Emerging Infectious Diseases
[Volume 5 No.3 / May - June 1999]

Research

Tuberculosis in the Caribbean: Using Spacer Oligonucleotide Typing to
Understand Strain Origin and Transmission

Christophe Sola, Anne Devallois, Lionel Horgen, Jrme Masetti, Ingrid
Filliol, Eric Legrand, and Nalin Rastogi
Institut Pasteur de Guadeloupe, Pointe  Pitre, Guadeloupe

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      We used direct repeat (DR)-based spacer oligonucleotide typing
      (spoligotyping) (in association with double-repetitive
      elementpolymerase chain reaction, IS6110-restriction fragment
      length polymorphism [RFLP], and sometimes DR-RFLP and
      polymorphic GC-rich sequence-RFLP) to detect epidemiologic
      links and transmission patterns of Mycobacterium tuberculosis
      on Martinique, Guadeloupe, and French Guiana. In more than a
      third of the 218 strains we typed from this region, clusters
      and isolates shared genetic identity, which suggests
      epidemiologic links. However, because of limited epidemiologic
      information, only 14.2% of the strains could be directly
      linked. When spoligotyping patterns shared by two or more
      isolates were pooled with 392 spoligotypes from other parts of
      the world, new matches were detected, which suggests imported
      transmission. Persisting foci of endemic disease and increased
      active transmission due to high population flux and
      HIV-coinfection may be linked to the recent reemergence of
      tuberculosis in the Caribbean. We also found that several
      distinct families of spoligotypes are overrepresented in this
      region.

Sequencing of the Mycobacterium tuberculosis H37Rv genome (1) has
facilitated the study of the biodiversity of M. tuberculosis around the
world (2). We investigated M. tuberculosis epidemiology and biodiversity in
the Caribbean region, where sequencing data are not available. Caribbean
islands possess independent and shared characteristics that justify
investigation of both the molecular epidemiology (3) and the evolutionary
history of M. tuberculosis (4). We used spacer oligonucleotide typing
(spoligotyping), double-repetitiveelement (DRE) polymerase chain reaction
(PCR) and IS6110-restriction fragment length polymorphism (RFLP) (5-7) to
understand the molecular epidemiology and reconstruct the phylogeny of M.
tuberculosis. The first step was to demonstrate that the chosen molecular
methods differentiated isolates with respect to their molecular clonality.
The second step was to demonstrate that under proper epidemiologic
circumstances, clonality implies active transmission. To eliminate sample
bias, we used all data for Guadeloupe during a 3-year period (1994-96) and
for Martinique and French Guiana during a 2-year period (1995-96).

Table 1. Demographic characteristics of Guadeloupe, Martinique, and        
French Guiana                                                              
-------------------------------------------------------------------------  
                                                                           
Data                    Guadeloupe   Martinique    French       Total      
                                                   Guiana
------------------------------------------------------------------------- 
Surface area (km2)          1,705       1,100       91,000      93,805    
Population (1990)         387,034     359,579      114,808     861,421     
Population (1996)         422,290     388,340      151,780     962,410     
Density (inhabitants          248         353            2          10
  /km(sup 2))                                                                                            
Birth rate/1,000                                                           
inhabitants                  16.8          14.4         29.2        17.8      
AIDS(sup a)                 700           383          561       1,644    
HIV-positive (%)(sup b)       1.4           0.8          1.8         1.3       
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(sup a)Cumulated cases on June 30, 1996.                                         
(sup b)On the basis of 7,087 serologic tests performed in departmental 
clinics in 1994.

M. tuberculosis may have developed through the domestical of cattle, and if
so, M. bovis was its most probable precursor (8). On the basis of
restricted allelic diversity, the speciation of M. tuberculosis is
estimated to have occurred 15,000 to 20,000 years ago (4). It is thought
that the spread of tuberculosis (TB) began in Europe around the middle ages
and reached the new world in the 16th century, Africa in the 19th century,
and only recently, remote regions such as Papua-New Guinea (mid-20th
century) and the Amazon (last quarter of the 20th century) (8). The study
of the genetic biodiversity of M. tuberculosis might help reconstruct this
evolutionary scenario. Previous work from our laboratory showed that a
significant proportion of patients had conserved (or ancient) strains of
tubercle bacilli (6), and analysis based on multiple genetic markers showed
genetic relatedness between some clusters of bacilli within Guadeloupe (9).

