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Journal of Clinical Microbiology, April 2001, p. 1559-1565, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1559-1565.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Diversity of Mycobacterium
tuberculosis in Sicily Based on Spoligotyping and Variable Number
of Tandem DNA Repeats and Comparison with a Spoligotyping Database
for Population-Based Analysis
Christophe
Sola,1,*
Severine
Ferdinand,1
Caterina
Mammina,2
Antonino
Nastasi,3 and
Nalin
Rastogi1
Unité de la Tuberculose et des
Mycobactéries, Institut Pasteur, F-97165 Pointe-à-Pitre
Cedex, Guadeloupe,1 and Department of
Hygiene and Microbiology, "G. D'Alessandro" University, I-90127
Palermo,2 and Department of Public
Health, University of Florence, I-50134
Florence,3 Italy
Received 27 November 2000/Returned for modification 20 January
2001/Accepted 3 February 2001
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ABSTRACT |
In a previous study, we proposed to associate spoligotyping and
typing with the variable number of tandem DNA repeats (VNTR) as an
alternative strategy to IS6110-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on
tuberculosis. The aim of the present study was to further evaluate this
PCR-based typing strategy and to describe the population structure of
Mycobacterium tuberculosis in another insular setting,
Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping
and VNTR typing. All isolates were independently genotyped by the
standard IS6110-RFLP method, and clustering results between
the three methods were compared. The totals for the clustered isolates
were, respectively, 15, 60, and 82% by IS6110-RFLP,
spoligotyping, and VNTR typing. The most frequent spoligotype included
type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of
all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The
combination of spoligotyping and VNTR typing results reduced the number
of clusters to 43% but remained superior to the level of
IS6110-RFLP clustering (ca. 15%). All but one
IS6110-defined cluster were identified by the combination
of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by
IS6110-RFLP. Reinterpretation of previous
IS6110-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was
previously missed. Although less discriminative than
IS6110-RFLP, our results suggest that the use of the
combination of spoligotyping and VNTR typing is a good screening
strategy for detecting epidemiological links for the study of
tuberculosis epidemiology at the molecular level.
| |
INTRODUCTION |
DNA fingerprinting is an important
tool for differentiating the clinical isolates identified as
Mycobacterium tuberculosis at the subspecies level and for
studying the epidemiology of tuberculosis in a community (11,
32). Although restriction fragment length polymorphism (RFLP)
analysis based on the insertion sequence IS6110 is
considered a "gold standard" among DNA fingerprinting methods (23, 30), a number of limitations and drawbacks of
IS6110 fingerprinting have been demonstrated, both at the
theoretical level and at the practical level (18). First,
its discriminatory power for isolates with fewer than six
IS6110 copies is low and, consequently, secondary typing
using another independent genetic marker is often required
(3). Second, the existence of IS6110 insertion
preferential loci or ipl (4, 17, 34) raises an important issue; since IS6110 typing relies on random
IS6110 integration into the genome, the clonality between
two isolates by IS6110 typing, even in high-copy-number
isolates, may not always indicate recent transmission of tuberculosis
(8). Third, IS6110-RFLP is a labor-intensive
and time-consuming methodology, and adequate intralaboratory or
interlaboratory comparison between profiles is a difficult task
requiring intensive input and expertise from investigators. Thus, in
order to improve the comparison of RFLP profiles, specialized softwares
are needed (22). For these reasons, the worldwide
implementation of IS6110-RFLP technique remains difficult
and alternative, rapid, simple, and cost-effective genotyping methodologies are required. The advent of "spoligotyping," an alternative PCR-based technique based on the polymorphism of the direct
repeat (DR) locus gave promising results; however, it has been shown to
be less discriminative than IS6110-RFLP (15). In this context, the association of two PCR-based techniques was suggested as a potential alternative to IS6110-RFLP
(9). Subsequently, the association of spoligotyping and
double-repetitive-element PCR (DRE-PCR), a procedure based on the
detection of inter-IS6110-PGRS (polymorphic GC-rich
sequence) polymorphism (6) was proposed as an alternative
to IS6110-RFLP for epidemiological studies of tuberculosis
(19, 26). Nevertheless, DRE-PCR reproducibility was
recently shown to be suboptimal (16). Consequently, an
optimal association of two PCR-based genotyping methods to study
tuberculosis epidemiology still remains an open question. Aside from
spoligotyping and DRE-PCR (26), ligation-mediated PCR and
spoligotyping (1), spoligotyping and typing with the
variable number of tandem DNA repeats (VNTR) (5), and
recently spoligotyping and PGRS-typing (35) have also been
proposed as alternatives to IS6110-RFLP.
