False detection of Coxiella burnetii-what is the risk?

The insertion sequence IS1111 is used formolecular detection of Coxiella burnetii but also found in Coxiella-like endosymbionts of ticks, presenting a risk of false positive detection of C. burnetii. Limited IS1111 sequences from Coxiella-like bacteria restrict in silico assessment of IS1111 assays. However, Coxiella-like bacteria detectable by IS1111 assays appear to be rare in tick populations, limiting the impact of false positives on C. burnetii prevalence estimations. C. burnetii can be distinguished from Coxiella-like bacteria usingC. burnetii SNP genotyping assays. Such assays can be used for detection, but are best used as post hoc tests given the extreme sensitivity of assays that target the multiple copy IS1111. Recently, Duron (2015) demonstrated the presence of IS1111 in Coxiella-like endosymbionts of ticks. This work adds to our knowledge of sequence diversity within IS1111 from Coxiellalike endosymbionts. Duron et al. (2015) also show that Coxiellalike endosymbionts are widespread among tick species. Detection assays for the Q fever pathogen, C. burnetii, often target portions of IS1111 (Kim et al. 2005; Klee et al. 2006; Loftis et al. 2006; Panning et al. 2008; de Bruin et al. 2011); the repetitive nature of IS1111 providesmultiple targets for primer annealing, resulting in unparalleled detection sensitivity. Given the presence of IS1111 in Coxiella-like bacteria, Duron and colleagues (Duron 2015; Duron et al. 2015; Jourdain et al. 2015) raised the concern that these C. burnetii detection assays may lead to misidentification with Coxiella-like bacteria. Furthermore, these authors demonstrate that an IS1111 based assay does indeed amplify Coxiella-like bacteria (Jourdain et al. 2015). While these findings have an important bearing on C. burnetii research, current evidence suggests that in most situations, the overall risk of false detection of C. burnetii is low and can be mitigated with current methods. Sequence alignment of haplotypes presented byDuron (2015) and Vilcins, Old and Deane (2009) that include primer and probe sequences from popular IS1111 qPCR assays demonstrate substantial gaps in our knowledge about diversity of this genomic region among Coxiella-like bacteria (Fig. 1 and Supplemental Files). In particular, the region (∼570bp) sequenced by Duron (2015) covers the primer and probe regions of only one assay. These 10 haplotypes are highly diverse and only 3 (from Rhipicephalus decoloratus, Bothriocroton auruginans and Ornithodoros maritimus) show matches at primer/probe regions likely to result in amplification. Thus, out of 115 tick samples known to be positive for endosymbiont bacteria (Duron 2015), only 4 would test positive with this assay. Jourdain et al. (2015) detected positive results in four additional tick species, but did not publish the primer/probe sequences. While we do not know the likelihood of other IS1111 assays detecting Coxiella-like bacteria, it is reasonable to expect that the diversity exhibited in this region can be extrapolated to flanking regions, causing highly variable amplification. Undoubtedly, additional diversity in IS1111 exists among Coxiella-like bacteria, and future sequencing will provide better estimates of the degree to which various assays differentiate between C. burnetii and Coxiella-like bacteria. Current evidence therefore suggests that cross-reactivity does indeed occur for one assay at an unknown region (Jourdain et al. 2015), is likely for another (Fig. 1), and possible for others. The works by Duron (2015); Duron et al. (2015); Jourdain et al. (2015) are based on tick species known to harbor Coxiella-like endosymbionts. Unless environmental surveys for C. burnetii contain a high proportion of these species, few samples are likely to test positive for Coxiellalike bacteria using IS1111 assays. However, given the low prevalence of C. burnetii in ticks, a few false positives can have a large proportional impact on prevalence estimations. For positive identification of C. burnetii, Duron and colleagues (Duron 2015; Duron et al. 2015; Jourdain et al. 2015) suggest testing other targets in addition to IS1111. They demonstrate that other targets (GroEL/htpB, p1, scvA) are also contained in