Development of a multiplexed qPCRs-based approach for the diagnosis ofDirofilaria immitis, D. repens,Acanthocheilonema reconditumand the others filariosis

Abstract

27Background: The frequent canine filariosis are caused by zoonotic filarial nematodes 28 called Dirofilaria immitis, D. repens and Acanthocheilonema reconditum (Spirurida: 29 Onchocercidae). The absence of reliable diagnostic tools to diagnose and discriminate 30 between these infections as well as their different forms constitutes a major obstacle to 31 their control. The serological diagnosis of heartworm disease has recently shown both 32 sensitivity and specificity problems. 33Findings: Herein, we developed and set up a novel molecular approach for an improved 34 detection of the occult and non-occult filarioses, especially those caused by A. 35 reconditum, D. immitis and D. repens as well as their differential diagnosis based on 36 qPCRs assays. This approach designated the "Combined multiplex approach", proceeds 37 as follows: Filaria and wolbachia identification using the newly customized 28S-based 38 pan-filarial and 16S-based pan-wolbachia qPCRs, respectively, followed by the fast 39 typing method of positive samples using the triplex qPCR targeting A. reconditum, D. 40 immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and that of 41 D. repens. The analytical sensitivity of the newly qPCR systems was confirmed by the 42 detection limit of wolbachia and filaria DNA ranged from 5E -1 to 1.5E -4 mf/ml of blood 43 with an R² higher than 0.99, Cohen's Kappa agreement ranged from 0.98 to 1. The 44 approach was complemented by a pan-filarial COI and pan-Wolbachia ftsZ PCR for the 45 identification of other filarial parasites and their Wolbachia, respectively. 46 When tested on clinical samples, the results are as follows: 29.2 % (49/168) tested 47 positive to at least filariae or wolbachiae DNA. 19 samples of them tested positive for 48 filarial DNA, 9 for wolbachia DNA and 21 for both. Filarial species and Wolbachia 49 genotype were also identified by the combined multiplex approach from all the positive 50 samples. The single DNA of D. immitis was identified in 12 samples, D. repens in 7, and 51 3 A. reconditum in 15 samples, the co-infection was observed in 5 samples, 4 for both 52Dirofilaria and one harbored the three species. Therefore, 22 samples were positive for 53 Wolbachia endosymbiont of D. immitis, 3 for that of D. repens and 5 for both genotypes. 54A newly duplex qPCR developed for the differential diagnosis of heartworm and French 55 heartworm (Angiostrongylus vasorum) was successfully validated in vitro. However, no 56 DNA of this latter was detected in canine blood samples used in this study. The 57 immunochromatographic test for dirofilariasis antigen during evaluation before and after 58 thermal pretreatment of sera showed substantial agreement (K=0.6) and weak agreement 59 (K=0.15), respectively. 60 Conclusion:The proposed molecular tool targeting filarial genes and associated 61 Wolbachia genes is a reliable tool for the exploration and diagnosis of occult and non-62 occult canine filariasis. We believe that the current diagnosis of heartworm based on...

Detection of Canine Vector-Borne Filariasis and Their Wolbachia Endosymbionts in French Guiana

“…This tool had gained an important increase in research area [41,42]. In addition, several studies have recently associated the molecular detection of Wolbachia in the diagnosis of canine filariasis [9,10,27].…”

“…This represents a limitation of this study, which decrease the accuracy of our results in terms of sensitivity and specificity. Likewise, the molecular assays are able to detect the DNA from a micro-fragment of microfilaria with an analytical sensitivity threshold of 1.5 × 10 −4 mf/mL [27].…”

“…First, all samples were tested using the combined multiplex approach, recently developed for the detection of filarioid and Wolbachia DNAs [27]. The screening was processed as follows: (i) the exploration of filarial and Wolbachia DNAs using, respectively, the pan-filarial [Pan-Fil 28S] and the pan-Wolbachia [All-Wol-16S] qPCRs (…”

“…First, all samples that tested positive for filarial DNA were subjected to PCR amplification and sequencing analysis using the following systems: The standard (Pan-Nematoda) PCR [9] was used to amplify 1194 pb from the small subunit rRNA gene and the filarial specific (Pan-fil COI) PCR [27] targeting 509 pb from the Cytochrome c oxidase subunit I (cox1) gene. The third PCR was developed to amplify 497-570 pb from the 12S rDNA gene of filarial nematodes (Table 1), while all Wolbachia-positive samples were tested using the standard (W16S-Spec) PCR [30] targeting 438 pb fragment from the 16S rDNA.…”

“…These features constitute make its a suitable target for the diagnosis of filarial infection, especially when occurring in dead-end hosts as is the case of D. immitis in humans and cats [24,25]. Recent studies showed that the combined detection of Wolbachia and filarioid DNA improves the diagnosis of these infections [26,27].…”