Infection, Genetics and Evolution 77 (2020) 104059

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Short communication

Genetic diversity of Mycobacterium tuberculosis clinical isolates from HIV-TB T

patients from two public hospitals at Bogotá, Colombia
Magda Beltrán-Leóna, Juan Germán Rodríguez-Castilloa, Thierry Zoziob, Nalin Rastogib,
⁎
Martha I Murciaa,
a
Universidad Nacional de Colombia, Facultad de Medicina, Departamento de Microbiología, Grupo MICOBAC-UN, Colombia
b
WHO Supranational TB Reference Laboratory, TB and Mycobacteria Unit, Institut Pasteur de la Guadeloupe, Guadeloupe, France

A R T I C LE I N FO A B S T R A C T

Keywords: The co-infection of TB/HIV is an increasing problem for public health worldwide. In Colombia, of 13.871
HIV-TB confirmed cases of TB in 2016 (prevalence of 0,028%) 14% correspond to HIV co-infection. However, we have
Genetic diversity scarce information regarding genetic diversity of strains infecting HIV patients. In this study, we carried-out an
Mycobacterium tuberculosis active search of cases of TB in 356 HIV-infected individuals, who were enrolled in two Public Hospitals at
Bogotá-Colombia
Bogotá-Colombia, between 2014 and 2015. We found 49 patients with HIV-TB co-infection. Genetic char-
acterization of Mycobacterium tuberculosis (Mtb) isolates from these patients showed a predominance of three
major sub-lineages: Haarlem (n = 26), LAM (n = 12) and T (n = 11). Remarkably, the most predominant pattern
in the present study (SIT62/H1, n = 11) is very specific to this country. Indeed, taking in account distribution in
countries with at least 3% of SIT62/H1, 36% of all such patterns collected worldwide were from Colombia.
Furthermore, Colombia alone is responsible for almost all the SIT62/H1 strains in South America, suggesting a
successful transmission of this genotype inside TB/HIV population from Colombia.

1. Introduction
Human immunodeficiency virus (HIV) infection has a key role in the
susceptibility to various acquired infections, mainly tuberculosis.
Indeed, the risk to be infected and diseased by tuberculosis (TB) in-
creases by 20–37-fold in HIV infected individuals, unlike non-HIV-in-
fected individuals who have a risk of 5–10% after infection (Getahun
et al., 2010). Of the 10.4 million incident cases of TB in 2016, an es-
timated 1.0 million were attributable to HIV infection, becoming a
worldwide concern (WHO. Global TB Report, 2018). In Colombia,
13,871 cases of tuberculosis were reported to the Public Health Sur-
veillance System (Sivigila) in 2016; out of which, 14.0% (1939 cases)
corresponded to coinfection with the HIV virus, meanwhile the re-
corded incidence was 25.7 cases per 100,000 inhabitants (López-Perez,
2016).
Genetic diversity of Mtb infecting TB-HIV patients is scarcely stu-
died in Colombia (Castro et al., 2017; Realpe et al., 2014; Hernández
et al., 2008) These studies represent a period (1995–2012) and only one
study involves all the country. To contribute to this knowledge, we
genotypically characterized 49 Mtb isolates from a cohort of 356 HIV-
patients enrolled in two public hospitals in Bogotá-Colombia, between
2014 and 2015. We compared this information with data from other
neighboring countries, and with the Mtb genotypes isolated from HIV-
negative patients in Colombia. Analysis of this data conform a baseline
work to this regard and provide important information about the TB
lineages circulating in the HIV population in Bogotá-Colombia.
2. Materials and methods
The study was approved by The Santa Clara Hospital, Simón Bolivar
Hospital, and Universidad Nacional de Colombia ethical Committees.
This is a descriptive cross-sectional study. We analyze the circulating
genotypes of Mtb isolated from HIV-positive patients with clinical
suspicion or a history of previous TB. Informed written consent was
required to participate in the study. To maintain the anonymity of the
patients, informed consents and surveys conducted were in the custody
of the principal investigators, An active search for cases of TB was
carried out in 356 patients infected with HIV, enrolled from October
2014 to November 2015. Demographic and clinical data were collected
in a survey that was performed on each patient. The samples of these
patients were collected according to the symptomatology and medical
criteria.

