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BMC Infectious Diseases

BioMed Central

Open Access

Research article

Mycobacterium tuberculosis ecology in Venezuela: epidemiologic
correlates of common spoligotypes and a large clonal cluster
defined by MIRU-VNTR-24
Edgar Abadía1, Monica Sequera3, Dagmarys Ortega4, María Victoria Méndez1,
Arnelly Escalona1, Omaira Da Mata2, Elix Izarra5, Yeimy Rojas1,
Rossana Jaspe1, Alifiya S Motiwala6, David Alland6, Jacobus de Waard2 and
Howard E Takiff*1
Address: 1Laboratorio de Genética Molecular, CMBC, Instituto Venezolano de Investigaciones Cientificas (IVIC), 1020A Caracas, Venezuela,
2Laboratorio de Tuberculosis, Instituto de Biomedicina, Universidad Central de Venezuela, (UCV), Caracas, Venezuela, 3UCGEI/LaBDEI,
Universidad de Carabobo, Valencia, Venezuela, 4Centro Amazónico de Investigaciones y Control de Enfermedades Tropicales (CAICET), Puerto
Ayacucho, Venezuela, 5Instituto Nacional de Estadistica, (INE), Caracas, Venezuela and 6Center for Emerging Pathogens, The University of
Medicine and Dentistry of New Jersey, Newark, New Jersey, USA
Email: Edgar Abadía - eabadia@gmail.com; Monica Sequera - moniquita4@gmail.com; Dagmarys Ortega - dagmarys@gmail.com;
María Victoria Méndez - mvmendez@ivic.ve; Arnelly Escalona - leeuwenhoek@hotmail.com; Omaira Da Mata - odamata78@yahoo.com;
Elix Izarra - elixjosei@yahoo.es; Yeimy Rojas - yeimyrojas@hotmail.com; Rossana Jaspe - rjaspe@ivic.ve;
Alifiya S Motiwala - ghadiaah@umdnj.edu; David Alland - allandda@umdnj.edu; Jacobus de Waard - jacobusdeward@gmail.com;
Howard E Takiff* - htakiff@ivic.ve
* Corresponding author

Published: 6 August 2009
BMC Infectious Diseases 2009, 9:122

doi:10.1186/1471-2334-9-122

Received: 4 January 2009
Accepted: 6 August 2009

This article is available from: http://www.biomedcentral.com/1471-2334/9/122
© 2009 Abadía et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Tuberculosis remains an endemic public health problem, but the ecology of the TB
strains prevalent, and their transmission, can vary by country and by region. We sought to
investigate the prevalence of Mycobacterium tuberculosis strains in different regions of Venezuela. A
previous study identified the most prevalent strains in Venezuela but did not show geographical
distribution nor identify clonal genotypes. To better understand local strain ecology, we used
spoligotyping to analyze 1298 M. tuberculosis strains isolated in Venezuela from 1997 to 2006,
predominantly from two large urban centers and two geographically distinct indigenous areas, and
then studied a subgroup with MIRU-VNTR 24 loci.
Results: The distribution of spoligotype families is similar to that previously reported for
Venezuela and other South American countries: LAM 53%, T 10%, Haarlem 5%, S 1.9%, X 1.2%,
Beijing 0.4%, and EAI 0.2%. The six most common shared types (SIT's 17, 93, 605, 42, 53, 20)
accounted for 49% of the isolates and were the most common in almost all regions, but only a
minority were clustered by MIRU-VNTR 24. One exception was the third most frequent overall,
SIT 605, which is the most common spoligotype in the state of Carabobo but infrequent in other
regions. MIRU-VNTR homogeneity suggests it is a clonal group of strains and was named the
"Carabobo" genotype. Epidemiologic comparisons showed that patients with SIT 17 were younger
and more likely to have had specimens positive for Acid Fast Bacilli on microscopy, and patients
with SIT 53 were older and more commonly smear negative. Female TB patients tended to be

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younger than male patients. Patients from the high incidence, indigenous population in Delta
Amacuro state were younger and had a nearly equal male:female distribution.
Conclusion: Six SIT's cause nearly half of the cases of tuberculosis in Venezuela and dominate in
nearly all regions. Strains with SIT 17, the most common pattern overall may be more actively
transmitted and SIT 53 strains may be less virulent and associated with reactivation of past
infections in older patients. In contrast to other common spoligotypes, strains with SIT 605 form
a clonal group centered in the state of Carabobo.

Background
There has evolved a genre of publications describing
aspects of the molecular epidemiology of tuberculosis in
varied populations of diverse countries [1-4]. While many
of the earlier studies were coupled with epidemiologic
investigation to identify routes and risk factors for transmission [5-8], most recent publications describe strain
variation and the dynamics in the local population structure of Mycobacterium tuberculosis. While this change
reflects an increasing interest in the phylogeny of M. tuberculosis and its correlation with factors underlying the
dynamics in the tuberculosis epidemic, it is also related to
the techniques used. Where IS6110 RFLP typing distinguishes between strains [9], spoligotyping [10] and
sequence polymorphisms [11-13], are suitable for large
studies on phylogeny and identification of genotype families. The more recent technique of minisatellite analysis
[14], MIRU-VNTR 24 loci, is less technically demanding
than the IS6110 RFLP typing technique, can discriminate
at strain level, and is also promising for use in phylogenetic studies [15]. However, this technique is laborious in
its manual form, and fairly expensive when 24 loci are
analyzed with an automated sequencer [16]. As spoligotyping is a robust, cheap, and easy-to-perform technique
for large scale studies, this is a pragmatic choice, which
can be complemented by the selective use of MIRU-VNTR
analysis with 24, 15, 12 or fewer loci [17] to discriminate
genotypes within clusters of strains showing identical spoligotypes.
In Venezuela, the average national incidence of tuberculosis is moderate, approximately 34 per 100,000 http://
www.who.int/globalatlas/predefinedreports/tb/
PPDF_Files/ven.pdf. However, the incidence is two to
three times higher in some isolated Amerindian populations, likely the result of prolonged deficiencies in control
programs, although improvements over the past few years
have made strains available for this study. A recent publication described the spoligotype patterns of M. tuberculosis
strains in Venezuela based on analysis of 670 isolates collected in a nationwide drug resistance survey [18]. While
this study identified the predominant spoligotypes in the
country, it did not include epidemiologic data, nor infor-

