Trypanosoma caninum n. sp. (Protozoa: Kinetoplastida) isolated from intact skin of a domestic dog ( Canis familiaris) captured in Rio de Janeiro, Brazil.

An unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15.2%) and DMEM (9.2%) media containing 5% FCS, with a mean length of these forms of 43.0 and 36.0 mum, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24salpha rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.

Accuracy of an ELISA and indirect immunofluorescence for the laboratory diagnosis of American tegumentary leishmaniasis.

We compared the accuracy of ELISA and indirect immunofluorescence (IIF) using Leishmania braziliensis and L. major-like antigens and antigens from the Bio-Manguinhos kit for serological diagnosis of American tegumentary leishmaniasis (ATL). Cut-off values were defined by the area under the receiver-operating characteristic curve. For ELISA, statistical analyses revealed better accuracy [95.7% sensitivity, 100% specificity, 100% positive predictive value (PPV), 97.5% negative predictive value (NPV)] and reliability [intraclass correlation coefficient (ICC): 0.940] for L. braziliensis antigen compared with L. major-like antigen (78.7% sensitivity, 82.8% specificity, 73.3% PPV, 86.6% NPV, ICC: 0.833). ELISA optical density values obtained for both antigens were higher in mucosal forms of ATL. For IIF, sensitivity and specificity were 81.5 and 86.2%, respectively, for the L. braziliensis antigen, compared with 95.4 and 77.7% for the L. major-like antigen and 75.4 and 89.2% for the Bio-Manguinhos kit. No difference in the specificity of the IIF test was observed between antigens, whereas sensitivity differed between the L. braziliensis and L. major-like antigens and the Bio-Manguinhos kit. Parallel ELISA and IIF testing increased sensitivity, irrespective of the antigen employed, and serial testing increased overall specificity. These results support the recommendation that ELISA employing L. braziliensis antigen be used as a diagnostic tool for suspected cases of ATL in L. braziliensis-endemic areas.

Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil.

We report here the first case of co-infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil. Isoenzyme characterisation identified the parasites isolated in culture from the cutaneous lesion as L. (V.) braziliensis and the isolates from blood and lymph node as L. (L.) chagasi. PCR analysis using specific primers followed by molecular hybridisation for direct Leishmania species identification in tissue fragments confirmed the presence of L. (V.) braziliensis DNA in the cutaneous lesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. This report emphasises the importance of identification of Leishmania species infecting seropositive dogs in endemic areas, and the consequent re-assessment of control and epidemiological surveillance measures for the control of leishmaniasis, as is the case in Brazil.

American cutaneous leishmaniasis in two cats from Rio de Janeiro, Brazil: first report of natural infection with Leishmania (Viannia) braziliensis.

We describe the isolation of Leishmania (Viannia) braziliensis from two female cats with American cutaneous leishmaniasis in Rio de Janeiro, Brazil. The isolates were identified as L. (V.) braziliensis by isoenzyme electrophoresis.

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.

[Epidemiological aspects of canine American tegumentary leishmaniasis in the Municipality of Paraty, State of Rio de Janeiro, brazil]. / Aspectos epidemiológicos da leishmaniose tegumentar americana em cães, no Município de Paraty, Estado do Rio de Janeiro, Brasil.

American tegumentary leishmaniasis (ATL) is endemic in the State of Rio de Janeiro, with the highest incidence observed in the municipalities along the Southern coast. A total of 169 human cases were reported from 1993 to 1995 in the municipality of Paraty, representing 15% of all case reports in the State of Rio de Janeiro during that period. From March to December, 1996, a survey was conducted in endemic american tegumentary leishmaniasis (ATL) areas (Praia Grande, Taquari, Várzea do Machado, and Graúna) in Paraty. 326 households with 215 pet dogs were surveyed. The initial study consisted of taking venous blood samples to perform RIFI and ELISA tests. The canine skin test was performed during the same visit and was positive in 8.8% of dogs. Of the 215 dogs, 5 presented lesions suspected of ATL; biopsies and the vacuum aspiration technique were performed on these cases. Correlating the study areas with the results obtained, some dogs presented sub-clinical infection, highly relevant to studies on canine ATL infection in the State of Rio de Janeiro.

Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC. The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

N-methylglucamine antimonate (SbV+): intralesional canine tegumentary leishmaniasis therapy.

