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      Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails

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          Abstract

          Angiostrongylus cantonensis is a zoonotic nematode parasite causing human eosinophilic meningitis (or meningoencephalitis) worldwide. A closely related species, Angiostrongylus malaysiensis, might also be a human pathogen. Larvae were obtained from land snails in Lao PDR, Cambodia, Myanmar and Thailand. We sequenced two nuclear gene regions (nuclear ribosomal ITS2 and SSU rRNA) and a portion of one mitochondrial gene (COI) from these larvae. Angiostrongylus cantonensis and A. malaysiensis were identified. This is the first report of the molecular identification of the two Angiostrongylus species in Lao PDR, Cambodia and Myanmar. The regional distributions of the two species broadly overlap. Phylogenetic relationships were inferred including data from Angiostrongylus species deposited in public databases. All the gene regions we sequenced have potential value in distinguishing between species of Angiostrongylus. The COI gene exhibited the greatest intraspecific variation in the study region (five haplotypes in A. cantonensis and four in A. malaysiensis) and might be suitable for more detailed phylogeographic studies.

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          Neuro-angiostrongyliasis: unresolved issues.

          Angiostrongylus cantonensis, the rat lungworm, probably evolved with its hosts, members of the genus Rattus and closely related species, in south-east Asia. Since its first discovery in rats in China and in a case of human infection in Taiwan, the parasite has been found to infect humans and other mammals across a wide and ever-increasing territory, which now encompasses much of south-east Asia, Melanesia, Polynesia and eastern Australia. It has also established a foothold in Africa, India, the Caribbean and south-eastern USA. This dispersal has been a direct result of human activity, and in some cases has been linked with the spread of the African giant land snail, Achatina fulica. However, this snail is not critical to the extension of the parasite's range, as numerous other indigenous molluscan species serve as adequate intermediate hosts; the importance of Achatina to the life cycle may have been over-emphasized. In Australia, the parasite is established along parts of the east coast, and the presence of an indigenous close relative, Angiostrongylus mackerrasae, suggests a long association of the parasite with its local rat hosts, a situation analogous to that of Angiostrongylus malaysiensis in south-east Asia. These three Angiostrongylus species share virtually the same life cycle, but only A. cantonensis has been confirmed to be a human pathogen.
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            Eosinophilic meningitis caused by Angiostrongylus cantonensis: report of 17 cases.

            To describe two outbreaks of Angiostrongylus cantonensis infection that occurred in Kaohsiung, Taiwan, during 1998 and 1999, and to characterize the source of the outbreaks and the clinical manifestations of the disease. We performed a retrospective cohort study among Thai laborers with eosinophilic meningitis who ate raw snails (Ampullarium canaliculatus), as well as an environmental surveillance of larvae in snails. We enrolled 17 Thai laborers in whom severe headache and eosinophilia developed within 4 to 23 days after eating raw snails. Twelve (71%) developed eosinophilic meningitis. Third-stage larvae were found in the cerebrospinal fluids of 2 patients and in all 12 tested snails. Specific antibodies to A. cantonensis were detected in serum from 16 of the patients and in cerebrospinal fluid from 5 of the patients. Central nervous system manifestations included headache (n = 17 [100%]), fever (n = 11 [65%]), Brudzinski's sign/stiff neck (n = 11 [65%]), hyperesthesia (n = 3 [18%]), cranial nerve palsy (n = 2 [12%]), diplopia (n = 2 [12%]), and ataxia (n = 1 [6%]). Laboratory findings included peripheral eosinophilia (n = 15 [88%]) and cerebrospinal fluid eosinophilia (n = 12 [71%]); elevated immunoglobulin (Ig) E levels (n = 13 [100%]); and transient increases in white blood cell count (n = 7 [41%]) and in serum levels of creatine kinase (n = 7 [41%]), transaminase (n = 3 [18%]), and lactate dehydrogenase (n = 2 [12%]). The severity of illness and eosinophilia were correlated with the number of ingested snails. Meningeal and basal ganglion enhancement was noted on magnetic resonance imaging in several patients. Treatment with mebendazole combined with glucocorticosteroids appeared to shorten the course of the infection, but not the number of relapses. The eosinophil count fell to normal within 3 months, but IgE levels remained elevated for as long as 6 months. All patients recovered with minimal neurologic sequelae. Eosinophilic meningitis caused by A. cantonensis should be considered in patients who have headache or central nervous system manifestations after eating raw snails.
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              Phylogenetic relationships of rat lungworm, Angiostrongylus cantonensis, isolated from different geographical regions revealed widespread multiple lineages.

