphism

(RFLP) analysis, based on the IS6110 sequence, is the

reference genotyping method for M. tuberculosis (Van

Soolingen et al. 1997). However, this method is tech-

nically demanding and time-consuming, thus making it

unsuitable for real-time epidemiological intervention. A

method variation, an amplied-fragment length poly-

morphism (AFLP) simplied test, can be carried out by

using only one restriction enzyme (XhoI), one double

strand adapter, and one PCR primer (Viader-Salvadó et

al. 2009).

Currently, one of the most promising PCR-based geno-

typing methods is a variable number of tandem repeats

(VNTR) assay that makes use of mycobacterial inters-

persed repetitive units (MIRU). This technique is based

on the variability found in 12 specic MTB loci inters-

persed throughout the mycobacterial genome (Supply et

al. 2001; Kremer et al. 2005; Supply 2005; Supply et al.

2006). Recently, MIRU-VNTR genotyping approaches

with 15 or 24 loci (Supply et al. 2006; Alonso-Rodriguez

et al. 2008; Alonso-Rodriguez et al. 2009) have been

evaluated and applied in molecular epidemiological ty-

ping in mycobacteria.

The purpose of this study was to determine genetic varia-

bility, circulating the lineages and discrimination power

of two PCR-based techniques: AFLPs and MIRU-VNTR

(15 loci) on GenoType® MTBC-conrmed MTB clini-

cal isolates.

MATERIALS AND METHODS

Clinical isolates.- Sixty-three clinical isolates were

collected. Fifty-seven isolates, corresponding to 53

patients, were identied as belonging to the Mycobac-

terium tuberculosis complex. Isolates were part of the

Microbiology Department collection at the Hospital Ge-

neral Universitario de Valencia, España. The collection

included six M. africanum isolates from three patients.

Isolates for this study were collected from 2008 to 2011.

Clinical samples were classied as respiratory (n=35;

55.6%) and extra-pulmonary (n=28; 44.4%). For each

assay, the Mycobacterium tuberculosis ATCC 25177 re-

ference strain was used as a control. Clinical and epide-

miological data from patients are shown in Supplemen-

tary Information 1.

Genetic diversity of M. tuberculosis

Jiménez et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

Molecular species-level identication of isolates by

GenoType® MTBC.- Molecular identication was per-

formed for the entire collection as well as for the My-

cobacterium tuberculosis ATCC 25177 control strain.

Isolates were inactivated using heat-ultrasound methods,

and their processing was as follows: 1) DNA extraction

from the clinical isolates; 2) multiplex-PCR with biotin

labeled primers; and, 3) reverse hybridization.

PCR procedure.- For the amplication assay, 5 µl of the

inactivated bacterial supernatant was mixed with 5 µl of

amplication buer (10X), 2 µl of MgCl

(25mM), 3 µl

of sterile distilled water, 0.2 µl of HotStart-Taq®DNA

polymerase and 35 µl of PMN Mix (Primer/Nucleotide

mix). The thermocycler (A®TEFACTindustriekulture)

program consisted of: i) 95 °C for 15 minutes; ii) ten

cycles of 30 seconds at 95 °C and 2 minutes at 58°C; iii)

twenty cycles of 25 seconds at 95 °C, 40 seconds at 53

°C and 40 seconds at 70 °C; and iv) one nal step at 70

°C for 8 minutes.

Hybridization assays.- Auto-LiPA (Innogenetics N.V.,

Ghent, Belgium) was the automated system used for am-

plicon hybridization. The instrument requires 20uL of

sample, and the protocol includes chemical denaturation,

a hybridization step for biotin probes binding to a mem-

brane, and a stringent wash step and acid phosphatase

staining (AP). Interpretation of results was performed

according to the manufacturer’s instructions.

Simplied Amplied Fragment Length Polymor-

phism (AFLP).- AFLP was performed with the entire

collection and the Mycobacterium tuberculosis ATCC

25177 control strain, following the protocols published

by Viader-Salvadó (2009), with modications based on

Gaafar (2003).

