Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil

Abstract

Since the first report of the genus Leishmania, many species have been described. In Brazil, human leishmaniasis has been associated with eight Leishmania species, of which seven are responsible for cutaneous leishmaniasis (CL). In some endemic areas, CL is associated with only one species; however, in other areas, such as the Amazon, the etiology of CL can be assigned to many species. The multitude of highly similar Leishmania species in Brazil makes it difficult to develop an appropriate method of typing them. Most Leishmania species were first described based on epidemiological and biological characteristics, and these were later corroborated by Multilocus Enzyme Electrophoresis (MLEE), the gold standard technique for identifying Leishmania species. In an attempt to overcome the limitations of MLEE, many PCR-based methods have been developed and used for parasite identification. In the present study, we analyzed the sequence of the hsp70 gene in Leishmania species associated with human leishmaniasis in Brazil. This analysis led to the identification of restriction enzymes that could be used for PCR-RFLP-based identification. The results obtained were in complete agreement with those obtained by MLEE, suggesting that PCR-RFLP analysis of hsp70 could soon replace MLEE for routine Leishmania typing.

Introduction

Leishmaniasis is caused by at least 20 species of parasites from the genus Leishmania that are transmitted via the bite of various phlebotomine sandfly species. Factors that influence the prevalence of the disease's modalities include the many species of Leishmania and their variants, the insect vectors, the reservoirs and hosts, and the highly diverse ecosystem (Lainson and Shaw, 1987).

In Brazil, cutaneous leishmaniasis (CL) can be caused by six Leishmania (Viannia) species, as well as L. (Leishmania) amazonensis and occasionally L. (L.) infantum (syn. L. (L.) chagasi). The visceral manifestation of this disease is mainly caused by L. (L.) infantum. Species belonging to the L. (Viannia) subgenus are responsible for the most severe forms of the cutaneous disease, such as the oropharingeal mucosal form, and they have been implicated in disease relapse. L. amazonensis, while presenting as a mild form, is responsible for a diffuse form of the illness that does not respond to treatment. In the L. (Viannia) subgenus, two species are of the utmost importance: L. braziliensis is the main CL etiological agent and is widely distributed throughout the country, while L. guyanensis is an important species that is restricted to the north of the Amazonian basin, where it is the most prevalent species in this region. However, other L. (Viannia) species also have an important role in the epidemiology of the disease, which might involve, for example, differences in treatment response (Arevalo et al., 2007). In addition, the recent spread of species like L. naiffi (Pratlong et al., 2002, Kato et al., 2008), L. lainsoni (van der Meide et al., 2008, Tojal da Silva et al., 2006, Arevalo et al., 2007), and L. shawi (Felinto de Brito et al., 2009) indicates a change in leishmaniasis scenarios in the Americas.

The need for species identification in cases of leishmaniasis is very clear. Most Leishmania species have been described by considering clinical, epidemiological, and biological characteristics (Lainson and Shaw, 1987), and some can be distinguished by Multilocus Enzyme Electrophoresis (MLEE) (Cupolillo et al., 1994, Thomaz-Soccol et al., 1993) or other immunological, biochemical, or molecular methods (Cupolillo et al., 1995, Grimaldi et al., 1991, Botilde et al., 2006, Rotureau et al., 2006). MLEE is still considered the gold standard method for Leishmania species identification. However, this method has the disadvantage of the need to isolate and cultivate the parasites, which may impede and/or delay identification. For several L. (Viannia) species, successful isolation and maintenance of the parasite in culture is known to be very difficult (Herwaldt, 1999, Lainson and Shaw, 1987). Furthermore, although MLEE has been used by many authors to identify Leishmania species, the results obtained cannot be compared, as the methodologies used and/or the enzymes assayed are different.

Since the first use of polymerase chain reaction (PCR) for Leishmania detection (Rodgers et al., 1990), several methods have been developed to discriminate among the described Leishmania species (Cupolillo et al., 1995, Marfurt et al., 2003, Mauricio et al., 2006, Rotureau et al., 2006, Tintaya et al., 2004, Victoir et al., 1998). Considering both isolated strains and clinical material, one of the most promising methods has been PCR amplification of the internal transcribed spacer 1 of the rDNA gene (ITS1) and of the small subunit of the rDNA plus the ITS1 (Schonian et al., 2003, Rotureau et al., 2006). Potentially promising results were also obtained using the heat shock protein hsp70 as a PCR target; however, the authors concluded that the method was not able to discriminate all L. (Viannia) species (Garcia et al., 2004, Garcia et al., 2005, Garcia et al., 2007).

In the present study, we evaluated the nucleotide sequence of the hsp70 gene of several Leishmania species and/or isolates. Restriction enzymes able to distinguish among the species were selected, and PCR-RFLP profiling was conducted for these Leishmania species. Finally, we present this PCR-RFLP analysis of the hsp70 gene approach as a useful method for typing Leishmania species in Brazil.