RESUMO
Failure of treatment of cutaneous leishmaniasis with antimonial drugs and miltefosine is frequent. Use of oral combination therapy represents an attractive strategy to increase efficacy of treatment and reduce the risk of drug resistance. We evaluated the potency of posaconazole, itraconazole, voriconazole, and fluconazole and the potential synergy of those demonstrating the highest potency, in combination with miltefosine (HePC), against infection with Leishmania (Viannia) panamensis. Synergistic activity was determined by isobolograms and calculation of the fractional inhibitory concentration index (FICI), based on parasite quantification using an ex vivo model of human peripheral blood mononuclear cells (PBMCs) infected with a luciferase-transfected, antimony and miltefosine sensitive line of L. panamensis. The drug combination and concentrations that displayed synergy were then evaluated for antileishmanial effect in 10 clinical strains of L. panamensis by reverse transcription-quantitative (qRT-PCR) of Leishmania 7SLRNA. High potency was substantiated for posaconazole and itraconazole against sensitive as well as HePC- and antimony-resistant lines of L. panamensis, whereas fluconazole and voriconazole displayed low potency. HePC combined with posaconazole (Poz) demonstrated evidence of synergy at free drug concentrations achieved in plasma during treatment (2 µM HePC plus 4 µM Poz). FICI, based on 70% and 90% reduction of infection, was 0.5 for the sensitive line. The combination of 2 µM HePC plus 4 µM Poz effected a significantly greater reduction of infection by clinical strains of L. panamensis than individual drugs. Orally administrable miltefosine/posaconazole combinations demonstrated synergistic antileishmanial capacity ex vivo against L. panamensis, supporting their potential as a novel therapeutic strategy to improve efficacy and effectiveness of treatment.
Assuntos
Antiprotozoários , Leishmania guyanensis , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Azóis/farmacologia , Humanos , Leucócitos Mononucleares , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêuticoRESUMO
Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania (Viannia) braziliensis was highly prevalent (69/71), and Leishmania (Viannia) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.
Assuntos
Leishmaniose Cutânea , Recombinases , Animais , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/veterinária , Peru/epidemiologia , Floresta Úmida , Leitura , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Control of cutaneous leishmaniasis by public health systems in the Americas relies on case identification and treatment. Point-of-care diagnostics that can be performed by health workers within or near affected communities could effectively bring the health system to the resource-limited sites providing early diagnosis and treatment, reducing morbidity and the burden of disease. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was undertaken to evaluate the diagnostic test performance of Isothermal Recombinase Polymerase Amplification (RPA) targeting Leishmania kinetoplast DNA, coupled with a lateral flow (LF) immunochromatographic strip, in a field setting and a laboratory reference center. Minimally invasive swab and FTA filter paper samples were obtained by community health workers and highly trained technicians from ulcerated lesions of > 2 weeks' evolution from 118 patients' ≥ 2 years of age in the municipality of Tumaco, Nariño. Extracted DNA was processed by RPA-LF at a reference center or in a primary health facility in the field. Evaluation was based on a composite "gold standard" that included microscopy, culture, biopsy and real-time polymerase chain reaction detection of Leishmania 18S rDNA. Standard of care routine diagnostic tests were explored as comparators. Sensitivity and specificity of RPA-LF in the reference lab scenario were 87% (95%CI 74-94) and 86% (95%CI 74-97), respectively. In the field scenario, the sensitivity was 75% (95%CI 65-84) and specificity 89% (95%CI 78-99). Positive likelihood ratios in both scenarios were higher than 6 while negative likelihood ratios ranged to 0.2-0.3 supporting the usefulness of RPA-LF to rule-in and potentially to rule-out infection. CONCLUSIONS/SIGNIFICANCE: The low complexity requirements of RPA-LF combined with non-invasive sampling support the feasibility of its utilization by community health workers with the goal of strengthening the diagnostic capacity for cutaneous leishmaniasis in Colombia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04500873.
