, 2005). For the ‘SFG’ set, a mean cycle threshold (Ct) value below 35 indicates the sample is

positive, and a Ct value above 35 indicates the sample is positive if another set is positive and/or a sequence is obtained and/or serology is positive. Thus, samples are run in duplicate using sets targeting two different genes. From January 2009 to December 2009, the set ‘RAF-plasmid’ was used to detect R. africae; its target gene is located on a plasmid of the species. Following recent R. africae genome sequencing, it was reported that this plasmid might be unstable. check details To avoid false-negative results, we designed a new primer and probe set targeting a non-plasmidic gene. Consequently, the set ‘RAF’ was used to detect R. africae in clinical samples from January 2010 to December 2010. We retrospectively collected data for the molecular diagnosis

of rickettsioses from January 2009 to December 2010 to assess the usefulness of this strategy. Except for the ‘SFG’ set, which had been previously described (Socolovsch et al., 2010), the sets were found to be specific for the corresponding rickettsial species both in silico and in vitro, when tested against a panel of 30 rickettsial strains (Fig. 1a). Sensitivity was also evaluated using 10-fold serial dilutions (Fig. 1b). A total of 643 clinical specimens corresponding to 465 different patients were received at the FNRC from January 2009 to December 2010. Among these, find more 204 originated from locally hospitalized patients, 218 from other French hospitals and 43 from international hospitals. Forty-five positive qPCRs

were obtained: 31/150 cutaneous biopsies, 8/42 cutaneous swab specimens, 2/223 total blood samples and 4/94 serum samples. The first molecular screening of SFG Rickettsia using the set labelled ‘SFG’ was positive for 44 samples; the 45th sample was positive using the set labelled ‘TG’, which detects TG Rickettsia. Among 45 positive results, 11 were obtained from locally hospitalized Orotic acid patients, 32 from other French hospitals and two from international hospitals. A final diagnosis of R. africae was obtained for 15 samples (13 cutaneous biopsies, two eschar swabs) corresponding to 15 different patients with a diagnosis of ATBF; five samples were positive for the sets ‘SFG’ and ‘RAF-plasmid’, and 10 samples were positive for the sets ‘SFG’ and ‘RAF’. A final diagnosis of R. conorii was obtained for nine samples corresponding to nine different patients with a diagnosis of MSF; eight samples (cutaneous biopsies) were positive for the sets ‘SFG’ and ‘RCO’. One remaining sample (serum) was positive for the set ‘SFG’ and negative for ‘RCO’; a final diagnosis of R. conorii was obtained using conventional PCR followed by sequencing. A final diagnosis of R. honei was obtained for one sample (serum) corresponding to a patient whose final diagnosis was FISF (Murphy et al., 2011); it was positive for the set ‘SFG’, and a final diagnosis of R.