The aim of the present investigation was to apply PCR-based genotyping
methods on Sicilian isolates previously subjected to IS6110-RFLP typing (20) and to compare their
discriminative power to IS6110-RFLP. The discriminative
power of each method or each association of methods was calculated by
using the Hunter-Gaston Index (HGI [12]). Moreover, some
considerations on the genotype population structure of M. tuberculosis in this area were drawn in the light of these new
molecular findings. Our results show that the use of a combination of
independent PCR-based genotyping methods is a reliable although
less-discriminative strategy for performing molecular epidemiologic
studies on tuberculosis. We also show that each method produces a very
coherent set of data indicative of the clonal structure of M. tuberculosis strain population and that molecular clustering may,
in certain epidemiological contexts, further elucidate tuberculosis epidemiology.
| |
MATERIALS AND METHODS |
Patients.
The population studied included 106 patients (29 women and 77 men; male/female sex ratio of 2.65). The mean ages among
the female group were 45.6 years, excluding two children, and 42.6 years if these two patients are included. The mean age among the male
group was 40 years if four children and infants are included among the
patients and 42 years if they are excluded. In both populations (males
and females), two age groups of 20 to 29 and 50 to 59 years were the
most represented. This bimodal distribution may underline most of the
reactivation cases that occurred among the age group of 50 to 59 years,
whereas "recent" transmission cases were mostly limited to the age
group of 20 to 29 years. In Italy, the incidence rate has remained
stable between 1995 and 1996 at approximately 9 cases per 100,000 population, underlining the need for a continued surveillance and a
more effective control strategy (20).
Clinical isolates and DNAs.
A collection of 106 clinical
isolates was collected during a period of approximately 5 years (June
1994 to February 2000). Most of these isolates had been previously
characterized by IS6110-RFLP (20). All DNAs
were prepared by using the classical cetyltrimethylammonium bromide
method (33).
Genotyping methods.
Southern blotting with labeled
IS6110 DNA was performed as previously described using an
internationally agreed protocol (30). Briefly, M. tuberculosis DNA was digested with PvuII,
electrophoresed, and hybridized with a labeled 245-bp PCR-generated
probe. Each Southern blot included DNA from M. tuberculosis
strain 14323 as an external standard (30). Autoradiographs
of the Southern blots were compared by using Gel Manager software
(Biosystematica, Tavistock, United Kingdom), and RFLPs were clustered
by the unweighted pair group method using arithmetic averages (UPGMA
[24]).
Spoligotyping was performed using in-house prepared membranes according
to a published protocol (15). A Biodyne C membrane (Pall
Biosupport, Portsmouth, England) having 43 covalently bound oligonucleotides was prepared essentially as previously published (15) with slight modifications (31); 6 spacers were derived from M. bovis BCG sequence, and 37 spacers were derived from sequences present in the M. tuberculosis H37Rv DR locus. Briefly, the Biodyne C membrane was
incubated for 10 min in 12 ml of a freshly prepared 16% (wt/vol) EDAC
(1-ethyl-3-3-dimethyl aminopropyl carbodiimide). It was then rinsed in
deionized water for 2 min and placed in a Miniblotter 45 system
(Immunetics, Cambridge, Mass.). The first and last slots were filled
with 150 µl of diluted black ink. The 43 remaining slots were filled
with 150 µl of an amino-linked oligonucleotide solution in 500 mM
NaHCO3 (pH 8.4) as reported previously (15).