Assuntos
Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Cromatografia de Afinidade , Colômbia , Estudos Transversais , Primers do DNA/genética , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Adulto JovemRESUMO
A controlled clinical trial was carried out to assess the mortality and repellency of a new topical combination of f ipronil-permethrin (Effitix® Virbac, Mexico) against Rhodnius prolixus in dogs. Ten medium-size dogs (10−15kg) with short hair were used. The dogs were exposed to 8 adult triatomines once weekly for 7 weeks. On the control day (D0), the dogs were exposed to the insects without treatment. On D7, the dogs were immediately treated with a spot-on 2.2ml pipette containing 134mg of fipronil and 1200mg permethrin after exposure to the insects. The dose was repeated after 4 weeks following the manufacturer's instructions. Repellency at D0 was, 0 % and the insects had a high blood content. After 12h post-contact, repellency was 86.3 % and slowly decrease though D21 and D28. On D7, none of the insects survived after 3h of feeding on the treated dogs. On D14, D35 and D42, all insects died within 12h post-feeding, whereas no mortality was observed in the control D0 (P < 0.05). The results of this study indicated that administration of the product following the manufacturer's instructions was efficacious at inducing rapid mortality of R. prolixus and therefore could be useful to prevent the transmission of American trypanosomiasis in dogs.
Assuntos
Rhodnius , Doença de Chagas , Permetrina , Cães , InseticidasRESUMO
A pivotal strategy to decrease the risk of visceral leishmaniasis in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum (L. chagasi). Immunotherapy is a viable approach to treat sick dogs because cell-mediated immunity is the principal defense mechanism against L. infantum. Domperidone is an immune-stimulatory drug increasingly used in veterinary medicine as a prophylactic or immunotherapeutic agent. Domperidone treatment has shown to prevent overt disease or improve the clinical condition of infected dogs. However, veterinarians should be aware of the potential cardiotoxicity of domperidone when given together with drugs that inhibit CYP450s liver enzymes or those that prolong the QT interval. On the other hand, learning whether domperidone treatment significantly decreases dog infectivity to sand fly vectors is of capital importance since this result should have a palpable impact on the infection risk of humans living in regions endemic for visceral leishmaniasis.
Assuntos
Doenças do Cão/tratamento farmacológico , Doenças do Cão/parasitologia , Domperidona/uso terapêutico , Antagonistas de Dopamina/uso terapêutico , Leishmaniose Visceral/veterinária , Animais , Cães , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/transmissão , Fatores de RiscoRESUMO
Chagas disease is a lingering Public Health problem in Latin America with â¼5.7 million people infected with Trypanosoma cruzi. Transmission is still taking place in most countries of the Americas, including the United States. Dogs are frequently infected with T. cruzi and its high infection prevalence is associated with increased risk of Chagas disease in humans. The city of Mérida in the Yucatan peninsula is endemic for Chagas disease and canines are frequently infected with T. cruzi. The objective of this study was to evaluate the performance of a qualitative point of care (POC) molecular test (RPA-LF, recombinase polymerase amplification-lateral flow) developed in our laboratory for identifying infected dogs. We used retrospective samples of dogs that came for consultation because of cardiac alterations and proved to be infected with T. cruzi as determined by enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative PCR (qPCR). The analytical sensitivity indicated that RPA-LF amplified T. cruzi DNA in samples containing almost equal to one to two parasites per reaction. Serial twofold dilutions of T. cruzi epimastigotes showed that the test had 95% (19/20) repeatability at concentrations of two parasites per reaction. The test showed no cross reactivity with human DNA or other protozoan parasites (Trypanosoma rangeli, Leishmania spp., and Plasmodium spp.). RPA-LF had the capacity to amplify all discrete typing units (DTUs I-VI) of T. cruzi that circulate in domestic or extradomestic environments. The RPA-LF had 93.2% (95% confidence interval 87.2-98.1) sensitivity and excellent agreement with qPCR used as gold standard (Cohen's Kappa test = 0.963). ELISA was positive in 96.6% (85/88) of dogs, which together with the molecular tests confirmed the frequent contact with infected triatomine bugs in the city of Mérida. These preliminary results on the diagnostic efficacy of the RPA-LF deserve further large-scale field testing of this POC test for T. cruzi infection in endemic areas.
