Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Diterpenos de Copaifera reticulata Ducke com atividade larvicida contra Aedes aegypti (L.) (Diptera, Culicidae)

The objective of this study was to evaluate the larvicidal activity of diterpenoids obtained from the oil-resin of Copaifera reticulata against Aedes aegypti larvae, the principal vector of dengue and urban yellow fever. Four diterpenes were obtained from oil-resin extraction with organic solvents and subsequent chromatographic and spectroscopic procedures allowed to isolation and identification of these compounds as 3-b-acetoxylabdan-8(17)-13-dien-15-oic acid (1), alepterolic acid (2), 3-b-hidroxylabdan-8(17)-en-15-oic acid (3), and ent-agatic acid (4). Each compound was previously dissolved in dimethylsulphoxide, and distilled water was added to obtain the desired concentrations. Twenty larvae of third instars were placed into plastic beckers, containing the solution test (25 mL), in a five repetitions scheme, and their mortality, indicated by torpor and darkening of the cephalic capsule, was recorded after 48h. Probit analyses were used to determine lethal concentrations (LC50 and LC90) and their respective 95% confidence intervals. This study showed that only diterpenoids 1 and 2 exhibited larvicidal properties with LC50 of 0.8 ppm and 87.3 ppm, respectively, revealing the former as the most toxic compound against third instars of Ae. aegypti. Therefore, this compound seems to be an interesting source for new metabolite to be exploited.

Immunoperoxidase technique using an anti-Leishmania (L.) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs

Canine visceral leishmaniasis (CVL) is recognizable by characteristic signs of disease and is highly lethal. The infection, however, may be quite inapparent in some seropositive dogs, and this has raised the polemic question as to whether or not such animals can be a source of infection for Lutzomyia longipalpis, the vector of American visceral leishmaniasis (AVL). In this study we have examined 51 dogs with acute CVL from an AVL area in Pará State, northern Brazil, and compared the parasite density, amastigotes of Leishmania (L.) infantum chagasi, in the skin, lymph node and viscera of symptomatic with that of nine asymptomatic but seropositive dogs (IFAT-IgG). Post-mortem biopsy fragments of these tissues were processed by immunohistochemistry, using a polyclonal antibody against Leishmania sp. The X² and Mann Whitney tests were used to evaluate the means of infected macrophage density (p < 0.05). There was no difference (p > 0.05) in the skin (10.7/mm² x 15.5/mm²) and lymph node (6.3/mm² x 8.3/mm²), between asymptomatic and symptomatic dogs, respectively. It was higher (p < 0.05), however, in the viscera of symptomatic (5.3/mm²) than it was in asymptomatic (1.4/mm²) dogs. These results strongly suggest that asymptomatic or symptomatic L. (L.) i. chagasi-infected dogs can serve as a source of infection, principally considering the highest (p < 0.05) parasite density from skin (10.7/mm² x 15.5/mm²), the place where the vetor L. longipalpis takes its blood meal, compared with those from lymph node (6.3/mm² x 8.3/mm²) and viscera (1.4/mm²x 5.3/mm²).

