RESUMO
Accurate detection of viable Leishmania parasites is critical for evaluating visceral leishmaniasis (VL) treatment response at an early timepoint. We compared the decay of kinetoplast DNA (kDNA) and spliced-leader RNA (SL-RNA) in vitro, in vivo, and in a VL patient cohort. An optimized combination of blood preservation and nucleic acid extraction improved efficiency for both targets. SL-RNA degraded more rapidly during treatment than kDNA, and correlated better with microscopic examination. SL-RNA quantitative polymerase chain reaction emerges as a superior method for dynamic monitoring of viable Leishmania parasites. It enables individualized treatment monitoring for improved prognoses and has potential as an early surrogate endpoint in clinical trials.
Assuntos
DNA de Cinetoplasto , Leishmaniose Visceral , RNA Líder para Processamento , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , DNA de Cinetoplasto/genética , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/análise , Animais , Leishmania/genética , Antiprotozoários/uso terapêutico , BiomarcadoresRESUMO
Cryptosporidium spp. and Cystoisospora belli are monoxenic protozoa that have been recognized as the causative agents of chronic diarrhea in immunocompromised individuals, especially HIV-infected subjects. The objective of this study was to evaluate the frequency of these intestinal protozoa in HIV-positive patients in the Triângulo Mineiro region of Brazil and to correlate the presence of these infections with clinical, epidemiological and laboratory data of the patients. Oocysts were detected in stool samples of 10 (16.9%) of the 59 patients studied, while Cryptosporidium spp. were present in 10.1% (6/59) and C. belli in 6.7% (4/59). The frequency of these parasites was higher among patients with diarrheic syndrome and CD4+ T lymphocyte counts < 200 cells/mm 3 , demonstrating the opportunistic characteristic of these infections. A significant association was observed between the lack of adherence to antiretroviral therapy and the presence of Cryptosporidium spp. and/or C. belli. Parasitism with Cryptosporidium spp. was more frequent in February and April, the months following the period of high rainfall. The same was not observed for C. belli. Genetic characterization of two isolates led to the identification of Cryptosporidium parvum, one of the main species associated with the zoonotic transmission of cryptosporidiosis.
Cryptosporidium spp. e Cystoisospora belli são protozoários monoxenos reconhecidos como agentes causadores de diarréia crônica em indivíduos imunocomprometidos, especialmente aqueles infectados pelo HIV. Os objetivos deste estudo foram o de avaliar a frequência destes protozoários em pacientes HIV - positivos na região do Triângulo Mineiro, Brasil, e correlacionar a presença destas infecções com dados clínicos, epidemiológicos e laboratoriais dos pacientes. Oocistos foram detectados em amostras fecais de 10 (16,9%) dos 59 pacientes estudados, sendo 10.1% (6/59) das amostras positivas para Cryptosporidium spp. e 6,7% (4/59) das amostras positivas para C. belli. A frequência destes parasitos foi maior entre pacientes com síndrome diarreica e contagem de linfócitos T CD4+ < 200 cells/mm 3 , o que demonstra o caráter oportunista destas infecções. Foi observada uma associação significativa entre a falta de aderência à terapia antiretroviral e a presença de Cryptosporidium spp. e/ou C. belli. Parasitismo por Cryptosporidium spp. foi mais frequente em fevereiro e abril, meses subsequentes ao período chuvoso. O mesmo não foi observado para C. belli. A caracterização genética de dois isolados levou à identificação de Cryptosporidium parvum, uma das principais espécies associadas com a transmissão zoonótica da criptosporidiose.
Assuntos
Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Brasil/epidemiologia , Criptosporidiose/diagnóstico , Criptosporidiose/parasitologia , Cryptosporidium/classificação , Fezes/parasitologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA de Protozoário/análise , RNA Ribossômico/análiseRESUMO
Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.
