Species diversity causing human cutaneous leishmaniasis in Rio
Branco, state of Acre, Brazil
Anna Christina Tojal da Silva1, Elisa Cupolillo2, Angela Cristina Volpini2, Roque Almeida3 and Gustavo Adolfo
Sierra Romero1
1 Nucleo de Medicina Tropical, Universidade de Brasılia, Campus Darcy Ribeiro, Brasılia, DF, Brazil2 Laboratorio de Pesquisas em Leishmanioses, Departamento de Imunologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil3 Servico de Imunologia, Hospital Universitario Professor Edgar Santos, Universidade Federal da Bahia, Salvador, Brazil
Summary objective Information on Leishmania species diversity in western Brazilian Amazon and the clinical
picture of human cutaneous leishmaniasis it causes is scarce. We describe clinical findings, diagnostic
procedures and identification of Leishmania species in patients from that region.
methods The sample consisted of 50 patients, prospectively evaluated for epidemiological and clinical
characteristics by means of a structured questionnaire. Conventional and molecular tools were applied
to confirm the parasitological diagnosis and identify the species responsible for the disease.
results Patients were predominantly male (76.5%) and living in rural areas. Median average age was
18 years1 and median average disease evolution was 8 weeks. For the diagnostic procedures of leishm-
anin skin test, direct visualization of amastigotes in dermal scrapings and parasite culture of aspirates of
the ulcer border were positive for 98%, 52% and 34%, respectively. Molecular methods applied to
DNA extracted from skin biopsies of the 50 patients yielded 100%, 82% and 44% positivity by PCR
minicircle kDNA, PCR-RFLP ITS1rDNA and PCR-glucose-6-phosphate (G6P), respectively. Fourteen
samples from 13 patients were successfully isolated and identified. Multilocus enzyme electrophoresis,
PCR-RFLP ITS1rDNA and PCR-G6P permitted identification of the Leishmania species responsible for
the aetiology of American tegumentary leishmaniasis in 60% of the examined patients: 16 Leishmania
(Viannia) braziliensis, 12 Leishmania (Viannia) lainsoni, 1 Leishmania (Viannia) guyanensis and 1
putative hybrid of Leishmania (Viannia) naiffi and L. (V.) lainsoni.
conclusion The clinical and epidemiological behaviour of cutaneous leishmaniasis in Acre, Brazil, is
similar to other Amazon scenarios previously described; however Acre’s complex parasite diversity may
be contributed to the concomitant circulation of at least three distinct Leishmania species. The imple-
mentation of control interventions in the studied area must take into consideration the possibility of
various expected phlebotomine vectors and reservoirs.
keywords cutaneous leishmaniasis, Leishmania (Viannia) braziliensis, Leishmania (Viannia) lainsoni,
Leishmania (Viannia) naiffi, western Brazilian Amazon, genetic diversity
Introduction
American tegumentary leishmaniasis (ATL) is a zoonotic
disease caused by parasites belonging to the Leishmania
genus transmitted by sand fly bites. The widely dissemin-
ated disease afflicts individuals mainly in tropical and
subtropical regions. Many clinical presentations have been
described involving skin and the mucosal surface of the
upper airway, and the most common syndrome is charac-
terized by localized cutaneous disease. In Brazil, the genetic
diversity among the Leishmania parasites is great; at least
seven Leishmania species have been described as the
aetiological agent of human cutaneous disease: Leishmania
(Viannia) braziliensis, Leishmania (Viannia) guyanensis,
Leishmania (Viannia) shawi, Leishmania (Viannia) lain-
soni, Leishmania (Viannia) naiffi, Leishmania (Viannia)
lindenbergi and Leishmania (Leishmania) amazonensis
(Grimaldi et al. 1989; Shaw 1994; Silveira et al. 2002).
From 1980 to 2003, 552 059 cases of ATL were officially
reported in Brazil with an annual incidence from 3.83 to
22.94 cases per 100 000 inhabitants (Ministerio da Saude
2004). Knowledge on the geographical distribution of each
species causing ATL is scarce for some regions, such as the
state of Acre, in the western Brazilian Amazon, where the
disease is endemic (Figure 1). The Amazon region is
especially interesting because of its biological diversity of
Tropical Medicine and International Health doi:10.1111/j.1365-3156.2006.01695.x
volume 11 no 9 pp 1388–1398 september 2006
1388 ª 2006 Blackwell Publishing Ltd
potential vectors and reservoirs, which may quite possibly
favour the sympatric circulation of various Leishmania
species (Grimaldi et al. 1989; Lainson et al. 1994). From
1980 to 2003, reported cases from Acre increased from 41
to 1298, calling the attention of health officers to the
problem that is considered a public health challenge
because of its diagnostic, therapeutic and follow-up com-
plexity. Limited clinical and demographic data on cases
from Acre have been published from the information in
notification files, and there are no prospective studies
aiming at parasite species identification in that region (Silva
et al. 1999).