The membrane was removed and inactivated with 100 mM NaOH for 10 min
and then rinsed twice in 250 ml of 2× SSPE (1× SSPE is 0.18 M NaCl,
10 mM NaH2PO4, and 1 mM EDTA [pH 7.7])-0.1% sodium dodecyl sulfate (SDS) at 60°C for 5 min and twice in 20 mM
EDTA (pH 8) for 15 min at room temperature. It was kept at 4°C in
sealed plastic bags until use. A premix was prepared for 44 PCR
samples, with each final sample containing PCR reagents (49 µl) and
DNA (1 µl). Each tube contained 5 µl of Tth polymerase reaction Mix 10x (Eurobio, Les Ulis, France), 7 µl of 50 mM
MgCl2, 0.8 µl of a deoxynucleoside triphosphate mix
(dNTPs; 25 mM), 1 µl of each primer (DRa and biotinylated DRb, 20 pmol/µl), 0.5 U of Tth polymerase (Eurobio), and ultrapure
water to obtain a final volume of 49 µl. Diluted DNA (from 50 to 300 ng of DNA in 1 µl) was added to the tubes. The samples were subjected
after a 3-min denaturation to 96°C to 35 amplification cycles
(96°C, 1 min; 54°C, 1 min; 72°C, 30 s) in a first-generation
Perkin-Elmer Thermocycler (Perkin-Elmer, Norwalk, Conn.), followed by a
final extension step at 72°C for 10 min. The membrane was washed
twice for 5 min in 250 ml of 2× SSPE-0.1% SDS and placed in the
Miniblotter. Then, 30 µl of each PCR product was diluted into 135 µl of 2× SSPE-SDS 0.1%, heat denatured for 10 min at 100°C, and
frozen on ice for 5 min. Each slot was filled with the diluted PCR
product. A 1-h hybridization at 60°C in an oven was performed, and
the membrane was washed twice for 5 min at 60°C in 250 ml of 2×
SSPE-0.5% SDS. Detection was done after a 45-min to 1-h incubation of
the membrane in 14 ml of 2× SSPE 0.5% SDS containing 5 µl of
streptavidin-POD conjugate (Roche Biochemicals, Meylan, France). It was
followed by two 10-min washings at 42°C in 2× SSPE-0.5% SDS, two
5-min washings in 2× SSPE at room temperature, and a final 2-min
incubation in 20 ml of ECL Detection Liquids (Enhanced
Chemo-Luminescence Detection Kit; Amersham, Buckinghamshire, England).
The autoradiograms were developed using standard photochemicals after
10 min to 2 h of exposition on ECL Hyperfilms (Amersham).
VNTR typing was performed as described previously with slight
modifications (
7). PCR was performed in a total volume of
30 µl containing 3 µl of 10x recombinant
Taq buffer
(AP-Biotech,
Uppsala, Sweden), 2 mM MgCl
2, a 30 nM
concentration of each primer,
a 500 µM concentration of each of the
four dNTPs, 3 µl of dimethyl
sulfoxide, 0.6 U of recombinant
Taq (AP-Biotech), and 50 to 200
ng of DNA sample. Reactions
were run in a Perkin-Elmer 9600 Thermocycler.
An initial denaturation
of 12 min at 95°C was followed by 35 cycles
of denaturation at 94°C
for 30 s, annealing at 58°C for 1 min,
and extension at 72°C
for 2 min, with a final extension step at
72°C for 10 min. An aliquot
of the reaction tubes was run on a
3% Metaphor gel (FMC Bioproducts,
Rockland, Maine). Molecular
weight standards (100-bp ladder or
PhiX-
HaeIII; AP-Biotech) were
run every four to
five lanes. The molecular weight determination
of PCR fragments was
performed using Taxotron software (Taxolab;
PAD Grimont, Institut
Pasteur, Paris, France) on images digitized
using the Video-Copy system
(Bioprobe, Montreuil, France). Once
the length of the PCR fragments was
precisely calculated, the
number of copies for each exact tandem repeat
(ETR) was deduced
according to a previously published scheme
(
7) and documented
as a five-digit number representing
allele profiles ETR-A to ETR-E.
Analysis of spoligotyping results.
