Fernanda Santos de Oliveira

Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: evaluation by two molecular markers.

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.

Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil.

The present study was developed in the urban area of Paracatu, an endemic city for the American visceral leishmaniasis in Brazil. A six-month canine survey was performed with 6295 domiciled dogs in 28 districts in that area and showed that 4.2% of those (267 dogs) were positive for VL by ELISA and IFAT serum assays. Prevalence ratios for canine VL varied between 1.2% and 16.1%, depending on the district under investigation. Fifteen dogs - 80% of which were clinically asymptomatic for VL - were submitted to a more detailed study that comprised direct parasitological examination and Leishmania kDNA amplification of tissue samples as well as two PCR-RFLP methods using myelocultures. Leishmania amastigotes or Leishmania DNA were detected in all dogs but one. The infecting species of Leishmania was identified in about 50% (7/15) of the sample dogs: Leishmania (Leishmania) chagasi in two of them and, unexpectedly, Leishmania (Leishmania) amazonensis in the remaining five. Three months after the end of confiscation and elimination of the VL-seropositive dogs in the 28 districts of Paracatu, a systematic entomological survey was performed in five of them. Six hundred and sixty five (665) phlebotomine sand flies were captured in total, from which 89.5% were identified as Lutzomyia longipalpis. The population density of that species increased during the rainy season. Other thirteen (13) species of phlebotomine sand flies were captured at varying percentages from 0.2 to 5.0%. It is worth noting that L. longipalpis females were predominantely intradomicile when compared to males, suggesting that the VL transmission cycle in Paracatu may be occurring inside home.

kDNA minicircle signatures of Leishmania (Viannia) braziliensis in oral and nasal mucosa from mucosal leishmaniasis patients.

Polymerase chain reaction (PCR) and low-stringency single-specific primer PCR (LSSP-PCR) analyses were used to detect Leishmania (Viannia) braziliensis DNA and investigate kDNA signatures of parasite populations present in oral and nasal mucosa lesions from mucosal leishmaniasis patients. A total of 25 samples from 22 patients were processed by specific PCR/hybridization assays. Parasite DNA was detected in all samples analyzed. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was also investigated by LSSP-PCR. Similar kDNA signatures were observed in parasites recovered from nasal and oral mucosa lesions of the same patient. In contrast, genetically divergent profiles were detected in lesions from patients biopsied at different times within a period of 1 year. This is the first work to report genetic typing of L. (V.) braziliensis directly from human oral and nasal mucosal lesions.

American tegumentary leishmaniasis caused by Leishmania ( Viannia ) braziliensis: assessment of parasite genetic variability at intra- and inter-patient levels

BackgroundThe genetic variability of Leishmania (Viannia) braziliensis was assessed at intra and interpatient levels of individuals with different clinical manifestations of American tegumentary leishmaniasis (ATL).MethodsFifty-two samples, of which 13 originated from cutaneous lesions and 39 from mucosal lesions, provided by 35 patients, were examined by low-stringency single-specific-primer PCR (LSSP-PCR) and phenetic analysis. Genetic variability of L. (V.) braziliensis, in kinetoplast DNA (kDNA) signatures, was compared both from different patients and from different lesions of the same patient. Phenetic analysis was performed to evaluate the degree of heterogeneity of the kDNA minicircles. In order to evaluate inter and intrapatient L. (V.) braziliensis genetic variability, the percentage of shared bands and analysis of the coefficients of similarity were analyzed.ResultsDifferent genetic profiles, representing kDNA signatures of the parasite, were obtained by LSSP-PCR analysis of each sample. Phenetic analysis grouped genetic profiles of different levels of differentiation from more similar to most divergent. The percentage of shared bands at the inter and intrapatient levels was 77% and 89%, respectively. Comparison of the average inter and intrapatient coefficients of similarity and their standard deviations were statistically significant (p < 0.001).ConclusionGenetic variability at the intrapatient level was less pronounced than that between different patients. A conceptual model was proposed to better understand the complexity at both levels.

Genetic polymorphism in Leishmania (Viannia) braziliensis detected in mucosal leishmaniasis of HIV-infected and non-HIV-infected patients

The genetic polymorphism of Leishmania (Viannia) braziliensis detected in cases of mucosal leishmaniasis (ML) from HIV-infected and non HIV-infected patients was evaluated. Nine samples from three HIV-infected patients and five samples from five non HIV-infected patients were analysed by polymerase chain reaction (PCR), low-stringency single-specific primer PCR (LSSP-PCR) and phenetic analysis. The presence of L. (V.) braziliensis DNA was detected in all samples by specific PCR assay. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was investigated by LSSP-PCR. Phenetic analysis grouped the genetic profiles into two distinct clusters, which discriminated between samples obtained from HIV-infected and non HIV-infected patients. In two HIV-infected patients, identical genetic profiles were detected in lesions biopsied at different times after the treatment of the initial lesion. Interestingly, genetically divergent profiles were detected in the cutaneous and mucosal lesions of the same HIV-infected patient collected at the same time. This is the first work comparing genetic polymorphism of L. (V.) braziliensis in cases of mucosal leishmaniasis from HIV-infected and non HIV-infected patients.

PCR Associated with Molecular Hybridization Detects Leishmania (Viannia) braziliensis in Healthy Skin in Canine Tegumentary Leishmaniasis

Abstract:  Tegumentary leishmaniasis (TL) is a zoonotic disease that affects humans and domestic dogs. In Brazil, TL is considered endemic, and Leishmania (Viannia) braziliensis is the prevalent species causing this disease. There is debate about the role of dogs (Canis familiaris) as domestic reservoirs in the transmission cycle of TL. To date, classical parasitological techniques, including parasite isolation in culture media, have been able to detect parasites only from cutaneous lesions. In this study, we detected L. (V.) braziliensis DNA in intact skin fragments collected from 3 naturally infected dogs from the state of Rio de Janeiro, Brazil, with the use of PCR techniques associated with molecular hybridization. The detection of parasitic DNA in this anatomical site is an important finding vis-à-vis the importance of the domestic dogs in endemic areas of TL.

Canine Cutaneous Leishmaniasis: Dissemination and Tissue Tropism of Genetically Distinct Leishmania (Viannia) braziliensis Populations

Little is known regarding the internal dissemination of initial cutaneous lesions and tissue tropism of Leishmania (Viannia) braziliensis populations in naturally infected dogs. The aim of this study was to investigate genetic polymorphisms of L. (V.) braziliensis populations in different anatomic sites of naturally infected dogs by using polymerase chain reaction (PCR) and low-stringency single specific primer-PCR (LSSP-PCR) techniques. The amplified products were analyzed by LSSP-PCR to investigate the genetic variability of the parasite populations present in different anatomical sites. Twenty-three out of the 52 samples gave PCR-positive results. The existence of L. (V.) braziliensis strains that remained restricted to cutaneous lesions and others showing characteristics of dissemination to internal organs and healthy skin was observed. LSSP-PCR and numerical analyses revealed that parasite populations that do not disseminate were genetically similar and belonged to a separate phenetic cluster. In contrast, populations that showed spreading to internal organs displayed a more polymorphic genetic profile. Despite the heterogeneity, L. (V.) braziliensis populations with identical genetic profiles were observed in popliteal and cervical lymph nodes of the same animal. Our results indicate that infection in dogs can be manifested by dissemination and tissue tropism of genetically distinct populations of L. (V.) braziliensis.