Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Acre, Brazil

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.




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




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.




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

-RB

O-0

31

L.

(V.)

l.N

D14

M1

Low

erli

mb

4R

ound

ulc

erY

es

MH

OM

/BR

/2002/N

MT

-RB

O-0

32

L.

(V.)

l.N

D30

F5

Low

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mb/u

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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




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

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[email protected]

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




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




Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Acre, Brazil

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