Demographics and Molecular Typing Methods

Guadeloupe,        Table 2. Epidemiologic and clinical data for 410
Martinique, and    tuberculosis (TB) cases reported to the health
French Guiana      authorities from 1994 to 1996 in the French
have major         West Indies
demographic        ---------------------------------------------------
differences
(Table 1).                            Guade-  Marti-  French   Total
                                      loupe   nique  Guiana
Guadeloupe and     ---------------------------------------------------
Martinique are
densely populated  TB cases             136     89     185      410
islands in the     Pleuropulmon         115     71     156      342
center of Lesser     -nary cases
Antilles; they     Total TB              11      8.2    40.6     14.20
have similar         incidencea
population sizes   Sex ratio              1.7    1.9     1.5      1.65
(417,000 and       PDR to INH (%)         4.4    2.2     3.5      3.4
388,000) and       PDR to RIF (%)         1.4     -       -        -
areas (1,705 
(km(sup 2)         Foreign-born TB       24.5   20.3    68       43.2
and 1,100 km(sup 2)  patients (%)
and homogeneous    TB-HIV coin-          28     19      25       24.6
populations (93%   fection (%)b
French
nationals).        ---------------------------------------------------
French Guiana is   (sup a)New cases/100,000 inhabitants, 1994 to 1996.
sparsely           (sup b)Total number of known HIV-positive cases among
populated          TB patients was 101. However, foreign-born patients
(150,000), its     represented 50% of all the TB-HIV coinfected patients
inhabitants        in Martinique, 60% in Guadeloupe, and 80% in French
scattered          Guiana. The % shown is a minimal estimation as HIV
throughout a       serology results were available only for 49% of
large territory    patients in Martinique, 75% in Guadeloupe, and 80%
(91,000 km2).      in French Guiana. 
Guadeloupe and     PDR, primary drug resistance; INH, isoniazid; RIF
Martinique are     rifampin.
characterized by   
an insular         
population,        
whereas French     
Guiana, which
borders Surinam and Brazil, is populated by persons of diverse geographic
and ethnic origin (many immigrants from South and Central America). The
distribution of TB cases among these three French overseas territories
reflects their individual demographics. In Guadeloupe and Martinique, 27%
and 25% of all TB cases, respectively, were imported, while in French
Guiana, 68% of all cases were imported (10). The basic epidemiologic data
(incidence, age, sex, nationality, HIV coinfection) for all TB cases
reported to health authorities describe a 3-year period (1994-96) (Table
2).

Of 136 TB cases reported in Guadeloupe, 107 had smear- or culture-positive
isolates, with culture available in 100 cases; we typed 95 (95%) of 100
isolates. Of 52 cases reported in Martinique, 41 had smear- or
culture-positive isolates, and a culture was available in 34 cases. We
typed 31 (91%) isolates. Of 124 cases reported in French Guiana, 96 had
culture- or smear-positive isolates, with a culture available in 76 cases;
we typed 73 (96%) of 76 isolates, along with three that grew in 1995 but
came from pathologic specimens received in 1994.

The isolates were first studied by spoligotyping (11), which is based on
polymorphism of the DR locus of M. tuberculosis (12); spoligotyping results
were analyzed by using the Recognizer and Taxotron software (Institut
Pasteur, Paris). The 1-Jaccard Index was calculated, and strains were
compared by the unweighted pair-group method using arithmetic averages
(UPGMA) (13) or by the neighbor-joining method (14). Secondary typing was
performed by DRE-PCR (15) or IS6110-RFLP (Table 3) (5). To determine
clonality between isolates, we used the following algorithm: strains were
considered clonal only when a combination of spoligotyping plus IS6110-RFLP
or DRE-PCR indicated they were identical. Data from DR-RFLP (12) and
SmaI-RFLP that used a polymorphic GC-rich probe (PGRS) (16) and available
epidemiologic information are also included in Table 3.

