Abstract
Purpose
Human ocular dirofilariasis is a zoonotic disease caused by several species of filarioid helminths of the genus Dirofilaria. The aim of this study was to further re-examine five preserved specimens previously isolated from patients with ocular dirofilariasis by molecular means.
Methods
Four of the examined helminths had been stored in unbuffered formaldehyde solution for more than eight years; whereas, the fifth helminth was stored in ethanol buffer for more than two years. For the four specimens stored in formaldehyde, different methods of DNA recovery and amplification were applied and investigated for their efficiency in DNA extraction and PCR amplification. However, the DNA extraction and PCR amplification were successful only for the ethanol-preserved helminth.
Results
The genetic identification of the ethanol-preserved specimen as Dirofilaria repens (D. repens) and its phylogenetic position based on the analysis of mitochondrial 12S ribosomal RNA, nuclear 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit one sequences are reported in the present paper. To our knowledge, these are the only deposited sequences related to D. repens that have been isolated in Greece.
Conclusions
Routine laboratory diagnosis is based on phenotypic characteristics of the helminthic parasites, but more accurate diagnosis requires molecular identification. Although the specimens preserved in formalin buffers may be a potential source for the enrichment of parasite genome databases, the DNA recovery of such samples is a challenging task.
References
Anderson RC (2000) Nematode parasites of vertebrates. Their development and transmission, 2nd edn. CABI Publishing, Wallingford. https://doi.org/10.1079/9780851994215.0000
Otranto D, Dantas-Torres F, Brianti E, Traversa D, Petrić D, Genchi C, Capelli G (2013) Vector-borne helminths of dogs and humans in Europe. Parasit Vectors 6:16. https://doi.org/10.1186/1756-3305-6-16
Pampiglione S, Rivasi F (2000) Human dirofilariasis due to Dirofilaria (Nochtiella) repens: an update of world literature from 1995 to 2000. Parassitologia 42:231–254
Beaver PC (1989) Intraocular filariasis: a brief review. Am J Trop Med Hygc 40:40–45. https://doi.org/10.4269/ajtmh.1989.40.40
Kersten RC, Locastro AJ, Eberhard ML, Spaulding AG, Kulwin DR (1994) Periorbital dirofilariasis. Ophthal Plast Reconstr Surg 10:293–296. https://doi.org/10.1097/00002341-199412000-00015
Sathyan P, Manikandan P, Bhaskar M, Padma S, Singh G, Appalaraju B (2006) Subtenons infection by Dirofilaria repens. Indian J Med Microbiol 24:61–62. https://doi.org/10.4103/0255-0857.19899
Bhat KS, Kotigadde S, Vishwanatha Bhat K, Pare P (2014) Subconjunctival dirofilariasis: case studies with review of literature. Trop Parasitol 4:119–121. https://doi.org/10.4103/2229-5070.138541
Simón F, López-Belmonte J, Marcos-Atxutegi C, Morchón R, Martín Pacho JR (2005) What is happening outside North America regarding human dirofilariasis? Vet Parasitol 133:181–189. https://doi.org/10.1016/j.vetpar.2005.03.033
Gorezis S, Psilla M, Asproudis I, Peschos D, Papadopoulou C, Stefaniotou M (2006) Intravitreal dirofilariasis: a rare ocular infection. Orbit 25(1):57–59. https://doi.org/10.1080/01676830500499224
Capelli G, Genchi C, Baneth G, Bourdeau P, Brianti E, Cardoso L, Danesi P, Fuehrer HP, Giannelli A, Ionică AM, Maia C, Modrý D, Montarsi F, Krücken J, Papadopoulos E, Petrić D, Pfeffer M, Savić S, Otranto D, Poppert S, Silaghi C (2018) Recent advances on Dirofilaria repens in dogs and humans in Europe. Parasit Vectors 11:663. https://doi.org/10.1186/s13071-018-3205-x