mation on where in Venezuela each strain was obtained.
Also, because it was based on data from spoligotyping
alone, it could not discriminate clonal clusters.
The Laboratorio de Tuberculosis of the Instituto de Biomedicina and the Laboratorio de Genética Molecular at
IVIC, both in Caracas, have been working in collaboration
with the National TB Control Program and regional labs
in the Amazonas, Carabobo and other states, to implement routine cultures to improve TB diagnosis and control. The M. tuberculosis isolates described herein were
collected from 1997 through 2006, and while this group
of strains could be described as a convenience sample, it
is likely to be representative of the country as a whole. It
includes large numbers of isolates from the two biggest
cities in the center of the country – Caracas and Valencia,
as well as from two rural, largely Amerindian populations
– Delta Amacuro and Amazonas, located at widely separated geographical poles, and also a smaller number of
isolates from other regions (Figure 1 and Additional file
1). Data on strains from the Amazonas [19], Carabobo
[20], and Delta Amacuro [21] states that have been
described separately but are included here in a broader,
national analysis.
The strains were first analyzed by spoligotyping, whereafter a subgroup of strains with the most prevalent spoligotypes were further analyzed with MIRU-VNTR 24 loci
typing. We show the geographic distribution of spoligotypes, including a large, geographically centered clonal
outbreak, and associate the spoligotype results with epidemiologic parameters to suggest that particular genotypes
of M. tuberculosis are emerging in Venezuela while others
may be disappearing.

Results
Clustering Analysis of Spoligotypes
A total of 300 different spoligotype patterns were recognized among the 1298 strains subjected to analysis, of
which 201 were unique patterns, and 99 were shared with
other isolates, constituting clusters (Additional file 2).
Thirty-two clusters comprised only two cases. Of the 300
patterns, 173 were not found in SpolDB4 [10], and of

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BMC Infectious Diseases 2009, 9:122

Valencia
467 isolates
2003- 2006

http://www.biomedcentral.com/1471-2334/9/122

Maracay
9 isolates
2005

Caracas
572 isolates
1998- 2006

Sucre
54 isolates
2004- 2006

Delta
Amacuro
84 isolates
1999- 2006

Apure
12 isolates
2005- 2006

Amazonas
100 isolates
1997- 2004

Figure 1
Map of Venezuela showing the geographic origin of isolates included in the study
Map of Venezuela showing the geographic origin of isolates included in the study.

these 32 were shared and 141 were unique types. Of the
127 spoligotypes that have counterparts in SpolDB4, 9
patterns have been found exclusively in Venezuela: ST
1696, belonging to the family LAM5, 21 isolates; ST 1702,
LAM5, 10 isolates; ST 375, LAM5, 7 isolates; ST 1692, ×1,
5 isolates; ST 1698, U, 3 isolates; and one isolate each of
ST 1104, T5; 1700, T1; ST 1711 LAM2; and ST 1718, X1LAM9.

The six most common spoligotypes (Figure 3) were
Shared Types (SIT's) 17, 93, 605, 42, 53 and 20, which
together accounted for 49% of the total isolates. The single most common pattern, SIT 17, was found in 242 isolates (18.6%). The most common spoligotypes overall
were also the most common in each region, with SIT 17
being the first or second most frequently isolated in all
regions studied.

The most common spoligotype families were LAM (53%),
T (10.6%), and Haarlem (5%), but 17.9% could not be
classified (Figure 2). Using the program SPOTCLUST to
predict the most likely family, a similar distribution was
obtained for the 32 cluster spoligotypes (91 isolates) not
found in SpolDB4 (Additional file 2).

Although SIT 605 is relatively uncommon in most
regions, it was the third most common spoligotype overall because the collection includes 467 strains (36% of the
total) from the state of Carabobo, where it is the most
common pattern. It is present in SpolDB4, but the only
two strains with SIT605 described outside of Venezuela

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X 1.2%
S 1.9%
Haarlem
5.0%

Beijing 0.4%
EAI 0.2%

U 9.7%

T 10.6%

NF 17.9%

http://www.biomedcentral.com/1471-2334/9/122

LAM 53.0%

FNDS 6.9%
LAM 53.0%
NF 17.9%
T 10.6%
U 9.7%
Haarlem 5.0%
S 1.9%
X 1.2%
Beijing 0.4%
EAI 0.2%

A

EAI 3.4%
S 6.5%
Haarlem
6.9%
T 16.4%

LAM 59.9%

LAM 59.9%
T 16.4%
Haarlem 6.9%
S 6.5%
EAI 3.4%
FNDS 6.9%

B

Figure 2
Distribution of Spoligotype Families
Distribution of Spoligotype Families. A. Distribution of Venezuelan M. tuberculosis biogeographic families defined by
SpolDB4. B. Distribution of Venezuelan M. tuberculosis spoligotypes absent in SpolDB4 and identified at a family level by SPOTCLUST. NF= Not found in SpolDB4. FNDS= Family not described in SpolDB4.
were isolated in New York in patients from Colombia (Jeffrey Driscoll, Natalia Kurepina and Barry Kreiswirth, personal communication), which shares a long border with
Venezuela.
In SpolDB4, SIT 605 is not clearly assigned to a major genotype family, but the absence of spacers 21 to 24 and 33
to 36 would place it in the LAM family, even though it also
lacks spacers 31 – 32 and 37 – 40. To confirm its designation as LAM, 45 chromosomal SNPs [13] were analyzed
for 23 strains, including 4 SIT 605, 4 SIT 17 and 4 SIT 93.
SIT 605 was found to belong to SCG v-ST7, as were the SIT
17, SIT 93 strains, as well as the other 11 strains with different LAM spoligotypes (data not shown). If SIT 605 is
classified as a LAM strain, this family would account for a
total of 60.2% of the isolates in this study.
MIRU-VNTR 24 Loci Analysis
To determine the genotype similarity of strains sharing
identical spoligotype patterns, we performed MIRU-VNTR
24 loci analysis on a subgroup of strains representing the
major spoligotype clusters (Figure 4). MIRU-VNTR confirmed the spoligotyping designation in all strains. Surprisingly, while only occasional strains with SIT 17 or SIT
93 shared MIRU-VNTR 24 patterns, all SIT 605 strains
were identical for at least 21 loci, even if the strains had
been isolated at distinct geographic regions, suggesting
that SIT 605 comprises a clonal group of strains. Even the
two SIT 605 strains isolated in New York also appeared to
be part of this clone, based on previous MIRU 12 results
(Jeffrey Driscoll personal communication). We propose