Assuntos
Grânulos Citoplasmáticos/genética , RNA de Protozoário/análise , RNA de Transferência/análise , Trypanosoma cruzi/genética , Grânulos Citoplasmáticos/química , RNA de Protozoário/genética , RNA de Transferência/genéticaRESUMO
Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.
Assuntos
Antiprotozoários/farmacologia , Cetoconazol/farmacologia , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , /metabolismo , Regulação para Cima/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/análise , RNA de Protozoário/análise , /genéticaRESUMO
The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0 percent (6/197) among cattle and 10.7 percent (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.
O objetivo deste estudo foi avaliar a ocorrência de Cryptosporidium, em animais domésticos de propriedades rurais ao redor de fragmentos de mata Atlântica de interior no município de Teodoro Sampaio, por exame convencional de flutuação em solução de sacarose, seguido de caracterização molecular dos parasitas através do sequenciamento dos produtos amplificados na PCR do gene SSU rRNA. Foram coletadas amostras fecais de animais domésticos criados para subsistência e estimação nas propriedades rurais do entorno de três fragmentos florestais. Amostras de bovinos (197), equinos (63), suínos (25), ovinos (11) e cães (28) foram coletadas de 98 propriedades rurais. A ocorrência de Cryptosporidium para a espécie bovina foi de 3,0 por cento (6/197); para os cães, de 10,7 por cento (3/28); e para os demais animais os resultados foram negativos. Todas as sequências obtidas das seis amostras de bovinos apresentaram identidade molecular com Cryptosporidium andersoni, enquanto as sequências oriundas de amostras de fezes de cães revelaram-se similares ao C. canis. A ocorrência do Cryptosporidium entre os animais estudados foi baixa. Diante dos resultados do presente estudo, o ciclo zoonótico da criptosporidiose parece ter menos importância nesta região. A presença de espécies de Cryptosporidium ainda pouco relatadas em humanos pode ser, por outro lado, importante para a fauna silvestre, uma vez que estes animais podem ser considerados como uma fonte de infecção e disseminação deste protozoário. O impacto e a magnitude da infecção de C. andersoni, em ruminantes selvagens, e de C. canis, em cães silvestres, deve ser avaliado em estudos futuros, com intuito de verificar a real importância dessas espécies nesta região.
Assuntos
Animais , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Animais de Estimação/parasitologia , RNA de Protozoário/análise , Brasil , Saúde da População Rural , ÁrvoresRESUMO
The initial encounter of Leishmania with its host's immune system is important in the outcome of infection. Previous studies have shown that PBMCs from healthy volunteers (HV) exposed to Leishmania differ in IFN-γ production. We have expanded such observations evaluating the profile and kinetics of cytokines (IFN-γ, IL-12p70, IL-10, IL-13), chemokines (CCL5, CCL3, CCL4, CXCL10), and chemokine receptors (CCR1,CCR5, CXCR3, CCR4) in vitro L. amazonensis-stimulated of HV's PBMCs. HVs were divided in groups of high (HR) or low (LR) IFN-γ responders. In both groups, HR and LR, after L. amazonensis infection there was a predominance of IL-10 and IL-13 over IFN-γ production, while IL-12 was produced in similar amount. Regarding chemokines, a more striking difference was observed for CCL3 expression that was lower at 12 hours and 48 hours post infection in LR than in HR. Interestingly, a downregulation of CCR5 and a greater expression of CCR4 were found in low IFN-γ responders. These data suggest that early after L. amazonensis infection there is a cytokine milieu dominated by IL-13 and IL-10, and despite of this environment, IFN-γ is produced, supporting the complexity of the response. It is noteworthy that the pattern of immune response is mounted in first hours after Leishmania stimulation, with the definition of the differentiation of Th1 versus Th2 cells. It remains to be determined if such an in vitro difference has an in vivo counterpart in terms of susceptibility to infection.
