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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 1005-1008
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 1005-1008
IS6110 Fingerprinting
of Sensitive and Resistant Strains (1991-1992) of Mycobacterium tuberculosis
in Colombia
Jorge Enrique Gómez-Marin/+,Clara
Inés León Franco*, Martha Inirida Guerrero*, Leen Rigouts**, Françoise
Portaels**
Centro de Investigaciones Biomedicas,
Facultad de Medicina, Universidad del Quindio, Armenia (Q), Colombia *Laboratorio
de Micobacterias, Subdirección de Investigación y Desarrollo,
Instituto Nacional de Salud, Bogotá, Colombia **Laboratory of Mycobacteriology,
Institute of Tropical Medicine, Antwerpen, Belgium
+Corresponding author. Fax:
+57-67-460168. E-mail : jegomezmarin@hotmail.com
Received 19 December 2001
Accepted 7 August 2002
Code Number: oc02225
The standardized method to study
the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium
tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle
region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 ±
3. Similarity between strains was of 60% in 81% of strains and this tended to
be correlated with geographic origin. For the first time M. tuberculosis
without IS6110 bands in restriction fragment length polymorphism analysis
was found in Colombia. Additional studies are necessaries in order to best characterize
the situation in relation to human immunodeficiency virus epidemic and recent
changes in tuberculosis control program.
Key words: IS6110 - Mycobacterium
tuberculosis - Colombia
In Colombia are reported near of
11,000 new cases of tuberculosis each year (Victoria 1999). The incidence of
new cases has been shown to be in decrease, however this can be explained by
a reduction in the search of new case (Victoria 1999). There are an urgent need
to best define effective measures in order to stop transmission in particular
conditions where restriction fragment length polymorphism (RFLP) analysis can
plays a helpful role. RFLP is based on the detection of the species specific
insertion sequence IS 6110 that allows differentiation of strains belonging
to the Mycobacterium tuberculosis complex (van Soolingen et al. 1991).
IS6110 is stable enough to provide identical fingerprints for mycobacterial
strains isolated from one patient and from patients belonging to small clusters
of infection (van Soolingen et al. 1991). Most studies with IS 6110 fingerprinting
have focused on tracing routes of transmission of M. tuberculosis in
outbreaks. The expanded use of fingerprinting have identified sources of infection
and routes of transmission that were previously not known or suspected (Crawford
et al. 1993, Geneiwen et al. 1993). Findings of this sort could help in developing
certain types of policies or actions to more effective control measures. In
addition, some data have been published and indicate that there are an association
between fingerprinting types and their geographic origin (Hermans et al. 1995).
These studies have shown that it is possible to distinguish between isolates
originating from different locations.
Screening of all isolates from a
particular geographical area would identify related isolates and provide the
starting point for contact investigations. In 1993, international consensus
was reached on a standardized method of M. tuberculosis RFLP analysis
opening the way to interlaboratory comparison (van Embden et al. 1993). However,
in a Medline search using MESH term IS6110, appeared 77 papers dealing with
use of RFLP for IS6110 in Europe, 103 from United States, 35 from Africa and
only 17 articles from Latin America. Some papers from Latin America characterized
the polymorphims in general and human immunodeficiency virus (HIV) population
(Escalante et al. 1998, Ferrazoli et al. 2000, Suffys et al. 2000, Diaz et al.
2001, Yang et al. 2001). In Colombia, two previous published works (Gomez-Marin
et al. 1995, Henao et al. 1999) reported RFLP patterns of IS6110 in M. tuberculosis
strains in two particular regions: Quindio (center west region department) and
Guaviare (eastern region department). The objective of the present work was
to enlarge and complete these previous studies and to characterize IS6110 polymorphism
of M. tuberculosis strains obtained in different geographical areas in
Colombia. The group was a sample of 53 from a total of 121 strains collected
during a survey on initial drug resistance in different regions of Colombia
performed at the National Institute of Health of the Republic of Colombia during
1991-1992. All the patients were smear positive. The methodology used to select
this group of patients has been described in detail previously (Laszlo &
Kantor 1995).
Samples were decontaminated using
the Kudoh's method and cultured on Kudoh's modified Ogawa medium and Giraldo's
modified Stonebrink medium at 37°C (Orozco et al. 1985). Culture identification
was made by the CDC's criteria (Kent & Kubric 1985) and drug susceptibility
testing by the proportion method of Cannetti et al. (1969) for isoniazide (INH,
0.2 µg/ml), rifampicine (RPM, 40 µl/ml), ethambutol (EMB, 2 µg/ml)
and streptomicine (SM, 4 µg/ml). Resistance was defined according to the
past history of treatment referred by the patient. Some of the results of the
drug sensitivity testing were confirmed in the WHO-Colaborator Center in Ottawa
(Canada) or in the Institute of Tropical Medicine in Antwerpen. Strains were
sent to the Institute in Antwerpen on L-J medium for fingerprinting.