naming this strain the "Carabobo" genotype. Furthermore, ST 1698, whose spoligotype pattern differs from SIT
605 by the absence of one additional spacer, was found by
MIRU-VNTR 24 to belong to the SIT 605 "Carabobo" genotype.
Correlation with Epidemiologic Parameters
Epidemiologic data was not available for all of the
patients from whom the 1298 strains had been isolated,
but for several parameters the information was recorded
for at least 25% of the 1298 patients. Overall, of those
patients with available data, the mean age was 38 years,
68% were between 15 and 45 years of age, while 29%
were over 45, and just 3% were less than 15 years old.
Sixty nine percent were men, 74% had a positive clinical
specimens, 88% had pulmonary disease, and 75% had
cavitations according to the chest X-rays (data not
shown). Venezuelans made up 93% of patients and foreign-borns just 7%. The distribution of spoligotype patterns in isolates from the foreign born population did not
appear different from that of native Venezuelan patients.

Comparisons of patients in different regions revealed that
the average patient age was similar for the two urban areas
– 39 years for Carabobo and 37.8 years for Caracas – and
36.8 years for the Amazon region (Table 1). However it
was much younger, 32.6 years, for patients from the indigenous Delta Amacuro region. Also, while males made up
about 71% of patients from Caracas, 70% from Carabobo
(69%) [20], and 64% from Amazonas [19], males comprised only 54% of the patients from Delta Amacuro [21].

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Rank
1
2
3
4
5
6
7
8
9
10
11
12
13

SIT
17
93
605
42
53
20
50
1696
34
51
376
33
47

14

4

15
16
17

64
106
1702

18

NA

19
20
21
22
23

62
960
1691
162
194

24
25
26
27
28
29
30
31
32
33
34
35
36

291
375
1694
866
1
86
150
167
334
905
1692
1905
NA

Family
LAM2
LAM5
U
LAM9
T1
LAM1
Haarlem3
LAM5
S
T1
LAM3
LAM3
Haarlem1
LAM3- S
conver
LAM6
U?
LAM5
LAM2
*0.82
Haarlem1
LAM5
LAM2
LAM9
LAM2LAM4
T1
LAM5
LAM5
LAM9
Beijing
T1
LAM9
T1
T1
U
X1
T1
T1 *1,00

http://www.biomedcentral.com/1471-2334/9/122

Spoligotype
■■□■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■□■■■■■■■□□□□■■■■■■□□□□□□□□□□■■■
■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■
■■□■■■■■■■■■■■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□■□□□□■■■■■■■
■■■□□□■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■□□■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■□□□□
□■■■■■■■□□□■■■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■□□□■■■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■■■■■■□□□□□□■□□□□■■■■■■■

Total
242
129
93
85
53
35
22
21
20
19
16
13
13

Total
%
18,6
9,9
7,2
6,5
4,1
2,7
1,7
1,6
1,5
1,5
1,2
1
1

Cs
572
44
115
50
17
42
29
19
15
3
16
14
8
6
3

Cb
467
36
65
52
70
31
12
7
3
18
3
3
6
7
10

□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■■■□□□□■■■■■■■

12

0,9

11

10
10
10

0,8
0,8
0,8

5
2
3

10

0,8

■■■■■■■■■■■■■■■■■■■■■■■■■□□□□□□■□□□□■■■□■■■
■□□■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■□■■■■■■■■■□■□■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■■□□□□■■□■■■■
■■□■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■□■■■

9
9
9
8
8

0,7
0,7
0,7
0,6
0,6

■■■■■■■■■■■■■■■■■■■■□■■■■■■■■■■■□□□□■■■■■■■
□□□■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
□■□■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■□■■■■■■■■■■■■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■
□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■■■■
■■■■■■■■■■■■■■■■■■■■■□■■■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■□■■■■■□□□□■■■■■■■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□■■□□□□■■■■■■■
■□■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■
■■□■■■■■■■■■□■■■■■■■□□□□■■■■■■□□□□□□■■■■■■■
■□□□□■■■■□□■□■■■■□■■■■■■■■■■■■■■□□□□■■■□■■■
■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□■■□□□□■■■■■■■
■■■■■■■■■■■■■■■■■■■■□■■■■■■■■■■■□□□□■■□■■■■

7
7
7
6
5
5
5
5
5
5
5
5
5

0,5
0,5
0,5
0,5
0,4
0,4
0,4
0,4
0,4
0,4
0,4
0,4
0,4

Su
54
4,2
7
9
4
5
1

Ap
12
0,9
3
3

Ar
9
0,7
2
1
1
1

1
1

1

1
1

1

■■□■■■■■■■■■□■■■■■■■□□□□■■■■■□□■□□□□■■■■■□□

4
9
5
1

Am
100
7,7
27
7
1
2
1
3
2

1
2

1

■■■■■■■■■■■■■■■■■■■■□□□□■■■■□■■■□□□□■■■■■■■
■■■■■■■■□□□■■■■■■■■□□□□□□□□□□□□□□□□□□□■■■■■
□□□□■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■

Da
84
6,5
23
7

1

7
2

2
9
4
2
7
5
5
5
5

3

8
8

2

5

5
2
2

6
4

2
1

1

1
5

1
3
5
1
2

4
2
3

1

3
5
5

Figure 3
Geographic Distribution of the Most Common Spoligotypes in Venezuela
Geographic Distribution of the Most Common Spoligotypes in Venezuela. Spoligotypes found in at least 5 isolates,
ranked by number of isolates, with SIT, Spoligotype family (SpoDB4), geographic distribution, and percentage of total strains
(1298). Cs = Caracas, Cb= Carabobo State, Da = Delta Amacuro State, Am = Amazonas State; Suc = Sucre State, Ap = Apure
State, and Ar = Aragua State. For spoligotypes not represented in SpolDB4, the "*" next to the family designation indicates the
probability of belonging to that family as determined by the program SPOTCLUST.