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

In Amazonian Brazil, the Cebus apella monkey (Primates: Cebidae) has been associated with the enzootic cycle of Leishmania (V.) shawi, a dermotropic parasite causing American cutaneous leishmaniasis (ACL). It has also been successfully used as animal model for studying cutaneous leishmaniasis. In this work, there has been investigated its susceptibility to experimental Leishmania (L.) infantum chagasi-infection, the etiologic agent of American visceral leishmaniasis (AVL). There were used ten C. apella specimens, eight adult and two young, four males and six females, all born and raised in captivity. Two experimental infection protocols were performed: i) six monkeys were inoculated, intra-dermal via (ID), into the base of the tail with 2 x 10(6) promastigotes forms from the stationary phase culture medium; ii) other four monkeys were inoculated with 3 x 10(7) amastigotes forms from the visceral infection of infected hamsters by two different via: a) two by intravenous via (IV) and, b) other two by intra-peritoneal via (IP). The parameters of infection evaluation included: a) clinical: physical exam of abdomen, weigh and body temperature; b) parasitological: needle aspiration of the bone-marrow for searching of amastigotes (Giemsa-stained smears) and promastigotes forms (culture medium); c) immunological: Indirect fluorescence antibody test (IFAT) and, Delayed-type hypersensitivity (DTH). In the six monkeys ID inoculated (promastigotes forms) all parameters of infection evaluation were negative during the 12 months period of follow-up. Among the four monkeys inoculated with amastigotes forms, two IV inoculated showed the parasite in the bone-marrow from the first toward to the sixth month p.i. and following that they cleared the infection, whereas the other two IP inoculated were totally negative. These four monkeys showed specific IgG-antibody response since the third month p.i. (IP: 1/80 and IV: 1/320 IgG) toward to the 12th month (IP: 1/160 and IV: 1/5120). The DTH-conversion occurred in only one IV inoculated monkey with a strong (30 mm) skin reaction. Considering these results, we do not encourage the use of C. apella monkey as animal model for studying the AVL.

Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

In this study, we report on the safety and skin delayed-type hypersensitivity (DTH), responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG), Montanide ISA 720 (MISA) or Monophosphoryl lipid A (MPLA) in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels were also measured and these were compared with DTH. Only those animals immunized with alum-BCG reacted adversely to the inoculum by producing ulcerative erythematous skin indurations. Non-parametric analysis of variance followed by a post-test showed significantly higher DTH responses in the MISA+Ag group compared with other immunized groups (p < 0.001). The MPLA+Ag group indicated significantly lower DTH responses to the sonicate antigen compared with the AlBCG+Ag group. There was a significant correlation between the DTH and cytokine responses (p < 0.0001). Based on this study we conclude that Leishmania donovani sonicate antigen containing MISA 720 is safe and is associated with a strong DTH reaction following immunization.

Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection

This study examined the susceptibility of peritoneal macrophage (PM) from the Neotropical primates: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus and Callimico goeldii to ex vivo Leishmania (L.) infantum chagasi-infection, the etiological agent of American visceral leishmaniasis (AVL), as a screening assay for evaluating the potential of these non-human primates as experimental models for studying AVL. The PM-susceptibility to infection was accessed by the PM-infection index (PMI) at 24, 72 h and by the mean of these rates (FPMI), as well as by the TNF-α, IL-12 (Capture ELISA) and Nitric oxide (NO) responses (Griess method). At 24h, the PMI of A. azarae infulatus (128) was higher than those of C. penicillata (83), C. goeldii (78), S. sciureus (77) and C. jacchus (55). At 72h, there was a significant PMI decrease in four monkeys: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) and C. jacchus (55/12), with exception of C. goeldii (78/54). The FPMI of A. azarae infulatus (82.5) and C. goeldii (66) were higher than C. jacchus (33.5), but not higher than those of C. penicillata (60.5) and S. sciureus (57.5). The TNF-a response was more regular in those four primates which decreased their PMI at 24/72 h: C. jacchus (145/122 pg/mL), C. penicillata (154/130 pg/mL), S. sciureus (164/104 pg/mL) and A. azarae infulatus (154/104 pg/mL), with exception of C. goeldii (38/83 pg/mL). The IL-12 response was mainly prominent in A. infulatus and C. goeldii which presented the highest FPMI and, the NO response was higher in C. goeldii, mainly at 72 h. These findings strongly suggest that these New World primates have developed a resistant innate immune response mechanism capable of controlling the macrophage intracellular growth of L. (L.) i. chagasi-infection, which do not encourage their use as animal model for studying AVL.

Molluscicidal and Mosquitocidal Activities of the Essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L.

