Photodynamic antimicrobial chemotherapy (PACT) using phenothiazine derivatives as photosensitizers against Leishmania braziliensis

Background and Objective Cutaneous and mucocutaneous leishmaniasis are diseases characterized by skin or mucosal manifestations. In the new world, Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis, condition that may evolve to the mucocutaneous form. The therapeutic arsenal routinely employed to treat infected patients is unsatisfactory, especially for pentavalent antimonials, treatment recommended by the WHO, as they are often highly toxic, poorly tolerated and of variable effectiveness. This work aimed to evaluate in vitro the effectiveness of photodynamic antimicrobial chemotherapy as a new approach for the treatment of leishmaniasis. Materials and Methods A laser (??=?660?nm, 40?mW, 4.2?J/cm2, and 8.4?J/cm2, CW) associated to phenothiazine's derivatives (5 and 10?mu g/ml, toluidine blue O, methylene blue, or phenothiazine) on the promastigote forms of L. braziliensis in a single session. Samples were removed and analyzed in a hemocytometer 72?hours after PACT and viability of the parasites was assessed in quadruplicates. Results An important decrease in the number of viable parasites on all treated groups in comparison to their controls was observed as all tested compounds lead to significant parasite lethality being the highest lethality achieved with 10?mu g/ml of TBO. No lethality was observed on groups treated with laser or with any of the compounds separately. Conclusions TBO presented higher parasite lethality in comparison to MB with impressive reduction from 1?hour to 5?minutes of pre-incubation time. Lasers Surg. Med. 44: 850855, 2012. (c) 2012 Wiley Periodicals, Inc.

Sandflies (Diptera, Psychodidae) from forest areas in Botucatu municipality, central western São Paulo State, Brazil

Abstract Background The study of the distribution and ecology of sandfly species is essential for epidemiological surveillance and estimation of the transmission risk of Leishmania spp. infection. Findings In the present study, sandflies were captured in native fragmented forest areas in Rubião Júnior district, Botucatu municipality, São Paulo state, Brazil, between September 2001 and January 2005. A minimum of two automatic light traps were installed per night from 6 pm to 8 am, in different months, resulting in approximately 900 collecting hours. During this period, 216 sandfly specimens of sixteen species were captured. Pintomyia monticola and Brumptomyia guimaraesi were the most abundant with 56 specimens (25.93%) captured per species, followed by Pintomyia fischeri 28 (12.96%) and Psathyromyia pascalei 18 (8.33%). Other captured species were Lutzomyia amarali, Sciopemyia sordellii, Psathyromyia aragaoi, Nyssomyia whitmani, Migonemyia migonei, Pintomyia bianchigalatiae, Pintomyia misionensis, Brumptomyia carvalheiroi, Brumptomyia cardosoi, Brumptomyia cunhai, Brumptomyia nitzulescui, Brumptomyia brumpti and Brumptomyia spp. represented by 58 (26.85%) specimens. Conclusions Although less frequently found, the presence of Pintomyia fischeri, Nyssomyia whitmani and Migonemyia migonei, known vectors of Leishmania braziliensis, indicates risk of American cutaneous leishmaniasis occurrence. Moreover, the absence of Lutzomyia longipalpis-the main vector of Leishmania infantum chagasi, which is the agent of American visceral leishmaniasis-suggests that there is no risk of introduction and establishment of this disease in the studied area.

Leishmaniose visceral americana: avaliação dos parâmetros da capacidade vetorial de Lutzomyia longipalpis em área urbana do muncípio de Panorama, São Paulo, Brasil