The standardized method (van Embden
et al. 1993) was applied to all isolates. Briefly about 10 mg of bacteries were
suspended in 500 µl TE buffer (10 mM, Tris HCl pH 8.0, 1 mM EDTA). After
lysis with lysozyme, proteinase K and SDS (Sodium Dodecyl Sulfate), the DNA
was extracted with CTAB (cetyltrimethylammoniumbromide). The DNA was digested
with PvuII, and the fragments were separated by electrophoresis on a
0.8% agarose gel followed by a DNA transfer to a charged nylon membrane by vacuum-blotting.
The membrane was hybridized with a 245 bases pair long probe which is directed
against the segment at right of cut site of PvuII in the IS6110. The
detection of the peroxidase labeled probe was realized by the Enhanced Chemoluminiscence
System (ECL, Amersham, UK) and registered on hypersensitive films. To obtain
accurate standardization of RFLP results two internal molecular weight markers
(supercoiled DNA ladder/PvuII and PhiX174HaeIII) were included
in each sample and two external markers on each membrane. The last consisted
of lambdaHindIII to check the migration during electrophoresis and DNA
of Mt14323 M. tuberculosis reference strain with a known fingerprint.
These internal markers were used during the computer assisted analyses.
The DNA fingerprint patterns of the
mycobacterial analysis were analyzed and compared by a computer assisted system
(Gelcompar 4.2 software, Kortrijk, Belgium) in order to construct dendrograms
based on similarity coefficients. IS6110 groups were formed based on a similarity
coefficient greater than 60%.
In total, 53 isolates of M. tuberculosis
derived from 53 unrelated patients from 14 regions in Colombia were studied.
The results of IS6110 characterization are shown in the Figure.
We found that two couples of isolates from Valle region had identical IS6110
pattern. Thus, 25% (4/16) of the strains in Valle department originated two
clusters that probably were originated from two index cases of recent transmission.
However, we were unable to obtain the epidemiological data concerning these
cases. By using the criteria of similarity coefficient greater than 60% it were
formed seven groups of IS6110 related strains (Figure).
The distribution of groups by department and type of resistance is shown in
the Table. Group A have a large number
of strains located in western departments (Valle, Risaralda and Antioquia),
only one isolate from this group was obtained in Caribbean region. Also, a large
group of strains from Antioquia were in group F. In total, 43 of 53 isolates
(81%) were grouped based on this similarity coefficient. No correlation was
found between RFLP types and the phenotypes of resistance to various antituberculous
drugs. Each group included resistant and sensitive isolates.
The mean number in all strains that
showed some IS6110 band was 10 ± 3. The number of copies ranged of 0 to
18. Four isolates were found without IS6110 copies and were originating from
four different regions in Colombia (Antioquia, Valle, Choco and Tolima). The
number of isolates with low number of copies (less than 4) was 6 of 53 isolates
studied (11%).
The two clusters of strains with
identical fingerprinting in present study occurred in Valle, the region with
the greater number of isolates included. The percent of isolates that were related
with probably recent transmission (25%) in Valle strains is near to the 14%
found in Quindio (Gómez-Marin et al. 1995). However, the percentage is
in contrast with the rate found in Guaviare where were found 31 isolates that
formed cluster between 55 isolates (56%) that were studied (Henao et al. 1999).
The situation in Gauviare is particular because most of cases were originated
from indigenous people that have a rate of incidence of 558 cases/100.000 habitants,
a frequency 100 times greater than in non-indigenous population.
Dendrograms are used to establish
IS6110 degrees of similarity between isolates. Thus, in a study in Tanzania
45% of non-identical isolates were included in three big groups with similarity
coefficients that ranged between 70 to 96 % (Yang et al. 1995) and in Tegucigalpa
(Honduras) where 30% of non identical isolates have similarity coefficients
greater than 90% with an incidence of tuberculosis (TB) of 84/100,000 habitants
(Pineda-Garcia et al. 1997). We selected a coefficient of similarity greater
than 60% to establish groups of IS6110 patterns. Although the stability of IS6110
element is high, it is by nature transposable and the highly related but non
identical patterns reflect the result of evolution of IS6110 through generations
of tuberculosis infections in the country. When groups of isolates were located
geographically, it was observed that in some groups DNA fingerprint patterns
tended to be in accordance with geographical origins of the isolates. This was,
obviously, more evident in groups formed by a number of isolates greater than
two or three. Thus, in Antioquia region 5 of 11 isolates (45%) were classed
in Group F. This is evidence that coefficient similarity can be used to reflect
the common past of IS6110 exchange between diverse migratory and selective forces.