Surprisingly, female patients had significantly lower mean
ages overall, and also in Caracas and in Delta Amacuro
(Table 1).
There were no significant differences when all patients in
clusters were compared with all patients with non-clustered isolates. However, Table 2 shows that patients
whose isolates had the most common spoligotype, SIT 17,
were significantly younger compared to patients with
other spoligotype strains (mean ages 34 vs 39 years, p =
0.0008). The SIT 17 patients also had lower mean ages
when the comparisons were stratified for each of the three
regions with the most isolates, but the differences did not

quite reach statistical significance (Caracas 35 vs 38.4, p =
0.077; Carabobo 34.5 vs 39.8 p = 0.076; and Amazonas
30.6 vs 39.1, p = 0.056). In contrast, patients with SIT 53
had a significantly higher mean age compared to all other
patients (48.2 vs 37.6, p = 0.001), or compared within the
Caracas cohort (50.25 vs 37.1, p = 0.0002), the source of
most SIT 53 patients with epidemiologic data. The mean
ages for patients in the other large clusters weren't significantly different from the overall group (data not shown).
As seen in Table 3, patients with SIT 17 isolates were also
more likely to have had clinical specimens positive for
Acid Fast Bacilli (AFB) by microscopy when compared

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Spoligotype

24 MIRU- VNTR
ST

spoligo+mvla

Lineage

Strain Code

4348

4156

4052

3690

3192

3171

3007

2996

2687

2531

2461

2401

2347

2165

2163b

2059

1955

1644

960

802

580

577

424

154

43

41

42

40

39

38

37

36

34

35

33

32

31

30

29

27

28

26

25

24

23

22

20

21

19

18

17

16

15

13

14

12

11

10

9

8

mvla

7

5

6

4

3

2

1

100

spoligo

90

80

70

mlv a-spoligo

2

3

4

2

4

3

3

2

2

3

2

4

2

2

5

1

5

3

2

3

3

6

2

2

.53

T1

14_0

2

2

4

2

4

3

3

2

2

5

3

4

2

2

5

1

5

3

3

3

2

5

2

2

.53

T1

156

T1

15_0

2

2

4

2

2

3

4

2

2

4

2

4

2

2

5

1

5

3

3

3

3

5

2

2

.53

2

2

4

2

2

3

4

2

2

4

2

4

2

2

5

1

5

3

3

3

3

5

2

2

.53

T1

16H1

T1

31I7

2

2

4

2

2

3

4

2

2

4

2

4

2

2

5

1

5

3

3

3

3

4

2

2

.53

2

2

4

2

2

3

4

2

2

4

2

4

2

2

5

1

5

3

3

5

3

4

2

2

.53

T1

14A8

2

2

4

2

1

3

3

2

2

3

3

4

2

2

6

1

5

3

3

3

4

4

2

2

.53

T1

32I6

2

4

3

2

5

3

3

3

2

4

3

4

4

2

5

1

5

2

3

3

3

8

3

2

.53

T1

6G3

2

4

4

2

1

4

2

3

2

4

2

4

1

1

6

1

5

3

5

3

2

1

2

2

.42

LAM9

11_0

LAM9

15A5

2

5

4

2

1

4

2

3

2

4

2

4

1

1

6

1

5

3

5

3

2

1

2

2

.42

2

4

4

2

1

4

2

3

2

2

2

4

1

2

6

1

4

3

3

2

2

1

2

2

.42

LAM9

19C2

LAM2

96_0

2

3

4

2

1

4

2

3

2

3

2

4

1

2

6

1

5

3

3

3

2

1

2

2

.17

2

4

4

2

1

4

2

3

2

3

2

4

1

2

6

1

5

3

3

3

2

2

2

2

.17

LAM2

33

2

4

4

2

1

4

2

3

2

3

2

4

1

2

6

1

6

3

3

3

2

3

2

2

.17

LAM2

11E_

2

4

4

2

1

4

2

3

2

3

2

4

1

2

6

1

6

3

3

3

2

3

2

2

.17

LAM2

11G3

2

4

4

2

1

4

2

3

2

3

1

4

1

2

6

1

6

3

3

3

2

3

2

2

.17

LAM2

31D6

LAM2

14E_

2

4

4

2

1

3

2

3

2

3

2

4

1

2

6

1

6

3

3

3

2

3

2

2

.17

2

4

4

2

1

3

2

3

2

3

2

4

1

2

6

1

6

3

3

3

2

3

2

2

.17

LAM2

37

LAM2

10

2

3

4

2

1

5

2

3

2

3

1

4

1

2

5

1

7

3

3

2

2

3

2

2

.17

2

4

4

2

1

4

2

3

2

1

1

3

1

2

6

1

6

3

2

2

2

1

1

2

.53

T1

14F1

1

3

4

2

1

4

3

3

2

2

2

4

1

2

6

1

5

3

3

3

2

5

3

2

.42

LAM9

12D2

1

3

4

2

0

3

2

3

2

2

2

4

1

2

6

1

5

3

3

4

2

6

2

2

.42

LAM9

142-0

3

3

4

2

1

5

2

3

2

2

2

4

1

1

6

1

4

3

3

3

2

5

3

2

.42

LAM9

4088

2

4

4

2

1

4

2

3

2

3

2

4

1

1

6

1

4

3

5

3

2

5

2

2

.93

LAM5

14C2

2

4

4

2

1

4

2

3

2

3

2

4

1

1

6

1

4

3

5

3

2

5

2

2

.93

LAM5

14H1

LAM5

19C5

2

4

4

2

1

4

2

3

2

3

2

4

1

1

6

1

4

3

5

3

2

5

2

2

.93

2

4

4

2

1

4

2

3

2

3

2

4

1

1

6

1

4

3

5

3

2

6

2

2

.93

LAM5

14A3

LAM5

32C1

2

4

4

2

1

4

2

3

2

3

2

4

1

1

6

1

4

3

5

3

2

6

2

2

.93

2

4

4

2

1

4

2

2

2

3

2

4

1

1

6

1

4

3

5

3

2

5

2

2

.93

LAM5

18C1

2

4

4

2

1

4

2

3

2

2

2

4

1

1

6

1

4

3

5

3

2

6

2

2

.93

LAM5

11F7

2

4

4

2

1

3

2

3

2

3

2

4

1

1

6

1

4

3

5

3

2

6

2

2

.93

LAM5

40_0

2

4

4

2

1

4

2

3

1

3

2

4

1

1

6

1

5

3

5

3

2

6

2

2

.93

LAM5

33B7

2

4

4

2

1

4

2

3

2

4

2

4

1

1

6

1

4

2

5

3

2

6

2

2

.93

LAM5

14C8

1

4

4

2

1

4

2

3

2

3

2

4

1

1

6

1

3

3

5

2

2

6

2

2

.17

LAM2

49_0

LAM9

13H3

2

2

4

2

1

4

2

3

2

4

2

4

1

1

6

1

4

3

5

3

2

6

2

2

.42

2

4

4

2

1

4

2

3

2

4

2

4

1

2

6

1

7

3

3

3

2

9

2

2

.17

LAM2

25C7

2

4

3

2

1

4

2

3

2

4

2

4

1

2

6

1

7

3

3

2

2

8

2

2

.17

LAM2

34F1

2

4

4

2

1

4

2

3

2

4