Table 3. Molecular fingerprinting and epidemiologic information from
spoligotyping-defined clusters shown in Figure 7.(sup a)
----------------------------------------------------------------------------
                    No. of strains typed   Summary of
                             by             results
Spoligo-  No. of                          obtained by      Clinical and
type      strains  IS6110   DR     DRE     molecular   epidemiologic data,
no.     harboring   RFLP    RFLP    PCR      typing             origin,
meth. 1  this type meth.2  meth.3 meth.4    methods(sup b)  observations
----------------------------------------------------------------------------
     1        2        2      2       2  Different by  Found in Surinam and
                                         meth. 2,      Guadeloupe
                                         Beijing IS6110
                                         pattern
     2        9        9      2       8  No subcluster 2 patients from
                                         by PGRS-RFLP: hospital A and 2
                                         all strains   from B + 2 patients
                                         identical by  with same surnames
                                         meth. 2 and 4
                                         IS-type J and
                                         by PGRS-RFLP
     3        3        3      1       3  2/3 strains   Found in Surinam and
                                         identical by  Guadeloupe, 2
                                         meth. 2, 3,   patients from
                                         and 4, IS-typehospital A
                                         P
     5        2        1      1       2  2/2 strains   Found in Martinique
                                         identical by  and Guadeloupe, no
                                         meth. 2 and 4,evident
                                         IS type not   epidemiologic link
                                         yet defined
   12         2        2      2       1  One spacer    Very common pattern,
                                         difference    represent both
                                         with type 14, active transmission
                                         identical by  and reactivation
                                         meth. 2 and 3,
                                         IS-type A
   13         2        2      2     ND   One spacer    Suspicion of
                                         difference    cross-contamination
                                         with type 14, (sampling in the
                                         identical by  same hospital in 3
                                         meth. 2 and 3,days)
                                         IS-type A
   14       15       15      13       8  Identical by  Very common pattern,
                                         meth. 2 and 3,represent both
                                         identical by  active transmission
                                         method 4 (one and reactivation
                                         band), IS-type
                                         A
   15         2        2      2       2  2/2 strains   Imported cluster
                                         identical by  (Surinam or
                                         meth. 2, 3,   Dominican Republic)
                                         and 4, IS-type
                                         C
   17         6        6      1       5  Subclustered  17B found in 2
                                         by PGRS-RFLP: Guadeloupean
                                         17B, 2 strainspatients
                                         identical by  hospitalized in same
                                         meth. 2, 3 andhospital B
                                         4, IS-type N
   29         5        5      4       5  5/5 strains   3 of 5 Guadeloupean
                                         identical by  patients
                                         meth. 2, 3,   hospitalized in same
                                         and 4, IS-typehospital B
                                         B
   30        2         2      2       2  2/2 strains   2 patients from the
                                         identical by  same part of
                                         meth. 2, 3,   Guadeloupe
                                         and 4
   31         2        2      1       1  2/2 strains   2 Found in French
                                         different by  Guiana and in
                                         meth.         Guadeloupe, no
                                                       epidemiologic link
   33         2      ND      ND       2  2/2 strains   Found in French
                                         different by  Guiana, no
                                         meth. 4       epidemiologic link
   34         2        2      1       2  2/2 strains   Patients from French
                                         identical by  Guiana, suspected to
                                         meth. 4,      be epidemiologically
                                         method 2:     linked
                                         inconclusive,
                                         under
                                         investigation
   42         3        3      1       3  3/3 strains   Found in Guadeloupe
                                         different by  (one patient) and
                                         meth. 2 and 4 French Guiana (2
                                                       patients), no link
   44         2        2     ND       2  2/2 strains   2 patients from St.
                                         identical by  Maarten (couple)
                                         meth. 2 and 4
   45         6       1      ND       4  4/6 strains   5 patients from
                                         identical by  Martinique, one from
                                         meth. 4. (2:  Guadeloupe, under
                                         pending)      investigation
   46         3       1      ND       3  2/3 strains   Found in a
                                         identical by  Martinique and a
                                         meth. 4       Guadeloupe patient,
                                                       under investigation
   50(sup c) 29       16       8     20  Subclustered  Imported clusters
                                         by meth. 2, 4 (Hati), other links
                                         and PGRS-RFLP,under investigation
                                         IS-type E (3
                                         pat.) and F (2
                                         pat)
   51         4        3      2       3  2/4 strains   Imported cluster
                                         identical by  (Hati) for 2
                                         meth. 2 and 4 patients, other
                                         (2: pending)  links under
                                                       investigation
   53c      29       10       8     20   Subclustered  Patients from
                                         by meth. 2, 4 cluster T come from
                                         and PGRS,     the same ward of
                                         IS-type K (2  hospital A, 1996
                                         pat.) and T (3
                                         pat.)
   61         2        2      1       1  2/2 strains   Found in French
                                         identical by  Guiana in
                                         PGRS-RFLP, IS Guadeloupe, under
                                         results: underinvestigation
                                         investigation
   63        2         2      1       2  2/2 strains   2 patients from
                                         identical by  hospital A in
                                         meth. 2 and 4,Guadeloupe, under
                                         IS-type R     investigation
   64         1        1     ND       1  ***           2 isolates from one
                                                       single patient
   65         2      ND      ND     ND   (Results      Found in French
                                         pending)      Guiana, under
                                                       investigation
   66         2        2     ND       2  2/2 strains   Found in French
                                         identical by  Guiana, same surname
                                         meth. 2 and 4
   67         2        1     ND       2  2/2 strains   Found in French
                                         identical by  Guiana, under
                                         meth. 2 and   investigation
                                         PGRS-RFLP
   68         2        1     ND     ND   (Results      Found in one
                                         pending)      Martinique patient
                                                       and in Barbados,
                                                       under investigation
----------------------------------------------------------------------------
(sup a)Of 218 isolates typed, 145 isolates were grouped in 27 distinct
spoligo-defined clusters, which were further analyzed by one or more typing
methodsIS6110-RFLP (meth. 2), DR-RFLP (meth. 3), and DRE-PCR (meth.4), and
sometimes PGRS-RFLP when DRE-PCR or IS6110-RFLP results were inconclusive
or unavailable.
(sup b)Isolates with matching spoligotypes and matching IS6110 patterns (meth. 1
and 2) or with matching spoligotypes and matching DRE-PCR patterns (meth. 1
and 4) were considered to make up a cluster of epidemiologically associated
strains.
(sup c)Noninformative spoligotype patterns that lack any discriminating power.
*** Two clinical isolates from a single patient.
RFLP, restriction fragment length polymorphism; DR, direct repeat; DRE,
double-repetitive element; PCR, polymerase chain reaction; PGRS,
polymorphic GC-rich probe; ND, not done.

Fig. 1. Distribution of
tuberculosis cases reported to
health authorities in Guadeloupe,
Martinique, and French Guiana,		[fig]
1994-96, by nationality. DomR =
Dominican Republic; Dom =
Commonwealth of Dominica.

