Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: Evaluation by two molecular markers
Introduction
American tegumentary leishmaniasis (ATL) is caused by Leishmania species of the subgenera Leishmania (Viannia) and Leishmania (Leishmania), which are transmitted to man and other domestic and wild mammals by the bite of sandflies, and is distributed from the south of the United States to the north of Argentina. In Brazil, ATL is found in all states and has shown a high incidence over the last 20 years. The main clinical forms of ATL are cutaneous leishmaniasis, mucosal or mucocutaneous leishmaniasis, and diffuse cutaneous leishmaniasis (Marzochi, 1992).
In the State of Rio de Janeiro, L. (V.) braziliensis is the most prevalent species that has so far been implicated in the cycle of ATL. However, more recently an autochthonous case of diffuse leishmaniasis due to L (L) amazonensis has been reported (Azeredo-Coutinho et al., 2007). Transmission mainly occurs in periurban areas where the primitive rain forest vegetation is being replaced with disorderly human occupation. In this context, adaptation of the vector Lutzomyia intermedia to the domiciliary and peridomiciliary environment has been observed, as well as the presence of infected humans, dogs and horses (Aguilar et al., 1987; Barbosa-Santos et al., 1994, Marzochi, 1992, Marzochi and Marzochi, 1994). The most frequent clinical manifestation is a recent single or double cutaneous ulcer located in areas exposed to the bites of sandflies, which shows a favorable response to antimonial therapy. Multiple or localized skin lesions in areas normally covered by clothing or lesions of long duration, mucosal lesions, an unfavorable response to antimonial treatment, and co-infection with Mycobacterium are eventually observed (Matos et al., 2005, Schubach et al., 2005).
It is known that the clinical manifestations of the disease (Alexander and Russell, 1992, Berman, 1997, Marzochi and Marzochi, 1994) and the response to treatment are related in part to the Leishmania species involved (Grogl et al., 1992, Navin et al., 1992). However, little is known about the importance of biological variability within the same Leishmania species. Studies conducted in Rio de Janeiro suggest that only one zymodeme (Z 27) of L. (V.) braziliensis is involved in the disease in humans and animals (Cupolillo et al., 2003). Several methods can be applied to molecular epidemiology and to the study of genetic variability. Multilocus enzyme electrophoresis (MLEE) is widely used for the taxonomic characterization of Leishmania species (Cupolillo et al., 1994, Momen et al., 1985, Pacheco et al., 1999) but is limited because the marker is less polymorphic. Some techniques among others, are more frequently applied to the study of genetic diversity in trypanosomatids, including the restriction fragment length polymorphisms of kDNA (RFLP) analysis (Tojal Silva et al., 2006), random amplified polymorphic DNA (RAPD) analysis (Hanafi et al., 2001, Ishikawa et al., 2002, Martinez et al., 2003, Pacheco et al., 2005) and low stringency single specific primer—polymerase chain reaction (LSSP-PCR) analysis (Ferreira et al., 2007).
The objective of the present study was to determine the genetic variability among human ATL isolates in the State of Rio de Janeiro using MLEE as taxonomic tool and, RAPD and LSSP-PCR as polymorfic molecular markers to compare the panel of isolates obtained from lesions of patients with rare clinical manifestations of the disease with that of isolates obtained from patients with manifestations typically found in the State.
Section snippets
Samples and patients
A total of 34 Leishmania isolates obtained from tegumentary lesions of 32 patients living in the State of Rio de Janeiro and diagnosed at Centro de Referência em Leishmanioses, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz (CRLeish/IPEC/FIOCRUZ) were studied.
Group I consisted of 25 Leishmania isolates obtained from 23 patients with the following clinical and epidemiological characteristics of ATL rarely reported in this region: co-infection with Mycobacterium sp.,
Results
All of the 34 human samples analyzed by MLEE demonstrated similar electromorphic profiles which were similar to that of L. (V.) braziliensis reference strain (MHOM/BR/75/M2903). No isoenzymatic variants were observed between parasites isolated from groups I and II, or between isolates obtained from initial and reactivated lesions.
For RAPD analysis, all conditions such as reagents and DNA concentrations of the isolates and thermocycling conditions were the same during all experiments. Three
Discussion
The aim of the present investigation was to evaluate the genetic variability between Leishmania samples studied by RAPD and LSSP-PCR obtained from lesions of patients with typical or atypical clinical characteristics of ATL seen at the IPEC-Fiocruz outpatient clinic, Rio de Janeiro, Brazil.
The most frequent manifestation of ATL is a single cutaneous ulcer located in uncovered areas of the body, which presents a circular shape, elevated and well-defined borders and a granular base (Oliveira-Neto
Acknowledgments
This work received financial support from Instituto Kinder do Brasil (IKB), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ). C.Baptista benefited from funding by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Coordenação de Aperfeiçoamento do Pessoal de Ensino). A. Schubach is investigator of CNPq, Brazil. We also thank the Reference Center of Leishmaniasis—IPEC/Fiocruz
References (37)
- et al.
