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      Molecular characterization of human Dirofilaria isolates from Kerala

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          Abstract

          Background & objectives:

          Human dirofilariosis is a well-recognized zoonosis caused by several species of the genus Dirofilaria. The disease is prevalent among canines and human beings in Kerala. The objective of the present study was to confirm the human Dirofilaria isolates by molecular characterization.

          Methods:

          The worms or segments obtained from human sources were subjected to diagnostic polymerase chain reaction (PCR) targeting the cytochrome oxidase subunit 1 ( COI) and 5S ribosomal RNA ( rRNA) genes of Dirofilaria repens. The amplicons were sequenced and analyzed.

          Results:

          The filariid nematodes recovered from ocular as well as subcutaneous tissue of human origin were identified as D. repens based on PCR targeting COI as well as 5S rRNA genes. The phylogenetic analysis of the COI gene nucleotide sequence obtained in the present study showed that D. repens shared the closest evolutionary relationship with D. honkongensis.

          Interpretation & conclusions:

          Molecular identification of D. repens isolated from human source assumes significance from the point of zoonotic threat of this mosquito-borne nematode. Phylogenetic analysis revealed a close evolutionary relationship with Asian isolate of D. honkongensis. Timely detection and treatment of infection in dogs, together with mosquito control, should be an integral part of the control strategy of this disease.

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          Most cited references22

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          Human and animal dirofilariasis: the emergence of a zoonotic mosaic.

          Dirofilariasis represents a zoonotic mosaic, which includes two main filarial species (Dirofilaria immitis and D. repens) that have adapted to canine, feline, and human hosts with distinct biological and clinical implications. At the same time, both D. immitis and D. repens are themselves hosts to symbiotic bacteria of the genus Wolbachia, the study of which has resulted in a profound shift in the understanding of filarial biology, the mechanisms of the pathologies that they produce in their hosts, and issues related to dirofilariasis treatment. Moreover, because dirofilariasis is a vector-borne transmitted disease, their distribution and infection rates have undergone significant modifications influenced by global climate change. Despite advances in our knowledge of D. immitis and D. repens and the pathologies that they inflict on different hosts, there are still many unknown aspects of dirofilariasis. This review is focused on human and animal dirofilariasis, including the basic morphology, biology, protein composition, and metabolism of Dirofilaria species; the climate and human behavioral factors that influence distribution dynamics; the disease pathology; the host-parasite relationship; the mechanisms involved in parasite survival; the immune response and pathogenesis; and the clinical management of human and animal infections.
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            Discrimination between six species of canine microfilariae by a single polymerase chain reaction.

            Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.
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              A novel Dirofilaria species causing human and canine infections in Hong Kong.

              Dirofilariasis is globally the commonest manifestation of zoonotic filariasis. We report the detection of a novel canine species causing human and canine dirofilariasis in Hong Kong. Three human cases occurring over 10 months were identified, one presenting with cervical lymphadenopathy, one with an abdominal subcutaneous mass, and one with a subconjunctival nodule. Transected worms recovered from the resected abdominal subcutaneous mass were morphologically compatible with Dirofilaria. The cox1 gene sequences of the three human isolates were identical; however, they were only 96.2% and 89.3% identical to the cox1 gene of Dirofilaria repens and Dirofilaria immitis, respectively. Sequencing of the 18S-ITS1-5.8S gene cluster was successful in the intact worm, and the nucleotide sequences were 94.0% and 94.9% identical to those of D. repens and D. immitis, respectively. Screening of the blood samples from 200 dogs and 100 cats showed the presence of the novel Dirofilaria species in 3% (6/200) of the dogs' but none of the cats' blood samples. Nucleotide sequences of the cox1 gene and 18S-ITS1-5.8S gene clusters of the dogs' samples were identical to those in the human samples. The sera of canines infected by this novel Dirofilaria species were negative when tested with the SNAP 4Dx D. immitis detection kit, except in the case of dogs with a mixed infection with D. immitis as detected by PCR. The results from this study suggest that this novel Dirofilaria species is a cause of filarial infection in humans and dogs in Hong Kong. We propose to name this Dirofilaria species "Candidatus Dirofilaria hongkongensis."
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                Author and article information

                Journal
                Indian J Med Res
                Indian J. Med. Res
                IJMR
                The Indian Journal of Medical Research
                Medknow Publications & Media Pvt Ltd (India )
                0971-5916
                October 2017
                : 146
                : 4
                : 528-533
                Affiliations
                [1 ] Department of Veterinary Biochemistry, College of Veterinary & Animal Sciences, Thrissur, India
                [2 ] Department of Veterinary Parasitology, College of Veterinary & Animal Sciences, Thrissur, India
                Author notes
                Reprint requests: Dr Bindu Lakshmanan, Department of Veterinary Parasitology, College of Veterinary & Animal Sciences, Mannuthy, Thrissur 680 651, Kerala, India e-mail: bindul@ 123456kvasu.ac.in
                Article
                IJMR-146-528
                10.4103/ijmr.IJMR_1425_15
                5819036
                29434068
                3d4350f8-5f3c-49f7-996e-01d4fb69fd21
                Copyright: © 2018 Indian Journal of Medical Research

                This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

                History
                : 07 September 2015
                Categories
                Original Article

                Medicine
                5s ribosomal rna gene,cytochrome oxidase subunit 1 gene,dirofilaria repens,dirofilariosis,human,polymerase chain reaction

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