2

4

1

2

6

1

6

3

4

2

2

8

2

2

.17

LAM2

34F9

2

4

4

2

1

5

2

3

2

3

1

4

1

2

5

1

7

3

3

2

2

8

2

2

.17

LAM2

31I4

2

2

4

2

1

4

2

3

2

4

2

4

1

2

5

1

5

3

3

2

2

8

2

2

.17

LAM2

11F4

2

3

4

2

1

4

2

3

2

4

2

4

1

2

6

1

5

3

3

3

2

6

2

2

.17

LAM2

13C7

LAM2

39_0

2

3

4

2

1

4

2

3

2

4

2

4

1

2

6

1

5

3

3

3

2

5

2

2

.17

2

3

4

2

1

4

1

3

2

4

2

4

1

2

6

1

6

3

3

3

2

6

2

2

.17

LAM2

72_0

LAM2

7_06

2

3

4

2

1

4

1

3

2

3

2

4

1

2

6

1

6

3

3

3

2

6

2

2

.17

2

3

4

2

1

4

2

3

2

3

2

4

1

2

6

1

5

3

3

3

2

7

2

2

.17

LAM2

21D5

2

3

4

2

1

4

2

3

2

3

2

4

1

2

6

1

5

3

3

3

2

7

2

2

.17

LAM2

33A1

2

3

4

2

1

4

2

3

2

3

2

4

1

2

6

1

5

3

3

3

2

6

2

2

.17

LAM2

35C9

2

5

5

2

1

4

2

3

2

2

2

4

1

2

6

1

5

3

3

3

2

6

2

2

.17

LAM2

10_0

LAM2

29F3

2

4

4

2

1

4

2

3

2

1

2

4

1

2

6

1

6

3

3

3

2

8

2

2

.17

2

4

4

2

3

4

2

2

2

3

2

4

1

2

6

1

3

3

3

3

2

8

2

2

.17

LAM2

34E_

LAM9

20C1

2

4

7

2

1

2

1

3

2

4

2

4

1

1

6

1

4

2

5

3

2

6

2

2

.42

2

4

2

2

1

4

2

3

2

4

2

4

2

2

6

1

5

3

1

3

1

7

2

2

.42

LAM9

20B6

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

1

2

2

.605

LAM

78_0

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

1

2

2

.605

LAM

96_0

1

2

5

2

6

4

3

3

2

2

1

4

1

2

6

1

5

3

3

2

2

2

2

2

.605

LAM

45_0

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

3

3

3

2

2

1

2

2

.605

LAM

41_0

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.1698

LAM

110_

LAM

158-0

2

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.1698

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

LAM

115_

LAM

130_

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

LAM

13G6

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

LAM

144_

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

LAM

151_

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

LAM

32F4

LAM

49_0

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

1

2

5

2

6

4

3

3

2

2

2

4

1

2

6

1

5

3

3

2

2

6

2

2

.605

LAM

77_0

2

.605

LAM

USA

2

.605

LAM

20F5

1
1

2
2

5

6

4

3

2

1

4

3

2
3

2

6
2

2

4

1

2

1

5

3

6

1

5

3

2
3

2

2

6

2

Figure 4
Dendrogram of Venezuelan M. tuberculosis strains
Dendrogram of Venezuelan M. tuberculosis strains. Dendrogram combining MIRU-VNTR and Spoligotype results from
representative strains generated by Bionumerics software (Applied Maths) using the unweighted pair group method of averages (UPGMA). MIRU patterns with 3 or fewer changes are considered clonal. Inside the box are the SIT 605 strains defined as
clonal by spoligo and by 24 MIRU- VNTR loci. Only MIRU 12 results were available for the strains of SIT 605 isolated in New
York.
overall, or just within the Caracas cohort, but the differences were only significant compared to other Caracas
patients (SIT 17 vs non-SIT 17, all patients: OR = 1.47, p
= 0.14; Caracas: OR = 1.96, p = 0.04). Within the Caracas
group, specimens from patients with the SIT 53 spoligo-

type were less likely be AFB positive, but the difference
was not statistically significant (OR = 0.68, p = 0.55).
However, there were only 20 Caracas SIT 53 patients with
AFB microscopy results. The difference was more marked
when AFB positivity was compared between Caracas
Page 6 of 12
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BMC Infectious Diseases 2009, 9:122

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Table 1: Comparison of mean ages of TB patients whose isolates were included in this study

Overall
N° of individuals
Individuals with age data
Mean age
(years)
N° of females
% Females
Female with
age data
Mean Female age
(years)
N° of males
% Males
Males with
age data
Mean Male Age
Difference M/F
Mean age
p value

Amazonas

Caracas

Carabobo

Delta Amacuro

Sucre

Apure

1298
766
38.0
(37–39)
304
31
241

100
100
36.8
(33–41)
36
36
36

572
410
37.8
(36–39)
151
29
118

467
212
40.0
(37–41)
66
30
66

84
22
31.2
(23–39)
33
46
13

54
15
44.7
(34–55)
11
24
6

12
7
42.6
(27–58)
4
33
2

36.2
(34–38)
670

38.5
(32–45)
64

35.1
(32–38)
373

37.6
(33–42)
157

24.5
(15–34)
38

48.8
(30–67)
34

8

525

64

292

156

9

9

5

38.8
(37–40)
0.05

35.8
(31–41)
0.52

38.8
(37–41)
0.03

39.6
(37–42)
0.39

40.9
(28–54)
0.05

42.0
(30–54)
0.54

The mean ages are shown for the entire group and also separated by region and gender. In each category, the age was available only for the number
of patients indicated with age data. Confidence intervals are shown in parentheses. Determination of mean ages, confidence intervals and statistical
significance using the Student's two -tailed T test assuming equal variance were performed within Excel. Statistically significant p values are shown in
bold.

patients with SIT 17 and SIT 53, but still failed to reach
statistical significance (OR = 2.44, p = 0.14).

