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RESUMEN La pandemia de la Covid 19 forzó a las facultades de medicina a optar por una metodología virtual de enseñanza por la suspensión de las prácticas presenciales con pacientes reales en los establecimientos de salud, debido al confinamiento social y riesgo de contagio. Objetivo Determinar las ventajas y desventajas percibidas por los estudiantes de medicina en relación con la transición de las prácticas presenciales en hospitales a las sesiones virtuales de aprendizaje en un curso de semiología en una facultad de medicina de Lima, Perú. Material y métodos Estudio descriptivo de corte transversal realizado en estudiantes del 4to año de la carrera de Medicina. Los datos se recolectaron mediante una encuesta virtual no validada. Resultados 94 estudiantes respondieron la encuesta (tasa de respuesta: 51,1%). Las ventajas percibidas fueron: 57,4% tener mayor tiempo para estudio teórico y 38,3% en ahorro de tiempo en transporte y movilidad. Las desventajas fueron: 42,6% falta de contacto con pacientes reales y 39,4% no poder realizar una historia clínica adecuada. En cuanto al logro de objetivos de aprendizaje, el 72,3% consideró que logró identificar los problemas de salud del paciente, mientras que el 24,4% afirmó que logró realizar una adecuada historia clínica, y sólo el 9,6% que logró realizar un examen físico completo en pacientes. Conclusión La modalidad virtual de enseñanza permitió a los estudiantes tener más tiempo para revisar aspectos teóricos del curso, pero limitó la adquisición de habilidades prácticas, como realizar una anamnesis adecuada, presentar historias clínicas y examinar pacientes.
SUMMARY The COVID-19 pandemic forced the school of medicines to opt for a virtual teaching modality due to the suspension of face-to-face activities imposed by the lockdown. Objective To determine the advantages and disadvantages of the virtual teaching modality perceived by the students in an introduction to clinical medicine course of a school of medicine in Lima, Peru. Methods A virtual non-validated survey was circulated among fourth year medical students. Results 94 studentes answered the survey (51%). Perceived advantages were to have more time to study (57.4%) and saving time in transportation (39.4%). The disadvantages were lack of contact with real patients (42.6%) and not to be able to obtain a clinical history from patients (39.4%). The 72.3% of students were able to identify the medical problems of patients, but only 24.4% were able to obtain an adequate clinical history and just 9.6% performed an adequate physical examination. Conclusion The virtual teaching modality allowed the student to have more time for self-study but limited their abilities to obtain a clinical history and to perform a physical examination.
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Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.
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It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-γ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-γ/IL-10 and IFN-γ/TGF-ß in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-γ/IL-10 and IFN-γ/TGF-ß ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids.
Assuntos
Proteínas de Insetos/imunologia , Leishmaniose Visceral/prevenção & controle , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Psychodidae/imunologia , Proteínas e Peptídeos Salivares/imunologia , Vacinas de DNA/imunologia , Animais , Cricetinae , Feminino , Proteínas de Insetos/genética , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leishmaniose Visceral/imunologia , Fígado/parasitologia , Linfonodos/imunologia , Masculino , Glicoproteínas de Membrana/genética , Mesocricetus , Proteínas de Protozoários/genética , Psychodidae/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas e Peptídeos Salivares/genética , Baço/imunologia , Baço/parasitologia , Fator de Crescimento Transformador beta/metabolismo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
BACKGROUND: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-ß. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression. CONCLUSIONS/SIGNIFICANCE: Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.
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Transmissão de Doença Infecciosa/prevenção & controle , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Psychodidae/química , Proteínas e Peptídeos Salivares/imunologia , Vacinas de DNA/imunologia , Animais , Cricetinae , Feminino , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/transmissão , Linfonodos/patologia , Masculino , Mesocricetus , Vacinas Protozoárias/administração & dosagem , Saliva/química , Saliva/imunologia , Proteínas e Peptídeos Salivares/isolamento & purificação , Pele/parasitologia , Pele/patologia , Vacinas de DNA/administração & dosagemRESUMO
Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-gamma/TGF-beta ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-gamma was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-gamma at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.
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Proteínas de Insetos/imunologia , Insetos Vetores/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Psychodidae/imunologia , Proteínas e Peptídeos Salivares/imunologia , Animais , Cricetinae , Modelos Animais de Doenças , Humanos , Imunidade , Mordeduras e Picadas de Insetos/imunologia , Proteínas de Insetos/genética , Insetos Vetores/parasitologia , Interferon gama/metabolismo , Vacinas contra Leishmaniose/uso terapêutico , Leishmaniose Visceral/imunologia , Plasmídeos/genética , Psychodidae/parasitologia , Proteínas e Peptídeos Salivares/genética , VacinaçãoRESUMO
Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva 1912) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho 1939) (Diptera: Psychodidae) are vectors of American cutaneous leishmaniasis in several endemic regions of Brazil. We analyzed the external morphological aspects of the immature stages of these two vectors by using scanning electron microscopy. In general, the larval stages of the two species are morphologically similar, although some differences were noted. Detailed examination of the eggs of both species revealed similar exchorionic ornamentations of unconnected parallel ridges. The larval head capsules are well defined, heavily sclerotized, and bear prominent chewing mouthparts. The abdominal segments are easily recognized by the presence of prolegs on their ventral surfaces. The morphology of the anal lobe on the terminal abdominal segment differs between the two species. We found the following three types of sensillae inserted on the antennae: (1) clavate basiconic; (2) small, blunt coeloconic; and (3) multipourous clavate coleoconic. In addition; five subtypes of trichoid sensillae were found on the larval body: (1) long, (2) short, (3) curved long, (4) brush-like, and (5) weakly brush-like. The caudal filaments located on the last abdominal segment were recognized as long trichoid sensillae. We observed pores on the surface of the clavate coelonic sensillae and on the caudal filaments that presumably function as chemoreceptors. The larvae of the two species show similarities in the lobular-form antennae of L1 larvae, which changes to digitiform in second instar (L2), L3, and L4. This study demonstrated that the external surface of the eggs and larvae of Lu. intermedia and Lu. whitmani are morphologically similar, but they can be distinguished by details in the microanatomy observed by scanning electron microscopy.