Introdução: A leishmaniose visceral (LV) é um importante problema de saúde pública no Brasil, com cerca 3000 mil casos notificados anualmente. Nos últimos anos, a LV tem ampliado sua distribuição em vários estados do país, associada principalmente aos processos socioambientais, antrópicos e migratórios. A LV é causada pela infecção com Leishmania infantum chagasi, transmitida, principalmente, por Lutzomyia longipalpis (Diptera: Psychodidae). Este flebotomíneo apresenta ampla distribuição nas Américas, todavia, evidências sugerem que se constitui em um complexo de espécies crípticas. A dinâmica de transmissão da LV é modulada por fatores ecológicos locais que influenciam a interação entre populações do patógeno, do vetor e dos hospedeiros vertebrados. Portanto, o estudo das variáveis associadas a esta interação pode contribuir para elucidar aspectos dos elos epidemiológicos e contribuir para a tomada de decisões em saúde pública. Objetivo: Avaliar parâmetros relacionados à capacidade vetorial da população de Lu. longipalpis presente em área urbana do município de Panorama, estado de São Paulo. Métodos: Foram realizadas capturas mensais durante 48 meses para avaliar a distribuição espaço-temporal de Lu. longipalpis e investigar a circulação de Le. i. chagasi. Também foram realizados os seguintes experimentos com o vetor: captura-marcação-soltura-recaptura para estimar a sobrevida da população e a duração do seu ciclo gonotrófico, a atratividade dos hospedeiros mais frequentes em áreas urbanas, a proporção de repasto em cão, infecção experimental e competência vetorial. Resultados: Observou-se que no município de Panorama, Lu. longipalpis apresentou as frequências mais elevadas na estação chuvosa (entre outubro e março), maior densidade em áreas com presença de vegetação e criação de animais domésticos, locais aonde também foi demonstrada a circulação natural de espécimes de Lu. longipalpis infectados com Le. i. chagasi. Além disto, foi corroborado que a população de Lu. longipalpis apresentou hábito hematofágico eclético, altas taxas de sobrevivência e que foi competente para transmitir o agente da LV. Nos experimentos de laboratório foi evidenciada a heterogeneidade na infecção de fêmeas de Lu. longipalpis desafiadas a se alimentarem em cães comprovadamente infectados por L. i. chagasi e o rápido desenvolvimento do parasita neste vetor natural. Conclusões. As observações do presente estudo corroboram a capacidade vetora de Lu. longipalpis para transmitir a Le. i. chagasi e ressaltam a importância da espécie na transmissão do agente etiológico da LV. Ações de manejo ambiental, educação e promoção à saúde são recomendadas às autoridades municipais para diminuir o risco potencial de infecção na população humana e canina, considerando-se o elevado potencial vetor de Lu. longipalpis e a presença de condições que favorecem a interação dos componentes da tríade epidemiológica da LV.

Estudo sobre a participação de roedores na cadeia de transmissão de Leishmania infantum (Protozoa: Trypanosomatidae) no Rio Grande do Norte

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted by the bite of the sand flies Lutzomia longipalpis.The main domestic reservoir is the dog, while foxes and opposums are the known wild reservoirs. However, identification of natural infections with L. infantum in rodents appears for need of investigating the participation of these rodents how source of infection of the parasite. In the present work the Leishmania infantum infection was investigated in rodents captured in Rio Grande do Norte, aiming at to offer subsidies to the understanding of the epidemic chains of LVA in the State. Thirteen Galea spixii were distributed in four groups, being G1 the group control with four animals and the others, G2, G3 and G4, with three animals each. Those animals were intraperitoneally inoculated with 107 promastigotas of L. infantum and accompanied for, respectively, 30, 90 and 180 days. Weekly the animals were monitored as for the corporal weight and rectal temperature. At the end of each stipulated period the animals were killed. Blood were used for determination of the parameters biochemical and haematological, PCR, ELISA, microscopic examination and cultivation in NNN medium. Liver, spleen and lymph node were used in Giemsa-stained impression and cultivation in NNN medium. Liver and spleen fragments were still used in PCR and histopathological, respectively. At the same time 79 rodents of the species Rattus rattus, Bolomys lasiurus, Oligoryzomys nigripis, Oryzomys subflavus and Trichomys apereoides were captured in the Municipal districts of Brejinho, Campo Grande, Coronel Ezequiel, Passa e Fica and Vázea for identification of natural infection with L. infantum. Evidence of infection was checked by direct examination of Giemsa-stained impression of liver, spleen and blood and culture of these tissues in NNN medium. Antibodies were researched by ELISA. They were not found differences among the weigh corporal final, rectal temperature and biochemical and haematological parameters of the Galea spixii controls and infected. The rectal temperature of the animals varied from 36OC to 40OC. For the first time values of the haematocrit (33,6% to 42,8%), hemoglobin (10,2 to 14,5g/dl), erythrocyts number (4,67x106 to 6,90x106/mm3), total leukocytes (0,9x103 to 9,2x103/mm3), platelets (49x103 to 509x103/mm3) total proteins (1,56 to 6,06 g/dl), albumin (1,34 to 3,05 g/dl) and globulins (0,20 to 3,01 g/dl) of the Galea spixii were determined. The lymphocytes were the most abundant leucocytes. Infection for L. infantum was diagnosed in two animals euthanasied 180 days after the infection. In one of the animals was also identified antibodies anti-Leishmania. The parasite was not found in none of the five other species of rodents captured. Galea spixii are resistant to the infection for L. infantum and they are not good models for the study for visceral leishmaniose, although they can act as infection sources. More studies are necessary to determine the paper of the rodents in the epidemic chain of transmission of the visceral leishmaniose in the State of Rio Grande do Norte