Assuntos
Cardiomiopatia Chagásica/veterinária , Doenças do Cão/parasitologia , Reação em Cadeia da Polimerase/veterinária , Trypanosoma cruzi/genética , Animais , Cardiomiopatia Chagásica/epidemiologia , Cardiomiopatia Chagásica/parasitologia , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Doenças do Cão/patologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , México/epidemiologia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real , Estudos RetrospectivosRESUMO
A pivotal strategy to decrease the risk of visceral leishmaniasis in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum (L. chagasi). Immunotherapy is a viable approach to treat sick dogs because cell-mediated immunity is the principal defense mechanism against L. infantum. Domperidone is an immune-stimulatory drug increasingly used in veterinary medicine as a prophylactic or immunotherapeutic agent. Domperidone treatment has shown to prevent overt disease or improve the clinical condition of infected dogs. However, veterinarians should be aware of the potential cardiotoxicity of domperidone when given together with drugs that inhibit CYP450s liver enzymes or those that prolong the QT interval. On the other hand, learning whether domperidone treatment significantly decreases dog infectivity to sand fly vectors is of capital importance since this result should have a palpable impact on the infection risk of humans living in regions endemic for visceral leishmaniasis.
Assuntos
Animais , Cães , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/terapia , Leishmaniose Visceral/transmissão , Domperidona/uso terapêuticoRESUMO
We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.
Assuntos
Inflamação/sangue , Leishmaniose Cutânea/sangue , Necrose/sangue , Infiltração de Neutrófilos , Animais , Cricetinae , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/parasitologia , Inflamação/fisiopatologia , Leishmania/patogenicidade , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/fisiopatologia , Macrófagos/patologia , Necrose/parasitologia , Necrose/fisiopatologia , Neutrófilos/patologia , Óxido Nítrico/metabolismo , Carga Parasitária , Espécies Reativas de Oxigênio/metabolismo , Estados UnidosRESUMO
Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti-Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 µM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 µM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl2. The combination of disulfiram with ZnSO4 resulted in a 1.8-fold increase in IVTI against L. donovani. This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis.
Assuntos
Cloretos/farmacologia , Dissulfiram/farmacologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Tiram/farmacologia , Tripanossomicidas/farmacologia , Compostos de Zinco/farmacologia , Sulfato de Zinco/farmacologia , Animais , Cátions Bivalentes , Linhagem Celular , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Hep G2 , Humanos , Concentração Inibidora 50 , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Modelos AnimaisRESUMO
Dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis (VL) but current serological methods are not sensitive enough to detect all subclinically infected animals, which is crucial to VL control programs. Polymerase chain reaction (PCR) methods have greater sensitivity but require expensive equipment and trained personnel, impairing its implementation in endemic areas. We developed a diagnostic test that uses isothermal recombinase polymerase amplification (RPA) to detect Leishmania infantum. This method was coupled with lateral flow (LF) reading with the naked eye to be adapted as a point-of-care test. The L. infantum RPA-LF had an analytical sensitivity similar to real time-PCR, detecting DNA of 0.1 parasites spiked in dog blood, which was equivalent to 40 parasites/mL. There was no cross amplification with dog or human DNA or with Leishmania braziliensis, Leishmania amazonensis, or Trypanosoma cruzi. The test also amplified Leishmania donovani strains (N = 7). In a group of clinically normal dogs (N = 30), RPA-LF detected more subclinical infections than rK39 strip test, a standard serological method (50% versus 13.3% positivity, respectively; P = 0.005). Also, RPA-LF detected L. infantum in noninvasive mucosal samples of dogs with a sensitivity comparable to blood samples. This novel molecular test may have a positive impact in leishmaniasis control programs.
