DNA Sequencing Confirms the Involvement of Leishmania (L.) Amazonensis in American Tegumentary Leishmaniasis in the State of São Paulo, Brazil

INTRODUCTION American tegumentary leishmaniasis (ATL) represents one of the most important public health issues in the world. An increased number of autochthonous cases of ATL in the Northeastern region of São Paulo State has been documented in the last few years, leading to a desire to determine the Leishmania species implicated. METHODS PCR followed by DNA sequencing was carried out to identify a 120bp fragment from the universal kDNA minicircle of the genus Leishmania in 61 skin or mucosal biopsies from patients with ATL. RESULTS DNA sequencing permitted the identification of a particular 15bp fragment (5’ …GTC TTT GGG GCA AGT... 3’) in all samples. Analysis by the neighbor-joining method showed the occurrence of two distinct groups related to the genus Viannia (V) and Leishmania (L), each with two subgroups. Autochthonous cases with identity to a special Leishmania sequence not referenced in Genbank predominated in subgroup V.1, suggesting the possible existence of a subtype or mutation of Leishmania Viannia in this region. In the subgroup L.2, which showed identity with a known sequence of L. (L.) amazonensis, there was a balanced distribution of autochthonous and non-autochthonous cases, including the mucosal and mucocutaneus forms in four patients. The last observation may direct us to new concepts, since the mucosal compromising has commonly been attributed to L. (V.) braziliensis, even though L. (L.) amazonensis is more frequent in the Amazonian region. CONCLUSIONS These results confirm the pattern of distribution and possible mutations of these species, as well as the change in the clinical form presentation of ATL in the São Paulo State.


INTRODUCTION
Leishmaniasis persists as a concern of public health. American tegumentary leishmaniasis (ATL) has been listed among the six most prevalent infectious-parasitic diseases in the world, with a growing number of cases in Brazil. 1 It is characterized by cutaneous, mucosal, and mucocutaneous forms, caused by the subgenus Viannia (V) and Leishmania (L). L. (V.) braziliensis predominates in a wider geographical distribution than other species in the subgenus Viannia in Brazil. In the subgenus Leishmania, L. (L.) amazonensis is primarily observed in the Amazonian forest region. Its distribution has recently increased, with autochthonous cases described in the southeastern region, where the human disease is relatively rare and presents in the localized and diffuse cutaneous forms. 2,3 Molecular biology data have demonstrated the complexity of the population structure of the Leishmania species and their vectors. Studies have demonstrated the occurrence of genetic polymorphisms among strains of L. (V.) braziliensis and L. (L.) amazonensis. 4-6 Dujardin et al. (2002) suggested that leishmania rearrange and adequately adjust their genome structure for the purpose of fitness and pathogenicity. 7 Since an increased number of ATL autochthonous cases has been diagnosed in the Northeastern region of the State of São Paulo (SP), it has become important to identify the Leishmania species involved as well as its pathogenecity. 1,8 For this proposal, PCR has been utilized with great specificity and sensibility. [9][10][11][12]

MATERIALS AND METHODS
Sixty-one patients --each with a clinical epidemiological and laboratory diagnosis of ATL attended at the Dermatology outpatient clinic at the Hospital of Clinics, Faculty of Medicine of Ribeirão Preto, University of São Paulo, between January 1993 and June 2002 --were included in the study after giving informed consent to participate. All patients and procedures were approved by the Ethics Committee (HCRP n.4134/02).

Skin and/or mucosal biopsy
Skin or mucosal biopsies were obtained from the border of the ulcerated lesion using a 4 mm punch under aseptic conditions and with local anesthesia. The specimen was frozen at -70ºC for later PCR processing.

Sample preparation for PCR
A 2 mm fragment of the frozen biopsy was placed in 1 mL of proteinase K (PK) buffer (0.1 M Tris, pH 8.0, 0.1 M EDTA, 1% SDS, and PK at a final concentration of 20 mg/ mL). The samples were incubated at 55ºC overnight and then heated to 95 o C for 10 min for PK inactivation. Phenol and chloroform extraction and ethanol precipitation were then performed. The precipitated DNA was then resuspended in 30 µL of distilled water.

Polymerase chain reaction (PCR)
The The PCR products were separated by electrophoresis in 1.5% agarose gel with 1X TBE (Tris-borateethylenediaminetetraacetic acid) buffer and stained with ethidium bromide.

