Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum
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BACKGROUND: Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. METHODS: We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearman's correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. RESULTS: mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98-1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively. CONCLUSIONS: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.
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D. repens affects dogs and other carnivores, causing subcutaneous dirofilariasis.
“D. repens affects dogs and other carnivores, causing subcutaneous dirofilariasis [44,45].”
From Emergent and Neglected Equine Filariosis in Egypt: Species Diversity and Host Immune Response (2022) · cites this paper as [45]
The Background identifies dogs as natural hosts of D. repens and says the parasite causes subcutaneous infection. It does not state that D. repens affects other carnivores, so that part of the claim is not supported by our copy. (LLM, conf=0.86)
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- Authors
- Laidoudi, Y., Davoust, B., Varloud, M., Niang, E. H. A., Fenollar, F., & Mediannikov, O.
- Affiliations
- Aix-Marseille Université (FR)
- Corresponding author
- Mediannikov, Oleg
- ORCID
- Venue
- Parasites & Vectors
- Published
- 2020-06-22
- Volume
- 13
- Issue
- 1
- Pages
- 319-319
- ISSN
- 1756-3305 (ISSN-L)
- PMC
- PMC7309989
- OpenAlex
- W3035815136
- Cited by
- 74
- References
- 59
- Citations by year
- 2026: 9 · 2025: 11 · 2024: 8 · 2023: 9 · 2022: 16 · 2021: 10 · 2020: 10 · 2019: 1
- FWCI
- 1.48
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Parasites & Vectors (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- HAL (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Zoetis
- Agence Nationale de la Recherche (10 -IAHU-03, ANR-10-IAHU-0003, ANR-10IAHU-03, ANR-10)
- Topics
- Health Sciences › Medicine › Infectious Diseases › Parasitic Diseases Research and Treatment
- Health Sciences › Veterinary › Small Animals › Helminth infection and control
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Research on Leishmaniasis Studies
- Keywords
- Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum, Wolbachia, multiplex real-time PCR, zoonotic filariasis, Angiostrongylus vasorum, occult infection, molecular diagnosis, 28S rRNA
- MeSH terms
- Angiostrongylus, Animals, Antigens, Helminth, Bacterial Proteins, Diagnosis, Differential, Dog Diseases, Dogs, Filariasis, Filarioidea, Genotype, Sensitivity and Specificity, Helminth Proteins, Wolbachia, Molecular Diagnostic Techniques, Multilocus Sequence Typing, Real-Time Polymerase Chain Reaction
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