DNA Extraction.- Two full bacterial loops were dissol-

ved in 400 µL TE (1X) buer in 2ml microfuge tubes,

followed by heat inactivation at 80 °C for 20 minutes. A

50µL (10mg/ml) lysozyme volume was added, and the

tubes were incubated at 37 °C for 18 hours. Then, 75µL

of SDS 10% (w/v) and 20µL of proteinase K (20 mg/ml)

were added to the mix and the tubes were heated at 65

°C for 10 minutes. Next, 100µL of 5M NaCl and 100µL

of CTAB/NaCl were added and the solution was incu-

bated at 65 °C for 10 minutes. For DNA precipitation,

750µL of chloroform: isoamyl alcohol (24:1) was used;

the vial was then vigorously shaken and centrifuged at

13000 rpm for 8 minutes. The aqueous phase was re-

moved into a new tube, cold isopropyl alcohol in 1:0.6

(V/V) ratio was added and the mixture was homogenized

by inversion, stored at -20 °C for 30 minutes, centrifu-

ged at 13000 rpm for 15 minutes, and the supernatant

removed. Finally, the obtained pellet was washed with

1mL of cold 70% ethanol, followed by inversion mixing

and centrifugation at 13000 rpm for 5 minutes (twice).

The pellet was left to dry, and 50µL of TE (1X) buer

was then added. The elution was incubated at 37 °C for 1

hour and stored at -20 °C.

Enzymatic Digestion.- Genomic DNA (200ng) was di-

gested with 10 U of XhoI (Roche) for 2 hours at 37 °C in

a 25µL reaction containing 2.5µL 10X buer and sterile

ultra-pure water. Digested DNA was maintained at 4 °C.

Oligonucleotides: XA-1 (GTAGACTGCGTACATGCA)

and XA-2 (TCGATGCATGTACGCAGT) at a stock

concentration of 25µM were diluted in PCR 10X buer

to a nal volume of 50µL. For hybridization, this mixtu-

re was heated at 90 °C for 5 minutes and cooled down to

4° C in a thermal cycler (A®TEFACT industriekulture)

with a ramp rate of 1 °C/min. Double strand adaptors

obtained were preserved at 4 °C for a week.

Adaptors Ligation.- Digested DNA (15µL) was mixed

with 2.5 µM adaptors and 1U of T4 DNA ligase (Roche)

in a nal volume of 25µL, and incubated at 12 °C for 17

hours. Next, T4 DNA ligase was thermally inactivated

at 65 °C for 10 minutes. Finally, 40µL of distilled water

was added and eluted DNA was stored at -20 °C.

PCR.- PCR reactions of 25µL were assembled as fo-

llows: PCR buer 1X, 1.5 mM MgCl2, 200µM dNTPs,

6µM XP-G primer (TGCGTACATGCATCGAGG), 2 U

Hot Star Taq DNA polymerase (HAIN Lifescience) and

4, 2µL of the digested-ligated DNA solution. The ther-

mocycler program consisted of: i) initial denaturation at

72 °C for 2 minutes; ii) sixteen cycles of 20 seconds at

94 °C, 30 seconds at 65 °C and 2 minutes at 72°C; iii)

nineteen cycles of 30 seconds at 94 °C, 30 seconds at 50

°C and 2 minutes at 72 °C; and iv) a nal extension step

of 30 minutes at 60 °C with a subsequent permanent hold

at 4 °C. Amplicons were visualized by agarose horizon-

tal electrophoresis in a 1.5% (w/v) agarose gel with TBE

1X buer. Electrophoresis runs were set at 55 volts for

2h30min, and products were visualized by staining with

a 0.5µg/mL ethidium bromide solution (Sigma-Aldrich

Co) for 20 minutes. A molecular weight marker (1Kb)

was used in all assays. Identication of the bands was

performed with the automated system CHEMI HR (SY-

NGENE). Two negative controls were used: i) DNA wi-

thout digestion-ligation control, in order to identify any

band related to non-specic hybridization of the primer

into genomic DNA; and, ii) blank PCR reaction (negati-

ve control) without DNA, to recognize contamination or

primer-related artifacts.

Relative size of the fragments was calculated with the

software Bio-1D++ and similarity of the isolates was

determined with Dice’s coecient in order to obtain ho-

REMCB 39 (1): 63-71, 2018

mology matrixes and dendograms. For AFLP analysis,

genetically identical isolates were expected to have si-

milar patterns in terms of number and size of fragments;

dissimilar isolates were expected to show dierent pat-

terns (Gaafar et al. 2003).

MIRU-VNTR. Based on its high discrimination power

(Comas et al. 2009) a 15 loci MIRU-VNTR assay (4,

26, 40, 10, 16, 31, 42, 43, ETR A, 47, 52, 53, QUB-1

1b, 1955, QUB-26) was selected based on the protocols

recommended by Supply (2005).

