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Journal of Clinical Microbiology, October 2000, p. 3572-3576, Vol. 38, No. 10
Regional Tuberculosis Genotyping
Laboratory1 and Departments of
Medicine,2
Microbiology/Immunology,4
Pathology,5 and
Anatomy,6 University of Arkansas for
Medical Sciences, and Central Arkansas Veterans Healthcare
System, Arkansas Department of Health,3 Little
Rock, Arkansas
Received 22 March 2000/Returned for modification 10 July
2000/Accepted 18 July 2000
Several genetic loci have been utilized to genotype isolates of
Mycobacterium tuberculosis. A shortcoming of the most
commonly used method, IS6110 fingerprinting, is that it
does not adequately discriminate between isolates having few copies of
IS6110. This study was undertaken to compare pTBN12
fingerprinting of polymorphic GC-rich repetitive sequence genes and
spoligotyping of the direct repeat locus as secondary typing procedures
for M. tuberculosis isolates having fewer than six copies
of IS6110. A total of 88 isolates (100% of the isolates
with fewer than six copies of IS6110 isolated in Arkansas
during 1996 and 1997) were included in this study. Among the 88 isolates, 34 different IS6110 patterns were observed, 10 of
which were shared by more than 1 isolate, involving a total of 64 isolates. The 64 isolates were subdivided into 13 clusters (containing
37 isolates) and 27 unique isolates based on a combination of
IS6110 and pTBN12 fingerprinting and into 11 clusters
(containing 51 isolates) and 13 unique isolates based on a combination
of IS6110 fingerprinting and spoligotyping. Identical spoligotypes were found among isolates having different
IS6110 patterns, as well as among isolates showing
different pTBN12 patterns. In contrast, all isolates that had different
IS6110 patterns were found to be unique by pTBN12 typing.
The clustering rate was 73, 58, and 42%, respectively, for
IS6110 fingerprinting alone, IS6110 fingerprinting and spoligotyping combined, and IS6110 and
pTBN12 combined fingerprinting. The data indicate that the pTBN12
method has greater discriminating power among low-copy-number isolates than does spoligotyping.
DNA fingerprinting of
Mycobacterium tuberculosis strains based on the insertion
element IS6110 is an important new tool to differentiate
strains and to study the epidemiology of tuberculosis (8, 18,
30). Due to the relative stability of IS6110 within a
strain over time and the variable copy number and locations within the
genome (9, 20, 30), IS6110 DNA fingerprinting has
been beneficial in directing outbreak investigations (12, 13,
21), distinguishing between exogenous reinfection and relapse of
cured infection (25, 27), and confirming suspected laboratory cross-contamination (5, 6, 23). The major
limitation of the IS6110 fingerprinting method is its low
discriminating power for isolates with fewer than six copies of
IS6110. In previous large-scale investigations, from
one-fifth to one-third of the M. tuberculosis isolates were
found to be low-copy-number strains (3, 4, 7, 24, 28, 31).
Secondary typing methods to subgroup low-copy-number isolates have been
developed based on different short repetitive DNA sequences associated
with some degree of genetic diversity, including major polymorphic
tandem repeats, direct repeats (DR), polymorphic GC-rich repetitive
sequences (PGRS), and variable-number tandem repeats (10, 14, 15, 16, 17, 22). The most popular secondary typing methods are PGRS
fingerprinting using the recombinant plasmid pTBN12 and DR spoligotyping. To date, none of the evaluations of genotyping methods
have systematically compared the two methods with M. tuberculosis isolates having a low IS6110 copy number.
The DR locus, which is the basis for the spoligotyping method, consists
of a series of directly repeated sequence of 36 bp, with each DR being
separated by nonrepetitive unique spacer DNA of 34 to 41 bp in length.
Most M. tuberculosis strains contain a copy of
IS6110 at a site within the DR region. When the DR regions of several strains are compared, the order of spacers appears to be
about the same among all strains, but insertions or deletions of
spacers and DR do occur (15, 16, 17). The spoligotyping method involves PCR amplification of the DR locus and hybridization to
a series of oligonucleotides representing each of the unique spacer
sequences in the DR locus. The final result is an array of hybridizing
spots that can be readily analyzed in a word processing program. The
most attractive aspect of the method is the ability to rapidly
fingerprint strains without the need to subculture isolates for DNA
isolation. Furthermore, it can be applied to bacilli found in
concentrated sputum sediments when such specimens are smear positive,
reducing the time required to determine the secondary fingerprint by
approximately a month.
The PGRS method is similar to the standardized IS6110
fingerprinting in that it requires purified DNA for Southern blot
hybridization and banding pattern analysis. PGRS fingerprinting has
proven to be useful for differentiating M. tuberculosis
strains with fewer than six copies of IS6110 that could not
readily be differentiated by IS6110 fingerprinting (1,
4, 7, 10, 29). A better correlation between DNA fingerprinting
data and the results of conventional epidemiology was found when a
combination of the IS6110 and PGRS fingerprinting was
applied than when the IS6110 was used alone (7,
29). However, the difficulties in computerizing the analysis of
PGRS fingerprints due to the complexity of the fingerprint patterns
have limited the wide use of PGRS fingerprinting.