Species identification in ATL is important not only for
taxonomic purposes but also for prognostic issues, because
the response to treatment with pentavalent antimonial is
dependent upon parasite species (Navin et al. 1992;
Romero et al. 2001a). The gold standard for Leishmania
species identification is the multilocus enzyme electro-
phoresis (MLEE) approach that differentiates all parasites
causing ATL in Brazil (Rioux et al. 1990; Cupolillo et al.
1994). Monoclonal antibodies have been employed in
identification procedures as well as discrimination among
parasite species causing ATL, although less efficiently, as
there are no species-specific monoclonals available for
some of these species. Both techniques enable intraspecific
variation identification as extensively reported (Miles et al.
1981; McMahon-Pratt et al. 1982; Cupolillo et al. 1994;
Grimaldi & McMahon-Pratt 1996; Romero et al. 2002).
Molecular tools for species identification are under eval-
uation, and some techniques such as PCR and PCR-RFLP
are extremely promising (Castilho et al. 2003; Schonian
et al. 2003; Volpini et al. 2004).
The objective of our study is to contribute to the
understanding of ATL epidemiology in the state of Acre
located in the western Brazilian Amazon, by describing the
clinical findings, the positivity of diagnostic procedures and
the identification of Leishmania species responsible for
cases of cutaneous disease in this area.
Patients and methods
Patients
Our study was conducted in Rio Branco municipality
where 25% of the state’s ATL reported cases occurred in
the last 3 years. A convenience sample composed of
patients with suspected lesions was evaluated from March
to September 2002. Patients were invited to participate
during diagnostic consultation in the main State Laborat-
ory or during home visits by the National Foundation of
Health officers. Inclusion criteria were the presence of any
number of cutaneous lesions with more than 2 weeks of
evolution suggestive of leishmaniasis and a signed consent
agreement form. Exclusion criteria were the presence of
mucosal disease and children under 6 years old because of
the difficulties involved in invasive diagnostic procedures in
young children. This work was in accordance with
resolution 196/96 of the National Health Council of the
Ministry of Health of Brazil, which regulates research in
human beings. The Ethics Research Committee of the
Figure 1 Political map of Brazil and
neighbouring countries, detaching the stateof Acre with municipalities where patients
came from during the study period.
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
ª 2006 Blackwell Publishing Ltd 1389
University of Brasilia approved the study protocol. All
patients were treated following the recommendations of
the Brazilian Ministry of Health for leishmaniasis treat-
ment. ATL clinical cases were defined as patients with
clinically suspected lesions of more than 2 weeks evolution
plus positive leishmanin skin test or any positive parasito-
logical diagnostic test such as: direct visualization of
amastigotes, successful parasite isolation or amplification
of conserved kDNA region by the PCR described below.
Skin test
The Montenegro skin test was performed with an antigen
prepared as described formerly (Reed et al. 1986). Briefly,
0.1 ml of soluble components of L. (L.) amazonensis
promastigotes (MHOM/BR/86/BA 125 strain), with anti-
gen concentration of 250 lg/ml, was applied by intrader-
mic injection on the volar surface of the left forearm, and
the reaction was measured after 48–72 h. Induration
diameters ‡5 mm were considered positive.
Parasite isolation and visualization
Leishmania cultures of the ulcer border were collected
using the technique described previously (Romero et al.
1999a). Some patients underwent satellite lymph node
aspiration for the same purposes employing the procedures
described already (Romero et al. 1999b). Scarification of
the ulcer border was performed to obtain smears for direct
examination sought for amastigotes. Smears were fixed
with methanol, stained with Giemsa and observed via
optical microscopy (1000·).
Multilocus enzyme electrophoresis
Parasite identification was carried out by enzyme electro-
phoresis using 12 enzymatic loci as follows: hexokinase
(E.C.2.7.1.1), glucose-6-phosphate dehydrogenase
(G6PDH; E.C.1.1.1.49), isocitrate dehydrogenase
(E.C.1.1.1.42), malate deshydrogenase (E.C.1.1.1.37),
malic enzyme (E.C.1.1.1.40), acid phosphatase
(E.C.3.1.3.2), glucose phosphate isomerase (E.C.5.3.1.9),
nucleopeptidases 1 and 2 (E.C.3.2.2.1), proline peptidase
(E.C.3.4.13.9), phosphoglucomutase (PGM; E.C.1.4.1.9)
and 6-phosphogluconate dehydrogenase (6PGDH;
E.C.1.1.1.43); sample preparation and visualization pro-
cedures followed the protocols described by Cupolillo
et al. (1994). Seven reference strains of different Leish-
mania species were used as control for parasite identifica-
tion (Table 1). Isolates were evaluated using numerical
methods to determine their taxonomic positions and the
relationship among them. Briefly, the table containing the
electromorphs (alleles) positions was transformed in a
binary matrix, and then a similarity matrix was construc-
ted applying Jaccard’s coefficient to finally obtain the
phenogram using the UPGMA algorithm (NTSYS pro-
gram, version 1.70).