Individual PCR reactions (nal volume: 20µL) were

assembled for each locus as follows: PCR 1X buer,

3mM MgCl2 (MIRU 4, 26, 40, 47, 52, 53), 2mM MgCl2

(MIRU 10, 16, 31), 1.5mM MgCl2 (MIRU 42, 43,

ETRA, QUB-11b, 1955, QUB-26), 0.2mM dNTPs, 1M

betaine, 0.4 pmol primers, 0.5 U Platinum Taq DNA

Polymerasa (Invitrogen) and 2µL of genomic DNA

(5 ng/µL). Amplicons were separated in a 1.5% (w/v)

agarose gel electrophoresis with TBE 1X and a 0.5 µg/

mL ethidium bromide solution staining step. The size of

the fragments for each locus was calculated thorough

visual comparison with the molecular weight marker.

Allelic assignment was based on the methodology

proposed by Supply (2005), with a 15-digit numeric

code for each isolate. This code was then entered into

an on-line database platform (http://www.miru-vntrplus.

org/) to establish clonal complexes and lineages. The

criterion for a clonal complex in a clinical isolate was

the absence of variation in more than two loci.

Discrimination index.- Discrimination power analysis

of this methodology was performed according to Simp-

son’s diversity index (Dillon et al. 1993). A value of 0.9

is considered satisfactory in order to establish appropria-

te conclusions (Hunter and Gaston 1988).

RESULTS

Molecular species-level identication using Genotype®

MTBC showed the following (Figure 1): two clinical

isolates (3.17%) were identied as M. bovis BCG (posi-

tive probes 4, 7, 9, 10 and 13), and six isolates (9.52%) as

M. africanum (positive probes 4, 5, 6, 7 and 10). Control

strain ATCC 25177 plus 55 clinical isolates (87; 30%)

were identied as M. tuberculosis (positive probes 4, 5,

6, 7, y 8).

Simplied AFLP showed polymorphic restriction pro-

les with the XP-G primer. Consequently, restriction

patterns and classication of isolates according to their

homology was performed in order to establish AFLP

proles for the entire collection. Restriction fragments

obtained with XP-G primer uctuated from 7 to 12, with

sizes from 200 pb to 750 pb. Electropherogram analysis

allowed for identication of seven patterns (P1 to P7)

(Figure 2), in which the most prevalent were P1 and P2

Figure 1. Interpretation of GenoType® MTBC´s results

Genetic diversity of M. tuberculosis

Jiménez et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

with 35 (55.6%) and 17 (26.9%) isolates, respectively.

Out of the total isolate number, 52 (82.5%) belonged

to P1 and P2, followed by patterns P3, P4 and P5 with

three isolates (4.8% each) and P6 y P7 with one isolate

(1.59%). The M. tuberculosis ATCC 25177 control stra-

in restriction pattern was not present in the previously

described patterns and was not included in the analysis.

However, the control prole was consistently present in

all assays. Reproducibility of the method was evaluated

with AFLP (duplicate) characterization of ve random

isolates, which showed the same band pattern in each

experiment.

Dice’s coecient establishes the level of genetic simila-

rity among isolates that have dierent restriction patter-

ns. For the group P5 vs. P7, the Dice’s coecient was

71%, while for P1 vs. P2 and P2 vs. P4, it was 96%.

Data obtained allowed for the construction of a homolo-

gy dendogram as represented in Figure 3.

All isolates classied as M. africanum belonged to the

pattern P1, two isolates identied as M. bovis ssp. BCG

were included in the P5 group, and the remaining M.

tuberculosis isolates were distributed along all seven

patterns.

Temporal distribution of clusters showed that the most

prevalent cluster P1 was found in the years 2008, 2009,

2010 and 2011. Meanwhile, clusters P6/P7 were found in

the year 2009. Geographic scattering of clusters revealed

the presence of the P1 pattern in isolates from the three

study sites (Manises, Hospital General Universitario de

Valencia and Picassent´s Detention Centre). Patterns

P3, P4, P5, P6 and P7 were not found in the Detention

Centre.

Patterns from isolates recovered from patients who

contributed to this study with several samples showed

identical restriction proles, with only one exception

showing two dierent patterns in the P1 and P6 groups.

