Evaluation of targets for Strongyloides genus specific molecular diagnosis in experimental strongyloidiasis.

Strongyloides venezuelensis has been used in different experimental studies, such as those aimed at the evaluation of diagnostic techniques for human strongyloidiasis, mainly the molecular diagnosis. In this study, three regions (genus, 18S and 28S targets) of Strongyloides ribosomal DNA were evaluated for the molecular diagnosis of experimental strongyloidiasis. Rats were infected subcutaneously with 400 or 4000 S. venezuelensis infective larvae (400iL3 and 4000iL3), and kept for 35 days. Fecal samples were collected daily to count eggs per gram of feces (EPG) and to perform the polymerase chain reaction (PCR). Egg count started on the 5th day post-infection (pi) and ended on days 33 and 34 pi, in 400iL3 and 4000iL3 groups, respectively. Based in EPG, fecal samples were selected from days 2, 5, 8, 11, 15, 23 and 35 pi for DNA extraction; PCR (genus, 18S and 28S); and sequencing. The PCR-28S products showed higher values of identity (95-100%) in the database with the Strongyloides sequences. Therefore, it is possible to reinforce the application of PCR-28S in the diagnosis of experimental and human strongyloidiasis.

Microscopic and molecular evaluation of Strongyloides venezuelensis in an experimental life cycle using Wistar rats. / Evaluación microscópica y molecular de Strongyloides venezuelensis en un ciclo de vida experimental utilizando ratas Wistar

INTRODUCTION: Strongyloides venezuelensis is a nematode whose natural host is rats. It is used as a model for the investigation of human strongyloidiasis caused by S. stercoralis. The latter is a neglected tropical disease in Ecuador where there are no specific plans to mitigate this parasitic illness. OBJECTIVE: To evaluate the stages of S. venezuelensis in an experimental life cycle using Wistar rats. MATERIALS AND METHODS: Male Wistar rats were used to replicate the natural biological cycle of S. venezuelensis and describe its morphometric characteristics, as well as its parasitic development. Furthermore, the production of eggs per gram of feces was quantified using two diagnostic techniques and assessment of parasite load: Kato-Katz and qPCR. RESULTS: Viable larval stages (L1, L2, L3) could be obtained up to 96 hours through fecal culture. Parthenogenetic females were established in the duodenum on the fifth day postinfection. Fertile eggs were observed in the intestinal tissue and fresh feces where the production peak occurred on the 8th. day post-infection. Unlike Kato-Katz, qPCR detected parasitic DNA on days not typically reported. CONCLUSIONS: The larval migration of S. venezuelensis within the murine host in an experimental environment was equivalent to that described in its natural biological cycle. The Kato-Katz quantitative technique showed to be quick and low-cost, but the qPCR had greater diagnostic precision. This experimental life cycle can be used as a tool for the study of strongyloidiasis or other similar nematodiasis.

Introducción. Strongyloides venezuelensis es un nematodo cuyo huésped natural son las ratas. Se utiliza como modelo para la investigación de la estrongiloidiasis humana producida por S. stercoralis. Esta última es una enfermedad tropical desatendida que afecta al Ecuador, donde no existen planes específicos para mitigar esta parasitosis. Objetivo. Evaluar experimentalmente los estadios del ciclo de vida de S. venezuelensis utilizando ratas Wistar. Materiales y métodos. Se emplearon ratas Wistar macho para replicar el ciclo biológico natural de S. venezuelensis y describir sus características morfométricas y su desarrollo parasitario. Además, se cuantificó la producción de huevos por gramo de heces mediante dos técnicas de diagnóstico y valoración de carga parasitaria: Kato-Katz y qPCR. Resultados. Se obtuvieron estadios larvarios viables (L1, L2, L3) hasta las 96 horas del cultivo fecal. En el duodeno se establecieron hembras partenogenéticas a partir del quinto día de la infección. Se observaron huevos fértiles en el tejido intestinal inspeccionado y en las heces frescas, en las que el pico de producción ocurrió al octavo día de la infección. A diferencia del método Kato-Katz, la qPCR detectó ADN parasitario en días que usualmente no se reportan. Conclusiones. La migración larvaria de S. venezuelensis dentro del ratón en un ambiente experimental fue equivalente al descrito en un ciclo biológico natural. El método cuantitativo de Kato-Katz dio resultados inmediatos a más bajo costo, pero la qPCR tuvo mayor precisión diagnóstica. Este ciclo de vida experimental puede usarse como una herramienta para el estudio de la estrongiloidiasis u otras nematodiasis similares.

Strongyloidiasis in humans: diagnostic efficacy of four conventional methods and real-time polymerase chain reaction.