⁎
Corresponding author.
E-mail address: mimurciaa@unal.edu.co (M. I Murcia).

https://doi.org/10.1016/j.meegid.2019.104059
Received 11 February 2019; Received in revised form 26 September 2019; Accepted 29 September 2019
Available online 31 October 2019
1567-1348/ © 2019 Published by Elsevier B.V.
M. Beltrán-León, et al.
The samples of pulmonary and extra-pulmonary origin, which were
phenotypically tested for identification of Mtb complex by smear mi-
croscopy, blood culture, culture in MGIT™ liquid medium and
Lowenstein-Jensen (LJ) solid were retained, and cultures were con-
firmed to be Mtb by immunochromatographic detection of the MPT64
antigen (SD Bioline TB AG mpt64 rapid, Abbot). The susceptibility to
anti-TB drugs was determined by BACTEC MGIT™ SIRE and Genotype
MTBDRplus VER 2.0 (Hain Lifescience) using the manufacturer's in-
structions. The nucleic acid extraction process from colonies was car-
ried out using the GenoLyse® commercial method, in accordance with
the manufacturer's procedures, and stored at −20 °C until its use.
All positive Mtb samples were genotipically characterized by spo-
ligotyping according to the methodology decribed by Kamerbeek et al.
(Kamerbeek et al., 1997), and following the instructions provided by
the manunfacturer (Gentaur Molecular Products, Belgium; membranes
and reagents from Ocimum Biosolutions BV, and the enzyme Platinum™
Taq DNA-Polymerase). The results obtained were converted into binary
and octal codes and analyzed using the SITVIT2 proprietary database of
the Pasteur Institute of Guadeloupe (Demay et al., 2012). In this data-
base, Spoligotype International Type (SIT) designates patterns shared
by two or more isolates, whereas “orphan” designates patterns reported
for a single isolate. Major phylogenetic lineages were assigned ac-
cording to spoligotype signatures using this database.
Genotyping by 24-loci MIRU-VNTR of each clinical isolate was
performed according to the methodology proposed by Supply et al.
(Supply et al., 2006). Each locus was amplified individually by end-
point PCR using the Taq DNA polymerase QIAGEN 5 units/μl and the
concentration of all primers was modified to 0.2 pmol, except for MIRU
4, QUB-11b, Mtub21, QUB 26 which were used at 1.5 pmol and QUB
4156 at 2.5 pmol. The products were analyzed by electrophoresis in 2%
agarose gel and the molecular weight was determined by GeneSnap®
software version 6.07 GeneTools. The molecular weight values were
stored in Microsoft Excel®. The corresponding bands of each MIRU-
VNTR were interpreted as the number of copies based on a reference
table authorized by the Reference and Research Laboratory of the
Pasteur Institute of Guadeloupe (http://www.pasteur-guadeloupe.
fr:8081/SITVITDemo/) and the analysis were done using the tools
available on the web page MIRU-VNTRplus (www.miruvntrplus.org).
We also performed a comparison of the predominant SITs found in
the present study with those reported previously in Colombia and
neighboring countries (n = 8313) in the international database, split as
Colombia (excluding the present study, n = 1199 strains), Peru
(n = 1282), Brazil (n = 4489), Paraguay (n = 226), Argentina
(n = 1381), and Venezuela (n = 935).
The evolutionary relationships among all the observed spoligotypes
(n = 49) were obtained by drawing minimum spanning trees (MSTs) in
function of lineages, patient's demographic data, and MIRU loci for-
mats. A tree connects each pattern based on degree of changes required
to go from one allele to another. The structure of the tree is represented
by branches (continuous vs. dashed and dotted lines) and circles re-
presenting each individual pattern. Note that the length of the branches
represents the distance between patterns while the complexity of the
lines (continuous, gray dashed and gray dotted) denotes the number of
allele/spacer changes between two patterns: solid lines, 1 or 2 or 3
changes (thicker ones indicate a single change, while the thinner ones
indicate 2 or 3 changes); gray dashed lines represent 4 changes; and
gray dotted lines represent 5 or more changes. The size of the circles is
proportional to the total number of isolates in our study, illustrating
unique isolates (smaller nodes) versus clustered isolates (bigger nodes),
while the numbers represent major SITs.
Using the SITVIT2 database, we also made a detailed phylogenetical
comparison of data generated in this investigation vs. pooled data from
Colombia which included n = 1248 M. tuberculosis strains with spoli-
