RESUMO
Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.
Assuntos
DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Strongyloides stercoralis/isolamento & purificação , Estrongiloidíase/diagnóstico , Adulto , Animais , DNA de Helmintos/genética , Humanos , Larva , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Strongyloides stercoralis/genética , Estrongiloidíase/parasitologiaRESUMO
The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.
Assuntos
Babesia bovis/imunologia , Babesiose/veterinária , Interações Hospedeiro-Parasita/imunologia , Lipídeos/imunologia , Macrófagos/imunologia , Transdução de Sinais , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Antifosfolipídeos/sangue , Babesia bovis/química , Babesia bovis/patogenicidade , Babesiose/imunologia , Babesiose/parasitologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Leucócitos Mononucleares/citologia , Lipídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologiaRESUMO
Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.
Assuntos
Babesia bovis/imunologia , Babesia bovis/patogenicidade , Lipídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Receptor 2 Toll-Like/imunologia , Animais , Babesia bovis/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Dinoprostona/biossíntese , Indução Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Merozoítos/efeitos dos fármacos , Merozoítos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Virulência/efeitos dos fármacosRESUMO
Infection with a myotropic Trypanosoma cruzi clone induces maternal fertility failure. In the current work, we evaluated whether reduction of maternal parasitaemia before mating has beneficial effects on pregnancy outcome. Female mice were subjected to benznidazole (Bz) treatment after infection. On day 30 of therapy, mating was assessed and pregnancy outcome was determined on day 14 of gestation. Fetal resorptions diminished in T. cruzi-infected Bz-treated mice compared with T. cruzi-infected untreated mice. This was in agreement with the reduction in the blood/solid tissue parasite load and with the percentage of necrotic foci in placental samples from T. cruzi-infected Bz-treated females. To study eventual changes in the immune homeostasis of T. cruzi-infected Bz-treated mice, activation of the immune system was evaluated at the end of Bz therapy and before mating. We found specific IgG1 reduction resulting in a predominance of specific IgG2a, reduced numbers of CD69+ CD4+ cells and diminished frequency and numbers of CD44+ T cells. Concanavalin A-stimulated splenocytes from T. cruzi-infected Bz-treated mice produced higher amounts of IFN-gamma than T. cruzi-infected untreated mice. These results indicate that reduction of maternal parasite load improves pregnancy outcome. These findings correlate with a favourable modulation of the immune response.
Assuntos
Doença de Chagas/parasitologia , Parasitemia/parasitologia , Complicações Parasitárias na Gravidez/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Doença de Chagas/tratamento farmacológico , Feminino , Regulação da Expressão Gênica , Receptores de Hialuronatos/metabolismo , Imunoglobulina G/sangue , Interferon gama/metabolismo , Lectinas Tipo C , Camundongos , Nitroimidazóis/uso terapêutico , Parasitemia/tratamento farmacológico , Placenta/parasitologia , Gravidez , Complicações Parasitárias na Gravidez/tratamento farmacológico , Resultado da Gravidez , Baço/citologia , Baço/metabolismo , Fatores de TempoRESUMO
Biomphalaria peregrina was found to be naturally infected with cercariae of Zygocotyle lunata in a pond of the Zoological Garden of Buenos Aires. Mice and chicks were fed metacercariae and gravid adults recovered. Eggs recovered from mice feces were used for experimental infections. Laboratory-reared B. peregrina and 4 other Biomphaluria spp. were successfully infected with Z. lunata miracidia. Biomphaleria glabrata was refractory. Species of Helisoma, the only intermediate hosts of Z. lunata so far reported, have never been found in Argentina. Species of Biomphalaria may be intermediate hosts of Z lunata in the southern region of the Parana River Basin.
Assuntos
Biomphalaria/parasitologia , Doenças das Aves/transmissão , Vetores de Doenças , Paramphistomatidae/fisiologia , Infecções por Trematódeos/veterinária , Animais , Argentina , Doenças das Aves/parasitologia , Aves , Galinhas , Camundongos , Infecções por Trematódeos/parasitologia , Infecções por Trematódeos/transmissãoRESUMO
Pathology of chronic Chagas' disease involves peripheral nervous system (PNS) compromise. A high prevalence of antibodies reacting with nervous system antigens has been found in the sera of patients and infected animals, although their physiological role in mediating PNS tissue damage is unknown. Here, we demonstrate that epineural injection of sera from Trypanosoma cruzi infected mice affects the characteristics of the sciatic nerve action potential (SNAP) depending on the parasite strain. Sera from mice infected with the reticulotropic/neurotropic RA strain with reactivity against sciatic nerve (RA/Ne+ sera) induced delays on latency and diminished amplitudes 4 days after injection. Sera from mice infected with the myotropic CA-I strain failed to affect SNAP. Purified immunoglobulin (Ig)G from RA/Ne+ also diminished the amplitude of SNAP. Deposits of IgG labelling axonal fibres and/or myelin sheaths were detected in nerves injected with RA/Ne+ sera. No major histological damage or parasite DNA was found in those nerves. The SNAP changes after sera injection were similar to those observed in mice injected with trypomastigotes in the epineurum 17 days before and in chronically infected animals. This investigation suggests that autoantibodies triggered as a consequence of T. cruzi infection are able to mediate, at least in part, the electrophysiological abnormalities observed in PNS during the course of Chagas' disease.
