Dataset Information

Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

ABSTRACT: ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1-10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.

SUBMITTER: Martinez-Sernandez V

PROVIDER: S-EPMC4954672 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

Martínez-Sernández Victoria V Orbegozo-Medina Ricardo A RA González-Warleta Marta M Mezo Mercedes M Ubeira Florencio M FM

PLoS neglected tropical diseases 20160720 7

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. Ho ...[more]

PMID: 27438470

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Project description:Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop-mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross-sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens' parasitological examination was performed by using the direct wet mount and formalin-ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica-positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51-0.72 and p < .001). According to the results of chi-square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3- to 4-year-old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.

Project description:Intestinal capillariasis is a parasitic zoonosis caused by the tiny nematode parasite Capillaria philippinensis. It is a major health problem that may lead to death if not diagnosed and treated appropriately. The difficulties in the diagnosis of C. philippinensis highlight the importance of developing accurate, sensitive, and specific methods for early diagnosis. This study aimed to detect the presence of C. philippinensis infection among 42 clinically suspected patients with certain criteria that are highly suggestive of capillariasis and to compare the diagnostic yield of microscopy, copro-ELISA, and PCR for the detection of copro-DNA. Sociodemographic characteristics and clinical data were also described for the infected group. Out of 42 patients, 10 were microscopically positive, 40 samples were positive by copro-ELISA, nested PCR detected 35 positive cases, with total detection rates of 23.8%, 95.2%, and 83.3% using direct microscopic examination, copro-ELISA, and PCR, respectively. The majority of positive cases were females, middle-aged people, and people from rural areas. The real number of cases infected with C. philippinensis may far exceed those estimated using microscopy. The diagnosis by copro-ELISA for the detection of C. philippinensis coproantigen and by nested PCR to identify parasite DNA revealed a higher number of positive cases. Using ELISA for the detection of coproantigen is a sensitive test that identifies the infection, yet it is not specific. Copro-DNA offers a satisfactory sensitive and specific method for the detection of infection in clinically suspected patients. The most susceptible individuals to C. philippinensis infection are females, middle-aged people, and people of low social standards. Intestinal capillariasis needs to be considered in patients who present with symptoms of chronic diarrhea and hypoalbuminemia because if these cases are left undiagnosed and untreated, they may suffer from lethal complications.

Project description:Taenia solium, the cause of neurocysticercosis (NCC), has significant socioeconomic impacts on communities in developing countries. This disease, along with taeniasis is estimated to infect 2.5 to 5 million people globally. Control of T. solium NCC necessitates accurate diagnosis and treatment of T. solium taeniasis carriers. In areas where all three species of Taenia tapeworms (T. solium, Taenia saginata and Taenia asiatica) occur sympatrically, conventional microscope- and copro-antigen based diagnostic methods are unable to distinguish between these three Taenia species. Molecular diagnostic tools have been developed to overcome this limitation; however, conventional PCR-based techniques remain unsuitable for large-scale deployment in community-based surveys. Moreover, a real-time PCR (qPCR) for the discrimination of all three species of Taenia in human stool does not exist. This study describes the development and validation of a new triplex Taq-Man probe-based qPCR for the detection and discrimination of all three Taenia human tapeworms in human stools collected from communities in the Central Highlands of Vietnam. The diagnostic characteristics of the test are compared with conventional Kato Katz (KK) thick smear and copro-antigen ELISA (cAgELISA) method utilizing fecal samples from a community based cross-sectional study. Using this new multiplex real-time PCR we provide an estimate of the true prevalence of taeniasis in the source population for the community based cross-sectional study.Primers and TaqMan probes for the specific amplification of T. solium, T. saginata and T. asiatica were designed and successfully optimized to target the internal transcribed spacer I (ITS-1) gene of T. solium and the cytochrome oxidase subunit I (COX-1) gene of T. saginata and T. asiatica. The newly designed triplex qPCR (T3qPCR) was compared to KK and cAgELISA for the detection of Taenia eggs in stool samples collected from 342 individuals in Dak Lak province, Central Highlands of Vietnam. The overall apparent prevalence of taeniasis in Dak Lak province was 6.72% (95% confidence interval (CI) [3.94-9.50]) in which T. solium accounted for 1.17% (95% CI [0.37-3.17]), according to the T3qPCR. There was sympatric presence of T. solium, T. saginata and T. asiatica. The T3qPCR proved superior to KK and cAgELISA for the detection and differentiation of Taenia species in human feces. Diagnostic sensitivities of 0.94 (95% credible interval (CrI) [0.88-0.98]), 0.82 (95% CrI [0.58-0.95]) and 0.52 (95% CrI [0.07-0.94]), and diagnostic specificities of 0.98 (95% CrI [0.94-1.00]), 0.91 (95% CrI [0.85-0.96]) and 0.99 (95% CrI [0.96-1.00]) were estimated for the diagnosis of taeniasis for the T3qPCR, cAgELISA and KK thick smear in this study, respectively.T3qPCR is not only superior to the KK thick smear and cAgELISA in terms of diagnostic sensitivity and specificity, but it also has the advantage of discriminating between species of Taenia eggs in stools. Application of this newly developed T3qPCR has identified the existence of all three human Taenia tapeworms in Dak Lak province and proves for the first time, the existence of T. asiatica in the Central Highlands and the south of Vietnam.

