Development of a multiplexed qPCRs-based approach for the diagnosis of Dirofilaria immitis, D. repens, Acanthocheilonema reconditum and the others filariosis

Abstract

BackgroundThe frequent canine filariosis are caused by zoonotic filarial nematodes called Dirofilaria immitis, D. repens and Acanthocheilonema reconditum (Spirurida: Onchocercidae). The absence of reliable diagnostic tools to diagnose and discriminate between these infections as well as their different forms constitutes a major obstacle to their control. The serological diagnosis of heartworm disease has recently shown both sensitivity and specificity problems. FindingsHerein, we developed and set up a novel molecular approach for an improved detection of the occult and non-occult filarioses, especially those caused by A. reconditum, D. immitis and D. repens as well as their differential diagnosis based on qPCRs assays. This approach designated the "Combined multiplex approach", proceeds as follows: Filaria and wolbachia identification using the newly customized 28S-based pan-filarial and 16S-based pan-wolbachia qPCRs, respectively, followed by the fast typing method of positive samples using the triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and that of D. repens. The analytical sensitivity of the newly qPCR systems was confirmed by the detection limit of wolbachia and filaria DNA ranged from 5E-1 to 1.5E-4 mf/ml of blood with an R{superscript 2} higher than 0.99, Cohens Kappa agreement ranged from 0.98 to 1. The approach was complemented by a pan-filarial COI and pan-Wolbachia ftsZ PCR for the identification of other filarial parasites and their Wolbachia, respectively. When tested on clinical samples, the results are as follows: 29.2 % (49/168) tested positive to at least filariae or wolbachiae DNA. 19 samples of them tested positive for filarial DNA, 9 for wolbachia DNA and 21 for both. Filarial species and Wolbachia genotype were also identified by the combined multiplex approach from all the positive samples. The single DNA of D. immitis was identified in 12 samples, D. repens in 7, and A. reconditum in 15 samples, the co-infection was observed in 5 samples, 4 for both Dirofilaria and one harbored the three species. Therefore, 22 samples were positive for Wolbachia endosymbiont of D. immitis, 3 for that of D. repens and 5 for both genotypes. A newly duplex qPCR developed for the differential diagnosis of heartworm and French heartworm (Angiostrongylus vasorum) was successfully validated in vitro. However, no DNA of this latter was detected in canine blood samples used in this study. The immunochromatographic test for dirofilariasis antigen during evaluation before and after thermal pretreatment of sera showed substantial agreement (K=0.6) and weak agreement (K=0.15), respectively. ConclusionThe proposed molecular tool targeting filarial genes and associated Wolbachia genes is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariasis. We believe that the current diagnosis of heartworm based on antigen detection should be always confirmed by qPCR-based essays; the heat-pretreatment of sera is useless and strongly discouraged.