PCR and RFLP
Biopsies of the ulcer border were obtained with disposable
4 mm sterile punches and stored at )20 �C until the DNA
extraction procedure. DNA was extracted from biopsies
using the WizardTM Genomic DNA Purification System
(Promega, Madison, WI, USA) following manufacturer’s
instructions. In order to identify the species incriminated as
the ATL aetiological agent in the studied region, mainly
involving patients where parasite isolation was unsuccess-
ful, different PCR-based methods were employed: (1) PCR
to amplify Leishmania conserved region of minicircle
kDNA (mkDNA) of 120 base pairs that are Leishmania-
genus-specific (PCR mkDNA), adhering to exactly the
protocol previously described (Volpini et al. 2004), (2)
PCR-RFLP of ITS1 region from rDNA (Schonian et al.
Table 1 International code and zymo-
demes assignment of the Leishmania refer-
ence strains employed in this study�
International code IOC/Z� Species
MHOM/BR/75/M4147 23 Leishmania (Viannia) guyanensisMHOM/BR/75/M2903 27 Leishmania (Viannia) braziliensisIFLA/BR/67/PH8 07 Leishmania (Leishmania) amazonensisMHOM/BR/74/PP75 01 Leishmania (Leishmania) chagasiMHOM/BR/81/M6426 15 Leishmania (Viannia) lainsoniMDAS/BR/79/M5533 36 Leishmania (Viannia) naiffiMCEB/BR/84/M8408 26 Leishmania (Viannia) shawi
�The strains are deposited at the CLIOC – Colecao de Leishmania do Instituto OswaldoCruz (registration no. WDC 731, WFCC World Data Center on Microorganisms Directory
for identification procedures).
�IOC/Z: zymodeme of the Instituto Oswaldo Cruz Leishmania collection.
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
1390 ª 2006 Blackwell Publishing Ltd
2003), employing in each reaction 0.20 lm of each primer,
1.5 U of Taq polymerase and 1.5 mm MgCl2, followed by
one cycle of 4 min at 94 �C and 35 cycles of 94 �C for 30 s,
53 �C for 30 s and 72 �C for 40 s then finally an extension
cycle of 72 �C for 8 min and (3) PCR of the glucose-
6-phosphate (G6P) gene with combinations of primers
described as L. (Viannia) specific, non-L. (V.) braziliensis
and specific to L. (V.) braziliensis (Castilho et al. 2003).
The PCR reactions were conducted introducing some
modifications in the previously described protocols. In the
experiment directed towards discrimination between
L. (Viannia) and non-L. (Viannia) parasites, 50 pmol/ll of
the ISVC primer and 10 pmol/ll of the ISVA, 2 U of Taq
polymerase and 2 mm MgCl2 was utilized. An initial PCR
cycle at 94 �C for 4 min followed by 30 cycles of 94 �C for
30 s, 60 �C for 1 min and 72 �C for 30 s followed by one
final cycle at 72 �C for 8 min was adopted. The PCR cycle
used for the reactions, which employed primers to
discriminate between L. (V.) braziliensis and non-L. (V.)
braziliensis, consisted of one initial step for 4 min at 94 �Cand then 30 cycles of 94 �C at 30 s, 60 or 62 �C (for
ISVC + ISVB and ISVC + ISVNB, respectively) for 1 min
and 72 �C for 30 s with a final step of 8 min at 72 �C.
Positive PCR controls containing DNA from reference
strains of L. (V.) braziliensis, L. (V.) lainsoni, L. (V.)
guyanensis and L. (V.) naiffi (Table 1) together with a
negative control with no DNA were included in each
reaction set. In order to avoid the risk of PCR contamin-
ation, all reactions aiming at mkDNA amplification were
performed as described in Volpini et al. (2004). Briefly, (a)
the extraction, preparation of the PCR mix and amplifi-
cation were conducted in different rooms with separate
reagents, plastics and equipment and (b) the use of Ready-
To-GoTMsans PCR Beads (GE Healthcare, Piscataway, NJ,
USA) instead of the hand-made PCR reagent mix.
After PCR amplification, the amplicons corresponding to
the mkDNA were digested with restriction enzyme ApaLI
(PCR-RFLP mkDNA) and subjected to 6% polyacrylamide
gel electrophoresis (Volpini et al. 2004). The PCR products
of ITS1rDNA were digested with restriction enzyme
Sau3AI, subsequently exposed to electrophoresis in the
GenePhor apparatus (GE Healthcare) using a 12.5%
polyacrylamide gel (GeneGel; GE Healthcare). Amplicons
from the G6P gene were visualized in 6% polyacrylamide
gel electrophoresis. All gels were silver stained (DNA silver
staining kit; GE Healthcare).