Assuntos
DNA de Cinetoplasto/isolamento & purificação , Doenças do Cão/parasitologia , Leishmania/isolamento & purificação , Leishmaniose Visceral/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Sistemas Automatizados de Assistência Junto ao Leito , Animais , Antígenos de Protozoários/isolamento & purificação , Argentina/epidemiologia , DNA de Cinetoplasto/genética , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas de Protozoários/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
In the Americas there are between 4,500 and 6,800 annual cases of severe visceral leishmaniasis, and mortality is estimated to range between 7 and 10%. However, underreporting and subclinical infections mask the real epidemiological importance of visceral leishmaniasis. Control efforts, which have typically focused on insecticide spraying of sand fly vectors and dog culling, have yielded disparate results. Nevertheless, thousands of dogs are sacrificed each year in countries endemic for visceral leishmaniasis. Additionally, current guidelines of leishmaniasis control programs have banned dog treatment with drugs of human use while therapy with other drugs resulted in high rates of relapses. Society requires that control programs take a more humanitarian approach aimed at limiting dog culling. There is an urgent need to promote responsible dog-ownership and support research on: a) novel veterinary therapies, b) low-cost molecular diagnosis of canine visceral leishmaniasis, and c) determination of dog infectivity threshold for proper reservoir management.
Assuntos
Temas Bioéticos , Doenças do Cão/parasitologia , Doenças do Cão/terapia , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/veterinária , Direitos dos Animais , Animais , Doenças do Cão/epidemiologia , Cães , Humanos , América Latina/epidemiologia , Leishmaniose Visceral/epidemiologiaRESUMO
Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.
Assuntos
Arginase/metabolismo , Leishmaniose Visceral/imunologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Receptor IGF Tipo 1/agonistas , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Células Th2/imunologia , Animais , Arginase/genética , Linhagem Celular , Células Cultivadas , Progressão da Doença , Indução Enzimática/efeitos dos fármacos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interleucina-4/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Leishmania donovani/patogenicidade , Leishmania donovani/fisiologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Mesocricetus , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT6/agonistas , Fator de Transcrição STAT6/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Transdução de Sinais/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Células Th2/parasitologiaRESUMO
Leptospirosis en el humano o síndrome de Weil se caracteriza por ictericia progresiva, hemorragias de curso variable, insuficienciarenal o compromiso pulmonar (1). A pesar de los estudios realizados en algunos modelos animales sobre patogenicidad y virulencia de aislados de Leptospira, procedentes de casos humanos (2,3),se desconoce la virulencia y patogenicidad deaislados procedentes de pacientes colombianos.
Assuntos
Humanos , Leptospira , Síndrome de Weill-MarchesaniRESUMO
Introducción. Los caninos son el principal reservorio domestico de la leishmaniasis visceral en el Nuevo y Viejo mundo. La reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real para la medición de citocinas caninas no ha sido implementada para el estudio de la leishmaniasis visceral. Objetivo. Estandarizar la cuantificación relativa de IFN-g, IL-4, IL-10, IL-12p40 y IL-12p35 caninas utilizando reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real. Materiales y métodos. Células mononucleares de sangre periférica de perros Fox-Hound fueron estimuladas con ConA, LPS y extracto de Staphylococcus aureus. El ARN fue utilizado en la reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real de un solo paso para optimizar las concentraciones de iniciadores y sondas especificas de cada citocina, generar curvas estándar, confirmar la eficiencia de amplificación de las citocinas y del normalizador (18S ARNr) y cuantificar la expresión de ARN. El método comparativo Ct fue utilizado para determinar los niveles relativos de expresión de ARN en las muestras, expresado como el incremento en el número de veces comparado con los controles. Resultados. El coeficiente de regresión para las curvas estándar y las eficiencias de amplificación de las citocinas y el normalizador, indicaron que la cuantificación fue confiable en un amplio rango de concentraciones de ARN. La activación de células mononucleares de sangre periférica resultó en un incremento en la expresión de IFN-g (132), IL-4 (8.8), IL-10 (7,2), y IL-12p40 (275), relativo a células control. La expresión basal de IL-12p35 fue también detectada. Conclusión. Esta metodología, comparada con los métodos convencionales disponibles para la medición de citocinas, ofrece varias ventajas y podría ser utilizada en estudios sobre inmunopatogenia e inmunidad en leishmaniasis visceral canina.