Twenty five adult dogs of three municipalities of the State of Rio de Janeiro, Brazil, that had been naturally infected by L. (V) braziliensis were treated with N-methylglucamine antimonate (Glucantime). Nine of the animals (36.0%) presented ulcerated skin lesions, twelve (48.0%) had mucosal lesions and four (16.0%) had multiple lesions. In some cases the mucosal lesions were associated to skin lesions. A dose of 85 mg SbV+ or 1 ml of the drug was intralesionally administered to the dogs. The animals were divided into three groups according to the amount of necessary doses (between one and three) for the complete healing of the lesions. The dogs were observed for six months after the third group received the last dose. Within this period two animals perished. Serial antibody evaluation through IFAT has shown that in 14 samples (63.3%) the titers have remained unaltered, in four of them (16%) there has been a decrease in two titers and in five of them (21%) serology was negative. Nineteen of the dogs (86.6%) had their lesions completely healed. The authors suggest intralesional therapy be the first choice of treatment of canine tegumentary leishmaniasis due to its effectiveness.

Parasite genotypically related to a monoxenous trypanosomatid of dog's flea causing opportunistic infection in an HIV positive patient.

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a panel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulantis a trypanosomatid found in the dog's flea.

Epidemiological survey on canine population with the use of immunoleish skin test in endemic areas of human American cutaneous leishmaniasis in the state of Rio de Janeiro, Brazil.

A survey for canine tegumentary leishmaniasis (CTL) has been carried out between 1986 and 1993 in seven endemic localities for American cutaneous leishmaniasis in the State of Rio de Janeiro. 270 dogs have been examined for their clinical aspects, the development of delayed hypersensitivity (DHS) with Immunoleish antigen and with immunofluorescent antibody research of IgG (IF). 28.2% of them had ulcer lesions and 3.3% had scars. The lesions consisted of single (39.5%) and mucocutaneous lesions (31.6%), multiple cutaneous (25.0%) and mucocutaneous lesions associated with cutaneous ulcers (4.0%). Twelve (15.8%) isolates from biopsies were analyzed by zimodeme and schizodeme and identified as L. (V) braziliensis. The overall prevalence of canine infection that was evaluated with the skin test was of 40.5% and with IF it was of 25.5%. Both tests showed a high positive rate with relation to the animals with mucosal lesions, as in the case of human mucocutaneous leishmaniasis. The comparison of the two tests showed the skin test to have a better performance although there was no statistical difference (p > 0.05) between them. The proportional sensitivity and specificity was of 84.0% and 74.0%, respectively. The Immunoleish skin test and IF are useful tools to be employed in CTL field epidemiological surveys.

Cutaneous scars in American tegumentary leishmaniasis patients: a site of Leishmania (Viannia) braziliensis persistence and viability eleven years after antimonial therapy and clinical cure.

Two former patients treated for the cutaneous form of American tegumentary leishmaniasis were reviewed eight and 11 years, respectively, following clinical cure. We were able to isolate Leishmania parasites in a culture of material from the two scar biopsies, and in one of them the parasite was characterized as Leishmania (Viannia) braziliensis. In both cases, the histopathology revealed discreet hyperceratosis and a slight infiltrate of mononuclear cells surrounding and on the walls of the surface and deep dermal vessels. No amastigotes were seen on immunohistochemical or histopathologic examination. The Montenegro skin test result and the in vitro lymphoproliferative response to Leishmania antigen were positive, but no specific IgG and IgM antibodies were detected. Otorhinolaryngologic examination showed no macroscopic alteration in the mucosae. These findings are important for the evaluation and criteria of post-treatment cure.

[Intestinal parasite contamination of vegetables sold at supermarkets in the city of Rio de Janeiro]. / Estudo da contaminação por enteroparasitas em hortaliças comercializadas nos supermercados da cidade do Rio de Janeiro.

Parasitological examinations were performed in material derived from 220 vegetables to detect cysts and eggs of intestinal parasites. This material was collected from supermarkets in northern and southern areas and from CEASA (Central Supply Market--40 vegetables) in Rio de Janeiro city. Vegetables proceeding from the northern areas presented a greater degree of contamination mostly of helminth eggs; lettuce was the most contaminated vegetable. The authors suggest that differences found between the northern and southern areas of the city may be due to either packing or manipulation by shop assistants and consumers.

Bancroftian filariasis in two urban areas of Recife, Brazil: pre-control observations on infection and disease.