TB Epidemiology

Epidemiologic data for the three territories have been described in
detail (Table 2) (10). The mean incidence of cases per 100,000
inhabitants for the period studied was 11 in Guadeloupe, 8.2 in Martinique,
and 40.6 in French Guiana. The sex ratio varied from 1.5 in French Guiana
to 1.9 in Martinique. In each territory, the highest proportion of 
TB cases was in persons more than 65 years old; most were reactivated 
infections. The highest number of cases was in adults 25 to 44 
years old; most were new cases due to active disease
transmission and the high rate of TB-HIV coinfections. Pleuropulmonary
disease was most common (80% to 85% of cases), and drug resistance was
rare; primary resistance to isoniazid was 2.2% to 4.4%; single drug
resistance to rifampin was not observed in Martinique and French Guiana and
was limited to 1.4% of cases in Guadeloupe. Four cases of
multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid
and rifampin) were diagnosed; these cases of secondary drug-resistance were
caused by noncompliance to standard antituberculosis chemotherapy. In
addition, the rate of TB-HIV coinfection in these three territories was
high (19% of total TB cases in Martinique, 25% in French Guiana, and 28% in
Guadeloupe). Coinfected patients were most likely men 25 to 44 years of age
and of foreign origin (1 of 2 TB-HIV coinfected patients was a foreign
national in Martinique, compared with 6 of 10 in Guadeloupe and 8 of 10 in
French Guiana). These figures agree with the epidemiologic data concerning
the distribution of patients on the basis of their nationality (Figure 1).

						[fig]
In the        Fig. 2. Nomenclature of the spoligotype database (from 1 to
last 20       69) based on published spoligotypes (n = 393) and on
years, TB     spoligotypes generated during this investigation (n = 218).
incidence     The corresponding hybridization patterns for oligonucleotides
has           1 to 43 (black square, hybridizing; empty square,
decreased     nonhybridizing) are shown. Type 1 is unique for the Beijing
considerably  type pattern, whereas type 69 is unique for the Manila type
in these      pattern. Bold characters illustrate patterns that have so far
territories.  been noticed only in Caribbean and neighboring Central
For           American isolates (specific types). Unbolded characters
example,      illustrate patterns common to those reported elsewhere
the           (ubiquitous types), whereas italicized characters with an
incidence     asterisk illustrates patterns not yet found in the Caribbean.
of new        BCG and H37Rv spoligotypes are shown as controls.
cases in
Guadeloupe,   
36 per
100,000
inhabitants in 1975, declined to 10 per 100,000 inhabitants in the late
1980s. If the same trend continued, TB would vanish by 2000; however, this
decline has slowed and incidence has remained at 10 to 12 per 100,000 since
1989. This reversal of decline is linked to poor economic conditions,
increase in unemployment, increase in drug and alcohol abuse, and
persistent immigration from countries with a high incidence of TB and
ongoing HIV epidemics.

The Spoligotype Database

To define predominant genotypes and trace the origin of strains and their
potential movement, we compared spoligotypes of Caribbean isolates with
those of other geographic regions. We built a database of 610 spoligotypes
(218 from our own investigation and 392 from other countries) with Excel
software. The database contains 167 patterns from Goyal et al. (17); 118
from Kamerbeek et al. (11); 106 from Goguet et al. (18); and a single
pattern, named the Manila family, from Douglas et al. (19). In this
database, 69 spoligotypes shared by more than two patients in any region of
the world were numbered types 1 to 69 (Figure 2): types 1-54 denote 395
spoligotypes that were initially ordered from empty to full squares read
from left to right, and types 55 to 69 represent 15 new shared types in
chronologic order and correspond to 215 spoligotypes investigated later.
The nomenclature is temporary until international guidelines are
established. Fourteen shared types might be specific for the Caribbean and
bordering Central American regions (patterns 5, 12, 13, 14, 15, 17, 29, 30,
54, 63, 64, 66, 67, 68 [bolded in Figure 2]); 23 shared types were found
both in the Caribbean region and in other parts of the world (in regular
font in Figure 2). The remaining 32 patterns were not present in the
Caribbean (highlighted by an asterisk in Figure 2).

                             Fig. 3. A dendrogram represents
                             spoligotyping results of 95 Mycobacterium
                             tuberculosis isolates from Guadeloupe (shared
                             patterns are shown by bold characters). From
                             top to bottom; type 53, ubiquitous; type 44,
                             described by Goguet et al. (two cases) (18);
                             type 54, which does not appear, is found only
                             in isolate 94142, a pattern also found in
                             French Guiana; type 37, which also does not
                             appear, described by Kamerbeek et al. (three
                             cases) (11); type 50, ubiquitous; type 63,
                             specific; type 61, which does not appear is
                             found only in isolate 96131, a pattern also
                             found in Barbados and French Guiana; type 51,
  	[fig]                  described by Goguet et al. (one case) (18);
                             isolate 96095, a M. bovis BCG clinical
                             isolate; types 12 to 14, specific types; type
                             5, which does not appear, is found only in
                             isolate 95068, a pattern also found in
                             Martinique; type 31, which does not appear, is
                             found only in isolate 94112, a pattern also
                             found in French Guiana and already reported by
                             Goguet et al. (two cases) (18); type 46, which
                             does not appear, is found only in isolate
                             95087, a pattern also found in Martinique and
                             was already reported (18); type 17, specific;
                             type 30, specific; types 45 and 15, which do
                             not appear, are found only for isolates 96129
                             and 94127, respectively, and share patterns
                             with isolates in Martinique and Surinam; type
                             29, specific; type 2, previously reported by
                             Goguet et al. (one case) (18); type 3, also
                             found in Surinam, has been described by
                             Kamerbeek et al. (one case) (11); type 1,
                             which does not appear, is found only in 95076,
                             a pattern also found in Surinam, and described
                             (20) as the Beijing type.

