First case of multidrug-resistant tuberculosis caused by a rare "Beijing-like" genotype of Mycobacterium tuberculosis in Bogotá, Colombia.

Abstract

This report describes a first case due to a genetically distinct and relatively rare "Beijing-like" strain of Mycobacterium tuberculosis isolated from a 15 years old female patient who died shortly after the initiation of antituberculous therapy with second-line drugs. Positive cultures obtained from lung, kidney and adrenal glands upon autopsy were identified as Mycobacterium tuberculosis complex characterized by an identical 15-banded IS6110-RFLP pattern, and were found to be resistant to all the 4 first-line antituberculous drugs tested (rifampin, isoniazid, ethambutol and streptomycin). Spoligotyping followed by comparison with the SITVIT2 database revealed that the isolate belonged to a rare pattern identified as Spoligotype International Type SIT190, which represents only 1.7% of all the Beijing strains worldwide. We present data on its worldwide distribution and present an evolutionary scenario based on available MIRU typing data.

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First case of multidrug-resistant tuberculosis caused by a rare ‘‘Beijing-like’’
genotype of Mycobacterium tuberculosis in Bogota ´, Colombia
Martha I. Murciaa,*, Marina Manotasb, Yesica J. Jime ´neza, Johana Herna ´ndeza,
Maria Irene Cerezo Corte `sa, Lilia E. Lo ´pezc, Thierry Zoziod, Nalin Rastogid,**
aDepartamento de Microbiologı´a, Facultad de Medicina, Universidad Nacional de Colombia, Bogota ´, Colombia
bPediatric Pneumology, Hospital Central de la Policia, Bogota ´, Colombia
cSecretarı´a Distrital de Salud de Bogota ´, Colombia
dUnite ´ de la Tuberculose et des Mycobacte ´ries, Institut Pasteur de la Guadeloupe, Abymes, Guadeloupe
1. Introduction
Mycobacterium tuberculosis Beijing lineage described in 1995
(vanSoolingenetal.,1995),isanemergingpathogeninseveralareas
of the world, and a predominant endemic strain in others (Pfyffer
et al., 2001; European Concerted Action, 2006). It is frequently
associated with drug resistance and active transmission of
tuberculosis (TB) (Anh et al., 2000). Recent immigration in Western
Europe from high Beijing genotype prevalence areas such as China
and Russia is increasingly associated with the occurrence of the
Beijing/W genotype (Lari et al., 2004; Samper et al., 2005). This
observation has created a new challenge for TB control programs
worldwide, since Beijing strains are associated with large TB
outbreaks,increasedvirulenceandmultidrug-resistance(MDR),and
constitute a significant risk factor for TB treatment failure and
relapse (Lan et al., 2003). Nonetheless, little is know regarding the
disease caused by rare ‘‘Beijing-like’’ genotypes that differ from the
classical Beijing genotype by their spoligotype signature (Brudey
et al., 2006); the classical Beijing genotype is characterized by the
Spoligotype International Type SIT1 in the international database
SITVIT2 (at the time of this comparison, SITVIT2 contained a total of
6109 SIT1 strains as compared to only 385 strains classified in
various ‘‘Beijing-like’’ patterns). This paper describes the first killer
case due to a genetically distinct and relatively rare ‘‘Beijing-like’’
strain of M. tuberculosis isolated from a 15 years old female patient
from Buenaventura city port on the Colombian Pacific Coast, and
summarizes available data on its worldwide distribution and
possible evolution. We suggest that various ‘‘Beijing-like’’ sub-
lineages should be better studied to avoid the emergence of highly
virulent clones of tubercle bacilli. A thorough nationwide survey
might help evaluate its dissemination in Colombia.
