Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.
Abstract: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd 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. 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. The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd 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 5 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 Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. 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.
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Publication Date: 2020-12-01 PubMed ID: 33252651PubMed Central: PMC8492198DOI: 10.1093/cid/ciaa1791Google Scholar: Lookup
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Summary
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The research introduces a highly sensitive assay called AcanR3990 qPCR which is a developed tool for detecting Angiostrongylus cantonensis infections, a leading cause of eosinophilic meningitis in humans.
Research Methodology
- The researchers investigated the rat lungworm, Angiostrongylus cantonensis (Ac), a primary cause of eosinophilic meningitis in humans. The transmission happens when a person ingests the 3rd stage larvae of this parasite from their hosts such as snails, slugs or paratenic hosts.
- The team’s approach involved the in silico design, which is computer-based, of a more sensitive qPCR assay. This was done based on the recognition of tandem repeats predicted to be most abundant by using a tool known as the RepeatExplorer algorithm.
- Genomic DNA (gDNA) of Ac was utilized to establish the analytical sensitivity and specificity related to the ideal set of primers and probes. The newly developed assay was then applied to both clinical and environmental samples.
Outcomes and Findings
- The AcanR3990 assay proved highly efficient, showcasing a detection limit equivalent to a 1/100 000 dilution of a single 3rd stage larvae.
- Application of the assay to 127 CDC archived CSF samples from different geographic locations revealed its sensitivity. It was able to detect Ac in 49 out of 49 cases confirmed by the existing ITS1 qPCR. Interestingly, it also detected Ac in 15 out of 73 samples that had previously tested negative by ITS1 qPCR but had strong clinical suspicion for angiostrongyliasis.
- Further tests conducted on intermediate hosts (gastropods) and an accidental host (a horse showing symptoms) showed similar improvement in detection.
Applicability and Prospects
- The AcanR3990 assay displayed no cross-reactivity in 5 CSF samples from patients diagnosed with other diseases namely, neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis.
- Its only limitation was its inability to amplify genomic DNA from other related Angiostrongylus species, except for Angiostrongylus mackerrasae (Am), a neurotropic species restricted to Australia.
- Overall, the results pointed to AcanR3990 qPCR assay’s high sensitivity and specificity, suggesting its potential for broader applicability as a One Health detection method for Ac and Am.
Cite This Article
APA
Sears WJ, Qvarnstrom Y, Dahlstrom E, Snook K, Kaluna L, Baláž V, Feckova B, Šlapeta J, Modry D, Jarvi S, Nutman TB.
(2020).
AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.
Clin Infect Dis, 73(7), e1594-e1600.
https://doi.org/10.1093/cid/ciaa1791 Publication
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Researcher Affiliations
- Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.
- Parasitic Disease Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
- RML Genomics Unit, Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, Hiwaii, USA.
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, Hiwaii, USA.
- Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceske Budejovice, Czech Republic.
- Department of Ecology and Diseases of Zoo Animals, Game, Fish and Bees, Faculty of Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.
- Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceske Budejovice, Czech Republic.
- Department of Pathology and Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.
- Laboratory of Veterinary Parasitology, Sydney School of Veterinary Science, Faculty of Science, University of Sydney, Sydney, New South Wales, Australia.
- Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceske Budejovice, Czech Republic.
- Department of Pathology and Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.
- Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic.
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, Hiwaii, USA.
- Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.
MeSH Terms
- Angiostrongylus
- Angiostrongylus cantonensis / genetics
- Animals
- Horses
- Humans
- Meningitis
- Rats
- Strongylida Infections / diagnosis
Grant Funding
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases
- SEA-EUROPE JFS 2019-053 / Czech team
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