Results
Of the 51 patients studied, 50 had localized cutaneous
lesions and one had the disseminated disease. For the
purpose of counting and lesions localization, we excluded
the patient with the disseminated disease who had 30
lesions. There were 88 lesions observed in 50 patients: 53
lesions (60.2%) appeared in the lower limbs, 22 (25.0%) in
the upper limbs, 10 (11.4%) in the trunk and 3 (3.4%) in
the face and neck. Other clinical findings and demographic
characteristics are described in Table 2.
Fifty patients were submitted to an aspiration procedure
of the ulcer border, and Leishmania cultures were positive
in 17 patients (34%), four isolates were lost during
transportation. Eight patients were submitted to lymph
node aspiration, and parasite isolation was successful in
one. The positivity of diagnostic procedures is described in
Table 3.
Fourteen isolates were obtained from 13 patients. In one
patient, there was successful parasite isolation from the
skin ulcer and its satellite lymph node. All isolates were
identified by enzyme electrophoresis and belonged to 10
different zymodemes, nine of them new enzymatic profiles.
Table 2 Demographic and clinical findings of 51 patients withcutaneous leishmaniasis attended in Rio Branco, Acre, Brazil from
March to September 2002
Characteristics
Number of
patients or
medians
% of
quartiles
Male 39 76.5
Median age (years) 18 15 and 34
Living in rural areas 27 52.9Median disease
evolution (weeks)
8 4 and 12
Signs and symptomsRegional lymphadenopathy
at first examination
10 19.6
Itching 30 58.8
Fever 8 15.7Secretion 41 80.4
Table 3 Positivity of diagnostic procedures in patients with
cutaneous leishmaniasis attended in Rio Branco, Acre, Brazil from
March to September 2002
Diagnostic test
No. of
positives/tested
Positivity
(%)
Leishmanin skin test 49/50 98
Direct visualization of amastigotesin scrapings from ulcer border
26/50 52
Ulcer skin culture 17/50 34
Lymph node culture 2/8 25
PCR mkDNA 50/50 100PCR-RFLP ITS1rDNA 41/50 82
PCR-G6P 22/50 44
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
ª 2006 Blackwell Publishing Ltd 1391
Eight isolates were new zymodemes of L. (V.) braziliensis,
one isolate was identical to zymodeme IOC/Z23 of L. (V.)
guyanensis, three isolates corresponded to a new zymo-
deme of L. (V.) lainsoni and one isolate named MHOM/
BR/2002/NMT-RBO-OO4P was identified as a putative
hybrid between L. (V.) lainsoni and L. (V.) naiffi. This
strain presented the same profile of L. (V.) lainsoni in 9 of
the 12 enzymatic loci used, in one locus the pattern
observed could not discriminate between the two species,
and in 2 loci (6PGDH and PGM) the patterns were
suggestive of heterozygotes for a dimeric and monomeric
enzyme, respectively. A representative panel of electroph-
oretic patterns of these isolates is shown in Figure 2. The
dendogram (Figure 3) demonstrates the relation among 14
isolates belonging to 10 different zymodemes.
Biopsies of 50 patients were obtained and DNA was
extracted to perform different PCR-based methods useful
for the aetiological diagnosis of ATL. The results of PCR
mkDNA, PCR-RFLP ITS1 and G6P exhibited 100%, 82%
and 44% positivity, respectively. After the digestion of the
120 bp fragment, representing the amplification of the
mkDNA, it was possible to determine that all the samples
analysed represent species belonging to the L. (Viannia)
subgenus. The patterns obtained through PCR-RFLP ITS1
from 41 samples displayed 13 compatible patterns with the
reference strains of L. (V.) lainsoni and 28 with either
L. (V.) braziliensis or L. (V.) guyanensis, as this meth-
odology has not enabled discrimination between L. (V)
braziliensis and L. (V.) guyanensis yet. The results of 22
positives for G6P demonstrate that 16 were L. (V.)
braziliensis and 6 were non-L. (V.) braziliensis. These
results were compared and in agreement with those
obtained by MLEE.
The three methods together (MLEE, PCR-RFLP
ITS1rDNA and PCR-G6P) enabled identification of the
Leishmania species responsible for the aetiology in 60.8%
(31/50) of the patients who had tissue biopsies for
molecular diagnosis. In the sample studied, 12 L. (V.)
lainsoni, 16 L. (V.) braziliensis, 1 L. (V.) guyanensis and 1
putative hybrid between L. (V.) naiffi and L. (V.) lainsoni
were detected. In 11 samples, it was not possible to
determine the Leishmania species involved in the infection,
but it is certain that the patients were infected with L (V.)