2. Materials and methods
Sputum samples from a 15-year-old female patient diagnosed
with tuberculosis (TB) were received at the Mycobacteria
Laboratory of the School of Medicine, Universidad Nacional de
Colombia, for bacteriological confirmation of the disease. Later,
these were followed by various tissue samples (lung, kidney,
adrenal glands, brain, and heart) upon the autopsy of the same
Infection, Genetics and Evolution 10 (2010) 678–681
A R T I C L EI N F O
Article history:
Received 2 October 2009
Received in revised form 19 March 2010
Accepted 23 March 2010
Available online 31 March 2010
Keywords:
Mycobacterium tuberculosis
MDR-TB
Beijing genotype
Spoligotyping
Database
Multidrug-resistance
A B S T R A C T
This report describes a first case due to a genetically distinct and relatively rare ‘‘Beijing-like’’ strain of
Mycobacterium tuberculosis isolated from a 15 years old female patient who died shortly after the
initiation of antituberculous therapy with second-line drugs. Positive cultures obtained from lung,
kidney and adrenal glands upon autopsy were identified as Mycobacterium tuberculosis complex
characterized by an identical 15-banded IS6110-RFLP pattern, and were found to be resistant to all the 4
first-line antituberculous drugs tested (rifampin, isoniazid, ethambutol and streptomycin). Spoligotyp-
ing followed by comparison with the SITVIT2 database revealed that the isolate belonged to a rare
pattern identified as Spoligotype International Type SIT190, which represents only 1.7% of all the Beijing
strains worldwide. We present data on its worldwide distribution and present an evolutionary scenario
based on available MIRU typing data.
? 2010 Elsevier B.V. All rights reserved.
* Corresponding author at: Universidad Nacional de Colombia, Edificio 471
Facultad de Medicina, Oficina 304, Departamento de Microbiologı `a, Avenida 30
Calle 45 Ciudad Universitaria, Bogota `, Colombia. Tel.: +57 1 3165000x15016–
15014; fax: +57 1 3165000x15024.
** Corresponding author at: Unite ´ de la Tuberculose et des Mycobacte ´ries, WHO
Supranational TB Reference Laboratory, Institut Pasteur de la Guadeloupe, BP 484,
97183 Abymes Cedex, Guadeloupe. Tel.: +590 590 897661; fax: +590 590 89 69 41.
E-mail addresses: mimurciaa@unal.edu.co (M.I. Murcia),
nrastogi@pasteur-guadeloupe.fr (N. Rastogi).
Contents lists available at ScienceDirect
Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid
1567-1348/$ – see front matter ? 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2010.03.010

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patient after she died. Specimens were processed and cultured,
followed by bacterial identification using standard mycobacter-
iological procedures (Kent and Kubica, 1985). In total, 3 cultures
from lung (strain 091), adrenal glands (strain 093), and kidney
(strain 094), and 2 cultures from sputum (strains 254 and 255)
were identified as Mycobacterium tuberculosis complex using
catalase at room temperature, catalase at 68 8C, nitrate reduction
and the niacin test. Drug susceptibility testing to 4 first-line drugs
(rifampin, isoniazid, ethambutol, and streptomycin) was per-
formed using the 1% proportion method (Canetti et al., 1969).
DNA was extracted and subjected to molecular typing using
spoligotyping (Kamerbeek et al., 1997), 12-loci MIRU typing
(Supply et al., 2001) and IS6110-based restriction fragment length
polymorphism (RFLP) analysis of PvuII-digested bacterial DNAs
(van Embden et al., 1993). For IS6110-RFLP, M. tuberculosis H37Rv
was used as control while a mixture of PvuII-digested supercoiled
DNA ladder and HaeII-digested KX174 DNA was run as a ladder
(Lari et al., 1999). Spoligotypes in binary format were converted to
an octal code for comparison with the SITVIT2 proprietary
database of the Pasteur Institute of Guadeloupe. SITVIT2 is an
updated proprietary version of the previously released SpolDB4
database (Brudey et al., 2006). At the time of the present study,
SITVIT2 contained genotyping information on about 70,000 M.
tuberculosis clinical isolates from 160 countries of origin. In this
database, Spoligotype International Type (SIT) and MIRU Interna-
tional Type (MIT) designate spoligotypes and MIRU patterns
shared by 2 or more patient isolates, respectively, whereas
‘‘orphan’’ designates patterns reported for a single isolate. The
potential evolution of the SIT190 strains was studied by drawing a
minimum spanning tree (MST) of available MIRU patterns in the
SITVIT2 database (Bionumerics software version 3.5, Applied
Maths, Sint-Marteen-Latem, Belgium).