Fig. 4. A dendrogram illustrating spoligotyping results 
of 76 Mycobacterium tuberculosis isolates from French 
Guiana (shared patterns are shown in bold). Top to bottom; 
patterns 50 and 53, ubiquitous; types 54 and 36, which do not 
appear, are found only for isolates IPC99 and IPC57, 
respectively; type 34, a ubiquitous type that was limited 		[fig]
to French Guiana in this study; types 66 and 67, specific; 
type 42, ubiquitous; type 17, specific; type 33,
ubiquitous; type 51, ubiquitous; type 31, which does
not appear, is found only for isolate IPC23 and is shared
with 94112 in Guadeloupe; type 2, shared by four isolates in
Guadeloupe.

                     
Population Structure of M. tuberculosis in Guadeloupe

Of 95 isolates from Guadeloupe (Figure 3), 34 were a unique spoligotype, and
61 shared 13 patterns (six patterns were shared by only two isolates, and
seven patterns were shared by the remaining 49 [3 to 13 isolates per pattern]).
Patterns 2 (4 isolates), 14 (13 isolates), 29 (5 isolates), 50 (10 isolates), 
and 53 (12 isolates) were the major shared patterns from Guadeloupe and 
made up 72% of clustered isolates. The interpretation of the population 
structure from Guadeloupe shows the presence of important nodes on the 
dendrogram. As illustrated in Figure 3 (from bottom to top), three 
distinct patterns show strain 95076 (designated type 1 in our database and 
identical to the Beijing type commonly found in Asia [20]), the recently 
reported pattern 3 (11), and pattern 2 (18), respectively. Above the 
patterns 1-3 (pattern 1 is not shown) lies the previously undescribed 
pattern 29, which might be specific to our region. Two very different 
groups can be seen on the next node (the "lower" and "upper" groups). 
The "lower" group, which comprises 20 isolates and two shared 
patterns (17 and 30), shows a stepwise polymorphism. This
group contains many ubiquitous patterns (reported from at least three
geographic regions) shared by a variety of isolates from around the world
and concerns isolates 95016, 94105, and 94041 of shared types 20, 42, and
47, respectively. The "upper" group, which comprises the remaining 63
isolates and eight shared spoligotypes (types 12, 13, 14, 51, 63, 50, 44,
53), can be further divided into two subgroups: "upper ubiquitous," which
lies above M. bovis BCG strain 96095 and the "upper specific," which lies
below the BCG strain (Figure 3). The "upper specific" subgroup is
homogeneous (shared types 12, 13, 14, and strain 95068 of shared type 5)
and probably has been present in Guadeloupe for a long time (except in a
single isolate with pattern 5 from the neighboring island of Martinique).
The "upper ubiquitous" subgroup seems considerably closer to M. bovis BCG
than do other isolates of M. tuberculosis from Guadeloupe.

Population Structure of M. tuberculosis in French Guiana

The first population structure of M.    			[fig]
tuberculosis isolates from French       
Guiana is shown in Figure 4. Of 76
isolates, 30 had a unique spoligotype.  Fig. 5. A dendrogram illustrating
The remaining 46 shared a total of 11   31 spoligotyping results of
patterns: eight patterns (51, 33, 17,   Mycobacterium tuberculosis isolates
42, 67, 66, 65, 34) were shared by      from Martinique. From top to
only two isolates and three major       bottom, types 50 and 53,
patterns (pattern 2, 4 isolates;        ubiquitous; types 52 and 49 are
pattern 53, 9 isolates; pattern 50, 17  represented by a single isolate,
isolates) were shared by the remaining  respectively, M35 and M30, and are
30 isolates. The three major patterns   ubiquitous; isolates M10 and M7
represented 65% of clustered isolates   belong to specific types 17 and 68,
in French Guiana. Most clusters were    respectively found in Guadeloupe
common to those found in Guadeloupe,    and Barbados; M25, belongs to
as well as other regions of the world   specific type 5 observed in
(types 2, 17, 33, 34, 42, 50, 51, 53,   Guadeloupe; type 45, ubiquitous;
65), except two patterns that have      type 46, ubiquitous; isolate M23
been so far only reported from French   shares type 2, with isolates in
Guiana (types 66, 67). No isolate was   Guadeloupe and French Guiana.
of the Beijing type, which is
unexpected, considering the number of
persons of Chinese origin in French Guiana. A high degree of heterogeneity
was observed in French Guiana, which is not surprising given the large
surface area, high number of immigrants and persons of various ethnic
origins, and high TB incidence rate.

