JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1997, p. 2393–2397 Vol. 35, No.

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0095-1137/97/$04.0010
Copyright © 1997, American Society for Microbiology

NOTES

DNA Fingerprinting of Mycobacterium tuberculosis Strains from

Patients with Pulmonary Tuberculosis in Honduras
LELANY PINEDA-GARCIA,1–4 ANNABELLE FERRERA,1 AND SVEN E. HOFFNER3,4*
Department of Microbiology, Universidad Nacional Autonoma de Honduras, and Instituto Nacional Del Tórax,2
1

Tegucigalpa, Honduras, and Swedish Institute for Infectious Disease Control3 and Microbiology and
Tumorbiology Center, Karolinska Institute,4 Stockholm, Sweden
Received 23 December 1996/Returned for modification 12 March 1997/Accepted 9 May 1997

Mycobacterium tuberculosis isolates from 84 patients with pulmonary tuberculosis in Honduras were char-
acterized by restriction fragment length polymorphism analysis. Seventy-three different IS6110 patterns were
found; 63 of these were unique and 10 were shared by two to three strains each. Thus, no ongoing spread of
any specific clone of bacteria could be demonstrated.

Molecular genetic techniques, such as DNA fingerprinting
of Mycobacterium tuberculosis, offer new tools for understand-
ing the epidemiology of tuberculosis (2, 6, 8). Restriction frag-
ment length polymorphism (RFLP) analysis with the insertion
sequence IS6110 is useful for the characterization of M. tuber-
culosis, the identification of outbreaks, and, for example, trac-
ing nosocomial infections (1, 4, 5, 10). IS6110 is a 1,355-bp
compound that is specific for M. tuberculosis and that is present
in most strains at up to about 20 copies per genome (3, 9, 12,
15). Identical fingerprints for different isolates indicate recent
person-to-person transmission or a common source of infec-
tion.
This study was performed to investigate the relationship
among clinical isolates of M. tuberculosis from patients with
pulmonary tuberculosis in Honduras.
M. tuberculosis strains were isolated from sputum or bron-
choalveolar aspirates from 90 of 235 suspected tuberculosis
patients at the National Reference Hospital for Tuberculosis,
Instituto Nacional del Tórax in Tegucigalpa, Honduras, during
a 6-month period in 1994 and 1995 (11). Isolates from 84
patients (60 men and 24 women; age range 15 to 82 years)
could be analyzed by DNA fingerprinting.
All samples were examined by acid-fast microscopy (Ziehl-
Neelsen staining) and culture (Lowenstein-Jensen medium).
Identification and susceptibility testing of the isolated strains
were carried out as described previously (11). Extraction of
DNA, as well as the subsequent Southern blotting, hybridiza-
tion, and detection, was performed by standard methods (16).
The Gel Compar software, version 3.1b (Applied Maths
BVBA, Kortrijk, Belgium), was used to compare fingerprints
by the unweighted pair-group method of arithmetic averaging
with the Dice coefficient.
The IS6110 copy numbers in M. tuberculosis isolates from
Honduran patients ranged from 2 to 17, with 9 to 12 copies
being the most common, representing 55 strains. Seventy-three
different IS6110 RFLP patterns were found; 63 strains had
unique fingerprints, while isolates from two patients, or, in one
* Corresponding author. Mailing address: Swedish Institute for In-
fectious Disease Control, S-105 21 Stockholm, Sweden. Phone: 46 8
735 1415. Fax: 46 8 735 1358.
case, isolates from three patients shared 1 of 10 patterns (Ta-
ble 1).
Smear-positive samples were found for 64 (76%) of all pa-
tients, and an isolate derived from at least one smear-positive
patient was represented in each cluster of strains with identical
patterns (Table 2). Isolates derived from patients with smear-
positive disease were somewhat more common (17 of 21; 81%)
among strains belonging to a cluster than among the 63 strains
with unique patterns (46 of 63; 73%).
Identical or highly related strains were found much more
frequently in younger patients (age, 15 to 42 years) than older
patients (age, 43 to 82 years). Thirteen patients in the younger
age group were infected with strains belonging to one of six
clusters, and altogether, 20 of 56 strains had similarity co-
efficients of .90%. No identical strains were isolated from
the older patients, and only 2 of 25 strains from older pa-
tients were highly related. The higher prevalence of related
strains in the younger age group was statistically relevant
(P , 0.05).
Men were shown to be infected by related or identical strains
more often than women. Nine clusters with 18 strains and
altogether 28 highly (.90%) related strains were found among
the 60 isolates from men. No cluster and only six strains in
three related pairs were found among 24 isolates from women.
Resistant strains were isolated from 13 patients. In one case an
identical fingerprint was found for two multidrug-resistant iso-
lates from young men in Tegucigalpa with smear-positive tu-
berculosis. Apart from this cluster, drug-resistant strains were
represented in three pairs of strains with identical fingerprints.
In these pairs, one resistant and one susceptible isolate clus-
tered. Thus, no correlation was generally seen between drug
resistance and IS6110 patterns. For four patients, a subsequent
isolate was obtained after 13 to 15 months from patients in-
fected with drug-resistant M. tuberculosis isolates. Identical
band patterns were seen for isolates from two patients, and
very closely related fingerprints (one extra band was present in
one of the isolates) were found for isolates from the other two
patients. At the same time an increased level of drug resis-
tance was seen for isolates from three of the four patients
(Table 3).
Eleven of 76 tested patients were found to be human immu-
nodeficiency virus (HIV) positive. Only 5 of 11 strains isolated

2393
2394 NOTES J. CLIN. MICROBIOL.