3. Results and discussion
A 15 years old young female patient from Buenaventura city
port on the Colombian Pacific Coast was suspected to be suffering
from TB based upon a three-month clinical history (cough, fever,
astheniaandadynamia),tuberculinskintest(13 mm),andchestX-
ray done in Cali. The patient had a history of contact with a fellow
student who had TB family history, including father and brother
with history of sickle cells anemia. She also presented sickle cells
anemia. A relative advised that BCG vaccine was administered
during the neonatal period physical examination with a resulting
BCG scar. The initial results of Ziehl–Neelsen (ZN) staining of
sputum and bronchial wash were negative for acid-fast bacilli
(AFB). Treatment using directly observed therapy—short course
(DOTS) using isoniazid (INH), rifampin (RIF), pyrazinamide (PZA),
ethambutol (EMB), and additional folic acid, only improved the
condition transiently, followed by fever and deterioration in
general condition that prompted admission to the ‘‘Hospital
Central de la Policia Nacional’’ (HOCEN) in Bogota ´, Colombia on
November 9, 2007. The ZN staining of a 2nd sputum demonstrated
acid-fast bacilli AFB++. HIV co-infection was negative. The
treatment initiated in Cali was continued; the dosage was adjusted
based upon weight, resulting in a good clinical response. The
patient continued without fever during 10 days of treatment with
improvement of her general condition, but presented tendency to
hypotension andsupraventricular
insufficiency secondary to TB on the 11th day. Cortisol levels
were measured and corticoids treatment was initiated. Due to the
possibility of bacterial co-infection, antibiotic treatment was
initiated with broad spectrum antibiotics Vancomycin-Tazobac-
tam (VAN-TZB). The blood culture remained negative. Suspecting a
possibility of multidrug-resistant (MDR) TB, while awaiting the
culture-based results of M. tuberculosis drug-susceptibility, a
tachycardia, andadrenal
treatment with second line drugs (amikacin, ethionamide, D-
cycloserine and para-amino salicylic acid) was initiated. The
patient’s condition deteriorated. She was transferred to the
Pediatric Intensive Care Unit (PICU). She presented ventilation
failure, multiple organic failure, suprarenal failure, myocarditis
and myocardial dysfunction, followed by death on November 30,
2007.Anautopsy wasperformed, andtissuesamples (lung,kidney,
adrenal glands, brain, and heart) were investigated for the
detection of mycobacteria. ZN staining demonstrated AFB only
in the lung tissues. Histological examination of the lungs
demonstrated multiple granulomas, giant cells and fibrosis. The
specimens were macerated and concentrated by centrifugation
and cultured in the solid Ogawa Kudoh medium. In addition to the
2 positive cultures from previous sputum samples, 3 cultures from
lung, kidney and adrenal glands were later positive for M.
tuberculosis complex, all isolates being resistant to all the 4 first-
line antituberculous drugs: rifampin, isoniazid, ethambutol and
streptomycin.
Molecular typing by spoligotyping (Fig. 1A) and IS6110-RFLP
(Fig. 1B) showed that all the strains were strictly identical by the 2
methods; they shared the spoligotype pattern SIT190 correspond-
ing to a rare ‘‘Beijing-like’’ lineage in the SITVIT2 database, and
were characterized by an identical 15-banded IS6110-RFLP
pattern. At the time of this comparison, SIT190 represented a
total of 103 strains in the SITVIT2 database; i.e., only 1.7% of Beijing
strains sensu stricto (SIT 1, n = 6109), and 1/4 of all the ‘‘Beijing-
like’’ lineage strains (n = 385). The worldwide distribution of
SIT190 strains was essentially limited to: USA 57.3%; Thailand
22.3%, Russia 3.9%, Japan and Taiwan (2.9% each), Georgia and
Vietnam (1.9% each). Other countries totaled only 7% of the
worldwide distribution. Interestingly, no SIT190 strains were
found in any country of the South-America with the exception of a
single isolate from Cuba. The origin of the patients was known for
10 out of 59 cases from USA, and it corresponded to a cluster of
strainsisolatedfromKorean-bornpatientsintheWashingtonState
(M. Counter, T. Zozio, and N. Rastogi, unpublished results).