gotyping data (n = 1248); n = 483 strains with combined spoligo-
typing and 12-loci MIRU data; and n = 115 strains with similar data
that were isolated from patients with known HIV-serology.
2
Infection, Genetics and Evolution 77 (2020) 104059
Cluster analysis provides a way to estimate the proportion of recent
transmission. Estimating the proportion of cases due to recent trans-
mission was calculated using the “n-1 method” in which one case from
each cluster is assumed to be an index case, and the proportion of non-
index cases pn – 1, is used as a measure of recent transmission (Uys,
2012). We use the following formula: pn – 1 = (A – M) / A, where A is
the number of cases, M is the total number of clusters.
Statistical analysis was performed using the R software. Chi-squares
and P-values were calculated with Pearson's Chi-squared test. A p-value
between 0.05 and 0.07 was considered marginally significant. A p-
value < .05 was considered statistically significant. The discriminatory
power of genotyping techniques used were calculated by the Hunter-
Gaston index (HGDI) (Hunter and Gaston, 1988).
3. Results
In total, 356 patients were recruited who were hospitalized or at-
tended an outpatient clinic in the Simón Bolívar and Santa Clara hos-
pitals of the city of Bogotá. Of the total patients enrolled, 49 HIV-po-
sitive patients were simultaneously positive for Mycobacterium
tuberculosis complex. The average age of HIV diagnosis was 33.7 years
(range 18–61 years). The male-female ratio was 3.5:1. The age group
with the highest number of participants was between 25 and 35 years
old (42%). 44% of the patients lived in Bogotá, the others came from
different parts of the country, one from Brazil and the other from
Venezuela. 28% were street dwellers and 3% were deprived of their
freedom. 58% of patients took psychoactive substances and 61% con-
sumed alcoholic beverages. In addition, 44% of the study population
was in a state of malnutrition, 75% were not receiving antiretroviral
treatment (ART) and 88% had a CD4+ T lymphocyte count < 250/μl.
Finally, 28% of the patients died during the study.
49 Mtb positive isolates were recovered starting from a total 356
HIV-positive patients. Susceptibility tests using the phenotypic method
(BACTEC MGIT™ SIRE) showed that 46/49 (93.4%) were sensitive to
Rifampicin (R) and Isoniazid (H); 1/49 (2%) was mono-resistant to R,
and 2/49 (4%) were mono-resistant to INH. Detection of drug-re-
sistance mutations using the molecular method (GenoType
MTBDRplus) shown that 3/49 (6%) strains harbored drug-resistant
mutations to R, while none of the strains contained mutations con-
ferring resistance to INH.
The spoligotyping was performed on 49 isolates followed by 24-loci
MIRU-VNTRs and results obtained are detailed in Table 1. Briefly,
spoligotyping generated 16 distinct patterns grouped in 10 clusters (2
to 11 isolates per cluster) containing 45 of the 49 isolates (92%), and
the remaining 4 (8%) were unclustered; all but 1 of the patterns being
previously reported in the SIVIT2 database (Table 1). Three major
sublineages predominated in HIV/TB patients from Colombia: Haarlem
n = 26, LAM n = 12, and T n = 11. These were split in sublineages as
(Table 1): Haarlem split in H1 n = 14, H3 n = 12; LAM in LAM9 n = 7,
LAM2 n = 3, LAM1 n = 2; and T in T1 n = 9, T3 n = 1.
Description of all spoligotype patterns observed among HIV/TB
patients in Colombia and their global distribution in the SITVIT2 da-
tabase are showed in Table 2. The most predominant pattern in the
present study (SIT62/H1, n = 11) is very specific to this country since
taking in account distribution in countries with at least 3% of SIT62/
H1, 36% of all such patterns collected worldwide were from Colombia.
Indeed, Colombia alone is responsible for almost all SIT62/H1 strains in
South America; 38.5% of these strains are traced to South America in
the world, but almost all are from Colombia (Table 2). Same is also true
for another Haarlem sublineage (SIT727/H1, n = 2). Although it is not
a predominant pattern in this study, it is almost exclusively limited to
Colombia (64.3% of all isolates reported).
A comparison of all SITs found among HIV/TB isolates (n = 43/49
isolates) to previously recorded strains from Colombia (n = 1199)
showed that SITs encountered among HIV+ patients were already re-
ported from Colombia in the international database – with the
M. Beltrán-León, et al. Infection, Genetics and Evolution 77 (2020) 104059