TABLE 1. Clusters of 21 M. tuberculosis strains with identical RFLP patterns

No. of Smear
Strain no. Drug resistancea Sexc Age (yr) HIV statusb Area of Honduras
IS6110 copies resultb

H-1081/94 2d INH, RIF, SM Pos M 59 Neg San Isidro

H-23/95 2 Pos M 31 Neg San Pedro Sula

H-1233/94 4d Pos F 26 Not tested Potrerillos

H-1361/94 4 Pos M 56 Neg Tegucigalpa

H-1317/94 8 Pos M 25 Neg Aguan

H-1335/94 8 Pos M 20 Neg Tegucigalpa

H-1263/94 9 RIF Pos M 40 Neg Tegucigalpa

H-1063/94 9 Pos M 27 Pos Aguan

H-1303/94 9 Pos M 33 Pos Marcovia

H-136/95 9 Neg M Not known Neg Tegucigalpa

H-1216/94 10 Pos M 20 Neg Tegucigalpa

H-1333/94 10 Neg M 19 Pos Tegucigalpa

H-1284/94 11 Pos M 36 Neg Tegucigalpa

H-1220/94 11 Pos M 64 Neg Tegucigalpa

H-1274/94 12 Neg M 40 Pos Danli

H-1331/94 12 Pos M 19 Pos Tegucigalpa
H-1221/94 12 Pos F 32 Neg Tegucigalpa

H-44/95 12 INH, RIF Pos M 52 Neg San Antonio

H-1367/94 12 Pos M 33 Neg Tegucigalpa

H-1005/94 14 INH, RIF, SM, EB Pos M 23 Neg Tegucigalpa

H-1173/94 14 INH, RIF, SM, EB Neg M 29 Pos Tegucigalpa
a
INH, isoniazid (0.2 mg/liter); RIF, rifampin (2.0 mg/liter); SM, streptomycin (4.0 mg/liter); EB, ethambutol (5.0 mg/liter).
b
Pos, positive; Neg, negative.
c
M, male; F, female.
d
Too few bands were present to be sure of an epidemiological relationship between these strains.

from AIDS patients had unique IS6110 banding patterns, while Another patient was one of the young men with multidrug-
6 strains clustered with isolates from HIV-positive or HIV- resistant tuberculosis mentioned above. Among the isolates in
negative patients. Two of the three patients whose isolates this cluster, as in the three additional clusters of susceptible M.
belonged to the three-strain cluster were HIV-positive men. tuberculosis isolates, one strain was isolated from an HIV-
positive individual and the other was isolated from an HIV-
negative individual.
TABLE 2. Characteristics of the patients with tuberculosis with About 50% of all patients came from Tegucigalpa, the cap-
regard to IS6110 RFLP patterns ital city, or its surroundings. Nine of the 10 clusters of strains
Strains Strains
Total
Characteristics with 100% similarity with 90% similarity
no. of
related to patients (no. of strains/ (no. of strains/no.
patients TABLE 3. Drug susceptibility patterns of subsequent M.
no. of clusters) of groups)
tuberculosis strains isolated from four patients with drug-tuberculosis
Male 60 18/9 24/12 resistant
Female 24 0/0 6/3
Susceptibility to the followinga:
Strain no. Date of isolation
Agea INH RIF SM EB
,42 yr 56 13/6 20/11
.42 yr 25 0/0 2/1 H-1263/94 November 1994 R S S S
February 1996 R R S S
Tegucigalpa area 45 8/4 19/10
Other areas 39 2/1 6/3 H-1311/94 December 1994 R R S S
February 1996 R R S S
Drug-resistant 13 2/1 2/1
tuberculosis H-1346/94 December 1994 R R S S
February 1996 R R S R
Smear positive 64 14/7 26/14
Smear negative 20 0/0 0/0 H-44/95 January 1995 R R S S
February 1996 R R R R
HIV positive 11 2/1 2/1
a
INH, isoniazid (0.2 mg/liter); RIF, rifampin (2.0 mg/liter); SM, streptomycin
a
The ages of three patients were not known. (4.0 mg/liter); EB, ethambutol (5.0 mg/liter); S, susceptible; R, resistant.
VOL. 35, 1997 NOTES 2395