braziliensis or L. (V.) guyanensis, according to the PCR-
RFLP ITS1rDNA results. In 18% of the samples submitted
for molecular assays (9/50), it was only possible to
determine that the ATL was being caused by one of the
L. (Viannia) species, considering that only the PCR-RFLP
of the mkDNA yielded positive results. The putative hybrid
presented a profile identical to L. (V.) lainsoni by the PCR-
RFLP ITS1rDNA. Table 4 demonstrates the clinical and
diagnostic findings of the 31 patients whose Leishmania
575
57915
45 005
025
037
01113
65 004
02702
7g10
23 036
01356
601
803
504
002
913
65
Figure 2 One percent agarose gel showing some alleles observedfor the 6PGDH loci. Reference strains: 575 –L. (L.) amazonensis,579 –L. (L.) chagasi, 1545 –L. (V.) shawi, 1365 –L. (V.) naiffi,
1023 –L. (V.) lainsoni and 566 –L. (V.) braziliensis. The other
strains correspond to isolates from Rio Branco, Acre, Brazil.Arrow indicates the profile corresponding to the putative hybrid
(004) between L. (V.) naiffi and L. (V.) lainsoni.
0.00
L. guyanensis – Z23
L. braziliensis – Z27
0.25 0.50 0.75 1.00
100
83
L. shawi – Z26
013P – Z23 L. naiffi – Z36 L. lainsoni – Z15 027P044P027G036P004P – Z87
005P025P037P – Z79 018P – Z80 035P – Z81 040P – Z82 011P – Z83029P – Z84
100
90
Z86
Z78
Figure 3 Dendogram obtained through the
UPGMA algorithm and Jaccard’s similaritycoefficient.
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
1392 ª 2006 Blackwell Publishing Ltd
Tab
le4
Cli
nic
al,
dem
ogra
phic
and
dia
gnost
icfindin
gs
of
31
pati
ents
att
ended
inR
ioB
ranco
,st
ate
of
Acr
e,B
razi
l,w
ho
had
succ
essf
ul
para
site
iden
tifica
tion
thro
ugh
mult
ilocu
sen
zym
eel
ectr
ophore
sis
and/o
rm
ole
cula
rm
ethods
Isola
te/inte
rnati
onal
code
Spec
ies
IOC
/Z�
Age
(yea
rs)
Sex
No.
of
lesi
ons
Les
ion
site
Dis
ease
dura
tion
(wee
ks)
Les
ion
appea
rance
Lym
ph
node
involv
emen
t�
MH
OM
/BR
/2002/N
MT
-RB
O-0
05
L.
(V.)
b.
78
42
M2
Low
erli
mb
8R
ound
ulc
erN
ot
MH
OM
/BR
/2002/N
MT
-RB
O-0
08
L.
(V.)
b.
ND
23
M3
Low
erli
mb/u
pper
lim
b/t
hora
x4
Round
ulc
erN
ot
MH
OM
/BR
/2002/N
MT
-RB
O-0
09
L.
(V.)
b.
ND
7M
1A
bdom
en2
Round
ulc
erY
esM
HO
M/B
R/2
002/N
MT
-RB
O-0
11
L.
(V.)
b.
83
17
M2
Upper
lim
b20
Round
ulc
erN
ot
MH
OM
/BR
/2002/N
MT
-RB
O-0
12
L.
(V.)
b.
ND
14
M1
Low
erli
mb
4R
ound
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
15
L.
(V.)
b.
ND
6M
1L
ow
erli
mb
10
Round
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
17
L.
(V.)
b.
ND
18
M1
Hea
d9
Round
ulc
erY
esM
HO
M/B
R/2
002/N
MT
-RB
O-0
18
L.
(V.)
b.
80
14
M1
Face
4R
ound
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
22
L.
(V.)
b.
ND
29
F2
Low
erli
mb
16
Round
ulc
erN
ot
MH
OM
/BR
/2002/N
MT
-RB
O-0
23
L.
(V.)
b.
ND
38
M2
Low
erli
mb
12
Round
ulc
erY
esM
HO
M/B
R/2
002/N
MT
-RB
O-0
25
L.
(V.)
b.
78
14
M3
Upper
lim
b24
Round
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
28
L.
(V.)
b.
ND
21
M1
Low
erli
mb
8R
ound
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
29
L.
(V.)
b.
84
32
M2
Upper
lim
b8
Round
ulc
erN
ot
MH
OM
/BR
/2002/N
MT
-RB
O-0
35
L.
(V.)
b.
81
16
M1
Low
erli
mb
4R
ound
ulc
erY
esM
HO
M/B
R/2
002/N
MT
-RB
O-0
37
L.
(V.)
b.
79
16
M1
Thora
x3
Irre
gula
rulc
erN
ot
MH
OM
/BR
/2002/N
MT
-RB
O-0
40
L.
(V.)
b.
82
16
M1
Low
erlim
b7
Ver
ruco
us
Not
MH
OM
/BR
/2002/N
MT
-RB
O-0
45
L.
(V.)
b.
ND
25
M3
Low
erli
mb
4R
ound
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
10
L.
(V.)
l.N
D25
M3
Low
erli
mb/t
hora
x/a
bdom
en3
Round
ulc
erY
esM
HO
M/B
R/2
002/N
MT
-RB
O-0
14
L.