Population Structure of M. tuberculosis in Martinique

Of 31 isolates studied in Martinique (Figure 5), 19 had an unshared
spoligotype, and 12 shared 4 patterns (pattern 46, 2 isolates; pattern 45,
5 isolates; pattern 53, 2 isolates; pattern 50, 3 isolates). All the
clusters in Martinique were also found in Guadeloupe (45, 46, 50, and 53).
Pattern 45 is overrepresented in this population, which could suggest
active transmission of this clone of tubercle bacilli in Martinique. Other
patterns common in Guadeloupe are only poorly represented in Martinique:
type 2 (isolate M23), type 5 (isolate M25), and type 17 (isolate M10). Type
5 is specific only to Martinique and Guadeloupe. On the contrary,
Martinique shares some patterns with the rest of the world that are not
found in Guadeloupe or French Guiana (type 49, isolate M30; type 52,
isolate M35). Despite the small sample size of isolates from Martinique,
many isolates (clustered or unclustered) share published patterns from the
rest of the world (18 of 31 isolates studied).

Population Structure of M. tuberculosis in Other Caribbean Regions

      				   We also investigated 16 additional M.
         [fig]                   tuberculosis isolates (12 from Surinam and
                                 4 from Barbados). Although not
Fig. 6. A dendrogram             representative of the population studied,
illustrating spoligotyping       spoligotyping of these isolates allowed
results of 16 Mycobacterium      detection of some interesting links
tuberculosis clinical            (Figure 6). Fourteen unique types and one
isolates from Barbados and       shared type (pattern 53, two isolates)
Surinam. From top: type 53,      were found. Among the unclustered
ubiquitous; isolates Barb.3      isolates, strain 94030 from Surinam
and 94030 belong to specific     matched isolate 94127 from Guadeloupe
types 68 and 15,                 (type 15), strain 94018 from Surinam
respectively; isolates 94018,    matched type 19 from Holland (11), and
94020, 94034, and Barb.1         strain 94034 from Surinam matched isolate
belong to ubiquitous types       95047 (type 3) from Guadeloupe.
19, 1, 3, and 61,                Furthermore, two strains from Barbados
respectively.                    shared patterns with isolates from other
                                 geographic areas: Barb 3 to M7 (type 68)
from Martinique and strain Barb 1 to the pattern 61 from France (18).

Predominant Genotypes and Strain Origin and Transmission

The dendrogram representing the global structure of the population studied
(Figure 7) shows the potential historical or epidemiologic interregional
flux of M. tuberculosis between Caribbean and neighboring Central American
regions. Of the 218 isolates, 145 (66.5%) had 29 shared types. Twenty-five
isolates were not initially grouped but were clustered only when a
dendrogram of all 218 isolates was drawn. These interregional links defined
eight new types in the database (patterns 1, 5, 15, 31, 54, 61, 64, and 68)
for 15 isolates; the remaining 10 isolates enriched patterns already
present (Figure 7). These links, made by finding clonal strains in distant
geographic territories, do not necessarily define epidemiologic
relationships between these strains (21) but are unlikely to be due to
independent genetic evolution. The dendrogram is an indirect picture of the
common history of TB spread in this part of the world.

For example, spoligotype 2 was recently proposed to have originated in
Latin America (22). Our recent investigations favor this hypothesis as we
have traced this pattern in all three territories investigated (Martinique
[one isolate]), Guadeloupe [four isolates], and French Guiana [four
isolates]). Isolates from all nine patients were further investigated by
IS6110-RFLP, DRE-PCR, and SmaI-PGRS typing (23), and the results confirmed
the clonality of this specific cluster. Searching for this genotype in
other Latin American countries may help elucidate its origin and
distribution.

To define the clonality of the isolates and to    
be in accordance with the current practice for           [fig]
molecular typing of M. tuberculosis using         
spoligotyping as a first-line method (7, 11, 17,
18), we systematically performed a study of a     Fig. 7. A cumulative
spoligotyping-defined cluster by a second method  dendrogram for the 218
(IS6110-RFLP or DRE-PCR). When corroborated by    Caribbean isolates of
epidemiologic information, the clusters so        Mycobacterium
defined were considered evidence of ongoing       tuberculosis. New types
active transmission of TB (Table 3). However, in  not visible on individual
numerous cases, the epidemiologic data were not   dendrograms can now be
conclusive, and molecular clonality was not       observed (types 1, 5, 15,
considered direct proof of a link. Thus, despite  31, 54, 61, 64, and 68).
suspected links in 81 (37%) of 218 of cases on
the basis of molecular typing alone, a direct
epidemiologic link was demonstrated in 31 (14.2%) of 218 of cases. However,
future prospective studies including active case-finding and source tracing
may help increase the number of epidemiologically linked cases in our
region.

Distribution of Spoligotypes Bearing Geographic Specificities

To reconstruct the evolutionary tree of M. tuberculosis on the basis of 610
different spoligotypes (mostly from the United Kingdom, Holland, France,
the Caribbean, and the neighboring Central American region), we performed a
similarity search (Figure 2). Although the tree may not reflect the full
diversity of spoligotypes, it suggests the existence of distinct families
of spoligotypes with geographic specificities (Figure 8). Some strain
families may be related to specific populations, geographic regions, and
the history of TB spread. For example, the Beijing genotype, which is most
divergent in the tree shown in Figure 8 (type 1), would have undergone the
most extensive genetic evolution since the origin of M. tuberculosis. This
information is consistent with the mechanism of evolution of the DR locus,
which appears to proceed through losses of direct repeats (24,25).