MIRU-15 data from 63 clinical isolates showed the

presence of 25 unique patterns. Thirty-eight isolates

(60.32%) were grouped into 14 clonal complexes (CC1

to CC14), each of them with a minimum of two isolates/

cluster or a maximum of six isolates/cluster. An identical

MIRUtype was found in three strains/isolates of CC4

and two isolates of CC5. Clinical isolates/strains within

the study belonged to the following lineages: Haarlem

(36.51%; n= 23); Cammeroon (3.17%; n=2); S (30.16%;

n=19); LAM (19.05%; n=12); West African I (9. 52%;

n=6); EAI (1.59%; n=1).

Figure 2. Restriction AFLP proles obtained with the primer XP-G. Lane 1 and 10: M, 1Kb molecular weight marker. Lane 2 to 6: Clinical isolates

restriction pattern. Lane 7: Negative control, DNA without digestion-ligation. Lane 8: Negative control without DNA. Lane 9: Restriction prole

of M. tuberculosis ATCC 25177.

REMCB 39 (1): 63-71, 2018

Figure 3. Homology dendogram obtained with the primer XP-G.

Simpson’s diversity index was calculated in order to

establish the discriminatory power of the genotyping

methods. The values for AFLP and MIRU-VNTR were

0.6211 and 0.9785, respectively.

DISCUSSION

In this study, 63 clinical isolates of Mycobacterium tu-

berculosis complex were molecularly characterized and

identied to species level by an automated commercially

available assay (Genotype MTBC). Genotyping of the

entire collection was then performed through a simpli-

ed AFLP assay as well as by a 15-loci MIRU-VNTR

platform. Clustering, MIRUtypes and lineages were de-

ned, and eciency indexes were calculated for both

AFLP and MIRU-VNTR.

Microbiological diagnosis at species level is important

for targeted therapy. All M. bovis isolates are intrinsica-

lly resistant to pyrazinamide. M. bovis-BCG is used as

an immunogenicity stimulator in some diseases due to

its high antigenicity and immunogenic activity. In these

cases, it is essential to know if the species isolated is M.

bovis-BCG or another species within the Mycobacterium

tuberculosis complex, which will require immediate

treatment.

In 2008, Somoskovi used the automated GenoType®

MTBC technique to assess its discriminatory power for

Mycobacterium tuberculosis complex isolates. Thirty-

ve reference strains, along with 157 clinical isolates and

79 positive smear samples, were included in the study.

The results showed high sensitivity and specicity for

the MTBC assay, with 93.2% of the samples belonging

to the Mycobacterium tuberculosis complex, with the

exception of M. canetti and M. africanum type 2 (Richter

et al. 2004) .

In our study, DNA from all clinical isolates and M. tu-

berculosis 25177 control strain hybridized to specic

probes corresponding to specic Mycobacterium tuber-

culosis 87,3%), M. bovis BCG (3,17 %), or M. africanum

9,5 %) AFLP proles. The technique is relatively easy to

perform once optimized. Visualization and interpretation

of band proles are straightforward and, in our opinion,

suitable for routine mycobacteriology lab work. Controls

are included in the test package which allow for proper

eciency monitoring. This test has been evaluated by

other authors, such as Richter, with results that support

the use of the assay as a easy routine diagnostic tool for

species identication of the Mycobacterium tuberculosis

complex.

In the AFLP assay, XhoI restriction endonuclease (Via-

der-Salvadó et al. 2009) cuts the insertion sequence

IS6110, which results in a fragment pattern that depends

on sequence variability. In general, the typing scheme is

robust, and the restriction patterns (7 to 12 bands) ob-

tained were stable over time. Similar results were found

in Viader-Salvadó’s study. Distribution of clinical isola-

tes in dierent proles conrmed that M. tuberculosis

was present in all patterns; M. africanum was present in

the P1 group, while M. tuberculosis BCG was present

in the P5 group. AFLP’s Simpson’s diversity index of

0.621 limits the assay’s value as a full genotyping tool

(Hunter and Gaston 1988). In our opinion, the technique,

due to its capability to show clonality, its relative straigh-

tforwardness, and its highly discriminating XP-C primer,

would be suitable for M. tuberculosis isolates screening

rather than for full, in-depth genotyping. It is worth no-

ting that the XP-C primer could be combined with XP-G

(Goulding et al. 2000) for polymorphisms identication.

MIRU-VNTR (15 loci) is a PCR-based methodology

Genetic diversity of M. tuberculosis

Jiménez et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

that allows full genotyping of M. tuberculosis complex

isolates. It is more ecient and has shorter turnaround

times than AFLP. Also, MIRU-VNTR has a better discri-

mination index (HGDI 0.9785) and allows for the iden-

tication of related lineages. Analysis of MIRUtypes

showed 25 ungrouped isolates and 14 clonal complexes

or clusters, based on the criterion of 2 loci variation. Clo-

nal complex 1 was comprised of isolates 55, 58, 57, 52,

59 and 56 (as dened by the GenoType® MTBC assay).

