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Rapid and Visual Detection of Trichinella Spp. Using a Lateral Flow Strip-Based Recombinase Polymerase Amplification (LF-RPA) Assay.

ABSTRACT: Trichinella spp., are amongst the most widespread parasitic nematodes, primarily live in the muscles of a wide range of vertebrate animals and humans. Human infection occurs by ingestion of raw or undercooked meat containing Trichinella larvae. Accurate diagnosis of Trichinella spp. infection in domestic animals is crucial for the effective prevention and control of human trichinellosis. In the present study, a simple, rapid and accurate diagnostic assay was developed combining recombinase polymerase amplification and a lateral flow strip (LF-RPA) to detect Trichinella spp. infection. The LF-RPA assay targets Trichinella spp. mitochondrial small-subunit ribosomal RNA (rrnS) gene and can detect as low as 100 fg DNA of Trichinella strains, which was approximately 10 times more sensitive than a conventional PCR assay. The LF-RPA assay can be performed within 10-25 min, at a wide range of temperatures (25-45°C) and showed no cross-reactivity with DNA of other parasites and related host species of Trichinella. The performance of the LF-RPA assay in the presence of high concentration of PCR inhibitor was better than that of a conventional PCR assay. Results obtained by LF-RPA assay for the detection of experimentally infected mice were comparable to the results obtained by using a conventional PCR, achieving 100% specificity and high sensitivity. These results present the developed LF-RPA assay as a new simple, specific, sensitive, rapid and convenient method for the detection of Trichinella infection in domestic animals.

SUBMITTER: Li TT

PROVIDER: S-EPMC6348712 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:BackgroundControl of cutaneous leishmaniasis by public health systems in the Americas relies on case identification and treatment. Point-of-care diagnostics that can be performed by health workers within or near affected communities could effectively bring the health system to the resource-limited sites providing early diagnosis and treatment, reducing morbidity and the burden of disease.Methodology/principal findingsA cross-sectional study was undertaken to evaluate the diagnostic test performance of Isothermal Recombinase Polymerase Amplification (RPA) targeting Leishmania kinetoplast DNA, coupled with a lateral flow (LF) immunochromatographic strip, in a field setting and a laboratory reference center. Minimally invasive swab and FTA filter paper samples were obtained by community health workers and highly trained technicians from ulcerated lesions of > 2 weeks' evolution from 118 patients' ≥ 2 years of age in the municipality of Tumaco, Nariño. Extracted DNA was processed by RPA-LF at a reference center or in a primary health facility in the field. Evaluation was based on a composite "gold standard" that included microscopy, culture, biopsy and real-time polymerase chain reaction detection of Leishmania 18S rDNA. Standard of care routine diagnostic tests were explored as comparators. Sensitivity and specificity of RPA-LF in the reference lab scenario were 87% (95%CI 74-94) and 86% (95%CI 74-97), respectively. In the field scenario, the sensitivity was 75% (95%CI 65-84) and specificity 89% (95%CI 78-99). Positive likelihood ratios in both scenarios were higher than 6 while negative likelihood ratios ranged to 0.2-0.3 supporting the usefulness of RPA-LF to rule-in and potentially to rule-out infection.Conclusions/significanceThe low complexity requirements of RPA-LF combined with non-invasive sampling support the feasibility of its utilization by community health workers with the goal of strengthening the diagnostic capacity for cutaneous leishmaniasis in Colombia.Trial registrationClinicalTrials.gov NCT04500873.

Dataset Information

Rapid and Visual Detection of Trichinella Spp. Using a Lateral Flow Strip-Based Recombinase Polymerase Amplification (LF-RPA) Assay.

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