DNA Sequencing
The PCR products were sequenced using the sense primer and the DNA Sequencing Kit Big Dye Terminator™ (Applied Biosystems) according to the manufacturer's instructions. Sequencing was performed with an ABI Prism 310™ Genetic Analyzer (Applied Biosystems).

Phylogenetic analysis
The similarities among the Leishmania sequences of the 61 samples were determined by the neighbor-joining (NJ) method. 14 This method uses a distance matrix between sequence pairs to generate dendograms. In this analysis, we included Leishmania sequences obtained from cultures (L. There were three samples of disseminated forms: n.34, a patient with a cutaneous form from Minas Gerais, which presented identity with subgroup V.2; n.17, a patient with a cutaneous form from Goiás co-infected with HIV; and n.103, a patient with a mucocutaneous form from the northeast region of the State of São Paulo. The latter two samples were assigned to the group L, having no correlation with a subgroup or identity with a reference sequence. It is important to note that in addition to n.17, n.38, a patient with mucocutaneous form from the State of Bahia, presented co-infection with HIV and was assigned to the subgroup V.2.

DISCUSSION
Since 1913, when Leishmania (V.) braziliensis was identified as the responsible parasite for epidemic ulcers in Bauru (a city in SP), that particular species has been implicated as the etiologic agent of ATL in SP. 2,16-20 Yoshida et al. (1979) 21 were the first to isolate leishmania from a wild animal, the Didelphis marsupialis aurita (identified as belonging to the subgenus Leishmania). In 1988, Tolezano et al. 18 isolated leishmania identified as Leishmania amazonensis from Akodon sp. (Rodentia, Cricetidae) captured in the Ribeira Valley region of SP. In agreement with Cupolillo et al., 22,23 the dendogram obtained by the NJ method consisted of two groups in which the distribution of the reference Leishmania was identified by subgenus Viannia (V) and Leishmania (L). In subgroup V.1, consisting exclusively of patients' sample sequences, 15 (93.75%) were autochthonous cases with the cutaneous form. It should be pointed out that six (40%) of the patients lived in Ribeirão Preto, SP. Surprisingly, the data in subgroup L.2 were obtained from 15 patients' sample sequences in addition to the reference sequence of L. amazonensis. Of these, seven (46.76%) corresponded to autochthonous cases.
Regarding the clinical forms, the cutaneous form showed uniform distribution amongst the groups. The mucosal and mucocutaneous forms also showed this distribution pattern. Four cases with the mucosal and mucocutaneous forms in subgroup L.2 were observed, which maintained identity with L. amazonensis. Of these, two patients represented autochthonous cases from the northeastern region of SP.
In Brazil, mucosal and mucocutaneous forms of leishmaniasis can be manifested exclusively as mucosal compromising or as a concomitant cutaneous lesion that may be concurrent or contiguous with the mucosal region, respectively, which are attributed to L. (V.) braziliensis. Nevertheless, L. (L.) amazonensis and L. (V.) guyanensis species can also be involved in mucosal form. 2 Of the four mucosal forms observed in subgroup L.2, only two showed a concomitant cutaneous lesion, with the possibility of mucosal compromise resulting from contiguity. The mucosal lesion was isolated in the other two cases, an observation that disagrees with past literature.
In our study, there were three cases with a disseminated form, one of them located in subgroup V.2, which maintains identity with the references L. braziliensis and L. guyanensis. The other cases, one cutaneous and the other a mucocutaneous form, were located in the group that identified with the subgenus Leishmania.
The autochthonous cases in subgroup L.2, which maintains identity with the L. amazonensis complex, showed an unexpected result, since this complex has been predominantly identified in the Amazon region. It is tempting to propose that in subgroup V.1, which contains strains from autochthonous cases (especially in the municipality of Ribeirão Preto) and does not identify with a particular Leishmania Viannia species, there may be a subtype or a mutation of the subgenus Viannia in the region under study. More extensive studies are needed in order to isolate and identify the Leishmania involved and to correlate it with wild vectors and hosts.
The primers used in the present study amplify a 120bp fragment in all Leishmania species. 24 It is important to note that the sense primer is complementary to the universal sequence of the kDNA minicircle. The initial intention was to sequence more common fragments of the kDNA minicircle in a search for mutations. These mutations were observed in several samples, perhaps explaining the diversity of the strains in the listed subgroups.