Table 1
Description of all spoligotype patterns observed among HIV/TB patients in Colombia and their global distribution in the SITVIT2 database.

* The 2 letter country codes are according to http://en.wikipedia.org/wiki/ISO_3166-1_alpha-2; countrywide distribution is only shown for SITs with ≥3% of a given
SITs as compared to their total number in the SITVIT database.
** Worldwide distribution is reported for UN Sub-regions with > 3% of a given SITs as compared to their total number in the SITVIT database.

Table 2
Comparison of predominant SITs found among HIV/TB patients in the present study (n = 49), as compared to all previously recorded isolates from Colombia
(n = 1199) and neighboring countries (n = 8313) in the international database.

* Proportions for a given SIT above 2% of all strains are shown in bold.

3
M. Beltrán-León, et al.
Fig. 1. MST drawn on all M. tuberculosis spoligotypes from Colombia (n = 1248) including the present study on HIV/TB isolates. (A) The colors of the circles indicate
the phylogenetic lineages encountered in Colombia, (B) MST showing strains from the present study on HIV/TB isolates (n = 49) represented within the global tree
observed for Colombian M. tuberculosis isolates.
exception of 2 patterns that are unique to HIV patients; SIT777/Ural-1
and SIT4108/T1 (Table 1 and Fig. 1). Both SIT777/Ural and SIT4108/
T1 certainly represent imported cases of such genotypes among HIV+
patients. Indeed, SIT4108/T1 (a single strain in a HIV+ patient) was
absent among all other countries shown in Table 2; and SIT777/Ural-1
(also a single strain in a HIV+ patient) was traced only to Peru n = 1,
and Brazil n = 3. A comparison with the neighboring countries
(n = 8313 strains) showed that Colombia is unique by the almost ex-
clusive presence of SIT62/H1 strains (and to a lesser extent of SIT727/
H1), as well as SIT777/Ural-1 and SIT4108/T1 among HIV/TB patients.
A split of spoligotype clusters among HIV/TB patients in Colombia
by MIRU-VNTRs showed that 2 clusters, namely SIT62/H1, n = 11, and
SIT50/H3, n = 10 were relatively very well preserved upon 12-loci
format, but ultimately split to smaller clusters by 15- and 24-loci for-
mats (Supplemental Table S1). This is expected, since the dis-
criminatory power calculated by the Hunter-Ganston index (HGDI)
(Hunter and Gaston, 1988) of the 24-loci MIRU-VNTRs (0.984) is far
greater than the spoligotyping technique (0.888).
The results of the “n-1 transmission rate” depend on the typing
formats used for calculation, if we calculate it by spoligotyping alone (9
clusters containing 43 strains), the “n-1 transmission rate” was 69.4%.
Meanwhile, if we use spoligotyping and MIRU12 (10 clusters containing
32 strains), the “n-1 transmission rate” is 44.9%. Finally, note that both
15- and 24-loci formats resulted in an identical split (9 clusters con-
taining 22 strains), with a “n-1 transmission rate” of 26%
(Supplemental Table S1).
MST drawn on all M. tuberculosis spoligotypes from Colombia
(n = 1248) including the present study on HIV/TB isolates is shown in
Fig. 1. The lineages found in the HIV/TB correspond mainly to
Haarlem, LAM and T as mentioned before; also reflect the major TB
lineages prevalent in Colombia. Remarkably, only two isolates were
unique to HIV/TB patients, SIT777/Ural-1 and SIT4108/T1. One may
4
Infection, Genetics and Evolution 77 (2020) 104059
notice that the SIT777/12-MIT45/Ural-1 strain cluster is very closely
linked to SIT50/12-MIT45/H3 and this relatedness is conserved despite
15- and 24-loci MIRU typing. Hence, we may conclude on its phylo-
genetical relatedness to H3 sublineage.
Whatever the MIRU format chosen, the main clusters/lineages were
very well-conserved – both on the MST drawn on all M. tuberculosis
