Development of a multiplexed qPCRs-based approach for the diagnosis ofDirofilaria immitis, D. repens,Acanthocheilonema reconditumand the others filariosis
Authors: Laidoudi Younes , Davoust Bernard , Varloud Marie , Niang El Hadji Amadou , Fenollar Florence , Mediannikov Oleg 
Published: November 2019

Background

The frequent canine filariosis are caused by zoonotic filarial nematodes calledDirofilaria immitis,D. repensandAcanthocheilonema 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.

Findings

Herein, we developed and set up a novel molecular approach for an improved detection of the occult and non-occult filarioses, especially those caused byA. reconditum,D. immitisandD. repensas 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 targetingA. reconditum, D. immitisandD. repens, and a duplex qPCR targetingWolbachiaofD. immitisand that ofD. repens. The analytical sensitivity of the newly qPCR systems was confirmed by the detection limit of wolbachia and filaria DNA ranged from 5E-1to 1.5E-4mf/ml of blood with an R² higher than 0.99, Cohen’s 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 theirWolbachia, 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 andWolbachiagenotype were also identified by the combined multiplex approach from all the positive samples. The single DNA ofD. immitiswas identified in 12 samples,D. repensin 7, andA. reconditumin 15 samples, the co-infection was observed in 5 samples, 4 for bothDirofilariaand one harbored the three species. Therefore, 22 samples were positive forWolbachiaendosymbiont ofD. immitis, 3 for that ofD. repensand 5 for both genotypes. A newly duplex qPCR developed for the differential diagnosis of heartworm and French heartworm (Angiostrongylus vasorum) was successfully validatedin 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.

Conclusion

The proposed molecular tool targeting filarial genes and associatedWolbachiagenes 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.