INTRODUCTION: Strongyloides stercoralis is an intestinal parasitic nematode that causes hyperinfection and/or a dissemination syndrome in hosts, which is often difficult to diagnose. This study aims to compare the diagnostic efficacy of four conventional methods used to diagnose strongyloidiasis with real-time polymerase chain reaction (qPCR) to detect S. stercoralis in fecal samples. METHODS: We analyzed 143 fecal samples collected from Colombian regions with varying degrees of risk for intestinal infections caused by S. stercoralis to assess the validity, performance, overall efficiency, and concordance of the qPCR using a direct stool test, modified Ritchie concentration technique, agar plate culture, and Harada-Mori technique as reference tests. RESULTS: While four fecal samples were positive for S. stercoralis using conventional methods, 32 were positive via qPCR. The diagnostic sensitivity of the qPCR was 75% [95% confidence interval (CI): 20.07-100%], whereas its specificity, negative predictive value, negative likelihood ratio, and Youden's J index were 78.42% (95% CI: 71.22-85.62%), 99.09% (95% CI: 96.86-100%), 0.32 (95% CI: 0.06-1.74), and 0.53, respectively. In addition, the estimated kappa index between the qPCR and the conventional methods was 0.12 (95% CI: -0.020-0.26). CONCLUSIONS: The diagnostic sensitivity of qPCR to detect strongyloidiasis is analogous to that of conventional parasitology methods, with an additional advantage of being capable of identifying the parasite DNA at low sample concentrations.

Strongyloidiasis in humans: diagnostic efficacy of four conventional methods and real-time polymerase chain reaction

Abstract INTRODUCTION: Strongyloides stercoralis is an intestinal parasitic nematode that causes hyperinfection and/or a dissemination syndrome in hosts, which is often difficult to diagnose. This study aims to compare the diagnostic efficacy of four conventional methods used to diagnose strongyloidiasis with real-time polymerase chain reaction (qPCR) to detect S. stercoralis in fecal samples. METHODS: We analyzed 143 fecal samples collected from Colombian regions with varying degrees of risk for intestinal infections caused by S. stercoralis to assess the validity, performance, overall efficiency, and concordance of the qPCR using a direct stool test, modified Ritchie concentration technique, agar plate culture, and Harada-Mori technique as reference tests. RESULTS While four fecal samples were positive for S. stercoralis using conventional methods, 32 were positive via qPCR. The diagnostic sensitivity of the qPCR was 75% [95% confidence interval (CI): 20.07-100%], whereas its specificity, negative predictive value, negative likelihood ratio, and Youden's J index were 78.42% (95% CI: 71.22-85.62%), 99.09% (95% CI: 96.86-100%), 0.32 (95% CI: 0.06-1.74), and 0.53, respectively. In addition, the estimated kappa index between the qPCR and the conventional methods was 0.12 (95% CI: -0.020-0.26). CONCLUSIONS: The diagnostic sensitivity of qPCR to detect strongyloidiasis is analogous to that of conventional parasitology methods, with an additional advantage of being capable of identifying the parasite DNA at low sample concentrations.

Molecular and Immnune Diagnosis: Further Testing for Human Strongyloidiasis.

INTRODUCTION: Detection of Strongyloides stercoralis larvae is particularly challenging because only a small number of larvae are released into the feces, regardless of infection stage. OBJECTIVE: Our objective was to apply conventional polymerase chain reaction (PCR) to the detection of S. stercoralis DNA in feces samples to evaluate its performance in samples of patients with strongyloidiasis and compare results with those of immunodiagnosis. METHODS: Stool, serum, and saliva samples were collected from each individual (n = 48) at the clinic hospital of the State University of Londrina, Brazil, for parasitological, immunological, and molecular tests. Stool samples were processed via parasitological methods. Serum samples were used for immunoglobulin G (IgG) detection and saliva samples for IgA detection by ELISA. RESULTS: For amplification by conventional PCR, two different primers were used: species specific (101 bp) and genus specific (392 bp). The results showed that 34 (97.1%) of the 35 copro-positive individuals for S. stercoralis were positive for serum IgG and 19 (54.3%) were positive for salivary IgA. Regarding molecular analysis, both primers (species and genus specific) demonstrated positivity in 100% of the samples, which was confirmed by sequencing the positive samples. CONCLUSION: Complementary examinations of the parasitological method demonstrated excellent results in the context of the diagnosis of strongyloidiasis, especially in asymptomatic patients with irregular larval release in the feces.

Comparison of parasitological, immunological and molecular methods for evaluation of fecal samples of immunosuppressed rats experimentally infected with Strongyloides venezuelensis.

