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Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

ABSTRACT: BACKGROUND: In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. multilocularis eggs with two highly sensitive DNA isolation and extraction methods combined with one conventional PCR and three real-time PCR methods for detection. METHODS: We performed a comparison of two extraction protocols; one based on sieving of faecal material and one using targeted DNA sampling. Four methods of molecular detection were tested on E. multilocularis-egg spiked fox faeces. RESULTS: There were significant differences between the multiplex PCR/egg sieving DNA extraction methods compared to the new DNA fishing method and the three real-time PCR assays. Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low. CONCLUSIONS: The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs. Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

SUBMITTER: Oines O

PROVIDER: S-EPMC4060867 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

Øines Øivind Ø Isaksson Mats M Hagström Åsa Å Tavornpanich Saraya S Davidson Rebecca K RK

Parasites & vectors 20140528

<h4>Background</h4>In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. ...[more]

PMID: 24885321

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Project description:BACKGROUND: Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p</=0.05), 35 genes were assigned to the functional gene ontology group <immune response>, while 3 associated with the functional group <intermediary metabolism>. Upregulated genes associated with <immune response> could be clustered into functional subgroups including <macrophages>, <APCs>, <lymphocytes, chemokines and regulation>, <B-cells> and <eosinophils>. Two downregulated genes related to <lymphocytes, chemokines and regulation> and <intermediary metabolism>, respectively. The <immune response> genes either associated with an <immunosupression> or an <immunostimulation> pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied methods. CONCLUSIONS/SIGNIFICANCE: Based on this set of biomarkers, new diagnostic targets have been made available to predict disease status and progression. These biomarkers may also offer new targets for immuno-therapeutic intervention.

Project description:Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples.A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74-0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65-88%), compared to the sensitivity (91-100%) of the new molecular diagnostic workflow.Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.

Project description:BackgroundEchinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.MethodsIn the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.ResultsThe multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/μl, 10 pg/μl and 5 pg/μl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.ConclusionsThe developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.

Dataset Information

Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

Publications

Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

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