Steam distillation of essential oils of aerial parts of Thymus capitatus and Marrubium vulgare L. collected at North cost of Egypt yielded 0.5% and 0.2%, respectively. Results of Gas chromatography-mass spectrometry analyses of the two samples identified 96.27% and 90.19% of the total oil composition for T. capitatus and M. vulgare, respectively. The two oil samples appeared dominated by the oxygenated constituents (88.22% for T. capitatus and 57.50% for M. vulgare), composed of phenols, mainly carvacrol (32.98%) and thymol (32.82%) in essential oil of T. capitatus, and thymol (34.55%) in essential oil of M. vulgare. It was evaluated the molluscicidal activity of T. capitatus and M. vulgare essential oils on adult and eggs of Biomphalaria alexandrina as well as their mosquitocidal activity on Culex pipiens. The LC50 and LC90 of T. capitatus essential oil against adult snails was 200 and 400 ppm/3hrs, respectively, while for M. vulgare it was 50 and 100 ppm/3hrs, respectively. Moreover, M. vulgare showed LC100 ovicidal activity at 200 ppm/24 hrs while T. capitatus oil showed no ovicidal activity. It was verified mosquitocidal activity, with LC50 and LC90 of 100 and 200 ppm/12hrs respectively for larvae, and 200 and 400 ppm/12hrs respectively for pupae of C. pipiens.

ANTIFUNGAL ACTIVITY OF Cymbopogon nardus (L.) Rendle (CITRONELLA) AGAINST Microsporum canis FROM ANIMALS AND HOME ENVIRONMENT

Dermatophytosis is a common zoonosis in urban centers. Dogs and cats have played an important role as its disseminators. Environmental decontamination is essential for the prevention of its propagation to humans and animals. However, sanitizers or disinfectants with antifungal activity, currently available, have high toxicity. The present study evaluated the in vitro effects of an extract of citronella (Cymbopogon nardus) on 31 Microsporum canis isolates from animals and home environments. Susceptibility tests were performed based on document M38-A2 (2008) of the Clinical and Laboratory Standards Institute with modifications for natural products. Although susceptibility variation was observed between the fungus tested, the concentrations that inhibited the growth of 50 and 90% of the microorganisms were low (19.5 and 78 µg/mL, respectively). Thus, this citronella extract showed potent fungistatic and fungicide activities against M. canis isolated from animals and home environments. Therefore, it could be an alternative for dermatophytosis prophylaxis in the home environment.

Tentativa de transmissão da Leishmania donovani pela picada do Lutzomyia longipalpis entre cães

Os Autores apresentam dados sôbre tentativas de transmissão experimental da Leishmania donovani pela picada de Lutzomyia longipalpis entre cães. Dois cães jovens sadios foram picados respectivamente por dois e sete flebótomos ricamente infectados e não adquiriram leishmaniose.

Attempts using cryotherapy to achieve more rapid healing in patients with cutaneous leishmaniasis due to L. braziliensis braziliensis

The use of cryotherapy as an adjunct to systemic antimonial therapy (Clucantime) was studied in 17 patients with a total of23 skin lesions of leishmaniasis in an area where L. braziliensis braziliensis is the species in circulation. Cryotherapy did not speed healing and has been discarded as an auxiliary therapeutic measure in our practice. However this technique may be suitable for species o/Leishmania causing more limited superficial lesions in man without the danger of metastasis.

Observações sobre calazar em Jacobina, Bahia. VI - Investigações sobre reservatórios silvestres e comensais