Definitive diagnosis of strongyloidiasis in humans is typically achieved by detection of larvae in fecal samples. However, limitations on sensitivity of parasitological methods emphasize the need for more robust diagnostic methods. The aim of this study was to compare the diagnostic value of three methods: eggs per gram of feces (EPG), coproantigen detection by enzyme linked immunosorbent assay (ELISA), and DNA detection by conventional polymerase chain reaction (PCR). The assays were performed at 0 and 5, 8, 13, 21 and 39 days post-infection (dpi) using fecal samples from experimentally infected immunocompetent and immunosuppressed rats. In immunocompetent rats, eggs were detected in feces on days 5, 8 and 13 dpi; coproantigen detection and PCR amplification were successful at all post-infection time points (5, 8, 13, 21 and 39 dpi). In immunosuppressed rats, eggs were detected at 5, 8, 13 and 21; coproantigen detection and PCR amplification were successful at all post-infection time points. In conclusion, these results suggest that coproantigen detection and PCR may be more sensitive alternatives to traditional methods such as EPG for diagnosis of Strongyloides venezuelensis infection.

Parasitological and molecular diagnosis in experimental Strongyloides venezuelensis infection.

Strongyloides venezuelensis is a parasitic nematode of rats which is frequently used as a model to study human and animal strongyloidiasis. The aim of this study was to evaluate the correlation between parasitological and molecular diagnosis in Strongyloides venezuelensis infection. PCR assays were used to detect S. venezuelensis DNA in fecal samples obtained from experimentally infected Rattus norvegicus. The results showed a higher sensitivity of the PCR assay in detecting the infection compared to parasitological methods.

Migratory route of Strongyloides venezuelensis in Lewis rats: comparison of histological analyses and PCR.

Strongyloides venezuelensis is a parasitic nematode that has been used as a model to study human and animal strongyloidiasis. In this study, we compared the sensitivity between traditional methodologies and PCR assay to characterize the dynamics of S. venezuelensis infection and its migration route in Lewis rats subcutaneously infected with 4000 L3. The dynamics of the infection was determined by counting the number of eggs and by detecting parasite deoxyribonucleic acid in faeces samples. Both techniques similarly detected the infection at day 6 after larvae inoculation. However, PCR performed with the genus primer showed higher sensitivity during the recovery phase. Histological analysis and PCR assay were then used to follow parasite tissue migration. S. venezuelensis migration route included the muscular fibers below the skin, the pulmonary alveoli and the small intestine vilosities. The sensitivity of these two techniques to detect parasite's presence in these tissues was statistically similar.

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats.

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100% sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100% specificity, whereas PCR sensitivity with the species primer decreased to 77.7%. In low infection, the sensitivity was 60% for EPG, 0% for PCR with the species primer and 90% for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100 percent sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100 percent specificity, whereas PCR sensitivity with the species primer decreased to 77.7 percent. In low infection, the sensitivity was 60 percent for EPG, 0 percent for PCR with the species primer and 90 percent for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Morphological and molecular characterization of Strongyloides ophidiae (Nematoda, Strongyloididae).

The aim of the present study is to report morphological data from parasitic female, rhabditoid and filarioid larvae, free-living female worms and eggs of Strongyloides ophidiae (Nematoda, Strongyloididae). In addition, a molecular DNA analysis was carried out using a pool of eight S. ophidiae parasitic females. Samples were obtained from the small intestine of Oxyrhopus guibei (Serpentes, Colubridae) collected in the municipality of Lençóis Paulista, State of São Paulo, Brazil. DNA amplification by polymerase chain reaction (PCR) resulted in a 350 bp band for samples containing S. ophidiae and Strongyloides venezuelensis DNA. Strongyloides ophidiae nucleotide sequence analysis showed 98% similarity with Strongyloides procyonis and 97% with Strongyloides cebus, Strongyloides stercoralis, Strongyloides fuelleborni and Strongyloides sp. from snakes.

The ubiquitin-proteasome system in Strongyloididae. Biochemical evidence for developmentally regulated proteolysis in Strongyloides venezuelensis.

Nematode parasites from the genus Strongyloides spp. are important pathogens of the intestinal mucosa of animals and humans. Their complex life cycles involve alternating developmental adaptations between larvae stages and the adult parthenogenetic female. Here, we report, primarily through homology-based searching, the existence of the major components of the ubiquitin-proteasome system in this genus, using the available EST data from S. ratti, S. stercoralis, and Parastrongyloides trichosuri. In this study, S. venezuelensis was used as our model organism for detection of proteasome activity and ubiquitinated substrates in cytosolic preparations from the L3 larvae and the adult female. Marked differences in proteasome capabilities were found when these two stages were compared. A preference for degradation of chymotryptic synthetic peptides was found in both stages with the adult exhibiting a higher rate of hydrolysis compared to the larvae. Due to the high evolutionary conservation of proteasome alpha subunits, an anti-human proteasome antibody was able to recognize proteasome subunits in these preparations by Western blotting, supporting the proposal that the activity of the ubiqutin-proteasome system is developmentally regulated in this nematode.