Although in an unrooted tree, the positions of patterns 50 and 53 correlate
well with the isolates most widely represented internationally (116 of 610
isolates studied; 54 of 218 isolates from the Caribbean and neighboring
Central American region and 62 of 392 published spoligotypes from different
parts of the world) and may constitute a candidate root for the
interpretation of distances between isolates.

Genetic Evolution of Tubercle Bacilli

                                  The genetic evolution of tubercle
          [fig]                   bacilli is closely associated with the
                                  past and present of its host.
Fig. 8. A preliminary             Consequently, human migration,
phylogenetic tree obtained by     population mixing, and other
the neighbor-joining algorithm    sociodemographic factors have long
on the basis of the 1-Jaccard     played an important role in the spread
index (Sj=a/a+c, where a is the   and subsequent evolution of the M.
number of simultaneously          tuberculosis genome. The insular model
positive characters and c is the  of the Caribbean, in which human
number of discrepancies), which   migration (hence the introduction of the
is not exhaustive for all         disease) was estimated to occur only
existing phylogenetic links. A    approximately 400 years ago, is
total of 70 shared spoligotypes   particularly interesting for discovering
were analyzed (69 types shown in  conserved strains of tubercle bacilli
Figure 2 and Mycobacterium bovis  and for detecting rare M. tuberculosis
BCG).                             genotypes. In this context, the DR locus
                                  is a unique chromosomal region
                                  characteristic of the M. tuberculosis
complex, which shows a high degree of polymorphism that involves homologous
recombination and IS-mediated transposition (24). The high degree of
internal homology within the DR region of M. tuberculosis is likely to
favor such genetic rearrangements. Similarly, transposition of IS6110 is
instrumental in generating new subclones of M. tuberculosis (26).

Following the principle of Dollo parsimony, which assumes that losses of
genes are much more common and likely than independent evolutionary origins
(27), the evolutionary process of M. tuberculosis can be speculated to have
involved the loss of DR repeats. However, the tree in Figure 8 does not
perfectly represent all phylogenetic links between isolates as the
mechanisms of loss of DR elements (by homologous recombination or
replication slippage) could involve simultaneous loss of >1 of the 43
building blocks. The present phylogenetic analysis will be extended to
other multiple genetic markers to include variables recently proposed by
Sreevatsan et al. (4). However, construction of such trees on the basis of
the simultaneous feeding and computer analysis of multiple mycobacterial
markers remains cumbersome and constitutes a priority research topic for
the studies of M. tuberculosis genome evolution.

---------------------------------------------------------------------------

Acknowledgments

      The authors thank F. Pfaff, L. Schlegel, P. Levett, and P. Prabhakar
for sending the cultures for identification to the Institut Pasteur,
Guadeloupe, and F. Prudent, M. Berchel, and K.S. Goh for their expert
technical help.

      This project was financed by research grants provided by Dlgation
Gnrale au Rseau International des Instituts Pasteur et Instituts
Associs, Institut Pasteur, Paris, and Fondation Franaise Raoul Follereau,
Paris, France.

      Dr. Sola is Charg de Recherche at the Tuberculosis and Mycobacteria
Unit at the Pasteur Institute of Guadeloupe, which is headed by Dr.
Rastogi. The authors work closely with public health agencies in
Guadeloupe, Martinique, and French Guiana to support the tuberculosis
control program. They provide expertise and training on the genetic
characterization and laboratory diagnosis of mycobacteria. Their current
research interests include molecular diagnostics and epidemiology, drug
resistance, and mechanisms of mycobacterial pathogenicity.

      Address for correspondence: Nalin Rastogi, Unit de la Tuberculose &
des Mycobactries, Institut Pasteur, Morne Jolivire, B.P. 484, F-97165
Pointe  Pitre-Cedex, Guadeloupe; fax: 590-893-880; e-mail:
rastogi@ipagua.gp.