Bancroftian filariasis is a major public health problem in the city of Recife in north-eastern Brazil. In some of its urban areas microfilaraemia prevalence reaches 14%. This study describes epidemiological characteristics, infection and disease, in 2 urban areas, Coque and Mustardinha, before control measures were applied. The parasitological survey was performed by a 'door-to-door' census covering 5563 subjects, aged between 5 and 65 years. Microfilaraemia was detected by the thick drop technique, using 45 microL of peripheral blood collected between 20:00 and 24:00. In both areas the prevalence of microfilaraemia was 10%, and males had higher prevalences of infection and disease than females. The prevalence of microfilaraemia was higher in the 15-24 and 25-34 years age groups in both sexes. Most microfilaria (mf) carriers (72.1% in Coque and 79.7% in Mustadrinha) had mf densities < 100/60 microL of blood. Females of reproductive age had significantly lower mf densities than males. The overall disease prevalence in both areas was 6.3%. Amongst the subjects who presented with chronic disease 15.7% were microfilaraemic. Chronic disease prevalence increased from 1.4% in the 5-14 years age group to 11.3% in the oldest age group. The most frequent clinical manifestation was hydrocele (5.4%), followed by lymphoedema (1.8%). The epidemiological pattern of filariasis in the populations studied was marked by high prevalence of microfilaraemia, low mf density, and relatively low prevalence of filarial disease considering the level of endemicity.

Bancroftian filariasis in two urban areas of Recife, Brasil: the role of individual risk factors.

Bancroftian filariasis is spreading in towns of endemic areas in Recife, northeastern Brazil, where it is a major public health problem. This paper deals with the prevalence of microfilaraemia and filarial disease in two urban areas of Recife, studying their association with individual characteristics and variables related to the exposure to the vectors. The parasitologic survey was performed through a "door-to-door" census and microfilaraemia was examined by the thick-drop technique using 45 microliters of peripheral blood collected between 20:00 and 24:00 o'clock. 2,863 individuals aged between 5 and 65 years were interviewed and submitted to clinical examination. Males aged between 15 and 44 years old presented the greatest risk of being microfilaraemic. Microfilaraemia was also significantly associated with no use of bednet to sleep. The risk of being microfilaraemic was greater among those who had lived in the studied areas for more than 5 years. The overall disease prevalence was 6.3%. Males presented the greatest risk of developing acute disease. The risk of developing chronic manifestations was also greater among males and increased with age. We found no association between time of residence, bednet use, microfilaraemia and acute and chronic disease. We may conclude that in endemic areas there are subgroups of individuals who has a higher risk of being microfilariae carriers due to different behaviours in relation to vector contact.

Leishmaniasis dissemineted by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays

Cutaneous disseminated lesions caused by Leishmania sp. were found in a pregnant mare (Equus cabalus) from a rural city in the State of rio de Janeiro, Brazil. Before delivering, treatment was undertaken by immunotherapy followed by chemotherapy. Histopatology and serology were performed during treatment, as well as the biochemical characterization of the parasite (L. braziliensis) that was isolated from one of the lesions

Leishmaniasis disseminated by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays.

Cutaneous disseminated lesions caused by Leishmania sp. were found in a pregnant mare (Equus cabalus) from a rural city in the State of Rio de Janeiro, Brazil. Before delivering, treatment was undertaken by immunotherapy followed by chemotherapy. Histopatology and serology were performed during treatment, as well as the biochemical characterization of the parasite (L. braziliensis) that was isolated from one of the lesions.

Tegumentary and visceral leishmaniases in Brazil: emerging anthropozoonosis and possibilities for their control.

The existence of a number of different species of Leishmania, the persistent increase in the infection rate of diseases caused by this parasite (tegumentary and visceral forms), the different epidemiological situations found in regions of both recent and older colonization, and the trend towards urbanization have led to the adoption of different strategies to control leishmaniases in Brazil. The control measures involve studies related to the parasite, vectors, sources of infection (animal and human), clinical aspects, geographical distribution, historical and socioeconomic factors, integration of health services, and adequate technologies for diagnosis, treatment, and immunoprophylaxis. Finally, successful control requires work with human communities, involving education, provision of information, health promotion, and participation of these communities in the planning, development, and maintenance of control programs.

Visceral leishmaniasis in Rio de Janeiro.

Visceral leishmoniosis has recently become established in the peri-urban areas of the city of Rio de Janeiro. Mauro Marzochi, Keyla Marzochi and Raimundo Carvalho here discuss its incidence, and consider the factors involved in its establishment and propagation, as well as environmental, human, social, economic and historical factors.

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 Montenegro's skin test (MST). A low prevalence (3.7%) of positive MST was encountered. The disease had been occuring 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 environmewnt