These factors can acts as positive selection factors. Evolutionary forces in
Colombia can include, among others, people of diverse ethnic origin, migratory
movements and a long history of vaccination with BCG vaccines that is compulsory
in our country in all newborns. In this context, it will be important to compare
results of present study performed during 1992 and isolates of recent years
where national BCG production and rates of vaccination have been reduced.
The number of IS6110 copies was evenly
distributed within a range of 0 to 18. The most frequent number of bands was
9. Notoriously, for the first time in Colombia we found some isolates without
IS6110 bands. The total of isolates with low number of copies (less than 4)
was 11%, that is a percent intermediate between regions of the world with an
important proportion of isolates with absence or low number of copies as 21%
in Vietnam (Yuen et al. 1993) or 24% in Guadeloupe, French West Indies (Sola
et al. 1997) or 26% in Tanzania (Yang et al. 1995) and regions with only 8%
in Denmark (Yang et al 1994) or 5% in Tunisia (Hermans et al. 1995) or 4% in
Cuba (Diaz et al 2001). It have been showed for others authors that isolates
with IS6110 lower than four require the use of additional genetic markers, such
as PGRS and DR, to establish the genetic relatedness (van Soolingen et al. 1993).
It seems that PvuII site of restriction in IS6110 can be methyled in
some strains and this can explains that can be missed during Southern blotting
(van Soolingen et al. 1996).
In conclusion, this study showed
that similarity between strains was of 60% in 81% of strains and this tended
to be correlated with geographic origin. However, due to the small number of
strains studied a new work is needed to include a representative number of strains
from all isolates that are found each year in Colombia. Also it would be necessary
to complement epidemiological investigation of cases with identical fingerprinting
that we were unable to perform due to the retrospective character of present
work. For the first time M. tuberculosis without IS6110 bands in RFLP
was found in Colombia and 11% of isolates had less than four bands. Additional
studies are necessaries in order to characterize the situation in relation with
HIV epidemic and recent changes in tuberculosis control program as the reduction
in BCG vaccination.
REFERENCES
- Cannetti G, Wallace Fox A, Khomenko
A, Mahler HT, Menon MK, Mitchison DA, Rist N, Ämelov NA 1969. Advances
in techniques of testing mycobacterial drug sensitivity and the use of sensitivity
test in tuberculosis control programs. Bull WHO 41: 21-43.
- Crawford JT 1993. Applications
of molecular methods to epidemiology of tuberculosis. 9th Forum in Microbiology.
Res Microbiol 144: 111-115. [ Medline
]
- Diaz R, Gomez R, Restrepo E, Rumbault
R, Sevy Court J, Valdivia JA, van Soolingen D 2001. Transmission of tuberculosis
in Havana, Cuba. A molecular epidemiological study by IS6110 restriction polymorphism
typing. Mem Inst Oswaldo Cruz 96: 437-443. [ Medline
] [ Lilacs
] [ SciELO
]
- Escalante P, Ramaswanny S, Sanabria
H, Soini H, Pan X, Valiente O, Musser JM 1998. Genotypic characterization
of drug resistant Mycobacterium tuberculosis isolates from Peru. Tuber
Lung Dis 79: 111-118. [ Medline
]
- Ferrazoli L, Palaci M, Marques
LR, Jamal LF, Afiume SB, Chimara E, Martins MC, Silva MA, Oliveira CA, Pelhares
MC, Spada DT, Riley LW 2000. Transmission of tuberculosis in an endemic urban
setting in Brazil. Int J Tuber Lung Dis 4: 18-25.
- Genewein A, Telenti A, Bernasconi
C, Mordasini C, Weiss S, Maurer AM, Rieder HL, Schopfer K, Bodmer T 1993.
Molecular approach to identifying route of transmission of tuberculosis in
the community. Lancet 342: 841-844.
- Gómez-Marin JE, Rigouts
L, Villegas LE, Portaels F 1995. Restriction fragment length polymorphism
(RFLP) analysis and tuberculosis epidemiology. Bull PAHO 29:
226-236.
- Henao G, de la Hoz F, León
CI, Ribón W, Guerrero MI 1999. Epidemiología clásica
y molecular de la tuberculosis en Guaviare 1997-1998. Informe Quincenal
Epidemiológico Nacional 4: 85-91.