In the present study we have tested 88 isolates having fewer than six
copies of IS6110 with both secondary typing methods and
compared the results with the IS6110 fingerprinting results and epidemiologic data from the corresponding patients. Thus, we
determined the discriminating power of the PGRS and DR spoligotyping methods when combined with IS6110 fingerprinting for
low-copy-number strains.
Study sample.
Eighty-eight isolates of M. tuberculosis were included in this study. The sample represents
100 percent of the isolates having fewer than six copies of
IS6110 encountered during 1996 and 1997. During this time
frame IS6110 fingerprints were obtained for 322 isolates
(98.5% of the total culture-positive cases). All the patients included
in the study were residents of Arkansas at the time of their diagnosis.
The records of the Arkansas Health Department were used to ascertain
the study population.
DNA fingerprinting and spoligotyping.
Isolates of M. tuberculosis were cultured on Lowenstein-Jensen medium.
Chromosomal DNA was extracted from the isolates with chloroform-isoamyl
alcohol as described previously (19). IS6110 fingerprinting and pTBN12 fingerprinting were performed according to
standard procedures (10, 26). Restriction endonucleases PvuII and AluI were used to cleave DNA for
IS6110 DNA fingerprinting and pTBN12 secondary typing,
respectively. The IS6110 probe used is a PCR product
complementary to the sequence on the right side of the PvuII
site within IS6110, extending from bp 568 to 1089. The PGRS
probe, designated pTBN12 probe, is a 3.4-kb insert of a PGRS contained
in the recombinant plasmid pTBN12. The preparation of the two probes
was as described previously (30). Both probes were labeled
with the enhanced chemiluminescence direct nucleic acid labeling system
(Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire,
England). Spoligotyping to detect 43 known spacer sequences in the DR
locus of M. tuberculosis was performed as described
previously (17). Approximately 20 ng of DNA from each isolate was used as a target for spoligotyping.
Analysis of genotyping results.
IS6110
fingerprints were analyzed by computer-assisted analyses using Bioimage
(Ann Arbor, Mich.) Whole Band Analyzer software (version 3.4).
Fingerprints were matched by the average linkage clustering method. Any
two fingerprints that were 100% matched by computer analysis at a band
deviation of 2.5% were considered to be identical. Fingerprints
generated by pTBN12 secondary typing were compared by visual
inspection. Bands that were larger than 1.6 kbp were taken into
consideration in the comparison. Spoligotyping results were read
manually. Results expressed as the presence (by using the letter
"O") or absence (by a dot) of each of the 43 spacer sequences were
entered into Microsoft Excel software and sorted by the software
according to the similarity of the patterns. Spoligotypes that matched
exactly were considered to be identical.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Spoligotyping and Polymorphic GC-Rich Repetitive
Sequence Fingerprinting of Mycobacterium tuberculosis
Strains Having Few Copies of IS6110
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Pattern designation. Each isolate was assigned an IS6110, a pTBN12, and a spoligotype pattern designation. Each different IS6110 pattern was designated with an arabic numeral, each new pTBN12 pattern was indicated with an uppercase letter, and a lowercase letter was used to designate each distinct spoligotype.
Epidemiological investigation. Demographic information, details of tuberculosis infection and disease, contact information, and social history of the patients were obtained by review of medical records and interview of the patients using an intensive standardized questionnaire (4). Patients who lived in the same household or had a close work or social contact were considered to have definite epidemiological links. Patients who had neither a close contact (personal contact) nor a casual contact (attending the same church or the same public places of any other type) were classified as having no epidemiologic links.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Diversity of DNA fingerprints.
Based on IS6110
fingerprinting alone, a total of 34 IS6110 fingerprint
patterns were observed among 88 low-copy-number isolates. Of the 34 patterns, 10 patterns are shared by more than 1 isolate, including a
total of 64 (73%) of the isolates studied. Of the 88 isolates, 40 different spoligotypes and 68 different pTBN12 patterns were seen.
Correlation among IS6110, pTBN12, and spoligotyping results
among the 64 isolates clustered by IS6110 is shown in Table
1.
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Clustering analysis of isolates.
Primary clustering analysis
was based on data from IS6110 fingerprinting. Subclustering
of isolates was based on a combination of (i) IS6110
fingerprinting and spoligotyping, (ii) IS6110 fingerprinting and pTBN12, and (iii) all three methods. Differences in clustering rate
were observed among the different combinations of typing methods (Table
2). A combination of IS6110
fingerprinting and spoligotyping did not decrease the clustering rate
significantly (P > 0.1) compared to the rate observed
with IS6110 alone. In contrast, a combination of
IS6110 and pTBN12 typing significantly increased the
discriminating power of genotyping, reducing the clustering rate
determined by IS6110 fingerprinting by 42% (P < 0.01). Using all three typing methods provided no significant change in clustering rate compared with data obtained with a
combination of IS6110 and pTBN12 fingerprinting. The size of
the largest cluster identified by IS6110-pTBN12 combined
typing is the same as that identified using a combination of all three
methods, equal to approximately half of the size of the largest cluster
determined by IS6110-spoligotyping and a third of the size
of the largest cluster determined based on IS6110 typing
alone.
