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      A combined human case of Dirofilaria ursi infection in dorsal subcutaneous tissue and Anisakis simplex sensu stricto (s.s.) infection in ventral subcutaneous tissue

      case-report

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          Abstract

          Background

          Dirofilaria ursi is a filarial nematode that parasitizes the subcutaneous tissues of the American black bear ( Ursus americanus) and Japanese black bear ( Ursus thiabetanus japonicus). D. ursi that has parasitized black bears has the potential to subsequently infect humans. In addition, extra-gastrointestinal anisakiasis is less common in Japan.

          Case presentation

          We report a case of ventral subcutaneous anisakiasis and dorsal subcutaneous dirofilariasis that was acquired in Fukushima, in the northern part of Japan. The patient was an 83-year-old Japanese female, and subcutaneous parasitic granulomas were present on her left abdomen (near the navel) and left scapula. A pathological examination of the surgically dissected tissue sections from each region demonstrated eosinophilic granulomas containing different species of parasites. To enable the morphological and molecular identification of these parasites, DNA was extracted from paraffin-embedded sections using DEXPAT reagent, and the cytochrome oxidase 2 (COX2), internal transcribed spacer 1 (ITS1), 5.8S and ITS2 regions of the Anisakis larvae, and the 5S rRNA region of the male Dirofilaria were sequenced. The PCR products were examined and compared with DNA databases. Molecular analysis of the COX2 and 5S rRNA sequences of each worm revealed that the nematode found in the ventral region belonged to Anisakis simplex sensu stricto (s.s.) and the male Dirofilaria found in the dorsal region was classified as D. ursi.

          Conclusion

          The present case showed a combined human case of D. ursi and A. simplex s.s. infections in subcutaneous tissues. The results of this study will contribute to the identification of unknown parasites in histological sections.

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          Most cited references20

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          Basic local alignment search tool.

          A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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            First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy

            Background Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted. Method We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis. Results The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin. Conclusion The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.
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              Anisakis simplex sensu stricto and Anisakis pegreffii: biological characteristics and pathogenetic potential in human anisakiasis.

              Anisakiasis is one of the most common fishborne helminthic diseases in Japan, which is contracted by ingesting the larvae of the nematode Anisakis spp. carried by marine fish. Anisakis simplex sensu stricto (s.s.) and A. pegreffii are the dominant species in fish caught offshore Japan. The present study aimed to identify the anisakid species infecting Japanese patients and determine whether there is any difference in the pathogenetic potential of A. simplex (s.s.) and A. pegreffii. In total, 41 and 301 Anisakis larvae were isolated from Japanese patients and chub mackerel (Scomber japonicus), respectively; these were subjected to molecular identification using polymerase chain reaction targeted at a ribosomal DNA internal transcribed spacer region. Chub mackerel larvae were further examined for survival in artificial gastric juice (pH 1.8) for 7 days and for invasiveness on 0.75% solid agar over a 24-h interval. All clinical isolates, including those of asymptomatic, acute, and chronic infections as well as those from the stomach, small intestine, colon, and stool, were identified as A. simplex (s.s.). Chub mackerel harbored A. simplex (s.s.) and A. pegreffii larvae, together with a few larvae of other anisakid species. A. simplex (s.s.) larvae from chub mackerel tolerated the artificial gastric juice better than A. pegreffii, with 50% mortality in 2.6 and 1.4 days, respectively. In addition, A. simplex (s.s.) penetrated the agar at significantly higher rates than A. pegreffii. These results show that A. simplex (s.s.) larvae have the potential to survive acidic gastric juice to some extent and penetrate the stomach, small intestine, or colon in infected humans.
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                Author and article information

                Contributors
                minori@koto.kpu-m.ac.jp
                skpatho@shirakawa-kosei.jp
                nozawa86@gmail.com
                unishigehiko@um.edu.my
                din85051@koto.kpu-m.ac.jp
                tnakaya@koto.kpu-m.ac.jp
                Journal
                Trop Med Health
                Trop Med Health
                Tropical Medicine and Health
                BioMed Central (London )
                1348-8945
                1349-4147
                1 November 2017
                1 November 2017
                2017
                : 45
                : 26
                Affiliations
                [1 ]ISNI 0000 0001 0667 4960, GRID grid.272458.e, Department of Infectious Diseases, , Kyoto Prefectural University of Medicine, ; Kyoto, Japan
                [2 ]Department of Pathology, Shirakawa Kosei General Hospital, Fukushima, Japan
                [3 ]ISNI 0000 0001 2308 5949, GRID grid.10347.31, Institute of Biological Sciences, Faculty of Science, , University of Malaya, ; Kuala Lumpur, Malaysia
                Article
                67
                10.1186/s41182-017-0067-4
                5664901
                29118653
                ecda017c-4e71-4af3-a32a-c80880f42df2
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 2 June 2017
                : 28 August 2017
                Categories
                Case Report
                Custom metadata
                © The Author(s) 2017

                Medicine
                helminthic subcutaneous infections,dirofilaria ursi,anisakis simplex sensu stricto (s.s.),a combined human case

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