Cutaneous leishmaniosis in naturally infected dogs in Paraná, Brazil, and the epidemiological implications of Leishmania (Viannia) braziliensis detection in internal organs and intact skin.

Environmental changes have occurred over the years, altering the eco-epidemiological pattern of leishmaniosis in the State of Paraná, Brazil, involving the pillars of the cycle (parasite, vectors, reservoir, and environment) and their interaction. Much has been discussed about the dog's role as a reservoir of the Leishmania (Viannia) braziliensis Vianna, 1911 transmission cycle. However, this question remains unanswered. The purpose of this study was to investigate, using parasitological and molecular methods, different samples in eight naturally infected dogs from an endemic rural locality where only L. (V.) braziliensis is present, and where human cases have been previously notified. Blood and biopsied organ samples from naturally infected dogs were analyzed by culture media, PCR, random amplified polymorphic DNA and sequencing methodologies. Only skin lesions from all dogs yielded positive cultures and when PCR was performed, L. (V.) braziliensis DNA was amplified from intact skin, peripheral blood, bone marrow, spleen, liver and lymph nodes. RAPD was also applied to isolates from the skin lesions, exhibiting the genetic variability of the parasite identified. To confirm which species of Leishmania was amplified in PCR, the sequencing method was performed, verifying 100% similarity with the Viannia subgenus. This study showed that L. (V.) braziliensis can spread to other sites besides the ulcerous lesions, such as intact skin, peripheral blood and internal organs, making it possibility for dogs to serve as active sources of parasite transmission. For definitive proof, xenodiagnostic test on intact skin of infected dogs, should be done.

Molecular detection of the blood meal source of sand flies (Diptera: Psychodidae) in a transmission area of American cutaneous leishmaniasis, Paraná State, Brazil.

The feeding behavior of sand flies provides valuable information about the vector/host interactions and elucidates the epidemiological patterns of American cutaneous leishmaniasis (ACL) transmission. The aim of this study was to identify the blood meal sources of sand flies in endemic areas of leishmaniasis in Paraná State through polymerase chain reaction (PCR) amplification of a prepronociceptin (PNOC) gene fragment and its subsequent DNA sequencing. Moreover, molecular assays were conducted to evaluate the sensitivity and reproducibility of the PNOC gene amplification. Besides that, a time-course digestion test of the blood using sand flies that fed artificially on BALB/c mice was performed. Of 1263 female sand flies collected in the field, 93 (3.6%) specimens were engorged and 27 allowed efficient amplification of the PNOC gene. These flies had fed on equine (Equus caballus), porcine (Sus scrofa) and canine (Canis lupus familiaris) species. The results also showed that the identification of the blood meal sources of the sand flies using the molecular method was directly linked to the level of digestion of the blood (time-course) and not to the amount of blood that had been ingested or to the presence of inhibitors in the blood.

A new focus of cutaneous leishmaniasis in the central area of Paraná State, southern Brazil.

We report a new endemic zone of cutaneous leishmaniasis (CL) in the central area of the State of Paraná (Municipality of Prudentópolis), in southern Brazil. This region was not previously considered endemic for CL, and this work constitutes the first report of CL endemicity there. Leishmaniasis was confirmed by smear, culture, and ELISA. Parasites were isolated and identified by random amplification of polymorphic DNA (PCR-RAPD). Phylogeographical analysis, based on two different criteria, was able to distinguish between RAPD profiles from different geographical regions. In total, 100 patients were diagnosed with leishmaniasis by culture and serology methods. The reported incidence rate was 4.32%. Of the 100 patients, 92% of the patients had single lesions, and 79.98% of these lesions were located on their limbs. The fact that 61% of patients were male rural workers points to an extradomiciliar type of transmission. In houses where human leishmaniasis was diagnosed, 29% of the dogs presented anti-Leishmania antibodies. A total of 1663 phlebotomines, representing 5 species, were captured in the studied area with CDC-like light minitraps. Lutzomyia intermedia s.l. was the most prevalent species (94.40%). The isolated parasites were grouped with Leishmania (V.) braziliensis. The epidemiological implications are discussed in the present article.

Molecular diagnosis of leishmaniosis in the Paraná state of southern Brazil.