- Hermans PWM, Messadi F, Guebrexabher
H, van Soolingen D, de Haas PEW, Heersma H, de Neeling H, Ayoub A, Portaels
F, Frommel D, Zribi M, van Embden JDA 1995. Analysis of the population structure
of Mycobacterium tuberculosis in Ethiopia, Tunisia and the Netherlands:
usefulness of DNA typing for global tuberculosis epidemiology. J Infect
Dis 171: 1504-1513. [ Medline
]
- Kent P, Kubica G 1985. Public
Health Mycobacteriology: A Guide for The Level III Laboratory, Center
for Diseases Control, Atlanta.
- Laslo A, Kantor IN 1995. Encuesta
por muestreo aleatorio de farmacorresistencia inicial en casos de tuberculosis
en America Latina. Bol Oficina Sanit Panam 119:226-235.
- Orozco LC, Quintero de R O, Ulloa
de M AI, Giraldo de BE, León CI, Naranjo N, Bernal M, Guerrero MI 1985.
Tuberculosis: Manual de procedimientos, Red Nacional de Laboratorios,
Instituto Nacional de Salud, Serie de Publicaciones Científicas No.
1, Bogotá.
- Pineda-Garcia L, Ferrera A, Hoffner
SE 1997. DNA fingerprinting of Mycobacterium tuberculosis strains from
patients with pulmonary tuberculosis in Honduras. J Clin Microbiol
35: 2393-2397. [ Medline
]
- Sola C, Horgen L, Seng- Goh K,
Rastogi N 1997. Molecular fingerprinting of Mycobacterium tuberculosis
on a Caribbean island with IS6110 and DR probes. J Clin Microbiol 5:
843-846.
- Suffys PN, Ivens ME, Rossetti
ML, Zahab A, Barroso EW, Barreto AM, Campos E, van Soolingen D, Kremer K,
Heerma H, Degrave WM 2000. Uselfulness of IS6110 restriction fragment polymorphism
typing of Brazilian strains of Mycobacterium tuberculosis and comparison
with an international fingerprint database. Res Microbiol 151: 343-351.
- van Embden JD, Cave MD, Crawford
JT, Dale JW, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnick
TM, Small PM 1993. Strain identification of Mycobacterium tuberculosis
by DNA fingerprinting: recommend-ations for a standardized methodology. J
Clin Microbiol 31: 406-409. [ Medline
]
- van Soolingen D, de Haas EW, Hermans
PW, Groenen PMA, van Embden JD 1993. Comparison of various repetitive DNA
elements as genetic markers for strain differentitation and epidemiology of
Mycobacterium tuberculosis. J Clin Microbiol 31: 1987-1995.
[ Medline
]
- van Soolingen D, de Haas P, Blumenthal
R Blumenthal RM, Kremer K, Sluijter M, Pijnenburg JE, Schouls LM, Thole JE,
Dessens-Kroon MW, van Embden JD, Hermans PW 1996. Host modification of PvuII
restriction site in Mycobacterium tuberculosis. J Bacteriol
178: 78-84
- van Soolingen D, Hermans PWM,
de Haas PEW, Sol DR, van Embden A 1991. The ocurrence and stability of insertion
sequences in Mycobacterium tuberculosis complex strains: evaluation
of IS dependent DNA polymorphisms as a tool in the epidemiology of tuberculosis.
J Clin Microbiol 29: 2578-2586.
- Victoria JE 1999. Epidemiologia
de la tuberculosis en Colombia. Informe Quincenal Epidemiológico
Nacional 4: 82-85.
- Yang ZH, de Haas PEW, van Soolingen
D, van Embden JDA, Andersen AB 1994. Restriction fragment length polymorphism
of Mycobacterium tuberculosis strains isolated from Greenland during
1992: Evidence of tuberculosis transmission between Greenland and Denmark.
J Clin Microbiol 32: 3018-3025.
- Yang ZH, Mtoni I, Chonde M, Mwasekaga
M, Fuursted K, Askgard DS, Bennedsen J, de Haas PEW, van Soolingen D, van
Embden JDA, Andersen AB 1995. DNA fingerprinting and phenotyping of Mycobacterium
tuberculoiss isolates from human immunodeficiency virus (HIV)-seropositive
and HIV-seronegative patients in Tanzania. J Clin Microbiol 33:
1064-1069.
- Yang ZH, Rendon A, Flores A, Medina
R, Ijaz K, Flaca J, Eisenach KD, Bates JH, Villareal A, Cave MD 2001. A clinic
based molecular epidemiological study of tuberculosis in Monterrey, Mexico.
Int J Tuber Lung Dis 5: 313-320.
- Yuen LKW, Ross BC, Jackson KM,
Dwyer B 1993. Characterization of Mycobacterium tuberculosis strains
from Vietnamese patients by southern blot hybridization. J Clin Microbiol
31: 1615-1618. [ Medline
]
Copyright 2002 Instituto Oswaldo
Cruz - Fiocruz
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