Introduction. Canines are the principal domestic reservoirs of visceral leishmaniasis in both the Old and New World. The development of highly sensitive and quantitative methods, such as real time reverse transcriptase polymerase chain reaction for measurement of canine cytokines has not been exploited in studies of visceral leishmaniasis. Objective. To standardize the relative quantification of canine IFN-g, IL-4, IL-10, IL-12p40 and IL-12p35 using real time reverse transcriptase polymerase chain reaction. Materials and methods. RNA was isolated from PBMCs from 1 yearold foxhounds and cultured with or without Con A, LPS or Staphylococcus aureus extract. This RNA was used in one-step real time reverse transcriptase polymerase chain reaction to optimize the concentrations of the cytokine primers and probes, generate standard curves for each cytokine, confirm equivalent amplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify the expression of the cytokine RNA. The comparative Ct method was used to determine the relative levels of gene expression in the samples, expressed as the fold-increase relative to the control samples. Results. The regression coefficient for the standard curves and the amplification efficiencies of the cytokine and normalizer RNA indicated that the quantification was reliable over a broad concentration range of input RNA. Relative to control cells, activation of PBMCs led to increased expression of IFN-g (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basal expression of IL-12p35 was also detected. Conclusion. This approach provides several advantages over conventional assays for cytokine measurement and can be exploited in the study of the immunopathogenesis and immunity in canine leishmaniasis.
Assuntos
Citocinas , Leishmaniose Visceral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T , Reservatórios de DoençasRESUMO
Introducción. A pesar de que la leishmaniasis cutánea canina ha sido reportada en varios focos de Sudamérica, no existe información publicada de Colombia. Objetivo. Se reportan dos casos hallados en la región de la costa Pacífica de este país, que se presentaron como una úlcera escrotal única en un perro y como dos úlceras en la cara externa de la oreja en un segundo individuo. Materiales y métodos. Los parásitos fueron aislados por cultivo en medio de Senekjie e identificados empleando anticuerpos monoclonales. La capacidad de los perros para transmitir parásitos a los flebótomos vectores ( Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia longipalpis, Lutzomyia youngi) se ensayó permitiendo que los insectos se alimentaran sobre el borde de las lesiones. Resultados. Ambos aislamientos se identificaron como Leishmania ( Viannia) braziliensis. No se detectaron infecciones durante la disección de los flebótomos alimentados. Una sola inyección peri- y sub-lesional de 1-2 ml de antimonio pentavalente en el perro con las lesiones auriculares resultó en la curación clínica a las 6 semanas post-tratamiento. Conclusiones. Estas observaciones sugieren que aunque los perros son susceptibles a L. braziliensis, su competencia como reservorio podría ser baja. Sin embargo, si estudios posteriores demuestran que los caninos tienen capacidad de reservorio para especies de L. Viannia, el tratamiento local de las lesiones podría ser una estrategia factible para disminuir el riesgo de infección humana en el peridomicilio, sin necesidad de sacrificar a los perros infectados.
Introduction. Although canine cutaneous leishmaniasis has been reported in several foci of South America, no published information from Colombia is available. Objective. We report on two cases found in the Pacific coast region of this country, which presented as a single scrotal ulcer in one dog, and two ulcers on the external surface of the ear in a second dog. Materials and methods. Parasites were isolated by culture in Senekjies culture medium and identified using monoclonal antibodies. The capacity of these dogs to transmit the parasites to sand fly vectors (Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia longipalpis, Lutzomyia youngi) was tested by allowing the flies to feed on the lesion borders. Results. Both isolates were identified as Leishmania (Viannia) braziliensis. No infections were detected upon dissection of engorged flies. A single peri-and sub-lesional injection of 1-2 ml of pentavalent antimony in the dog with ear lesions resulted in clinical cure 6 weeks post-treatment. Conclusions. These observations suggest that although dogs are susceptible to L. braziliensis, their reservoir competence could be low. However, if further studies indicate that canines are capable reservoir hosts of L. Viannia spp., the local treatment of lesions could become a feasible approach to diminish the risk of human infection in the peridomestic setting, without sacrificing infected dogs.