(V.)
l.N
D43
M1
Upper
lim
b3
Irre
gula
rulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
16
L.
(V.)
l.N
D41
F1
Hea
d6
Round
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
20
L.
(V.)
l.N
D10
F5
Low
erli
mb
2R
ound
ulc
erY
es
MH
OM
/BR
/2002/N
MT
-RB
O-0
21
L.
(V.)
l.N
D12
F1
Low
erli
mb
2R
ound
ulc
erY
esM
HO
M/B
R/2
002/N
MT
-RB
O-0
27
L.
(V.)
l.86
17
M3
Low
erli
mb
8R
ound
ulc
erY
es
MH
OM
/BR
/2002/N
MT
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Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
ª 2006 Blackwell Publishing Ltd 1393
parasites, including the putative hybrid, were identified by
any of the diagnostic methods.
Discussion
The increasing number of ATL cases in the state of Acre
may be ascribed to a number of factors such as the
improvement of the official surveillance system, migratory
movements for agricultural purposes of susceptible indi-
viduals to active transmission areas, progressive defores-
tation with negative impact on parasite, vector and
reservoir natural equilibrium and exploitation activities of
forest products such as fruits, rubber and lumber.
The clinical presentation of cutaneous leishmaniasis in
this study was similar to descriptions from other geo-
graphical regions of Brazil where the species belonging to
the Leishmania (Viannia) subgenus are responsible for the
majority of ATL cases (Grimaldi et al. 1989; Grimaldi &
Tesh 19932 ). The predominance of L. (V.) braziliensis or
L. (V.) guyanensis is also in agreement with the epide-
miology of ATL in Brazil where these species have been
associated with most of the cases described in the different
endemic regions (Grimaldi et al. 1989, Romero et al.
2001b). However, it is important to mention that the
relative proportion of each observed species in our study
might be inaccurate, as this work was based on a small,
non-random sample that may have excluded rare cases
caused by other species. In fact, some other species are quite
abundant in neighbouring countries (e.g. Bolivia and Peru
presenting several ATL cases associated with L. lainsoni
and Leishmania peruviana), which may quite possibly
affect the epidemiology of ATL in our studied region.
No clinical signs were specifically associated to any of
the species identified in our study reinforcing the fact that it
is almost impossible to identify the species causing the
disease based on clinical findings. The performance of
isolation procedures was lower than expected, if we
consider the experience with the same technique in other
regions where at least in 50% of the patients the parasite
was successfully isolated. There were no exclusion criteria
based on disease duration, which may be the determinant
factor to explain the results. In fact, patients with longer
disease more frequently had negative isolation procedures.
Despite our low unsuccessful Leishmania isolation, 13/50
(26%), which has also been reported by others in literature3
(Cuba et al. 1984; Weigle et al. 1987; Rodriguez et al.
1994), our data suggest a wide diversity of Leishmania,
with at least three different species plus a putative hybrid
circulating in the studied region. The diversity of genotypes
found was impressive with nine newly described zymo-
demes, seven of them belonging to the L. (V.) braziliensis
complex, confirming the great diversity of this species, as
already observed in other regions (Ishikawa et al. 2002;
Cupolillo et al. 2003).
The identification of one isolate as L. (V.) guyanensis,
belonging to the same zymodeme as the reference strain
(Cupolillo et al. 1994), which circulates in the north of the
Amazon River (Grimaldi et al. 1989; Romero et al.
2001a), suggests the possibility of this species spreading to
the western Brazilian Amazon. This patient had 5 weeks of
lesion evolution and had moved from Bolivia to Acre
7 months before contracting the disease. We did not
consider the possibility of the infection being contracted in
Bolivia; as far as we knew there were no reports of L. (V.)
guyanensis infection there4 .
New World parasites considered as hybrids have been
identified with monoclonal antibodies, MLEE, molecular
karyotyping and RAPD because of shared biochemical,
antigenic or genomic characteristics from two species
termed parental. These findings were previously described
with Old World parasites (Evans et al. 1987), however,
recently reported in the Americas (Bonfante-Garrido et al.
1992; Belli et al. 1994; Dujardin et al. 1995; Delgado et al.
1997). Of note is the fact that in this study, the first
description of Leishmania isolates exhibiting heterozygotic
profiles suggesting a hybrid pattern between L. (V.)
lainsoni and L. (V.) naiffi is introduced. Our results imply
that the hybrid parasite is genetically closer to L. (V.)
lainsoni than to L. (V.) naiffi. It is also important to
mention that we did not characterize any L. (V.) naiffi in
the sample examined by any of the adopted methodologies.
However, L. (V.) naiffi have been encountered infecting
humans and sylvatic animals in several areas of the
Brazilian Amazon region near the state of Acre (Naiff et al.
