Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum

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Laidoudi, Younes | Davoust, Bernard | Varloud, Marie | Niang, El Hadji Amadou | Fenollar, Florence | Mediannikov, Oleg

Edité par CCSD ; BioMed Central -

International audience. Background: Dirofilaria immitis,D. repensandAcanthocheilonema reconditumare the main causative agents of zoonotic canine filariosis. Methods: We developed a combined multiplex approach for filaria andWolbachiadetection using the28S-based pan-filarial and16S-based pan-WolbachiaqPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targetingA. reconditum,D. immitisandD. repens, and a duplex qPCR targetingWolbachiaofD. immitisandD. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitisandAngiostrongylus vasorum) and pan-filarialcox1 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 ofWolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitisand itsWolbachiawere considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachiaas well asA. reconditum DNA indicates true positive infections. Results: The detection limit forWolbachia and filariae qPCRs ranged from 5 x 10(-1) to 1.5 x 10(-4) 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 ofWolbachia. 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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