Assuntos
Cães , Doenças do Cão , Leishmania braziliensis , Leishmaniose Cutânea , ColômbiaRESUMO
INTRODUCTION: Although canine cutaneous leishmaniasis has been reported in several foci of South America, no published information from Colombia is available. OBJECTIVE: We report on two cases found in the Pacific coast region of this country, which presented as a single scrotal ulcer in one dog, and two ulcers on the external surface of the ear in a second dog. MATERIALS AND METHODS: Parasites were isolated by culture in Senekjie's culture medium and identified using monoclonal antibodies. The capacity of these dogs to transmit the parasites to sand fly vectors (Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia longipalpis, Lutzomyia youngi) was tested by allowing the flies to feed on the lesion borders. RESULTS: Both isolates were identified as Leishmania (Viannia) braziliensis. No infections were detected upon dissection of engorged flies. A single peri-and sub-lesional injection of 1-2 ml of pentavalent antimony in the dog with ear lesions resulted in clinical cure 6 weeks post-treatment. CONCLUSIONS: These observations suggest that although dogs are susceptible to L. braziliensis, their reservoir competence could be low. However, if further studies indicate that canines are capable reservoir hosts of L. Viannia spp., the local treatment of lesions could become a feasible approach to diminish the risk of human infection in the peridomestic setting, without sacrificing infected dogs.
Assuntos
Antiprotozoários/uso terapêutico , Doenças do Cão/tratamento farmacológico , Doenças do Cão/parasitologia , Leishmania braziliensis , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/veterinária , Meglumina/uso terapêutico , Compostos Organometálicos/uso terapêutico , Psychodidae/parasitologia , Animais , Colômbia , Cães , Antimoniato de MegluminaRESUMO
INTRODUCTION: Canines are the principal domestic reservoirs of visceral leishmaniasis in both the Old and New World. The development of highly sensitive and quantitative methods, such as real time reverse transcriptase polymerase chain reaction for measurement of canine cytokines has not been exploited in studies of visceral leishmaniasis. OBJECTIVE: To standardize the relative quantification of canine IFN-gamma, IL-4, IL-10, IL-12p40 and IL-12p35 using real time reverse transcriptase polymerase chain reaction. MATERIALS AND METHODS: RNA was isolated from PBMCs from 1 year-old foxhounds and cultured with or without Con A, LPS or Staphylococcus aureus extract. This RNA was used in one-step real time reverse transcriptase polymerase chain reaction to optimize the concentrations of the cytokine primers and probes, generate standard curves for each cytokine, confirm equivalent amplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify the expression of the cytokine RNA. The comparative Ct method was used to determine the relative levels of gene expression in the samples, expressed as the fold-increase relative to the control samples. RESULTS: The regression coefficient for the standard curves and the amplification efficiencies of the cytokine and normalizer RNA indicated that the quantification was reliable over a broad concentration range of input RNA. Relative to control cells, activation of PBMCs led to increased expression of IFN-gamma (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basal expression of IL-12p35 was also detected. CONCLUSION: This approach provides several advantages over conventional assays for cytokine measurement and can be exploited in the study of the immunopathogenesis and immunity in canine leishmaniasis.