Discussion
We have used Spoligotyping to analyze M. tuberculosis
strains isolated in different parts of Venezuela, and while
the collection could be described as a convenience sample, without proportionate geographic or temporal distribution, it likely reflects the prevalence of strains in the
entire country, as the spoligotypes are generally the same
as those found in a previous study based on a proportional nationwide sampling to determine prevalence of
drug resistance [18]. There are a total of approximately
6500 cases of tuberculosis reported per year in Venezuela,
so for the ten years during which strains were collected,
the sample represents only ~2% of registered cases, but
5% for Caracas and a larger percentage for Valencia and
the Carabobo state of which it is the capital.
As in other studies in South America [18,22,23] the LAM
family predominates, accounting for 53% of all strains,
and 60% if, as suggested by the SNP results, SIT 605 is also
considered LAM. Although 300 different spoligotype patterns were found, only 6 accounted for almost 50% of the
isolates, and these patterns were the most common in
nearly all the regions sampled. In contrast, 201 patterns
were found in only one isolate, and 173 (57.7%) of these
were not present in SpolDB4. Nine of the 127 patterns
that are represented in SpolDB4 have been found exclusively in Venezuela, and a tenth, SIT 605, has been isolated outside of Venezuela only in New York, in two

immigrants from the neighboring country of Colombia.
This last spoligotype, SIT 605, is perhaps the most surprising finding in this study, because unlike the other common spoligotypes such as SIT 17 and 93, which contain a
number of distinct genotypes when analyzed by MIRUVNTR 24 loci, SIT 605 appears to represent a large clonal
cluster that is geographically centered around the city of
Valencia and the state of Carabobo [20], but encompasses
almost all the SIT 605 strains examined, including the two
isolated in New York. The previous study [18] of the
molecular epidemiology of M. tuberculosis strains in Venezuela found several isolates with SIT 605, but as that study
was based on spoligotyping without geographic data, the
focal and clonal nature of this genotype could not have
been detected. We propose that the SIT 605 strains be
termed the "Carabobo" genotype.
Phylogeographic analysis of spoligotypes have identified
a number of regionally originating patterns or clades
whose isolates have similar patterns with RFLP IS6110
and appear to represent strains with a common lineage.
The most notorious example is SIT 1, or Beijing, which
presumably originated in Asia and has spread widely [24].
Other examples are the SIT 33/F11/LAM clade [25] first
described in South Africa, the SIT 60/F15/LAM4 KZN
clade associated with XDR-TB in Kwazulu Natal, South
Africa [26], the SIT 61/LAM10 Cameroon clade, SIT 59/
LAM11/ZWE/MERU, SIT 21/CAS1-Kili, SIT 26/CAS1Dehli, SIT 41/LAM7-Tur, and SIT 19/Manila/EAI2
[10,27]. These clades are not homogeneous, but include
several related MIRU-VNTR and RFLP IS6110 patterns,

Page 7 of 12
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BMC Infectious Diseases 2009, 9:122

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Table 2: Comparison of mean ages of TB patients with SIT 17 or SIT 53 spoligotype patterns

Overall

Caracas

Carabobo

Amazonas

Delta Amacuro

Sucre

Apure

N° of isolates
% with age
Mean age

1298
59
37.6
(36–39)

572
72
37.8
(36–39)

467
45
39.0
(37–41)

100
100
36.8
(33–41)

84
25
32.6
(24–41)

54
28
44.7
(34–55)

12
58
42.6
(27–58)

SIT 17
% with age
Mean age

242
64
34
(32–36)

115
66
35
(32–38)

65
52
34
(30–39)

27
100
31
(24–37)

Non SIT 17
% with age
Mean age

1056
58
39
(38–40)
P = 0.0008

457
73
38
(37–40)
P = 0.077

402
44
40
(37–42)
P = 0.076

73
100
39
(34–44)
P = 0.056

SIT 53
% with age
Mean age

53
42
48
(42–57)

29
69
50
(42–58)

Non SIT 53
% with age
Mean age

1245
60
38
(36–39)
P = 0.001
1004
59
38.63
(37–40)
P = 0.003

544
72
37
(36–39)
P = 0.0002
413
76
37.7
(36–39)
P = 0.0005

17 vs non 17

53 vs non 53
Non 53 non 17
% with age
Mean age
53 vs non 53 non 17

The table shows the mean age of all patients with age data in the study as a single cohort and also divided by region. Statistical comparisons are
shown between patients with SIT 17 and patients with other spoligotypes and, separately, between SIT 53 and other spoligotypes. Confidence
intervals are shown in parentheses. Mean values, confidence intervals, and P values with the Student's two-tailed T test assuming equal variance
were performed within Excel. Statistically significant p values are shown in bold.

some of which may comprise more localized clonal clusters. However, the fact that these spoligotype families
have a wide distribution, and some are associated with
MDR outbreaks, suggests that they have selective advantages over other strains in their ability to cause disease and
be transmitted. It appears that SIT 17, and perhaps SIT 93,
might similarly be considered successful clades, as they
are responsible for 18.6% and 9.9%, respectively, of
tuberculosis cases in Venezuela, and have been found in
several other countries.
There are also reports of spoligotype patterns that are geographically limited and appear to be clonal by MIRU/
RFLP analyses, such as the SIT 2643 Haarlem3 strain in
Paraguay [22], but the number of isolates of these clonal
genotypes is generally small. An examination of SpolDB4
[10] shows several SIT's with more than 10 isolates limited to one country, or with a single additional isolate in
a separate country, perhaps from an emigrant (for exam-

ple, SIT's 210, 339, 1258, 1329, 1457, 1518, and 1898).
Similarly, in Additional file 2 there are, besides SIT 605,
other spoligo patterns that could represent clonal strains
because they are found only in particular regions of Venezuela, for example the putative LAM2 strain in position 18
(Figure 3). Several of these are currently being analyzed by
MIRU-24 loci. However, it is striking that the SIT 605/SIT
1698 Carabobo genotype is the most common spoligotype in a state with a population greater than 2 million,
accounting for 15% of all isolates, while it is relatively
uncommon in the other regions we sampled. However, of
only nine isolates obtained from the state of Aragua,
which borders Carabobo, one was SIT 605, so it is possible that this strain is also common in adjacent areas. As
the spoligotype for the Carabobo strain evolved with the
loss of spacers 31–32 and 37 – 40, other elements of the
genome could have, independently, evolved to gain an
unknown selective advantage over other local M. tuberculosis genotypes, but unlike SIT 17, there were no epidemi-