Assuntos
Humanos , Interações Hospedeiro-Parasita/imunologia , Imunidade Humoral/imunologia , /biossíntese , /biossíntese , Leishmania mexicana/imunologia , Leucócitos Mononucleares/parasitologia , Citocinas/biossíntese , DNA de Protozoário/análise , Ensaio de Imunoadsorção Enzimática , /imunologia , /imunologia , Leishmania mexicana/fisiologia , Leucócitos Mononucleares/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA de Protozoário/análiseRESUMO
This work aimed to study the T helper type 1/2 (Th1/Th2) cytokine profile in a co-infection murine model of Plasmodium chabaudi chabaudi and Leishmania infantum. Expression of interferon-gamma and interleukin-4 (IL-4) was analyzed, in spleen and liver of C57BL/6 mice, by reverse transcriptase-polymerase chain reaction. High levels of IFN-gamma expression did not prevent the progression of Leishmania in co-infected mice and Leishmania infection did not interfere with the Th1/Th2 switch necessary for Plasmodium control. The presence of IL-4 at day 28 in co-infected mice, essential for Plasmodium elimination, was probably a key factor on the exacerbation of the Leishmania infection.
Assuntos
Animais , Feminino , Camundongos , Interferon gama/análise , /análise , Leishmania infantum/imunologia , Plasmodium chabaudi/imunologia , Células Th1/imunologia , /imunologia , Modelos Animais de Doenças , Interferon gama/genética , /genética , Leishmaniose Visceral/complicações , Leishmaniose Visceral/imunologia , Fígado/imunologia , Fígado/parasitologia , Malária/complicações , Malária/imunologia , Parasitemia/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/análise , RNA de Protozoário/análise , Baço/imunologia , Baço/parasitologiaRESUMO
OBJETIVO: Evaluar en condiciones de laboratorio la sensibilidad y especificidad de una prueba rápida de diagnóstico (OptiMAL), basada en tiras inmunorreactivas para detectar Plasmodium vivax en pacientes febriles del sur de Chiapas, México. MATERIAL Y MÉTODOS: Entre diciembre de 2000 a abril de 2002 se investigó la presencia de parásitos en muestras sanguíneas de 893 pacientes por examen microscópico de gotas gruesas teñidas con Giemsa (prueba de referencia). Otra gota de sangre de la misma punción fue empleada en las tiras inmunorreactivas para investigar la presencia de pLDH del parásito. Los resultados discordantes se resolvieron por PCR del gen de la subunidad ribosomal 18S del parásito para descartar infección. RESULTADOS: OptiMAL mostró una sensibilidad de 93.3 por ciento y especificidad de 99.5 por ciento, con valores predictivo positivo y negativo de 96.5 y 98.9 por ciento, respectivamente. La intensidad de las reacciones en las tiras OptiMAL correlacionaron con la densidad parasitaria (r=0.601, p=0.0001). CONCLUSIONES: La prueba rápida presentó sensibilidad y especificidad aceptables para detectar P. vivax en condiciones de laboratorio y podría ser útil para el diagnóstico de paludismo en operaciones de campo en México.
Assuntos
Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Malária Vivax/diagnóstico , Plasmodium vivax/isolamento & purificação , Malária Vivax/parasitologia , México , Parasitemia/diagnóstico , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , RNA de Protozoário/análise , Fitas Reagentes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis
Assuntos
Animais , Impressões Digitais de DNA/métodos , DNA de Protozoário/análise , Entamoeba histolytica/genética , Reação em Cadeia da Polimerase/métodos , RNA de Protozoário/análise , DNA Complementar , Entamoeba histolytica/patogenicidade , Expressão Gênica , Técnica de Amplificação ao Acaso de DNA Polimórfico , Virulência/genéticaRESUMO
An in vitro translation system has been prepared from Plasmodium falciparum by saponin lysis of infected-erythrocytes to free parasites which were homogeneized with glass beads, centrifuged to obtain a S-30 fraction followed by Sephadex G-25 gel filtration. This treatment produced a system with very low contamination of host proteins (<1). The system, optimized for Mg2+ and K+, translates endogenous mRNA and is active for 80 min which suggests that their protein factors and mRNA are quite stable.