Project description:BACKGROUND:Cattle rearing in Cameroon is both economically and culturally important, however parasitic diseases detrimentally impact cattle productivity. In sub-Saharan Africa bovine fasciolosis is generally attributed to F. gigantica, although understanding of Fasciola species present and local epidemiology in individual countries is patchy. Partly limited by the lack of representative surveys and understanding of diagnostic test perfromance in local cattle populations. The aims of this paper were to determine the Fasciola species infecting cattle, develop a species specific serum antibody ELISA, assess the performance of the ELISA and use it to assess the prevalence of F. gigantica exposure in two important cattle-rearing areas of Cameroon. RESULTS:A random sample of Fasciola parasites were collected and were all identified as F. gigantica (100%, CI:94.0-100%, n?=?60) using RAPD-PCR analysis. A F. gigantica antibody ELISA was developed and initially a diagnostic cut-off was determined using a sample of known positive and negative cattle. The initial cut-off was used as starting point to estimate an optimal cut-off to estimate the best combination of sensitivity and specificity. This was achieved through sampling a naturally infected population with known infection status (cattle slaughtered at Bamenda abattoir, North West Region (n?=?1112) and Ngaoundere abattoir, Vina Division, Adamawa Region (n?=?776) in Cameroon). These cattle were tested and results analysed using a Bayesian non-gold standard method. The optimal cut-off was 23.5, which gave a sensitivity of 65.3% and a specificity of 65.2%. The prevalence of exposure to F. gigantica was higher in cattle in Ngaoundere (56.4% CI: 50.2-60.0%) than Bamenda (0.6% CI: 0.0-1.4%). CONCLUSION:Fasciola gigantica was identified as the predominant Fasciola species in Cameroon. Although the sensitivity and specificity F. gigantica antibody ELISA requires improvement, the test has shown to be a potentially useful tool in epidemiological studies. Highlighting the need for better understanding of the impact of F. gigantica infections on cattle production in Cameroon to improve cattle production in the pastoral systems of Central-West Africa. This paper also highlights that non-gold standard latent class methods are useful for assessing diagnostic test performance in naturally-infected animal populations in resource limited settings.

Project description:BackgroundThe accumulation of misfolded proteins appears as a fundamental pathogenic process in human neurodegenerative diseases. In the case of synucleinopathies such as Parkinson's disease (PD) or dementia with Lewy bodies (DLB), the intraneuronal deposition of aggregated alpha-synuclein (?S) is a major characteristic of the disease, but the molecular basis distinguishing the disease-associated protein (?SD) from its normal counterpart remains poorly understood. However, recent research suggests that a prion-like mechanism could be involved in the inter-cellular and inter-molecular propagation of aggregation of the protein within the nervous system.ResultsOur data confirm our previous observations of disease acceleration in a transgenic mouse line (M83) overexpressing a mutated (A53T) form of human ?S, following inoculation of either brain extracts from sick M83 mice or fibrillar recombinant ?S. A similar phenomenon is observed following a "second passage" in the M83 mouse model, including after stereotactic inoculations into the hippocampus or cerebellum. For further molecular analyses of ?SD, we designed an ELISA test that identifies ?SD specifically in sick mice and in the brain regions targeted by the pathological process in this mouse model. ?SD distribution, mainly in the caudal brain regions and spinal cord, overall appears remarkably uniform, whatever the conditions of experimental challenge. In addition to specific detection of ?SD immunoreactivity using an antibody against Ser129 phosphorylated ?S, similar results were observed in ELISA with several other antibodies against the C-terminal part of ?S, including an antibody against non phosphorylated ?S. This also indicated consistent immunoreactivity of the murine ?S protein specifically in the affected brain regions of sick mice.ConclusionsPrion-like behaviour in propagation of the disease-associated ?S was confirmed with the M83 transgenic mouse model, that could be followed by an ELISA test. The ELISA data question their possible relationship with the conformational differences between the disease-associated ?S and its normal counterpart.

Dataset Information

Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

Publications

Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

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