Durante os anos de 1982 a 1986, a investigação sobre mamíferos comensais e silvestres, da periferia da cidade de Jacobina, Bahia, mostrou, ao lado do escasso número de exemplares, uma reduzida variedade específica dessa fauna. Capturou-se apenas 11 espécies, entre as quais, predominou o Didelphis albiventris, que abrangeu 44% dos 213 espécimens capturados. Entre os 193 com exames já concluídos, 84 eram exemplares de D. albiventris e 2 estavam infectados pela Leishmania donovani senso lato, 1 por L. mexicana amazonensis, 1 por L. braziliensis, subespécie e 3 por Trypanosoma cruzi Também foram observadas formas suspeitas de serem amastigotas de leishmanics, nos esfregaços de órgãos de 3 exemplares de Dasyprocta aguti, 1 Cercomys cunicularius - e 1 Oryzomys eliurus. 0 restante dos exemplares, inclusive 14 de Lycalopex vetulus, estava negativo para flagelados. Apesar de reforçado por outros indicadores epidemiológicos, como a predominância específica, a freqüência domiciliar, a atratividade para a vetora Lutzomyia longipalpis, e a concomitância com casos humanos nos mesmos locais, o índice de 2,3% de infecção natural do Didelphis albiventris, não autoriza a conclusão definitiva de ser o marsupial o mais importante reservatório natural da leishmaniose visceral em Jacobina.

Leishmaniose cutânea experimental: II - aspectos evolutivos da infecção no primata Cebus apella (Cebidae) pela Leishmania (V.) Braziliensis e L. (L.) Amazonensis

Objetivando avaliar o potencial do primata C. apella como modelo experimental da leishmaniose cutânea, produzida pela L. (V.) braziliensis e L. (L.) Amazonensis , inocularam-se, via intradérmica, 3 X 10(6) de promastigotas dessas leishmanias, em 8 sítios da cauda de 10 espécimens desse primata, 5 deles com a L. (V.) braziliensis e outros 5 com a L. (L.) Amazonensis . Posteriormente, às inoculações, o exame semanal dos animais e biópsias mensais, revelaram os seguintes resultados relativos a cada parasita: a) L. (V.) braziliensis : o período de incubação foi de 15-20 dias; aos 30 dias evidenciaram-se lesões pápulo-eritematosas, que evoluíram para nódulos ao fim de 60 dias; no 3.° mês, notou-se ulceração espontânea destas lesões e, no 4° mês, deu-se o início da reparação das lesões ulceradas, culminando com a cura em um dos animais após 5 meses, em dois após 6 meses, noutro após 7 meses e, no último, após 10 meses. Quanto ao parasitismo nas lesões, foi demonstrado nos 5 animais, até 90 dias; depois disto, somente em 2 até 120 dias e, por fim, até 180 dias apenas naquele que curou depois de 10 meses, b) L. (L.) Amazonensis : o período de incubação foi de 20 dias; aos 30 dias notou- se lesões pápulo-eritematosas, que também evoluíram para nódulos ao fim de 60 dias, porém, a partir do 3.° mês, estas lesões regrediram rapidamente ao fim de 90 dias, quando não mais detectou-se o parasita na pele dos animais. Em relação aos testes de Montenegro, somente 2 dos 5 animais infectados com a L. (V.) braziliensis reagiram ao teste, 60 e 90 dias após as inoculações. Os resultados observados permitiram confirmar a infectividade do C. apella a estas leishmanias e, também, reforçar a indicação desse primata como modelo experimental da leishmaniose cutânea causada por estes parasitas.

Verificação da eficácia de diferentes inseticidas no controle ambiental de Tunga penetrans (L., 1758)

Referências sobre o controle de Tunga penetrans a nível ambiental têm sido feitas, sem contudo se estabelecer critérios para avaliar a eficácia dos produtos. Neste trabalho usamos as variações das prevalências obtidas antes e após o controle químico, como instrumento de avaliação. Foram testados os piretróides cipermetrina na formulação de concentrado emulsionável, CE, (Cymperator 25CE) a 0,2% e 0,5%; deltametrina na formulação de concentrado emulsionável CE, (K-Othrine 25CE) a 0,6% e deltametrina na formulação de concentrado de suspensão ou flowable concentrate, SC, (K-Othrine 50SC) a 0,3%. O resultado deste trabalho não se baseia apenas na performance dos produtos testados, verificada através da variação de prevalência, mas também em outros aspectos relevantes em saúde pública como a relação custo/beneficio; subsídios fundamentais em campanhas de saúde pública.