References

  1. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al.
     Deciphering the biology of Mycobacterium tuberculosis from the
     complete genome sequence. Nature 1998;393;537-44.
  2. van Helden PD. Bacterial genetics and strain variation. Novartis
     Foundation Symposium 1998;217:178-94.
  3. Small PM, van Embden JDA. Molecular epidemiology of tuberculosis. In:
     Bloom BR, editor. Tuberculosis: pathogenesis, protection and control.
     Washington: American Society for Microbiology; 1994, p. 569-82.
  4. Sreevatsan S, Pan X, Stockbauer KE, Connell ND, Kreiswirth BN, Whittam
     TS, et al. Restricted structural gene polymorphism in the
     Mycobacterium tuberculosis complex indicates evolutionarily recent
     global dissemination. Proc Natl Acad Sci U S A 1997;97:9869-74.
  5. van Embden JDA, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B,
     et al. Strain identification of Mycobacterium tuberculosis by DNA
     fingerprinting: recommendations for a standardized methodology. J Clin
     Microbiol 1993;31:406-9.
  6. Sola C, Horgen L, Goh KS, Rastogi N. Molecular fingerprinting of
     Mycobacterium tuberculosis on a Caribbean island with IS6110 and DRr
     probes. J Clin Microbiol 1997;35:843-6.
  7. Sola C, Horgen L, Masetti J, Devallois A, Goh KS, Rastogi N.
     Spoligotyping followed by double-repetitive element PCR as rapid
     alternative to IS6110-fingerprinting for epidemiological studies of
     tuberculosis. J Clin Microbiol 1998;36:1122-4.
  8. Stead WM, Eisenach KD, Cave MD, Beggs ML, Templeton GL, Thoen CO, et
     al. When did Mycobacterium tuberculosis infection first occur in the
     New World? Am J Respir Crit Care Med 1995;151:1267-8.
  9. Sola C, Horgen L, Devallois A, Rastogi N. Combined numerical analysis
     based on the molecular description of Mycobacterium tuberculosis by
     four-repetitive sequence-based DNA typing sequence. Res Microbiol
     1998;149:349-60.
 10. Rastogi N, Schlegel L, Pfaff F, Jeanne I, Magnien C, Lajoinie G, et
     al. La tuberculose dans la Rgion Antilles-Guyane: situation
     epidemiologique de 1994  1996. Bulletin Epidmiologique Hebdomadaire
     1998;11/98:45-7.
 11. Kamerbeek J, Schouls L, van Agterveld M, van Soolingen D, Kolk A,
     Kuijper S, et al. Simultaneous detection and strain differentiation of
     Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin
     Microbiol 1997;35:907-14.
 12. Hermans PWM, van Soolingen D, Bik EM, de Haas PEW, Dale JW, van Embden
     JDA. Insertion element IS987 from Mycobacterium bovis BCG is located
     in a hot-spot integration region for insertion elements in
     Mycobacterium tuberculosis complex strains. Infect Immun
     1991;59:2695-705.
 13. Sneath PHA, Sokal RR. Numerical taxonomy: the principles and practices
     of classification. San Francisco (CA): W.H. Freeman & Co.; 1973.
 14. Saitou N, Nei M. The neighbor-joining method: a new method for
     reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.
 15. Friedman CR, Stoeckle MY, Johnson WD, Riley LW.
     Double-repetitive-element PCR method for subtyping Mycobacterium
     tuberculosis clinical isolates. J Clin Microbiol 1995;33:1383-4.
 16. Poulet S, Cole ST. Characterization of the highly abundant polymorphic
     GC-rich repetitive sequence (PGRS) present in Mycobacterium
     tuberculosis. Arch Microbiol 1995;163:87-95.
 17. Goyal M, Saunders NA, van Embden JDA, Young DB, Shaw RJ.
     Differentiation of Mycobacterium tuberculosis isolates by
     spoligotyping and IS6110 restriction fragment length polymorphism. J
     Clin Microbiol 1997;35:647-51.
 18. Goguet de la Salmonire Y, Li HM, Torrea G, Bunschoten A, van Embden
     JDA, Gicquel B. Evaluation of spoligotyping in a study of the
     transmission of Mycobacterium tuberculosis. J Clin Microbiol
     1997;35:2210-4.
 19. Douglas JT, Qian L, Montoya JC, Sreevatsan S, Musser JT, van Soolingen
     D, et al. Detection of a novel family of tuberculosis isolates in the
     Philippines. 97th general meeting of the American Society for
     Microbiology, Washington: American Society for Microbiology Press;
     1997. p. 572.
 20. van Soolingen D, Qian L, de Haas PEW, Douglas JT, Traore H, Portaels
     F, et al. Predominance of a single genotype of Mycobacterium
     tuberculosis in countries of East Asia. J Clin Microbiol
     1995;33:3234-8.
 21. Yang Z, Barnes PF, Chaves F, Eidenach KD, Weis SE, Bates JH, et al.
     Diversity of DNA fingerprints of Mycobacterium tuberculosis in the
     United States. J Clin Microbiol 1998;36:1003-7.
 22. Prodinger WM, Pavlic M, Pedroza JC, Allenberger FJ. Tracing of a
     cluster of tuberculosis infections [abstract U-56]. 98th General
     Meeting of the American Society for Microbiology, Washington: American
     Society for Microbiology Press; 1998. p. 504.
 23. Jasmer RM, Ponce de Leon A, Hopewell PC, Alarcon RG, Moss AR, Paz A,
     et al. Tuberculosis in Mexican-born persons in San Francisco:
     reactivation, acquired infection and transmission. Int J Tuberc Lung
     Dis 1997;1:536-41.
 24. Groenen PMA, Bunschoten AE, van Soolingen D, van Embden JDA. Nature of
     DNA polymorphism in the direct repeat cluster of Mycobacterium
     tuberculosis; application for strain differentiation by a novel typing
     method. Mol Microbiol 1993;10:1057-65.
 25. van Soolingen D, de Haas PEW, Hermans PWM, Groenen PMA, van Embden
     JDA. Comparison of various repetitive DNA elements as genetic markers
     for strain differentiation and epidemiology of Mycobacterium
     tuberculosis. J Clin Microbiol 1993;31:1987-95.
 26. Bifani PJ, Plykaitis BB, Kapur V, Stockbauer K, Pan X, Lutfey ML, et
     al. Origin and interstate spread of a New York City
     multidrug-resistant Mycobacterium clone family. JAMA 1996;275:452-7.
 27. Meyer A. In: Harvey PH, Leigh Brown AJ, Maynard Smith J, editors. New
     uses for new phylogenies. London: Oxford University Press; 1996. p.
     322-40.

Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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