Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts

Parasitology ◽  
2020 ◽  
pp. 1-8
Author(s):  
Susan I. Jarvi ◽  
Elizabeth S. Atkinson ◽  
Lisa M. Kaluna ◽  
Kirsten A. Snook ◽  
Argon Steel
Keyword(s):  
Lateral Flow ◽  
Angiostrongylus Cantonensis ◽  
Lateral Flow Assay ◽  
Diagnostic Methods ◽  
Qpcr Assay ◽  
Recombinase Polymerase Amplification ◽  
Intermediate Hosts ◽  
Infection Level ◽  
Specialized Equipment ◽  
The Central Nervous System

Abstract Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai‘i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies μL−1 to ~1 copy μL−1. All three assays consistently detected 50–100 copies μL−1 in triplicate and qPCR was able to detect ~13 copies μL−1 in triplicate. RPA-EXO was able to detect 25 copies μL−1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL−1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

scholarly journals A New Rapid Method for the Authentication of Common Octopus (Octopus vulgaris) in Seafood Products Using Recombinase Polymerase Amplification (RPA) and Lateral Flow Assay (LFA)

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1825
Author(s):  
Amaya Velasco ◽  
Graciela Ramilo-Fernández ◽  
Françoise Denis ◽  
Luís Oliveira ◽  
Peter Shum ◽  
...  
Keyword(s):  
Value Chain ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Recombinase Polymerase Amplification ◽  
Octopus Vulgaris ◽  
Gene Markers ◽  
Ring Trial ◽  
Seafood Products ◽  
Specialized Equipment ◽  
Common Octopus

The common octopus (Octopus vulgaris) is a highly valued cephalopod species which is marketed with different grades of processing, such as frozen, cooked or even canned, and is likely to be mislabeled. Some molecular methods have been developed for the authentication of these products, but they are either labor-intensive and/or require specialized equipment and personnel. This work describes a newly designed rapid, sensitive and easy-to-use method for the detection of Octopus vulgaris in food products, based on Recombinase Polymerase Amplification (RPA) and a detection using a Lateral Flow assay (LFA). After studying several gene markers, a system of primers and nfo-probe was designed in the COI (Cytochrome Oxidase I) region and was successfully tested in 32 reference samples (covering 14 species) and 32 commercial products, after optimization. The method was also validated in a ring trial with eight European laboratories and represents a useful tool for food authenticity control at all levels of the value chain.

The life history of the rat lung-worm, Angiostrongylus cantonensis (Chen) (Nematoda: Metastrongylidae).

1955 ◽  
Vol 3 (1) ◽  
pp. 1 ◽  
Author(s):  
MJ Mackerras ◽  
DF Sandars
Keyword(s):  
Pulmonary Arteries ◽  
Blood Stream ◽  
The Body ◽  
Angiostrongylus Cantonensis ◽  
Intermediate Hosts ◽  
Anterior Portion ◽  
Third Stage ◽  
History Of ◽  
The Central Nervous System ◽  
The Right

Adult Angiostrongylus cantonensis live in the pulmonary arteries. Unsegmented ova are discharged into the blood stream, and lodge as emboli in the smaller vessels. First-stage larvae break through into the respiratory tract, migrate up the trachea, and eventually pass out of the body in the faeces. Slugs (Agriolimax laevis) act as intermediate hosts. Two moults occur in the slug, and third-stage larvae appear about the 17th day. The larvae remain within the two cast skins until freed in the stomach of the rat by digestion. They then pass quickly along the small intestine as far as the lower ileum, where they leave the gut and become blood-borne. They congregate in the central nervous system, and have been found there 17 hr after ingestion. The anterior portion of the cerebrum is the most favoured site, and here the third moult takes place on the sixth or seventh day and the final one between the 11th and 13th days. Young adults emerge on the surface of the brain from the 12th to 14th day, and spend the next 2 weeks in the subarachnoid space. From the 28th to 31st days they migrate to the lungs via the venous system, passing through the right side of the heart to their definitive site in the pulmonary arteries. The prepatent period in the rat usually lies between 42 and 45 days.

scholarly journals AcanR3990 qPCR: a novel, highly sensitive, bioinformatically-informed assay to detect Angiostrongylus cantonensis infections