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Leishmaniose Cutânea/transmissão , Psychodidae/ultraestrutura , Animais , Insetos Vetores , Larva/ultraestrutura , Microscopia Eletrônica de Varredura , Óvulo/ultraestruturaRESUMO
Experiments in animals verified that phlebotomine saliva enhances Leishmania infection, and vaccination with saliva prevents disease. We have shown that individuals from an endemic area of visceral leishmaniasis displayed robust antibody responses to saliva from the vector Lutzomyia longipalpis, which correlated with anti-parasite cell-mediated immunity. Here, we explored human anti-saliva responses following exposure to sand flies, using an in vivo bite model in which normal volunteers were exposed four times to 30 laboratory-reared Lu. longipalpis. Following the third exposure, normal volunteers developed diverse dermatological reactions at the site of insect bite. Serum from normal volunteers displayed high levels of anti-salivary gland sonicate IgG1, IgG4 and IgE as well as several salivary gland proteins. Furthermore, following in vitro stimulation with salivary gland sonicate, there was an increased frequency of CD4(+)CD25(+) and CD8(+)CD25(+) T cells as well as IFN-gamma and IL-10 synthesis. Strikingly, 1 year after the first exposure, PBMC from the volunteers displayed recall IFN-gamma responses that correlated with a significant reduction in infection rates using a macrophage-lymphocyte autologous culture. Together, these data suggest that human immunization against sand fly saliva is feasible and recall responses are obtained even 1 year after exposure, opening perspectives for vaccination in man.
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Proteínas de Insetos/imunologia , Insetos Vetores/imunologia , Leishmaniose Visceral/prevenção & controle , Psychodidae/imunologia , Saliva/imunologia , Adulto , Animais , Western Blotting , Citocinas/sangue , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Memória Imunológica , Leishmaniose Visceral/transmissão , Masculino , Proteínas e Peptídeos Salivares/imunologia , Linfócitos T/imunologiaRESUMO
Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.
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Quimiocina CCL2/genética , Quimiotaxia , Macrófagos/fisiologia , Psychodidae/imunologia , Saliva/imunologia , Animais , Regulação da Expressão Gênica , Inflamação/etiologia , Leishmania/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade da EspécieRESUMO
Sand flies inject saliva into the mammalian host when probing for a blood meal. Understanding the initial vertebrate reactions against sand fly saliva is important for possible interventions because these insects transmit diseases to humans and other animals. Little is known of these reactions to New World sand flies. Repeated exposure of BALB/c mice to Lutzomyia longipalpis bites leads to local inflammatory cell infiltration comprised of neutrophils, macrophages, and eosinophils. Total IgG and IgG1 antibodies react predominantly with three major protein bands (45, 44, and 16 kD) of the insect saliva by Western blot. The injection of immune serum previously incubated with salivary gland homogenate induced an early infiltration with neutrophils and macrophages, suggesting the participation of immune complexes in triggering inflammation.
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Anticorpos/imunologia , Mordeduras e Picadas de Insetos/imunologia , Macrófagos/imunologia , Psychodidae/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Saliva/imunologia , Linfócitos T/imunologiaRESUMO
Antibody responses to salivary gland sonicate (SGS) from Lutzomyia longipalpis were investigated using serum samples from individuals living in an area where visceral leishmaniasis is endemic. Individuals were classified into 2 groups, according to the alteration of their responses to Leishmania chagasi antigen over the course of 6 months. Group 1 included children who experienced anti-L. chagasi seroconversion from negative to positive; group 2 included children who experienced delayed-type hypersensitivity (DTH) response to L. chagasi antigen conversion from negative to positive. Individuals who experienced seroconversion against L. chagasi antigens did not have increased anti-saliva antibody response, whereas those who developed a positive anti-L. chagasi DTH response had increased immunoglobulin (Ig) G, IgG1 and IgE anti-SGS antibody levels. Despite wide variation, serum samples from individuals in group 2 recognized more bands in SGS than did those from individuals in group 1. This simultaneous appearance of anti-saliva humoral response and anti-L. chagasi cell-mediated immunity supports the hypothesis that induction of immune response against SGS can facilitate induction of a protective response against leishmaniasis.