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Table 3: Comparison of specimens whose cultures grew isolates with SIT 17 or SIT 53 for percentage that were positive for Acid Fast
Bacilli by microscopy

All

Total
% with
data
Positive
Negative
% positive
Comparison
Odds ratio

All w/o
SIT 17

All SIT 17

CCS all

CCS w/o SIT 17

CCS SIT 17

CCS w/o SIT 53

CCS SIT
53

1298
48

1054
48

244
50

572
53

457
53

115
51

543
52

29
69

463
164
74

366
139
72
All:
17 vs non 17
OR= 1.47
(0.91–2.43)
P = 0.14

97
25
80

178
125
59
CCS:
17 vs non 17
OR= 1.96
(1.06–3.7)
P = 0.04

136
108
56

42
17
71
CCS:
53 vs non 53
OR= 1.46
(0.57–3.7)
P = 0.55

168
115
59

10
10
50
CCS:
17 vs 53
OR= 2.44
(0.84–7.11)
P = 0.14

Comparisons were made based on the patients with microscopy data. The percentage of specimens that were AFB positive was compared between
those that grew strains with either spoligotype SIT 17 or SIT 53, and those from which strains with all other patterns were isolated. Comparisons
were made for all specimens in the study, and separately, for those obtained in Caracas (CCS). Two by two table comparisons with odds ratios and
Yates corrected two-tail chi square values were calculated using OpenEpi http://www.openepi.com. Statistically significant p values are shown in
bold

ologic correlations suggesting higher virulence. At least six
SIT 605 patients were present or former inmates in a particular prison in Carabobo state, and it appears that other
SIT 605 patients had contact with people that were
interned in that prison, so this institution could play a
role in its local dissemination, as described for other
prison-associated clonal outbreaks [28,22,29].
Although at least one SIT 605 isolate was found to be
Multi-Drug Resistant [18], the great majority of SIT 605
isolates were pan-sensitive. Our study found only a very
few Beijing isolates, some of them lethal MDR strains
(National Tuberculosis Program, unpublished data), but
fortunately they do not appear to have been transmitted
extensively within the Venezuelan population [30]. At
least one of the Beijing strains was isolated from a Peruvian national [31].
The comparisons of epidemiologic and clinical parameters revealed that in the indigenous Warao population in
Delta Amacuro [21] tuberculosis occurs at younger ages
(mean of 32.6 years) than in the rest of the population
(mean of 38–39 years), and unlike the 71% male predominance in the cities, there is a nearly equal male:female
distribution (54%). This pattern is consistent with very
active spread within this isolated Amerindian community
[21] that has the highest TB incidence in Venezuela and
amongst the lowest life expectancies. The data from the
largely Amerindian tuberculosis population in the Amazonas states [19] suggests a similar, but less marked trend,
with an average age of 36.8 that is 64% male. More
polemical are the findings suggesting that SIT 17 may be
more actively transmitted because patients with isolates
belonging to the SIT 17 cluster tended to be younger and
were more likely to have AFB positive specimens. Low

patient age is considered characteristic of strains being
actively transmitted [32]. In contrast, SIT 53 may be less
virulent because patients with this spoligotype tended to
be older and may have more AFB negative sputa, but the
AFB trend did not reach statistical significance, and the
older age, while reaching statistical significance, was
based on only 21 SIT 53 patients and needs to be confirmed in subsequent studies.
These associations provoke a number of questions. If SIT
53 were really less virulent, why is it still the sixth most
common spoligotype, causing 4% (53) of cases? Could it
have been a very common strain in the past, that is now
more apt at latency and reactivation than person-to-person transmission, and will its prevalence decrease over
time? SIT 93 is the second most common spoligotype,
found in 10% of all isolates, but why isn't it also associated with clinical parameters suggesting virulence and
transmission? Finally, preliminary MIRU-VNTR analysis
suggests that all of the most common spoligotype clusters,
except for SIT 605, contain several different strain genotypes. Therefore, for the epidemiologic associations with
SIT 17 or SIT 53 to make sense, it must be assumed that
the strains with these spoligotypes derived from a common ancestor with genetic characteristics that were maintained even as the MIRU-VNTR patterns evolved, as seems
true for globally dispersed lineages such as Beijing.
Finally, it must be recalled that clinical data was not available for many of the patients whose isolates comprised
this study, so although all data were collected before spoligotyping results were known, the epidemiologic associations with SIT 17 could be subject to biases due to regional
differences in patient admission or data recording. We
attempted to reduce this possibility through regional stratification, and found that associations for SIT 17 persisted,

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although not always reaching statistical significance. If
subsequent studies with more complete data confirm that
the SIT 17 or SIT 53 spoligotypes, or the SIT 605 Carabobo
genotype, are associated with particular clinical disease
characteristics, the challenge will be to identify the molecular basis for these apparent differences.

for Acid Fast Bacilli by microscopy were cultured, but
some regions, such as Carabobo, also cultured specimens
from patients that were symptomatic and had abnormal
chest X-rays or were otherwise highly suspicious for having TB. In Caracas all specimens from patients with respiratory symptoms for more than two weeks were cultured.

Conclusion

Spoligotyping
Spoligotyping was performed as previously described by
Kamerbeek [34]. The spacer sequences contained in the
direct repeat locus were detected by hybridization onto
either homemade or commercially prepared spoligotyping membranes. DNAs from M. tuberculosis H37Rv and M.
bovis BCG were used as positive controls, and spoligotyping was repeated until results were unambiguous. Each
spoligotype was converted to octal format [35] and compared with the International Spoligo Database, SpolDB4
[10]. SPOTCLUST, an on-line program developed by Vitol
et al. [36], was used to tentatively assign phylogeographic
families to spoligotype patterns not present in SpolDB4.