The objective of the present study was to establish a polymerase chain reaction (PCR) technique for the diagnosis of cutaneous and mucocutaneous leishmaniosis from autochthonous cases in the state of Paraná in southern Brazil as well as imported cases. We sought to determine its utility and accuracy compared with smears and present culture methods. To standardize PCR samples, skin and mucosal punch biopsies from human lesions were performed on patients living in different regions of the Paraná state (76 cases) and other endemic areas of Brazil and Argentina (7 cases). For PCR standardization, two pairs of primers (MP1L/MP3H and B1/B2) were utilized for amplification of the conserved sequences in the minicircle of kinetoplast DNA (kDNA) for the Leishmania braziliensis complex. Two other primer pairs (b1/b2 and a1/a2) were species-specific for L. (V.) braziliensis and L. (V.) amazonensis, respectively. After differential diagnosis, eight patients had clinical diagnosis of the cutaneous ulcer changed to others pathologies such as syphilis, baso-cellular carcinoma, varicose ulcer, ecthyma and paracoccidioidomycosis. Of the 75 patients with cutaneous (CL) and mucocutaneous (MCL) lesions who provided samples, 47 (46 CL + 1 MCL) were diagnosed with leishmaniosis by smear and 57 (52 LC + 5 MCL) were diagnosed by culture methods. In contrast, our PCR technique presented higher accuracy when compared to the direct examination and culture of parasites. PCR is applicable both for CL where all 61 lesions were diagnosed, and MCL where 12 of 14 lesions were diagnosed. This molecular biology technique is also a faster and more specific diagnostic method compared with present parasitological procedures.

Comparing the efficacy of chlorine, chlorine dioxide, and ozone in the inactivation of Cryptosporidium parvum in water from Parana State, Southern Brazil.

In the present work, assays were performed to compare the efficacy of hypochlorous acid, chlorine dioxide, and ozone in the inactivation of Cryptosporidium oocyst in public water supply from Brazilian South conditions. Experiments were carried out in samples containing 2 x 10(4) oocysts/ml of C. parvum purified from feces of experimentally contaminated calves. An in vitro excystation method was used to evaluate oocysts' viability and to determine the inactivation rates of hypochlorous acid at 2 ppm, chlorine dioxide at 1, 2, and 5 ppm, and ozone at the doses of 0.18, 0.24, 0.36, 0.48, and 1.44 mg/l. By using hypochlorous acid, the maximum inactivation rate obtained was 49.04% after 120 min. Chlorine dioxide at 5 ppm inactivated 90.56% of oocysts after 90 min of contact. Ozone was the most effective product, rendering an inactivation of 100% with the concentration of 24 mg/l. Resistance of Cryptosporidium to the usual disinfectants and the need for more effective water treatments to prevent waterborne diseases in Brazil are discussed in this manuscript.

Eco-epidemiological survey of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in Ribeira Valley River, Paraná State, Brazil.

Leishmaniasis is endemic since last century in Adrianópolis Municipality, Ribeira Valley and is a serious public health. A study carried out during 1993-2003 on epidemiological surveys conducted in rural communities showed 339 new cases of cutaneous leishmaniasis (CL) detected from four municipalities (Adrianópolis, Cerro Azul, Doutor Ulysses and Rio Branco do Sul). A larger prevalence of cutaneous lesions was observed in rural workers (36%), women with domestic activities (18%), and younger students (31%). Multiple lesions were noticed in 53% of patients, but only one case of mucocutaneous leishmaniasis was reported. Twenty stocks were isolated from patients with characteristics lesions and were identified as Leishmania (Viannia) braziliensis using multi-locus enzyme electrophoresis (MLEE) and Random Amplified DNA (RAPD). In Phlebotominae survey, five species were obtained. Lutzomyia intermedia sl. represented 97.5% in peridomiciliar area and 100% in domicile. A canine serological survey made (Indirect Immunofluorescence Antibody Test, IFAT and Enzyme Linked Immunosorbent Assay, ELISA) in six rural county of Adrianópolis Municipality during 1998-1999 showed that 15.1% (24/159) of dogs were sera reactive. No lesions were observed in dogs and no parasite was isolated from lymph node aspirates and biopsies. In wild reservoirs study, only seven animals (Cricetidae, Desmodus sp. and edentates) were captured, but no parasites were found in culture from deep organs. The paper presents results of our 10 years study on cutaneous leishmaniasis survey in the Ribeira River Valley, East Region of Paraná State, Brazil. Environment changes in this region are also discussed.

[Epidemiological and clinical study of 332 cases of cutaneous leishmaniasis in the north of Parana State from 1993 to 1998]. / Estudo das características epidemiológicas e clínicas de 332 casos de leishmaniose tegumentar notificados na região norte do Estado do Paraná de 1993 a 1998.

Cases of cutaneous leishmaniasis (CL) in northern Parana State have been reported since the 1950's, but towards the end of the 1980's there has been an increase in the number of human infections. From 1993 to 1998, a study was carried out in an attempt to define the epidemiological profile of leishmaniasis in this region. A total of 316 cases of CL were reported from 35 municipal districts and 16 imported cases. Most cases of CL notified (70.8%) were in the age group of 15 to 49 years old and most of these were males (61.2%). Of the 332 positive patients, 66.8% presented single classic lesions and 31.1% multiple lesions. Most of these lesions were located in the lower limbs (47.7%) while 26.7% were in the arms and 16% on the face. Thirty-two Leishmania stocks were isolated and identified by isoenzymatic characterization using 13 enzymatic systems as Leishmania (Viannia) braziliensis.