Assuntos
Doença de Chagas/imunologia , Nervo Isquiático/fisiologia , Potenciais de Ação , Animais , Antígenos de Protozoários , Axônios/imunologia , Doença de Chagas/complicações , Doença de Chagas/parasitologia , DNA de Protozoário/análise , Modelos Animais de Doenças , Humanos , Soros Imunes , Imunoglobulina G/análise , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C3H , Bainha de Mielina/imunologia , Doenças do Sistema Nervoso Periférico/etiologia , Nervo Isquiático/parasitologia , Trypanosoma cruzi/genéticaRESUMO
Biomphalaria straminea snails from Argentina fail to shed cercariae even if exposed to high doses of Schistosoma mansoni EC miracidia. Alternative explanations for this failure are that miracidia are unable to penetrate the snail's epithelium or that the miracidia are killed by the snail's defense system. To discriminate between these 2 possibilities, B. straminea snails were individually exposed to increasing doses of miracidia. Susceptible B. glabrata were used as controls. Exposed snails were fixed 12 hr after exposure, and histological sections of the whole specimens were examined. Miracidia were seen to penetrate the epithelium of B. straminea and B. glabrata at similar rates (14.7%), independent of the exposure level. Regardless of the miracidial dose, 94% of the penetrating miracidia appeared encapsulated by the B. straminea defense system, whereas in B. glabrata, only 42% of the miracidia underwent encapsulation. These results show that resistance of B. straminea to S. mansoni EC strain is due to an efficient defense system that destroys miracidia once they have penetrated.
Assuntos
Biomphalaria/parasitologia , Schistosoma mansoni/imunologia , Animais , Argentina , Biomphalaria/imunologia , Suscetibilidade a Doenças , Vetores de Doenças , Interações Hospedeiro-Parasita , Camundongos , Contagem de Ovos de Parasitas , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/transmissãoRESUMO
To investigate hepatitis C virus (HCV) and GBV-C/hepatitis G virus (HGV) genotype prevalence among HCV-infected porphyria cutanea tarda (PCT) patients, 19 HCV-infected patients with associated PCT were studied. A control group of 53 age-matched HCV-infected patients without associated PCT was selected. Eighteen of the 19 serologically positive HCV-PCT patients showed HCV RNA in serum. Genotype 1b was the most prevalent among both HCV-PCT patients (72.2%; 13/18) and age-matched HCV controls (50.9%; 27/53). Such different genotypic prevalence failed to reach statistical significance (chi(2) with Yates' correction, p = 0.19). The single HCV-PCT patient without detectable HCV RNA was also infected with genogroup 3 GBV-C/HGV. This GBV-C/HGV RNA prevalence (5.3%) among HCV-PCT patients is not statistically different from that observed among Argentine blood donors (5.5%; 11/200). To our knowledge, these results show for the first time the molecular epidemiology of both HCV and GBV-C/HGV associated to PCT in America.
Assuntos
Flaviviridae/genética , Hepacivirus/genética , Hepatite C/complicações , Hepatite Viral Humana/complicações , Porfiria Cutânea Tardia/complicações , Adulto , Idoso , Argentina/epidemiologia , Feminino , Genótipo , Hepatite C/epidemiologia , Hepatite Viral Humana/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , RNA Viral/genéticaRESUMO
For the diagnosis of Chagas' disease, the trans-sialidase inhibition assay was able to resolve the results for samples with borderline results, to detect as positive 60% of samples that were negative by conventional serology, and to discriminate idiopathic from chagasic megaviscera or cardiopathy. No cross-reaction with sera from patients with other diseases was observed.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Glicoproteínas/antagonistas & inibidores , Neuraminidase/antagonistas & inibidores , Animais , Anticorpos Antiprotozoários/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Brasil/epidemiologia , Doença de Chagas/sangue , Doença de Chagas/epidemiologia , Doença de Chagas/imunologia , Reações Cruzadas , Glicoproteínas/imunologia , Humanos , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/imunologia , Malária/sangue , Malária/imunologia , Neuraminidase/imunologia , Testes de Neutralização/métodos , Paraguai/epidemiologia , Sensibilidade e Especificidade , Sífilis/sangue , Sífilis/imunologia , Trypanosoma cruzi/imunologiaRESUMO
There is agreement today about the role that the characteristics of the population of Trypanosoma cruzi play in the pathogenesis of the different clinical forms of Chagas disease. In our laboratory, we have studied the outcome of the infection of mice with two populations with polar biological behaviour: RA and CA-I. We have demonstrated that the neuromuscular damage is, in part, mediated by different T cell subsets. We have also observed that the T cell phenotype responsible for the pathology and the targetted tissues depend on the parasite population. Although we found no differences regarding the reactivity of IgG to native nerve structures in sera from mice infected with either strain, it is presumed that the humoral response would play an additional role in the development of strain-dependent neuromuscular pathology since passive transfer of sera from mice infected with RA triggered alterations of the nerve action potential whereas sera from CA-I-infected mice did not. We have also detected a reduction in the fertility of female mice infected with CA-I/K98, whereas females infected with RA showed no difference in comparison with uninfected controls. However, congenital transmission was only observed in mice infected with RA. The differences observed in fertility, in newborn survival, and in the number of fetal resorptions in mice infected with the myotropic strain could be attributed to the uterine inflammatory response, since no estrous alterations were observed between infected and control groups.