The 12-loci MIRU pattern of one of the strains from the present
investigation (isolate 254) was determined (224326153223), and
corresponded to an orphan pattern in the SITVIT2 database
(designated as orphan 1 in Fig. 1C and D). When it was pooled with
available MIRU data on all SIT190 strains in the SITVIT2 database
(Fig. 1C), a sub-division of SIT190 strains in 12 distinct patterns
containing 22 strains was observed; 4 orphans, 1 strain each of
MIT16, MIT84, MIT139 and MIT988, 2 of MIT17, 3 of MIT95, 4 of
MIT570, and 5 of MIT857. In the MST illustrated in Fig. 1D, the
evolution is supposed to happen starting from MIT17 (MIRU
pattern223325173533)whichisthemostconservedMIRUpattern
for SIT190 in SITVIT2 database. In this figure, the central node
made up of MIT17 (n = 2 strains) does not represent the biggest
cluster among SIT190 strains, however it does represent the
maximum number of branching (8 branches); interestingly, MIT17
is the most predominant pattern for the classical Beijing type
(SIT1) in the SITVIT2 database, representing 19.5% of the isolates
worldwide. Phylogenetically our strain (orphan 1) happens to be
the farthest from the major cluster group and with the most
variable loci as compared to MIT17. It probably derived from
MIT95 which is characteristic of the Beijing genotype (indeed 100%
of the MIT95 strains belong to the Beijing lineage in SITVIT2
without any homoplasy with other lineages).
Although Beijing-like strains of M. tuberculosis have not
received much attention for the time being, this study shows that
‘‘Beijing-like’’ sub-lineages could emerge into highly virulent
clones of tubercle bacilli. Unfortunately, no additional demo-
graphic, epidemiologic or clinical information other than the
geographic origin of the SIT190 isolates was available in SITVIT2, a
fact that did not allow concluding if the high virulence and
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multidrug resistance observed in our study is only episodic or
represents a frequent or recurrent feature of the SIT190 sublineage
organisms.
Beijing genotype (van Soolingen et al., 1995) which is a
predominant endemic strain in China, Japan, and Russia, today
constitutes an emerging pathogen in several other areas of the
world (Brudey et al., 2006). Frequently associated with active TB
transmission, large outbreaks, increased virulence, and drug
resistance, the Beijing genotype has been shown as a significant
risk factor for treatment failure and relapse (Anh et al., 2000; Lan
et al., 2003; European Concerted Action, 2006). In a previous report
that identified a few isolates of the Beijing genotype among
chronic, drug-resistant TB patients in Colombia in the year 1998,
the authors suggested that it was rather associated to clinical and
programmatic mismanagement than community outbreak (Laser-
son et al., 2000). The popular belief remains that both Colombia
and rest of the South America are relatively untouched by the
Beijing strains so far. Although it has been reported in small
numbers in Peru, Argentina, Brazil, Paraguay, Colombia, Ecuador,
andVenezuela(Aristimun ˜oetal.,2007;Ritaccoetal.,2008),littleis
know regarding its exact prevalence in Latin and South America.
Furthermore, hardly anything is known regarding the much rare
‘‘Beijing-like’’ isolates in Latin America and the Caribbean. A
thorough nationwide survey might help know the exact level of M.
tuberculosis drug resistance in Colombia, as well as evaluate the
dissemination of both Beijing and ‘‘Beijing-like’’ variants. Detailed
investigations using 24-loci MIRUs, extended-spacer spoligotyp-
ing, regions of deletions (RD), and SNPs would help shed light on
the origin, evolution and global spread of TB caused both by the
Beijing and ‘‘Beijing-like’’ variants.
Acknowledgments
We thank the Hospital Central de la Policia, Bogota ´, for their
fruitful collaboration. The SITVIT2 project at Institut Pasteur de
GuadeloupeisfundedbytheEuropeanRegionalDevelopmentFund,
European Commission (ERDF/FEDER, A34-05), and the Regional
Counsil of Guadeloupe (Biodiversity project, CR08/031380).
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Fig. 1. Molecular characterization of the rare ‘‘Beijing-like’’ genotype of M. tuberculosis in Bogota ´-Colombia. (A) The spoligotyping membrane performed on DNAs from
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