1991; Gil et al. 2003), and the dissemination of this species
throughout the South American countries has already been
demonstrated (Pratlong et al. 2002). The genetic mecha-
nisms involved in the formation of Leishmania hybrid
parasites remain unclear. Whether Leishmania parasites
presenting hybrid profiles are contributing to or are a result
of the genetic diversity detected in the area is another point
that needs to be addressed.
The fact that all biopsies were positive for PCR
mkDNA was not surprising as all were obtained from the
lesions of suspected ATL patients, and the sensitivity of
the technique is recognized. Although it has recently been
announced that human DNA can be amplified when some
primers targeted to Leishmania kinetoplast are applied
(Vergel et al. 2005), we excluded the possibility of false-
positive results. Previous studies utilizing the same prim-
ers employed herein displayed negative results when
human DNA obtained from patients presenting non-
leishmaniasis diseases was tested (Pirmez et al. 1999;
Marques et al. 2001). Furthermore, the PCR-RFLP
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
1394 ª 2006 Blackwell Publishing Ltd
patterns observed after digestion with ApaLI as well as
other restriction enzymes (Volpini A.C. & Cupolillo E.
2006, unpublished data5 ) were identical to those witnessed
when promastigote DNA from Leishmania species was
used. The positivity of PCR-ITS1rDNA (82%) and PCR-
G6P (44%) markers were low compared with mkDNA,
but altogether MLEE, PCR-RFLP ITS1rDNA and PCR-
G6PDH were able to identify the species involved in the
infection of 30 samples submitted to molecular diagnostic
methods.
The presence of L. (V.) lainsoni in the study region
should be explained by the dissemination of this species, as
a number of cases were reported in Peru and Bolivia, both
countries bordering the state of Acre (Lucas et al. 1998;
Martinez et al. 2001). The four isolates (two from the same
patient) identified as L. (V.) lansoni by MLEE belonged to
the same zymodeme (IOC/Z86); however they differed
from the reference strain of L. (V.) lainsoni (IOC/Z15) that
was isolated in the Eastern Brazilian Amazon region. A
comparison of L. (V.) lainsoni isolates from Acre, Peru and
Bolivia by MLEE could give more information about the
relationship and dispersion of these parasites. Disease
acquired outside the borders of the state of Acre was
considered during the interview prior to the diagnostic
procedure. In all the patients examined, only three declared
exposure outside the state of Acre. One patient had travel
history to Bolivia and the second one travelled to the state
of Roraima, Brazil. Both patients did not have positive
species identification of their Leishmania parasites and it
was possible only to determine that they were infected by
L. (Viannia) species. The third was infected with L. (V.)
guyanensis, as already commented.
The genetic diversity of Leishmania parasites from
patients presenting similar disease in the state of Acre is
important to reinforce the need of species identification
for individual purposes. The isolation of L. (V.) brazi-
liensis, a recognized cause of mucosal disease (Marsden
1985), could be relevant for prognostic counselling after
healing of cutaneous disease. The external validity of our
results is limited by the non-random sampling and the
exclusion of suspected mucosal disease; however, the
observed parasite diversity indicates that the scenario for
implementation of interventions could be complex in spite
of the actual relative proportion of cases that might be
attributed to each Leishmania species. Because of this
relevant parasite diversity, it would be reasonable to
expect heterogeneity of transmission cycles including
various phlebotomine vectors and reservoirs. Control
measures based on vector behaviour will be extremely
complex considering the peculiarities of each species, and
human exposure prevention could be difficult in a
region where public policies stimulate the permanence of
people in forested areas practising activities with low
impact on natural environment.
Acknowledgements
The research was funded by the following Brazilian
research agencies: the National Council for Scientific and
Technological Development of the Ministry of Science and
Technology (CNPq/MCT process 475106/-1-6; Elisa
Cupolillo is a CNPq fellow researcher) and the Carlos
Chagas Filho Research Foundation of the state of Rio de
Janeiro. The English version of the article was reviewed
and revised by Mitchell Raymond Lishon.
Table Available on Request: Electromorphs (alleles)
presented in each Leishmania (Viannia) zymodeme de-
tected infecting patients in Acre State, Brazil.
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Corresponding Author Gustavo Adolfo Sierra Romero, Nucleo de Medicina Tropical, Universidade de Brasılia, Campus Darcy
Ribeiro, Asa Norte, CP 04517, Brasılia, DF 70904-970, Brazil. Tel.: +55-61-3273-5008; Fax: +55-61-3273-2811; E-mail:
La diversite des especes responsables chez l’homme de leishmanioses cutanees a Rio Branco, Bresil
objectif Il y a peu d’informations sur la diversite d’especes des Leishmania en Amazonie bresilienne de l’ouest et sur la diversite des manifestations
cliniques de la leishmaniose cutanee humaine. Nous decrivons dans cet article les resultats cliniques, les procedures diagnostiques et d’identification des
especes de Leishmania rencontrees chez des patients de cette region.
