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Journal of Clinical Microbiology, March 2001, p. 1175-1177, Vol. 39, No. 3
Department of Clinical Microbiology and
Infectious Diseases, The Hebrew University-Hadassah Medical
School,1 and Department of Tuberculosis
& AIDS, Ministry of Health,2 Jerusalem, Israel
Received 16 August 2000/Returned for modification 22 October
2000/Accepted 9 January 2001
Restriction fragment length polymorphism analysis with
IS6110 and DR-r probes was used to study 69 Mycobacterium tuberculosis isolates obtained from Israeli
patients and new immigrants from the former Soviet Union and Ethiopia.
DNA fingerprinting identified unique patterns for almost all isolates,
indicating that most patients were infected with a unique strain
imported from their country of origin and that their latent infection
was reactivated in Israel.
In recent years, the incidence of
tuberculosis has increased worldwide (13). The incidence
of tuberculosis also increased in Israel due to immigration from
high-incidence countries (such as the former Soviet Union and Ethiopia)
(3). For the last decade, the incidence of tuberculosis
among Israelis born in Israel or living in the country for many years
is 4 cases per 100,000 people, whereas the tuberculosis rates of recent
immigrants from the former Soviet Union (38 to 172/100,000) and
Ethiopia (500 to 3,000/100,000) are much higher (3). From
1990 to 1996, Israel absorbed more than 750,000 immigrants, one-seventh
of the whole population. The proportion of drug-resistant
Mycobacterium tuberculosis strains in Israel was greatly
influenced by these waves of immigration (14). Typing of
M. tuberculosis strains has improved the understanding of
tuberculosis transmission (1, 4, 6, 7, 10-12, 17, 21).
Restriction fragment length polymorphism (RFLP) can differentiate strains of M. tuberculosis, particularly when insertion
sequence IS6110 probes are used (2, 4, 6, 12,
15-18). This is a standardized method (16)
commonly used for analyzing the molecular epidemiology of tuberculosis.
We present here the first RFLP analysis of strains obtained in Israel
from Ethiopian, Russian, and local Israeli patients.
Sixty-nine M. tuberculosis isolates were chosen (55 from the
Israeli mycobacterium reference laboratory, A. Felix Public Health Laboratories, Tel Aviv, and 14 from the mycobacterial laboratory of
Hadassah Hospital, Jerusalem, Israel) to include isolates of Ethiopian,
former Soviet Union, and Israeli origin, as well as different
antibiotic susceptibility patterns. Isolates were cultured from
patients with pulmonary tuberculosis between 1992 and 1996 and
identified as M. tuberculosis complex with the Accu Probe kit (Gen-Probe, San Diego, Calif.). Seventeen isolates were excluded: nine with identical RFLP patterns shown to be a laboratory
contamination and eight originating from the same patients or family.
Twenty percent of the isolates were from patients born in Israel or
long-term residents, 39% were from former Soviet Union immigrants, and
32% were from Ethiopian immigrants. There was no information on the country of origin for 9% of the isolates. Fifty-five percent of the
isolates were susceptible to all antimycobacterial drugs tested, 39%
were resistant to at least one antimycobacterial drug, and there was no
information on drug susceptibility for 6% of the isolates. Isolates
were coded, so that the patients' country of origin and the
antimicrobial susceptibility patterns of the isolates were revealed
only after molecular typing was performed.
RFLP analysis was done as described by van Embden et al.
(16). The probes used were the 245-bp PCR product of the
right IS6110 arm (19), and the 36-bp DR-r
oligonucleotide that detects direct repeats (8). The
IS6110 DNA probe was prepared by PCR using INS-1 and INS-2
primers (19). The DR-r oligonucleotide (5'-GTCGTCAGACCCAAAACCCCGAGGGGACGGAAAC-3') was synthesized
(DNA synthesizer 394; Applied Biosystems Inc.). Probes were labeled with digoxigenin (Roche Molecular Biochemicals, Roche Diagnostics GmbH)
by random priming (IS6110 probe) or by 3'-end labeling
(DR-r). Isolates were cultivated on Lowenstein-Jensen slants, and
chromosomal DNA was extracted using 50 µl of lysozyme (10 mg/ml) and
75 µl of sodium dodecyl sulfate-proteinase K and
cetyltrimethylammonium bromide-NaCl mixtures (16). DNAs
were digested with 20 U of PvuII, which cleaves the
IS6110 sequence once. Restriction fragments were separated
by 0.8% agarose gel electrophoresis (19), transferred to
nylon membranes (Roche Molecular Biochemicals), hybridized to the
probes described above, and detected by chemiluminescence (DIG
system; Roche Molecular Biochemicals). DNAs from M. tuberculosis H37Rv and BCG 1173P2 Pasteur strains were included as
controls. The fingerprinting patterns were compared by visual
examination. Clustering was defined as the occurrence of two or more
isolates from different patients that showed an identical pattern for
at least six IS6110 bands. When there were less than 6 IS6110 bands, clustering was defined as the occurrence of
similar RFLP patterns using both IS6110 and DR-r as probes.