Six spoligotypes accounted for 49% of the 1298 M. tuberculosis Venezuelan isolates analyzed, and the most common overall were the most common in all regions
examined. The patients with isolates showing spoligotype
SIT 17 tended to be younger and more likely to have had
AFB positive specimens, suggesting that these strains are
being actively transmitted. In contrast, SIT 53 may be
more common in cases of reactivation, as the patients
with these strains tended to be older and may be less likely
to be AFB positive. MIRU-VNTR 24 showed that only a
minority of strains in the most common spoligotypes
were clustered, with the exception of SIT 605 strains,
which appear to be clonal. SIT 605 was named the "Carabobo" genotype because it is the most common spoligotype in this state, but infrequently seen in other regions of
Venezuela. The only two strains with SIT 605 described
outside of Venezuela were isolated in New York in
patients from Colombia, and also belong to the SIT 605
cluster. Overall, female patients had lower mean ages than
male patients. Tuberculosis patients in the Amerindian
Warao population in the state of Delta Amacuro had a
lower mean age and a more equal male:female distribution, possibly reflecting the active transmission that gives
this population the highest incidence of tuberculosis in
the country.

Patient data, including age, sex, nationality, HIV status,
AFB positivity of the original clinical specimen, treatment
history, and chest X-ray information were included, when
available, in the spreadsheets that contained the spoligotyping results.
MIRU-VNTR Genotyping
Twenty four loci were amplified using multiplex PCR and
run with Rox labeled Map Marker 1000 size standards
(Bioventures) on ABI 3130 xl sequencers as described
[14,16,37,38]. Sizing of the PCR fragments and assignments of MIRU-VNTR alleles were done with the Gene
Mapper software package (PE Applied Biosystems).

Methods
M. tuberculosis strains
The 1298 clinical isolates in this report were obtained
from an equal number of different patients between 1997
and 2006 (Additional file 1) as part of an ongoing study
of M. tuberculosis strains from different geographic regions
of Venezuela (Figure 1). They are largely from two large
urban regions, Caracas (pop. > 5 million), the national
capital (n = 572), and Valencia (pop. >1.5 million), capital of the state of Carabobo (Pop. > 2.2 million) (n = 467),
and two relatively isolated states with large and distinct
Amerindian populations, Delta Amacuro – where the
Warao inhabit the wide delta of the Orinoco River (n =
84) and Amazonas (n = 100), home to many Amerindian
ethnicities. Smaller numbers of strains were also obtained
from the states of Apure (n =12), Aragua (n = 9) and Sucre
(n = 54). The isolates were obtained by culturing sputa or
non-pulmonary clinical specimens using either the Petroff
method with Löwenstein – Jensen slants or the Kudoh
method with Ogawa slants [33] The criteria for culturing
clinical specimens varied by region. All that were positive

Cluster analysis
Spoligotyping and MIRU-VNTR profiles were also
recorded as character data and analyzed using Bionumerics software (Applied Maths) or MIRU-VNTR plus, an on
line program developed by Allix-Béguec et al [39]. Dendrograms were generated by using the categorical character option and the unweighted pair group method of
averages (UPGMA) clustering method.
SNP Analysis
A subset of strains was analyzed for a set of 45 informative
SNPs as described [13].
Statistical analysis of epidemiologic data
The clinical and epidemiologic characteristics of patients
belonging to each of the six most common spoligotype
clusters were compared between these clusters and also
against all patients not in the analyzed cluster. Comparisons of sputum positivity were done as two by two tables
using the Yates corrected two-tail chi-square test on the

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OpenEpi website http://www.openepi.com. Mean age calculations, confidence intervals and age comparisons with
the two-tailed T Test, assuming equal variance, were performed within Excel. A p value of < 0.05 was considered
statistically significant.

SIT 605 strains isolated in New York; Drs. Hermes Piñango and Alexander
Barrios for help with phlyogenetic and statistical analyses; the Caracas and
National Tuberculosis Programs, especially Dr. Beatriz Fernandez, Dr. Mercedes España and Dr. Alexis Guilarte for support and collaboration
throughout this study; and Dr. Dick van Soolingen for critically reading the
manuscript.

Competing interests

References

The authors declare that they have no competing interests.

1.

Authors' contributions
EA performed and analyzed spoligotyping, analyzed epidemiologic data and helped draft the manuscript; MS isolated strains, collected epidemiologic data, and performed
spoligotyping; DO isolated strains and collected epidemiologic data; MVM performed and analyzed MIRU-VNTR24; AE and ODM isolated strains and performed spoligotyping; YR and RJ isolated strains, performed and analyzed spoligotyping; ASM and DA performed and
analyzed SNP studies; EI helped with the statistical analysis of the epidemiologic data; JdW directed strain isolation
and spoligotyping and helped to conceive the study; HET
conceived and directed the study, analyzed the data and
wrote the manuscript.

Additional material

2.

3.

4.

5.

6.

Additional file 1
Distribution of strains included in the study by year and region of isolation. Cs = Caracas, Cb = Carabobo State, Da = Delta Amacuro State,
Am = Amazonas State; Suc = Sucre State, Ap = Apure State, and Ar =
Aragua State.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712334-9-122-S1.doc]

Additional file 2
All Spoligotypes found in the study. Spoligotypes found in the study,
ranked by number of isolates, with SIT, Spoligotype family (SpoDB4),
geographic distribution, and percentage of total (1298). Cs = Caracas, Cb
= Carabobo State, Da = Delta Amacuro, Am = Amazonas State; Suc =
Sucre, Ap = Apure, and Ar = Aragua state. The "*" next to a family designation indicates the probability of belonging to that family as determined by the program SPOTCLUST.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712334-9-122-S2.doc]

7.

8.

9.
10.

11.

12.

Acknowledgements

13.

This work was funded by FONACIT Proyecto G-2005000393, FONACIT
Agenda de Salud Proyecto 20010001851 and LOCTI project "Las Cepas de
Tuberculosis Mas Virulentas de Venezuela".
The authors thank: Dr. Philip Supply for generous help and support setting
up the MIRU-VNTR technique; Drs. Nalin Rastogi and Christophe Sola for
help in setting up spoligotyping and membrane preparation; Drs. Jeffrey
Driscoll, Natalia Kurepina and Barry Kreiswirth for unpublished data on the

14.

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