Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid

Abstract

BackgroundAngiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve the accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the only other neurotropic Angiostrongylus species, which is native to Australia. Methodology/Principal FindingsCerebrospinal fluid (CSF) from dogs (2020-2022) with a presumptive diagnosis of A. cantonensis infection were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR. A newly designed LAMP assay targeting AcanR3390 in a diagnostic laboratory setting was directly compared to the reference ultrasensitive qPCR for determination of presence of A. cantonensis DNA to aid the diagnosis of CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa=0.81, 95%CI 0.69-0.92; n=93) and rapid single-step lysis of archived CSF samples (Kappa=0.77, 95%CI 0.59-0.94; n=52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. Conclusions/SignificanceThe Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in CSF of dogs and performs comparably to laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for effective diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension for humans. Authors summaryA potentially fatal disease, neural angiostrongyliasis, is caused by the rat lungworm (Angiostrongylus cantonensis). The parasite migrates into the spinal cord and brain of accidental hosts, such as humans and dogs, after ingestion of infective larvae. Recently, an ultrasensitive molecular assay which can detect tiny fragments of the parasites DNA was developed and has been used for confirmatory diagnosis. Although this assay outperforms previously developed assays, it requires clean DNA with specialised equipment in a laboratory setting. There is an urgent need for an alternative diagnostic method which is sensitive and portable, for deployment in the field and in the hospitals in remote areas or in low-income countries. The authors developed a fast and portable loop-mediated isothermal amplification (LAMP) assay that compares favourably to the ultra-sensitive PCR assay when tested using cerebrospinal fluid from dogs on the Australian east coast with presumptive neural angiostrongyliasis. Considering a One Health approach to diagnostics, this assay enables portable emergency diagnostics equally suitable to humans, dogs and wildlife. The newly developed assay will also enable water supplies to be screened, as well as crustaceans and molluscs used as potential food sources, for presence of the parasite.