strains from Colombia (n = 483), as well as the present study (n = 49)
(Fig. 2). In the MST drawn by combined spoligotyping and MIRU data
on HIV/TB isolates (n = 49) in function of lineages, one can observe 4
large clusters (H1, H3, T, LAM9). Phylogenetically, LAM9 is distant
from the other 3 clusters (which are closer to each other) (Supplemental
fig. S1) independently of the MIRU format. Note that a better resolution
of clusters of HI, H3 and T1 lineage is observed following typing with
MIRU15 or MIRU24 loci formats. Combined spoligotyping and 12-loci
MIRU based MST on M. tuberculosis isolates with known HIV-serology
(n = 115) in function of lineages, patient's demographic data, and drug-
resistance is shown in the supplemental fig. S2.
Unknown signature lineage strains (n = 6) were significantly asso-
ciated with drug-resistance (including MDR/XDR cases), and some were
phylogenetically close to H1/H3 sublineages (Supplemental fig. S2).
These should be closely monitored to see if they do not represent
emergence of new drug-resistant clones. Despite representing the lar-
gest proportion as a sublineage, H1 and H3 strains (which are pre-
dominant in the capital city Bogotá) show little MIRU-based diversity,
suggesting a relatively recent expansion of these strains. Note that re-
garding the patient's HIV serology, the lineage H3 was present ex-
clusively in HIV/TB patients (Supplemental fig. S2).
Statistical comparison of demographic parameters vs. lineages
among HIV/TB isolates for 4 major sublineages encountered in the
present study shows that the profile of M. tuberculosis strains infecting
the HIV+ patients is gender-related (p < .05), but not age-related
(p = .28) (Supplemental Table S2 and supplemental fig. S1).
M. Beltrán-León, et al.
Fig. 2. MST drawn on combined spoligotyping and 12-loci MIRU data available on M. tuberculosis strains from Colombia (n = 483) including the present study on
HIV/TB isolates. (A) The colors of the circles indicate the phylogenetic lineages encountered in Colombia, (B) HIV/TB isolates (n = 49) represented within the global
spoligo/12-loci MIRU tree drawn for Colombian isolates.
We also performed a statistical comparison of parameters (age,
gender and lineage) vs. HIV-serology results available for a total of 115
patients (HIV- n = 47; HIV+ n = 68). HIV serology results varied in
function of age (p < .05) and genotypic lineages infecting the patients
(p < .05), but not gender. Indeed, if one analyzes the relationship
between Haarlem H1 / H3 sublineages and the HIV-serology, an HIV+
patient is more likely to be infected with a H3 than H1 sublineage of M.
tuberculosis (p = .03). (Supplemental Table S3 and supplemental fig.
S2).
4. Discussion
We characterized M. tuberculosis isolates from 49 HIV/TB patients
from a cohort of 356 HIV-patients from two public Hospitals in Bogotá,
Colombia. The present study showed a higher proportion of men, under
the age of 45 years co-infected, which is in agreement with what was
previously reported for both TB and HIV co-infection (WHO, 2017;
Murcia-Aranguren et al., 2001). Factors such as alcohol consumption
and psychoactive substances have been associated with TB in HIV po-
sitive patients (WHO, 2017; Peñuela-Epalza et al., 2006; WHO, 2019),
these conditions are present in > 50% of the patients studied. Likewise,
the CD4+ T lymphocyte count lower than 250 cells/μl which is closely
related to TB and its complications (Jones et al., 1993), significantly
increasing the risk of early mortality. Despite the availability of anti-
retroviral drugs, there was a very low adherence to treatment, attrib-
uted mainly to drug addiction conditions; in fact, 75% of the patients
were not consuming ART, increasing the probabilities not only to ac-
quire TB, but also an opportunistic infection and consequently, in-