A infecção por Leishmania infantum chagasi altera o metabolismo lipídico do hospedeiro

American visceral leishmaniasis (AVL), caused by Leishmania infantum chagasi (L.i.chagasi), stands as a public health problem in Brazil, with human and canine cases related in all states..Lipid metabolism can be modified in several status of infection. For example, experimental studies show that the cholesterol is necessary to internalization and replication of L.i.chagasi in macrophages through caveolar domains. Patients with AVL present low levels of cholesterol and a visible triglycerides increase. This work aimed to evaluate the lipid metabolism in several post-infection status by L.i.chagasi, including individuals with symptomatic infection (AVL), and asymptomatic. The levels of cholesterol, triglycerides, HDL and reactive C protein, were measured. Individuals with AVL were compared with individuals with assymptomatic infection and presented low levels of total cholesterol (128 ± 6.180 mg/dL vs. 158 ±5.733 mg/dL, p=0.0001), HDL (29 ± 1.746 mg/dL vs. 37 ± 1.647 mg/dL, p=0.0001), increased levels of triglycerides (149.5 mg/dL ± 12.72 vs. 78.00 ± 10.43 mg/dL, p=0.0095) and higher levels of reactive C protein (1.750± 0.4939 mg/dL vs. 0.40 ± 0.1707 mg/dL; p=0.0001). The expression of genes related to lipid metabolism, such as LXR-a, LXR-b, PPAR-a, PPAR-d, PPAR-g and APOE was evaluated by real time PCR. A reduction in the expression of those genes was found in the group of AVL patients corroborating the serum levels of the metabolites earlier quantified. Our findings suggest a modulation of metabolism of lipids, in the chronic phase of AVL, this could facilitate the survival of leishmania, due to the known reduction on the ability of macrophages in presenting antigens efficiently to the T cells due to the reduction in the cholesterol available, it results in a subversion of the host immunity.

Spread of Phlebotominae in temperate climates: province of Córdoba, Argentina

We report the presence of the competent vector for Leishmania spp, Migonemyia migonei, and the Evandromyia cortelezzii-sallesi complex south of its known distribution in the central temperate region of Argentina, in the province of Córdoba. The persistence of this phlebotomine in the northern border of the province, its association with a case of cutaneous leishmaniasis, and the new record in the outskirts of the city of Córdoba, the second most populated in the country, strengthens the need for regular vector surveillance and a case detection-sensitive health system in vulnerable regions, even in temperate climates.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii (SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Active surveillance of canine visceral leishmaniasis and american trypanossomiasis in rural dogs from non endemic area

A leishmaniose visceral canina (LVC) e a tripanossomíase americana são importantes zoonoses para a saúde pública que encontram no cão o principal reservatório doméstico para o homem. O trabalho procurou estimar a prevalência de anticorpos circulantes anti-Trypanosoma cruzi e anti-Leishmania spp., em amostras de cães provenientes da zona rural do município de Botucatu, SP. Durante a campanha de vacinação antirrábica canina da zona rural do município, foram coletadas 689 amostras de soro e processadas pela técnica de imunofluorescência indireta. Os testes sorológicos revelaram a ausência de anticorpos anti-Leishmania spp. e, na pesquisa dos anticorpos anti-T. cruzi, foram detectados 3 (0,4%) cães.

A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase

A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.

Phlebotomine (Diptera: Psychodidae) survey and some considerations about epidemiological aspects of canine leishmaniasis in Rio Claro city, São Paulo, Brazil.

Between 2000 and 2004, 170 sand flies were captured in urban or peri-urban areas of the Rio Claro city, SP, Brazil. Nyssomyia neivai, the main LTA vector species in São Paulo territory, corresponded to 124 specimens (72,94%) of the total number of captured insects. Incidence of American Cutaneous (LTA) and Visceral leishmaniasis (LVA) is increasing in human and dogs in São Paulo State. Rio Claro city is located in the center-east region of the State. During 2001 and 2003, two cases of alochtonus canine LVA and an autochthonous case of canine LTA were locally diagnosed. Results indicate the risk of LVA establishment in this region. Possible role of alternative vectors in leishmaniasis transmission is discussed.

Neutrophil extracellular traps identification in tegumentary lesions of patients with paracoccidioidomycosis and different patterns of NETs generation in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clinical and epidemiological aspects of feline leishmaniasis in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.