Assuntos
Interferon gama/análise , Interleucinas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Cães , Interferon gama/biossíntese , Interferon gama/genética , Interleucinas/biossíntese , Interleucinas/genética , Leishmaniose Visceral/imunologia , Leucócitos Mononucleares/química , RNA Mensageiro/análiseRESUMO
Lutzomyia evansi is the vector of Leishmania chagasi in northern Colombia. Differences in feeding success were revealed, when this phlebotomine sand fly was fed on five species of small mammal hosts from an endemic focus of visceral leishmaniasis. In each trial, 50 female sand flies were provided access to similar-sized depilated areas of the hind foot of each of 44 individual mammals and allowed to feed for 30 minutes. The number of engorged sand flies was counted at the end of each trial and compared among host species by analysis of variance and Tukey's multiple comparisons test. Sand flies fed least successfully on Sciurus granatensis, a common squirrel in the endemic area. It has not been found infected with L. chagasi. Intermediate numbers of sand flies engorged on Heteromys anomalus and Zygodontomys brevicauda, but these two mammals have not been found infected with L. chagasi and are not expected to be important in transmission. Sand flies fed most successfully on Didelphis marsupialis and Proechimys canicollis. These are the two most abundant mammals in the endemic area and frequently are infected. Results provided further evidence that these two species are the wild mammals with the greatest impact on transmission of L. chagasi in northern Colombia.Key words: Phlebotomines, Leishmania, reservoirs, Colombia, attraction.Éxito de la alimentación de Lutzomyia evansi (Diptera: Psychodidae) expuestos experimentalmente a reservorios mamíferos pequeños en un foco endémico de Leishmania chagasi en el norte de Colombia.
Assuntos
Leishmania , Psychodidae , Reservatórios de Água , Colômbia , GambásRESUMO
We determined that the site of inoculation (foot or snout) influences the clinical evolution and immune responses of hamsters infected with Leishmania (Viannia) panamensis. Hamsters infected in the snout showed (i) a more rapid and severe lesion evolution at multiple time points (P < 0.05), (ii) a more extensive inflammatory infiltrate and tissue necrosis, (iii) a higher tissue parasite burden, (iv) a higher antibody titre (P < 0.01), but lower antigen-specific spleen cell proliferative response (P = 0.02), and (v) a slower response to anti-leishmanial drug treatment (P < 0.002). In both inoculation groups there was co-expression of type 1 (IFN-gamma and IL-12) and some type 2 (IL-10 and TGF-beta, but not IL-4) cytokines in the cutaneous lesions and spleen. Early in the course of infection, hamsters infected in the snout showed higher expression of splenic IL-10 (P = 0.04) and intra-lesional IFN-gamma (P = 0.02) than foot infections. No expression of IL-12p40 or IL-4 was detected. During the chronic phase, snout lesions expressed more IFN-gamma (P = 0.001), IL-12p40 (P = 0.01), IL-10 (P = 0.009) and TGF-beta (P = 0.001), and the level of expression of each of these cytokines correlated with lesion size (P < or = 0.01). These results suggest that the site of infection influences the clinical outcome in experimental cutaneous leishmaniasis, and that the expression of macrophage-deactivating type 2 cytokines and/or an exaggerated type 1 proinflammatory cytokine response may contribute to lesion severity.
Assuntos
Citocinas/biossíntese , Leishmania guyanensis , Leishmaniose Mucocutânea/imunologia , Dermatopatias/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Cricetinae , Citocinas/genética , Modelos Animais de Doenças , Interferon Tipo I/metabolismo , Leishmaniose Mucocutânea/parasitologiaRESUMO
Lutzomyia evansi is the vector of Leishmania chagasi in northern Colombia. Differences in feeding success were revealed, when this phlebotomine sand fly was fed on five species of small mammal hosts from an endemic focus of visceral leishmaniasis. In each trial, 50 female sand flies were provided access to similar-sized depilated areas of the hind foot of each of 44 individual mammals and allowed to feed for 30 minutes. The number of engorged sand flies was counted at the end of each trial and compared among host species by analysis of variance and Tukey's multiple comparisons test. Sand flies fed least successfully on Sciurus granatensis, a common squirrel in the endemic area. It has not been found infected with L. chagasi. Intermediate numbers of sand flies engorged on Heteromys anomalus and Zygodontomys brevicauda, but these two mammals have not been found infected with L. chagasi and are not expected to be important in transmission. Sand flies fed most successfully on Didelphis marsupialis and Proechimys canicollis. These are the two most abundant mammals in the endemic area and frequently are infected. Results provided further evidence that these two species are the wild mammals with the greatest impact on transmission of L. chagasi in northern Colombia.