creasing physical and immunological deterioration (Beltrán-León et al.,
2019).
5
Infection, Genetics and Evolution 77 (2020) 104059
In Colombia, genotyping studies have been conducted in the general
population and have had a low percentage of HIV positive patients
(Castro et al., 2017; Realpe et al., 2014; Hernández et al., 2008). Ac-
cording to the results obtained using spoligotyping and 12-loci MIRU,
we found predominant sub-lineages Haarlem (n = 26), LAM (n = 12)
and T (n = 11), which were previously reported in HIV/TB patients
from Colombia and correspond to the major genotypes prevalent in the
country, all of them belonging to the lineage 4/Euro-American (Realpe
et al., 2014; Cerezo et al., 2012; Nieto et al., 2012; Puerto et al., 2015).
This result supports the hypothesis that HIV/TB patients in Colombia
were infected by the more prevalent strains. Regarding other countries
in South America, for example in Peru, Sheen P. et al., observed that the
sublineage that predominated in these patients was LAM with a pre-
valence of 26% (Sheen et al., 2013), while in Argentina the Haarlem
sublineage prevailed with 60% in this same population (Ritacco et al.,
2012), promoting the hypothesis of adaptation processes between the
circulating strains and specific population features (Gagneux et al.,
2006). However, we found a spoligotype not detected previously in
Colombia or in another country shown in Table 2, the SIT4108/T1, a
single strain in a HIV+ patient; the presence of this unique strain in this
patient requires a more exhaustive study to determine its origin. Similar
situation occurs with the strain SIT777/Ural-1 (also a single strain in a
HIV+ patient) which was traced only to Peru n = 1, and Brazil n = 3.
Equally remarkable in this study, is the exclusive presence of SIT62/H1
strains (and to a lesser extent of SIT727/H1) among HIV/TB patients
from Bogotá-Colombia, and their absence in patients from other Latin-
American countries, suggesting a successful transmission of this geno-
type in this setting.
Regarding the estimation of recent transmission rate calculated
using the “n-1 method”, the result depends on the typing methods; in
M. Beltrán-León, et al.
our study, the format spoligotyping-15 or 24 MIRU-VNTR got lowest
value (26%), indicating a recent transmission event on the HIV+ po-
pulation from Bogotá. Finally, unknown signature lineage strains
(n = 6) are significantly associated with drug-resistance (including
MDR/XDR cases), and some are phylogenetically close to H1/H3 sub-
lineages. These should be closely monitored to see if they do not re-
present emergence of new drug-resistant clones.
The relationship between a patient with HIV-positive status and the
susceptibility to be infected by a specific genotype, including the drug-
resistant genotype, is an open question to resolve, but will be required
large sample size and a study design very robust, two requirements not
covered in this study.
In conclusion, the present genotypic study of Mtb in HIV-positive
patients, represents a baseline for the control of TB in people living with
HIV in Colombia. More studies are needed at the national level to
evaluate the dynamics of TB transmission in this important vulnerable
population, which will allow the control of outbreaks related not only
in hospitals but also in places with high risk such as prisons, schools and
shelters.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.meegid.2019.104059.
Acknowledgments
To the people responsible for the HIV and Tuberculosis programs of
the Hospital Santa Clara and Simón Bolivar, for their love, respect and
help during the process of taking samples. This project was supported
by COLCIENCIAS grant, 696-2013, Colombia.
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