M. africanum , as depicted in the phylogenetic tree of this

complex, corresponds to the 6 AFRI 1 lineage, which is

in agreement with other studies (Comas et al. 2009).

Clonal complex 5 comprised isolates 61 (CFS) and 62

(gastric juice), with identical MIRU type from one neo-

nate. This result points to the possible presence of the

same genotype in dierent bodily sites. Isolate 60, which

grew from a sample from the patient’s father, showed the

same MIRU type, which supports a direct transmission

path. Isolates 20 (sputum) and 33 (BAL), were grouped

into the clonal complex 13. Both isolates corresponded

to the same patient with dierent sample collection da-

tes. Isolates identied as Mycobacterium bovis BCG pre-

sent dierent MIRU types within clonal complexes 6 and

12. According to the AFLP assay, these isolates group to

the P5 pattern.

Lineage 4 was the most prevalent, with the following

distribution: Haarlem (36.51%); Cammeroon (3.17%); S

(30.16%); and LAM (19.05%). Lineage 6 (West African

I 9.52%) and lineage 1 (EAI1, 59%) were also present.

MIRU-VNTR displayed 14 clusters while AFLP assays

showed only seven. For instance, M. africanum isolates

are present in cluster 1 and pattern group P7. This could

valuable and of interest for lineage-level typing and epi-

demiology studies.

Based on a classical epidemiology point of view, this

study’s limitation is the lack of isolates, which reduces

the level of genetic variation identication in a popula-

tion. However, it is appropriate for the technical valida-

tion of genotyping tools. Additionally, the used of AFLP

and MIRU-VNTR have advantages and disadvantages

that aect the genotypication process. AFLP has lower

costs and requires time (laborious) and trained personal,

while MIRU VNTR is more expensive, but more accu-

rate than AFLP.

CONCLUSION

MIRU-VNTR (15 loci) showed a better discrimination

index than the simplied AFLP assay. Since MIRU-VN-

TR might be rened for better discrimination (24 loci

system) and its sensitivity and specicity may readily

improve by coupling the assay to spoligotyping, a com-

prehensive typing of MTBC isolates might be feasible.

Simplied AFLP would be useful for initial isolates

screening or in low resources settings.

