Manoel P. Oliveira-Neto

Genetic Polymorphism and Molecular Epidemiology of Leishmania (Viannia) braziliensis from Different Hosts and Geographic Areas in Brazil

ABSTRACT Numerical zymotaxonomy and variability of the internal transcribed spacers (ITS) between the small and large subunits of the rRNA genes were used to examine strain variation and relationships in natural populations of Leishmania (Viannia) braziliensis. A total of 101 strains from distinct hosts and Brazilian geographic regions were assigned to 15 zymodemes clustered in two major genetic groups. The great number of isolates (48.5%) placed in zymodeme IOC/Z-27 were collected on the Atlantic coast. The high molecular diversity found in populations in the Amazon Basin was related to the great number of sandfly vector(s) in that region. The results of the restriction fragment length polymorphism analysis of the ITS depicted considerable intraspecific variation. Genotypic groups A, B, and C contained 39, 40, and 22 isolates, which were divided into 16, 10, and 15 genotypes, respectively. The genetic polymorphism observed demonstrates the degree of diversity of L. (V.) braziliensis strains from different regions where they are endemic. The results reinforce the clonal theory for Leishmania parasites showing the genetic diversity of this pathogen and an association of L. (V.) braziliensis genotypes with specific transmission cycles, probably reflecting an adaptation of different clones to the vector species involved.

An outbreak of american cutaneous leishmaniasis (Leishmania braziliensis braziliensis) in a periurban area of Rio de Janeiro city, Brazil: clinical and epidemiological studies

From July 1984 to September 1986, 105 cases of American cutaneous leishmaniasis were studied in a locality closely situated to an urbanized area of the city of Rio de Janeiro, Brazil. Settlement in this area was established at least 20 years ago but the first cases were noted six months prior to the beginning of this study. Cases were almost exclusively cutaneous and ulcerated, with one to six months of evolution. Montenegros skin tests were positive in all cases and anti-Leishmania antibodies were detected by indirect immunofluorescence test in 74.3% of the patients. Parasites were demonstrated in 69.5% of cases. Domestic animals were easily found infected: 32% of the examined dogs and 30.8% of the examined equines were positive to the presence of Leishmania in cutaneous ulcerated lesions. Parasite isolates from human, dog and equines were immunologically characterized and identified as L. b. braziliensis. 73.0% of the sandfly population were Lutzomyia intermedia mainly caught on human baits and on domestic animals. Our observations suggest that this is an area of recent established L. b. braziliensis infection and that transmission probably occurs indoors or outdoors close to the houses.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Flow cytometric analysis of cellular infiltrate from American tegumentary leishmaniasis lesions.

Background  CD4+ and CD8+ T lymphocytes play different roles in the outcome of leishmaniasis. However, T‐cell distribution in lesions shows significant variability in in situ immunocytochemical studies.

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

IFNG +874T/A polymorphism is not associated with American tegumentary leishmaniasis susceptibility but can influence Leishmania induced IFN-γ production

BackgroundInterferon-gamma is a key cytokine in the protective responses against intracellular pathogens. A single nucleotide polymorphism (SNP) located in the first intron of the human IFN-γ gene can putatively influence the secretion of cytokine with an impact on infection outcome as demonstrated for tuberculosis and other complex diseases. Our aim was to investigate the putative association of IFNG+874T/A SNP with American tegumentary leishmaniasis (ATL) and also the influence of this SNP in the secretion of IFN-γ in vitro.MethodsBrazilian ATL patients (78 cutaneous, CL, and 58 mucosal leishmaniasis, ML) and 609 healthy volunteers were evaluated. The genotype of +874 region in the IFN-γ gene was carried out by Amplification Refractory Mutational System (ARMS-PCR). Leishmania-induced IFN-γ production on peripheral blood mononuclear cell (PBMC) culture supernatants was assessed by ELISA.ResultsThere are no differences between +874T/A SNP frequency in cases and controls or in ML versus CL patients. Cutaneous leishmaniasis cases exhibiting AA genotype produced lower levels of IFN-γ than TA/TT genotypes. In mucosal cases, high and low IFN-γ producers were clearly demonstrated but no differences in the cytokine production was observed among the IFNG +874T or A carriers.ConclusionOur results suggest that +874T/A polymorphism was not associated with either susceptibility or severity to leishmaniasis. Despite this, IFNG +874T/A SNP could be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released by CL patients, although it could not prevent disease development. On the other hand, it is possible that in ML cases, other potential polymorphic regulatory genes such as TNF-α and IL-10 are also involved thus interfering with IFN-γ secretion.

American cutaneous leishmaniasis associated with HIV infection: report of four cases

A report of four cases of American cutaneous leishmaniasis associated with HIV infection is presented.

Leishmaniasis recidiva cutis in New World cutaneous leishmaniasis

Background Leishmaniasis recidiva cutis (LRC) is rare in New World leishmaniasis. Only seven cases have been reported so far.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

BackgroundConcomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function.MethodsTo address this issue we analyzed CD4+ T absolute counts and the proportion of CD8+ T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy.ResultsWe found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4+ T cell counts under 200 cells/mm3, differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm3). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8+ T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects.ConclusionsLeishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.