methodes Notre echantillon de patients etait compose de 50 patients, prospectivement evalues sur leurs caracteristiques epidemiologiques et cliniques
au moyen d’un questionnaire pre-etabli. Des outils conventionnels et moleculaires ont ete utilises pour confirmer le diagnostic parasitologique et pour
identifier les especes responsables de la maladie.
resultats Les patients etaient principalement de sexe masculin (76.5%) et vivaient dans une zone rurale. L’age moyen etait 18 ans. L’evolution
mediane de la maladie etait de 8 semaines. Les resultats des tests cutanes a la leishmanine, des examens directs de visualisation des formes amastigotes
dans les sucs dermiques obtenus apres scarification et la mise en culture des serosites prelevees en bordure d’ulceration etaient respectivement positives
dans 98%, 52% et 34% des cas. Les tests moleculaires sur ADN extrait a partir de biopsies de peau etaient respectivement positifs chez les 50 patients a
100%, 82% et 44% par les techniques PCRmkDNA, PCR-RFLP ITS1rDNA et PCR G6P. 14 echantillons provenant de 13 patients ont ete isoles et
identifies avec succes. Grace aux techniques par MLEE, PCR-RFLP-ITS1rDNA et PCR-G6P, nous avons identifie les especes de Leishmania responsables
des manifestations cutanees pour 60% des patients examines. Il s’agissait de: 16 cas de L. (V.) braziliensis, 12 cas de L. (Viannia) lainsoni, 1 cas de
L. (V.) guyanensis et 1 cas parent hybride de L. (V.) naiffi et de L. (V.) lainsoni.
conclusion Les caracteristiques epidemiologiques et les manifestations cliniques des leishmanioses cutanees dans l’etat de l’Acre au Bresil, sont
semblables a celles des regions d’Amazonie precedemment decrites. Toutefois au moins 3 especes differentes circulent dans l’etat de l’Acre. Dans cette
region, la mise en œuvre de mesure de controle doit prendre en compte la diversite des vecteurs et des reservoirs impliques.
mots cles Leishmaniose cutanee, Leishmania (Viannia) braziliensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Amazone bresilienne
occidentale, diversite genetique
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
ª 2006 Blackwell Publishing Ltd 1397
Diversidad de especies causantes de leishmaniasis cutanea en Rio Branco, Brasil
objetivo La informacion sobre la diversidad de especies de Leishmania en el Amazonas occidental de Brasil y del panorama clınico que la leishmaniasis
cutanea humana causa es escasa. Describimos los hallazgos clınicos, los procedimientos de diagnostico y la identificacion de las especies de Leishmania
en pacientes de esa region.
metodos La muestra consistio en 50 pacientes, prospectivamente evaluados por caracterısticas epidemiologicas y clınicas, por medio de un cuestio-
nario estructurado. Se aplicaron herramientas moleculares y convencionales para confirmar el diagnostico parasitologico e identificar las especies
responsables de la enfermedad.
resultados Los pacientes fueron predominantemente del sexo masculino (76.5%), y habitantes de zonas rurales. La mediana de edad fue 18 anos y la
mediana de evolucion de la enfermedad fue de 8 semanas. En el procedimiento de diagnostico para la prueba cutanea de leishmaniasis, la directa
visualizacion de amastigotes en raspados dermicos y cultivo de parasitos de aspiraciones del borde de las ulceras fueron positivos para 98%, 52% y 34%
respectivamente. Los metodos moleculares aplicados para el ADN extraıdo de las biopsias de piel de los 50 pacientes produjeron 100%, 82% y 44% de
positividad para PCR mkDNA, PCR-RFLP ITS1rDNA y PCR G6P, respectivamente. 14 muestras de 13 pacientes fueron exitosamente aisladas e
identificadas. MLEE, PCR-RFLP-ITS1rDNA y PCR-G6P permitio la identificacion de las especies de Leishmania responsables por la etiologıa de ATL
en 60% de los pacientes examinados: 16 L. (V.) braziliensis, 12 L. (Viannia) lainsoni, un L. (V.) guyanensis y un hıbrido putativo de L. (V.) naiffi y L.
(V.) lainsoni.
conclusiones El comportamiento clınico y epidemiologico de la leishmaniasis cutanea en Acre, Brasil, es similar a otros escenarios amazonicos
previamente descriptos. No obstante, la complejidad de la diversidad parasitaria en Acre puede contribuir a la circulacion concomitante de por lo menos
tres especies distintivas de Leishmania. La implementacion de intervenciones de control en las areas estudiadas debe tomar en consideracion la
posibilidad de varios flebotominos vectores y reservorios.
palabras clave leishmaniasis cutanea, Leishmania (Viannia) braziliensis, Leishmania (viannia) lainsoni, Leishmania (Viannia) naiffi, Amazonas
occidental brasilero, diversidad genetica
Tropical Medicine and International Health volume 11 no 9 pp 1388–1398 september 2006
A. C. Tojal et al. Leishmania diversity in Brazil
1398 ª 2006 Blackwell Publishing Ltd
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