FIG. 1. Dendrogram showing the relationship of IS6110 banding patterns of Honduran M. tuberculosis strains isolated from 45 patients with pulmonary tuberculosis
in the Tegucigalpa area.
2396 NOTES
TABLE 4. Largest clusters of identical strains
No. of strains in largest cluster/total
Source
no. of strains (%)
Reference
Guadeloupe 12/51 (24) 14
Ethiopia 28/154 (18) 9 one could expect to find large clusters of identical strains. In
San Francisco 30/191 (16) 13
New York City 12/104 (12) 1
Denmark 19/186 (10) 17
Colombia 2/27 (7.4) 7 with pulmonary tuberculosis. Thus, 3.6% of strains belonged
Tunisia 10/190 (5.3) 9 to the largest cluster, which could be compared to earlier
Honduras 3/84 (3.6) Present study
The Netherlands 3/153 (2.0) 9 were in the largest cluster ranging from 2% in The Nether-
having identical fingerprints were isolated from patients from
the Tegucigalpa area; 6 of these clusters included isolates from
patients in other parts of the country. Moreover, many of the
isolates from the Tegucigalpa area had similar RFLP patterns;
11 (30%) of a total of 37 nonidentical strains belonged to eight
groups of highly related strains (similarity coefficient, .90%).
A dendrogram showing the relationship of these 45 strains is
presented in Fig. 1. Among the isolates from the 39 patients
living outside the capital area, only 4 (11%) of 37 nonidentical
strains belonged to pairs of highly related strains. The higher
prevalence of identical or highly related strains in the Teguci-
galpa area was statistically significant (P , 0.01).
DNA fingerprinting of M. tuberculosis isolates from selected
populations in various geographical areas has been used to
describe the ongoing spread of certain strains, e.g., drug-resis-
tant strains. An international standard epidemiological marker
(IS6110) and technique (a standard RFLP assay) and a stan-
dard data processing system (Gel Compar software) make
possible the pooling and comparison of the results obtained in
different studies (16). Isolates of M. tuberculosis which share
identical IS6110 fingerprints are most likely epidemiologi-
cally linked, and patients infected with these isolates were
probably infected from a related or a common source, while
patients infected with strains with unique banding patterns
more likely have relapses or were infected from separate
sources (1, 5). Thus, in a population with a high percentage
of M. tuberculosis strains belonging to identical clusters, a
significant ongoing spread of infection must be suspected
(13).
The present study in Honduras is the first in this region in
which epidemiological fingerprinting has been used. In Hon-
duras, with a population of 5 million, the incidence of tuber-
culosis is high (84/100,000 population), and drug-resistant
forms of the disease are common (11). A high degree of di-
versity was seen; 75% of the strains had unique IS6110 pat-
terns. Ten small clusters of two or three strains each with
identical fingerprints were detected. Isolates in two of the
clusters had an IS6110 copy number of four or less, which is
considered too low to prove that the strains are closely
related; thus, further studies would be necessary to demon-
strate that these strains are identical. It can be noted that
the four-copy pattern found in isolates from two patients
seems very similar to the pattern A recently reported among
isolates from 24% of patients in Guadeloupe, French West
Indies (14).
Drug-resistant tuberculosis and HIV are prevalent problems
in many developing countries. In Honduras, however, no
clonal spread of tuberculosis among AIDS patients or patients
with drug-resistant tuberculosis could be demonstrated. On the
other hand, drug-resistant strains shared the same fingerprints
with susceptible strains in 3 of 10 clusters. Thus, it seems that
J. CLIN. MICROBIOL.
drug-resistant tuberculosis in Honduras emerged in a number
of unrelated patients (who were perhaps noncompliant with
their therapy) rather than from an ongoing spread of initially
resistant M. tuberculosis strains.
In a population with a rapid ongoing spread of tuberculosis,
this study the largest cluster comprised only 3 strains among a
total of 84 tested isolates from the same number of patients
reports, with the proportion of strains whose fingerprints
lands to 24% of strains in the Caribbean island of Guade-
loupe (Table 4).
The major findings in this study were the high degree of
diversity and the lack of any certain clone infecting, for exam-
ple, patients with drug-resistant tuberculosis or AIDS. How-
ever, identical or highly related strains were seen more often in
certain groups, such as men, younger patients, smear-positive
patients, patients from the Tegucigalpa area, and AIDS pa-
tients. Clustering of RFLP patterns is considered to be related
to an ongoing transmission of infection (13). Thus, the result
could indicate that most of the recent transmission takes place
among patients belonging to these groups.
We acknowledge the technical assistance of Solomon Gebremichael,
SIIDC, and Jeanett Bauer, State Seruminstitute, Copenhagen, Den-
mark. We also thank Ada Pavón, Carlos Alvarado, and the medical
staff at the Instituto Nacional del Tórax.
This work was supported by the Karolinska International Research
and Training Programme and EU project BMH1-CT93-1614.
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