Spoligotyping results
were analyzed as previously described using Excel (Microsoft,
Cupertino, Calif.) and Taxotron (27) softwares. The
dendrogram was constructed by using UPGMA after pairwise comparison of
strains by calculation of the Jaccard Index (13, 24).
Pattern designation.
Each IS-clustered isolate was assigned
to its IS6110-defined cluster as previously published
(20). Spoligotypes described only once were designated as
"orphans." Spoligotyping-defined shared-types were designated for
each clinical isolate after comparison of spoligotyping results to a
proprietary database of spoligotyping, which contained 4,500 individual
spoligotype patterns of M. tuberculosis representative of
more than 50 countries. A limited version of this database was recently
described (28). VNTR results are given under the form of a
five digit format as reported previously (5).
| |
RESULTS |
Spoligotyping of the Sicilian M. tuberculosis clinical
isolates.
We first characterized the collection of 106 DNAs from
Sicilian M. tuberculosis by spoligotyping in a blind study.
A total of 104 results were obtained that identified 56 unique
patterns. A total of 63 clinical isolates were clustered (60% of total
isolates) in 15 clusters, whereas 41 clinical isolates harbored unique
profiles. The designation of the spoligotype was attributed by
comparison of the results to those contained in a database containing
4,500 spoligotype patterns from 50 countries (Fig.
1 and Table
1). New shared types were created when
the patterns were not found into the database (types 272 to 278) either
for new observed shared types (types 272, 273, 275, and 277) or when an
orphan isolate from this study matched an orphan isolate from our
database (types 274, 276, and 278). The distribution of the 63 clustered isolates was as follows: six clusters of 2 isolates (Fig. 1;
clusters for types 4, 213, 272, 273, 275, and 277), three clusters of 3 isolates (types 52, 71, and 159), one cluster of 4 isolates (type 33), three clusters of 5 isolates (types 34, 47, and 50), and two large clusters of 13 and 10 isolates, respectively (types 42 and 53). To
these 63 clustered isolates, we may also add 16 clinical isolates belonging to a previously identified shared type, according to the
database, but being unique in Sicily (Table 1). Thus, the total number
of unclustered isolates (isolates for which no matching pattern was
detected in the database) dropped to 25 instead of 41 previously (24%
instead of 40%). A dendrogram showing the population structure of
M. tuberculosis clinical isolates assessed was constructed by pairwise comparison of isolates using the Jaccard Index and the
UPGMA algorithm (Fig. 1).
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FIG. 1.
Dendrogram and schematic representation of spoligotyping
and VNTR typing results obtained with 104 clinical isolates of M. tuberculosis from Sicily. The scale represents the dissimilarity
index according to computation of the Jaccard Index and use of the
UPGMA algorithm. Columns A, isolate number; B, spoligotype description;
C, VNTR typing results; D, shared type identification number; E,
IS6110-RFLP cluster.
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TABLE 1.
Shared type designations, origins, and references for 16 orphan clinical isolates from Sicily that matched patterns in our
databasea
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VNTR typing of the Sicilian M. tuberculosis clinical
isolates.
The same clinical isolates were independently studied by
VNTR typing, and the results are shown in column C of Fig. 1.
Thirty-three unique types were observed upon VNTR typing with a total
of 84 clustered isolates found in 15 clusters (81% of total isolates). The most frequently observed genotype was allele combination 22433 (15 clinical isolates). This is positively correlated to the most prevalent
spoligotyping-defined major clade characterized by the absence of
spacers 21 to 24 and 33 to 36 (type 42 and derived shared types 33, 213, 273, and 275, plus two orphan isolates (strains 98 and 108), that
altogether represent more than 30% of the isolates. The second and
third most prevalent genotypes are, respectively, allele combinations
32333 and 32433 (13 and 12 clinical isolates). The first combination is
linked to spoligotypes 47 and 50, which belong to the Haarlem family of
tubercle bacilli (five isolates each or 4.8%) and which may be
tentatively defined by the simultaneous absence of spacer 31 and
spacers 33 to 36 and VNTR profile 32333 (16). A third
clade totaling 11 isolates (10%) was characterized by the simultaneous
absence of spacers 9 and 10 and spacers 33 to 36 (shared types 34 and
71 plus two orphan isolates M11 and 109) and was found to be linked to
the first VNTR allele (exact tandem repeat A [ETR-A] equal to four
copies). No Beijing type (spoligotype 1, VNTR allele 42435) was
observed in Sicily to date, whereas the ubiquitous shared type 53 (VNTR
variable) accounted for 10% of the clinical isolates. The description
of other less-represented spoligotypes were as follows: spoligotypes
types 44 (isolate 140), 150 (isolate 89), 159 (isolates 15, 99, and
152), 162 (isolate 100), and 169 (isolate C5) were identified in an
earlier study performed in Verona, Italy (1). Other types
were found in the United States or in Cuba, which may be explained by
the past history of emigration of Sicilians in the 1950s (Table 1).