2020 ◽  
Author(s):  
William J Sears ◽  
Yvonne Qvarnstrom ◽  
Eric Dahlstrom ◽  
Kirsten Snook ◽  
Lisa Kaluna ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Tandem Repeats ◽  
Limit Of Detection ◽  
Clinical Syndrome ◽  
Angiostrongylus Cantonensis ◽  
Qpcr Assay ◽  
Analytical Sensitivity ◽  
Intermediate Hosts ◽  
Highly Sensitive ◽  
Its1 Rdna

Abstract Background Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3 rd stage larvae from primary hosts, snails and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. Methods In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. Results The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100,000 dilution of a single 3 rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in five CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for A. mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. Conclusion These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

scholarly journals Identification of Mycoses in Developing Countries

2019 ◽  
Vol 5 (4) ◽  
pp. 90 ◽  
Author(s):  
Amir Arastehfar ◽  
Brian L. Wickes ◽  
Macit Ilkit ◽  
David H. Pincus ◽  
Farnaz Daneshnia ◽  
...  
Keyword(s):  
Developing Countries ◽  
Real Time ◽  
Real Time Pcr ◽  
Fungal Infections ◽  
Point Of Care ◽  
Fungal Species ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Diagnostic Methods ◽  
Diagnostic Tools

Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.

Key significance of DNA-target size in lateral flow assay coupled with recombinase polymerase amplification

2020 ◽  
Vol 1102 ◽  
pp. 109-118 ◽  
Author(s):  
Irina V. Safenkova ◽  
Alexandr V. Ivanov ◽  
Elvira S. Slutskaya ◽  
Alexey V. Samokhvalov ◽  
Anatoly V. Zherdev ◽  
...  
Keyword(s):  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Recombinase Polymerase Amplification ◽  
Target Size

scholarly journals A comparative evaluation of three methods for the rapid diagnosis of cryptococcal meningitis (CM) among HIV-infected patients in Northern Malawi

2020 ◽  
Vol 32 (1) ◽  
pp. 3-7 ◽  
Author(s):  
Master Chisale
Keyword(s):  
Diagnostic Tests ◽  
Target Population ◽  
Rapid Diagnosis ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Diagnostic Methods ◽  
Gram Stain ◽  
Cross Sectional ◽  
Timely Initiation ◽  
India Ink

IntroductionCryptococcal meningitis (CM) is the most common systemic fungal infection in patients with HIV infection. Rapid diagnosis and timely initiation of antifungal therapy are key to reducing mortality rate associated with CM. This study aims to evaluate the ability of four different diagnostic tests (Gram stain, India ink, and two types of commercial lateral flow assay [LFA]) to identify CM-positive patients and to compare the sensitivity and specificity of these tests.MethodsThis was a prospective cross-sectional study on diagnostic tests accuracy conducted in Northern Malawi. The target population was HIV-infected adult patients presenting with features of meningitis. Four types of diagnostic tests were conducted: India ink, Gram stain, and two types of commercial lateral flow assay (LFA) (Immy, Inc., OK, USA and Dynamiker Biotechnology (Tianjin) Co., Ltd), Singapore). Culture was conducted as the reference standard.ResultsA total of 265 samples were collected. The rate of positive CM detection ranged from 6.4% (using India ink) to 14.3% (using LFA). India ink exhibited the lowest sensitivity of 54.8% (95% confidence interval [CI]: 36.0%–72.7%), followed by Gram stain (61.3%; 95% CI: 42.2%–78.2%). The Dynamiker LFA exhibited the highest sensitivity of 100.0% (95% CI: 90.0%–100.0%) but a lower specificity (97.0%; 93.9%–98.8%) compared to the Immy LFA (98.3%; 95% CI: 95.7%–99.5%). ConclusionLFA diagnostic methods have the potential to double the detection rate of CM-positive patients in resource-limited countries such as Malawi. As such, LFAs should be considered to become the main diagnostic tests used for CM diagnostics in these countries. Our data indicate that LFAs may be the best method for diagnosing CM and exhibits the highest diagnostic accuracy as it has shown that it outperforms cell culture, the current gold standard.