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Correlation between epidemiological data and fingerprinting results. Epidemiological data were obtained for 67 (76%) of the 88 patients included in this study. The correlation between epidemiological data and fingerprinting results was studied among the isolates for which epidemiological data were available. There were no known epidemiological links among patients whose isolates were unique by IS6110 fingerprinting-spoligotyping (31 isolates). Of the 43 patients who had unique isolates according to IS6110-pTBN12 typing, two were epidemiologically linked. Epidemiological links were found in 11 (46%) of 24 isolates clustered by IS6110-pTBN12 typing and in 13 of the 35 (37%) isolates that were clustered by IS6110 fingerprinting-spoligotyping (Table 1).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In a previous study, a combination of IS6110 fingerprinting and spoligotyping reduced the clustering rate among 249 M. tuberculosis isolates having low IS6110 copy number from 84% (determined solely based on IS6110 fingerprints) to 55% (2). In the present investigation, all low-copy-number isolates found in a population-based study in Arkansas during a 2-year period were examined. A systematic comparison among the three typing methods showed that pTBN12 typing provides the best discrimination for low-copy-number isolates. Combined typing with IS6110 and pTBN12 decreases significantly the clustering rate, while combining IS6110 and spoligotyping does not change the clustering of isolates significantly; combining all three of the methods only slightly decreases the clustering rate over that achieved with IS6110 and pTBN12. These data indicate pTBN12 fingerprinting is better than spoligotyping with regard to identifying different strains of M. tuberculosis.
Multiple tandem repeats of the PGRS are present in about 100 genes in the M. tuberculosis genome (11). This is reflected in the diversity of PGRS fingerprints among clinical isolates of M. tuberculosis with low IS6110 copy numbers. Further improvement of the pTBN12 typing method could be directed towards increasing the resolution of complex PGRS-containing restriction fragments by electrophoresis and in developing more-specific PGRS probes. Use of additional DR spacer sequences in the spoligotyping technique may increase the differentiating power of spoligotyping.
In this study, all isolates that had unique IS6110 fingerprints had unique pTBN12 patterns. Since spoligotyping is more rapid and easier to perform than is pTBN12 fingerprinting, screening of low-copy-number isolates by spoligotyping prior to pTBN12 typing would reduce the tedious work associated with pTBN12 secondary typing and analyzing pTBN12 fingerprints. Thirteen (20%) isolates that were in IS6110 clusters were identified as unique by both spoligotyping and pTBN12 fingerprinting. Thus, the number of isolates requiring pTBN12 subtyping could have been reduced by 20% if spoligotyping had been used as a screening technique prior to pTBN12 typing.
Among the 17 isolates with two copies of IS6110, pTBN12 subtyping subdivided the spoligotype cluster into three subclusters and four unique isolates. Epidemiologic links were found in two of the pTBN12 subclusters but in none of the pTBN12 unique patterns in that spoligotype cluster. Epidemiologic links were found in three other IS6110 clusters that were not subdivided by pTBN12, so in these cases, spoligotyping was as useful as pTBN12. Epidemiologic links were absent among the isolates that were unique by spoligotyping and pTBN12 fingerprinting except for a link between two isolates in one spoligotype cluster that was subdivided by pTBN12.
Even though the spoligotyping is less discriminatory than pTBN12 fingerprinting it allows one to differentiate unique isolates and leads to the fast detection and direct typing of M. tuberculosis in clinical specimens. One can envision that this would be a practical approach to genotyping in laboratories outside the research setting where multiple typing methods are not available. As described in a previous report (14a), spoligotyping could be used for initial screening, and then isolates with identical spoligotypes could be sent to a specialized genotyping laboratory for further differentiation with IS6110 and pTBN12 fingerprinting. In laboratories where all three typing methods are available, spoligotyping could be used as the first-line secondary typing method to reduce the number of isolates requiring pTBN12 fingerprinting. A combination of two or three methods would be necessary for the differentiation of low-copy-number isolates in a detailed epidemiologic study.
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ACKNOWLEDGMENTS |
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This study was supported in part by the Centers for Disease Control and Prevention, National Tuberculosis Genotyping and Surveillance Network cooperative agreement.
We thank Bill Starrett and Michael Freeland for their excellent technical assistance in laboratory work during the study. We are also indebted to Don Cunningham and Stewart Matthews for their help in obtaining isolates and interviewing patients.
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FOOTNOTES |
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* Corresponding author. Mailing address: Central Arkansas Veterans Healthcare System, Medical Research Service, LR/151, 4300 West 7th St., Little Rock, AR 72205. Phone: (501) 257-4829. Fax: (501) 664-6748. E-mail: cavedonald{at}exchange.uams.edu.
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Journal of Clinical Microbiology, October 2000, p. 3572-3576, Vol. 38, No. 10
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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