Célia Maria Ferreira Gontijo

Leishmaniose visceral no Brasil: quadro atual, desafios e perspectivas

No Brasil, a importância da leishmaniose visceral reside nao somente na sua alta incidencia e ampla distribuicao, mas tambem na possibilidade de assumir formas graves e letais quando associada ao quadro de ma nutricao e infeccoes concomitantes. A crescente urbanizacao da doenca ocorrida nos ultimos 20 anos coloca em pauta a discussao das estrategias de controle empregadas. Neste artigo foram analisados os principais aspectos biologicos, ambientais e sociais que influenciaram no processo de expansao e urbanizacao dos focos da doenca. Os metodos disponiveis para o diagnostico e tratamento nao apresentam a eficacia e aplicabilidade desejadas, embora avancos promissores tenham sido alcancados com as pesquisas de novos testes diagnosticos e drogas terapeuticas. As medidas de controle da doenca ate agora implementadas foram incapazes de eliminar a transmissao e impedir a ocorrencia de novas epidemias. E feita uma breve analise destas medidas e dos desafios a serem enfrentados. A prevencao da doenca nos caes atraves da imunoprofilaxia aparece como uma alternativa para o controle. Uma nova vacina para caes, ja testada em campo, esta sendo industrializada e sera comercializada no Brasil a partir de 2004. Apesar da existencia de inumeros estudos sobre a leishmaniose visceral humana e canina, muitas lacunas ainda precisam ser preenchidas.

Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

Leishmania (Viannia) braziliensis is the predominant species infecting patients with American cutaneous leishmaniasis in the State of Minas Gerais, Southeast Brazil

Skin biopsies from 53 patients with American cutaneous leishmaniasis (ACL) from the State of Minas Gerais, Brazil, were used for a characterization of the Leishmania parasites. A pair of primers flanking the conserved region of the Leishmania minicircle kDNA was used to obtain amplified DNA via the polymerase chain reaction. The amplified products were subsequently hybridized with Leishmania subgenus-specific radiolabeled probes. Parasites from 49 out of 53 samples (92.5%) were characterized as belonging to the subgenus Viannia and four (7.5%) as belonging to the subgenus Leishmania. Clinical, epidemiological and molecular evidence allow us to conclude that Leishmania (V.) braziliensis and Leishmania (L.) amazonensis are the species present in the patients studied and that L. (V.) braziliensis is the predominant species in the State of Minas Gerais, Brazil.

Epidemiological aspects of American Cutaneous Leishmaniasis in a periurban area of the metropolitan region of Belo Horizonte, Minas Gerais, Brazil

In order to characterize the epidemiology of American Cutaneous Leishmaniasis (ACL) in a periurban area of the municipality of Sabará in the metropolitan region of Belo Horizonte (MRBH), an area until then considered free of the disease, a cross sectional survey was undertaken in 1990. The survey of the population consisted of 1119 interviews and 881 clinical examinations using Montenegros skin test (MST). A low prevalence (3.7%) of positive MST was encountered. The disease had been occurring in the area for about 20 years in the form of sporadic cases. The predominant species of sandfly both in domestic areas and nearby areas of secondary vegetation was Lutzomyia whitmani. A canine survey of delayed hypersensitivity to the antigen P10,000 identified only one dog with a positive reaction out of 113 examined. The transmission of ACL in MRBH was confirmed. The occurrence of the disease in women, children and individuals with no contact with forest areas as well as the presence of potential vector species in the domiciliar environment, suggests the transmission of the disease in this environment.

Naturally Infected Lutzomyia Sand Flies in a Leishmania-Endemic Area of Brazil

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.

Epidemiological studies of an outbreak of cutaneous leishmaniasis in the Rio Jequitinhonha Valley, Minas Gerais, Brazil.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.

Concurrent cutaneous, visceral and ocular leishmaniasis caused by Leishmania (Viannia) braziliensis in a kidney transplant patient

Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia) braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V.) braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.

Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil.

ABSTRACT Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (=Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (=Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

Leishmaniose tegumentar na Região Metropolitana de Belo Horizonte: aspectos clínicos, laboratoriais, terapêuticos e evolutivos (1989-1995)

This study investigated clinical, laboratorial, therapeutic and prognostic aspects of American cutaneous leishmaniasis in Belo Horizonte in 358 patients with cutaneous leishmaniasis (CL) and 25 with mucocutaneous leishmaniasis (MCL). Compared to CL patients, the MCL patients reported longer duration of disease and higher frequency of other diseases, suggesting that debilitation caused by leishmaniasis or other conditions might contribute to activation and/or mucous dissemination of the parasite. The sensitivity of skin test, indirect immunofluorescence reactions and direct detection of parasites was 78.4, 79.3 and 68.3%, respectively. The treatment with meglumine antimoniate presented 100% efficacy, but 59% patients had side-effects. During two years of follow-up, there were 32/318 relapses after successful treatment. Most relapses (31/32) were of CL patients treated with 15 mg Sb 5+ /kg/day. The negative response to skin test was the only factor associated with a significant threefold increased risk of relapse. Higher dose or longer duration of treatment might improve the prognosis in these patients.This study investigated clinical, laboratorial, therapeutic and prognostic aspects of American cutaneous leishmaniasis in Belo Horizonte in 358 patients with cutaneous leishmaniasis (CL) and 25 with mucocutaneous leishmaniasis (MCL). Compared to CL patients, the MCL patients reported longer duration of disease and higher frequency of other diseases, suggesting that debilitation caused by leishmaniasis or other conditions might contribute to activation and/or mucous dissemination of the parasite. The sensitivity of skin test, indirect immunofluorescence reactions and direct detection of parasites was 78.4, 79.3 and 68.3%, respectively. The treatment with meglumine antimoniate presented 100% efficacy, but 59% patients had side-effects. During two years of follow-up, there were 32/318 relapses after successful treatment. Most relapses (31/32) were of CL patients treated with 15 mg Sb5+/kg/day. The negative response to skin test was the only factor associated with a significant threefold increased risk of relapse. Higher dose or longer duration of treatment might improve the prognosis in these patients.

Diagnosis of Canine Leishmaniasis in the Endemic Area of Belo Horizonte, Minas Gerais, Brazil by Parasite, Antibody and DNA Detection Assays

Canine leishmaniasis caused by Leishmania chagasi (L. infantum) is found throughout the South American continent, including Brazil, and dogs are considered to be the main reservoir host for this parasite. To support the implementation of a diagnostic protocol for surveillance of the disease in the region of Belo Horizonte (Minas Gerais, Brazil) we have compared the sensitivity and specificity of two serological tests, indirect immunofluorescent antibody test (IFAT) and direct agglutination test (DAT), with the combination of direct microscopy–culture–PCR as the gold standard, using samples obtained from 103 dogs in the city of Belo Horizonte, Minas Gerais. The currently used standard serodiagnostic test, IFAT, had a sensitivity of 100% and its specificity was 74% compared to the gold standard of the study. The sensitivity and specificity of the DAT were 100% and 91%, respectively. On the basis of this study it is recommended to change from the IFAT to DAT for the serodiagnosis of canine leishmaniasis because of the superior specificity of the test combined with its user-friendliness.