Kalogeropoulos CD, Stefaniotou MI, Gorgoli KE, Papadopoulou CV, Pappa CN, Paschidis CA (2014) Ocular dirofilariasis: a case series of 8 patients. Middle East Afr J Ophthalmol 21(4):312–316. https://doi.org/10.4103/0974-9233.142267
Taleb-Hossenkhan N, Bhagwant S, Gourrege N (2013) Extraction of nucleic acids from ancient formalin- and ethanol-preserved specimens of the tapeworm Bertiella studeri: which method works best? J Parasitol 99:410–416. https://doi.org/10.1645/GE-3197.1
Suzuki J, Kobayashi S, Okata U, Matsuzaki H, Mori M, Chen KR, Iwata S (2015) Molecular analysis of Dirofilaria repens removed from a subcutaneous nodule in a Japanese woman after a tour to Europe. Parasite 22:2. https://doi.org/10.1051/parasite/2015002
Shedlock AM, Haygood MG, Pietsch TW, Bentzen P (1997) Enhanced DNA extraction and PCR amplification of mitochondrial genes from formalin-fixed museum specimens. Biotechniques 22:394–400. https://doi.org/10.2144/97223bm03
Gilbert MT, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M (2007) The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS ONE 6:e537. https://doi.org/10.1371/journal.pone.0000537
Golenberg EM, Bickel A, Weihs P (1996) Effect of highly fragmented DNA on PCR. Nucleic Acids Res 24:5026–5033. https://doi.org/10.1093/nar/24.24.5026
Shi SR, Datar R, Liu C, Wu L, Zhang Z et al (2004) DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution. Histochem Cell Biol 122:211–218. https://doi.org/10.1007/s00418-004-0693-x
Yilmaz E, Fritzenwanker M, Pantchev N, Lendner M, Wongkamchai S, Otranto D, Kroidl I, Dennebaum M, Le TH, Le Anh T, Ramünke S, Schaper R, von Samson-Himmelstjerna G, Poppert S, Krücken J (2016) The mitochondrial genomes of the zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) Honkongensis provide evidence for presence of Cryptic Species. PLoS Negl Trop Dis 10(10):e0005028. https://doi.org/10.1371/journal.pntd.0005028
Dietrich D, Uh B, Sailer V, Holmes EE, Jung M, Meller S, Kristiansen G (2013) Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition. PLoS ONE 10:e77771. https://doi.org/10.1371/journal.pone.0077771
Wu L, Patten N, Yamashiro CT, Chui B (2002) Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues. Appl Immunohistochem Mol Morphol 10:269–272. https://doi.org/10.1097/00129039-200209000-00015
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. https://doi.org/10.1093/nar/22.22.4673
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704. https://doi.org/10.1080/10635150390235520
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Michalski ML, Bain O, Fischer K, Fischer PU, Kumar S, Foster JM (2010) Identification and phylogenetic analysis of Dirofilaria ursi (Nematoda: Filarioidea) from Wisconsin black bears (Ursus americanus) and its Wolbachia endosymbiont. J Parasitol 96:412–419. https://doi.org/10.1645/GE-2208.1
Purzycka JK, Olewiecki I, Soltyszewski I, Pepinski W, Janica J (2006) Efficiency comparison of seven different Taq polymerases used in hemogenetics. Int Congr Ser 1288:719–721. https://doi.org/10.1016/j.ics.2005.09.073
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Ethical Approval
The worms were not collected for the purpose of this study but were removed from the patients by the ophthalmologists to treat ocular dirofilariasis and they were sent to the Microbiology laboratory for further light microscopy identification. The present study is a retrospective study of the worms collected through years.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Bozidis, P., Sakkas, H., Pertsalis, A. et al. Molecular Analysis of Dirofilaria repens Isolates from Eye-Care Patients in Greece. Acta Parasit. 66, 271–276 (2021). https://doi.org/10.1007/s11686-020-00257-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11686-020-00257-4