              We conducted a pilot survey of genetic variation of A. cantonensis using small subunit (SSU) ribosomal (r) RNA and mitochondrial cytochrome c oxidase subunit I (coxI) gene sequences. Two distinct SSU genotypes (G1 and G2) were identified among 17 individual A. cantonensis worms from 17 different geographical localities in Japan, Mainland China, Taiwan, and Thailand. The partial coxI sequences were determined for 83 worms from 18 different geographical localities from Japan, Mainland China, Taiwan, and Thailand. Phylogenetic analysis showed eight distinct coxI haplotypes (ac1 to ac8). In 16 out of 18 localities, only a single coxI haplotype was found. However, in two localities, two coxI haplotypes coexisted. The common haplotypes found were: haplotype ac1 (Tokyo, Chiba, Kanagawa, Amamioshima Island, and Taichung), haplotype ac2 (Ishikawa, Shenzhen, and Lianjiang), haplotype ac5 (the Okinawa and the Ogasawara Islands), and haplotype ac7 (Miyagi, Aichi, and Kanagawa). Each of these regions is separated from the others by high mountain ranges or oceans. In addition, the lower genetic variation and particular geographical distribution of A. cantonensis in each location could indicate a founder effect, which may have resulted from multiple independent origins, and suggests that haplotypes migrated from endemic areas via human-related transportation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 August 2016
                2016
                : 11
                : 8
                : e0161128
                Affiliations
                [1 ]Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
                [2 ]Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
                [3 ]Faculty of Medicine, Mahasarakham University, Mahasarakham, Thailand
                [4 ]Laboratory Unit, Centre of Malariology, Parasitology and Entomology, Ministry of Health, Vientiane, Lao People's Democratic Republic
                [5 ]Department of Microbiology, University of Medicine 2, Yangon, Myanmar
                Australian Museum, AUSTRALIA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceived and designed the experiments: RR PMI WM.

                • Performed the experiments: RR OS LS IP SL WPA.

                • Analyzed the data: RR OS TT WM.

                • Contributed reagents/materials/analysis tools: PMI WM.

                • Wrote the paper: RR WM.

                • Critically reviewed the manuscript: RR PL PMI WM.

                Author information
                http://orcid.org/0000-0002-6726-3759
                Article
                PONE-D-16-07018
                10.1371/journal.pone.0161128
                4981448
                27513930
                ea418fac-b113-44e3-83d1-18619c841596
                © 2016 Rodpai et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 February 2016
                : 29 July 2016
                Page count
                Figures: 4, Tables: 4, Pages: 12
                Funding
                Funded by: The Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission, Thailand through the Health Cluster (SHeP-GMS)
                Award ID: NRU592014
                Award Recipient :
                Funded by: The Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program
                Award ID: grant no. PHD/0053/2556
                Award Recipient :
                Funded by: The Postdoctoral Training Program Graduate School and Khon Kaen University
                Award ID: grant no. 58101
                Award Recipient :
                Funded by: The TRF Senior Research Scholar Grant, Thailand Research Fund
                Award ID: grant number RTA5880001
                Award Recipient :
                Funded by: The TRF Senior Research Scholar Grant, Thailand Research Fund
                Award ID: grant number RTA5880001
                Award Recipient :
                This work was supported by: 1. The Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission, Thailand, through the Health Cluster (SHeP-GMS) Award Number: NRU592014, Recipient: Wanchai Maleewong, PhD. 2. The Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program Award Number: grant no. PHD/0053/2556, Recipient: Rutchanee Rodpai. 3. The Postdoctoral Training Program Graduate School and Khon Kaen University Award Number: grant no. 58101, Recipient: Oranuch Sanpool. 4. The TRF Senior Research Scholar Grant, Thailand Research Fund Award Number: grant number RTA5880001, Recipient: Wanchai Maleewong, PhD, and Pewpan M. Intapan. We certified that all the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript and the revised manuscript has not and will not be submitted for publication elsewhere, have reviewed and approved by all authors, and concurred in the revised submission by the corresponding author.
                Categories
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                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Ribosomal RNA
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                Ribosomal RNA
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