scholarly journals Effective Diagnosis of Foot-And-Mouth Disease Virus (FMDV) Serotypes O and A Based on Optical and Electrochemical Dual-Modal Detection

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 841
Author(s):  
Yun-Jung Hwang ◽  
Kyung-Kwan Lee ◽  
Jong-Won Kim ◽  
Kwang-Hyo Chung ◽  
Sang-Jick Kim ◽  
...  
Keyword(s):  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Lateral Flow ◽  
Mouth Disease ◽  
Lateral Flow Assay ◽  
Diagnostic Methods ◽  
Fmdv Serotypes ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Fmdv Type

Foot-and-mouth disease virus (FMDV) is a highly contagious disease that affects cloven-hoofed animals. The traditional diagnostic methods for FMDV have several drawbacks such as cross-reactivity, low sensitivity, and low selectivity. To overcome these drawbacks, we present an optical and electrochemical dual-modal approach for the specific detection of FMDV serotypes O and A by utilizing a magnetic nanoparticle labeling technique with resorufin β-d-glucopyranoside (res-β-glc) and β-glucosidase (β-glc), without the use of typical lateral flow assay or polymerase chain reaction. FMDV serotypes O and A were reacted with pan-FMDV antibodies that recognize all seven FMDV serotypes (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3). The antigen–antibody complex was then immobilized on magnetic nanoparticles and reacted with β-glc-conjugated FMDV type O or type A antibodies. Subsequently, the addition of res-β-glc resulted in the release of fluorescent resorufin and glucose owing to catalytic hydrolysis by β-glc. The detection limit of fluorescent signals using a fluorescence spectrophotometer was estimated to be log(6.7) and log(5.9) copies/mL for FMDV type O and A, respectively, while that of electrochemical signals using a glucometer was estimated to be log(6.9) and log(6.1) copies/mL for FMDV type O and A, respectively. Compared with a commercially available lateral flow assay diagnostic kit for immunochromatographic detection of FMDV type O and A, this dual-modal detection platform offers approximately four-fold greater sensitivity. This highly sensitive and accurate dual-modal detection method can be used for effective disease diagnosis and treatment, and will find application in the early-stage diagnosis of viral diseases and next-generation diagnostic platforms.

scholarly journals Recombinase Polymerase Amplification and Lateral Flow Assay for Ultrasensitive Detection of Low-Density Plasmodium falciparum Infection from Controlled Human Malaria Infection Studies and Naturally Acquired Infections

2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Albert Lalremruata ◽  
The Trong Nguyen ◽  
Matthew B. B. McCall ◽  
Ghyslain Mombo-Ngoma ◽  
Selidji T. Agnandji ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Reverse Transcription ◽  
Malaria Infection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Recombinase Polymerase Amplification ◽  
Low Density ◽  
Blood Samples ◽  
Human Malaria ◽  
Controlled Human Malaria Infection

ABSTRACT Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

Development of a reverse-transcription recombinase polymerase amplification assay with a lateral flow assay for rapid detection of avian orthoavulavirus 1

2021 ◽  
Vol 33 (2) ◽  
pp. 308-312
Author(s):  
Jindai Fan ◽  
Wenxian Chen ◽  
Yuanyuan Zhang ◽  
Zhixiang Liu ◽  
Xiaoming Li ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Newcastle Disease ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Recombinase Polymerase Amplification ◽  
Control Measures ◽  
Economic Losses ◽  
Resource Limited ◽  
Simple Detection

Newcastle disease is an avian infectious disease caused by avian orthoavulavirus 1, also known as Newcastle disease virus (NDV). This disease has caused significant economic losses to the poultry industry worldwide. The rapid and simple detection of NDV infection is crucial to inform the appropriate control measures. We developed a reverse-transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow assay (LFA) for NDV detection. The RPA assay can be completed at 37°C within 20 min, and the RPA result can be visualized by the LFA within 5 min. The NDV RT-RPA-LFA detected NDV specifically with no cross-reactivity with other pathogens. The detection limit of NDV cDNA with our RT-RPA-LFA was 3.34 × 10−3 ng/μL. Consequently, the RT-RPA-LFA showed good potential for the detection of NDV infection in the field, especially in resource-limited settings.

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