The RFLP patterns of the 25 drug-susceptible isolates from patients
living in different localities of Israel differed from each other (Fig.
1).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.1175-1177.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Epidemiology of Mycobacterium
tuberculosis Infection in Israel
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FIG. 1.
IS6110 DNA fingerprint patterns of 25 drug-susceptible M. tuberculosis strains obtained from the
Israeli mycobacterium reference laboratory, A. Felix Public Health
Laboratories, Tel Aviv. M. bovis BCG 1173 P2 (P2) was used
as a reference strain. PvuII-digested chromosomal DNA was
hybridized with a 245-bp PCR fragment of IS6110. The
positions of molecular size markers (in kilobases) are given to the
left of the gel.
Fourteen M. tuberculosis isolates (10 susceptible to all
antimycobacterial drugs and 4 resistant) from patients living in Jerusalem but originating from countries representing the diversity found in Israel exhibited different IS6110 RFLP patterns
(Fig. 2).
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Thirteen drug-resistant M. tuberculosis isolates (11 from
new immigrants from the former Soviet Union) were examined. Eight of
these were multidrug resistant (MDR). Only the RFLP patterns of
isolates 4 and 6 and isolates 8 and 10 were similar (Fig.
3), emphasizing the low rate of
clustering among M. tuberculosis strains in Israel (4 of 52 [7.7%]). Isolates 8 and 10 were MDR with identical susceptibility
patterns, whereas isolates 4 (MDR) and 6 (streptomycin resistant) had
different susceptibility patterns. No obvious contact could be found
between these cases.
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The IS6110 copy number of the isolates ranged from 1 to 14 (mean, 7 copies). No correlation was found between the IS6110 copy number and the origin of the patient or antibiotic susceptibility. Three isolates had a single copy of IS6110. The size of the band for isolate 12 was similar to that of BCG, which always has a single copy of IS6110 (Fig. 2). In a situation where it is impossible to differentiate between M. tuberculosis with a single IS6110 copy and BCG, RFLP with an unrelated probe is recommended. RFLP using a DR-r probe was performed and demonstrated that this isolate was identical to 1173 BCG-P2 strain (data not shown).
Israel has a unique epidemiological situation, where immigrants from many countries with varied incidence of tuberculosis concentrate in a relatively small area. RFLP analysis of 52 isolates of M. tuberculosis showed a considerable diversity, suggesting that most patients examined were infected with unique strains. RFLP revealed a 92% genetic diversity, a rate higher than that observed in other developed countries with populations of various geographical origins (1, 7). Our results indicate that each immigrant imported the mycobacteria from his or her country of origin and the disease reactivated in Israel, soon after arrival. This RFLP study does not indicate any trend of cross infection of tuberculosis between new immigrants themselves during their stay at immigration centers or between new immigrants and local Israelis. However, the selection of the isolates was performed retrospectively and the number of strains analyzed was limited. A larger prospective study over a longer period of time is required to determine whether this epidemiological trend continues. The polymorphism among the isolates from Ethiopian immigrants was unexpectedly high, as they came from a country with a high prevalence of tuberculosis where the polymorphism is usually low (2, 5, 9, 17). This finding is even more surprising, because many of these immigrants lived for a prolonged period in crowded conditions before coming to Israel. The low rate of clustering indicates that tuberculosis among the study population results mainly from reactivation of latent infection.
Our experience and that of others (20) suggest that M. tuberculosis strains have a low penetration-diffusion pattern in the human population.
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ACKNOWLEDGMENTS |
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We thank Anita Lavy of the Israeli mycobacterium reference laboratory, A. Felix Public Health Laboratories, Tel Aviv, and Andrea Rajs from the laboratory of mycobacteria, Hadassah Hospital, Jerusalem, Israel, for providing clinical specimens.
This work was supported in part by a grant from the Israeli Ministry of Health and by a grant from the Center for the Study of Emerging Diseases. The work was performed in the Peter A. Krueger P3 laboratory with the generous financial support of Nancy and Lawrence E. Glick.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Clinical Microbiology and Infectious Diseases, The Hebrew University-Hadassah Medical School, P.O. Box 12000, Jerusalem 91120, Israel. Phone: 972-2-6776594. Fax: 972-2-6419545. E-mail: rahavg{at}md2.huji.ac.il.
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Journal of Clinical Microbiology, March 2001, p. 1175-1177, Vol. 39, No. 3
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.1175-1177.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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