As such, developing laboratories must conduct formal verification to establish performance characteristics before results can be used for decisions regarding patient care. purposes. Keywords:Angiostrongyliasis, Diagnosis, Eosinophilic meningitis, Molecular methods, Serological methods == Introduction == Angiostrongylus cantonensisis a leading cause of eosinophilic meningoencephalitis. Angiostrongyliasis is often suspected when eosinophilic pleocytosis is observed in cases of acute meningitis, particularly if accompanying risk factors, such as residence or travel to an area known to be endemic for the parasite, are also present.1,2A more conclusive diagnosis can be established by direct observation of the parasite in cerebrospinal fluid (CSF) or by using laboratory tests specific forAngiostrongylus-specific antibodies or parasite DNA. Direct observation ofA. cantonensisin the DDX3-IN-1 CSF is not common.3,4Third stage larvae may be present in the brain and CSF one to three weeks after infection. Third stage larvae are slender, measuring 460520m long by 2227m wide, with a rhabditoid esophagus and a tail that constricts before tapering to a blunt end.5Juvenile worms, which may be seen in brain biopsy and also in the vitreous of the eye, can be morphologically distinguished from other nematodes, such asGnathostoma spinigerum, that might be found in the CSF, based on size. The parasite continues to grow in the human host, and may rarely approach the size of adult worms found in rats.6 == Antibody and Antigen Detection == BecauseA. cantonensisis not reliably observed in the CSF of patients with angiostrongyliasis, laboratory diagnosis has historically relied on immunodiagnostic methods to detect parasite specific antibodies. Immunodiagnostic methods for angiostrongyliasis were employed in Mouse monoclonal to CD152 the 1960s soon afterA. cantonensiswas determined to be the probable etiologic agent of eosinophilic meningitis in Asia and the Pacific. An early diagnostic test was an intradermal test based on a skin reaction to adultA. cantonensisextracts.3Results were indicative of angiostrongyliasis if the reaction to adultA. cantonensisextracts was three times greater than the reactions to a phosphate buffer control and toGnathostoma spinigerum, Paragonimus westermani, Dirofilaria immitis, andToxocara canis. Positive reactions were frequently elicited in asymptomatic individuals or patients with other parasitic infections.7 Various laboratory methods that focused on detection ofAngiostrongylus-specific antibodies were developed in the 1970s, including indirect haemagglutination, complement fixation, indirect fluorescent antibody staining of frozen worm sections, lipid extracts of adultA. cantonensis, and latex agglutinations tests.8Enzyme linked immunosorbent assay (ELISA) methods were developed in the late 1970s and used crude antigen extracts prepared from young adultA. cantonensis.9Yen and Chen described ELISAs using partially purifiedA. cantonensisextracts prepared from either juvenile or adultA. cantonensis.10To reduce non-specific reactions, immunoadsorption was used to remove cross-reacting antigens ofToxocara canis, Ascaris lumbricoides, andClonorchis sinensis. Both the juvenile worm and the adult worm preparations performed similarly in these experiments; neither preparation performed with significantly greater sensitivity or specificity than DDX3-IN-1 the other.10 Specificity continued to plague antibody detection methods so scientists sought to identify individual protein antigens that might be more specific than total worm extracts. Immunoblot studies demonstrated that serum antibodies from most patients with angiostrongyliasis specifically recognized the 29 kDa and 31 kDa proteins that are present in adult worm preparations.1115In one report, the 31 kDa protein was more specific than the 29 kDa protein, with minimal cross reactivity from antibodies generated by other commonly encountered tissue-invading helminths.13Specific antibodies are present in serum and CSF so either specimen can be used for immunodiagnosis; however, detection of serum antibodies to the 31 kDa protein was reportedly more sensitive than CSF antibodies in one study.10 Purification of the 31 kDa protein by electroelution from SDS-polyacrylamide gels resulted in a highly sensitive and specific ELISA.16The purified 31 kDa protein was used to develop a dot blot assay for use in regional hospitals in Thailand.17An inter-laboratory evaluation of the dot blot assay proved that the method was easy to perform and results were reliable and reproducible across nine regional hospital laboratories.17The purified 31 kDa antigen has subsequently been incorporated into a multiplex assay for diagnosis of angiostrongyliasis with success.18 Although most studies have focused on the 29 kDa and DDX3-IN-1 31 kDa antigens, detection of antibodies to otherA. cantonensisproteins may also prove to be equally sensitive. Monoclonal antibodies have been used to purify a 204 kDa protein from subadults (stage 5) that was 91% sensitive and 98% specific in patients with eosinophilic meningitis.19 Several studies have evaluated specific immunoglobulin subclass responses, either toA. cantonensiscrude somatic extracts or, in one study, to the 29 kDa protein specifically.20,21Specific IgG1 was the most.