The interaction of Leishmania species with macrophages
Advances in Parasitology
(1992) - et al.
Diagnosis of New World leishmaniasis: specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA
Acta Tropica
(1992) - et al.
Epidemiological studies of an outbreak of cutaneous leishmaniasis in the Rio Jequitinhonha Valley, Minas Gerais, Brazil
Acta Tropica
(2002) - et al.
Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex
Experimental Parasitology
(2001) - et al.
PCR-based diagnosis for detection of Leishmania in skin and blood of rodents from an endemic area of cutaneous and visceral leishmaniasis in Brazil
Veterinary Parasitology
(2005) - et al.
Genotypic polymorphisms in experimental metastatic dermal leishmaniasis
Molecular and Biochemical Parasitology
(1995) - et al.
Recurrent lesions in human Leishmania braziliensis infection-reactivation or reinfection?
Lancet
(1990) - Aguilar, C.M., Rangel, E.F., Grimaldi Filho, G., Momen, H., 1987. Human, canine and equine leishmaniasis caused by...
- Azeredo-Coutinho, R.B.G., Conceição-Silva, F., Schubach, A., Cupolillo, E., Quintela, L.P., Madeira, M.F., Pacheco,...
- et al.
Leishmaniasis disseminated by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays
Memórias do Instituto Oswaldo Cruz
(1994)
Lymphadenopathy associated with Leishmania braziliensis cutaneous infection
The American Journal of Tropical Medicine and Hygiene
Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years
Clinical Infectious Diseases
A general classification of New World Leishmania using numerical zymotaxonomy
The American Journal of Tropical Medicine and Hygiene
Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil
Journal of Clinical Microbiology
Discrimination of Leishmania braziliensis variants by kDNA signature produced by LSSP-PCR
The Journal of Parasitology
American cutaneous leishmaniasis
Revista da Sociedade Brasileira de Medicina Tropical
Drug resistance in leishmaniasis: its implication in systemic chemotherapy of cutaneous and mucocutaneous disease
The American Journal of Tropical Medicine and Hygiene
Genetic variation in populations of Leishmania species in Brazil
Transactions of the Royal Society of Tropical Medicine and Hygiene
Cited by (28)
In vitro characterization of Leishmania (Viannia) braziliensis isolates from patients with different responses to Glucantime<sup>®</sup> treatment from Northwest Paraná, Brazil
2016, Experimental ParasitologyCitation Excerpt :The cutaneous and mucosal forms of ATL are associated with different Leishmania species including the Leishmania (Viannia) braziliensis, which is present in many regions of Central and South America (Basano and Camargo, 2004). This species exhibits intraspecific genetic polymorphisms (Cupolillo et al., 2003; Oliveira et al., 2004) and various clinical manifestations, ranging from simple cutaneous lesions (which may heal spontaneously) to mucosal lesions (Grimaldo and Tesh, 1993; Baptista et al., 2009; Queiroz et al., 2012). Saraiva et al. (1998) described a high frequency of mucosal involvement in cases of human disease with a particular zymodeme.
Genetic polymorphism in Leishmania (Viannia) braziliensis detected in mucosal leishmaniasis of HIV-infected and non-HIV-infected patients
2012, Transactions of the Royal Society of Tropical Medicine and HygieneCitation Excerpt :Different polymorphic molecular markers have also been applied to molecular epidemiology and to the study of genetic variability of Leishmania. Recently, our group reported the application of the low-stringency single specific primer–PCR (LSSP–PCR) technique towards investigating intraspecific polymorphism of the variable region of kDNA minicircles of L. (V.) braziliensis from ATL patients.13,14 The aims of the present study were: to diagnose infections directly from patient biopsies and to assess the genetic polymorphism of L. (V.) braziliensis detected in cases of mucosal leishmaniasis from HIV-infected and non-HIV-infected patients.
kDNA minicircle signatures of Leishmania (Viannia) braziliensis in oral and nasal mucosa from mucosal leishmaniasis patients
2010, Diagnostic Microbiology and Infectious DiseaseCitation Excerpt :This finding corroborates previous studies in which multiple PCR-based techniques were unable to detect a relationship between genetic polymorphism among Leishmania strains and clinical manifestation (Cuervo et al., 2004; De Oliveira et al., 2007). Such result is also consistent with recent work from our group reporting the detection of 9 different genotypes by LSSP-PCR in lesions from patients with typical or atypical clinical characteristics of ATL; no relationship was found between genetic profile and clinical condition (Baptista et al., 2009). It is well established that Leishmania parasites circulate in nature as a set of heterogeneous subpopulations, and the demonstration of multiclonal origin of some Leishmania strains has already been reported (Pacheco et al., 1990).
Characterization of Differentially Abundant Proteins Among Leishmania (Viannia) braziliensis Strains Isolated From Atypical or Typical Lesions
2022, Frontiers in Cellular and Infection Microbiology