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Supplementary information

Supplementary information 1

Case Age Gender Area Diagnosis Type of sample Date Sample No. Code

1 47 M G Disseminated TB - HIV Urine 5/2/2010 9496177 MTB 01

2 32 M G Pulmonary tuberculosis Sputum 11/11/2010 14174751 MTB 02

3 23 F G Pulmonary tuberculosis Sputum 30/9/2010 14127700 MTB 03

4 78 F G Unknown Lymph node biopsy 10/6/2010 14044424 MTB 04

5 35 M G Pulmonary tuberculosis Sputum 23/10/2010 14153418 MTB 05

6 85 F G Disseminated TB Wound exudate 17/7/2009 9470934 MTB 06

7 30 F G Pulmonary tuberculosis BAL 10/9/2009 9477086 MTB 07

8 31 F G Disseminated TB Adenopathy 15/10/2009 9481517 MTB 08

9 35 F G Disseminated TB Sputum 14/1/2011 14235729 MTB 09

10 35 M G Pulmonary tuberculosis Sputum 30/12/2010 14222957 MTB 10

11 0 F G Pulmonary tuberculosis Gastric Juices 3/9/2009 9476264 MTB 11

12 0 F G Contact with TB-bacillus Gastric Juices 15/7/2009 9470679 MTB 12

13 69 M G Pulmonary tuberculosis with COPD BAL 13/11/2009 9485304 MTB 13

14 74 M G Hydronephrosis Urine 18/9/2009 9478063 MTB 14

15 31 M G Lymph node tuberculosis FNAB 28/8/2009 9475664 MTB 15

16 34 M G Lymph node tuberculosis FNAB 14/7/2009 9470491 MTB 16

17 42 M G Nódulo pulmonar Lung biopsy 21/12/2009 9490687 MTB 17

18 33 F G Pulmonary tuberculosis Sputum 14/12/2009 9489825 MTB 18

19 30 M G Disseminated TB Feces 26/9/2009 9479042 MTB 19

20 45 M G Pulmonary tuberculosis Sputum 6/8/2010 14087416 MTB 20

21 40 M G Quality Control SEIMC Sputum 31/3/2010 236 BCG 01

22 31 M G Pulmonary tuberculosis Sputum 3/7/2009 9469182 MTB 21

23 51 F G Pulmonary tuberculosis Sputum 7/10/2009 9480524 MTB 22

24 38 F G Diarrhea- HIV Feces 20/7/2009 9471285 MTB 23

25 31 F G Pulmonary tuberculosis Sputum 9/7/2009 9469914 MTB 24

26 38 M G TB meningitis CSF 4/3/2009 9454044 MTB 25

27 72 F G Mycobacterial cervical lymphadenitis Wound exudate 8/7/2009 9469823 MTB 26

28 66 M G Paravertebral abscess FNAB 2/7/2009 9469079 MTB 27

29 63 M G Pulmonary tuberculosis Sputum 16/2/2010 9497624 MTB 28

30 108 F G Pulmonary tuberculosis Sputum 16/9/2009 9477732 MTB 29

31 79 M G Pulmonary tuberculosis Sputum 22/12/2009 9490819 MTB 30

32 33 F G Lymph node tuberculosis FNAB 28/4/2010 14003097 MTB 31

33 45 M G Pulmonary tuberculosis BAL 13/7/2010 14071550 MTB 32

34 18 M G Pulmonary tuberculosis Sputum 24/10/2010 14153513 MTB 33

35 43 F G Urinary tract tuberculosis (UTB) Urine 12/3/2010 4600656 MTB 34

36 29 M G Pulmonary tuberculosis Sputum 1/6/2010 14035630 MTB 35

37 41 M G Lymph node tuberculosis -HIV Pus 23/3/2010 4601738 MTB 36

38 85 M G Disseminated bacillus Calmette-Guérin (BCG) Blood 3/9/2009 9476339 BCG 02

39 39 M G Pulmonary tuberculosis Sputum 18/6/2010 14052210 MTB 37

40 43 F G Lymph node tuberculosis Lymph node biopsy 21/5/2010 14026279 MTB 38

41 52 M G Pulmonary tuberculosis Sputum 20/5/2010 14024949 MTB 39

42 17 M G Diarrhea- HIV Feces 26/6/2009 9468328 MTB 40

43 40 F G Psoas Abscess Abdominal biopsy 18/3/2009 9455833 MTB 41

44 33 F G Lymph node tuberculosis Ulcer exudate 25/3/2010 4602201 MTB 42

45 25 M G Mycobacterial cervical lymphadenitis Surgical exudate 6/3/2009 9454335 MTB 43

46 45 F G Pulmonary tuberculosis- HIV Sputum 10/7/2010 14069689 MTB 44

47 65 M G Pleural effusion Pleural fluid 5/7/2010 14064998 MTB 45

48 45 M G Diarrhea- HIV Feces 11/10/2010 14138743 MTB 46

49 30 F G Lymph node tuberculosis Lymph node biopsy 18/5/2010 14022448 MTB 47

50 23 M G Pulmonary tuberculosis- HIV Sputum 28/10/2010 14159216 MTB 48

51 31 M G Pulmonary tuberculosis Sputum 17/4/2009 9250266 MTB 49

52 35 M C Pulmonary tuberculosis Sputum 1/3/2009 9055525 AFRI 01

53 51 M G Pulmonary tuberculosis Sputum 26/1/2010 9494804 MTB 50

54 79 M G Pulmonary tuberculosis Gastric Juices 27/5/2009 9464327 MTB 51

55 21 F C Pulmonary tuberculosis Sputum 1/11/2008 8116631 AFRI 02

56 35 M C Pulmonary tuberculosis Sputum 1/4/2009 9064005 AFRI 03

57 44 F C Abdominal mass Peritoneal fluid 1/5/2010 1083364 AFRI 04

58 21 F C Pulmonary tuberculosis Sputum 1/11/2008 8116629 AFRI 05

59 21 F C Pulmonary tuberculosis Sputum 1/11/2008 8116630 AFRI 06

60 24 M G Pulmonary tuberculosis Sputum 5/4/2011 14323266 MTB 52

61 0 M G Disseminated TB CSF 1/4/2011 14321197 MTB 53

62 0 M G Disseminated TB Gastric Juices 2/4/2011 14321259 MTB 54

63 24 F G Pulmonary tuberculosis BAL 23/4/2010 736 MTB 55

Patient