Finally, three isolates (isolates M28, 106, and 147) were shown to
harbor a high exact tandem repeat-A (ETR-A) allele (>4), a
characteristic spoligotype (absence of spacers 29-32 and 34), and a low
IS6110 copy number (<5). All of these characteristics were
recently described in a cluster of clinical isolates from Guinea-Bissau
(14). By spoligotyping, these strains were determined to
belong to a larger clade of a likely Afro-Asian bovine descent
(28). Indeed, a cross-examination of their demographical
records confirmed that these cases belonged to patients originating in
Asia and/or Africa and may constitute imported tuberculosis cases.
Combination of spoligotyping and VNTR typing.
The
IS6110-RFLP results of most of the isolates were described
in an earlier study. This study showed that the most prevalent clinical
isolates in Sicily harbored 8 to 11 copies of IS6110 (20). Table 2 summarizes the
correlation observed between previously defined IS6110-RFLP
clusters and clustering results obtained in this study, as well as
previous epidemiological findings. In all but one case, the results
obtained by the three molecular methods were in total agreement, a
finding which confirms the clonality of the isolates studied. These
results suggest recent tuberculosis transmission; however, traditional
epidemiological investigation did not reveal a direct link between the
patients (Table 2). In one case, two IS6110-RFLP-clustered
strains were further subdivided by spoligotyping. This result suggests
that an unusual mutation event happened in the DR locus and that these
two isolates, although very closely related genetically, are not
epidemiologically linked. This result concerns a single case of
clustering of two pulmonary tuberculosis cases involving patients of 47 and 69 years, respectively, that were not involved in a recent
tuberculosis transmission chain. DRE-PCR of these two isolates further
confirmed that these two cases were not epidemiologically linked. On
the other hand, DRE-PCR confirmed the clonality of the 16 other
IS6110-defined clustered strains (results not shown).
The discriminative power of VNTR typing and spoligotyping was
calculated by using the HGI (
12). According to this
method,
this index should be superior to 90% for a typing method to be
taken as efficient. In the present study, the HGI was, respectively,
93.9 and 96.75% for VNTR typing and spoligotyping. When they were
used
in combination, the HGI increased to 99.1%, which is close
to the HGI
of 99.7% obtained with IS
6110-RFLP (Table
3).
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TABLE 3.
Comparison of discriminatory power of three different
typing systems used individually and in various combinations
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| |
DISCUSSION |
In a previous study, we proposed the association of spoligotyping
and VNTR typing as an alternative to IS6110-RFLP for
molecular epidemiological studies on tuberculosis (5). The
aim of the present investigation was to study this typing strategy in
another geographical and epidemiological setting, Sicily, and to
describe the population structure of M. tuberculosis strains
in this region of Italy. Thus, a collection of 106 DNA isolates was
independently genotyped by three methods. The totals of the clustered
isolates were, respectively, 15, 60, and 82% by IS6110-RFLP
typing, spoligotyping, and VNTR typing. The combination of
spoligotyping and VNTR typing results reduced the number of clustered
isolates to 43%. All but one IS6110-defined cluster were
identified by the combination of spoligotyping and VNTR typing,
suggesting that the use of a combination of PCR-based, independent
genotyping methods is a reliable genotyping strategy for performing
molecular epidemiologic studies on tuberculosis. Indeed, the
differences observed in clustering level between spoligotyping-VNTR
typing versus IS6110-RFLP typing suggest that these loci
evolve with different molecular clocks. For example, the
IS6110 molecular clock has been shown to be faster than the
one of spoligotyping (15, 16).
In Sicily, the molecular epidemiology of M. tuberculosis
strains was previously assessed by IS6110-RFLP during a
4-year period (20). Although this study led to the
detection of microepidemics in the community, the data suggested that
the rate of disease caused by reactivation largely exceeded the rate
caused by recent transmission. Our study confirms the former study by
validating all but one previously defined IS6110-RFLP
cluster. It also extends the description of the genetic diversity of
Sicilian M. tuberculosis clinical isolates by including
spoligotyping and VNTR typing data, thus allowing us to place the
Sicilian M. tuberculosis genotyping data within an Italian
historical and geographical framework. As shown in Table 1,
similarities between Cuban, American, and Italian spoligotypes were
detected, which may be tentatively explained by the past migration
history from Sicily toward the Italian peninsula or the American
continent. Inversely, similarities between African or Indian and
Sicilian strains appear likely to be an expression of more recent
population migratory flows to Sicily. In this study, new molecular
characteristics of Sicilian M. tuberculosis bacilli were
identified which could mirror the influence of the complex nature of
Sicilian peopling history on the epidemiology of tuberculosis. For
example, a major family of M. tuberculosis isolates (type 42) accounted for 26% of total isolates (Fig. 1) and appears to possess a high biogeographical specificity in our spoligotyping database for Latin America, the Caribbean, and Mediterranean Europe. It
is highly significant that type 42 was not present in any of the 1,283 M. tuberculosis isolates recently described in the United States (25). Indeed, this result suggests that the high
prevalence of this cluster in Europe may be due to a remnant clone that
mirrors a historically prevalent clade in this region. Continuous entry and leaving of M. tuberculosis strains, along with their
human hosts, in the Sicilian population contributes to heterogeneity and, consequently, increases the epidemiologic predictive value of
finding a molecular cluster by reducing the probability of clustering
by chance. Thus, the demographic instability of Sicily (migration into
and from the island) and the concentration of the population in large
urban centers such as Palermo may contribute to an enhanced reliability
of molecular typing compared to a large and stable rural community such
as the one found in Arkansas (2). On the other hand, a low
prevalence area and an unstable sociodemographic context are conditions
where traditional case findings and epidemiological inquiries are more
laborious and poorly effective. All of these characteristics fit
completely with the epidemiological and demographic situation in the
province of Palermo, thus validating clustering via molecular methods.
When we consider methodological issues, a multistep screening strategy
combining two highly reproducible but moderately discriminative PCR-based genotyping techniques (spoligotyping and VNTR typing) is an
efficient two-step strategy for potentially detecting epidemiologic links in a given community. This multistep screening approach is also
interesting because it gradually increases the discriminatory power and
consequently retains fewer and fewer strains for subsequent analysis.
It also provides a population genetics view of tuberculosis genotype
prevalence in a given setting by easily linking local and global
epidemiologic issues with the use of spoligotype and VNTR databases.
Last, but not least, our results indicate the coherence of molecular
typing results using independent genetic markers underlying the clonal
structure of tubercle bacillus populations. In the future, the study of
various independent molecular clocks of M. tuberculosis
genomes through individual as well as combined numerical phylogenetic
analysis will help shed light on the monophyletic versus the poly-
and/or paraphyletic hypothesis of tuberculosis origin.
| |
ACKNOWLEDGMENTS |
This work was supported by the Délégation
Générale au Réseau International des Instituts
Pasteur et Instituts Associés and the Fondation Française
Raoul Follereau, Paris, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
la Tuberculose et des Mycobactéries, Institut Pasteur de
Guadeloupe, Morne Jolivière, BP 484, F-97165 Pointe-à-Pitre
Cedex, Guadeloupe. Phone: 590-897-665. Fax: 590-893-880. E-mail:
csola{at}pasteur.gp.
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Journal of Clinical Microbiology, April 2001, p. 1559-1565, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1559-1565.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
